Starch enzymatic hydrolysis, structural, thermal and rheological properties of pigeon pea (Cajanus cajan) and dolichos bean (Dolichos lab-lab) legume starches

1 Starch enzymatic hydrolysis, structural, thermal and rheological properties of pigeon pea (Cajanus cajan) and dolichos bean (Dolichos lab-lab) legume starches Belén A. Acevedoa,b, Marina Villanuevab, María G. Chavesa, María V. Avanzaa, Felicidad Rondab* aFaculty of Natural Sciences and Surveying, National Northeast University (UNNE), Institute of Basic and Applied Chemistry of Northeast Argentina, IQUIBA-NEA, UNNECONICET, Avenida Libertad 5460 Corrientes, 3400, Argentina. bCollege of Agricultural and Forestry Engineering, University of Valladolid, Av. Madrid 57, 34004 Palencia, Spain * Corresponding author: [email protected] Abstract Cajanus Cajans and Dolichos lab-lab legume starches from Argentine cultivars were investigated under a technological and nutritional point of view. Their physico-chemical, structural, thermal and the rheological properties of their gels were evaluated. Rice (RS) and potato (PS) starches were included as references. In vitro digestibility from Englyst method was also evaluated. Legume starches had the highest amylose content and the most stable chemical structure. Their rapidly digestible starch and starch digestibility rate index were very low, similar to PS, and 5-fold lower than RS. They had a much higher slowly digestible starch content than PS. Legume starches showed the highest gel stability versus heating and stirring and an intermedium pasting temperature between RS and PS. They formed viscoelastic gels at 6% concentration with stronger elastic-like behavior and higher yield stress than references. Our results indicate these legumes represent an efficient starch source to provide tailor-made properties to food/industrial applications. Keyword: legume starch; gel rheological properties; in vitro starch enzymatic hydrolysis; DSC, TGA, structural properties 1. Introduction Starch is a macro-constituent in many raw materials and processed foods with particular importance from both nutritional and functional point of view. The role of starch in many formulated products is as texture modifier, contributing in many quality aspects of processed foods (Villanueva et al., 2018a). Starch selection for industrial use is made by considering its availability and its physicochemical characteristics and quantitative and qualitative make-up, which vary depending on the source of starch. The starches from cereals (corn, wheat and 2 various types of rice), tubers or roots (potato and tapioca) are frequently used by industries; however, the continuous expansion of this market demands new starches sources, with novel properties or additional applications. On the other hand, the global market for starch is projected to reach US$ 75.4 billion by 2022 from a forecast of US$ 53 billion in 2016 for the global and regional markets including North America, Europe, Asia-Pacific, Latin America and rest of world (Research & Markets, 2016). Legumes are a potential source of starch with interesting features, little exploited up to now. The species of legumes like pigeon pea (PP; Cajanus cajans) and dolichos bean (DB; Dolichos lab-lab) are cultivated in East and South Africa, East and South Asia and South America (Kaur & Shandu, 2010). In the north-east of Argentina, these legumes are cultivated by small and medium farmers and are primarily used for human consumption. PP and DB present a starch content of 49–50% and different amylose/amylopectin ratios (Acevedo et al., 2013). In general, legumes promote slow and moderate postprandial glucose and insulin responses, with low glycemic index values, which are quite relevant characteristics for patients diagnosed with diabetes, some types of cancer, colon disease, obesity and cardiovascular diseases (Nayak et al., 2014). Rheological and thermal properties as well as nutritional and structural characteristics determine the potential application of a starch. Several works were developed to increase the potential use of legume starches from India ( Singh et al., 2008; Kaur & Shandu, 2010) or Nigeria (Agunbiade & Longe 1999). However, to best of our knowledge, the characteristics of DB (DBS) and PP (PPS) starches from South American cultivars have never been studied so far. Thus, the aim of this paper was to study the structural, physicochemical, in vitro digestibility, thermal behavior (pasting profiles, phase transitions and thermal mass loss) and rheological properties of the gels made from starches isolated from Argentinean legumes in order to establish their technological and nutritional functionality. Rice and potato starches were used as references and analyzed in parallel to the legume starches. 2. Materials and methods 2.1 Materials Pigeon pea (PP) (Cajanus cajans L.) and dolichos bean (DB) (Dolichos lab lab L.) were supplied by the “Estación Experimental El Sombrero-Corrientes” (Instituto Nacional de Tecnología Agropecuaria-INTA) Argentina. Shrunken, discolored and insect infested legumes seeds were eliminated, sun dried and then the legumes were kept in a hermetic vessel stored at 10 °C until use. The commercial rice starch (RS) and the commercial potato starch (PS) were provided by Beneo-Remy n. v. (Leuven-Wigjmaal, Bélgica) and Ferrer Alimentation SA (Barcelona, Spain), respectively. 3 2.2 Starch isolation and its proximal analysis Legume starches were extracted by using the method of Schoch and Maywald (1968). Samples were hermetically stored in polyethylene bags. Moisture content (Method 925.10), ash (Method 923.03), fat (Method 920.39) and protein content (Method 920.87) were measured with the standard methods of AOAC (AOAC, 1990). Amylose content was determined with the Megazyme assay kit (Megazyme International Ireland Ltd., Bray, Ireland). 2.3 Morphological properties Dried, finely ground starch samples were mounted on an aluminum stub with double-sided tape and coated with a thin film of gold. The samples were observed under a scanning electron microscope (model JEOL, 5800 LV, Tokio, Japan) at 15 kV. 2.4 X-ray diffraction The diffraction assessment was developed using the method described by Villanueva et al. (2018b). 2.5 In vitro digestibility of starch In vitro starch digestibility was measured according to Englyst et al. (1992) with modifications (Abebe et al., 2015). The glucose released at 20 min (G20) and 120 min (G120) and the total glucose (TG) were determined by the glucose oxidase colorimetric method. Rapidly digestible starch RDS=0.9·(G20−FGS), slowly digestible starch SDS=0.9·(G120−G20), resistant starch ReS=0.9·(TG−G120), total starch TS=0.9·(TG−FGS) and rapidly available glucose RAG=G20 were calculated. Starch digestion rate index (SDRI), which expresses the amount of RDS in the sample as a percentage of the TS content, was also computed. Each sample was measured at least in triplicate. 2.6 Differential scanning calorimetry (DSC) Thermograms of starch samples were determined using a differential scanning calorimeter (DSC-822e Mettler Toledo AG, Schwerzenbach, Switzerland) following the method described by Villanueva et al. (2018b). The retrogradation of starch was evaluated in the samples previously gelatinized and stored in the DSC pans for 7 days at (4±2) °C following the same temperature scan. The enthalpy (ΔH), onset temperature (To), end-set temperature (Te), peak temperature (Tp) and the difference Te−To (ΔT) were established in both scans, at 0 and 7 days. Samples were run in duplicate. 2.7 Thermoviscous tests: Pasting profiles Samples (6 g starch/100g) were analyzed following Acevedo et al. (2013) using a Kinexus Pro+ rheometer (Malvern Instruments Ltd, Malvern, UK) supplied with starch pasting cell 4 and controlled by rSpace software. The starch concentration was selected after determining that it was above the least gelation concentration (Sathe et al., 1981). Parameters recorded were pasting temperature (PT), peak viscosity (PV), trough viscosity (TV), final viscosity (FV), breakdown viscosity (BV) and setback viscosity (SV). 2.8 Thermogravimetric analysis (TGA) Thermogravimetric analyses were conducted with a TGA/SDTA-851e (Mettler Toledo, Schwerzenbach Switzerland) from 50°C to 600°C at a heating rate of 10°C/min under a nitrogen flow of 60 mL/min. A final isothermal step at 600 °C for 5 min under an air flow of 60 mL/min was performed. Samples (10 mg) were weighed into aluminum oxide pans. Each run was repeated at least twice. 2.9 Rheological properties: Viscoelastic and flow behavior of starch gels Flow and dynamic tests of starch gels were performed with Kinexus-Pro+ rheometer (Malvern Instruments Ltd, Malvern, UK) with parallel plate geometry (40mm diameter) of serrated surface and with 1mm of working gap. The gel samples were made following the same procedure described for thermoviscous tests. Just at the end of the pasting test the gels were removed from the canister and placed between the plates, the sample excess was removed and the sample was left to rest for 5 min to allow relaxation. Frequency sweeps were carried out from 20 to 0.1Hz in the linear viscoelastic region (LVR). The limit of the LVR (τmax) was determined according to Acevedo et al. (2013). Frequency sweep data were fitted to potential equations as described by Acevedo et al. (2013). The coefficients of the potential equation G’1, G’’1, and (tan 𝛿)1, represent the elastic and viscous moduli and the loss tangent at a frequency of 1 Hz. The a, b and c exponents quantify the dependence degree of dynamic moduli and the loss tangent with the oscillation frequency. Flow tests were carried out at 25ºC and shear rates in the range 1 to 500 s-1. To establish gels thixotropy shear rate cycles (up-down) were carried out and between the up and down phases, the maximum shear rate (500s-1) was applied for 2 min. The thixotropy degree of the gels was established from the area of the hysteresis loop. The experimental data from both, the up and down shear rate scans, were fitted to Herschel-Bulkley model: 𝜏 = 𝜏0+ 𝐾 · 𝛾𝑛 Where: τ=shear stress (Pa), γ=shear rate (s-1); τ0=yield stress (Pa), K=consistency coefficient (Pa·sn) and n=flow behaviour index. Samples were measured in duplicate. 2.10 Statistical analysis In order to check the statistical importance of observed variability, one-way ANOVA was applied and the least significant difference (LSD) was established at the importance level p=0.05. Statistical analysis was performed by using Statgraphics Centurion v.16 (Bitstream, Cambridge, MN, USA) software. 5 3. Results and discussion 3.1 Chemical composition of starch The yield and the chemical composition of starches are shown in Table 1. The yield of DBS (35.3%) was lower than that of PPS (38.2%). Singh et al. (2004) reported the yield of several legume starches ranging from 18 to 45%. These differences could be associated to species and cultivars, starch granule associations with other constituents, physiological state of the seed, and the estimation methods (Wani et al., 2016). Legumes showed intermedium water contents between those of RS (9%) and PS (14%). Differences in the equilibrium water content of starches indicate the diversity in their chemical composition and the different number of hydrophilic sites, which influence their water holding ability (Wani et al., 2016). PPS and DBS showed higher values of fat (0.63–1.04%), protein (3.08–4.81%) and ash (1.03–1.23%) contents than RS and PS. These results, in particular the protein content, are related to the extraction method used. PPS showed the highest amylose/starch ratio (26 %) followed by DBS (21%), PS (20%) and RS (15.8%). Table 1. Yield, chemical composition and starch fractions, TS, RAG and SDRI values of starches. PPS: pigeon pea starch, DBS: dolichos bean starch, RS: rice starch, PS: potato starch. dm: dry matter. TS: total starch, RDS: rapidly digestible starch, SDS: slowly digestible starch, ReS: resistant starch, RAG: rapidly available glucose, and SDRI: starch digestion rate index Data are the mean ± standard deviation. Values with a letter in common in the same row are not significantly different (p<0.05) PPS DBS RS PS Yield (%) 38.24±0.14b 35.32±0.28a - - Moisture (%) 12.00±0.03c 11.76±0.04b 9.92±0.01a 14.90±0.01d Fat (% dm) 1.04±0.03d 0.63±0.02c 0.42±0.02b 0.15±0.01a Ash (% dm) 1.03±0.05b 1.23±0.03c 0.21±0.01a 0.32±0.02a Protein (% dm) 4.81±0.17c 3.08±0.42b 0.56±0.02a 0.20±0.01a Amylose (% starch) 26.24±1.07c 21.18±0.13b 15.81±1.18a 9.60±0.46b TS (% dm) 86.4±3.5a 89.5±2.2ab 98.3±1.9b 95.8±2.8ab RDS (% dm) 9.9±2.2a 10.8±1.2a 54.5±1.5b 7.3±0.5a SDS (% dm) 23.6±2.3bc 19.9±2.3b 28.0±1.7c 7.4±0.8a ReS (% dm) 54.1±1.9b 57.7±2.1b 17.6±2.5a 81.4±3.6c RAG (% dm) 10.3±2.5a 12.0±1.4a 60.6±1.7b 8.1±0.5a SDRI (%) 11.4±0.9b 12.1±1.2b 55.4±2.0c 7.6±0.4a 6 3.2 Morphological properties PPS and DBS, showed oval/elliptical shape and smooth surface, similar to PS (Figure S1). However, legume starches had remnants of protein matrix not present in PS due to the extraction method used. RS showed the well-known polyhedral morphology with the smallest granule size (4.81x4.51μm) while PPS and DBS showed an intermediate size (14.4x13.4μm and 19x14.3μm, respectively). PS presented the greatest heterogeneity of granule sizes (46.5x31.3μm and 15.3x13.8μm). Figure S1. SEM pictures of starches at magnifications of 5000x. A) pigeon pea starch, B) dolichos bean starch, C) rice starch, D) potato starch. 3.3 X-Ray Diffraction PPS and DBS showed C-type (mixture of A and B-type) diffraction pattern characteristic of legumes, with diffraction peaks at 5.6°, 15°, 17.2°, and 23.2° (Figure S2), consistent with earlier reports for legumes (Kaur & Sandhu, 2010). RS showed the typical A-type pattern ( =15.2°, 17.0°, 18.0°, and 23.1°) while PS presented a B-type pattern 5°, 16.7°, 22° and 24°). Both, A and B structures, are composed of ordered arrays of double helices, and the polymorphic variation is based on packing differences of the hexagonically arranged chain duplexes; the B structure is more spacious, accommodating larger amounts of water (Lawal & Adebowale, 2005) compatible with equilibrium water content values informed. 7 The relative crystallinity measured from diffraction intensity was 54% and 48% for PPS and BDS respectively, lower than that found in RS and PS (64.4% and 55.9%). This is related to the content of amylopectin and the crystal size of the starches (Singh et al., 2008). 010 20 30 40 10000 20000 30000 40000 Intensity Angle (2 theta) Figure S2. X-ray diffraction patterns of starches. PPS: pigeon pea starch, DBS: dolichos bean starch, RS: rice starch, PS: potato starch. 3.4 Starch fractions analysis Table 1 summarizes the starch fractions obtained according to their ability to be attacked by digestive enzymes. The minor TS content found in PPS and DBS (86–90%) versus RS and PS (96–98%) agrees with their higher protein and fat contents. The RDS fraction of PPS (10%) and DBS (11%) was significantly (p≤0.05) lower than RS (55%) and similar to PS (7%). The low RDS content of these legume starches makes them particularly interesting for people suffering from type I diabetes. The larger granule diameter in legumes and potato than in rice starches, as assessed by SEM, would explain their lower specific surface area and their slower hydrolysis kinetics by the action of digestive enzymes The data is in agreement with predictions that smaller starch granules result in a higher glycaemic index which is highly correlated with RDS content (Englyst et al., 2003). The highest SDS content was also found for RS (28%) followed by PPS (24%) and DBS (20%). The lowest value was for PS (7.4%). SDS is generally the most desirable form of dietary starch and it is completely, but more slowly, digested in the small intestine (Englyst et al., 1992). Legume starches combined the double advantage of having a low RDS content (similar to PS) and a high SDS content (similar to RS). SDS molecular structure is related to amylopectin unit chain length distribution. The resistant starch content (ReS) found in starches was in the following order: PS>DBS>PPS>RS. ReS has been defined as the sum of starch and the product of starch degradation that are not absorbed in the small intestine, but it is fermented in the large 8 intestine of healthy individuals (Englyst et al., 1992). Total digestibility (TD=RDS+SDS) of starches was 32.8%, 30.7%, 82.5% and 14.7% for PPS, DBS, RS and PS, respectively; RS was highly attacked by digestive enzymes, which could also be related to the small size of the starch granule that increases the specific surface for the substrate-enzyme contact (Abebe et al., 2015). In addition, the A X-ray diffraction pattern of RS also facilitates its enzymatic hydrolysis unlike the type B and C diffraction patterns (Sivoli et al., 2007). RAG and SDRI showed the same tendency than RDS (Table 1). Free glucose content of all starch samples was always below the detection limit. 3.5 Differential scanning calorimetry Gelatinization and retrogradation thermal parameters of starches are shown in Table 2. PPS, DBS and RS showed similar values of Tpgel (67.6°C, 67.9°C and 67.1°C respectively) higher than PS (63.5°C), in agreement with the thermal stability of the starches measured by TGA. Differences in this transition temperature may be attributed to the degree of crystallinity of starch granules, which provides structural stability and makes the granule more resistant towards gelatinization (Ronda & Roos, 2008). An inverse relationship between starch gelatinization temperatures and short length chain amylopectin in starches from different Indian wheat varieties was reported by Singh et al. (2010). It seems that short chains of amylopectin are not able to form stable double-helical structures because of their shorter lengths, being easily disrupted by the heat at lower temperature. DBS exhibited the widest gelatinization peaks, with the highest ΔΤgel values (18°C), evidencing its major heterogeneity of crystalline regions within the starch granule (Kaur & Shandu, 2010). PPS and DBS had gelatinization enthalpies of 10.6 and 12.5 J/g, similar to RS but significantly lower than PS (16.9 J/g). This suggests that adjacent amylopectin chains are more strongly associated within PS native granule (Hoover et al., 1997). Starches with longer branch chain length, such as potato, display larger enthalpy changes, indicating larger amounts of energy were needed to gelatinize crystallites of longer chain lengths (Singh et al., 2010). Starch retrogradation after storage of gelatinized samples at 4°C for 7 days was studied. The melting temperature of the recrystallized amylopectin decreased from 11.5°C to 3.5°C with respect to the initial gelatinization temperature of the samples, suggesting that smaller and/or lesser perfect crystalline regions were formed during storage (Ronda & Roos, 2008). PPS and DBS showed melting enthalpies of recrystallized amylopectin (8.1–7.4 J/g), similar to PS and significantly higher than RS (1.9 J/g), denoting a higher degree of retrogradation in legume than in rice starches after 7 days of storage. ΔHret reflects the thermal energy associated with crystallite melting and dissociation/unraveling of double helices formed during storage, influenced by the amylopectin chain length distribution (Singh et al., 2010). Additionally, RS presented a reversible second endotherm in both, gelatinization and retrogradation scans, at a temperature close to 97°C and with an enthalpy of 2.4 and 2.5 J/g starch, respectively. The peak appeared at the same temperature and with similar enthalpy than in the first/gelatinization scan. This peak has been related to the amylose–lipid complex 9 dissociation that happens at this temperature when the water content is high enough (>60–70 g/100g) (Villanueva et al., 2018b). This transition was not found in PS or either in the legume starches, which confirms the absence of monoacyl lipids in these starches. Singh et al. (2010) registered cooling cycles at -10ºC/min after the heating scans of wheat starches and found exothermic peaks related to the amylose-lipid complex reassociation. Subsequent heating cycles showed an increase of 5ºC in the amylose-lipids dissociation temperature attributed to structural changes in amylose-lipids as result of successive heating/cooling cycles resulting in more stable crystalline polymorphs that dissociated at higher temperatures than amorphous amylose-lipids complex, more susceptible to degeneration. In our case, the dissociation temperature of the complex did not vary between successive cycles, demonstrating that the reassociation did not occur in a more stable crystalline structure. Table 2. Temperatures and enthalpies of phase transitions (from DSC tests) and pasting properties (from thermoviscous tests) of the starches. PPS: pigeon pea starch, DBS: dolichos bean starch, RS: rice starch, PS: potato starch. All enthalpy values are referred to weight of dry matter. To, Tp, Te: onset, peak and endset gelatinization temperatures; Tgel: gelatinization temperature range (Te – To), Hgel: enthalpy of gelatinization, Tpam-lip and Ham-lip: peak temperature and enthalpy of amylose-lipid complex dissociation measured during the gelatinization scan; Tpret and Hret: peak temperature and melting enthalpy of recrystallized amylopectin measured in the second scan (after 7 days of storage); Tret: retrogradation temperature range. PV: peak viscosity, TV: trough viscosity, BV: breakdown viscosity, FV: final viscosity, SV: setback viscosity, PT: pasting temperature Data are the mean ± standard deviation. Values with a letter in common in the same row are not significantly different (p<0.05). Sample PPS DBS RS PS To (°C) 61.41±0.13c 59.78±0.32a 60.51±0.11b 59.80±0.09a Tp (°C) 67.56±0.16bc 67.92±0.46c 67.12±0.01b 63.54±0.01a Te (°C) 74.91±1.70b 77.65±1.74c 73.94±0.01b 69.10±0.21a Tgel (ºC) 13.50±1.83a 17.87±1.72b 13.43±0.10a 9.31±0.30a Hgel (J/g) 10.58±0.81a 12.47±0.57b 11.27±0.75ab 16.91±0.28c Tpam-lip (°C) nd nd 97.09±0.02 nd Ham-lip (J/g) nd nd 2.41±0.16 nd Tpret (°C) 57.96±0.77b 56.36±0.76b 52.12±0.04a 60.11±0.54c Tret  (°C) 30.27±1.50b 24.09±1.70ab 19.16±0.24a 23.97±3.20ab Hret (J/g) 8.07±0.13b 7.41±0.79b 1.93±0.53a 9.21±0.14b PV (mPa·s) 638±4b 854±7c 554±5a 5779±57d TV (mPa·s) 638±4b 854±7c 425±7a 1667±102d BV (mPa·s) 0±0a 0±0a 130±4b 4112±45c FV (mPa·s) 862±12a 1018±7b 863±2a 2397±21c SV mPa·s 224±8a 164±14a 445±1b 730±81c PT (°C) 83.9±0.2c 80.6±0.4b 91.2±1.1d 67.9±0.6a 16 McPherson, A. E. & Jane, J. (1999). Physicalchemical properties of selected root and tuber starches. Carbohydrate Polymers, 40, 57-70. Nayak, B., Berrios, J. & Tang, J. (2014). 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