Enliven: Journal of Dietetics Research and Nutrition

An analysis of shape and size of the granules of starches isolated from the edible portion of eight unripe banana genotypes was performed. The study aimed at investigating the potential of banana starches as ingredients for industrial purposes. It also aimed at investigating their in vitro digestibility in relation to their morphometric characteristics. The granular size distribution and granular morphometric characteristics were determined by diffraction Laser sizing (LDS), light polarized microscopy (LPM), and scanning electronic microscopy (SEM) techniques. Results reveal differences in the starch shape and size without any correlation between the granular profile and the genomic group. Granule shape includes symmetrical and asymmetrical spheres, symmetrical and asymmetrical shell-shapes, tubular-shapes, irregular and ellipsoidal-truncated shapes. Some granules exhibited smooth shapes, whereas others exhibit faceted surfaces. It was also observed some variation in the starch hydrolysis among samples, which was most pronounced in the BB banana starch genotype. The variation of the granular shape and size could be considered as a key for the differentiation of the banana genotypes. Such variation could affect the digestibility of the starches, even gelatinized. Thus, the granular distribution histogram can be used as an indicator of the potential uses of the starch from bananas. Among potential industrial uses, the isolated starch from the BB genotype can be used for paper and cosmetic industry according to its granule small size and uniformity. The other evaluated starches have granular size and shape variations that have to be sorted in relation to their functional properties and how they could affect the process within industrial formulations.

Introduction

There is a tremendous potential for the profitable commercial use of tropical starches. However, considerable research and product development is required to properly exploit starch materials [1]. Among others, in the tropical area, there are numerous varieties of banana with high potential for starch production. A solution for the diversification of the botanical sources of starches including banana starches, could be to investigate their physical and functional properties [2-5]. If some physico-chemical features are helpful to differentiate the banana cultivars [6-7], some agro-morphological variations are often used to recognize genotypes among Musa acuminata and Musa balbisiana. Considered to be natural crossing of the two previous genomic groups, a combined group made of acuminata and balbisiana species usually exhibits characteristics that combine their morphological characters [8-9]. In consequence, diploïds (AA, BB or AB genomes), triploids (AAA, BBB, AAB, or ABB genomes), and tetraploids (AAAA, and ABBB genomes) are growing in nature.

 

Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids [10], varying greatly in form, size and functionality, between and within botanical species. For instance, starch granules diameter can range from 1 to 100 ?m, as a function of the botanical source [11]. Banana starch granules from the various hybrids differ in size, while being irregular in shape, and appear as elongated ovals with ridges by microscopy [3].

 

Starch morphometric differences, not only provide diverse properties, but also induce diverse behaviors during processing due to their inner inconsistency [12]. These morphometric variations could affect its bioavailability, since the granule size of starch can affect the rate of enzymatic-hydrolysis of starch granules [13-14].

 

Granule morphology and particle size distribution of starches has a direct influence on material properties such as reactivity or dissolution rate, stability in suspension, delivery efficiency, texture and feeling, appearance, ability to flow and handling, viscosity, packing density, and porosity. It is of consensus that the average particle size on the food systems is known to influence properties related to processing (e.g. gelatinization, water and reagents absorption and solubility) [15-17], and nutritional value (e.g. starch digestion rate) [18-21] of human foods and animal feeds. Since the size and shape of the granules of starch are among important factors in the determination of the potential starch uses, its granule size and particle size distribution must be measured.

 

Optimizing the particle size distribution in suspensions can produce up to 50-fold reductions in shear viscosity, facilitating pumping, mixing and transportation in the fuel, concrete, paint and food industries [22]. For example, in food industry, as reported by Campbell et al. [23], small granules (< 2.0 µm) can be used as fat substitutes due to its similar size with the lipid beads; the size also influence the wet-milling extraction. Same authors [23] have also pointed out that other applications in which the size of the granules is important is on the production of biodegradable plastic films and carbonless fax papers. Moreover, the smallest granules can be used as a vehicle in cosmetics. The starch suitability for the above-mentioned applications is often determined by granule size.

 

Several techniques to determine particle size distribution can be used, including laser light scattering, light microscopy and SEM, sieving, sedimentation analysis, permeability of a powder column, and electrical sensing zone technique. Those techniques measure different parameters and each one have its advantages and disadvantages; therefore, the choice of the technique will largely depend on the application. Laser diffraction particle size analyzer has proved to be an effective tool for providing accurate and precise particle size measurement, because its requires little time for analysis, cover a wide size range, and require small sample amount, facilitating detailed studies of particle size populations. Laser diffraction particle size analyzers provide indirect size measurements of spherically equivalent particles, based on the principle that particles of a given size diffract light through a given angle that increases logarithmically with decreasing size [24].

 

On the other hand, a researcher dedicated to microscope techniques is able to identify starches from different botanical sources [25-29]. Among others, the scanning electron microscope (SEM) is a microscope that produces images from the starch by scanning it with a focused beam of electrons. Both sources of electrons, those ones from the equipment and from the starch, interact and produce signals that contain information about the surface of the sample topography and composition. It helps to describe parameters as dimension, forms and porous presence of the granular structure of the starch. Indeed, as postulated before, each one defines the functional properties of the starch. With the help of these tools, scientists are slowly beginning to build up a picture of how starch is constructed and how it is behaving at the food and other systems.

 

The goal of this study was then to determine morphologies and granular size distribution of isolated starches from different bananas genotypes, relating them to their in vitro digestibility in order to suggest some food applications.

 

Material and Methods

Materials

Eight isolated and purified starches from different genotypes of bananas belonging from the field collection at the Nacional de Investigaciones Agrícolas (INIA), estado Aragua, Venezuela were studied. Three samples were from acuminata ascendance (AA, AAA, AAAA genomes), three from acuminata and balbisiana ascendance (AAB, ABB, and AAAB genomes), and just two from balbisiana (BB, BBB genomes).

 

Methods

Sample preparation

Fresh bananas at full green stage of maturity equivalent to a Cavendish-like Grade 1 [30] were cleaned and rinsed with a large amount of tapwater and wiped for starch isolation. The edible portion was cleaned, peeled and sliced into 2.5 inche pieces. The sliced portions were immediately immersed in 1% of a citric acid solution in order to avoid enzymatic browning prior to further processing.

 

Starch isolation and purification

The starch isolation was performed on independent batches of approximately 1-2 kg of each the starches were isolated and purified using the procedure described by Pérez et al. [31] with some modifications. After slicing, pieces were milled during 3 min at high speed using a Waring blender with small volumes of distilled water. This grinding and screening operation were repeated four times. The resulting slurry was sieved consecutively through a 200-mesh muslin cloth sieve, and centrifuged at 500xg during 20 min. After removing the mucilaginous layer, the sediment was washed several times by suspension in distilled water and centrifuged until it appeared to be free of non-starch material. The sediment was then oven-dried at 45°C during 24 hours. The dried starch was blended, passed through a 60-mesh sieve, and stored at room temperature in sealed plastic bags.

 

Particle Size Distribution (PSD): Laser Diffraction Sizing (LDS)

Particle size distribution was studied at room temperature by laser diffraction by means of a laser light scattering Malvern Mastersizer 2000. Few milligrams of native starch powder were fed directly into the measuring cell where was submitted to ultrasound (LADD Mod. Sonicor SC-T56 60kHz) during 5-10 min. Volume distribution of the diffraction equivalent sizes was determined using the Fraunhofer scattering theory, while considering that native starch granules were opaque [5,32]. Aggregated starches were also sonicated in a 2% solution of sodium dodecil sulfate (SDS).

 

Granule morphology: Scanning Electron Microscopy (SEM) and Polarized Light Microscopy (PLM)

Starch samples were sprayed on a metal plate previously covered with double sided adhesive tape and shadowed under vacuum with gold-palladium. Starch granules were examined with a scanning electron microscope (Hitachi S-2400) at 20kV accelerating voltage [25]. Those starches former presumed as aggregate were also measured using polarized light microscopy. This analysis was performed on a NIKON Optiphot 2 microscope with a polarized filter, and with a Nikon FX - 35DX camera attached, according to methodology originally described by Sivoli et al. [26]. Starches could be seen under the microscope by placing a small amount of the starch powder on the slides, adding a drop of distilled water and covering it with a glass in order to increase the refractive index of the sample and to obtain better images.

 

For the quantitative analysis of granular sizes, a random sample of 100 granules on 50x magnification surface area were observed and measured for major and minor diameters. The larger and smaller diameter (average) ratio was also measured in order to compare the elongation of the granules.

 

In vitro starch digestibility

In vitro starch digestibility of the gelatinized starch suspension was analyzed [33]. A ?-amylase (1200 UI/mg and 27 mg of proteins/ml) from porcine pancreas preparation were used (A3176, Sigma Chemical Co., St. Louis, USA). About 700mg of dry starch were suspended into 50 ml of a sodium and potassium phosphate buffer (0.5 M pH 6.9) and homogenized. The starch suspension was gelatinized during 20 min into boiling water bath under continuous stirring. The suspension was then cooled to 37°C. About 4 mg/ml of the ?-amylase solution diluted in the phosphate buffer were mixed to gelatinized starch suspensions and incubated at 37°C for an hour. Sampling were carried out in triplicate at various time (5, 15, 30 and 60 min) in addition to the initial condition where no enzyme was used. A standard curve was prepared using pure dry maltose from the 0?2 mg/ml range, in addition to the use of a control made of pure potato starch. The 3,5-dinitrosalicylic acid method (DNS) was used, while adding 0.2 ml of sample, plus 0.8 ml of distilled water and 1 ml DNS solution in boiling water bath for 10 min, prior to cooling at room temperature and reading at 540 nm against a DNS blank. The extent of the hydrolysis was computed as the percentage of dry hydrolyzed starch (mg of maltose/100 mg of pure starch).

 

Statistical analysis

Results were analyzed using statistical standard parameters such as mean and standard deviation from replicates of the samples.

 

Results and Discussion

The results of the analysis of size and shape of the starch granules from the different banana genotype are shown in Table 1 and in Figures 1 to 4. Results showed differences in sizes and shapes among all genotype sources. As can be seen, in the starches granules measured by SEM exhibited a wide range of sizes, ranging from 3 to over 80 µm, differently shaped (Table 1), which are in agreement with values reported by several authors [2,34-41] for green banana starch isolated from different genotypes. The frequent shape was those shell-like. The largest rang of size was observed in starch from the AAB genome (10-80 ?m), and the smallest ones were at the starches from AAAA genome (2-14 ?m), and the BB genome (4-16 ?m) [2,40,41]. Some author has pointed out that the granular size and shape varied with the ripening stage [35]. However, as it is revealed here, deferences must be due to the botanical source.

Starch from Banana clone	Range of size	Shape
	(µm)
AA genome Dessert banana (Titiaro)	8-36	Small round, and ellipsoidal, and large shell, and ellipsoidal shaped.
AAA genome Dessert banana (Grande Naine)	6-28	Small tubular-like and largest ellipsoidal-truncated and shell-like.
AAAA genome Dessert banana (FHIA 17)	2-14	Small round and oval-truncated, and largest oval truncated shaped
BB genome Dessert banana (Balbisiana)	4-16	Small round and medium shell-shaped.
BBB genome Dessert banana (Let chan kut)	10-48	Small round, medium like-shell, and largest triangular-shaped.
AAB genome Cooking banana (Hartón)	10-80	Small round, and tubular-like, and largest shell-like.
ABB genome Dessert banana (Topocho)	12-56	Small round and tubular and largest ellipsoidal-truncated and shell shaped.
AAAB genome Dessert banana (FHIA 1)	4-46	Small shell-shaped and largest tubular

Table 1. Morphometry of isolated starches from banana genotypes measured by polarized light microscopy

Figure 2. Photomicrographs (PLM: left side, and SEM: right side) of isolated starches from banana (diploid , triploid and tetraploid from acuminate genome) : granules and particle size distribution (laser).

Table 2 combine the granule size distribution (%) of isolated starches from banana genotype measured by laser light diffraction technique, and Figure 1 shown the histogram of the granular distribution of each genotype. As expected, the largest population of granules was located at 7-40 µm range [2,36,38,39,41]. AA and ABB genomes exhibited the highest fraction of small granules with sizes below 7 ?m, whereas the BB starch granule exhibited the lowest fraction of granule below 7 ?m, despite that BB starch granule exhibited the lowest fraction of granule below 7 ?m , despite that BB genome starch granule was revealed as the smallest granule average by SEM. All banana starches showed the characteristic eccentric Maltese crosses, indicating that the isolation method used yielded intact native starch granules (Figures 2, 3 and 4; left side).

Genome/Size ranging	AA	AAA	AAAA	BB	BBB	AAB	ABB	AAAB
<7µm	9.6±0.4	8.5±0.6	8.3±0.6	1.4±0.1	7.8±0.1	7.2±0.1	8.7±0.9	7.5±1.1
7-20µm	38.4±4.6	46.6±0.4	32.7±1.1	40.0±0.1	31.5±0.1	26.36±0.1	47.4±9.7	24.7±1.0
20-40µm	42.2±1.5	43.3±4.2	47.3±1.5	26.3±0.1	44.7±0.1	55.2±0.1	39.7±6.9	50.2±0.1
> 40µm	8.8±2.2	4.0±1.0	11.7±0.2	32.3±0.1	16.0±0.1	11.2±0.1	4.2±0.8	17.7±2.2

Table 2. Granular size distribution (%) of isolated starches from banana genotype measured by laser diffraction technique.

Figure 2 is comparing the morphometric characteristic of starches isolated from banana with acuminata genotype (diploid, triploid and tetraploid genomes) by microphotographs of PLM and SEM, and curves of the granular distribution sizes. As can be seen, there are differences in shapes (Table 1) and sizes, which is plotted by the curve.

 

Figure 3 representes the morphometric characteristics of two starches isolated from balbisiana genotype (diploid and triploid genomes). 90% of granular size distribution fluctuated in the 0.1-40 µm range for both populations. On the one hand, the diploid BB starch exhibited the lowest starch fraction of the smallest size (0.1?7µm) and the highest fraction of the starch (16%) above 40 µm (Figures 2 to 4). The highest fraction of the starch population above 40 µm revealed by laser diffraction analysis was most probably due to its agglomeration. Such hard agglomeration was so strongly bound that neither the reagent nor the mechanical force used for conditioning the sample prior to laser diffraction analysis were effective for dis-agglomeration. In the other hand, the BBB starch exhibited small rounded and medium shell-shaped granules, and the largest triangular-shapes. The low granule range (from 4 to 16 µm) of the BB starch genotype and regularity of its size rendered the resource attractive with desirable feature for chemical papers as used for copying and fax, and as well as for cosmetic industry.

 

Some other starches exhibited polymodal granular distribution, suggesting that the granules can be sorted into more than one size range, and shape (Figure 3). Banana starch, as found in this study, exhibited a distribution of both large and small granules, and with variations in granular shapes. Granule shapes included symmetrical and asymmetrical spheres, symmetrical and asymmetrical shell-shapes, tubular-shapes, and irregular and ellipsoidal-truncated shapes. Besides that, some granules exhibited smooth shapes, whereas others showed faceted surfaces.

 

Figure 4 compares the granular population of the isolated starches from acuminata and balbisiana genomic groups through the representation of the AAB, ABB and AAAB genotypes. The AAB and ABB genome`s starches exhibited small rounded and tubular granules, as well as the largest shell-like ones with size variations. In fact, AAB genotype exhibited the largest variation by SEM as illustrated in Table 1 with a highest fraction of granules with sizes above 40 ?m (Table 2). Contrary to AAB genotype, the ABB triploid exhibited the lowest fraction of granules above 40 µm (Figure 4) and highest fraction of granules of the smallest granule size average in the 0.1 to 7 µm range. The AAAB starch exhibited the smallest granule size average with small shell-shapes and large tubular granules varying from 4 to 46 ?m.

 

Digestibility of native starches has been attributed to the interplay of many factors, and the being the granular size is one of them [3]. The extent of the hydrolysis of the cooked banana starches studied here fluctuated from 0 to 68% (Figure 5) and all of them exhibited lower digestibility extent to that of the standard curve. Except for balbisiana ancestry (BB and BBB), starches showed slight differences in their rates of hydrolysis up to nearly 50 minutes of incubation. The diploid and triploid clones from balbisiana (BB and BBB) exhibited atypical starch hydrolysis behavior, when compared with the six other starches.

It has been reported that dessert banana starch was appreciably more resistant to ?-amylase and glucoamylase attack than corn starch [36]. Cooking banana starches had the least resistance to ?-amylase digestion, which could be due to the resistant starch (RS) content [36]. It has been shown that banana starch has important content of resistant starch (RS) [41-42]. Among other contributions, according to the morphometric variations in starch granules observed, an effective contribution of the shape and granular size could be suggested as earlier illustrated in Figure 1 and 3. Some investigations may later confirm the variation of the morphometric characteristics here observed and specific potential for industrial uses, based on a larger number of accessions belonging to various genomic groups [36, 39-41].

 

Conclusion

There exist wide differences in size and shape as a function of the banana genotype, but the granular profile was not correlated with the genotype. However, the granular shape variation of the starches can be typified as a functional property for various uses in the industry. The histogram of the distribution of the population seemed a potential indicator for some uses of the starch from banana, including the BB isolated starch for paper and cosmetic industry according to its small size and size and shape uniformity. The other evaluated starches exhibited significant variations in their granular sizes and shapes, which may be considered for specific functional properties within manufactured items.

 

Acknowledgments

Special thanks are due to Instituto Nacional de Investigaciones Agrícolas (INIA-Aragua), Maracay, Venezuela. For supplying the fresh material, and to the local stakeholders in Ocumare, Aragua state, Venezuela for the identification and the delivery of fresh material of genotypes, donated by the INIA, available in their crops. The authors would like also to thank to the Consejo de Desarrollo Científico y Humanístico (CDCH) de la Universidad Central de Venezuela for the financial support for this study, through the projects N°(s): 0376072009/1-2 and 03.32.3873.97-00-04. Authors gratefully acknowledge the fund provided by the Programa de Cooperación de Postgrado (PCP) France-Venezuela, between the CIRAD at Montpellier, France and Instituto de Ciencia y Tecnología de Alimentos (ICTA), Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.

 

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