Banana starch and molecular shear fragmentation dramatically increase structurally driven slowly digestible starch in fully gelatinized bread crumb

1 Banana starch and molecular shear fragmentation dramatically increase structurally driven slowly digestible starch in fully gelatinized bread crumb Laura Román1,4, Manuel Gómez1, Bruce R. Hamaker4, Mario M. Martinez2,3,4* 1Food Technology Area. College of Agricultural Engineering, University of Valladolid, 34004 Palencia, Spain. 2School of Engineering, University of Guelph, Guelph, ON N1G 2W1, Canada. 3Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada. 4Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University, IN 47907, USA. *Corresponding author e-mail: [email protected]; Abstract The role of native (NB) and extruded (EB) banana starch, and a 1:1 native:extruded banana starch composite (MB), in slowing down the starch digestibility of bread crumb and crust was investigated. During extrusion, the molecular weight of banana starch was reduced from 2.75x108 to 4.48x106 g/mol (HPSEC-MALS-RI). Results showed a slowly digestible starch (SDS) increase from 1.09 % (control) to 4.2, 6.6, and 7.76 % in NB, MB and EB crumbs (fully gelatinized), respectively. DSC data attributed this occurrence to the formation of supramolecular structures upon storage involving amylopectin branches (especially those from fragmented amylopectin in EB). The hedonic sensory test showed no differences in overall liking between MB, EB and control, validating feasibility of including banana in the formulation. For the first time, this study shows a molecular size reduction as a strategy to manufacture selected starches that result in highly gelatinized baked products rich in structurally driven SDS. Keywords: banana starch; extrusion; digestion; retrogradation; gluten-free bread 2 1 Introduction Dietary glycemic carbohydrates that result in small excursions in postprandial plasma glucose and insulin concentrations are associated with improved insulin secretion and sensitivity and enhanced glycemic control (Vinoy, Laville, & Feskens, 2016). On the other hand, recurrent postmeal high blood glucose levels have been linked to an increased risk of cardiovascular events and type two diabetes mellitus (Ceriello & Colagiuri, 2008). Starch, the major polysaccharide related to postprandial glycaemia, is hydrolyzed by salivary and pancreatic α-amylase in the mouth and small intestine of humans, and then to glucose by the mucosal brush border α-glucosidases. The rate and extent to which starch is digested by these enzymes will greatly influence differences in the glycemic response of starch-based products. Since bread constitutes the highest proportion of carbohydrates (mainly starch) to the daily dietary intake in many parts of the world (Cust et al., 2009), successful strategies that decrease the digestibility of fully gelatinized starch might impact the prevalence of metabolic syndrome associated diseases. In baked goods, the rate of starch digestibility has been correlated with the degree of starch gelatinization (Englyst, Vinoy, Englyst, & Lang, 2003). The amorphous structure of gelatinized starch results in a greater availability of α-amylase binding sites, which makes the substrate more susceptible to enzyme hydrolysis. Most of starches have been reported to gelatinize approximately in the range from 54 to 78 oC in excess of water (≥ 20% water) (Vamadevan, Bertoft, & Seetharaman, 2013). Therefore, considering that the moisture content of bread crumb is always ≥ 35% (Martinez, Roman, & Gomez, 2018), where temperatures ≥ 70 oC are reached (Thorvaldsson & Skjoldebran, 1998), a complete starch gelatinization in high moisture baked goods, such as bread, is expected to occur. In fact, Primo-Martin, van Nieuwenhuijzen, Hamer, and van Vliet (2007) and Martinez et al. (2018) reported a complete starch gelatinization in wheat bread crumb. Furthermore, Martinez et al. (2018) reported a complete starch gelatinization in candeal bread, also known as Fabiola or Bregado, with only 45% dough hydration (close to the minimum amount of water possible to make bread). Retrogradation is the recrystallization process of glycan chains in gelatinized starch, during which dispersed starch molecules begin to re-associate through H-bonding. Albeit retrogradation has been reported as one reason for bread staling (Gray, & BeMiller 2003), it is also known to result in a reduction of the rate and extension of starch digestion, depending on the main constituent involved. Thus, amylose double 3 helices are known to be enzymatically resistant and yield resistant starch (RS) [Patel et al., 2017], whereas retrograded amylopectin has been attributed to the formation of slowly digestible starch (SDS) [Cui & Oates, 1997; Zhang, Sofyan, M., & Hamaker, 2008]. Based on previous evidence indicating that cereal starches containing high proportions of amylopectin with long chains (Benmoussa, Moldenhauer, & Hamaker, 2007), or amylopectin with longer chains (Zhang, et al. 2008), result in a higher amount of slowly digestible starch in fully gelatinized material, Martinez, Li, Okoniewska, Mukherjee, Vellucci, and Hamaker (2018) investigated starch molecular structuredigestibility relationships in fully gelatinized starches. These authors reported for the first time two potential factors that may result in slowly digestible supramolecular structures during storage, namely: 1) double helices between A and B1 chains (unsubstituted and substituted short chains with a similar degree of polymerization, DP < ~36, respectively) of an average length ≥ 15.5 Glucose Units (perhaps involving internal long chains) that also are prone to forming intermolecular associations and; 2) interactions of starch molecules with lower molecular size that may be more mobile and easily aligned during retrogradation. However, the effect of starch molecules with both significantly small size and long amylopectin chains resulting in structurally-driven slowly digestible starch has not been studied. Banana starch has been reported to have amylopectin with small size (Yoo & Jane, 2002) and a low proportion of short chains (Zhang & Hamaker, 2012). It can be added into bakery products as purified starch or starch-predominant flour particles after simple liquid-solid extraction (Zhang, Whistler, BeMiller, & Hamaker, 2005) or after simple processing involving drying and grinding (Segundo, Román, Gómez, & Martínez, 2017) of banana pulp, respectively. When banana bunches arrive at central collection stations, bananas too small for shipping are removed, along with those that have damaged or spoiled areas. These rejected bananas are normally disposed of improperly and their successful industrial use would significantly reduce fruit food waste. Based on the aforementioned mechanistic understanding provided by Martinez et al. (2018), we hypothesized that: 1) banana starch, an untapped food by-product, would result in breads with a significant increase in slowly digestible starch and; 2) that the reduction of its molecular size through shear scission by means of extrusion (Zhang et al., 2015) would further slowdown the digestion rate of the starch glycemic fraction in breads. The objective of this study was, therefore, to identify a starch material that results in slowly digestible starch from fully gelatinized material. To do so, gluten-free breads served as 4 an ideal high-moisture baked system (and therefore with extensive starch gelatinization) to investigate the effect of native and extruded banana starch on their in vitro starch digestion and physical and sensory properties. The starch digestion fractions and degree of starch gelatinization and retrogradation were analyzed in the crust and the crumb separately to provide insights about the spatial location and origin of slowly digestible starch. 2 Materials and Methods 2.1 Materials Atlantic rice flour (9.58 % moisture, 7.43 % protein, 0.11 % RS, 12.49 % SDS) was provided by Molendum Ingredients (Zamora, Spain), Miwon maize starch (8.07 % moisture, non-detected protein, 0.76 % RS, 63.44 % SDS) was purchased from Daesang Co. (Seoul, Korea) and banana starch from green lady finger bananas (6.30 % moisture, 3.17 % protein, 42.21 % RS, 8.70 % SDS) was purchased from Natural Evolution (Walkamin, Queensland, Australia). Starchy materials (including extruded banana flour) were characterized according to AACC methods (AACC, 2015) for moisture (4415.02), protein content (46-30.01) with a Leco TruSpec device (Leco, St. Joseph, MI, USA) and resistant starch content (32-40.01) using RS Megazyme assay kit. SDS was analyzed as detailed in section 2.2.5. Particle size was measured with a laser diffraction particle size analyzer (Mastersizer 3000, Malvern Instruments, Ltd., Worcestershire, UK). The mean diameter of equivalent volume d(4,3), which indicates the central point of the volume distribution of the particles, was recorded. The plot of the particle size distribution is included as Fig. 1A. Thermal properties were analyzed in triplicate as reported by Roman, Gomez, Li, Hamaker and Martinez (2017) using a differential scanning calorimeter Q-20 (TA instruments, Crawley, UK) equipped with a RCS 40 refrigerated cooling system (supplementary material 1 and 3). Furthermore, the pasting profile of the starchy materials (supplementary material 2) was obtained following the standard method 61-02.01 (AACC, 2015) with a Rapid Visco Analyser (RVA-4) (Perten Instruments Australia, Macquarie Park, Australia). The ingredients used for bread making were VIVAPUR 4KM HPMC (Hydroxypropyl Methylcellulose, JRS, Rosenberg, Germany), Saf-Instant dry yeast (Lesaffre, Lille, France), salt (Union Salinera de España, Madrid, Spain), sucrose (Azucarera, AB, Madrid, Spain), Abrilsol sunflower oil (Ourense, Spain) and tap water. For performing starch in vitro digestion through the Englyst test, amyloglucosidase (EC 3.2.1.3) from 5 Aspergillus niger, 300 U/mL, pepsin (EC 3.4.23.1) from porcine stomach mucosa, 8002500 U/mg solid, pancreatin from porcine pancreas, 8 x USP Specification, and guar gum were purchased from Sigma Chemical Co. (St. Louis, MO). Glucose assay reagents were from Megazyme International Ireland Ltd. (Wicklow, Ireland). 2.2 Methods 2.2.1 Extrusion process The extrusion of banana starch (final moisture of 3.33 %) was performed in a Krupp Werner and Pfleiderer ZSK-25 twin screw extruder (Ramsey, NJ, USA) with corotating and closely intermeshing screws of 25 mm diameter and length to diameter (L/D) equal to 25. The extruder was equipped with 5 barrel sections heated to 40, 60, 90, 120 and 130 ± 1 ºC from the feeding to the die, respectively. The screw speed was maintained constant to 200 rpm. A combination of feeding, transporting, compression and mixing elements was used to provide a moderate-shear screw configuration that resulted in 330 KJ/Kg of specifical mechanical energy (SME). Feed input flow rate was 5 Kg/h with a water addition to obtain a total moisture content of 22 %. Banana starch was extruded through two circular dies of 4 mm diameter (each). Extruded banana starch was dried overnight at 55 oC by convection air and then ground with a pin mill. After milling, extruded banana starch (3.29 % protein, 0.84 % RS, 42.09 % SDS) contained a mean particle size [d(4,3)] of 299 µm, which was similar to the d(4,3) of native banana starch (268 µm) due to the presence of particles formed by starch aggregates (Fig. 1A,B). 2.2.2 Native and extruded banana starch molecular structure Starch structure of native banana starch was analyzed in triplicate using the method reported by Cave, Seabrook, Gidley, and Gilbert (2009) and Vilaplana, Hasjim, & Gilbert, (2012) with minor modification as reported previously in Martinez et al. (2018). Analyses were performed using a size exclusion chromatography (SEC) system (Agilent 1260 series, Agilent Technologies, Waldbronn, Germany) equipped with a refractive index detector (RID, 1260 RID, Agilent, Agilent Technologies, Waldbronn, Germany). The length of short (A and B1) and long internal (B2 andB3,with average DP 42-48 and 69-75, respectively) amylopectin branches is denoted as XAP1 and XAP2, respectively, and the molar ratio of long to short amylopectin chains is represented as hAp2/Ap1. The amylose content of banana starch was determined from the SEC molecular size distribution of debranched starch (supplementary material 4) as the ratio of the area under the curve (AUC) of amylose branches to the AUC of overall amylopectin and 6 amylose branches (Vilaplana et al., 2012). The chain length distribution of extruded banana starch was not analyzed since it has been reported not to be altered after extrusion (Zhang et al., 2015). The molecular weight (Mw) of banana starch molecules was measured in triplicate using a multi-angle light scattering detector (MALS, Dawn Heleos, Wyatt Technology, Santa Barbara, CA, USA) containing a K5-cell. 20 µL of starch [previously dissolved in DMSO/LiBr as described in Martinez et al. (2018) for the analysis of the size of fully branched starch] were injected into GRAM 30 and 3000 columns (PSS GmbH, Mainz, Germany) connected in series (0.3 mL/min at 80 ºC). Data was analyzed with ASTRA software (version 4.72.03, Wyatt Technology Corporation, Goleta, CA, USA) and using the Berry second order plot procedure. This procedure involves an extrapolation of the function √(K*c/Rθ) to zero scattering angle θ [as sin2 (θ/2)]. K* is a constant depending on the wavelength of the light and refractive index increment of the starch and Rθ is the ‘Rayeligh excess ratio’, which is a measure of the ratio of the intensity of excess light scattered compared to pure solvent at an angle θ to that of the incident light intensity (Harding, Adams, & Gillis, 2016). The specific refractive index increment (dn/dc) was assumed to be 0.066 mL/g as usually reported for starch dissolved in DMSO (Zhong, Yokoyama, Wang, & Shoemaker, 2006) and the second viral coefficient (A2) was assumed to be negligible (Yoo & Jane, 2002). 2.2.3 Dough preparation and bread-making Breads were made with water (105 g/100 g flour-starch mixture), instant dry yeast (3 g/100 g), salt (2 g/100 g), oil (6 g/100 g), HPMC (2 g/100 g) and white sugar (5 g/100 g). The base starchy source was comprised by a 1:1 mixture of maize starch and rice flour (control). Native banana starch (NB), extruded banana starch (EB) and a 1:1 mixture of native and extruded banana starches (MB) were used as a replacement of 20 % of the base starchy material. In all makings, water temperature was held between 20 and 22 °C for yeast dissolution before its incorporation. All the ingredients were mixed for 8 min at speed 2 with a flat beater (K45B) in a Kitchen Aid 5KSM150 mixer (Kitchen Aid, Michigan, USA). Subsequently, 150 g of dough were placed in small aluminum molds (109×159×38 mm) and fermented at 30 °C and 90 % relative humidity for 60 min. After fermentation, doughs were baked in an electric modular oven for 40 min at 190 °C. After baking, the loaves were demolded after 5 min and let to cool down for a total time of 60 min before their storage in sealed polyethylene bags at 20 ºC for 24 h. All the elaborations were prepared in duplicate. 7 2.2.4 Microstructural analysis of bread Bread and banana starch photomicrographs were taken with Quanta 200FEI (Hillsboro, Oregon, USA) environmental scanning electron microscope (ESEM). Photomicrographs were taken in high vacuum mode. Crumb and crust samples were pictured from a parallel slant (lengthwise section showing the thickness of the crust), whereas crust was also imaged from a perpendicular slant (supplementary material 5 and 6). In order to better assess crumb macrostructure, bread slices were pictured with a Canon EOS 1300D (Tokyo, Japan). 2.2.5 Moisture content, starch thermal transitions and slowly digestible and resistant starch content in bread crumb and crust Crumb, taken from the center of a bread slice, and crust samples, obtained by scratching the bread surface, were analyzed for moisture content according to approved method 44-15.02 (AACC, 2015). The starch thermal transitions were measured as in Martinez et al. (2018), although results from the degree of starch gelatinization must be interpreted differently since, in the present study, a pre-gelatinized starch source is added into the recipe. Thus, DSC measures the reduction of the endothermal peak attributed to native amylopectin double helices between dough and bread (crumb or crust), which indicates the degree of starch gelatinization produced during baking (DGB). If all the starch added into the recipe was in a native state, the degree of starch gelatinization during baking would be equal to the percent of gelatinized starch in the final product (g gelatinized starch/100 g starch). However, in this case, results must be interpreted as a reduction of native starch (or an increase of gelatinized starch) during baking, which does not necessarily represent the final percent of gelatinized starch in the final product. DSC thermograms of the control dough and bread crumb and crust are reported as an example in supplementary material 3. DGB was calculated as: DGB (%) = (1 − 𝛥𝐻𝐷 𝛥𝐻𝐵)∗100 where ΔHD is the enthalpy of a portion of dough and ΔHB is the enthalpy of crumb or crust samples, respectively. The content of slowly digestible and resistant starch in flours/starches as well as in crumb and crust samples were analyzed as in Martinez et al. (2018), who followed the procedure described in Englyst, Kingman and Cummings (1992), and the AACCI approved method 32-40.01 (AACC, 2015) with the RS assay kit from Megazyme 8 (Megazyme International, Wicklow, Ireland) for slowly digestible and resistant starch fractions, respectively. Each sample was analyzed in triplicate. 2.2.6 Specific volume of breads and crumb texture Bread volume was determined using a laser sensor with the Volscan Profiler (Stable Micro Systems, Godalming, UK). The volume measurements were performed on three loaves of each elaboration. The specific volume was calculated as the ratio of bread volume to its mass. Crumb texture was measured after 24h storage with a TA-XT2 texture analyzer (Stable Microsystems, Surrey, UK) equipped with the “Texture Expert” software. A 25-mm diameter cylindrical aluminum probe was used in a “Texture Profile Analysis” (TPA) double-compression test to penetrate up to 50% of the sample depth at a test speed of 5 mm/s, with a 30 s delay between the two compressions. Hardness (g), cohesiveness, springiness and resilience were calculated from the TPA curve. Texture analyses were performed on two 30 mm central slices from two loaves from each elaboration. 2.2.7 Consumer testing Hedonic sensory evaluation of gluten-free breads was conducted with 83 volunteers, between 16-65 years of age and from various socioeconomic backgrounds. Consumer test was conducted at a sensory laboratory in individual booths. Breads were assessed for their appearance, odor, flavor, texture, and overall liking on a nine-point hedonic scale. The scale ranged from “extremely like” to “extremely dislike” corresponding with highest and lowest scores of “9” and “1”, respectively. Samples were presented on white plastic dishes coded with four-digit random numbers and served in random order. Water was available for rinsing. For the evaluation of bread appearance, a coded loaf of each bread type was presented. Samples were tested 24 h after baking. 2.2.8 Statistical analysis Differences among results were studied by analysis of variance (one-way ANOVA). Fisher’s least significant difference (LSD) was used to describe means with 95% confidence intervals. The statistical analysis was performed with the Statgraphics Centurion XVI software (Statpoint Technologies, Inc., Warrenton, USA). 3 Results and Discussion 3.1 Moisture content, degree of starch gelatinization and amylopectin retrogradation in crumb and crust 9 The moisture content ranged from 45.87 to 49.85% and from 18.40 to 21.21% in crumb and crust samples, respectively (Table 1). It is well known that, during baking, faster moisture transfer phenomena occur at the bread surface resulting in the development of a drying zone (Luyts et al., 2013), also known as crust, which will be a barrier for further heat and moisture transfer (Thorvaldsson & Skjoldebran, 1998). In contrast, as soon as bread is removed from the oven, water rapidly migrates from the interior to the crust layer (Gorton, 2009). Results indicated a greater water migration from the crumb to the crust in EB, as seen by the significantly lower and higher moisture values in crumb and crust, respectively (Table 1). This might be attributed to two simultaneous factors: 1) a lower water retention capacity (loose bound water) of extruded banana starch (as suggested with the low pasting curve shown in supplementary material 2), as a consequence of starch fragmentation (Fig. 2) and/or; 2) a lower bread specific volume that may facilitate a faster transfer of water to the crust (i.e., shorter path for water to reach the bread surface). The starch gelatinization in crumb during baking (DGB) was complete for all breads (Table 2). Conversely, DGB was limited in the crust, ranging from 5.56 % in NB to 8.48 % in EB. The fast water evaporation suffered on the bread surface as the result of the high oven temperatures causes a rapid water depletion enough to hinder starch from gelatinization in the crust (Primo-Martín et al., 2007). Interestingly, NB crust displayed a lower DGB than the control, which suggests that the higher onset temperature of starch gelatinization (To) of banana starch (71.74 ºC) compared to that from rice (67.57 ºC) and maize (62.12 ºC, supplementary material 1) is enough to result in a significant reduction of starch gelatinization in the crust (even with a 20 % replacement). On the other hand, the higher DGB in EB crust could be explained by the higher moisture content in the crust over the course of baking due to the higher water migration from the crumb to the crust as discussed before. This would result in higher availability of water for the starch to gelatinize. During cooling and storage, amylopectin branches of gelatinized starch tend to reassociate with other amylose and amylopectin chains through the formation of double helices or aggregates of double helices (Klucinec & Thompson, 2002). The melting of these structures can be detected by DSC (Martinez et al. 2018) and in this study, crumbs of breads stored for 1 day after baking exhibited the onset of this transition between 43 ± 1 ºC and 68 ± 1 ºC (supplementary material 3). The addition of banana starch resulted in an increase of the enthalpy for retrograded amylopectin in crumb (Table 2), although 16 Acknowledgments L. R. and M. G acknowledge the financial support from the European Regional Development Fund (FEDER) and the Spanish Ministry of Economy and Competitiveness (Project AGL2014-52928-C2). L. R. would like to thank the University of Valladolid for her pre-doctoral fellowship. All authors are grateful to Molendum Ingredients for supplying the rice flour. References AACC (2015). Approved methods of the American Association of Cereal Chemists, Methods 44-15.02 (moisture), 32-40.01 (resistant starch), 46–30.01 (protein), 61-02.01 (rapid visco analysis), 11th ed. American Association of Cereal Chemists, St. Paul, Minnesota. Benmoussa, M., Moldenhauer, K. A., & Hamaker, B. R. (2007). Rice amylopectin fine structure variability affects starch digestion properties. Journal of agricultural and food chemistry, 55, 1475-1479. Camire, M. E., Camire, A., & Krumhar, K. (1990). Chemical and nutritional changes in food during extrusion. Critical Reviews in Foods Science and Nutrition, 29, 35-57. Cave, R. A., Seabrook, S. A., Gidley, M. J., & Gilbert, R. G. (2009). Characterization of starch by size-exclusion chromatography: the limitations imposed by shear scission. Biomacromolecules, 10, 2245-2253. Carrillo-Navas, H., Guadarrama-Lezama, A. Y., Vernon-Carter, E. J., García-Díaz, S., Reyes, I., & Alvarez-Ramírez, J. (2016). Effect of gelatinized flour fraction on thermal and rheological properties of wheat-based dough and bread. Journal of food science and technology, 53, 3996-4006. Ceriello, A., & Colagiuri, S. (2008). International Diabetes Federation guideline for management of postmeal glucose: a review of recommendations. Diabetic Medicine, 25, 1151-1156. Cui, R., & Oates, C. G. (1997). The effect of retrogradation on enzyme susceptibility of sago starch. Carbohydrate Polymers, 32, 65-72. Cust, A. E., Skilton, M. R., van Bakel, M. M., Halkjaer, J., Olsen, A., Agnoli, C., et al. (2009). Total dietary carbohydrate, sugar, starch and fibre intakes in the European Prospective Investigation into Cancer and Nutrition. European Journal of Clinical Nutrition, 63, S37-S60. 17 Emaga, T. H., Andrianaivo, R. H., Wathelet, B., Tchango, J. T., & Paquot, M. (2007). Effects of the stage of maturation and varieties on the chemical composition of banana and plantain peels. Food Chemistry, 103, 590-600. Englyst, K. N., Vinoy, S., Englyst, H. N., & Lang, V. (2003). Glycaemic index of cereal products explained by their content of rapidly and slowly available glucose. British Journal of Nutrition, 89, 329-339. Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, S33-S50. Bi, Y., Zhang, Y., Jiang, H., Hong, Y., Gu, Z., Cheng, L., ... & Li, C. (2017). Molecular structure and digestibility of banana flour and starch. Food Hydrocolloids, 72, 219-227. Gray, J. A., & Bemiller, J. N. (2003). Bread staling: molecular basis and control. Comprehensive Reviews in Food Science and Food Safety, 2, 1-21. Gorton, L. A. (2009) Fundamental Bakery Dough Processes. In E. J. Pyler & L. A. Gorton (Eds.), Baking Science & Technology Fourth Edition. Volume II: Formulation & Products (pp. 1-136). Sosland Publishing Company, Kansas City, MO. Harding, S. E., Adams, G. G., & Gillis, R. B. (2016). Molecular weight analysis of starches: Which technique?. Starch‐Stärke, 68, 846-853. Klucinec, J., & Thompson, D. (2002). Amylopectin nature and amylose-to-amylopectin ratio as influences on the behavior of gels of dispersed starch. Cereal Chemistry, 79, 2435. Lau, E., Soong, Y. Y., Zhou, W., & Henry, J. (2015). Can bread processing conditions alter glycaemic response? Food Chemistry, 173, 250-256. Le-Bail, A., Leray, D., Lucas, T., Mariani, S., Mottollese, G., & Jury, V. (2011). Influence of the amount of steaming during baking on the kinetic of heating and on selected quality attributes of bread. Journal of Food Engineering, 105, 379-385. Luyts, A., Wilderjans, E., Haesendonck, I. V., Brijs, K., Courtin, C. M., & Delcour, J. A. (2013). Relative importance of moisture migration and amylopectin retrogradation for pound cake crumb firming. Journal of Food Engineering, 108, 128-134. Martinez, M. M., Li, C., Okoniewska, M. Mukherjee, N., Vellucci, D., & Hamaker, B. R. (2018). Structure-digestion relationships of fully gelatinized starch: a novel approach to slow down the starch digestibility in hydrothermally-treated foods. Carbohydrate Polymers, 197, 531-539. 18 Martinez, M. M., & Gómez, M. (2017). Rheological and microstructural evolution of the most common gluten-free flours and starches during bread fermentation and baking. Journal of Food Engineering, 197, 78-86. Martinez, M. M., Roman, L., & Gómez, M. (2018). Implications of hydration depletion in the in vitro starch digestibility of white bread crumb and crust. Food Chemistry, 239, 295-303. Martinez, M. M., Oliete, B., Román, L., & Gómez, M. (2014). Influence of the addition of extruded flours on rice bread quality. Journal of Food Quality, 37, 83-94. Patel, H., Royall, P. G., Gaisford, S., Williams, G. R., Edwards, C. H., Warren, F. J., ... & Butterworth, P. J. (2017). Structural and enzyme kinetic studies of retrograded starch: Inhibition of α-amylase and consequences for intestinal digestion of starch. Carbohydrate Polymers, 164, 154-161. Pongjaruvat, W., Methacanon, P., Seetapan, N., Fuongfuchat, A., & Gamonpilas, C. (2014). Influence of pregelatinised tapioca starch and transglutaminase on dough rheology and quality of gluten-free jasmine rice breads. Food Hydrocolloids, 36, 143150. Primo-Martín, C., van Nieuwenhuijzen, N. H., Hamer, R. J., & van Vliet, T. (2007). Crystallinity changes in wheat starch during the bread-making process: Starch crystallinity in the bread crust. Journal of Cereal Science, 45, 219-226. Roman, L., Gomez, M., Li, C., Hamaker, B.R., & Martinez, M.M. (2017). Biophysical features of cereal endosperm that decrease starch digestibility. Carbohydrate Polymers, 165, 180-188. Roman, L., de la Cal, E., Gomez, M., & Martinez, M. M. (2018). Specific ratio of Ato B-type wheat starch granules improves the quality of gluten-free breads: optimizing dough viscosity and Pickering stabilization. Food Hydrocolloids. https://doi.org/10.1016/j.foodhyd.2018.04.034 Sarawong, C., Gutiérrez, Z. R., Berghofer, E., & Schoenlechner, R. (2014). Effect of green plantain flour addition to gluten‐free bread on functional bread properties and resistant starch content. International Journal of Food Science and Technology, 49, 1825-1833. Segundo, C., Román, L., Gómez, M., & Martínez, M. M. (2017). Mechanically fractionated flour isolated from green bananas (M. cavendishii var. nanica) as a tool to increase the dietary fiber and phytochemical bioactivity of layer and sponge cakes. Food Chemistry, 219, 240-248. 19 Thorvaldsson, K., & Skjöldebrand, C. (1998). Water diffusion in bread during baking. LWT-Food Science and Technology, 31, 658-663. Vamadevan, V., Bertoft, E., & Seetharaman, K. (2013). On the importance of organization of glucan chains on thermal properties of starch. Carbohydrate Polymers, 92, 1653-1659. Vilaplana, F., Hasjim, J., & Gilbert, R. G. (2012). Amylose content in starches: Towards optimal definition and validating experimental methods. Carbohydrate Polymers, 88, 103-111. Vinoy, S., Laville, M., & Feskens, E. J. (2016). Slow-release carbohydrates: growing evidence on metabolic responses and public health interest. Summary of the symposium held at the 12th European Nutrition Conference (FENS 2015). Food and Nutrition Research, 60, 31662. Yoo, S. H., & Jane, J. L. (2002). Molecular weights and gyration radii of amylopectins determined by high-performance size-exclusion chromatography equipped with multiangle laser-light scattering and refractive index detectors. Carbohydrate Polymers, 49, 307-314. Zhang, G., Sofyan, M., & Hamaker, B. R. (2008). Slowly digestible state of starch: mechanism of slow digestion property of gelatinized maize starch. Journal of Agricultural and Food Chemistry, 56, 4695-4702. Zhang, P., & Hamaker, B. R. (2012). Banana starch structure and digestibility. Carbohydrate polymers, 87, 1552-1558. Zhang, P., Whistler, R. L., BeMiller, J. N., & Hamaker, B. R. (2005). Banana starch: production, physicochemical properties, and digestibility-a review. Carbohydrate Polymers, 59, 443-458. Zhang, B., Dhital, S., Flanagan, B. M., Luckman, P., Halley, P. J., & Gidley, M. J. (2015). Extrusion induced low-order starch matrices: Enzymic hydrolysis and structure. Carbohydrate Polymers, 134, 485-496. Zhong, F., Yokoyama, W., Wang, Q., & Shoemaker, C. F. (2006). Rice starch, amylopectin, and amylose: molecular weight and solubility in dimethyl sulfoxide-based solvents. Journal of Agricultural and Food Chemistry, 54(6), 2320-2326. 20 Table 1. Effect of native and extruded banana starch on physical properties of breads Sample Specific volume (mL/g) Hardness (g) Springiness Cohesiveness Resilience Crumb moisture (g/100 g) Crust moisture (g/100 g) Appearance Odor Flavor Texture Overall liking Control 4.29c ± 0.15 622b ± 79 0.99a ± 0.02 0.38b ± 0.02 0.16b ± 0.01 49.36b ± 0.12 18.95a ± 0.01 6.6c ± 1.4 5.5a ± 1.6 4.9a ± 1.7 5.4b ± 1.8 5.5ab ± 1.5 Native Banana 5.34d ± 0.13 304a ± 18 0.99a ± 0.05 0.45c ± 0.02 0.21c ± 0.02 49.85c ± 0.17 18.96a ± 0.06 6.5c ± 1.4 6.0b ± 1.4 5.3ab ± 1.6 5.8b ± 1.6 6.0b ± 1.4 Mixture Banana 3.57b ± 0.07 1051c ± 146 1.00a ± 0.00 0.36ab ± 0.02 0.14a ± 0.01 48.90b ± 0.01 18.40 a ± 0.09 5.9b ± 1.6 6.1b ± 1.4 5.1ab ± 1.6 5.3b ± 1.7 5.6ab ± 1.3 Extruded Banana 2.51a ± 0.03 3604d ± 558 1.02a ± 0.02 0.34a ± 0.03 0.16b ± 0.01 45.87a ± 0.52 21.21b ± 0.76 4.2a ± 2.0 6.2b ± 1.4 5.5b ± 1.6 4.7a ± 1.8 5.4a ± 1.5 Values followed by the same letters within each parameter indicate no significant differences (p≤0.05). 21 Table 2. Degree of starch gelatinization and amylopectin retrogradation of bread crumb and crust. Crumb Crust Degree of starch gelatinization during baking, DGB (%) ΔHr (J/g) Degree of starch gelatinization during baking, DGB (%) ΔHr (J/g) Control 100a ± 0.00 2.79a ± 0.13 7.16b ± 0.56 n.d. Native Banana 100a ± 0.00 3.01a ± 0.08 5.56a ± 0.74 n.d. Mixture Banana 100a ± 0.00 3.38b ± 0.07 7.47bc ± 0.55 n.d. Extruded Banana 100a ± 0.00 3.47b ± 0.16 8.48c ± 0.99 n.d. Values followed by the same letters within each parameter indicate no significant differences (p≤0.05). n.d. non detectable. ΔHr, enthalpy for retrograded amylopectin. 22 Figure 1. Particle size distribution (A) of starchy ingredients and SEM pictures of native and extruded banana samples (B). Black circles highlight aggregates of banana starch granules. A) 23 B) 24 Figure 2. HPSEC-MALS chromatogram of native and extruded banana starch. 25 Figure 3. Pictures of central slices of bread. a) Control bread; b), bread made with 20% native banana starch (NB); c) bread made with 20% of a 1:1 mixture of native and extruded banana starch (MB) and d) bread made with 20% extruded banana starch (EB).