Pasting, thermal and structural properties of haricot beans flour (Phaseolus vulgaris, L.) as affected by variety and germination

ABSTRACT

This research aimed to verify how the germination method affects the pasting, thermal and structural properties of haricot bean flours to expand their potential for application in various food products. The pasting properties of flours showed considerable variation due to variety, germination, and interaction between variety and germination. Onset, peak and end of gelatinization temperature and enthalpy of haricot bean flours ranged with germination from 65.4 to 67.9, 70.6 to 73.3, 77.0 to 79.4 C, and 3.5 to 4.9 J/g, respectively. The Crystallinity index of 632 G flour was higher than that of 119 G and 125 G flour. The data revealed that the soaked and germinated haricot bean flour showed major diffraction peak values of the 2θ at 18.25°,18.40°,18.25°, 17.68°, 18.74°, and 18.34°, respectively for 119S, 119 G, 632S, 632 G,125S, and 125 G. FT-IR spectroscopy of four samples did not show any variations in spectra, but a change in the intensity of bands was observed. The SEM images reveal that starch granules were surrounded by a protein matrix.

KEYWORDS:

• Haricot beans flour
• germination
• structural properties

Introduction

Legumes have been considered the most significant food source, especially for low-income groups of people in developing countries,^[1] containing a relatively high amount of proteins.^[2] Dry beans are the dicotyledonous seed of plants belonging to the Leguminosae family, which are widely cultivated worldwide due to their global adaptability and drought tolerance.^[3] Among the commonly consumed dry beans, haricot beans (Phaseolus vulgaris L.) occupy a significant place in human nutrition in the East and Great Lakes regions of Africa.^[4] However, their role appears to be limited because of several factors including the presence of anti-nutritional compounds in the seeds which may have adverse effects on human nutrition, low protein, and starch digestibility,^[5] and poor mineral bioavailability.^[6] Transforming the beans into flour aids in improving the nutritional requirements of consumers, especially in developing countries.^[7] The germination processes are easy and cost-effective treatments given to legumes to modify macronutrient structure,^[8] improve nutritional, functional, and biological properties, as well as removing antinutritional factors such as tannin, phytic acid, oxalate, and enzyme inhibitors.^[9,10] The consumption of grain sprouts has become common among people interested in improving and keeping their health by altering dietary habits. Due to the health benefits of germination, different studies have reported on the incorporation of germinated seeds into various food manufacturing ingredients, such as pasta,^[11] crackers,^[12] beverages,^[13] muffins,^[14] and yogurt.^[15] Depending on the food products, the addition of legumes in a germinated form has different effects.^[16] Eventhough the compositional changes of haricot beans during germination have been investigated to a certain degree, the research performed on changes in the thermal and structural properties of flour during the germination of haricot beans is limited. It is based on this background that this work was undertaken to investigate the effect of germination on pasting, thermal, morphological, FTIR, and XRD properties of flours from three varieties of haricot bean seeds.

Material and methods

Sample preparation

Haricot bean (Phaseolus Vulgaris L.) varieties (SER 119, SAB 632, and SER 125) were collected from Awash Melkasa Agricultural Center based on the facts that they have shown to have a high production percentage, disease tolerance, short period to ripe and easy to adopt in Ethiopia.^[17] The soaked and germinated haricot bean flour was prepared according to previously done methods.^[10] The resulting haricot bean flours were placed in plastic bags, sealed, and stored at 4°C in a desiccator until needed for further analysis.

Pasting properties

The pasting properties of the flours were measured using a Rapid Visco Analyzer (Perten RVA 4800, PerkinElmer, Sweden). An aqueous dispersion of flour on a 14% moisture basis (12.28%, w/w; 28.5 g total weight) was equilibrated at 50°C for 1 min, heated at the rate of 12.2°C/min to 95°C, held for 2.5 min, cooled to 50°C at the rate of 11.8°C/min and again held at 50°C for 2 min. A constant paddle rotational speed (160 rpm) was used throughout the entire analysis, except for rapid stirring at 960 rpm for the first 10 s to disperse the sample.^[18]

Thermal properties

The thermal properties of flours were analyzed using DSC (SKZ1052B, Hunan, China) equipped with a thermal analysis data station. A 10 mg sample was weighed into a 40 µl capacity aluminum pan and 20 µl distilled water was added with the help of a Hamilton micro syringe. Pans were hermetically sealed and allowed to stand for 1 h at room temperature before heating in DSC. The DSC was calibrated using indium, and an empty aluminum pan was used as a reference. Sample pans were heated at a rate of 10°C/min from 20 to 180°C, and thermal parameters viz. onset (To), peak (Tp), conclusion (Tc) temperature, and enthalpy (DH) were calculated from the DSC curves.^[18]

Fourier transform infrared analysis

The spectra of the samples were recorded using Fourier transform infrared (FTIR) spectrometer system (Nicolet is50 ABX, Thermofisher Scientific, German) coupled to an attenuated total reflection (ATR) accessory. Analyses were carried out at room temperature, and spectra were acquired in the range of 4000–500 cm⁻¹ at a resolution of 4 cm^−1.[19]

Xrd

Samples were loaded into the XRD sample holder, and XRD pattern was obtained using an X-ray diffractometer (XRD-7000, Shangai Drawel scientific instrument co., Ltd., China) equipped with a divergence slit, operating at 40 kV and 40 mA at a scan speed of 1^o/min. The relative crystallinity (RC) was calculated using the areas of the crystalline and amorphous regions on the X-ray diffractogram.^[20]

Scanning electron microscopy

The flour was placed on an adhesive tape attached to a circular aluminum specimen stub and then coated vertically with gold palladium. The images of samples were captured at an accelerator potential of 10 kV using a scanning electron microscope (JCM-6000 plus, Jeol Ltd., Korea).^[18]

Statistical analysis

All analyses were conducted in triplicates observations except FTIR and SEM analysis, and data obtained were subjected to analysis of variance (ANOVA) using software package SAS version 9.0 (SAS Institute, Inc., Cary, North Carolina, USA). Tukey’s HSD test at the significance level of 5% (P < .05) was used to determine significant differences among means.

Results and discussion

Pasting properties

The pasting properties of the haricot bean four samples are presented in Table 1. Significant differences were observed in pasting characteristics of flours due to variety, processing method and interaction between them. While the non-germinated 119S flour had the highest peak viscosity value (1832 ± 187 cp) but flour from germinated SER125 variety had the lowest peak viscosity value (564 ± 36cp).

Table 1. Effects of interaction between particle size and pretreatment on pasting properties of pumpkin flour.

Flour Sample	Parameters
	Peak viscosity (cp)	Trough (cp)	Breakdown (cp)	Final viscosity (cp)	Setback (cp)	Peak time (Min)	Pasting Temp (℃)
119S	1832±187^a	1401±166^a	430±21^a	3516±692^.a	2114±525^a	7.20±0.04^a	79.6±0.0.6^abc
119G	696±288^b	654±294^b	42±6^c	1098±390^b	444±96^c	6.9±0.1^cd	77.±2^.cd
632S	1084±448^b	863±388^ab	221±60^b	1734±616^b	871±228^bc	7.1±0.2^ab	82.3±0.6^a
632G	878.±56^b	689±55^b	190.0±0.8^b	2123±265^b	1434±321^ab	7.03±0.03^abc	81.7±0.1^ab
125S	1176±120^b	1062±126^ab	114±6^c	2226±438^b	1164±312^bc	7.0±0.1^bcd	79.±0.1^bc
125G	564±36^b	522±42^b	42±6^c	1344±168^b	822±210^bc	6.8±0.1^d	75.±2.0^d
Effect
Variety(V)	***	***	***	***	***	***	***
Process (P)	***	***	***	***	***	***	***
Interaction (V*P)	***	***	***	***	***	***	***

Note: Values are mean ± SD. Means that sharing the same superscript letters in columns are not significantly different from each other (Tukey’s HSD test, p < .05). *** Significant effect at p< .05, NS, Not significant. 119S, SER 119 variety Soaked; 119G,SER 119 variety germinated; 632S, SAB 632 variety soaked; 632G,SAB 632 variety germinated; 125S, SER125 variety soaked;125G= SER125 variety germinated; cp, centipoise

Peak viscosity is indicative of the strength of the paste, which is made from gelatinization during processing in food applications. It also reflects the extent of granule swelling.^[21] As revealed in Table 1, peak and breakdown viscosities decreased with the germination. The decrease in peak viscosity with germination may be attributed to protein and starch degradation or debranching to simpler units by the enzymes activated during the germination,^[22] similar to Enyinnaya, Adewuyi, & Oneh^[23] and Obalolu & Cole^[24] report on malted maize and blends of cowpea and soybean flour, respectively. The high breakdown viscosity is indicative of the low ability of the flour to withstand heating and shear stress during cooking.^[25] Flour from germinated SAB 632 seeds (190.0 ± 0.8 cp) had higher breakdown viscosity than 119 G (42. ± 6) and 125 G (42 ± 6) flour samples. High breakdown viscosity for 632 G flour reflects its less resistance to the shear, and the starch granules have more susceptibility to disintegration than starch granules from other flours under study. Breakdown viscosity value measures the ease with which the swollen granules can be disintegrated.^[26] Trough viscosity values ranged from 522 ± 42to 1401 ± 166 cp, with the highest values recorded by 119S and the lowest by 125 G flour sample. As reported by Enyinnaya et al.^[23] and Liu et al.,^[27] the decrease in trough viscosity with germination may be attributed to starch degradation, which caused a decrease in viscosity value. Trough viscosity is the minimum viscosity value in the constant temperature phase of the RVA profile and measures the ability of the paste to withstand breakdown during cooling. There is no significant difference (P < .05) in the final viscosity among flour samples except for the 119S flour sample with the highest value (3516 ± 692), and also final viscosity decreases with germination. The observed low values of final viscosity for the germinated flours might be due to the attendant breakdown of starches, which is associated with the increase in amylase activity during seed germination of the legumes.^[28] Final viscosity indicates the ability of a material to form a viscous paste after cooking and cooling.^[29] The soaked bean (control) flour had significantly (P ≤ .05) the highest setback viscosity with the highest values attained by the 119S flour sample (2114 ± 525cp) and the SER119 germinated flour had the least setback of 444 ± 96cp. The lower setback values of germinated bean flour indicates its lowest rate of retrogradation and this will be useful in the prolonged shelf life of the flour.^[30] In line with this finding Acevedo et al.^[31] reported a decrease in setback viscosity of pigeon pea, Dolichos bean, and jack bean flours after germination. The highest setback viscosity of 119 G flour indicates its higher tendency to retrograde than other flours. According to Wani, Sogi, & Gill,^[32] retrogradation results from hydrogen bonding between starch molecules that have hydroxyl and hydrogen acceptor sites. Table 1 shows a significant difference (p < .05) among flour pasting temperatures. The pasting temperature ranged from 75–82.3°C, the lowest for 125 G and the highest for 632S flour samples. Pasting temperatures of all three flours decreased upon germination. This might be due to the enzymatic degradation of cell wall-wrapped protein and starch, in pulse seeds facilitating starch and protein to contact and absorb water, which increases the heat transfer rate.^[8] The reduction in pasting temperature during germination was in agreement with the decrease in Tc of gelatinization. Low pasting temperature translates to low energy cost and other component stability.^[28] According to this finding, pasting temperature results are lower than Felker et al.^[33] for the raw navy, black, and pinto bean flours (between 80 and 83°C) and higher than 72°C for chickpea flour. Peak time ranged from 6.8 to 7.20 min, with the non-germinated SER 119 variety having the highest peak time (7.20 min) while germinated SER 125 variety had the lowest peak time (6.8 min). The peak time indicates the minimum temperature required to cook the flour. Peak time values reported in this study are higher than the peak time (5.13 to 5.80 min) reported by Adebowale et al.,^[29] implies that it needs extended times for forming pastes and hence longer processing times. Generally, the activated amylases and proteases during germination might cause a decline in the pasting viscosity of flours, due to the degradation of starch chains and disruption of disulfide linkages, respectively^[22,34] Especially the protein degradation and loss of structural integrity in flours increased the mechanical breakability of the swollen starch granules, which caused a substantial decline in pasting viscosity.^[22]

Thermal properties

Values are mean ± SD. Means that sharing the same superscript letters in columns are not significantly different from each other (Tukey’s HSD test, p < .05). *** Significant effect at p < .05, NS, Not significant. 119S, SER 119 variety Soaked; 119 G,SER 119 variety germinated; 632S, SAB 632 variety soaked; 632 G,SAB 632 variety germinated; 125S, SER125 variety soaked;125 G= SER125 variety germinated;To, onset temperature; Tp, peak temperature; Tc, conclusion temperature; DH, gelatinization enthalpy.

The thermal properties of haricot bean flour were significantly affected by the varieties and germination as well as by the interaction between varieties and germination (p < .05) Table 2. But the difference between the temperatures at the beginning and end of gelatinization is not too large, signifying a low variability of the thermal properties of flour molecules. The thermal properties indicated that the hydrogen bond of starch in germinated flour could be broken down at a temperature lower than that of control flours (Table 2). To, Tp, Tc and ΔHgel for haricot bean flours ranged between 61 ± 1–66 ± 2, 67. ± 2–71.7 ± 0.2, 78 ± 2–80 ± 2 oC, and 4.9 ± 0.3–7.8 ± 0.4 J/g, respectively. The value of onset temperature of 632S flour (66 ± 2°C) was significantly higher than that of the other flours. An insignificant change was observed in Tp and Tc of flour with germination. The germinated haricot bean flour samples showed low thermal properties than native flour. The decrease in thermal properties of flour during germination might be due to decreased fat content, which covers the starch granules and thus become easily assessable for swelling.^[35] In addition, the reduction in Tc may be linked with the degradation of interactions between starch and protein/lipid during germination.^[22] The differences in thermal properties between different cultivars may be attributed mainly to the difference in starch properties, crystallinity, and protein content.^[36] Similar results of a decrease in thermal properties of chickpea and brown rice flour due to germination were reported by Xu et al.^[37] and Wu et al.,^[35] respectively.

Table 2. Effects of interaction between variety and processing method on thermal properties of haricot bean flour.

Flour Sample	Parameters
	To (^oC)	Tp (^oC)	Tc (^oC)	DH (J g^−1)
119S	64 ± 1^ab	69. ± 2^.a	78 ± 2^.a	7.8 ± 0.4^a
119 G	61 ± 1^b	67 ± 2^a	78. ± 2^.a	5.7 ± 0.3^b
632S	66 ± 2^a	71.7 ± 0.2^a	80 ± 2^.a	6.8 ± 0.4^a
632 G	63 ±^.ab	69 ± 2^.a	80 ± 2^a	4.9 ± 0.3^b
125S	65 ± 2^ab	70. ± 3^.a	79 ± 3^.a	7.3 ± 0.5^a
125 G	62 ± 2^ab	68 ± 2^a	79 ± 3^.a	5.3 ± 0.4^b
Effect
Variety (V)	NS	NS	NS	***
Process (P)	***	***	NS	***
Interaction (V*P)	***	NS	NS	***

Gelatinization enthalpy (ΔH) can be used to predict the energy required to break down the intermolecular hydrogen bonds of starch granules.^[38] Flour from soaked SER 119 seeds showed a higher ΔH value than flour from other cultivars. The ΔH of germinated flours decreases, suggesting the reduction of energy required to convert the chemical composition of bean flour from an ordered to a disordered form; also, it indicates that reduction in the number of double helical,^[39] which may be attributed to the degradation and consumption of starch during sprouting. According to Xu et al.,^[8] during germination, the partial hydrolysis of starch by the activated enzymes diminishes the intermolecular hydrogen bonds of starch, making them easily detached on heating. This result suggests that the gelatinization enthalpy values obtained were higher than those reported on lentil flours (3.35 to 3.70 J/g) by M. Xu et al.,^[8] and lower than chickpea flour (15.2 J g⁻1) by J. Huang et al.^[40]

Fourier transform infrared spectroscopy (FT-IR)

The soaked (control) and germinated haricot bean flour samples were analyzed by FT-IR spectroscopy to study the change in functional groups upon the interaction between variety and germination. The FT-IR spectroscopy of haricot bean flour samples did not show any variations in spectra, but a change in the intensity of bands was observed (Figure 1). The spectra obtained for all samples were similarly based on the form but differed in the intensity of the major peaks as presented in Figure 1. All tested flour samples showed high absorption spectra at the wavenumbers 3278.6, 2912.3, 1635.5, 1391.4, 1252.6, and 1008.3 cm⁻¹. The bands observed in the region 3278 cm⁻¹ in the spectra of the flour can be assigned to the stretching vibration of the – OH groups caused by the formation of hydrogen bonds in the^[41] starches and starch – proteins interaction and also from various compound like alcohol, phenols, and carboxylic acid.^[42] In the literature, it was highlighted that the spectral range between 3000 and 2800 cm⁻¹ corresponding to lipid compound, due to vibration produced by carbonyl group of triglyceride (C-H)^[16] The signal at 2,912 cm − 1 in a sample confirmed the development of more C─H bonds, possibly from unsaturated C-bonds. The spectral range between 1700 and 1500 cm − 1 corresponds to the protein content of the samples. The lengthening at 1635 cm⁻¹ indicated the presence of C=O stretching, which may be related to the amide I region^[16,43] The peaks at 1420 –1380 cm⁻¹ are assigned to C-H deformation vibrations. The signals in the region between 1200–900 cm⁻¹ are ascribed to C―O and C―C stretching vibrations of the polysaccharide molecules.^[44,45]

Figure 1. FTIR spectra of haricot bean flour: 119S, SER 119 variety Soaked; 119G,SER 119 variety germinated; 632S, SAB 632 variety soaked; 632G,SAB 632 variety germinated; 125S, SER125 variety soaked;125G= SER125 variety germinated.

X-ray diffraction (XRD)

XRD has been widely used to reveal the characteristics of the crystalline structure of starch granules.^[46] As exhibited in Table 3, the crystallinity index, diffraction peaks, peak intensity, and D-Spacing of all six flour samples ranged from 44–59.1%, 17.68–18.49°, 370.43 ± 1.01–537.6 ± 1.0, and 27.4 ± 0.8–28.6 ± 0. 5 nm, respectively. Both soaked and germinated haricot bean flours showed insignificant changes in diffraction peaks, suggesting no change in the type of crystalline structure due to variety and germination, as well as the interaction between them. It can be seen that the crystallinity index for the 125 G flour sample was higher (59.1 ± 0.9) than that of other flours. The variation of the crystallinity index of flour implies the differences in the extent of starch damage. According to Li et al.,^[47] during germination, the starch is hydrolyzed to provide energy, while the amorphous regions of the starch granules are hydrolyzed first, and then the crystalline regions are hydrolyzed so that the overall value in the calculation of the ratio decreased and the result of crystallinity increased. Figure 2 reveals that the soaked haricot bean flour showed strong intensities as compared to the germinated flour, with the 119S flour being the highest and 632 G the lowest.

Figure 2. XRD patterns of haricot beans flours: 119S, SER 119 variety Soaked; 119G,SER 119 variety germinated; 632S, SAB 632 variety soaked; 632G,SAB 632 variety germinated; 125S, SER125 variety soaked;125G= SER125 variety germinated.

Table 3. Effects of interaction between variety and processing method on XRD parameters of haricot bean flour.

Flour Sample	Parameters
	Cryistalinity index (%)	Peak diffraction (^o)	Peak intensity	D-spacing (nm)
119S	56±1^b	18.3±0.8^a	537.6±1.0^a	27.7±0.2^ab
119G	44±1^.d	18.4±0.4^a	457.83±0.72^b	27.5±0.3^ab
632S	56.±1^.b	18.25±0.14^a	370.43±1.01^e	27.7±0.4^ab
632G	57.5±0.3^ab	17.68±0.20^a	340.4±0.9^f	28.6±0. 5^a
125S	47.5±0.6^c	18.49±0.30^a	437±1^c	27.4±0.8^b
125G	59.1±0.9^a	18.34±0.23^a	412.80±1.11^d	27.6±0.5^ab
Effect
Variety (v)	***	NS	***	***
Processing (p)	NS	NS	***	NS
Interaction (v*p)	***	NS	***	***

Note: Values are mean ± SD. Means sharing the same superscript letters in columns are not significantly different from each other (Tukey’s HSD test, p < .05). *** Significant effect at p< .05, NS, Not significant. Values are mean ± SD. 119S, SER 119 variety Soaked; 119G,SER 119 variety germinated; 632S, SAB 632 variety soaked; 632G,SAB 632 variety germinated; 125S, SER125 variety soaked;125G= SER125 variety germinated

Scanning electron microscopy

Microstructures of the soaked and germinated haricot bean flours were examined by scanning electron microscopy (Figure 3) and the images reveal that some residual proteins deposited or fragments of protein matrix on the granule surfaces, which confirms that starch granules were surrounded by a protein matrix.^[3] In addition, it may be included mineral and fiber components, as reported by other workers.^[18] There were visible changes observed between the micrographs of the soaked and germinated flour samples of all varieties (Figure 3). This indicates that the germination could affect the shape of haricot bean flour granules. A similar finding on the structural change of sprouted onion powder and sprouted sorghum was observed by Majid, Dar, & Nanda^[48] and Yan et al.,^[49] respectively. The compositional changes during germination account for microstructural changes. This morphological change has an important influence on the flowability behavior of the powders

Figure 3. SEM of haricot bean flours: a) SAB 632 variety germinated; b) SAB 632 variety soaked; c) SER125 variety germinated; d) SER125 variety soaked; e) SER 119 variety germinated; f) SER 119 variety Soaked.

Conclusion

The study showed a significant effect of variety and germination on the pasting, thermal, XRD, FTIR, and SEM properties of haricot bean flour. The germination process decreases flour’s thermal and pasting properties, which could enhance its better utilization in the food industry due to the loosening structure caused by increased amylase activity. So the flour from germinated haricot bean seeds can provide a potential application for producing foods that require low viscosity compared to control flour. It was found that there were significant differences between the onset temperature and gelatinization enthalpy of the flours, with 632S flour showing the highest To and Tp, but 125 G flour exhibited the lowest ΔHgel. Germinated SER 125 and SAB 632 flour samples had high crystallinity index than other flours. This study may promote haricot bean flour utilization in the food industry.

Abbrevaition

119 G	=	Germinated SER 119 variety flour
119S	=	Soaked SER 119 variety flour
125 G	=	SER125 variety germinated flour
125S	=	Soaked SER125 variety flour
632 G	=	Germinated SAB 632 variety flour
632S	=	Soaked SAB 632 variety flour
DH	=	Gelatinization Enthalpy
Tc	=	Conclusion Temperature
To	=	Onset Temperature
Tp	=	Peak Temperature

Acknowledgments

The first author would like to acknowledge Wolkite University Food Process Engineering Department for providing us laboratory facilities.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the Minister of Education, Ethiopia [1].