# Banana starch breakdown in the human small intestine studied by electron microscopy

N. Faisant, D.J. Gallant, Brigitte Bouchet, Martine Champ

► To cite this version:

N. Faisant, D.J. Gallant, Brigitte Bouchet, Martine Champ. Banana starch breakdown in the human small intestine studied by electron microscopy. European Journal of Clinical Nutrition, 1995, 49, pp.98-104. hal-02712103

HAL Id: hal-02712103

https://hal.inrae.fr/hal-02712103

Submitted on 1 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Inventaire

<figure><img src="image_3.png" /></figure>

N°

<figure><img src="image_4.png" /></figure>

# Banana starch breakdown in the human small intestine studied by electron microscopy

N. Faisant, D. J. Gallant, B. Bouchet and M. Champ

Laboratoire de Technologie Appliquée à la Nutrition I.N.R.A., BP 1627, 44316 Nantes Cedex 03, France

**Objective:** To determine the origin of the poor digestibility of banana starch granules in the human small intestine.

**Design:** The subjects received the same experimental meal.

**Setting:** Nutrition Research Unit, Laënnec Hospital, CHU, Nantes.

**Subjects:** Six healthy young subjects.

**Interventions:** The digestion of raw green banana flour in the upper part of the gut was studied by the intubation technique. After ingestion of 30 g banana flour mixed with a complex meal, ileal samples were continuously collected during 14 h. In order to determine the structural nature of this resistant starch, the dried ileal samples were observed with scanning and transmission electron microscopy. Transmission electron microscopy was performed after treatment with periodic acid-thiosemicarbazide-silver nitrate.

**Results:** Banana starch proved very resistant to *in vivo* amylase hydrolysis since 84% of the starch ingested reached the terminal ileum. The microscopic observations showed that raw banana flour contained irregularly shaped dense starch granules with smooth surfaces. After their passage through the small intestine, starch granules appeared exocorroded, with porous surfaces, and some exhibited several irregular pits, crevices or holes by which the enzymes had penetrated and hydrolysed the inner part. Cell walls closely associated with starch granules could have hindered enzyme access to starch.

**Conclusions:** Encapsulation could be partly responsible for the low digestibility of starch in banana flour, together with the intrinsic resistance of banana starch granules.

**Sponsorship:** Supported by grants from the French Ministry of Agriculture and the Danone Group.

**Descriptors:** banana, digestion, human, microscopy, small intestine, starch

## Introduction

The bioavailability of starch in the upper part of the gut has been extensively studied during the last decade. It is now well known that a substantial fraction of dietary starch, referred to as 'resistant starch' (RS), is not digested and absorbed in the human small intestine. Three main intrinsic factors are responsible for this

resistance (Englyst, Kingman & Cummings, 1992): (i) physically inaccessible starch can escape enzymatic attack because of its entrapment in cell walls, as in the case of legumes or partly milled seeds and grains; (ii) some native or incompletely gelatinized starches, such as raw potato and banana starches, are intrinsically resistant to amylolysis; and (iii) retrograded starch, which is formed during the cooling of

Correspondence to: M. Champ.

Received 19 May 1994; revised 29 August 1994; accepted 2 September 1994
<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

gelatinized starches, is very resistant to digestion.

As most starchy foods are cooked before ingestion, few native starches are directly consumed, except in some biscuits in which the gelatinization of cereal starches is incomplete (Gallant, 1974), or in raw banana or hard-to-cook beans (Garcia, Lajolo & Swanson, 1993). Although native wheat starch is totally digested in the upper part of the gut of the rat (Björck *et al.*, 1986), unripened banana starch is very resistant to digestion *in vivo* in the rat and man as well as *in vitro* (Fuwa, Sugimoto & Takaya, 1979; Sugimoto *et al.*, 1980; Englyst *et al.*, 1992; Faisant *et al.*, in press). Starch content is high in unripened bananas (about 80% dry matter basis) but decreases during ripening when starch is hydrolysed and then converted into sucrose (Englyst & Cummings, 1986).

In *in vivo* conditions, part of the starch escaping digestion reaches the terminal ileum in the form of granules or insoluble fractions, whereas the rest was composed of soluble molecules (dextrins and oligosaccharides) being products of starch hydrolysis (Englyst & Cummings, 1986; Faisant *et al.*, in press). The purpose of the present work was to improve our understanding of the causes of native B-type starch resistance. Transmission (TEM) and scanning (SEM) electron microscopy were used to study banana starch granules from green banana flour after passage through the upper part of the gut.

## Materials and methods

### Preparation of banana flour

Raw banana flour was prepared from green bananas (*Musa paradisiaca sapientum*) originating from Martinique (French West Indies). Bananas were purchased locally prior to the customary gas treatment which induces ripening, when they were totally green and inedible as fruit. They were then peeled, cut in small slices, freeze-dried and milled (sieve: 3 mm). The total starch content of the banana flour was 77% [dry matter (DM) basis].

### Collection of human ileal samples

Ileal samples were collected by the intubation technique, as previously described (Faisant *et al.*, in press). Briefly, six volunteers were given a breakfast containing eggs, ham, sugar, oil,

yoghurt and 30 g of banana flour (i.e. 23 g of starch) as the only source of dietary polysaccharides (starch and non-starch). Banana flour was mixed together with yoghurt, oil and sugar. Ileal samples were then continuously aspirated for 14 h through a triple-lumen tube positioned the day before. Samples were pooled every 30 min, frozen in liquid nitrogen, stored at -70°C and later freeze-dried and gently milled. Total α-glucans were analysed in each sample and corrected for ileal flow rate as estimated by the dilution of the flow marker infused continuously 25 cm above the aspiration port. Total starch recovery, calculated as the sum of α-glucans recovered during 14 h, was 84% for banana starch. This fraction of undigested α-glucans contained about 87% insoluble starch granules and 13% soluble molecules produced from starch hydrolysis. For physical characterizations, aliquots of each 30-min dry fraction were pooled in order to prepare one representative sample for each subject of all the α-glucans that reached the terminal ileum during the 14 h period. The total starch content of those pooled ileal samples was about 25% (DM basis). Microscopy studies were performed on the pooled samples of two subjects.

### Microscopy studies

Transmission electron microscopy (TEM). In order to avoid material losses and heterogeneous distribution, aliquots of each dry sample were first embedded in 3% agar gel at temperatures below 50°C. When the agar had solidified, small cubes of the sample were cut and fixed for 1.5 h at 4°C in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), washed several times in buffer and then in deionized water. They were then treated for 1 h in a saturated solution of 2-4-dinitrophenylhydrazine in 15% acetic acid in order to block up free aldehydes. After several washes in deionized water, samples were oxidized for 30 min in 1% periodic acid aqueous solution, washed in deionized water, treated with a saturated solution of thiosemicarbazide, washed again and finally treated with 1% AgNO₃ aqueous solution according to Gallant (1974). Four-day treatment in silver nitrate was required to obtain good starch granule contrast. Samples were then washed, gradually dehydrated in an ethanol series and embedded in Epon 812. Sections <100 nm in thickness were examined
<figure><img src="image_7.png" /></figure>

under 80 keV using a JEOL 100S transmission electron microscope.

*Scanning electron microscopy (SEM).* Dry samples were stuck on brass stubs using double-sided carbon conductive adhesive tape and then ion-sputtered (gold deposit 25 nm) with JEOL JFC 1100. Bulk samples were examined at 10 keV under 2.10<sup>-12</sup> A using a JEOL JSM-840A scanning electron microscope.

## Results

### *Observations of raw banana flour*

Starch granules in raw banana flour were viewed as a mixture of irregularly shaped flat and elongated particles 10–50 µm in length (Fig. 1). The concentric shells ordinarily visible

in light microscopy were clearly apparent in TEM (Fig. 1d), but SEM provided only surface images showing little or no internal evidence of granule structure. Surfaces were smooth, but fragments from cell walls or cytoplasmic residues were often attached. Many aggregates were observed in which several granules were enclosed within fragmented cell walls. Starch granules were not extracted from the banana flour, and milling had not broken all the cell walls.

Observation of the intact tissues of raw banana showed that several granules were generally enclosed within a single cell (data not shown). The silver contrast in TEM studies indicated that these fragments were polysaccharide cell walls. Their close association with the granule surface was clearly apparent (Fig. 1a and c).

<figure><img src="image_8.png" /><figcaption>Fig. 1. Raw banana flour observed by SEM (a and b) and TEM (c and d). S = starch granule; CW = cell wall.</figcaption></figure>
<figure><img src="image_9.png" /></figure>

Observation of banana starch after in vivo
hydrolysis

In TEM and SEM studies, the extent of granule
degradation in the intestine was quite variable:
some granules appeared to be intact, whereas
others were greatly altered (Fig. 2).

SEM showed that the surface of most gran-
ules was superficially altered and porous, with
some granules exhibiting several irregular crev-
ices or scratches. Pores with more regular
shapes (Fig. 2b) were also randomly distributed
on the granule surface. TEM showed some

<figure><img src="image_10.png" /><figcaption>Fig. 2. Banana starch granules observed in ileal effluents of human after ingestion of banana flour by SEM (a, b and c) or TEM (d, e and f). S = starch granule; CW = cell wall.</figcaption></figure>
<figure><img src="image_11.png" /></figure>

granules that were partly hydrolysed internally but less damaged externally. Pinhole or crevice openings provided entries into the granules allowing hydrolysis of the internal structures (Fig. 2d and e).

When the external part of the starch granule was eroded, higher SEM magnification showed an interior composed of small, relatively spherical blocklets (Fig. 2c) arranged in concentric layers. This arrangement may have given the characteristic striated features apparent on some granule surfaces. However, this internal concentric organization with superimposed layers could not be observed in TEM since the staining procedure was optimized by action on oxidation kinetics in order to colour only the amorphous part. Generally, all the structures were quickly over-oxidized, and the staining showed no alternating shells (Fig. 1d) (Gallant & Guilbot, 1969; Gallant, 1974).

As starch granules were not isolated from the ileal contents, other structures were visible. In SEM studies on ileal samples, several fragments surrounding the granules (Fig. 2f) clearly protected them from hydrolysis. It was sometimes difficult to determine whether these fragments were cytoplasmic residues from banana parenchyma cells or endogenous material such as mucus or desquamative intestinal cells. However, the association of starch granules with polysaccharide fragments could also be seen in TEM studies after *in vivo* hydrolysis.

## Discussion

Our observations confirm previous results concerning the resistance of banana starch to hydrolysis (Fuwa *et al.*, 1979; Sugimoto *et al.*, 1980; Englyst & Cummings, 1986). Even after passage through the upper part of the gut, involving salivary amylase action, gastric retention under acidic conditions and pancreatic amylase action, banana starch granules were not greatly altered (84% recovery of α-glucans, but 73% recovery of the insoluble fraction).

The pattern of amylase attack on banana starch was comparable to that on potato starch (Gallant *et al.*, 1973; Sugimoto *et al.*, 1980). The granules first appeared to be only exocorroded, the surface having become porous and slightly pitted. The apparently random development of pinholes or crevices on the

granule surface suggests that there were no preferential sites of enzyme adsorption. Pores and crevices induced the formation of canals penetrating into the granules. Internal hydrolysis occurred only when the most external layers were cut into by enzyme activity, suggesting the presence of a more resistant external granule layer like that described for B-type starches such as potato starch (Gallant *et al.*, 1973, 1992). Endocorrosion was facilitated as soon as canals were formed. The small blocklets arranged in concentric shells, commonly attributed to the most crystalline part (Duprat *et al.*, 1980; Gallant *et al.*, 1992), appeared to be the most resistant structures.

Some granules showed irregular crevices or scratches on their surface which could hardly have been caused by enzymatic action alone. The role of chewing or other physical damage within the gastrointestinal tract could be involved. Physical damage during sample preparation which was then intensified by enzymatic action may also have played a part. Large granules such as those of raw potato and banana starches are known to be rather susceptible to mechanical alteration, especially after acidic action as in the stomach (Gallant, 1974). However, except for some small pits on the surface of raw banana starch granules, no crevices or scratches were ever observed in the initial product before ingestion. *In vivo* hydrolysis followed by freeze-drying may have weakened the granules.

The reasons for banana starch resistance to amylase hydrolysis are not clearly understood. B-type starches have a very compact and dense structure which gives specific swelling, solubility and pasting properties after heating in water. Such molecular associations, as well as granule size and absolute density, have no influence on their enzymatic susceptibility (Eggleston, Swennen & Akoni, 1992). Linnecar *et al.* (1993) concluded that the resistance of banana starch to digestion was not attributable to any physicochemical parameters such as crystallinity, X-ray pattern, temperature of gelatinization or amylose content. However, Lii, Chang & Young (1982) proposed that, as for potato starch, phosphorous could cross-link in starch granules and reinforce the granular structure. Accessibility, conditioned by the specific area and porosity, and enzyme ability to adsorb on the structures appear to be the
<figure><img src="image_12.png" /></figure>

main physicochemical factors differentiating native starch granule susceptibility (McGregor & McGregor, 1985). Thus, the most susceptible starches apparently possess ‘pores’ or a sponge-like internal structure of sufficient diameter to allow enzyme entry (Leach & Schoch, 1961). The smooth and dense surface of native banana starch granules could account in part for their resistance. However, it is more likely, as indicated by SEM and TEM studies (Gallant et al., 1992), that the B-type starch granule (as in the potato) is surrounded externally by a thick layer (several µm) of larger blocklets that impede enzyme action and reduce the rate of hydrolysis.

Apart from intrinsic factors responsible for the resistance of banana starch granules, another factor could have been involved in the present study. As many starch granules were surrounded by cell wall residues or enclosed within cell walls, these structures could also have hindered enzyme accessibility to granules. Thus, it is likely that both intrinsic resistance and encapsulation of starch granules

are responsible for the partial digestion of banana starch.

The hydrolysis of starch granules was very heterogeneous, although most presented porous features that should have increased accessibility to inner structures. However, as *in vivo* digestion was limited to transit time through the small intestine, some potentially digestible fractions could have entered the colon without being totally hydrolysed (Faisant et al., 1993).

This study indicates that initial exocorrosion of starch granules from banana flour renders their surface porous. Small canals are then formed by which enzymes can penetrate and digest the inner parts of the granules. However, cell walls are closely associated with the starch granules, so that encapsulation could also be involved in the low digestibility of banana starch.

*Acknowledgements*—This study was carried out as part of the EEC Flair program EURESTA and was supported by grants from the French Ministry of Agriculture and the Danone group.

## References

Björck I, Nyman M, Pedersen B, Siljeström M, Asp N-G & Eggum BO (1986): On the digestibility of starch in wheat bread. *Studies in vitro* and *in vivo*. *J. Cereal Sci.* **4**, 1-11.

Duprat F, Gallant D, Guilbot A, Mercier C & Robin JP (1980): L'amidon. In *Les polymères végétaux*, ed. Gauthier Villars, pp. 176-231.

Eggleston G, Swennen R & Akoni S (1992): Physicochemical studies on starches isolated from plantain cultivars, plantain hybrids and cooking bananas. *Starch* **44** (4.S.), 121-128.

Englyst HN & Cummings JH (1986): Digestion of the carbohydrate of banana (*Musa paradisiaca sapientum*) in the human small intestine. *Am. J. Clin. Nutr.* **44**, 42-50.

Englyst HN, Kingman SM & Cummings JH (1992): Classification and measurement of nutritionally important starch fractions. *Eur. J. Clin. Nutr.* **46**, Suppl. 2, S33-S50.

Faisant N, Champ M, Colonna P, Buléon A, Molis C, Langkilde A-M, Schweizer T, Fiourié B & Galmiche J-P (1993): Structural features of resistant starch at the end of the human small intestine. *Eur. J. Clin. Nutr.* **47**, 285-296.

Faisant N, Buléon A, Colonna P, Molis C, Lartigue S, Galmiche JP & Champ M (in press): Digestion of raw banana starch in the small intestine of healthy humans: structural features of resistant starch. *Br. J. Nutr.*

Fuwa H, Sugimoto Y & Takaya T (1979): Scanning electron microscopy of starch granules, with or without amylase attack. *Carbohydr. Res.* **70**, 233-238.

Gallant DJ (1974): Contribution à l'étude de la structure et de l'ultrastructure de l'amidon, Ph.D. Thesis, Univ. de Paris. CNRS AO No.10823.

Gallant DJ, Derrien A, Aumaitre A & Guilbot A (1973): Dégradation *in vitro* de l'amidon par le suc pancréatique. *Starch* **25**, 56-64.

Gallant DJ & Guilbot A (1969). Etude de l'ultrastructure du grain d'amidon à l'aide de nouvelles méthodes de préparation en microscopie électronique. *Starch* **21**, 156-163.

Gallant DJ, Bouchet B, Buléon A & Pérez S (1992): Physical characteristics of starch granules and susceptibility to enzymatic degradation. *Eur. J. Clin. Nutr.* **46**, Suppl. 2, S3-S16.

Garcia E, Lajolo FM & Swanson BG (1993): A comparative study of normal and hard-to-cook Brazilian common bean (*Phaseolus vulgaris*): ultrastructural and histochemical aspects. *Food Struct.* **12**, 147-154.

Leach HW & Schoch TJ (1961): Structure of the starch granule. II. Action of various amylase on granular starches. *Cereal Chem.* **38**, 34-46.

Lii CY, Chang SM & Young YL (1982): Investigation of the physical properties of banana starches. *J. Food Sci.* **47**, 1493-1497.

Linnecar KEM, Norton G, Neale RJ & Blanshard JMV
<figure><img src="image_13.png" /></figure>

(1993): Bioavailability of raw starches - causes of granule resistance to α-amylase attack. *Proc. 'Bioavailability 93'*, ed. U Schlemmer, Part 1, pp. 187-190.
B.F.E., Karlsruhe, Germany.
McGregor EA & McGregor AW (1985): A model for the

action of cereal alpha-amylases on amylose. *Carbohydr. Res.* **142**, 223-236.

Sugimoto Y, Fijita S, Takaya T & Fuwa H (1980): *In vivo* digestion of banana starch granules. *Starch* **32**, 209-294.