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Indigestible pyrodextrins prepared from corn starch in the presence of glacial acetic acid
Carbohydrate Polymers 188 (2018) 68–75
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Indigestible pyrodextrins prepared from corn starch in the presence of glacial acetic acid Chia-Long Lina, Jheng-Hua Linb, Han-Mei Zenga, Yuan-Hsi Wua, Yung-Ho Changa, a b
T
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Department of Food and Nutrition, Providence University, Taichung, 43301, Taiwan Department of Hospitality Management, MingDao University, Changhua, 52345, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords: Pyrodextrin Acetic acid Solubility Molecular weight distribution Indigestibility
This study was conducted to evaluate the feasibility of using acetic acid (0–2.5%) as the catalyst in the production of pyrodextrins from corn starch at 140–180 °C, and to elucidate the effects of catalyst concentration and pyrolytic temperature on the characteristics of indigestible pyrodextrins. In the absence of acetic acid, noticeable changes to the loss of birefringence, increased solubility, plateau of molecular weight reduction, decreased reducing sugar content and digestibility were observed at 180 °C. By using catalyst, the alterations in the aforementioned characteristics of pyrodextrins were intensified. Moreover, by using 0.5–1.0% catalyst, the substantial alterations in birefringence, solubility and molecular weight were observed concurrently for pyrodextrins formed at 170 °C. Further increasing the concentration of acetic acid to 1.0–2.5% decreased the intervening temperature to 160 °C. The findings suggest that indigestible pyrodextrins with desired characteristics could be tailored via using various combinations of catalyst concentration and pyrolytic temperature.
1. Introduction Benefits of increasing the consumption of dietary fiber have been associated with the lowering of blood pressure, the aid to regulating the secretion of insulin, the production of short chain fatty acids and the maintenance of homeostasis in gastrointestinal system (Anderson et al., 2009). Hence, a diet regime with high dietary fiber is beneficial to the reduction in the risk of non-communicable diseases, such as coronary heart disease, type-II diabetes, and intestinal disorders. To fulfil the consumers' demand for healthy processed foods, there is still a growing interest to the food industry in fortifying their products with dietary fiber and/or the physiological analogues (Kapusniak & Nebesny, 2017). Starch is not only an important energy source of man's diet but also an abundant polysaccharide occurring naturally. It consists of fractions varying in their susceptibility to digestive enzymes, namely rapidly digestible starch, slowly digestible starch and resistant starch (Englyst & Hudson, 1996). Among these fractions, resistant starch (RS) is analogous to dietary fiber in respect of its digestibility, which is resistant to digestive enzymes in the small intestine but is available to the microflora to ferment in the colon. The enzymatic resistance of starch arises from the protection of cell walls surrounding the starch granules (typeI), the morphology and crystallinity of the granules (type-II), the recrystallisation of starch after processing-induced gelatinization (type-
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III) and/or the alteration in the chemical structure of starch (type-IV) (Englyst, Liu, & Englyst, 2007). In light of versatility and cost-effectiveness, it is a common approach to meeting the commercial need for a starch with desired characteristics by modifying its structure (Tester & Karkalas, 2002). Production of type-IV RS is achieved commonly by chemical modifications, that is the chains of starch molecules are cross-bonded or the hydroxyl groups are substituted with functional groups (e.g. ether or ester) in accordance with the derivatizing agent used (Englyst et al., 2007). The formation of structure unrecognizable by the digestive enzymes contributes to the enzymatic resistance of modified starch. Along with the chemical approach, a change to the chemical structure of starch molecules can be induced in a physical fashion (Horton, 1965). When starch (with less than 5% moisture) is heated at elevated temperatures (135–218 °C) for prolong periods (10–12 h), the starch chains undergo a series of irreversible changes and the resulting pyrodextrins are of more resistance to α-amylase and β-amylase as compared to the starch used. During the course of pyrolysis, the browning of pyrodextrin, increase in its cold-water solubility and reduction in the viscosity also occur. By the inclusion of mineral acid as the catalyst for pyrolysis, pyrodextrins with low molecular weight, high solubility and low viscosity can also be prepared by heating starch at 100–200 °C for short periods (minutes to several hours) (Campechano-Carrera, Corona-Cruz,
Corresponding author at: Department of Food and Nutrition, Providence University, 200, Sec. 7, Taiwan Boulevard, Taichung, 43301, Taiwan. E-mail addresses: [email protected] (C.-L. Lin), [email protected] (J.-H. Lin), [email protected] (H.-M. Zeng), [email protected] (Y.-H. Wu), [email protected], [email protected] (Y.-H. Chang). https://doi.org/10.1016/j.carbpol.2018.01.087 Received 8 January 2018; Accepted 28 January 2018 Available online 01 February 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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undesired charring, glacial acetic acid was employed as a volatile organic catalyst with moderate dissociation constant, and was distributed in the gaseous form during the process of pyrolyzing corn starch hermetically. The obtained pyrodextrins were compared colorimetrically to those prepared in the presence of HCl as the conventional catalyst. Furthermore, the acetic acid-catalyzed pyrodextrins were analyzed for their physico-chemical properties and digestibility to evaluate the feasibility of adding more adjustability to the properties of pyrodextrins using of organic acid as an alternative to mineral acid.
Chel-Guerrero, & Betancur-Ancona, 2007; Kapusniak & Nebesny, 2017; Wang, Kozlowski, & Delgado, 2001). Structural analyses have revealed the complex nature of the obtained pyrodextrins as 1 → 2, 1 → 3 and 1 → 6 linkages were formed during the process (Bai & Shi, 2016; Ohkuma & Wakabayashi, 2001). The mechanism of starch pyrolysis has been postulated to depend on the temperature and concentration of acids applied (Wurzburg, 2006). In a pyrolytic condition where heat and duration were the main driving force, the cleavage of α-(1–4) glucosidic linkages of starch molecules occurred and the newly formed reducing ends transformed into glucosyl cations (Bai & Shi, 2016; Kroh, Jalyschko, & Häseler, 1996; Tomasik, Wiejak, & Pałasiński, 1989). The glucosyl cations might undergo intramolecular dehydration to form 1,6-anhydro glucans or intermolecular bonds formation with free hydroxyl groups of the adjacent chains. In the presence of acidic catalyst, the hydrolysis of α-(1–4) and probably α-(1–6) bonds was likely to be the dominant event at the initial stage of pyroconversion, followed by thermophilic reactions of transglucosidation and repolymerization (Singhal et al., 2008). Formation of glucosidic linkages other than α-(1–4) and α-(1–6) bonds resulting from the reaction of transglucosidation occurring in the course of manufacturing pyrodextrins has drawn a certain level of attention in respect of their resistance to digestive enzymes and potential benefits to mankind’s health. Ohkuma, Matsuda, Katta, & Hanno (1990) demonstrated that pyrodextrins prepared from potato starch at elevated temperatures (150–200 °C) in the presence of hydrochloric acid (HCl, 0.05–0.1% on dry starch basis) were resistant to the in vitro sequential hydrolysis of α-amylase, protease, and amyloglucosidase. The in vivo study showed that the obtained pyrodextrins were of low impact on glycaemia and insulin level. The indigestibility of pyrodextrins was attributed to the formation of 1 → 2 and 1 → 3 linkages during pyrolysis (Okuma & Matsuda, 2002). Not only from potato starch, indigestible pyrodextrins have also be prepared from the starches of corn (Kapuśniak & Jane, 2007; Okuma & Matsuda, 2002), high amylose corn mutant (Wang et al., 2001), sorghum, tapioca, sagu, cocoyam (Laurentin, Cárdenas, Ruales, Pérez, & Tovar, 2003) and legumes (Campechano-Carrera et al., 2007; OrozcoMartínez & Betancur-Ancona, 2004) in a similar fashion; the starches were pyrolyzed at the temperatures of 90–170 °C for 10–180 min with the use of not more than 0.2% HCl (on starch basis). The indigestibility of pyrodextrins, depending on the pyrolytic condition and the origin of starch, ranged diversely from minimum resistance (0.3%) to 67.8%. The solubility of pyrodextrins in water also varied widely, from minimum solubility (less than 1%) to 97.0%. Not limited to mineral acid, organic acids, such as citric acid and tartaric acid, could be used as the sole catalyst (Shin et al., 2007) or in cooperation with HCl (Jochym, Kapusniak, Barczynska, & Śliżewska, 2012; Kapusniak & Nebesny, 2017) in the production of pyrodextrin. The indigestibility of pyrodextrins was in the range of 19–40% and the solubility in water at 80 °C was more than 90%. It is apparent that the characteristics of pyrodextrins are intimately governed by the properties of starting material, pyrolytic temperature, heating rate, conversion duration and, if any, the quantify of acidic catalyst. When the addition of acid is desired, a diluted solution of mineral acid is introduced to starch slurry, followed by dehydration and pulverization before the commencement of pyrolysis (Wurzburg, 2006). To reduce the amount of water being introduced, diluted volatile mineral acid such as not more than 0.2% HCl (on starch basis) has commonly be used to spray onto the dry starch power (Campechano-Carrera et al., 2007; Kapuśniak & Jane, 2007; Laurentin et al., 2003; Okuma & Matsuda, 2002; Ohkuma et al., 1990; Orozco-Martínez & BetancurAncona, 2004), and its homogeneous distribution is of importance in minimizing the undesired charring resulting from uneven catalysis during the course of pyroconversion (Wurzburg, 1986). Distributing the volatile mineral acid uniformly is conceivably more imperative when a low dosage of the acid is intended to be used in the process. In this study, with a view to minimizing the localized catalysis leading to
2. Materials and methods 2.1. Materials Normal corn starch (Amyral food grade starch) was purchased from Tongaat Hulett Starch Ltd. (Isando, South Africa). All reagents were of analytical grade or liquid chromatography grade where appropriate. 2.2. Methods 2.2.1. Preparation of pyrodextrins Starch (10 g, db) was dried at 100 °C for 1 h to lower its moisture content to not more than 2%, and was transferred immediately to a bespoke polytetrafluoroethylene (PTFE) reaction vessel (250 ml) fitted with PTFE screw cap. For the purpose of distributing the acid in the form of gas, an aliquot of glacial acetic acid or concentrated HCl was pipetted accurately into a glass capsule with several pores on the circumferential wall. The final concentrations of acetic acid in starch were varied from 0.5 to 2.5% with an increment of 0.5%, and those of HCl were 0.05, 0.1 and 0.2%. The porous glass capsule was then fitted securely and perpendicularly to the orifice of the vessel. The hermetically sealed reaction vessel was incubated at the designated temperature for 3 h with continuous rotation at 100 rpm. The pyrolytic temperatures were varied from 140 to 180 °C with 10 °C interval for the use of acetic acid and from 120 to 140 °C for that of HCl. Upon completion, the vessel was cooled to the room temperature in an ice bath followed by the addition of 100 ml deionized water. The suspension was then neutralized to pH 7 with 3% NaOH solution, desalinated by using an adequate amount of mixed bed resin (Sigma M8157, Sigma-Aldrich Co., St. Louis, MO, USA) and filtered through a 100 mesh screen to remove the added ion-exchange resin. The pyrodextrin was then recovered by evaporating the collected filtrate to dryness at 60 °C. The yield was calculated as the mass ratio of obtained pyridextrin to the starch used. 2.2.2. Appearance and color Starch and pyrodextrins (approx. 1 g) were weighed and spread evenly on Petri dishes. The dishes were then photographed with a digital camera (SLT-A57, Sony Corp., Tokyo, Japan) against the aqua background. Color differences between the starch and pyrodextrins were quantified photoelectrically according to Orozco-Martínez and BetancurAncona (2004) with modifications. A colorimeter (Spectro Color Meter SE2000, Nippon Denshoku Industries Co. Ltd., Tokyo, Japan) was used to determine the Hunter color attributes of starch and pyrodextrins. The obtained color attributes of the pyrodextrins were compared to those of the starch and the color differences (ΔE) calculated as follows, ΔE = SQRT (ΔL2 + Δa2 + Δb2) where ΔL, Δa and Δb were the differences in the values of whiteness (L), redness-to-greenness (a) and yellowness-to-blueness (b) between starch and pyrodextrin, respectively. 2.2.3. Morphology Starch and pyrodextrins (approx. 0.1 g) were weighed into 50 ml 69
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equation of relative molecular weight on the retention volume. The regression equation was established using a series of pullulan standards with peak molecular weight ranging from 5.9 to 788.0 kDa (Standard P82, Showa Denko K.K., Kanagawa, Japan). For pyrodextrins, the same algorithm as that of starch was applied, while the F1 and F2 fractions were dissected at a constant retention volume (11.85 ml) obtained from the chromatogram of the starch.
glass beakers followed by the addition of 20 ml deionized water and 40 μl I2/KI solution (15.2 mg I2, 30.3 mg KI). Aliquots of the suspensions were spread on microscopic slides on which cover slips were then placed slowly to avoid the formation of bubbles. The morphology of the starch and pyrodextrins was observed with a light microscope (BX41, Olympus Corp., Tokyo, Japan). For birefringence observation, a polarizer was fitted to the light source of microscope and the addition of I2/KI solution was omitted from the specimen preparation.
2.2.6. Reducing sugar content Reducing sugar content of starch and pyrodextrins were determined colorimetrically as the capability of forming ferric ferrocyanide (Hizukuri, Shirasaka, & Juliano, 1983) with modifications. Samples (75 mg, db) were weighed accurately into 20 ml screw-capped glass vials. While stirring, 1.5 ml of water and 13.5 ml of DMSO aliquots were added sequentially by pipette. After sealing, the vials were placed in a boiling water bath for 1 h then at the room temperature overnight with continuous stirring. Aliquots (1 ml) of the glucan-DMSO solutions were pipetted into screw-capped tubes in triplicate followed by the addition of 0.5 ml each of potassium ferricyanide solution (1 mg ml‐1) and carbonate buffer (155 mM, [CO32−]/[HCO3−] = 0.414) containing 325 μg of potassium cyanide. After sealing and vortex mixing, the tubes were placed in a boiling water bath exactly for 15 min then cooled in running water for 10 min. Aliquots (2.5 ml) of ferric ammonium sulfate solution (3 mg ml−1 of 50 mM sulfuric acid) were pipetted into the tubes, the contents were mixed thoroughly after sealing, and the introduced air bubbles released periodically. After standing in the dark at the room temperature for 20 min, the absorbance of the solutions were measured at 715 nm against the reagent blank. For the establishment of a calibration curve, a set of glucose solutions with concentrations ranging from 1 to 5 μg ml‐1 was employed.
2.2.4. Solubility Solubility of starch and pyrodextrins at various temperatures was determined gravimetrically (Lin, Lee, & Chang, 2003) with modifications. Samples (0.1 g, db) were weighed in triplicate into screw-capped centrifuge tubes equipped with overhead stirring paddles. After the addition of 40 ml water, the tubes were immersed in a water bath set at the desired temperature (25, 50 and 90 °C) for 30 min with continuous stirring at 200 rpm. Upon completion and cooling to the room temperature in an ice bath, the tubes were centrifuged at 6000 rpm for 20 min and the supernatants decanted gently into 50 ml beakers without disturbing the sediment. The beakers were then placed in a fanassisted oven at 60 °C overnight followed by 130 °C for 1 h. The solubility was calculated as the mass ratio of solute to the initial sample. 2.2.5. Molecular weight Molecular weight distributions of starch and pyrodextrins were determined chromatographically (Chang, Lin, & Chang, 2006) with modifications. For starch and pyrodextrins with solubility less than 70% at 25 °C, samples (75 mg, db) were weighed accurately into 20 ml screw-capped glass vials. While stirring, 1.5 ml of water and 13.5 ml of dimethyl sulfoxide (DMSO) aliquots were added sequentially by pipette. After sealing, the vials were placed in a boiling water bath for 1 h then at the room temperature overnight with continuous stirring. Aliquots (2.1 ml) of the glucan-DMSO solutions were pipetted into centrifuge tubes and mixed thoroughly with 4 vols of ethanol. After centrifuging at 4000 × g for 10 min and discarding the supernatants carefully, the precipitated starch and pyrodextrins were re‐dissolved in 15 ml of water while heating in a boiling water bath for 1 h with continuous stirring. For pyrodextrins with solubility higher than 70% at 25 °C, without the aid of DMSO and centrifugation, the dextrins (75 mg, db) were dissolved in water (15 ml) while heating in a boiling water bath for 1 h. The solutions were then filtered via clean syringe filters (5 μm pore size) before injecting into a high performance size exclusion chromatograph (HPSEC) as below. The HPSEC was equipped with an inline degasser (ERC-3415α, ERC Inc., Saitama, Japan), isocratic pump (KP-12, Flom Corp., Tokyo, Japan), a manual injector (7725i, Rheodyne Inc., Berkeley, CA, USA) fitted with a 100 μl sample loop, three series-connected gel filtration columns (TSKgel PWH, TSKgel G5000PW and TSKgel G4000PW, Tosoh Corp., Tokyo, Japan), a triangle laser light-scattering detector (miniDAWN, Wyatt Tech., Santa Barbara, CA, USA) and a differential refractive index detector (DRI; Waters 410, Millipore Corp., Milford, MA, USA). The stationary phase was maintained at 70 °C and the mobile phase (0.1 M NaNO3 containing 0.02% NaN3) at a flow rate of 0.5 ml min−1. The weight-average molecular weight (Mw) of starch was calculated according to Lin et al. (2003) and the formula was as follows,
2.2.7. Digestible starch content Digestible α-glucans of starch and pyrodextrins were hydrolyzed by using of thermostable α-amylase and amyloglucosidase and the yielded glucose was colorimetrically quantified in the presence of glucose oxidase, peroxidase, 4‐aminoantipyrine and 4‐hydroxybenzoic acid (AACC, 2000). To aid the complete dissolution of starch and the pyrodextrins, DMSO was employed as described for the test objects potentially containing resistant starch. 3. Results and discussion 3.1. Yields, appearance and color characteristics Pyrolysis of corn starch using 0–2.5% acetic acid at 140–180 °C for 3 h formed pyrodextrins with yields of 93.5–98.4%, while the yields of pyrodextrins with 0.05–0.2% HCl at 120–140 °C were 73.9–98.9%. Horton (1965) reported that gaseous compounds, consisting of CO2, CO, water, volatile acids and volatile solids, were liberated from starch to which the temperatures of 180–210 °C were applied during the formation of pyrodextrins in the absence of acids. In this study, the temperatures used for pyroconversion were not higher than 180 °C, while the acidic catalysts were used. The concurrence of heat and acid might also give rise to a partial decomposition of starch into gaseous compounds and consequently to the observed decrease in the yield of pyrodextrins. This simultaneous effect of heat and acid was found to be particularly evident for pyrodextrins prepared under the extreme conditions of 0.2% HCl and 130–140 °C, and resulted in the yields of less than 90%. The pyrolysis at various temperatures in the presence of either HCl or acetic acid led to the changes in the appearance of pyrodextrins, and the extent of alteration in color was dependent on the type and concentration of the acid as well as the reaction temperature (Fig. 1). For pyrodextrins prepared with either acetic acid or HCl, the higher the pyrolytic temperature was, the more brownish the pyrodextrin
Mw = (WF1 (%) × MwF1 + WF2 (%) × MwF2)/100 where WF1 and WF2 were the weight percentages of fractions of amylopectin (F1) and low molecular weight molecules (F2), respectively, which were assigned by referencing the chromatogram of gyration radius against elution volume of starch (Chang, Lin, & Chen, 2006). The Mw of F1 fraction (MwF1) was calculated from the signals of lightscattering detector and DRI detector, where that of F2 fraction (MwF2) was computed from the first-order semi-logarithmic regression 70
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Fig. 1. Appearance of corn starch and pyrodextrins catalyzed by either hydrochloric acid or acetic acid at different concentrations and temperatures. The italic numeral placed beneath the photograph denotes the color difference (ΔE) between starch and the pyrodextrin.
complexes bore a resemblance to those of ΔE. The similarity in both changes may indicate the differences in color of iodine complex between starch and pyrodextrins arose from their different degrees of pyroconversion. Under polarized light, starch granules exhibited a typical birefringent pattern of Maltese cross in their center, while the birefringence was altered during pyrolysis. In analogous to the shifting in the color of pyrodextrin-iodine complexes, no considerable change in birefringence was observed for pyrodextrins formed at 140–150 °C despite the concentration of catalyst. When the temperature was raised to the range of 160–180 °C, the synergic effect of pyrolytic temperature and concentration of catalyst on birefringence began evident. Additionally, the temperature in connection with a major loss of birefringence was found to be in line with that of color shifting; 180 °C for the pyrolysis without catalyst, 170 °C for 0.5–1.0% acetic acid and 160 °C for 1.5–2.5% acetic acid. Formation of starch-iodine complex in blue-purple color reflected the occurrence of amylose and amylopectin molecules concurrently in the amorphous regions of the granules (Lin, Lii, & Chang, 2005; Tester, Karkalas, & Qi, 2004). With the positive correlation between the chain length of α-glucans and the λmax of their iodine complexes (Peymanpour et al., 2016) being borne in mind, the observed alteration in the color of pyrodextin-iodine complexes from blue-purple to redpurple then faint-pink indicates the cleavage of glucosidic linkages of starch chains occurred during the process of pyroconversion and thus led to the decrease in iodine binding capacity of pyrodextrin. This is in line with the structural change of starch during pyrolysis as postulated by Bai, Cai, Doutch, Gilbert, and Shi (2014). Besides, Wang et al. (2001) observed that the pyrodextrins prepared from native starches had higher enzymatic resistance than those of derivatied starches, and thus suggested that the amorphous region of starch being free from the steric hindrance of functional groups played an important role in the formation of indigestible pyrodextrins. In conjunction with the birefringent characteristics of pyrodextrins observed in this study, the pyrolytic reactions appeared to commence from the amorphous regions of starch granules then progressed into the crystalline regions when the temperature was increased. The disintegration of crystalline regions was further accelerated as the concentration of catalyst was increased.
appeared. Moreover, the appearance of the pyrodextrin was darkened with an increase in the concentration of catalyst. In terms of the type of catalyst, the alteration in the color of pyrodextrins prepared with HCl was more drastic than that prepared with acetic acid, and the ΔE were in the range of 1.2–70.5 for HCl-catalyzed pyrodextrins and 2.1–34.2 for those with acetic acid. Studies postulated that the change in the color of pyrodextrins was an indication of pyrolytic condition to which starch was exposed, and the increment of color difference implied the increased conversion degree of pyrodextrin (Ronald Terpstra, Woortman, & Hopman, 2010). As observed, HCl (0.05–0.2%) appears to be an effective catalyst in the formation of pyrodextrins at the temperatures between 120 °C and 140 °C. However, the spontaneous increase in ΔE and the decrease in yield probably due to the formation of char and volatile compounds can easily occur with a slight increase in the concentration of HCl within a tight temperature range. In contrast, the maximum increase in ΔE to 34.2 was observed only when the concentration of acetic acid was increased to 2.5% and the pyrolytic temperature from 140 °C to 180 °C. The results suggest that the glacial acetic acid could be an alternative acidic catalyst in the production of pyrodextrins when more adjustability to the properties of the obtained is desired, with the trade-off of a little higher conversion temperature. 3.2. Morphology Morphological structure of starch and acetic acid-catalyzed pyrodextrins was observed microscopically after staining with I2/KI solution. Their birefringent characteristics were also observed under polarized light without the use of staining agent during the specimen preparation. As shown in Fig. 2, starch used in the current study formed blue-purple complex with iodine, and the color of pyrodextrin-iodine complex changed to red-purple then faint pink with increasing pyrolytic temperature and catalyst concentration. In general, there was no appreciable alteration in color of iodine complex observed for pyrodextrins prepared at the temperatures of 140–150 °C. As the pyrolytic temperature increased to the range of 160–180 °C, the temperature effect began to be evident. Moreover, the temperature in connection with a substantial alteration in color was found to correlate inversely with the quantity of acetic acid used for pyrolysis. The altering temperature was 180 °C for the pyrolysis without catalyst, 170 °C for 0.5–1.0% acetic acid and 160 °C for 1.5–2.5% acetic acid. When compared to the appearance of starch and acetic acid-catalyzed pyrodextrins (Fig. 1), the pyrolysis-induced changes in color of iodine
3.3. Solubility Extents of solubilization of corn starch and acetic acid-catalyzed pyrodextrins in an excess amount of water at 25, 50 and 90 °C are 71
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Fig. 2. Light (upper) and polarized light (lower) micrographs of (a) corn starch before and after pyrolysis at 140 °C with 2.5% acetic acid, (b) acetic acid-catalyzed pyrodextrins prepared under the conditions specified (bar = 20 μm).
Fig. 3. Solubility at 25, 50 and 90 °C of corn starch (●) and acetic acid-catalyzed pyrodextrins prepared at 140–180 °C with 0% (▲), 0.5% (△), 1.0% (▼), 1.5% (▽), 2.0% (■) and 2.5% (□) acetic acid, respectively.
shown in Fig. 3. Taken all three temperatures as a whole, pyrodextrins exhibited a higher solubility than the starch. Solubility of pyrodextrins increased with increasing temperature of water used. The changes in solubility were from 1.0% to the range of 0.4–95.6%, 2.9% to 0.4–97.7% and 10.0% to 22.6–98.9% at the determination temperatures of 25, 50 and 90 °C, respectively. Furthermore, the solubility of pyrodextrins was found to increase synergistically with increasing pyrolytic temperature and ratio between catalyst and starch. This synergistic effect was particularly evident from the results determined at 25 and 50 °C. In the absence of acetic acid, an appreciable increment by approx. 20% in solubility was observed for the pyrodextrin prepared at 180 °C. By using of catalyst, the temperature, in connection with the rapid increase in solubility, reduced to 170 °C for 0.5–1.0% acetic acid and further to 160 °C when the concentration of catalyst increased to 1.5–2.5%. The increase in solubility of starch after pyroconversion was found to be in agreement with the alteration in color of starch complexing with iodine after pyrolysis showing in Fig. 2. This suggests that the
observed increase in solubility resulted from the cleavage of α-glucosidic linkages of starch during the process, and is well in line with other studies (Kapusniak & Nebesny, 2017; Kwon, Chung, Shin, & Moon, 2005). Moreover, studies have suggested that the extent of increase in solubility was correlated positively with the level of pyroconversion (Kapuśniak & Jane, 2007; Wang et al., 2001). As observed in this study, the reaction of degradation reflecting on the increase in solubility of starch after pyrolysis was of prominence, and the increasing extent was dependent on both of pyrolytic temperature and concentration of catalyst. This dependence was supported by the iodine binding capacity and birefringent properties of pyrodextrins discussed previously. 3.4. Molecular characteristics Molecular profiles of corn starch and pyrodextrins prepared by using 0.5–2.5% acetic acid at 140–180 °C were analyzed 72
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Fig. 4. High-performance size-exclusion chromatograms of molecular weight standards (●), corn starch (—) and acetic acid-catalyzed pyrodextrins prepared at 140 °C (–– ––), 150 °C (– –), 160 °C (– • –), 170 °C (– • • –), and 180 °C (• • • •) with 0–2.5% acetic acid, respectively.
Table 1 Weight-average molecular weight and reducing sugar content of corn starch and acetic acid-catalyzed pyrodextrins. Acetic acid (%, starch dry basis)
Mw (kDa) Starcha 0 0.5 1.0 1.5 2.0 2.5 Reducing sugar content (%, db) Starch 0 0.5 1.0 1.5 2.0 2.5 a
Temperature (°C) 140
150
160
170
180
119551 ± 4416 35783 ± 1110 2091 ± 75 921 ± 449 673 ± 102 496 ± 77 346 ± 120
– 9311 ± 480 605 ± 15 371 ± 85 426 ± 69 215 ± 106 264 ± 109
– 1978 ± 37 193 ± 44 101 ± 2 65 ± 5 60 ± 2 56 ± 3
– 351 ± 106 79 ± 26 42 ± 1 36 ± 4 25 ± 1 24 ± 2
– 197 ± 26 32 ± 1 21 ± 2 18 ± 1 20 ± 1 18 ± 2
0.13 0.17 0.33 0.26 0.35 0.42 0.43
– 0.23 0.42 0.27 0.39 0.45 0.55
– 0.33 0.49 0.52 0.51 0.50 0.58
– 0.43 0.56 0.65 0.50 0.52 0.65
– 0.24 0.48 0.27 0.22 0.31 0.18
± ± ± ± ± ± ±
0.01 0.01 0.08 0.04 0.01 0.04 0.04
± ± ± ± ± ±
0.01 0.16 0.08 0.18 0.23 0.04
± ± ± ± ± ±
0.01 0.13 0.14 0.21 0.09 0.12
± ± ± ± ± ±
0.02 0.06 0.07 0.08 0.30 0.18
± ± ± ± ± ±
0.06 0.04 0.09 0.12 0.04 0.02
Corn starch was used to determine its weight-average molecular weight and reducing sugar content, respectively.
shifting to a higher elution volume. The Mw of the starch was 119551 kDa and that of pyrodextrins in the range of 18–35783 kDa. In the absence of catalyst, the Mw of pyrodextrin reduced rapidly from 35783 kDa to 351 kDa when the pyrolytic temperature was increased from 140 °C to 170 °C, then levelled off at 197 kDa when the
chromatographically (Fig. 4 and Table 1). As evidenced by the chromatograms, starch exhibited a greater percentage of high molecular weight fraction (F1 fraction) than pyrodextrins. With increasing the pyrolytic temperature and the concentration of catalyst, the molecular weight distribution of pyrodextrins was narrowed with their peaks
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In addition, the digestible starch content of pyrodextrins was found to decrease with increases in the temperature and the concentration of catalyst used. It was also found that the temperature, in connection with a prominent decline in digestible starch content, tended to correlate with the concentration of catalyst. With the use of not more than 0.5% acetic acid, the prominent decrease in the enzymatic susceptibility of pyrodextrins was noticed at 180 °C. The intervening temperature was reduced to the range of 160–170 °C when a higher concentration of acetic acid was used. It is interesting to observe that the temperatures in connection with the considerable decrease in enzymatic susceptibility occurred at or before the temperatures at which a drastic decline of reducing sugar content was observed. These results suggest that an alteration in structural conformation leading to the resistance to α-amylase and amyloglucosidase probably due to its high numbers of 1 → 2, 1 → 3 and 1 → 6 linkages (Bai & Shi, 2016; Nunes et al., 2016; Okuma & Matsuda, 2002) was induced in the course of pyrolysis at 180 °C, irrespective of the concentration of acetic acid. The formation of this enzyme-resistant conformation would probably also occur between the temperature range of 140 °C and 170 °C even when the reducing sugar content appeared to increase with increasing the pyrolytic temperature. By collectively considering the results from the morphology (Figs. 1 and 2), solubility (Fig. 3) and molecular characteristics of pyrodextrins (Table 1), pyrolysis of starch tends to commence from the amorphous regions of starch granules. The cleavage of α-glucosidic linkages of starch chains and the transglucosidation of liberated fragments take place concurrently, while the former may be of prominence at low temperature. When the intervening temperature is reached, the degradation progresses towards the crystalline regions of the granules. At this point, the role of transglucosidation may be in parallel with that of depolymerization. When the pyrolytic temperature is increased further, the reaction of transglucosidation becomes dominant and results in the decrease in reducing sugar content and the further decline in digestible starch content. With the use of glacial acetic acid, the intervening temperature can be reduced.
temperature was further elevated to 180 °C. With 0.5–1.0% acetic acid, the appreciable decrease in Mw was observed to plateau out at the temperature of 170 °C; 2091 kDa to 101 kDa at 140–160 °C, and 79 kDa to 21 kDa at 170–180 °C. When the concentration of catalyst was in the range of 1.5–2.5%, the intervening temperature at which a steady decrease in the Mw commenced was further lowered to 160 °C; 673 kDa to 264 kDa at 140–150 °C, and 65 kDa to 18 kDa at 160–180 °C. The observed decrease in Mw suggests the reaction of degradation did occur during the course of pyrolysis, which is well in line with the mechanism of pyroconversion (Bai et al., 2014; Singhal et al., 2008) and supported by the increase in the solubility of pyrodextrin showing in Fig. 3. Additionally, the observations of substantial decrease in Mw at intervening temperature and the lowering of the temperate with increasing the concentration of catalyst might benefit from the use of acetic acid as the catalyst (H+ donor) with moderate dissociation constant. Besides, it is interesting to observe that the intervening temperature for the Mw of pyrodextrins is consistent with those of the birefringence (Fig. 2) and the solubility (Fig. 3). Reducing sugar content of corn starch and pyrodextrins were analyzed to aid in evaluating the effect of pyrolytic condition on the molecular properties of pyrodextrins. As shown in Table 1, the reducing sugar content of pyrodextrins was higher than that of the starch, suggesting the cleavage of α-glucosidic linkages of starch chains occurred during the course of pyrolysis (Kapuśniak & Jane, 2007). Further taken all the conditions as a whole, pyrodextrin showed an increase in its reducing sugar content as the pyrolytic temperature was raised, while a considerable decline of reducing sugar content was observed thereafter. At the pyrolytic temperature of 140 °C, the reducing sugar content of the obtained pyrodextrins was in the range of 0.17–0.43%. When the temperature was raised to 170 °C, the reducing sugar content increased to the range of 0.43–0.65%. As the temperature was raised further to 180 °C, decreasing in reducing sugar content at the range of 0.18–0.48% was observed. By collectively considering the observed decrease in Mw (Fig. 4), it is apparent that the depolymerization of starch occurred during pyrolysis in spite of the use of catalyst and the reaction temperature. Further supported by the continuous increases in the solubility of pyrodextrins with increasing the pyrolytic temperature (Fig. 3), it is conceivable that the depolymerization was an on-going event throughout the course of pyrolysis whether or not the intervening temperature had reached. When a high pyrolytic temperature (e.g. 180 °C) was applied, the transglucosidation and/or re-polymerization of the segmented dextrins probably became dominant and thus resulted in the observed decrease in reducing sugar content.
4. Conclusions Indigestible pyrodextrins were produced by pyrolyzing corn starch at 140–180 °C with glacial acetic acid (0–2.5%, w/w) as the catalyst. The acetic acid-catalyzed pyrolysis was of high yield, and the color of obtained pyrodextrins altered gradually from off-white to tan depending on the pyrolytic condition. Results of analyses on physicochemical properties and digestible starch content suggest that the properties of pyrodextrin can be tailored for its application by leveraging the volatility and low acid dissociation constant of glacial acetic acid as a H+ donor together with a wide range of feasible pyrolytic temperature. For instance, pyrodextrins of low digestibility and high solubility, prepared at 180 °C with 2.0–2.5% acetic acid, could be used to enhance the dietary fiber content of bottled beverages, and pyrodextrins of high molecular weight and moderate digestibility, obtained at 150–160 °C with 0.5–1.5% acid, to be included in the formulation of instant beverage as the sources of both calories and soluble dietary fiber. The intertwined effect of catalyst concentration and pyrolytic temperature on the characteristics of pyrodextrin has been revealed and discussed. In the same concentration of acetic acid, alterations to the loss of birefringence, considerably increased solubility and the plateau of molecular weight reduction have been observed at the same temperature. With increasing the catalyst concentration, the intervening temperature was reduced. Interestingly, the intervening temperatures for the changes to the decreased content of reducing sugar and the substantial decrease in digestibility were generally higher than that of birefringence, solubility and molecular weight. Thus, further research on the structural characteristics of pyrodextrins is of necessity for gaining a further in-depth insight into the complex mechanism of
3.5. Digestible starch content Corn starch and pyrodextrins were subjected to hydrolysis by thermostable α-amylase followed by amyloglucosidase to evaluate the pyrolysis-induced changes in the in vitro digestibility of starch. As shown in Table 2, pyrodextrins were less enzymatic susceptible than starch, changing from 99.0% to the range of 47.8–92.8% after pyrolysis. Table 2 Digestible starch content (%, db) of corn starch and acetic acid-catalyzed pyrodextrins. Acetic acid (%, starch dry basis)
Temperature (°C)
Starcha 0 0.5 1.0 1.5 2.0 2.5
99.0 92.8 87.6 83.8 77.6 76.6 73.4
a
140
150 ± ± ± ± ± ± ±
0.6 0.3 0.4 3.5 1.1 0.3 0.4
– 90.4 84.4 78.7 72.5 72.8 72.7
160
± ± ± ± ± ±
1.7 1.2 1.0 1.5 3.6 1.2
– 86.2 78.6 64.8 70.6 65.8 69.7
170
± ± ± ± ± ±
1.3 1.6 4.0 2.7 2.2 0.8
– 83.1 76.5 63.4 57.5 54.3 52.6
180
± ± ± ± ± ±
1.4 0.8 1.4 0.9 1.0 1.5
– 75.5 67.4 58.2 55.9 48.0 47.8
± ± ± ± ± ±
0.4 1.3 1.6 7.0 2.0 0.9
Corn starch was used to determine its digestible starch content.
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induced degradation of α-glucans. Part I: Analysis of oligomeric maltodextrins and anhydrosugars. Starch/Stärke, 48, 426–433. Kwon, S.-k., Chung, K. M., Shin, S. I., & Moon, T. W. (2005). Contents of indigestible fraction, water solubility, and color of pyrodextrins made from waxy sorghum starch. Cereal Chemistry, 82, 101–104. Laurentin, A., Cárdenas, M., Ruales, J., Pérez, E., & Tovar, J. (2003). Preparation of indigestible pyrodextrins from different starch sources. Journal of Agricultural and Food Chemistry, 51, 5510–5515. Lin, J.-H., Lee, S.-Y., & Chang, Y.-H. (2003). Effect of acid-alcohol treatment on the molecular structure and physicochemical properties of maize and potato starches. Carbohydrate Polymers, 53, 475–482. Lin, J.-H., Lii, C.-y., & Chang, Y.-H. (2005). Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid. Carbohydrate Polymers, 59, 507–515. Nunes, F. M., Lopes, E. S., Moreira, A. S., Simões, J., Coimbra, M. A., & Domingues, R. M. (2016). Formation of type 4 resistant starch and maltodextrins from amylose and amylopectin upon dry heating: A model study. Carbohydrate Polymers, 141, 253–262. Ohkuma, K., & Wakabayashi, S. (2001). Fibersol-2: A soluble, non-digestible, starch-derived dietary fibre. In B. V. McCleary, & L. Prosky (Eds.). Advanced dietary fibre technology (pp. 509–523). Oxford, UK: Blackwell Science. Ohkuma, K., Matsuda, I., Katta, Y., & Hanno, Y. (1990). Pyrolysis of starch and its digestibility by enzymes. Journal of the Japanese Society of Starch Science, 37, 107–114. Okuma, K., & Matsuda, I. (2002). Indigestible fractions of starch hydrolysates and their determination method. Journal of Applied Glycoscience, 49, 479–485. Orozco-Martínez, T., & Betancur-Ancona, D. (2004). Indigestible starch of P. lunatus obtained by proconversion: Changes in physicochemical properties. Starch/Stärke, 56, 241–247. Peymanpour, G., Marcone, M., Ragaee, S., Tetlow, I., Lane, C. C., Seetharaman, K., et al. (2016). On the molecular structure of the amylopectin fraction isolated from “highamylose” ae maize starches. International Journal of Biological Macromolecules, 91, 768–777. Ronald Terpstra, K., Woortman, A. J. J., & Hopman, J. C. P. (2010). Yellow dextrins: Evaluating changes in structure and colour during processing. Starch/Stärke, 62, 449–457. Shin, S. I., Lee, C. J., Kim, D.-I., Lee, H. A., Cheong, J.-J., Chung, K. M., et al. (2007). Formation, characterization, and glucose response in mice to rice starch with low digestibility produced by citric acid treatment. Journal of Cereal Science, 45, 24–33. Singhal, R. S., Kennedy, J. F., Gopalakrishnan, S. M., Kaczmarek, A., Knill, C. J., & Akmar, P. F. (2008). Industrial production, processing, and utilization of sago palm-derived products. Carbohydrate Polymers, 72, 1–20. Tester, R. F., & Karkalas, J. (2002). Starch. In E. J. Vandamme, S. De Baets, & A. Steinbüchel (Eds.). Polysaccharides II: Polysaccharides from eukaryotes (pp. 381–438). Hoboken, NJ, USA: Wiley-Blackwell. Tester, R. F., Karkalas, J., & Qi, X. (2004). Starch – composition, fine structure and architecture. Journal of Cereal Science, 39, 151–165. Tomasik, P., Wiejak, S., & Pałasiński, M. (1989). The thermal decomposition of carbohydrates: Part II. The decomposition of starch. Advances in Carbohydrate Chemistry and Biochemistry, 47, 279–343. Wang, Y.-J., Kozlowski, R., & Delgado, G. A. (2001). Enzyme resistant dextrins from high amylose corn mutant starches. Starch/Stärke, 53, 21–26. Wurzburg, O. B. (1986). Converted starches. In O. B. Wurzburg (Ed.). Modified starches: Properties and uses (pp. 17–40). Boca Raton, FL, USA: CRC Press. Wurzburg, O. B. (2006). Modified starches. In A. M. Stephen, G. O. Phillips, & P. A. Williams (Eds.). Food polysaccharides and their applications (pp. 87–118). Boca Raton, FL, USA: CRC Press.
pyrolysis. Declaration of interest None. Acknowledgment This work was supported by Ministry of Science and Technology, Taiwan (grant number MOST-104-2320-B-126-003-MY3). References AACC (2000). Method 76-13: Total starch assay procedure (Megazyme amyloglucosidase/αAmylase method)Approved methods of the American association of cereal chemists (10th ed.). St. Paul, MN, USA: the Association. Anderson, J. W., Baird, P., Davis, R. H., Jr., Ferreri, S., Knudtson, M., Koraym, A., et al. (2009). Health benefits of dietary fiber. Nutrition Reviews, 67, 188–205. Bai, Y., & Shi, Y.-C. (2016). Chemical structures in pyrodextrin determined by nuclear magnetic resonance spectroscopy. Carbohydrate Polymers, 151, 426–433. Bai, Y., Cai, L., Doutch, J., Gilbert, E. P., & Shi, Y.-C. (2014). Structural changes from native waxy maize starch granules to cold-water-soluble pyrodextrin during thermal treatment. Journal of Agricultural and Food Chemistry, 62, 4186–4194. Campechano-Carrera, E., Corona-Cruz, A., Chel-Guerrero, L., & Betancur-Ancona, D. (2007). Effect of pyrodextrinization on available starch content of lima bean (Phaseolus lunatus) and cowpea (Vigna unguiculata) starches. Food Hydrocolloids, 21, 472–479. Chang, Y.-H., Lin, J.-H., & Chang, S.-Y. (2006). Physicochemical properties of waxy and normal corn starches treated in different anhydrous alcohols with hydrochloric acid. Food Hydrocolloids, 20, 332–339. Chang, Y.-H., Lin, C.-L., & Chen, J.-C. (2006). Characteristics of mung bean starch isolated by using lactic acid fermentation solution as the steeping liquor. Food Chemistry, 99, 794–802. Englyst, H. N., & Hudson, G. J. (1996). The classification and measurement of dietary carbohydrates. Food Chemistry, 57, 15–21. Englyst, K. N., Liu, S., & Englyst, H. N. (2007). Nutritional characterization and measurement of dietary carbohydrates. European Journal of Clinical Nutrition, 61, S19–S39. Hizukuri, S., Shirasaka, K., & Juliano, B. O. (1983). Phosphorus and amylose branching in rice starch granules. Starch/Stärke, 35, 348–350. Horton, D. (1965). Pyrolysis of starch. In R. L. Whistler, E. F. Paschall, J. N. BeMiller, & H. J. Roberts (Eds.). Starch: Chemistry and technology (pp. 421–437). New York, NY, USA: Academic Press. Jochym, K., Kapusniak, J., Barczynska, R., & Śliżewska, K. (2012). New starch preparations resistant to enzymatic digestion. Journal of the Science of Food and Agriculture, 92, 886–891. Kapuśniak, J., & Jane, J.-l. (2007). Preparation and characteristics of enzyme-resistant pyrodextrins from corn starch. Polish Journal of Food and Nutrition Sciences, 57, 261–265. Kapusniak, K., & Nebesny, E. (2017). Enzyme-resistant dextrins from potato starch for potential application in the beverage industry. Carbohydrate Polymers, 172, 152–158. Kroh, L. W., Jalyschko, W., & Häseler, J. (1996). Non-volatile reaction products by heat-
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Indigestible pyrodextrins prepared from corn starch in the presence of glacial acetic acid Chia-Long Lina, Jheng-Hua Linb, Han-Mei Zenga, Yuan-Hsi Wua, Yung-Ho Changa, a b
T
⁎
Department of Food and Nutrition, Providence University, Taichung, 43301, Taiwan Department of Hospitality Management, MingDao University, Changhua, 52345, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords: Pyrodextrin Acetic acid Solubility Molecular weight distribution Indigestibility
This study was conducted to evaluate the feasibility of using acetic acid (0–2.5%) as the catalyst in the production of pyrodextrins from corn starch at 140–180 °C, and to elucidate the effects of catalyst concentration and pyrolytic temperature on the characteristics of indigestible pyrodextrins. In the absence of acetic acid, noticeable changes to the loss of birefringence, increased solubility, plateau of molecular weight reduction, decreased reducing sugar content and digestibility were observed at 180 °C. By using catalyst, the alterations in the aforementioned characteristics of pyrodextrins were intensified. Moreover, by using 0.5–1.0% catalyst, the substantial alterations in birefringence, solubility and molecular weight were observed concurrently for pyrodextrins formed at 170 °C. Further increasing the concentration of acetic acid to 1.0–2.5% decreased the intervening temperature to 160 °C. The findings suggest that indigestible pyrodextrins with desired characteristics could be tailored via using various combinations of catalyst concentration and pyrolytic temperature.
1. Introduction Benefits of increasing the consumption of dietary fiber have been associated with the lowering of blood pressure, the aid to regulating the secretion of insulin, the production of short chain fatty acids and the maintenance of homeostasis in gastrointestinal system (Anderson et al., 2009). Hence, a diet regime with high dietary fiber is beneficial to the reduction in the risk of non-communicable diseases, such as coronary heart disease, type-II diabetes, and intestinal disorders. To fulfil the consumers' demand for healthy processed foods, there is still a growing interest to the food industry in fortifying their products with dietary fiber and/or the physiological analogues (Kapusniak & Nebesny, 2017). Starch is not only an important energy source of man's diet but also an abundant polysaccharide occurring naturally. It consists of fractions varying in their susceptibility to digestive enzymes, namely rapidly digestible starch, slowly digestible starch and resistant starch (Englyst & Hudson, 1996). Among these fractions, resistant starch (RS) is analogous to dietary fiber in respect of its digestibility, which is resistant to digestive enzymes in the small intestine but is available to the microflora to ferment in the colon. The enzymatic resistance of starch arises from the protection of cell walls surrounding the starch granules (typeI), the morphology and crystallinity of the granules (type-II), the recrystallisation of starch after processing-induced gelatinization (type-
⁎
III) and/or the alteration in the chemical structure of starch (type-IV) (Englyst, Liu, & Englyst, 2007). In light of versatility and cost-effectiveness, it is a common approach to meeting the commercial need for a starch with desired characteristics by modifying its structure (Tester & Karkalas, 2002). Production of type-IV RS is achieved commonly by chemical modifications, that is the chains of starch molecules are cross-bonded or the hydroxyl groups are substituted with functional groups (e.g. ether or ester) in accordance with the derivatizing agent used (Englyst et al., 2007). The formation of structure unrecognizable by the digestive enzymes contributes to the enzymatic resistance of modified starch. Along with the chemical approach, a change to the chemical structure of starch molecules can be induced in a physical fashion (Horton, 1965). When starch (with less than 5% moisture) is heated at elevated temperatures (135–218 °C) for prolong periods (10–12 h), the starch chains undergo a series of irreversible changes and the resulting pyrodextrins are of more resistance to α-amylase and β-amylase as compared to the starch used. During the course of pyrolysis, the browning of pyrodextrin, increase in its cold-water solubility and reduction in the viscosity also occur. By the inclusion of mineral acid as the catalyst for pyrolysis, pyrodextrins with low molecular weight, high solubility and low viscosity can also be prepared by heating starch at 100–200 °C for short periods (minutes to several hours) (Campechano-Carrera, Corona-Cruz,
Corresponding author at: Department of Food and Nutrition, Providence University, 200, Sec. 7, Taiwan Boulevard, Taichung, 43301, Taiwan. E-mail addresses: [email protected] (C.-L. Lin), [email protected] (J.-H. Lin), [email protected] (H.-M. Zeng), [email protected] (Y.-H. Wu), [email protected], [email protected] (Y.-H. Chang). https://doi.org/10.1016/j.carbpol.2018.01.087 Received 8 January 2018; Accepted 28 January 2018 Available online 01 February 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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undesired charring, glacial acetic acid was employed as a volatile organic catalyst with moderate dissociation constant, and was distributed in the gaseous form during the process of pyrolyzing corn starch hermetically. The obtained pyrodextrins were compared colorimetrically to those prepared in the presence of HCl as the conventional catalyst. Furthermore, the acetic acid-catalyzed pyrodextrins were analyzed for their physico-chemical properties and digestibility to evaluate the feasibility of adding more adjustability to the properties of pyrodextrins using of organic acid as an alternative to mineral acid.
Chel-Guerrero, & Betancur-Ancona, 2007; Kapusniak & Nebesny, 2017; Wang, Kozlowski, & Delgado, 2001). Structural analyses have revealed the complex nature of the obtained pyrodextrins as 1 → 2, 1 → 3 and 1 → 6 linkages were formed during the process (Bai & Shi, 2016; Ohkuma & Wakabayashi, 2001). The mechanism of starch pyrolysis has been postulated to depend on the temperature and concentration of acids applied (Wurzburg, 2006). In a pyrolytic condition where heat and duration were the main driving force, the cleavage of α-(1–4) glucosidic linkages of starch molecules occurred and the newly formed reducing ends transformed into glucosyl cations (Bai & Shi, 2016; Kroh, Jalyschko, & Häseler, 1996; Tomasik, Wiejak, & Pałasiński, 1989). The glucosyl cations might undergo intramolecular dehydration to form 1,6-anhydro glucans or intermolecular bonds formation with free hydroxyl groups of the adjacent chains. In the presence of acidic catalyst, the hydrolysis of α-(1–4) and probably α-(1–6) bonds was likely to be the dominant event at the initial stage of pyroconversion, followed by thermophilic reactions of transglucosidation and repolymerization (Singhal et al., 2008). Formation of glucosidic linkages other than α-(1–4) and α-(1–6) bonds resulting from the reaction of transglucosidation occurring in the course of manufacturing pyrodextrins has drawn a certain level of attention in respect of their resistance to digestive enzymes and potential benefits to mankind’s health. Ohkuma, Matsuda, Katta, & Hanno (1990) demonstrated that pyrodextrins prepared from potato starch at elevated temperatures (150–200 °C) in the presence of hydrochloric acid (HCl, 0.05–0.1% on dry starch basis) were resistant to the in vitro sequential hydrolysis of α-amylase, protease, and amyloglucosidase. The in vivo study showed that the obtained pyrodextrins were of low impact on glycaemia and insulin level. The indigestibility of pyrodextrins was attributed to the formation of 1 → 2 and 1 → 3 linkages during pyrolysis (Okuma & Matsuda, 2002). Not only from potato starch, indigestible pyrodextrins have also be prepared from the starches of corn (Kapuśniak & Jane, 2007; Okuma & Matsuda, 2002), high amylose corn mutant (Wang et al., 2001), sorghum, tapioca, sagu, cocoyam (Laurentin, Cárdenas, Ruales, Pérez, & Tovar, 2003) and legumes (Campechano-Carrera et al., 2007; OrozcoMartínez & Betancur-Ancona, 2004) in a similar fashion; the starches were pyrolyzed at the temperatures of 90–170 °C for 10–180 min with the use of not more than 0.2% HCl (on starch basis). The indigestibility of pyrodextrins, depending on the pyrolytic condition and the origin of starch, ranged diversely from minimum resistance (0.3%) to 67.8%. The solubility of pyrodextrins in water also varied widely, from minimum solubility (less than 1%) to 97.0%. Not limited to mineral acid, organic acids, such as citric acid and tartaric acid, could be used as the sole catalyst (Shin et al., 2007) or in cooperation with HCl (Jochym, Kapusniak, Barczynska, & Śliżewska, 2012; Kapusniak & Nebesny, 2017) in the production of pyrodextrin. The indigestibility of pyrodextrins was in the range of 19–40% and the solubility in water at 80 °C was more than 90%. It is apparent that the characteristics of pyrodextrins are intimately governed by the properties of starting material, pyrolytic temperature, heating rate, conversion duration and, if any, the quantify of acidic catalyst. When the addition of acid is desired, a diluted solution of mineral acid is introduced to starch slurry, followed by dehydration and pulverization before the commencement of pyrolysis (Wurzburg, 2006). To reduce the amount of water being introduced, diluted volatile mineral acid such as not more than 0.2% HCl (on starch basis) has commonly be used to spray onto the dry starch power (Campechano-Carrera et al., 2007; Kapuśniak & Jane, 2007; Laurentin et al., 2003; Okuma & Matsuda, 2002; Ohkuma et al., 1990; Orozco-Martínez & BetancurAncona, 2004), and its homogeneous distribution is of importance in minimizing the undesired charring resulting from uneven catalysis during the course of pyroconversion (Wurzburg, 1986). Distributing the volatile mineral acid uniformly is conceivably more imperative when a low dosage of the acid is intended to be used in the process. In this study, with a view to minimizing the localized catalysis leading to
2. Materials and methods 2.1. Materials Normal corn starch (Amyral food grade starch) was purchased from Tongaat Hulett Starch Ltd. (Isando, South Africa). All reagents were of analytical grade or liquid chromatography grade where appropriate. 2.2. Methods 2.2.1. Preparation of pyrodextrins Starch (10 g, db) was dried at 100 °C for 1 h to lower its moisture content to not more than 2%, and was transferred immediately to a bespoke polytetrafluoroethylene (PTFE) reaction vessel (250 ml) fitted with PTFE screw cap. For the purpose of distributing the acid in the form of gas, an aliquot of glacial acetic acid or concentrated HCl was pipetted accurately into a glass capsule with several pores on the circumferential wall. The final concentrations of acetic acid in starch were varied from 0.5 to 2.5% with an increment of 0.5%, and those of HCl were 0.05, 0.1 and 0.2%. The porous glass capsule was then fitted securely and perpendicularly to the orifice of the vessel. The hermetically sealed reaction vessel was incubated at the designated temperature for 3 h with continuous rotation at 100 rpm. The pyrolytic temperatures were varied from 140 to 180 °C with 10 °C interval for the use of acetic acid and from 120 to 140 °C for that of HCl. Upon completion, the vessel was cooled to the room temperature in an ice bath followed by the addition of 100 ml deionized water. The suspension was then neutralized to pH 7 with 3% NaOH solution, desalinated by using an adequate amount of mixed bed resin (Sigma M8157, Sigma-Aldrich Co., St. Louis, MO, USA) and filtered through a 100 mesh screen to remove the added ion-exchange resin. The pyrodextrin was then recovered by evaporating the collected filtrate to dryness at 60 °C. The yield was calculated as the mass ratio of obtained pyridextrin to the starch used. 2.2.2. Appearance and color Starch and pyrodextrins (approx. 1 g) were weighed and spread evenly on Petri dishes. The dishes were then photographed with a digital camera (SLT-A57, Sony Corp., Tokyo, Japan) against the aqua background. Color differences between the starch and pyrodextrins were quantified photoelectrically according to Orozco-Martínez and BetancurAncona (2004) with modifications. A colorimeter (Spectro Color Meter SE2000, Nippon Denshoku Industries Co. Ltd., Tokyo, Japan) was used to determine the Hunter color attributes of starch and pyrodextrins. The obtained color attributes of the pyrodextrins were compared to those of the starch and the color differences (ΔE) calculated as follows, ΔE = SQRT (ΔL2 + Δa2 + Δb2) where ΔL, Δa and Δb were the differences in the values of whiteness (L), redness-to-greenness (a) and yellowness-to-blueness (b) between starch and pyrodextrin, respectively. 2.2.3. Morphology Starch and pyrodextrins (approx. 0.1 g) were weighed into 50 ml 69
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equation of relative molecular weight on the retention volume. The regression equation was established using a series of pullulan standards with peak molecular weight ranging from 5.9 to 788.0 kDa (Standard P82, Showa Denko K.K., Kanagawa, Japan). For pyrodextrins, the same algorithm as that of starch was applied, while the F1 and F2 fractions were dissected at a constant retention volume (11.85 ml) obtained from the chromatogram of the starch.
glass beakers followed by the addition of 20 ml deionized water and 40 μl I2/KI solution (15.2 mg I2, 30.3 mg KI). Aliquots of the suspensions were spread on microscopic slides on which cover slips were then placed slowly to avoid the formation of bubbles. The morphology of the starch and pyrodextrins was observed with a light microscope (BX41, Olympus Corp., Tokyo, Japan). For birefringence observation, a polarizer was fitted to the light source of microscope and the addition of I2/KI solution was omitted from the specimen preparation.
2.2.6. Reducing sugar content Reducing sugar content of starch and pyrodextrins were determined colorimetrically as the capability of forming ferric ferrocyanide (Hizukuri, Shirasaka, & Juliano, 1983) with modifications. Samples (75 mg, db) were weighed accurately into 20 ml screw-capped glass vials. While stirring, 1.5 ml of water and 13.5 ml of DMSO aliquots were added sequentially by pipette. After sealing, the vials were placed in a boiling water bath for 1 h then at the room temperature overnight with continuous stirring. Aliquots (1 ml) of the glucan-DMSO solutions were pipetted into screw-capped tubes in triplicate followed by the addition of 0.5 ml each of potassium ferricyanide solution (1 mg ml‐1) and carbonate buffer (155 mM, [CO32−]/[HCO3−] = 0.414) containing 325 μg of potassium cyanide. After sealing and vortex mixing, the tubes were placed in a boiling water bath exactly for 15 min then cooled in running water for 10 min. Aliquots (2.5 ml) of ferric ammonium sulfate solution (3 mg ml−1 of 50 mM sulfuric acid) were pipetted into the tubes, the contents were mixed thoroughly after sealing, and the introduced air bubbles released periodically. After standing in the dark at the room temperature for 20 min, the absorbance of the solutions were measured at 715 nm against the reagent blank. For the establishment of a calibration curve, a set of glucose solutions with concentrations ranging from 1 to 5 μg ml‐1 was employed.
2.2.4. Solubility Solubility of starch and pyrodextrins at various temperatures was determined gravimetrically (Lin, Lee, & Chang, 2003) with modifications. Samples (0.1 g, db) were weighed in triplicate into screw-capped centrifuge tubes equipped with overhead stirring paddles. After the addition of 40 ml water, the tubes were immersed in a water bath set at the desired temperature (25, 50 and 90 °C) for 30 min with continuous stirring at 200 rpm. Upon completion and cooling to the room temperature in an ice bath, the tubes were centrifuged at 6000 rpm for 20 min and the supernatants decanted gently into 50 ml beakers without disturbing the sediment. The beakers were then placed in a fanassisted oven at 60 °C overnight followed by 130 °C for 1 h. The solubility was calculated as the mass ratio of solute to the initial sample. 2.2.5. Molecular weight Molecular weight distributions of starch and pyrodextrins were determined chromatographically (Chang, Lin, & Chang, 2006) with modifications. For starch and pyrodextrins with solubility less than 70% at 25 °C, samples (75 mg, db) were weighed accurately into 20 ml screw-capped glass vials. While stirring, 1.5 ml of water and 13.5 ml of dimethyl sulfoxide (DMSO) aliquots were added sequentially by pipette. After sealing, the vials were placed in a boiling water bath for 1 h then at the room temperature overnight with continuous stirring. Aliquots (2.1 ml) of the glucan-DMSO solutions were pipetted into centrifuge tubes and mixed thoroughly with 4 vols of ethanol. After centrifuging at 4000 × g for 10 min and discarding the supernatants carefully, the precipitated starch and pyrodextrins were re‐dissolved in 15 ml of water while heating in a boiling water bath for 1 h with continuous stirring. For pyrodextrins with solubility higher than 70% at 25 °C, without the aid of DMSO and centrifugation, the dextrins (75 mg, db) were dissolved in water (15 ml) while heating in a boiling water bath for 1 h. The solutions were then filtered via clean syringe filters (5 μm pore size) before injecting into a high performance size exclusion chromatograph (HPSEC) as below. The HPSEC was equipped with an inline degasser (ERC-3415α, ERC Inc., Saitama, Japan), isocratic pump (KP-12, Flom Corp., Tokyo, Japan), a manual injector (7725i, Rheodyne Inc., Berkeley, CA, USA) fitted with a 100 μl sample loop, three series-connected gel filtration columns (TSKgel PWH, TSKgel G5000PW and TSKgel G4000PW, Tosoh Corp., Tokyo, Japan), a triangle laser light-scattering detector (miniDAWN, Wyatt Tech., Santa Barbara, CA, USA) and a differential refractive index detector (DRI; Waters 410, Millipore Corp., Milford, MA, USA). The stationary phase was maintained at 70 °C and the mobile phase (0.1 M NaNO3 containing 0.02% NaN3) at a flow rate of 0.5 ml min−1. The weight-average molecular weight (Mw) of starch was calculated according to Lin et al. (2003) and the formula was as follows,
2.2.7. Digestible starch content Digestible α-glucans of starch and pyrodextrins were hydrolyzed by using of thermostable α-amylase and amyloglucosidase and the yielded glucose was colorimetrically quantified in the presence of glucose oxidase, peroxidase, 4‐aminoantipyrine and 4‐hydroxybenzoic acid (AACC, 2000). To aid the complete dissolution of starch and the pyrodextrins, DMSO was employed as described for the test objects potentially containing resistant starch. 3. Results and discussion 3.1. Yields, appearance and color characteristics Pyrolysis of corn starch using 0–2.5% acetic acid at 140–180 °C for 3 h formed pyrodextrins with yields of 93.5–98.4%, while the yields of pyrodextrins with 0.05–0.2% HCl at 120–140 °C were 73.9–98.9%. Horton (1965) reported that gaseous compounds, consisting of CO2, CO, water, volatile acids and volatile solids, were liberated from starch to which the temperatures of 180–210 °C were applied during the formation of pyrodextrins in the absence of acids. In this study, the temperatures used for pyroconversion were not higher than 180 °C, while the acidic catalysts were used. The concurrence of heat and acid might also give rise to a partial decomposition of starch into gaseous compounds and consequently to the observed decrease in the yield of pyrodextrins. This simultaneous effect of heat and acid was found to be particularly evident for pyrodextrins prepared under the extreme conditions of 0.2% HCl and 130–140 °C, and resulted in the yields of less than 90%. The pyrolysis at various temperatures in the presence of either HCl or acetic acid led to the changes in the appearance of pyrodextrins, and the extent of alteration in color was dependent on the type and concentration of the acid as well as the reaction temperature (Fig. 1). For pyrodextrins prepared with either acetic acid or HCl, the higher the pyrolytic temperature was, the more brownish the pyrodextrin
Mw = (WF1 (%) × MwF1 + WF2 (%) × MwF2)/100 where WF1 and WF2 were the weight percentages of fractions of amylopectin (F1) and low molecular weight molecules (F2), respectively, which were assigned by referencing the chromatogram of gyration radius against elution volume of starch (Chang, Lin, & Chen, 2006). The Mw of F1 fraction (MwF1) was calculated from the signals of lightscattering detector and DRI detector, where that of F2 fraction (MwF2) was computed from the first-order semi-logarithmic regression 70
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Fig. 1. Appearance of corn starch and pyrodextrins catalyzed by either hydrochloric acid or acetic acid at different concentrations and temperatures. The italic numeral placed beneath the photograph denotes the color difference (ΔE) between starch and the pyrodextrin.
complexes bore a resemblance to those of ΔE. The similarity in both changes may indicate the differences in color of iodine complex between starch and pyrodextrins arose from their different degrees of pyroconversion. Under polarized light, starch granules exhibited a typical birefringent pattern of Maltese cross in their center, while the birefringence was altered during pyrolysis. In analogous to the shifting in the color of pyrodextrin-iodine complexes, no considerable change in birefringence was observed for pyrodextrins formed at 140–150 °C despite the concentration of catalyst. When the temperature was raised to the range of 160–180 °C, the synergic effect of pyrolytic temperature and concentration of catalyst on birefringence began evident. Additionally, the temperature in connection with a major loss of birefringence was found to be in line with that of color shifting; 180 °C for the pyrolysis without catalyst, 170 °C for 0.5–1.0% acetic acid and 160 °C for 1.5–2.5% acetic acid. Formation of starch-iodine complex in blue-purple color reflected the occurrence of amylose and amylopectin molecules concurrently in the amorphous regions of the granules (Lin, Lii, & Chang, 2005; Tester, Karkalas, & Qi, 2004). With the positive correlation between the chain length of α-glucans and the λmax of their iodine complexes (Peymanpour et al., 2016) being borne in mind, the observed alteration in the color of pyrodextin-iodine complexes from blue-purple to redpurple then faint-pink indicates the cleavage of glucosidic linkages of starch chains occurred during the process of pyroconversion and thus led to the decrease in iodine binding capacity of pyrodextrin. This is in line with the structural change of starch during pyrolysis as postulated by Bai, Cai, Doutch, Gilbert, and Shi (2014). Besides, Wang et al. (2001) observed that the pyrodextrins prepared from native starches had higher enzymatic resistance than those of derivatied starches, and thus suggested that the amorphous region of starch being free from the steric hindrance of functional groups played an important role in the formation of indigestible pyrodextrins. In conjunction with the birefringent characteristics of pyrodextrins observed in this study, the pyrolytic reactions appeared to commence from the amorphous regions of starch granules then progressed into the crystalline regions when the temperature was increased. The disintegration of crystalline regions was further accelerated as the concentration of catalyst was increased.
appeared. Moreover, the appearance of the pyrodextrin was darkened with an increase in the concentration of catalyst. In terms of the type of catalyst, the alteration in the color of pyrodextrins prepared with HCl was more drastic than that prepared with acetic acid, and the ΔE were in the range of 1.2–70.5 for HCl-catalyzed pyrodextrins and 2.1–34.2 for those with acetic acid. Studies postulated that the change in the color of pyrodextrins was an indication of pyrolytic condition to which starch was exposed, and the increment of color difference implied the increased conversion degree of pyrodextrin (Ronald Terpstra, Woortman, & Hopman, 2010). As observed, HCl (0.05–0.2%) appears to be an effective catalyst in the formation of pyrodextrins at the temperatures between 120 °C and 140 °C. However, the spontaneous increase in ΔE and the decrease in yield probably due to the formation of char and volatile compounds can easily occur with a slight increase in the concentration of HCl within a tight temperature range. In contrast, the maximum increase in ΔE to 34.2 was observed only when the concentration of acetic acid was increased to 2.5% and the pyrolytic temperature from 140 °C to 180 °C. The results suggest that the glacial acetic acid could be an alternative acidic catalyst in the production of pyrodextrins when more adjustability to the properties of the obtained is desired, with the trade-off of a little higher conversion temperature. 3.2. Morphology Morphological structure of starch and acetic acid-catalyzed pyrodextrins was observed microscopically after staining with I2/KI solution. Their birefringent characteristics were also observed under polarized light without the use of staining agent during the specimen preparation. As shown in Fig. 2, starch used in the current study formed blue-purple complex with iodine, and the color of pyrodextrin-iodine complex changed to red-purple then faint pink with increasing pyrolytic temperature and catalyst concentration. In general, there was no appreciable alteration in color of iodine complex observed for pyrodextrins prepared at the temperatures of 140–150 °C. As the pyrolytic temperature increased to the range of 160–180 °C, the temperature effect began to be evident. Moreover, the temperature in connection with a substantial alteration in color was found to correlate inversely with the quantity of acetic acid used for pyrolysis. The altering temperature was 180 °C for the pyrolysis without catalyst, 170 °C for 0.5–1.0% acetic acid and 160 °C for 1.5–2.5% acetic acid. When compared to the appearance of starch and acetic acid-catalyzed pyrodextrins (Fig. 1), the pyrolysis-induced changes in color of iodine
3.3. Solubility Extents of solubilization of corn starch and acetic acid-catalyzed pyrodextrins in an excess amount of water at 25, 50 and 90 °C are 71
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Fig. 2. Light (upper) and polarized light (lower) micrographs of (a) corn starch before and after pyrolysis at 140 °C with 2.5% acetic acid, (b) acetic acid-catalyzed pyrodextrins prepared under the conditions specified (bar = 20 μm).
Fig. 3. Solubility at 25, 50 and 90 °C of corn starch (●) and acetic acid-catalyzed pyrodextrins prepared at 140–180 °C with 0% (▲), 0.5% (△), 1.0% (▼), 1.5% (▽), 2.0% (■) and 2.5% (□) acetic acid, respectively.
shown in Fig. 3. Taken all three temperatures as a whole, pyrodextrins exhibited a higher solubility than the starch. Solubility of pyrodextrins increased with increasing temperature of water used. The changes in solubility were from 1.0% to the range of 0.4–95.6%, 2.9% to 0.4–97.7% and 10.0% to 22.6–98.9% at the determination temperatures of 25, 50 and 90 °C, respectively. Furthermore, the solubility of pyrodextrins was found to increase synergistically with increasing pyrolytic temperature and ratio between catalyst and starch. This synergistic effect was particularly evident from the results determined at 25 and 50 °C. In the absence of acetic acid, an appreciable increment by approx. 20% in solubility was observed for the pyrodextrin prepared at 180 °C. By using of catalyst, the temperature, in connection with the rapid increase in solubility, reduced to 170 °C for 0.5–1.0% acetic acid and further to 160 °C when the concentration of catalyst increased to 1.5–2.5%. The increase in solubility of starch after pyroconversion was found to be in agreement with the alteration in color of starch complexing with iodine after pyrolysis showing in Fig. 2. This suggests that the
observed increase in solubility resulted from the cleavage of α-glucosidic linkages of starch during the process, and is well in line with other studies (Kapusniak & Nebesny, 2017; Kwon, Chung, Shin, & Moon, 2005). Moreover, studies have suggested that the extent of increase in solubility was correlated positively with the level of pyroconversion (Kapuśniak & Jane, 2007; Wang et al., 2001). As observed in this study, the reaction of degradation reflecting on the increase in solubility of starch after pyrolysis was of prominence, and the increasing extent was dependent on both of pyrolytic temperature and concentration of catalyst. This dependence was supported by the iodine binding capacity and birefringent properties of pyrodextrins discussed previously. 3.4. Molecular characteristics Molecular profiles of corn starch and pyrodextrins prepared by using 0.5–2.5% acetic acid at 140–180 °C were analyzed 72
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Fig. 4. High-performance size-exclusion chromatograms of molecular weight standards (●), corn starch (—) and acetic acid-catalyzed pyrodextrins prepared at 140 °C (–– ––), 150 °C (– –), 160 °C (– • –), 170 °C (– • • –), and 180 °C (• • • •) with 0–2.5% acetic acid, respectively.
Table 1 Weight-average molecular weight and reducing sugar content of corn starch and acetic acid-catalyzed pyrodextrins. Acetic acid (%, starch dry basis)
Mw (kDa) Starcha 0 0.5 1.0 1.5 2.0 2.5 Reducing sugar content (%, db) Starch 0 0.5 1.0 1.5 2.0 2.5 a
Temperature (°C) 140
150
160
170
180
119551 ± 4416 35783 ± 1110 2091 ± 75 921 ± 449 673 ± 102 496 ± 77 346 ± 120
– 9311 ± 480 605 ± 15 371 ± 85 426 ± 69 215 ± 106 264 ± 109
– 1978 ± 37 193 ± 44 101 ± 2 65 ± 5 60 ± 2 56 ± 3
– 351 ± 106 79 ± 26 42 ± 1 36 ± 4 25 ± 1 24 ± 2
– 197 ± 26 32 ± 1 21 ± 2 18 ± 1 20 ± 1 18 ± 2
0.13 0.17 0.33 0.26 0.35 0.42 0.43
– 0.23 0.42 0.27 0.39 0.45 0.55
– 0.33 0.49 0.52 0.51 0.50 0.58
– 0.43 0.56 0.65 0.50 0.52 0.65
– 0.24 0.48 0.27 0.22 0.31 0.18
± ± ± ± ± ± ±
0.01 0.01 0.08 0.04 0.01 0.04 0.04
± ± ± ± ± ±
0.01 0.16 0.08 0.18 0.23 0.04
± ± ± ± ± ±
0.01 0.13 0.14 0.21 0.09 0.12
± ± ± ± ± ±
0.02 0.06 0.07 0.08 0.30 0.18
± ± ± ± ± ±
0.06 0.04 0.09 0.12 0.04 0.02
Corn starch was used to determine its weight-average molecular weight and reducing sugar content, respectively.
shifting to a higher elution volume. The Mw of the starch was 119551 kDa and that of pyrodextrins in the range of 18–35783 kDa. In the absence of catalyst, the Mw of pyrodextrin reduced rapidly from 35783 kDa to 351 kDa when the pyrolytic temperature was increased from 140 °C to 170 °C, then levelled off at 197 kDa when the
chromatographically (Fig. 4 and Table 1). As evidenced by the chromatograms, starch exhibited a greater percentage of high molecular weight fraction (F1 fraction) than pyrodextrins. With increasing the pyrolytic temperature and the concentration of catalyst, the molecular weight distribution of pyrodextrins was narrowed with their peaks
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In addition, the digestible starch content of pyrodextrins was found to decrease with increases in the temperature and the concentration of catalyst used. It was also found that the temperature, in connection with a prominent decline in digestible starch content, tended to correlate with the concentration of catalyst. With the use of not more than 0.5% acetic acid, the prominent decrease in the enzymatic susceptibility of pyrodextrins was noticed at 180 °C. The intervening temperature was reduced to the range of 160–170 °C when a higher concentration of acetic acid was used. It is interesting to observe that the temperatures in connection with the considerable decrease in enzymatic susceptibility occurred at or before the temperatures at which a drastic decline of reducing sugar content was observed. These results suggest that an alteration in structural conformation leading to the resistance to α-amylase and amyloglucosidase probably due to its high numbers of 1 → 2, 1 → 3 and 1 → 6 linkages (Bai & Shi, 2016; Nunes et al., 2016; Okuma & Matsuda, 2002) was induced in the course of pyrolysis at 180 °C, irrespective of the concentration of acetic acid. The formation of this enzyme-resistant conformation would probably also occur between the temperature range of 140 °C and 170 °C even when the reducing sugar content appeared to increase with increasing the pyrolytic temperature. By collectively considering the results from the morphology (Figs. 1 and 2), solubility (Fig. 3) and molecular characteristics of pyrodextrins (Table 1), pyrolysis of starch tends to commence from the amorphous regions of starch granules. The cleavage of α-glucosidic linkages of starch chains and the transglucosidation of liberated fragments take place concurrently, while the former may be of prominence at low temperature. When the intervening temperature is reached, the degradation progresses towards the crystalline regions of the granules. At this point, the role of transglucosidation may be in parallel with that of depolymerization. When the pyrolytic temperature is increased further, the reaction of transglucosidation becomes dominant and results in the decrease in reducing sugar content and the further decline in digestible starch content. With the use of glacial acetic acid, the intervening temperature can be reduced.
temperature was further elevated to 180 °C. With 0.5–1.0% acetic acid, the appreciable decrease in Mw was observed to plateau out at the temperature of 170 °C; 2091 kDa to 101 kDa at 140–160 °C, and 79 kDa to 21 kDa at 170–180 °C. When the concentration of catalyst was in the range of 1.5–2.5%, the intervening temperature at which a steady decrease in the Mw commenced was further lowered to 160 °C; 673 kDa to 264 kDa at 140–150 °C, and 65 kDa to 18 kDa at 160–180 °C. The observed decrease in Mw suggests the reaction of degradation did occur during the course of pyrolysis, which is well in line with the mechanism of pyroconversion (Bai et al., 2014; Singhal et al., 2008) and supported by the increase in the solubility of pyrodextrin showing in Fig. 3. Additionally, the observations of substantial decrease in Mw at intervening temperature and the lowering of the temperate with increasing the concentration of catalyst might benefit from the use of acetic acid as the catalyst (H+ donor) with moderate dissociation constant. Besides, it is interesting to observe that the intervening temperature for the Mw of pyrodextrins is consistent with those of the birefringence (Fig. 2) and the solubility (Fig. 3). Reducing sugar content of corn starch and pyrodextrins were analyzed to aid in evaluating the effect of pyrolytic condition on the molecular properties of pyrodextrins. As shown in Table 1, the reducing sugar content of pyrodextrins was higher than that of the starch, suggesting the cleavage of α-glucosidic linkages of starch chains occurred during the course of pyrolysis (Kapuśniak & Jane, 2007). Further taken all the conditions as a whole, pyrodextrin showed an increase in its reducing sugar content as the pyrolytic temperature was raised, while a considerable decline of reducing sugar content was observed thereafter. At the pyrolytic temperature of 140 °C, the reducing sugar content of the obtained pyrodextrins was in the range of 0.17–0.43%. When the temperature was raised to 170 °C, the reducing sugar content increased to the range of 0.43–0.65%. As the temperature was raised further to 180 °C, decreasing in reducing sugar content at the range of 0.18–0.48% was observed. By collectively considering the observed decrease in Mw (Fig. 4), it is apparent that the depolymerization of starch occurred during pyrolysis in spite of the use of catalyst and the reaction temperature. Further supported by the continuous increases in the solubility of pyrodextrins with increasing the pyrolytic temperature (Fig. 3), it is conceivable that the depolymerization was an on-going event throughout the course of pyrolysis whether or not the intervening temperature had reached. When a high pyrolytic temperature (e.g. 180 °C) was applied, the transglucosidation and/or re-polymerization of the segmented dextrins probably became dominant and thus resulted in the observed decrease in reducing sugar content.
4. Conclusions Indigestible pyrodextrins were produced by pyrolyzing corn starch at 140–180 °C with glacial acetic acid (0–2.5%, w/w) as the catalyst. The acetic acid-catalyzed pyrolysis was of high yield, and the color of obtained pyrodextrins altered gradually from off-white to tan depending on the pyrolytic condition. Results of analyses on physicochemical properties and digestible starch content suggest that the properties of pyrodextrin can be tailored for its application by leveraging the volatility and low acid dissociation constant of glacial acetic acid as a H+ donor together with a wide range of feasible pyrolytic temperature. For instance, pyrodextrins of low digestibility and high solubility, prepared at 180 °C with 2.0–2.5% acetic acid, could be used to enhance the dietary fiber content of bottled beverages, and pyrodextrins of high molecular weight and moderate digestibility, obtained at 150–160 °C with 0.5–1.5% acid, to be included in the formulation of instant beverage as the sources of both calories and soluble dietary fiber. The intertwined effect of catalyst concentration and pyrolytic temperature on the characteristics of pyrodextrin has been revealed and discussed. In the same concentration of acetic acid, alterations to the loss of birefringence, considerably increased solubility and the plateau of molecular weight reduction have been observed at the same temperature. With increasing the catalyst concentration, the intervening temperature was reduced. Interestingly, the intervening temperatures for the changes to the decreased content of reducing sugar and the substantial decrease in digestibility were generally higher than that of birefringence, solubility and molecular weight. Thus, further research on the structural characteristics of pyrodextrins is of necessity for gaining a further in-depth insight into the complex mechanism of
3.5. Digestible starch content Corn starch and pyrodextrins were subjected to hydrolysis by thermostable α-amylase followed by amyloglucosidase to evaluate the pyrolysis-induced changes in the in vitro digestibility of starch. As shown in Table 2, pyrodextrins were less enzymatic susceptible than starch, changing from 99.0% to the range of 47.8–92.8% after pyrolysis. Table 2 Digestible starch content (%, db) of corn starch and acetic acid-catalyzed pyrodextrins. Acetic acid (%, starch dry basis)
Temperature (°C)
Starcha 0 0.5 1.0 1.5 2.0 2.5
99.0 92.8 87.6 83.8 77.6 76.6 73.4
a
140
150 ± ± ± ± ± ± ±
0.6 0.3 0.4 3.5 1.1 0.3 0.4
– 90.4 84.4 78.7 72.5 72.8 72.7
160
± ± ± ± ± ±
1.7 1.2 1.0 1.5 3.6 1.2
– 86.2 78.6 64.8 70.6 65.8 69.7
170
± ± ± ± ± ±
1.3 1.6 4.0 2.7 2.2 0.8
– 83.1 76.5 63.4 57.5 54.3 52.6
180
± ± ± ± ± ±
1.4 0.8 1.4 0.9 1.0 1.5
– 75.5 67.4 58.2 55.9 48.0 47.8
± ± ± ± ± ±
0.4 1.3 1.6 7.0 2.0 0.9
Corn starch was used to determine its digestible starch content.
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