**3.2 Optical microscopy (OM)**

## *3.2.1 Cowpea*

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

were made with secondary electron detector.

before and after the starch isolation.

**3. Results and discussion**

for the three varieties of beans.

**3.1 Morphological characterization**

1 mol L<sup>−</sup><sup>1</sup>

were carried out at the National Center of Structural Biology and Bioimaging (Centro Nacional de Biologia Estrutural e Bioimagem - CENABIO) of the Federal University of Rio de Janeiro (UFRJ), using Zeiss microscope, model EVO MA10, tungsten filament, working distance of 10 mm, and voltage of −15 Kv. The images

Starch extraction was based on the method described by Wang and Wang (2004), with some modifications. The samples were ground in a laboratory mill (Perten, 3100) soaked in 0.1% NaOH solution in a ratio of 1:5 and allowed to stand for 20 hours. After dispersion, vigorous stirring in the blender was performed for 2 minutes. The resulting material was passed through a 63 μm sieve and centrifuged (Sorvall® RC 6 Plus centrifuge) at 1200 RPM for 5 minutes at room temperature (25°C ± 2). The supernatant was discarded, and the precipitate was resuspended in 0.1% NaOH solution and centrifuged again and the operation performed twice. The extracted starch was dispersed with distilled water and neutralized with

HCl to pH 6.5 and centrifuged. The sedimented material was resuspended

. The relative

in distilled water and centrifuged and the operation repeated twice. The resulting starch was oven-dried with air circulation at 40°C to 11% ± 0.5 humidity. The starch extraction yield was calculated on the difference between the dry flour masses

X-ray diffraction crystallinity tests were performed in the multiuser laboratory of the Chemistry Institute of the Federal University of Uberlândia (UFU). The diffractograms of the starches were obtained by an X-ray diffractometer (XRD-6000, Shimadzu, Brazil) in which the diffraction sweep region ranged from 5 to 30° with a

crystallinity (RC) of the starch granules was calculated by the software XRD-6000 v. 5.2. The RC values of all bean samples were evaluated by GraphPad Prism®

Common bean and raw cowpea grains can be visualized in **Figure 1**, being

Seed format characteristics and tegument coloration were within the range expected for species and varieties. Measurements of the materials studied revealed that the measurements were approximately 0.5–0.7 cm wide and 0.7–0.9 cm long

*Anatomical structure of bean pulse (A) cowpea, (B) black beans, and (C) carioca bean.*

target voltage of 30 kV and current of 30 mA. Scan speed was 1°min<sup>−</sup><sup>1</sup>

software, using the analysis of variance (ANOVA).

(A) cowpea, (B) black beans, and (C) carioca bean.

**34**

**Figure 1.**

**Figure 2** shows the optical microscopy of cowpea in cross section. Image A represents the integument of the raw grain prepared by manual cutting with an optical magnification of 50 x. Image B represents the tegument of the raw grain, prepared by the technique of infiltration in paraffin and serial cut with the help of a rotating microtome, both images with 50 x optical magnification. Image C refers to the cotyledon of the raw grain, and D refers to the cooked cotyledon under pressure; both images were observed using polarized light microscopy, with 10 x optical magnification, and slides prepared by freehand cut. Image E represents the cotyledon of the raw grain whose blade was prepared by freehand cut, and image F refers to cotyledon of the raw grain whose blade was prepared by serial cutting with the aid of a rotary microtome, preceded by paraffin infiltration; both images were obtained by optical microscopy with optical magnification of 50 x, using polarized light [22].

#### **Figure 2.**

*Cross section of cowpea: (A) seed coat of the raw seed cut by free hand (50 x), (B) seed coat of the raw seed, cut with microtome (50 x), (C) cotyledon of raw seed (10 x), (D) cotyledon of cooked seed (10 x), (E) cotyledon of freehand cut seed (50 x), and (F) cotyledon of the raw seed cut with microtome (50 x).*

In the integument images (A and B), three layers of tissues can be observed: epidermis, hypodermis, and lacunar parenchyma. The epidermis consists of two layers of flattened cells, with thickened walls. The hypodermis is composed of osteosclereids that are hourglass-shaped. The cells of the lacunar parenchyma have a shape close to the cylindrical, arranged with gaps between them. The thickness of the tegument is highly correlated with characters that reveal the size and shape of the seeds and water absorption capacity [23]. The images from SEM of raw whole cowpea seeds from Biaszczak et al. [24] revealed integument with an average thickness of about 90 μm, composed by palisade, glasshour and cells of lacunar parenchyma. In the tegument image whose material was previously infiltrated in paraffin (B), it is observed that the lacunar parenchyma presents rupture in the tissue, indicating that the presence of the reagents used in the embedding technique may have interfered in the sample structure, possibly due to the resumption of metabolic activities of seeds which broke their state of dormancy. Thus, the freehand cut allowed a better visualization of the structure, with the advantage of being a simple, efficient, and low-cost methodology, requiring no addition of organic solvents [23].

In the cotyledon images of the raw grain (C, E, and F) it is possible to perceive the great presence of amido within the amyloplasts, being coherent with the energy reserve function of this structure, since in beans as in other legumes, they constitute the main storage organs of the seed. The "Malta Cross" conformation of the starch resulting from the birefringence of the crystalline regions of the starch granule can be observed, as well as the characteristic spherical structure of the granule to that legume. Biaszczak et al. [24] reported that the cotyledon cells were rounded or elongated in the longitudinal axis, with an average size of 80 μm and had bimodal, elongated starch grains, firmly covered with protein material.

In the cooked grain image (D) the presence of starch grains exhibiting the characteristic "Malta Cross" is not observed, which evidences the loss of structural organization with the melting of the crystals. Such alteration is characteristic of the gelatinization process that occurs when the starch is submitted to temperatures higher than 50°C. Souza and Andrade [25] reported that after submission to temperatures above 75°C, there is no birefringence of starch grains of corn by optical microscopy on polarized light indicating loss of previously existing molecular ordering.

In the cotyledon image whose material was infiltrated in paraffin (E), the sharpness of the morphology of the starch granules is smaller than the image whose blade was prepared by free hand cut (F), suggesting once again that the technique manual cutting is more advantageous for bean seed samples.

#### *3.2.2 Black bean*

In **Figure 3**, optical microscopy of black beans in cross section can be observed. Image A represents the integument of the raw grain prepared by manual cutting with an optical magnification of 50 x. Image B represents the tegument of the raw grain, prepared by the technique of infiltration in paraffin and serial cut with the help of a rotating microtome, both images with 50 x optical magnification. Image C refers to the cotyledon of the raw grain, and D refers to the cooked cotyledon under pressure; both images were observed using polarized light microscopy, with 10 x optical magnification, and slides prepared by freehand cut. Image E represents the cotyledon of the raw grain, the blade of which was prepared by freehand cutting, and image F refers to the cotyledon of the raw grain, the blade of which was prepared by serial cutting with the aid of a rotary microtome; preceded by paraffin

**37**

**Figure 3.**

*microtome (50 x).*

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

infiltration the images were obtained by optical microscopy with 50 x optical

*Cross section of black beans: (A) seed coat of the raw seed, cut by free hand (50 x); (B) seed coat of the raw seed, cut with the help of a microtome (50 x); (C) cotyledon of raw seed (10 x); (D) cotyledon of boiled seed (10 x); (E) cotyledon of freehand cut seed (50 x); and (F) cotyledon of the raw seed, cut with the help of a* 

investigated the relative contributions of cotyledons and seed coats toward hardening of common beans were and the rate-limiting process which controls bean softening during cooking was determined. The authors suggested that the

In the same way as in cowpea bean microscopy (**Figure 2**), three layers of tissues can be observed in the integument images (A and B): epidermis, hypodermis, and lacunar parenchyma. However, in the integument image whose material was previously infiltrated in paraffin (B), the lacunar parenchyma shows rupture in the tissue. In the cotyledon images of the raw grain (C, E, and F), there is also a great presence of amido in the interior of the amyloplasts, its conformation of "Malta Cross," and the spherical structure of the granule. In the study by Ambigaipalan et al. [26], all black and carioca bean starch grains exhibited a strong birefringence pattern under polarized light, indicating that amylopectin crystallites are arranged radially within the bead at right angles to their interface with single reducing end group for the yarn. Weaker patterns would be indicative of double amylopectin helices disorganized within the crystalline lamellae of these grains. Chigwedere et al. [27]

magnification using polarized light [22].

*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*

#### **Figure 3.**

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

In the integument images (A and B), three layers of tissues can be observed: epidermis, hypodermis, and lacunar parenchyma. The epidermis consists of two layers of flattened cells, with thickened walls. The hypodermis is composed of osteosclereids that are hourglass-shaped. The cells of the lacunar parenchyma have a shape close to the cylindrical, arranged with gaps between them. The thickness of the tegument is highly correlated with characters that reveal the size and shape of the seeds and water absorption capacity [23]. The images from SEM of raw whole cowpea seeds from Biaszczak et al. [24] revealed integument with an average thickness of about 90 μm, composed by palisade, glasshour and cells of lacunar parenchyma. In the tegument image whose material was previously infiltrated in paraffin (B), it is observed that the lacunar parenchyma presents rupture in the tissue, indicating that the presence of the reagents used in the embedding technique may have interfered in the sample structure, possibly due to the resumption of metabolic activities of seeds which broke their state of dormancy. Thus, the freehand cut allowed a better visualization of the structure, with the advantage of being a simple, efficient, and low-cost methodology, requiring no addition of

In the cotyledon images of the raw grain (C, E, and F) it is possible to perceive the great presence of amido within the amyloplasts, being coherent with the energy reserve function of this structure, since in beans as in other legumes, they constitute the main storage organs of the seed. The "Malta Cross" conformation of the starch resulting from the birefringence of the crystalline regions of the starch granule can be observed, as well as the characteristic spherical structure of the granule to that legume. Biaszczak et al. [24] reported that the cotyledon cells were rounded or elongated in the longitudinal axis, with an average size of 80 μm and had bimodal, elongated starch grains, firmly covered with

In the cooked grain image (D) the presence of starch grains exhibiting the characteristic "Malta Cross" is not observed, which evidences the loss of structural organization with the melting of the crystals. Such alteration is characteristic of the gelatinization process that occurs when the starch is submitted to temperatures higher than 50°C. Souza and Andrade [25] reported that after submission to temperatures above 75°C, there is no birefringence of starch grains of corn by optical microscopy on polarized light indicating loss of previously existing molecular

In the cotyledon image whose material was infiltrated in paraffin (E), the sharpness of the morphology of the starch granules is smaller than the image whose blade was prepared by free hand cut (F), suggesting once again that the technique manual

In **Figure 3**, optical microscopy of black beans in cross section can be observed. Image A represents the integument of the raw grain prepared by manual cutting with an optical magnification of 50 x. Image B represents the tegument of the raw grain, prepared by the technique of infiltration in paraffin and serial cut with the help of a rotating microtome, both images with 50 x optical magnification. Image C refers to the cotyledon of the raw grain, and D refers to the cooked cotyledon under pressure; both images were observed using polarized light microscopy, with 10 x optical magnification, and slides prepared by freehand cut. Image E represents the cotyledon of the raw grain, the blade of which was prepared by freehand cutting, and image F refers to the cotyledon of the raw grain, the blade of which was prepared by serial cutting with the aid of a rotary microtome; preceded by paraffin

cutting is more advantageous for bean seed samples.

**36**

organic solvents [23].

protein material.

ordering.

*3.2.2 Black bean*

*Cross section of black beans: (A) seed coat of the raw seed, cut by free hand (50 x); (B) seed coat of the raw seed, cut with the help of a microtome (50 x); (C) cotyledon of raw seed (10 x); (D) cotyledon of boiled seed (10 x); (E) cotyledon of freehand cut seed (50 x); and (F) cotyledon of the raw seed, cut with the help of a microtome (50 x).*

infiltration the images were obtained by optical microscopy with 50 x optical magnification using polarized light [22].

In the same way as in cowpea bean microscopy (**Figure 2**), three layers of tissues can be observed in the integument images (A and B): epidermis, hypodermis, and lacunar parenchyma. However, in the integument image whose material was previously infiltrated in paraffin (B), the lacunar parenchyma shows rupture in the tissue.

In the cotyledon images of the raw grain (C, E, and F), there is also a great presence of amido in the interior of the amyloplasts, its conformation of "Malta Cross," and the spherical structure of the granule. In the study by Ambigaipalan et al. [26], all black and carioca bean starch grains exhibited a strong birefringence pattern under polarized light, indicating that amylopectin crystallites are arranged radially within the bead at right angles to their interface with single reducing end group for the yarn. Weaker patterns would be indicative of double amylopectin helices disorganized within the crystalline lamellae of these grains. Chigwedere et al. [27] investigated the relative contributions of cotyledons and seed coats toward hardening of common beans were and the rate-limiting process which controls bean softening during cooking was determined. The authors suggested that the

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

rate-determining process in bean softening relates to cell wall/middle lamella changes influencing pectin solubilization.

The presence of starch granules inside the amyloplasts is still observed in the cooked cotyledon image, suggesting that the cooking conditions were suitable for gelatinization, possibly due to a long storage period of the seeds, which characterizes an HTC phenomenon [19, 21].

### *3.2.3 Carioca bean*

**Figure 4** shows the optical microscopy of carioca beans in cross section. Image A represents the integument of the raw grain prepared by manual cutting with an optical magnification of 50 x. Image B represents the tegument of the raw grain, prepared by the technique of infiltration in paraffin and serial cut with rotating microtome, both images with 50 x optical magnification. Image C refers to the cotyledon of the raw grain, and D refers to the cooked cotyledon under pressure; both images are observed using polarized light microscopy, with 10 x optical magnification, and slides prepared by freehand cut. Image E represents the cotyledon of the raw grain whose blade was prepared by freehand cut, and image F refers to cotyledon of the raw grain whose blade was prepared by serial cutting with the aid of a rotary microtome, preceded by paraffin infiltration; both images were obtained by optical microscopy with optical magnification of 50 x, using polarized light [22].

#### **Figure 4.**

*Carioca bean cross section: (A) seed coat of the raw seed, cut by free hand (50 x); (B) seed coat of the raw seed, cut with microtome (50 x); (C) cotyledon of raw seed (10 x); (D) cotyledon of cocked seed (10 x); (E) cotyledon of freehand cut seed (50 x); and (F) cotyledon of the raw seed, cut with microtome (2.5 x).*

**39**

**Figure 5.**

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

were prepared by freehand cutting or rotating microtome.

Similar to the cowpea (**Figure 2**) and black bean microscopy (**Figure 3**), the layers of epidermis, hypodermis, and lacunar parenchyma can be observed in the tegument images (A and B). In the image of cotyledon of raw bean (C), a large amount of starch granules is also observed inside the amyloplasts, and in the image of cooked cotyledon (D), the absence of this structure is observed due to the phenomenon of gelatinization, also observed previously in the cowpea image (**Figure 2**). With 50 x optical magnification of cotyledon (images E and F), no notable differences were observed in the starch granules, between the blades that

**Figure 5** shows a cowpea endosperm SEM. In image A granules of starch attached to the cotyledon cell wall and its reniform shape (magnification 5.52 Kx) can be visualized, and in B bulges on the surface of the granule (magnification 30.36 Kx) can be perceived, both refer to raw samples. In the images of cooked samples with different optical amplifications (C 400 x and D 19.14 Kx), it is noticed that the starch granules were grouped, losing their crystalline structure due to

with the description of Agunbiade and Longe [29], which confirmed that grain lengths in all samples were mostly larger than their widths. With the enlargement

*(A) Cowpea raw endosperm SEM (5.52 Kx), (B) cowpea raw endosperm SEM (30.36 Kx) granule surface, (C) cooked and gelatinized cowpea cotyledon (400 x), and (D) cooked cowpea cotyledon (19.14 Kx).*

The morphological aspect of the starch observed in **Figure 5(A)** is in accordance

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

**3.3 SEM**

*3.3.1 Cowpea*

gelatinization [28].

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

Similar to the cowpea (**Figure 2**) and black bean microscopy (**Figure 3**), the layers of epidermis, hypodermis, and lacunar parenchyma can be observed in the tegument images (A and B). In the image of cotyledon of raw bean (C), a large amount of starch granules is also observed inside the amyloplasts, and in the image of cooked cotyledon (D), the absence of this structure is observed due to the phenomenon of gelatinization, also observed previously in the cowpea image (**Figure 2**). With 50 x optical magnification of cotyledon (images E and F), no notable differences were observed in the starch granules, between the blades that were prepared by freehand cutting or rotating microtome.

#### **3.3 SEM**

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

changes influencing pectin solubilization.

izes an HTC phenomenon [19, 21].

*3.2.3 Carioca bean*

rate-determining process in bean softening relates to cell wall/middle lamella

The presence of starch granules inside the amyloplasts is still observed in the cooked cotyledon image, suggesting that the cooking conditions were suitable for gelatinization, possibly due to a long storage period of the seeds, which character-

**Figure 4** shows the optical microscopy of carioca beans in cross section. Image A represents the integument of the raw grain prepared by manual cutting with an optical magnification of 50 x. Image B represents the tegument of the raw grain, prepared by the technique of infiltration in paraffin and serial cut with rotating microtome, both images with 50 x optical magnification. Image C refers to the cotyledon of the raw grain, and D refers to the cooked cotyledon under pressure; both images are observed using polarized light microscopy, with 10 x optical magnification, and slides prepared by freehand cut. Image E represents the cotyledon of the raw grain whose blade was prepared by freehand cut, and image F refers to cotyledon of the raw grain whose blade was prepared by serial cutting with the aid of a rotary microtome, preceded by paraffin infiltration; both images were obtained by optical microscopy with optical magnification of 50 x, using polarized light [22].

*Carioca bean cross section: (A) seed coat of the raw seed, cut by free hand (50 x); (B) seed coat of the raw seed, cut with microtome (50 x); (C) cotyledon of raw seed (10 x); (D) cotyledon of cocked seed (10 x); (E) cotyledon of freehand cut seed (50 x); and (F) cotyledon of the raw seed, cut with microtome (2.5 x).*

**38**

**Figure 4.**

#### *3.3.1 Cowpea*

**Figure 5** shows a cowpea endosperm SEM. In image A granules of starch attached to the cotyledon cell wall and its reniform shape (magnification 5.52 Kx) can be visualized, and in B bulges on the surface of the granule (magnification 30.36 Kx) can be perceived, both refer to raw samples. In the images of cooked samples with different optical amplifications (C 400 x and D 19.14 Kx), it is noticed that the starch granules were grouped, losing their crystalline structure due to gelatinization [28].

The morphological aspect of the starch observed in **Figure 5(A)** is in accordance with the description of Agunbiade and Longe [29], which confirmed that grain lengths in all samples were mostly larger than their widths. With the enlargement

#### **Figure 5.**

*(A) Cowpea raw endosperm SEM (5.52 Kx), (B) cowpea raw endosperm SEM (30.36 Kx) granule surface, (C) cooked and gelatinized cowpea cotyledon (400 x), and (D) cooked cowpea cotyledon (19.14 Kx).*

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

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.
