Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2007
Volume 10
Issue 3
Topic:
Food Science and Technology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Le Than J. , Błaszczak W. , Lewandowicz G. 2007. DIGESTIBILITY VS STRUCTURE OF FOOD GRADE MODIFIED STARCHES, EJPAU 10(3), #10.
Available Online: http://www.ejpau.media.pl/volume10/issue3/art-10.html

DIGESTIBILITY VS STRUCTURE OF FOOD GRADE MODIFIED STARCHES

Joanna Le Than1, Wioletta Błaszczak2, Grażyna Lewandowicz1
1 Department of Biotechnology and Food Microbiology, The August Cieszkowski Agricultural University of Poznan, Poland
2 Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Division of Food Science, Olsztyn, Poland

 

ABSTRACT

Digestibility of food grade modified starches were examined by the controlled enzymatic hydrolysis with the mixture of pancreatic alpha-amylase and glucoamylase. The structure and physicochemical properties of the experimental starch samples were examined by the Brabender rheological method, light microscopy, and X-ray diffractometry. It was found that investigated food grade modified starches revealed reduced digestibility-up to about 10%. The extent of decrease of digestibility depends on the degree of substitution of modified starches, but this correlation is not linear. Granular food grade modified starches reveal unchanged crystal structure, identical as native starch. The modification of starch changes its physicochemical properties i.e. pasting profile, gelatinisation temperature and solubility in water, however these changes do not correlate with digestibility. Changes in conformation of starch macromolecules in solution, caused by incorporation of modifying groups, hinder enzymatic action of amylases and cause a drop in digestibility of starch.

Key words: modified starches, structure, digestibility.

INTRODUCTION

In diet, starch is primarily considered as a source of energy and it mainly determines its nutritive importance. However, in many foodstuffs it escapes complete digestion by amylases of alimentary tract, which implicates their nutritional value. From this point of view, starch can be classified into three basic groups: rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) [7].

The digestibility of native starch depends on its botanical origin and significantly rises after gelatinisation [9, 15]. The chemical modification of starch may ultimately affect its rate and extent of digestion in the small intestine [17, 18]. It is especially important when creating so called “functional food” designed for groups of people suffering from diabetes or metabolic syndromes. Up to now it has been stated that the oxidation or dextrinisation of starch, its substitution with hydroxypropyl, acetyl or octenylsuccinate groups as well as crosslinking cause the decrease of digestibility [8, 17, 18, 19]. Unfortunately, the comparison of the data published by various research groups is difficult, due to the fact that obtained results are strongly influenced by the method applied for the determination of digestibility. The differences could be the effect of application merely different alpha-amylases (rabbit or human) [9]. Generally, all in vitro methods of the estimation of digestibility of starch consist in the simulation of digestion process in the human alimentary tract. However all these simulations are very imperfect due to the fact that only the activities of alphaamylase and glukoamylase are taken into consideration, and other enzymes occurring in enteron are omitted. As a source of enzymes are proposed not only pure enzymes – alpha-amylases and glucoamylases but also mixtures such as pancreatin [3, 8]. Older methods, for the digestion of starch, employed only one enzyme i.e. alpha-amylase. The amount of released reducing saccharides were determined spectrophotometrically with 3,5-dinitrosalicylicacid. The extent of the hydrolysis was expressed as a ratio of the amounts of released maltose to analysed starch [9, 19]. Essential progress was made during the research programme of the European Communitiy Commission of regarding resistant starch [2]. As a result of the work of EURESTA group the methods for the determination of rapid digestible starch (RDS), slow digestible starch (SDS) and resistant starch (RS) were proposed. The principle of the method base on hydrolysis of starch with the mixture of pancreatic alpha-amylase and glucoamylase followed by the measurement of the released glucose using glucose oxidase [7]. Rapidly digested and slowly digested starch are defined as the amount of glucose released after hydrolysis at the temperature of 37°C for a period of 20 and 100 min respectively. Resistant starch is the starch not hydrolysed after 120 min of incubation. The newest standards for the determination of resistant starch postulate the hydrolysis with the mixture of pancreatic alpha-amylase and glucoamylase at the temperature of 37°C for the time of 16 h [1, 4, 5].

The chemical modification of starch for food application is precisely restricted not only in terms of type of employed chemical reactions but also the extent of changes in starch macromolecules [6]. The above mentioned restrictions are recommended by Joint FAO/WHO Expert Committee on Food Additives (JECFA) with the aim of protecting consumers against intake of objectionable food. However changes in molecular and supermolecular structure of starch, caused by chemical modification and changing rate of digestion could be advantageous, for example in manufacturing functional food designed for the diabetics.

The aim of the work was to contribute to the understanding of the relationship between structure, physicochemical properties and the digestibility of food grade modified starches. Native starch was used as a reference material.

MATERIALS AND METHODS

Materials
Native potato starch Superior Standard as well as food grade modified starches listed below kindly provided by WPPZ Luboń SA. (Poland) were studied i.e.:

X-ray diffractometry
X-ray diffractometry was carried out with a TUR 62 Carl Zeiss X-ray diffractometer under the following conditions: X-ray tube CuKα (Ni filter); voltage 30 kV; current 15 mA; scanning from Θ = 2° to 18°. To avoid the influence of relative humidity on relative crystallinity, starch samples were placed in a desiccator and conditioned in the atmosphere of relative humidity of 92% for 48 h. The desiccator was filled with a sodium carbonate saturated water solution.

Microscopic examination
The starch samples were prepared for light microscopy examination using the smear method. The 8% starch suspension were heated at 90°C for 15 min. A drop of paste was placed on a microscope slide and, on cooling, the smear was stained with iodine (I2 in KI) and studied using an Olympus BX60 light microscope.

Viscosity measurements
The course of gelatinisation was monitored with a Brabender viscograph under the following conditions: measuring cartridge 0.07 Nm; heating/cooling rate 1.5° C/min; thermostating 30 min.

Chemical composition
The carboxyl as well as acetyl groups content of starch derivatives were determined according to Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommendation [6]. Carboxyl groups determination involves leaching of modified starch with mineral acid to convert carboxyl salts to the acid form. Cations and excess acid are removed by washing with water. The washed sample was gelatinised in water and titrated with standard alkali. Acetyl groups content determination consists in hydrolysing them with sodium hydroxide and titration of the excess of alkali with hydrochloric acid. Results compiled on tables are means of four measurements with standard deviation considered.

Digestibility
The rate of digestion of starch was determined by its hydrolysis with the mixture of pancreatic alpha-amylase and glucoamylase at the temperature of 37°C, at particular periods of time, followed by the measurement of the released glucose using glucose oxidase. Porcine pancreatic alpha-amylase type VI-B (Sigma) as well as glucoamylase AMG 300L (Novozymes) were used for the analyses. The amount of released glucose was determined colorimetrically at the λ = 500 nm using Liquick Cor-Glucose diagnostic kit (Cormay, Poland). Four replicates were made for each probe and standard deviation were calculated.

RESULTS AND DISCUSSION

Native potato starch, one of the most important sources of energy in human nutrition, was, as it could be expected, fully digested after 16 h (Table 1). It should be emphasised that almost full digestion of native starch was achieved already after 8 h. Similar digestibility, as well as digestion rate profile, revealed distarch phosphate E 1412 preparation. Distarch phosphate, as other cross-linked starches, is obtained as a result of very fine changes in molecular structure of starch i.e. on cross-linked bond occurs per 500–1000 anhydroglucose units [13, 14]. In other words, it could be expressed as a degree of substitution DS = 0.001 – 0.002. Starch modified by using other methods i.e. obtained by incorporation of carboxyl or acetyl groups revealed reduced digestibility-up to about 10%. The extent of this decrease was correlated with the degree of substitution of modified starch, but this correlation seems to be not linear. Slightly oxidised E 1403 starch containing 0.04 of carboxyl groups (what could be also expressed as DS = 0.0014) revealed the decrease of digestibility of about 5.6%. The increase of carboxyl groups content up to 0.1 % (DS = 0.036) caused the decrease of digestibility only of additional 1%. The lowest digestibility revealed acetylated starch E 1420 containing 1.7% of acetyl groups (DS = 0.065). Acetylated distarch adipate E 1422 containing 1.2% of acetyl groups (DS = 0.046) showed digestibility higher than E 1420 of about 2%. Acetylated distarch phosphate E 1414 containing the lowest amount of acetyl groups 0.8% (DS = 0.030) revealed relatively high digestibility, even higher than oxidised starches.

Table 1. The rate of digestion of chemically modified starches and modifying groups content

Starch preparation

Digestibility after different time of digestion [%]

Modifying groups content [%]

0.5 h

1.0 h

2.0 h

4.0 h

8.0 h

16.0 h

carboxyl

acetyl

Native

40.7±0.7

62.4±0.2

72.0±0.3

84.7±0.2

99.4±0.2

100.4±0.3

E1403

36.3±0.6

48.2±0.3

58.0±0.2

75.5±0.4

90.9±0.2

94.4±0.4

0.038±0.001

E1404

36.9±0.5

46.9±0.3

57.9±0.6

73.3±0.2

85.0±0.8

93.5±0.3

0.10±0.01

E 1412

39.1±0.8

62.6±0.7

73.0±0.6

82.2±0.3

96.8±0.7

100.1±0.1

E 1414

38.5±0.7

52.9±0.4

71.5±0.8

87.7±0.7

95.6±0.6

96.2±0.1

0.82±0.02

E 1420

30.7±0.6

43.7±0.3

53.0±0.1

65.9±0.7

88.4±0.5

90.9±0.2

1.71±0.02

E 1422

35.5±0.9

39.9±0.3

52.9±0.1

64.7±0.7

88.0±0.3

93.0±0.2

1.21±0.01

The data presented above pointed out that degree of substitution of modifying groups might not be the only reason for the decrease of digestibility, and some physicochemical or structural factors could be of important significance. Modification of starch for food application are fulfilled under very mild condition. Chemical reactions are carried out in water suspension, then the product is separated by filtration. Next, the filter cake is dispersed in water and filtered in order to removing by-products and unreacted raw materials. The purification procedure is repeated twice and then the product is dried in a flash drier [16]. As a consequence, these mild processing conditions did not cause any changes in crystal structure of the starch, what could be clearly observed at the Figures 1 and 2.

Fig. 1. X-ray diffraction pattern of: a – native potato starch; b – bleached starch E 1403; c – oxidised starch E 1404

Native potato starch revealed typical for tuber starches B type of X-ray diffraction pattern (Fig. 1). All modified starches show not only the same B type of X-ray diffraction pattern but also the same relative crystallinity (Fig. 1 and 2).

No changes in the crystal structure due to chemical modification pointed out that the structure of the granule did not influenced digestibility of starch. This conclusion is plain because starch is eaten after gelatinisation, and the determination of digestibility was also performed after gelatinisation. Probably, different level of digestibility is caused by the variability of interactions of water with modified starch macromolecules in solution.

Fig. 2. X-ray diffraction patterns of: a – distarch phosphate E 1412; b – acetylated distarch phosphate E 1414 ; c – acetylated starch E 1420; d – acetylated distarch adipate E 1422

Differently than in crystal structure, the changes in molecular structure of starch caused by chemical modification brought about significant differences in starch pasting characteristics (Fig. 3-5). When analysed the course of starch gelatinisation with Brabender viscograph it should be taken into consideration that the data gained does not depend only on applied temperature profile and measuring cartridge, but also on concentration of the investigated starch suspension.

Fig. 3. Brabender viscosity curves for 6% suspensions of native and oxidised starches

A typical concentration value usually applied to potato starch is 3.3%, and most of our measurements were done under such conditions. However hypochlorite modification (applied for manufacturing of the E 1403 and 1404 preparations) caused so significant decrease in the viscosity of starch that it was in that case immeasurably low. Consequently the pasting characteristics of oxidised starches E 1403 and E 1404 were determined at a concentration of 6%. Under these experimental conditions native potato starch (Fig. 3) revealed a high type of swelling characteristics, typical of tuber starch, with a rapid increase in viscosity within a narrow temperature range and the occurrence of a viscosity peak. Modified starches E 1403 and E 1404 revealed similar type of swelling characteristics. Drop in viscosity, mentioned above, depended on the degree of oxidation and was observed within almost all range of the measurement (Fig. 3). The only exception was final value of viscosity of the E 1403 preparation which was higher than in case of native starch. High type of swelling characteristics presented by all sorts of starch shown at fig. 1 pointed that process of dissolution were relatively easy and did not clarify changes in digestibility. Also a drop in viscosity accompanying the oxidation of starch could not be the reason of the decrease of digestibility.

Unlike oxidised starches characterised by a very low degree of substitution, acetylated starch E 1420 contained 1.7% of modifying groups, and revealed a course of swelling characteristics similar to native starch, accompanied by similar viscosity values (Fig. 4). It should be mentioned that acetylated starch revealed gelatinisation temperature significantly lower than native starch, which pointed to easier dissolving process. However this observation did not explain the differences in digestibility.

Fig. 4. Brabender viscosity curves for 3.3 % suspensions of native and acetylated starches

Cross-linked starch: distarch phosphate E 1412, acetylated distarch phosphate E 1414 containing 0.8% of acetyl groups, and acetylated distarch adipate E 1422 containing 1.2% of acetyl groups, were also investigated. These group of modified starch showed a changed type of swelling characteristic with a progressive rise in viscosity over a wide temperature range and lack of a viscosity peak (Fig. 5). The more restricted type of swelling characteristic indicated a more difficult dissolving of the starch and could be the reason of the reduction of digestibility. Paradoxically, distarch phosphate E 1412 revealing the most restricted type of swelling characteristics revealed almost unchanged digestibility in comparison with native starch. Consequently the solubility of starch could not be regarded as the most important factor influencing digestibility. Acetylated distarch adipate E 1422, standing out from other cross-linked types of starch with higher acetyl groups content, likewise with acetylated starch E 1420 it revealed lower pasting temperature. These observation indicated that the acetylation process caused a decrease in pasting temperature of starch, but did not explain the differences in digestibility.

Fig. 5. Brabender viscosity curves for 3.3% suspensions of cross-linked starches

Light microscopy investigation are a very useful tool for analysing starch – water interaction in solution [10, 11, 12]. It makes it possible, for example, to observe the mechanism of gelatinisation of starch and interaction between starch macromolecules and other substances present in water solution. Light microscopy pictures of all modified starches were presented in Figures 6-8. The images represents the structure of the solution formed in food products without sterilisation process. It is known that the gelatinisation process of starch started with swelling of starch granules and the leakage of amylose [11]. The pictures formed at the temperature of 90°C (Fig. 6 a) showed almost homogenous medium formed mainly with amylose and amylopectin solved in water with suspended swollen granules remnants. These remnants were formed mainly by amylopectin which is more difficult to dissolve. The images of hypochlorite modified starches (Fig. 6 b and 6 c) only slightly differed from that of native starch. The most important difference was that granule remnants were more gentle what was probably connected with the lower viscosity and consequently lower molecular mass of starches. The analysis of light microphotographs of oxidised starches did not explain the differences in digestibility.

Fig. 6. Light microscopy pictures of starch solutions
a. native potato starch
b. bleached starch E 1403
c. oxidised starch E 1404

Fig. 7. Light microscopy pictures of acetylated starch E 1420 solution

Light microscopy pictures of acetylated starch E 1420 (Fig. 7) represented a quite different image in comparison to native and oxidised starches. At the microphotograph of acetylated starch it was impossible to discriminate the amylose and amylopectin fractions. Neither amylose molecules formed the blue complex nor amylopectin the red one. All starch macromolecules created quite different brown complex. This observation seems to be crucial to understand the differences in digestibility of modified starches. Modifying acetyl groups changed the conformation of starch macromolecules so much that forming standard blue and violet complexes of starch with iodine became impossible. These relatively deep changes in the conformation of starch macromolecules probably hindered the enzymatic action of amylases and caused drop in the digestibility of starch. It could not be excluded that, also in the case of oxidised starches, the conformational changes of starch macromolecules hindered the enzymatic action of amylases, though these changes did not disturb forming complexes with iodine.

The light microscopy pictures of cross-linked starch (Fig. 8) indicate more difficult dissolving process of these preparations in comparison with native starch. The images showed mainly remnants of swollen granules. The suspending medium made from solution of amylose and amylopectin was more difficult to observe. Distarch phosphate E 1412 which revealed almost the same digestibility as the native starch seemed to be the most difficult to dissolve. Nevertheless, as it was stated before, solubility of starch could not be regarded as the most important factor influencing digestibility. Acetylated and cross-linked starch was better soluble in water than distarch phosphate. This observation pointed that incorporation of acetyl groups to starch macromolecules facilitated starch dissolving, but lowered their digestibility.

Fig. 8. Light microscopy pictures of cross-linked starch solutions: a – distarch phosphate E 1412; b – acetylated distarch phosphate E 1414; c – acetylated distarch adipate E 1422
distarch phosphate E 1412
acetylated distarch phosphate E 1414
acetylated distarch adipate E 1422

CONCLUSIONS

Investigated food grade modified starch revealed reduced digestibility – up to about 10%.
The extent of decrease of digestibility depends on the degree of substitution of modified starch, but this correlation is not linear.
Granular food grade modified starch reveals unchanged crystal structure, identical as native starch.
Modification of starch changes its physicochemical properties i.e. pasting profile, gelatinisation temperature and solubility in water. However these changes do not correlate with digestibility.
Changes in conformation of starch macromolecules in solution, caused by incorporation of modifying groups, hinder enzymatic action of amylases and cause drop in digestibility of starch.

ACKNOWLEDGMENTS

This work was partly supported by postgraduate students fellowship No US/60/06 funded in scope of the project “Fellowships for the best PhD Students in Wielkopolska Region conducting research in strategic areas supporting regional development” financed by the European Social Fund (75%) and Polish budget (25%).

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Accepted for print: 28.08.2007


Joanna Le Than
Department of Biotechnology and Food Microbiology,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 48, 60-627 Poznan, Poland
Phone: (+48 61) 846 60 05
Fax. (+48 61) 846 60 03

Wioletta Błaszczak
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences,
Division of Food Science, Olsztyn, Poland


Grażyna Lewandowicz
Department of Biotechnology and Food Microbiology,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 48, 60-627 Poznan, Poland
Phone: (+48 61) 846 60 05
Fax. (+48 61) 846 60 03
email: gralew@au.poznan.pl

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