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Trang 1The influence of time and storage temperature on resistant starch formation from autoclaved debranched banana starch
M Sa´nchez-Rivera a, L.A Bello-Pe´rez a,*
aCentro de Desarrollo de Productos Bio´ticos del IPN, Km 8.5 carr Yautepec-Jojutla, colonia San Isidro, apartado postal 24,62731 Yautepec, Morelos, Mexico
bDepto Graduados Inv Alimentos, Escuela Nacional de Ciencias Biolo´gicas, IPN, Carpio y Plan de Ayala, 11340 Me´xico, DF, MexicoReceived 10 February 2006; accepted 2 April 2006
Debranching and autoclaving processes of banana starch were carried out for obtaining a resistant starch-rich powder with functional characteristics Debranching was carried out using pullulanase for 24 h and the autoclaving was done at 121C for 30 min, the samples were then cooled down and stored between 24 and 48 h, and temperatures between 4 and 60C The resistant starch level increased due to the debranching and autoclaving processes The water absorption index values decreased when the storage time increased, pattern that agrees with the higher RS content The water solubility index (WSI) was affected by the storage temperature but not by the storage time The autoclaved sample was hydrolyzed to a lesser extent than native starch The RS-rich powder presented also crystallinity because the process of autoclaving and storage induced starch retrogradation The procedure proposed might be used for production of a RS-rich powder from banana starch with high RS level and functional properties.
2006 Elsevier Ltd All rights reserved.
Keywords: Banana starch; Resistant starch; Autoclaving; Functional properties; X-ray diffraction
1 Introduction
Carbohydrates constitute the main fraction of cereals, legumes, tubers and unripe fruits, accounting for up to 40–80% of the dry matter Starch and non-starch polysac-charides (dietary fiber) are the major carbohydrate constit-uents (Skrabanja, Liljerberg, Hedley, Kreft, & Bjorck,
sources for obtaining starch with better physicochemical and functional characteristics, and as raw material for development of new products In recent years, substantial progresses have been made in obtaining and characterize
Agama-Acebedo, Sa´yago-Ayerdi, & Moreno-Damian, 2000; Hoover, 2001; Spencer & Jane, 1999).
Banana is a climacteric fruit and, in Me´xico, is con-sumed when the fruit is ripe For this reason, high quanti-ties of fruit are lost during their commercialization due to poor postharvest handling Unripe banana starch is very resistant to digestion in the rat and man.Faisant, Bule´on et al (1995) and Faisant, Gallant, Bouchet, and Champ (1995) studied the in vivo digestibility of banana starch and the structural features of resistant starch (RS) from banana Various studies have demonstrated that resistant starch is a part of dietary starch, which is defined as the fraction of a starch, or the degradation products of that starch, that passes through the small intestine into the large intestine Within the large intestine, such indigested starch is fermented by gut bacteria, producing short-chain fatty acids, which confer a range of benefits for the gut health
0963-9969/$ - see front matter 2006 Elsevier Ltd All rights reserved.doi:10.1016/j.foodres.2006.04.001
Corresponding author Tel.: +52 7353942020; fax: +52 7353941896.E-mail address:labellop@ipn.mx(L.A Bello-Pe´rez).
www.elsevier.com/locate/foodresFood Research International 40 (2007) 304–310
Trang 2and are thought to reduce the risk of colon-rectal cancer (Asp, 1992; Sievert & Pomeranz, 1989; Topping & Clifton,
Cummings (1992), these indigestible starch fractions are classified as follows: (1) RS1 corresponds to physically inaccessible starches, entrapped in a cellular matrix, as in cooked legume seeds; (2) RS2 are native uncooked granules of some starches, such as those in raw potatoes and green bananas, whose crystallinity makes them scarcely suscepti-ble to hydrolysis; and (3) RS3, consists mainly of retro-graded starches, which may be formed in cooked foods that are kept at or below room temperature Recently, a fourth type (RS4), consists of certain fractions of
Pe´rez, & Tovar, 2003; Tovar, Herrera, Laurentı´n, Melito, & Pe´rez, 1999) Resistant starch can be found in both pro-cessed and raw food materials From these four types, RS3 seems to be particularly interesting because it preserves its nutritional characteristics when it is added as an ingredient to cooked foods (Rosin, Lajolo, & Menezes, 2002) RS3 is produced by gelatinization followed by retrogradation The formation of RS3 after retrogradation is due to increased interaction between starch components It has been shown that, after starch debranching, the linear chains can contribute to a high RS content (Berry, 1986) The degree of polymerization (DP) of the linear chains influences the retrogradation phenomena (Eerlingen & Del-cour, 1995) Previous studies have shown that a DP of 20 is
Bengs, & Jacobasch, 2000) The physiological importance of RS has been investigated in relation to reduction of the glycemic and insulinemic response to a food, as well as hypocholesterolemic and protective effects against colo-rectal cancer (Asp, Van Amelsvoort, & Hautvast, 1996; Cassidy, Binghman, & Cummings, 1994; de Deckere, Kloots, & van Amelsvoort, 1995; Jenkins et al., 1987) The most important effect is based on the high fermentation rate of retrograded RS3 to short-chain fatty acids (SCFA), with a high proportion of butyrate by action of the intesti-nal microflora (Sharp & Macfarlane, 2000) Resistant starches have been introduced in recent years as functional food ingredients important for human nutrition RS has
Zaks, & Gross, 1991) The RS development method by
Chiu et al (1994), involves gelatinization of a starch with an amylose content of more than 40%, enzymatic debran-ching of gelatinized starch, deactivation of the debrandebran-ching enzyme, and isolation of the resultant product either by drying, extrusion or crystallization by adding salt Strate-gies for the development of crop plants that contain RS have recently been reviewed (Morell, Konik-Rose, Ahmed, Li, & Rahman, 2004).
The aim of the present work was to evaluate the forma-tion of RS and some funcforma-tional characteristics by serial autoclaving and storage at different temperatures of deb-ranched banana starch.
2 Materials and methods 2.1 Starch isolation
Starch was isolated from unripe bananas by the method ofAparicio-Saguilan et al (2005).
2.2 Debranching of banana starch
Banana starch solution (25 g of starch in 100 ml of ace-tate buffer, pH 5.2 and 0.1 M) was gelatinized for 10 min in a boiling water bath (with stirring) then autoclaved (Yam-ato, Scientific, model SM 510 Tokyo, Japan) at 121C for 30 min, after this time, the gel was re-dissolved with 125 ml of acetate buffer The gel (10% w/v) was cooled to 50C and 10.6 U/g of pullulanase (activity of 462.4 ± 10.95 U/ml, where 1 U/ml = lmol of glucose/ml) (Promozyme D, Novozymes, Bagsyaerd, Denmark) was added The mix-ture was incubated with constant stirring for 24 h at 50C 2.3 Resistant starch production
for 30 min, cooled down and stored between 24 and 48 h,
freeze-dried and stored in closed glass containers The experimental design (22 factorial design), to evaluate the best storage conditions for resistant starch production from the autoclaved product, resulted in nine runs, includ-ing five replicates of the central point (Table 1) This exper-imental design was chosen due to the fact that the number of samples elaborated was small and it was possible to obtain tendencies in the range of values of the independent variables studied.
2.4 Resistant starch content
Resistant starch (RS) content was measured using the procedure ofGon˜i, Garcı´a-Diz, Man˜as, and Saura-Calixto (1996) The method had the following steps: removal of
incubation with a-amylase (Sigma A-3176, 37C, 16 h) to
Table 1
Experimental design to determine the influence of storage time andtemperature on resistant starch formation in autoclaved banana starchRunX1X2Temperature (C)Time (h)
Trang 3hydrolyze digestible starch, treatment of precipitates with 2 M KOH to solubilize RS, incubation with amyloglucosi-dase (Sigma A-3514, 60C, 45 min, pH 4.75), and determi-nation of glucose, using the glucose oxidase assay (Banks & Greenwood, 1971) RS was calculated as glucose· 0.9 2.5 Water absorption index and water solubility index
Water absorption index (WAI) and water solubility index (WSI) were measured according to a modified method ofSchoch (1964) A 0.4 g (dry basis) ground extru-date sample was mixed with 40 ml of water in a centrifuge tube and mixed with a vortex After heating for 30 min in a water bath at 30C, the heated solution was centrifuged at 3000g for 10 min The WAI and WSI were determined as: WAI = weight of sediment/weight of dry sample solids; WSI = (weight of dissolved solids in supernatant/weight of dry sample solids in the original sample)· 100.
2.6 Characterization of the sample with the highest resistant starch content
2.6.1 In vitro digestibility tests
Potentially available starch content was assessed
and Asp (1986) using Termamyl (48,700 units/ml, one unit will hydrolyze 1.0 mg of maltose from starch in
and amyloglucosidase (14.0 units/mg, at 25C with glyco-gen and standardized with BSA) (Boehringer, Mannheim) The in vitro rate of hydrolysis was measured using hog pancreatic amylase (15.8 units/mg of solid, one unit will hydrolyze 1.0 mg of maltose from starch in 3 min at pH
and Lundquist (1985); each assay was run with 500 mg available starch.
2.6.2 X-ray diffraction
Samples, before the analysis, were stored in desiccator with a relative humidity of 82% for obtaining a constant moisture content, and then analyzed between 2h = 2 and 2h = 60 with a step size 2h = 0.02 in an X-ray diffractom-eter (Philips PW 1710, The Netherlands) using Cu Ka radi-ation (k = 1.543), 50 kV and 30 mA The diffractometer was equipped with 18 divergence slit and a 0.1 mm receiv-ing slit.
2.6.3 Scanning electron microscopy
For SEM study, the samples were fixed to a conductive tape of copper of double glue, which was covered with a layer of coal of 20 nm thickness It was deposited at vac-uum with an evaporator in a JEOL JSMP 100 (Japan) elec-tron microscope Later on, it was covered with a layer of gold of 50 nm of thickness in the ionizer of metals JEOL This was observed in the microscope and registered photo-graphically Film pieces were mounted on aluminum stubs using a double-sided tape and then coated with a layer of
gold (40–50 nm), allowing surface and cross-section visual-ization All samples were examined using an accelerating voltage of 5 kV.
2.6.4 Statistical analysis
Results were expressed as the mean values ± standard error of the three separate determinations Comparison of means was performed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests using JMP (SAS Institute Inc., Versio´n 4.0.4) system Mul-tiple regression analysis was applied for evaluating the effect of time and temperature of storage on RS, WAI and WSI, and mathematical models were built up 3 Results and discussion
3.1 Effect of storage time and temperature on resistant starch (RS) formation
The RS content of the samples obtained from the
content in the control sample (i.e., 9.07%) with the content in the treatment samples, it was evident an increase in the RS content quite probably associated with debranching and the autoclaving process The highest storage tempera-ture had a negative effect on RS formation, showing the lowest RS values When starchy material was stored at high temperatures (i.e., 60C) and the glass transition temperature (Tg) was lower than the storage temperature, the material was in a rubbery state resulting in a slow ret-rogradation process (Slade & Levine, 1991) On the other hand, samples stored at 4 and 32C, independent of stor-age time, did not show significant differences in the RS con-tent This pointed out that Tgof banana starch might be
to develop products with appropriate RS levels with extended shelf life at room temperatures Statistical analy-sis showed that while storage time did not affect RS content (P = 0.798), temperature (T) significantly affected (P = 0.002) the RS formation The mathematical model that described RS formation under the conditions used in
Table 2
Resistant starch (RS), water absorption index (WAI) and water solubilityindex (WSI) in samples of autoclaved banana starch stored at differenttimes and temperaturesA
Temperature (C)Time (h)RS (%)WAI (%)WSI (%)
Trang 4this work presented a good determination coefficient (r2= 0.985):
RS¼ 41:24 0:2159T 6:73 103T2
Lehmann, Jacobasch, and Schmiedl (2002)determined RS in autoclaved native banana (Musa acuminata) finding val-ues between 5.9% and 6.5% These valval-ues are higher than the ones obtained in the present study.Liu et al (2003) re-ported a RS content of 18% in native corn starch, deb-ranched for 4 h with pullulanase and then autoclaved The RS concentration increased when debranching time in-creased to 48 h With high amylose corn starch an RS con-tent of 36.4% was obtained by an autoclaving process, but lower RS concentration was found when waxy corn starch was used as raw material (Escarpa, Gonza´lez, Man˜as, Gar-cı´a, & Saura, 1996) Therefore, with this kind of process the starch source plays an important role in determining RS production.
3.2 Water absorption index (WAI) and water solubility index (WSI)
Independent of storage temperature WAI values of sam-ples stored for 24 h were not different (P = 0.05) When the storage time increased WAI values decreased, a behavior that agreed with the higher RS content since the crystalliza-tion (i.e., retrogradacrystalliza-tion) that took place during storage reduced the water absorption capability of starch As men-tioned before, temperature did not show a significant effect on WAI values (P = 0.0711) and storage time had a qua-dratic effect on this parameter (P = 0.003) The following equation described the mathematical model that described this behavior as a function of the storage time (h) (r2= 0.90):
WAI¼ 10:39 11:625 102hþ 18:316 103h2
The storage temperature affected WSI (P = 0.029) but stor-age time did not show a significant effect (p = 0.7211) The
(r2= 0.72) where T is the storage temperature WSI¼ 22:31 þ 6:78 102Tþ 12:41 104T2
The WSI values were higher than the WAI, pointing out the higher amount of starch that was not transformed to RS and was solubilized during the WSI determination These results agree with the RS contents since samples showing the lowest RS levels also had the highest WSI values.
3.3 Starch digestibility of the sample with the highest RS content
The results obtained during the first step were used to choose a treatment with the highest RS content Therefore selecting the sample storage of 24 h at 4C.
The content of available starch (AS) in the powder obtained under these conditions was of 63.2 ± 1.38%, a low value considering that the sample was gelatinized (heat treatment) during the analysis The AS content assessed might be similar whether this powder were added to a food that is cook before consumption, the powder may be con-sidered as a low carbohydrate ingredient.
The results of the hydrolysis rate of the RS product obtained by autoclaving are shown inFig 1 The percent-age of hydrolysis observed in the autoclaved sample was lower than the one obtained with native starch After 30 min the autoclaved sample showed the highest percent-age of hydrolysis (i.e., 35%) followed by a plateau When
Trang 5the hydrolysis behavior of the autoclaving sample was compared with the one observed by native starch (maxi-mum hydrolysis of 75%), evidently debranching and auto-claving of the banana starch had a detrimental effect on starch hydrolysis The hydrolysis data agree with values determined for RS and AS.
3.4 X-ray diffraction pattern
Native banana starch provides an X-ray pattern that was a mixture between the A- and B-type polymorphs This behavior is also referred to C-type (seeFig 2) In general, legume starches and some tropical tuber starches display the C-type pattern, associated to a mixture of A- and B-type crystallinity within the starch granule (Spencer & Jane, 1999) Some peaks observed with banana starch were similar to those seen in cereal starches (A type) However, there are also differences that might indicate the presence of B-type crystals, i.e., the peak at 2h = 5.4 and the peak at 2h = 17 were more prominent than the peak at 2h = 18 and the peak at 2h = 23 was broader All these differences indicate that banana starch is a mixture of A- and
peaks of crystallinity in regions different from those observed in the native banana starch (see arrows), and oth-ers present in the native sample (i.e., 2h = 15 and 2h = 23) started to develop peaks due to starch chain
reor-ganization due to the retrogradation process The X-ray diffraction pattern showed in the RS sample agreed with the RS content, since the peaks observed were associated to the retrogradation phenomena and consequently to the RS formation.
3.5 Scanning electron microscopy
The microphotographs of RS rich-powder storage at different temperatures and times are shown inFig 3 The sample that was stored at 4C for 24 h (Figs 3a and b) observed a more compact structure (Fig 3a) than samples stored at higher temperatures (Figs 3c and e) This obser-vation might be associated to a higher level of cavities or channels in the matrix of starches stored at the highest tem-perature in contrast with the topological structure of starches stored at lower temperatures (see arrows in
Fig 3) The storage temperature had influence in the microscopic structure of the RS rich-powder since the stor-age temperatures that are above the glass transition
to the movement of starch chains and as a consequence, a rubbery state appears (Slade & Levine, 1991) as shown inFig 3f The samples that were stored at 4 and 32C pre-sented different structures (Figs 3b and d) due to the crys-tallin character present in those samples as assessed by the
Fig 2 X-ray diffraction pattern of native and autoclaved starch.308R.A Gonza´lez-Soto et al / Food Research International 40 (2007) 304–310
Trang 64 Conclusions
The RS level increased due to the debranching and autoclaving processes and the storage temperature had influence in the RS formation The mathematical model that describes RS formation under the conditions used in this work presented a good determination coefficient The water absorption index (WAI) values of the samples stored for 24 h were not significantly different indepen-dently of the storage temperature At longer storage times the WAI values decreased, these results are in agreement with the higher RS content The water solubility index
(WSI) values were higher than the WAI, since there was a high amount of starch present in the samples that was not resistant and could be solubilized A reduction in the hydrolysis percentage was obtained in the autoclaved sample compared with its native counterpart The RS-rich powder showed crystallinity due to reorganization of the starch chain by retrogradation The microstructure of the RS-rich powder had cavities or channels in the matrix and this was affected by the storage temperature Debran-ching and autoclaving processes can be used for RS-rich powder with high RS level and adequate functional properties.
Fig 3 Scanning electron microscopy of banana starch autoclaved and storage at different temperatures: (a) 4C; (b) 4 C; (c) 32 C; (d) 32 C; (e) 60 C;(f) 60C.
Trang 7The authors wish to acknowledge the economic support from CONACYT-Me´xico, CGPI-IPN, COFAA-IPN and EDI-IPN, LANFOODS (Sweden) and CYTED (XI.18) References
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