*3.3.2 Black bean*

**Figure 6** shows, by SEM, the black bean endosperm. In image A, the ellipsoid format of the starch granule is visualized (magnification 9.81 Kx), and in image B it is possible to observe the presence of cracks on the surface of the granule (magnification of 19.89 Kx), both refer to raw samples. The images of cooked samples (C magnification of 275 x and D and 29.01 Kx) reveal the stable structure of the black bean seed, suggesting a relation with the difficulties in its cooking (HTC phenomenon), requiring a longer cooking time for complete gelatinization, compared to the other samples studied [28].

#### **Figure 6.**

*(A) Black bean raw endosperm SEM (9.81 Kx), (B) black bean raw endosperm SEM (19.89 Kx) surface of granule, (C) cooked black bean cotyledon (275 x), and (D) unaccomplished gelatinization in cooked black bean cotyledon (29.01 Kx).*

**41**

**Figure 7.**

*(15.61 Kx).*

*Starch Granules from Cowpea, Black, and Carioca Beans in Raw and Cooked Forms*

starch grains were well defined and did not suffer any damage.

cells to soften or dissolve and separate the cells [19, 21].

*3.3.3 Carioca bean*

Ambigaipalan et al. [26], by SEM images, did not find the presence of cracks in black bean grains. Martínez-Preciado et al. [31] described the morphological structure of beans by SEM, observing that grains without the presence of fat had irregular oat-shaped starch granules with sizes of 10–40 μm in length and 10–25 μm in width, as well as small spherical beads of 10 μm. It was also observed that the

HTC phenomenon is one of the main obstacles to the consumption of beans grown in countries of Latin America and Africa, where ambient temperatures and relative humidity are high throughout the year, conditions that increase the possibility of occurrence of this phenomenon. At the microstructural level, the visible result of HTC seems to be related to the inability of the middle lamella of cotyledon

In **Figure 7**, the SEM of the carioca bean endosperm is observed. In image A, the ellipsoid formed of the starch granules (magnification 6.00 Kx) is observed, and in image B one perceives protrusions on the surface of the granule (magnification 15.61 Kx), both refer to raw samples. In the images of cooked samples (C 370 x and D 15.61 Kx), with different optical amplifications, it is noticed that the starch granules were grouped, losing their crystalline structure due to gelatinization [28]. In the isolated starch granule images by SEM from Wang and Ratnayake [31] study, there was no evidence of starch damage, with no visible cracks or notches in the surfaces, and the presence of foreign materials was also not observed. All cultivars presented spherical, oval, or elliptic forms with smooth surfaces. According to the authors, generally, *P. vulgaris* starch granules have similar morphologies

*(A) SEM of the raw carioca bean endosperm (6.00 Kx), (B) SEM of the endosperm of raw carioca bean (15.61 Kx), (C) cotyledon of cooked and gelatinized carioca bean (370 x), and (D) cotyledon of cooked carioca bean* 

*DOI: http://dx.doi.org/10.5772/intechopen.85656*

#### *Starch Granules from Cowpea, Black, and Carioca Beans in Raw and Cooked Forms DOI: http://dx.doi.org/10.5772/intechopen.85656*

Ambigaipalan et al. [26], by SEM images, did not find the presence of cracks in black bean grains. Martínez-Preciado et al. [31] described the morphological structure of beans by SEM, observing that grains without the presence of fat had irregular oat-shaped starch granules with sizes of 10–40 μm in length and 10–25 μm in width, as well as small spherical beads of 10 μm. It was also observed that the starch grains were well defined and did not suffer any damage.

HTC phenomenon is one of the main obstacles to the consumption of beans grown in countries of Latin America and Africa, where ambient temperatures and relative humidity are high throughout the year, conditions that increase the possibility of occurrence of this phenomenon. At the microstructural level, the visible result of HTC seems to be related to the inability of the middle lamella of cotyledon cells to soften or dissolve and separate the cells [19, 21].

### *3.3.3 Carioca bean*

*Legume Crops – Characterization and Breeding for Improved Food Security*

stability, due to the low percentage of water and oil absorption.

compared to the other samples studied [28].

of the starch grains of the same **Figure 5(B)**, it is possible to perceive the presence of protrusions. The authors also compared the structure of cowpea, pigeon pea (*Cajanus cajan* L.) and yam bean (*Sphenostylis stenocarpa* L.), perceiving these grooves visible only in cowpea, being scarce in the yam bean, and almost imperceptible in pigeon pea. According to the authors, the morphological and legume starch characteristics are good indicators to identify their botanical origin and to detect if they are contaminated or adulterated with starches from other sources. They also observed that cowpea, pigeon pea, and yam bean exhibited appreciable shelf life

Salgado et al. [30] observed that under the conditions in which their experiments were conducted, the morphological aspects of the starch grains were not influenced by the maturation stage of the grains. All presented a reniform shape, variable size between 11.8 μm and 26.7 μm, and smooth surface. Already the crystallinity pattern was higher in green beans than mature beans, as well as the percentage of resistant starch, whose test was based on the use of amylolytic enzymes.

**Figure 6** shows, by SEM, the black bean endosperm. In image A, the ellipsoid format of the starch granule is visualized (magnification 9.81 Kx), and in image B it is possible to observe the presence of cracks on the surface of the granule (magnification of 19.89 Kx), both refer to raw samples. The images of cooked samples (C magnification of 275 x and D and 29.01 Kx) reveal the stable structure of the black bean seed, suggesting a relation with the difficulties in its cooking (HTC phenomenon), requiring a longer cooking time for complete gelatinization,

*(A) Black bean raw endosperm SEM (9.81 Kx), (B) black bean raw endosperm SEM (19.89 Kx) surface of granule, (C) cooked black bean cotyledon (275 x), and (D) unaccomplished gelatinization in cooked black* 

**40**

**Figure 6.**

*bean cotyledon (29.01 Kx).*

*3.3.2 Black bean*

In **Figure 7**, the SEM of the carioca bean endosperm is observed. In image A, the ellipsoid formed of the starch granules (magnification 6.00 Kx) is observed, and in image B one perceives protrusions on the surface of the granule (magnification 15.61 Kx), both refer to raw samples. In the images of cooked samples (C 370 x and D 15.61 Kx), with different optical amplifications, it is noticed that the starch granules were grouped, losing their crystalline structure due to gelatinization [28].

In the isolated starch granule images by SEM from Wang and Ratnayake [31] study, there was no evidence of starch damage, with no visible cracks or notches in the surfaces, and the presence of foreign materials was also not observed. All cultivars presented spherical, oval, or elliptic forms with smooth surfaces. According to the authors, generally, *P. vulgaris* starch granules have similar morphologies

#### **Figure 7.**

*(A) SEM of the raw carioca bean endosperm (6.00 Kx), (B) SEM of the endosperm of raw carioca bean (15.61 Kx), (C) cotyledon of cooked and gelatinized carioca bean (370 x), and (D) cotyledon of cooked carioca bean (15.61 Kx).*

between their varieties but are very different from other starches such as tapioca and banana. The shape of the starch granules and size influence their functional properties, such as paste viscosity. A high viscosity is desirable for industrial uses, in which the purpose is the thickening function. Ambigaipalan et al. [26] did not find the presence of cracks in starch granules of carioca beans, as well as in black bean granules, both raw and evaluated by SEM.

Rupollo [17] analyzed by SEM the starch grains isolated from carioca beans stored for 360 days under three conditions: hermetically sealed at 5°C and atmosphere modified by nitrogen at 15°C and in a conventional atmosphere at 25°C. The author observed great similarity between the granules, even in different storage conditions of the seeds. However, the starch granules of the seeds stored in a conventional atmosphere at 25°C appeared to be more aggregate than the others. The influence of storage conditions on starch properties was verified through a joint data analysis, which is a multivariate technique used to evaluate how consumers develop preferences for products or services. The bean starch stored in a nitrogen-modified atmosphere at 15°C did not differ in solubility and gel properties compared to beans stored in a conventional atmosphere at 25°C. However, the gel properties of these two conditions differ from the hermetically packaged at 5°C, which presented lower crystallinity, as well as the swelling and heat power required for gelatinization. The grain starch stored in a nitrogen-modified atmosphere at 15°C, in turn, demonstrated lower crystallinity, swelling power, and heat required for gelatinization than grain stored in a conventional atmosphere at 25°C.

Vanier et al. [32] characterized starches from four common bean genotypes to use in production of biodegradable films. The authors observed that depending on the common bean genotype, a great variation on starch properties was found, which, in turn, clearly impacted on the characteristics of the starch-based films.

#### **3.4 X-ray diffraction**

The X-ray diffraction properties provide evidence of an ordered structure of the starch granule. The difference between crystallinities is associated with amylopectin, while amorphous regions are generally related to amylase. **Figure 8** presents the diffractograms of beans starches.

The starch isolated from black bean shows the highest peak values for the three evaluated. The diffraction angles 15, 17, and 23° represent the highest intensity peaks detected in the X-ray diffractograms, being even higher in 17° for all the analyzed starches in this work.

**Figure 8.** *Intensity of X-ray diffraction peaks of starch isolated from beans (A) cowpea, (B) black, and (C) carioca.*

**43**

**Table 1.**

*carioca.*

*\*CPS: counting per second.*

*Starch Granules from Cowpea, Black, and Carioca Beans in Raw and Cooked Forms*

crystallinity, verified in X-ray diffractograms, are shown in **Table 1**. The relative crystallinity (RC) was in descending order, black bean

lentil > smooth pea > pea > black beans > white beans.

association between the starch chains is observed [36].

HTC effect on grains stored in the conventional system.

**Bean Starch yield (%) Intensity (CPS\***

between the X-ray pattern of native starch and modified derivatives.

The yield of the starch isolation, the main peak intensities, and the relative

(10.64%) > cowpea (10.57%) > carioca bean (10.50%), having varied significantly,

According to Hoover and Ratnayake [35], differences in relative crystallinity between starches are affected by crystal size, amount of crystalline regions (influenced by amylopectin chain content and length), and orientation of double helices in the crystalline domains and by degree interaction between double helices. In their work, all starches showed a pattern of type C X-rays, typical of legumes. The peak at 2θ = 5.54 (characteristic of type B starches) was more pronounced in pinto bean and black bean starches. Relative crystallinity followed the order: pinto beans >

Type A pattern has the shortest amylopectin chain. Its structure is orthogonal and contains only eight molecules of water with few irregular connections, and amylose is distanced from amylopectin by an amorphous region, which is less dense and absorbs water more rapidly and is more susceptible to chemical and enzymatic modifications. In relation to the C pattern, a higher intensity of the diffractogram peak suggesting strong internal bonds of the molecules and a higher degree of

Lawal and Adebowale [37] analyzed the physicochemical characteristics and thermal properties of chemically modified porcine bean (*Canavalia ensiformis*) and observed, in addition to the conventional type C, an increase in the intensity of starch diluted in acid solution. The authors did not observe significant differences

Rupollo [17], evaluating the effects of storage conditions and time on the quality of carioca beans, observed that the starch of grains stored in a conventional atmosphere at 25°C were more influenced than the starch isolated from beans stored in modified atmosphere with nitrogen at 15°C, certainly due to the development of the

**15° 17° 23°**

**) RC (%)**

Pinto [38] evaluated carioca bean starch submitted to different treatments and observed the following sequence regarding the degree of relative crystallinity

Cowpea 16.04 1863 2214 1822 10.57 Black 16.85 1938 2358 1834 10.64 Carioca 30.24 1922 2306 1806 10.50

*Starch yield, main peaks intensity, and relative crystallinity of isolated starches from cowpea, black, and* 

Gernat et al. [33] analyzed X-ray diffractograms of *Vicia faba* and *Pisum sativum* (bean and pea, respectively) compared to corn and potato starches, which are types A and B, respectively. The authors found, by means of a linear regression method, pea starch composed of 38.6% of type B and 61.4% of type A and 17.0% of bean starch of type B and 83.0% of type A. Garcia and Lajolo [34] analyzed the changes of HTC in starch grains and found a very strong birefringence in starch grains from HTC beans, suggesting that the starch isolated from these grains has a higher degree

*DOI: http://dx.doi.org/10.5772/intechopen.85656*

considering the analysis of variance.

of crystallinity.

#### *Starch Granules from Cowpea, Black, and Carioca Beans in Raw and Cooked Forms DOI: http://dx.doi.org/10.5772/intechopen.85656*

The yield of the starch isolation, the main peak intensities, and the relative crystallinity, verified in X-ray diffractograms, are shown in **Table 1**.

The relative crystallinity (RC) was in descending order, black bean (10.64%) > cowpea (10.57%) > carioca bean (10.50%), having varied significantly, considering the analysis of variance.

Gernat et al. [33] analyzed X-ray diffractograms of *Vicia faba* and *Pisum sativum* (bean and pea, respectively) compared to corn and potato starches, which are types A and B, respectively. The authors found, by means of a linear regression method, pea starch composed of 38.6% of type B and 61.4% of type A and 17.0% of bean starch of type B and 83.0% of type A. Garcia and Lajolo [34] analyzed the changes of HTC in starch grains and found a very strong birefringence in starch grains from HTC beans, suggesting that the starch isolated from these grains has a higher degree of crystallinity.

According to Hoover and Ratnayake [35], differences in relative crystallinity between starches are affected by crystal size, amount of crystalline regions (influenced by amylopectin chain content and length), and orientation of double helices in the crystalline domains and by degree interaction between double helices. In their work, all starches showed a pattern of type C X-rays, typical of legumes. The peak at 2θ = 5.54 (characteristic of type B starches) was more pronounced in pinto bean and black bean starches. Relative crystallinity followed the order: pinto beans > lentil > smooth pea > pea > black beans > white beans.

Type A pattern has the shortest amylopectin chain. Its structure is orthogonal and contains only eight molecules of water with few irregular connections, and amylose is distanced from amylopectin by an amorphous region, which is less dense and absorbs water more rapidly and is more susceptible to chemical and enzymatic modifications. In relation to the C pattern, a higher intensity of the diffractogram peak suggesting strong internal bonds of the molecules and a higher degree of association between the starch chains is observed [36].

Lawal and Adebowale [37] analyzed the physicochemical characteristics and thermal properties of chemically modified porcine bean (*Canavalia ensiformis*) and observed, in addition to the conventional type C, an increase in the intensity of starch diluted in acid solution. The authors did not observe significant differences between the X-ray pattern of native starch and modified derivatives.

Rupollo [17], evaluating the effects of storage conditions and time on the quality of carioca beans, observed that the starch of grains stored in a conventional atmosphere at 25°C were more influenced than the starch isolated from beans stored in modified atmosphere with nitrogen at 15°C, certainly due to the development of the HTC effect on grains stored in the conventional system.

Pinto [38] evaluated carioca bean starch submitted to different treatments and observed the following sequence regarding the degree of relative crystallinity


#### **Table 1.**

*Legume Crops – Characterization and Breeding for Improved Food Security*

granules, both raw and evaluated by SEM.

**3.4 X-ray diffraction**

diffractograms of beans starches.

analyzed starches in this work.

between their varieties but are very different from other starches such as tapioca and banana. The shape of the starch granules and size influence their functional properties, such as paste viscosity. A high viscosity is desirable for industrial uses, in which the purpose is the thickening function. Ambigaipalan et al. [26] did not find the presence of cracks in starch granules of carioca beans, as well as in black bean

Rupollo [17] analyzed by SEM the starch grains isolated from carioca beans stored for 360 days under three conditions: hermetically sealed at 5°C and atmosphere modified by nitrogen at 15°C and in a conventional atmosphere at 25°C. The author observed great similarity between the granules, even in different storage conditions of the seeds. However, the starch granules of the seeds stored in a conventional atmosphere at 25°C appeared to be more aggregate than the others. The influence of storage conditions on starch properties was verified through a joint data analysis, which is a multivariate technique used to evaluate how consumers develop preferences for products or services. The bean starch stored in a nitrogen-modified atmosphere at 15°C did not differ in solubility and gel properties compared to beans stored in a conventional atmosphere at 25°C. However, the gel properties of these two conditions differ from the hermetically packaged at 5°C, which presented lower crystallinity, as well as the swelling and heat power required for gelatinization. The grain starch stored in a nitrogen-modified atmosphere at 15°C, in turn, demonstrated lower crystallinity, swelling power, and heat required

for gelatinization than grain stored in a conventional atmosphere at 25°C.

Vanier et al. [32] characterized starches from four common bean genotypes to use in production of biodegradable films. The authors observed that depending on the common bean genotype, a great variation on starch properties was found, which, in turn, clearly impacted on the characteristics of the starch-based films.

The X-ray diffraction properties provide evidence of an ordered structure of the starch granule. The difference between crystallinities is associated with amylopectin, while amorphous regions are generally related to amylase. **Figure 8** presents the

The starch isolated from black bean shows the highest peak values for the three evaluated. The diffraction angles 15, 17, and 23° represent the highest intensity peaks detected in the X-ray diffractograms, being even higher in 17° for all the

*Intensity of X-ray diffraction peaks of starch isolated from beans (A) cowpea, (B) black, and (C) carioca.*

**42**

**Figure 8.**

*Starch yield, main peaks intensity, and relative crystallinity of isolated starches from cowpea, black, and carioca.*

obtained with the different procedures: enzymatic hydrolysis > native starch > heating > ultrasound > low humidity heat treatment.
