Food Chemistry 155 (2014) 38–44
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Preparation and characterization of resistant starch III from elephant foot yam (Amorphophallus paeonifolius) starch Chagam Koteswara Reddy, Sundaramoorthy Haripriya ⇑, A. Noor Mohamed, M. Suriya Department of Food Science and Technology, Pondicherry Central University, Puducherry 605 014, India
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Article history: Received 1 July 2013 Received in revised form 26 November 2013 Accepted 11 January 2014 Available online 23 January 2014 Keywords: Elephant foot yam starch Enzymatic hydrolysis Physico-chemical properties Pullulanase Resistant starch
a b s t r a c t The purpose of this study was to assess the properties of resistant starch (RS) III prepared from elephant foot yam starch using pullulanase enzyme. Native and gelatinized starches were subjected to enzymatic hydrolysis (pullulanase, 40 U/g per 10 h), autoclaved (121 °C/30 min), stored under refrigeration (4 °C/24 h) and then lyophilized. After preparation of resistant starch III, the morphological, physical, chemical and functional properties were assessed. The enzymatic and retrogradation process increased the yield of resistant starch III from starch with a concomitant increase increase in its water absorption capacity and water solubility index. A decrease in swelling power was observed due to the hydrolysis and thermal process. Te reduced pasting properties and hardness of resistant starch III were associated with the disintegration of starch granules due to the thermal process. The viscosity was found to be inversely proportional to the RS content in the sample. The thermal properties of RS increased due to retrogradation and recrystallization (P < 0.05). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction In the food processing industries, starch contributes to important characteristics including thickening, gelling, consistency and shelf stability in a diverse range of diverse applications. Potato, rice and wheat starch have been exclusively employed for this purpose in transforming the microstructure and functionality of products in the food industries (Wheatley, Liping, & Bofu, 1996). Though starch contributes significantly to the quality and consistency of commercial products, it has a high glycemic index which often makes it unfavourable in terms of its effects to the consumer. In order to meet the growing demands of the consumers for functional foods, carbohydrates which can act as functional ingredients and have a beneficial effect to human health are favoured over carbohydrates with a high glycemic index. Resistant starch (RS) is one of the naturally occurring carbohydrate which is defined as the sum of starch and starch degradation products which cannot be digested in the small intestine of humans and when reached in the large intestine, it undergoes fermentation by the commencal Abbreviations: DHgel, enthalpy of gelatinization; DSC, differential scanning calorimetry; PHI, peak height index ; R, gelatinization range; RC, relative crystallinity; RS, resistant starch; SEM, scanning electron microscopy; SP, swelling power; T0, onset temperature; TC, conclusion temperature; TP, peak temperature; TPA, texture profile analyzer; WAC, water absorption capacity; WSI, water solubility index; XRD, X-ray diffractometer. ⇑ Corresponding author. Tel.: +91 9443701906; fax: +91 413 2654621. E-mail address: [email protected] (S. Haripriya). 0308-8146/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2014.01.023
intestinal microorganisms production, resulting in the production of short chain fatty acids (Annison & Topping, 1994). These short chain fatty acids can be partially absorbed in the small intestine and be a source of energy to the mucosal cells or can support the growth and metabolism of the colonic microbiota, with the undigested mass being excreted in the stool (Xue, Newman, & Newman, 1996). Besides its vital physiological role as a functional ingredient in lowering the risk of diet-related diseases, RS when compared with traditional insoluble fibres also has many favourable features for thea food industry. More specifically, RS is a natural white colour powder with bland taste and has acceptable appearance and texture (Sajilata, Singhal, & Kulkarni, 2006). RS is classified on the basis of its botanical source and processing methods (Englyst, Kingman, & Cummings 1992). RS can be classified in four types including RS1, physically inaccessible starch; RS2, native starch granules; RS3, retrograded starch and RS4, chemically modified starch (Sajilata et al., 2006). Sources of cereal grains, roots, tubers and legumes produce resistant starch through the process of cyclic heating, autoclaving and extrusion methods. Gelatinization and cooling processes which are generally referred to as annealing procedures, are the common methods used to enhance the formation of RS3 (retrograded starch) (Thompson, 2000). Ample studies are available on the RS of the cereals, pulses, and tubers, especially cassava and potato starch. One such starch source which is not explored commercially is Amorphophallus paeonifolius, which is also known as elephant foot yam, an herbaceous, perennial C3 crop. It is a tropical tuber which has originated
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from the south eastern Asian region and is extensively used in Indian cuisines (Ravi, Ravichandran, & Suja, 2009). Though several researchers have studied the flour and starch obtained from yam tubers in order to find new food applications (Okaka, Okorie, & Ozo, 1991) a limited number of studies on starch and resistant starch fr A. paeonifolius are available. Taking into account the need for RS3, as this would constitute a functional food ingredient, the study was designed to elucidate the preparation of starch and resistant starch (RS3) from elephant foot yam. The objectives of the study were to prepare RS3 retrograded starch from isolated starch of elephant foot yam (A. paeonifolius) and characterise its physico-chemical and functional properties.
2. Materials and methods 2.1. Materials The tuber of elephant foot yam (A. paeonifolius) was purchased from a local market. The resistant starch assay kit was purchased from Megazyme International (Ireland), whereas the enzymes used in the study included pullulanase from Bacillus acidopullulyticus (Promozyme 400L) and heat stable a-amylase from Bacillus licheniformis (Termamyl 120L), obtained from Sigma (USA).
2.2. Isolation of starch The starch was isolated from elephant foot yam (A. paeonifolius) according to the method of Amani, Buleon, Kamenan, and Colonna (2004). The tuber was peeled, cut into small pieces and immediately suspended in 0.1% (w/v) sodium metabisulphite solution. Then, the sample was homogenised with warring blender and suspended in a large amount of 4% NaCl. The slurry was filtered through a 100 lm sieve and the filtrate was centrifuged at 2660g for 15 min. This procedure was repeated for four times and the recovered white coloured starch was then oven dried at 48 h at 45 °C.
2.3. Preparation of resistant starch 2.3.1. Enzymatic hydrolysis of elephant foot yam starch Enzymatic hydrolysis of elephant foot yam starch was performed by using the procedure used by Polesi and Sarmento (2011) with a slight modification. The elephant foot yam starch (10% w/w db) was suspended in sodium acetate buffer (0.1 M and pH 5.3) and mixed with pullulanase enzyme (40 U/g dry starch), and the mixture incubated in a shaking water bath at 60 °C for 10 h. The sample was heated in a boiling water bath for 10 min to inactivate the enzyme. Starch gelatinization, prior to adding the enzyme, was performed by boiling the sample in a water bath for 10 min.
2.3.2. Preparation of resistant starch Starch samples, namely elephant foot yam starch (S1), native hydrolyzed by enzyme (S2) and gelatinized hydrolyzed by enzyme (S3) in suspensions (10% w/w db) were autoclaved at 121 °C for 30 min, cooled and kept at 4 °C for 24 h. The samples (S1, S2 and S3) were then lyophilized.
2.3.3. Determination of resistant starch content The amount of RS in the samples was analysed by using a Megazyme resistant starch assay kit, which is based on the Association of Official Analytical Chemists (AOAC) methods (2002.02).
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2.4. Scanning electron microscopy (SEM) The morphological characteristics of S1, S2 and S3 were evaluated using scanning electron microscope (HITACHI Model S-3000H) with a magnification 500 to 1500. The powdered samples were sprinkled on double-sided stick tape placed on aluminium stubs and were covered with a gold–palladium layer. 2.5. Physicochemical characteristics 2.5.1. Chemical composition The moisture content of S1 was recorded in terms of weight loss after heating at 130 ± 2 °C for 2 h using 2 g of sample. The amount of ash, protein, and fat were analysed according to AACC methods 08–01, 46–13 and 30–25, respectively. The amount of starch was analysed using the AOAC method 8.020. 2.5.2. Amylose content The total amylose content of the starch samples (S1, S2and S3) was analsyed according to the method described by Williams, Kuzina, and Hlynka (1970). Starch samples (20 mg) were added to 10 mL of 0.5 N KOH and the resultant suspension was thoroughly mixed. Subsequently, the dispersed sample was transferred into a 100 mL volumetric flask and diluted to the mark with distilled water. An aliquot (10 mL) was pipetted into a 50 mL volumetric flask and 5 mL of 0.1 N HCl were added followed by 0.5 mL of iodine reagent, and the solution made up to 50 mL with water; the absorbance was then measured at 625 nm. The measurement of amylose was determined from a standard curve developed using amylose as the standard. 2.5.3. Water absorption capacity (WAC) and water solubility index (WSI) The WAC and WSI of the samples (S1, S2 and S3) were analysed according to the procedure described by Anderson, Conway, Pfeifer, and Griffin (1969). Briefly, a sample of 0.5 g was mixed with 6 mL of distilled water, and centrifuged. The supernatant was heated at 30 °C with continuously stirring for 30 min in a water bath. The suspension was placed in a petridish and dried at 105 °C for 4 h to obtain the dry solids weight, and the wet sediment was weighed. The WSI and WAC were determined as: WSI = (weight of dry solids in supernatant/weight of dry sample) 100; WAC = weight of wet sediment/(weight of the dry sample-weight of the dry solids). 2.5.4. Swelling power (SP) The swelling power of the samples (S1, S2and S3) was anasyed using the method described by Nattapulwat, Purkkao, and Suwithayapan (2009). A sample (0.2 g) was dispersed in water (20 mL) to form a suspension. The suspension was heated to 85 °C in a water bath for 30 min with vigorous shaking every 5 min. The starch gel was then centrifuged at 2200 rpm for 15 min. The weight of the sediment was used to calculate the swelling power. The supernatant was dried and weighed to measure the amount of dissolved starch in the supernatant. The swelling power was determined as: swelling power = weight of sediment/(weight of dry starch weight of dissolved starch). 2.5.5. Pasting properties The viscoamylographic property of the samples (S1, S2 and S3) were performed with a Rapid Visco Analyser (RVA starch master 2, Newport Scientific, Warriewood, NSW, Australia) using 2 g of sample in 25 mL of water. The following parameters: paste temperature, peak viscosity, breakdown viscosity, final viscosity and setback viscosity were measured from the viscoamylographs.
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2.5.6. Textural characteristics The textural properties of RVA gels were studied using the method Kaur, Singh, McCarthy, and Singh (2007) using a texture profile analyzer (HDP/BS blade of texture analyzer (TA) TA–HD plus, Stable Micro Systems, Surrey). After 24 h incubation at 4 °C the gels formed from the RVA analysis were examined by the texture analyzer using probe No. 5. From the texture profile curve, hardness, cohesiveness, gumminess, adhesiveness, springiness, chewiness and stringiness were calculated. 2.5.7. XRD and relative crystallinity (RC) The X-ray patterns of the samples (S1, S2 and S3) were studied with a X-ray diffractometer (Shimadzu XRD 7000) according to the procedure described by Zobel (1964). The RC of the starches was quantitatively estimated following the method of Nara and Komiya (1983) using a software (Origin version 8, Microcal Inc., Northampton, MA, USA). The graphs were plotted between 2h of 4° to 30° and smoothed with the ‘‘Adjacent Averaging’’ tool. 2.5.8. Thermal properties The thermal properties of the samples (S1, S2 and S3) were examined using the method described by Gao, Li, Jian, and Liang (2011) based on differential scanning colorimetry (TA-Q20 DSC). The DSC curve which showed the onset (T0), peak (Tp), final (Tf), gelatinization temperatures and enthalpy (DH) were analysed using Universal Analysis 2000 3.9A software. 2.6. Statistical analysis The experiments were conducted in triplicate. The obtained data were submitted to one way ANOVA and Duncan’s Multiple Range Test (DMRT) (P 6 0.05) to analyse the significance of difference between the mean values of the samples using SPSS 18 software (SPSS Institute Inc., Cary, NC, USA). 3. Results and discussion 3.1. Morphological characteristics The SEM micrographs (Fig. 1) of S1 when compared to S2 and S3 differed significantly. SEM studies revealed that the elephant foot yam starch (S1) granules are round, elliptical and polygonal in shape with smooth surfaces, with no obvious effects or signs of damage on the surface. However, the granular structure of samples was destroyed after being cooked, debranched and dried. S2 and S3 showed a cohesive structure, leading to the loss of granular appearance and to a irregular shape, which is the consequence of gelatinization temperature where the coupled starch granules forms sponge like structure within the inner region of the retrograded starch (Ratnayake & Jackson, 2007). 3.2. Chemical composition The proximate composition in terms of moisture, ash, protein and fat in the isolated elephant foot yam starch (S1) was 8.5%, 2.3%, 0.9% and 0.2% respectively. The moisture content of S1 (8.5%) was comparatively lower when compared with cassava starch (10.2%) and arrow root starch (9.82%). Moisture content plays a significant role for flow and other mechanical properties (Raja & Sindhu, 2000). The moisture content of a starch powder is dependent upon the extraction procedure and drying process along with surrounding atmosphere humidity. The starch and amylose content in the extracted starch were 87.7% and 24.2%, respectively, on a dry weight basis. These parameters were compa-
rable with the proximate composition, and the starch and amylose content of other yam varieties (Wang, Yu, Liu, & Chen, 2008). 3.3. Physicochemical properties The amylose, RS content, SP, WAC and WSI of S1, S2 and S3 are shown in Table 1. The amylose content of S1 (24.21%) was found to be significantly lower than S2 (46.78%) and S3 (50.18%). Furthermore, S3 (50.18%) was found to be significantly greater than S2 (46.78%). The RS content of S3 (36.27%) was significantly higher when compared with S2 (30.67%) and S1 (17.98%) owing to the increased amylose content in samples S2 and S3 which are the resultant retrograded starch of elephant foot yam starch upon the treatment with pullulanase enzyme before and after gelatinization, respectively. The considerable increase of RS content in S3 followed by S2 would be attributed to the effect of pullulanase enzyme on debranching the a-(1–6) linkage of amylopectin (Leong, Karim, & Norziah, 2007); the latter is converted into small chain linear polysaccharides like amylose molecules, which form strong gel network through retrogradation (Polesi and Sarmento, 2011). The RS formed in S2 and S3 were subjected to retrogradation which ensures the conversion of RS2–RS3. The swelling power measures the tenacity of the bonds in the crystalline portion of the starch granule, showing the ease with which the starch will cook. Granules with greater crystalline areas along with stronger bonds in the crystalline regions swell less in cold water as well as when heated. The gels formed from these crystalline regions are weak and have greater tendency towards retrogradation because of the bonds. The swelling power of elephant foot yam starch at 85 °C (8.14%) was significantly differed from the swelling power of S2 at 85 °C (5.6%) and that of S3 at 85 °C (5.4%). The swelling power ascertains the magnitude of interaction among the starch chain within the regions of crystalline and amorphous nature. The distribution of molecular weight, degree of debranching, length of branches and the conformation of the molecules along with the ratio of amylose and amylopectin places a very important influence on the extent of water and starch interaction (Hoover, 2001). The observation of the decreased SP in S2 and S3, irrespective to the increase in temperature, could be attributed to the effect of gelatinization and the autoclaving process, which would have occurred during the preparation of S2 and S3. The properties of WAC and WSI of all starch samples were greatly dependent on the source, content of amylose/amylopectin, extraction procedure and thermal stability. WAC is a phenomenon linked to the wet heat treatment of starch samples. In this study, the gelatinization induced by heating and autoclaving significantly increased the WAC in S3 (6.04%) when compared to S1 (3.59%) and S2 (5.46%). The WAC of all the starch samples was comparable with other studies of RS from chick pea starch (Polesi and Sarmento, 2011). An increased water activity in retrograded starch was noted which is the result of a change in molecular structure or any other mechanisms leading to an easier mobility of the starch components, where leaching of starch was also noticed (Govindasamy, Campanella, & Oates, 1996). The parameter WSI is used as an indicator of the destruction of starch components. The WSI of S3 (13.89%) and S2 (12.06%) was significantly different from S1 (2.57%). The S2 and S3 have undergone an hydrolytic process when compared to S1. The enzymatic hydrolysis contributed to the increased water solubility in S2 and S3. 3.4. Pasting properties The pasting properties of all samples were determined using a rapid visco analyzer. The pasting property plays a vital role in the application of starch and resistant starch in food industries and these parameters are depended on the source, amount of
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Fig. 1. Scanning electron micrographs (SEM) of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
Table 1 Amylose, resistant starch, swelling power, water absorption capacity, water solubility index and relative crystallinity of S1, S2 and S3.# Parameter
Amylose (%) RS (%) SP (%) WAC (%) WSI (%) RC (%)
Type S1
S2
S3
24.21 ± 0.983c 17.98 ± 0.382c 8.14 ± 0.201a 3.59 ± 0.176c 2.57 ± 0.110b 19.37 ± 0.633c
46.78 ± 1.454b 30.67 ± 1.632b 5.67 ± 0.108b 5.46 ± 0.415a 12.06 ± 1.010a 25.12 ± 0.949b
50.18 ± 1.171a 36.27 ± 1.167a 5.48 ± 0.168b 6.04 ± 0.185a 13.89 ± 1.249a 27.84 ± 1.201a
RS, resistant starch; SP, swelling power; WAC, water absorption capacity; WSI, water solubility index; RC, relative crystallinity; S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
starch, and the interaction between molecules and testing conditions (Liu, Donner, Yin, Huang, & Fan, 2006). In the presence of water, the starch suspension was subjected to a thermal process, where the starch granules swell and thus the viscosity increases progressively. The viscoamylographs of S1, S2 and S3 are shown in Fig. 2 and Table 2. The pasting properties of S1 showed a significant difference with S2 and S3 (P < 0.05) which represents the typical pasting behaviour of native starch (Miao, Jiang, & Zhang, 2009). The pasting temperature was not detectable for S3 and the low pasting temperature for S2 (61.2 °C) could be due to the destruction of starch granules when subjected to autoclave at 121 °C during RS preparation which is also comparable with other studies like the RS from chickpea and red kidney bean starch (Polesi and Sarmento, 2011; Reddy, Suriya, & Haripriya, 2013). S2 and S3 show a lower amylographic pattern for parameters including peak viscosity, hold viscosity, final viscosity, break down and set back when compared with S1. This attribute could be a result of the
Fig. 2. Typical RVA starch pasting curves for of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
enzymatic hydrolysis process which improves the levels of short linear chain molecules and RS. The formation of starch gel ability was reduced in S2 and S3 when compared with S1 due to ithets autoclave process (Polesi and Sarmento, 2011; Gelenscer et al., 2008). 3.5. Textural properties After 24 h incubation at 4 °C, the retrograded starch pastes (RVA gels) of S1, S2 and S3 were examined with a texture profile analyzer and the obtained data are shown in Table 3. Higher peak viscosity and hardness was observed in S1 when compared with S2 and S3 (P < 0.05), which may be due to the presence of larger size starch granules and lower levels of amylose. The reduced hardness
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Table 2 Pasting properties of S1, S2 and S3: pasting temperature (°C), peak time (min), peak viscosity (cP), hold viscosity (cP), final viscosity (cP), break down (cP) and set back (cP).# Parameter
Pasting temp (°C) Peak time (min) Peak viscosity (cP) Hold viscosity (cP) Final viscosity (cP) Breakdown (cP) Setback (cP)
Table 3 Textural properties of S1, S2 and S3: hardness, cohesiveness, adhesiveness, gumminess, springiness, chewiness and stringiness.# Parameter
Type
Type S1
S1
S2
S3
89 ± 0.7a 4.42 ± 0.5c 1320.0 ± 31.320a 1248.33 ± 56.888a 1819.33 ± 30.171a 122.33 ± 4.725a 626.0 ± 7.151a
61.2 ± 0.5b 7.10 ± 0.33a 435.0 ± 55.865b 418.66 ± 29.022b 598.33 ± 60.351b 16.33 ± 1.527b 131.66 ± 3.511b
nd 5.30 ± 0.61b 238.0 ± 16.370c 228.0 ± 11.532c 275.0 ± 13.258c 13.33 ± 1.527c 42.0 ± 3.0c
S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch; nd, not detected. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
and the formation of gel ability of S2 and S3 is due to the partial hydrolysis of starch granules and also the destruction of granules by enzymatic hydrolysis and the autoclave process. The results elicited significant difference between all samples (S1, S2 and S3) with respect to the texture parameters including hardness, cohesiveness, adhesiveness, gumminess, springiness, chewiness and stringiness. The difference in the textural properties of all sample gels were influenced by rigidity in gelatinized starch, amylose content as well as interaction between the dispensed and continuous phase of the gel, which in turn is dependent on the amylose and amylopectin structure (Yamin, Lee, Pollak, & White, 1999). 3.6. X-ray diffraction The X-ray diffraction patterns of samples (S1, S2 and S3) are shown in Fig. 3. S1 gave an intermediate intensity peak at diffraction angles of 2h = 17.92° and a strong peak at 2h = 23.05°, with a weak peak at 14.7° showing an intermediate diffraction pattern A- and B-type (C-type). The S2 and S3 exhibited peaks at 16.85° and 16.95°, respectively, indicating a B-type crystallinity due to the retrogradation at low temperature. Similar crystalline patterns were also observed in wheat, corn and sago starch (Leong et al., 2007) when treated with pullulanase, and were autoclaved and
Hardness (N) Cohesiveness Adhesiveness (Ns) Gumminess (N) Springiness (s) Chewiness (Ns) Stringiness
0.506 ± 0.051a 0.323 ± 0.041b 13.04 ± 0.632b 0.17 ± 0.01a 1.14 ± 0.026a 0.127 ± 0.092a 5.973 ± 0.935a
S2 0.113 ± 0.005b 0.523 ± 0.035a 3.963 ± 0.241a 0.046 ± 0.011b 0.876 ± 0.096b 0.04 ± 0.01a 6.17 ± 0.238a
S3 0.093 ± 0.005b 0.536 ± 0.025a 4.04 ± 0.07a 0.046 ± 0.005b 0.903 ± 0.06b 0.0366 ± 0.005a 5.476 ± 0.434a
S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch; nd, not detected. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
retrograded. The type of crystallinity of samples is influenced by the chain length of amylopectin, growth temperature and fatty acids content (Gunaratne & Hoover, 2002). S3 gave a single broad peak when compared with S2 due to the recrystallization with retrogradation. The relative crystallinity of S1 (19.37%) and S2 (25.12%) were lower when compared with that of S3 (27.84%). The highest crystallinity of S3 could be due to the increased RS content, which could have been enhanced by gelatinization, enzymatic treatment and retrogradation (Table 1).
3.7. Thermal properties Generally, the endothermic transition of all types of starch samples are influenced by the interactions between amylose–amylose, amylose–amylopectin and the amylose–lipid contents (Shin, Byun, Park, & Moon, 2004). However in S1 there were no considerable levels of lipid present. The thermal properties of S1, S2 and S3 were influenced by the granule shape, amylopectin chain length and crystalline regions (Singh & Kaur, 2004). Thermal analyses of S1, S2 and S3 were performed by DSC and the data are shown in Table 4 and Fig. 4. The DSC profiles of the samples showed a low peak temperature for S1 and S2 when compared with S3, which is attributed to the gelatinization and retrogradation process. The
Fig. 3. X-ray diffraction pattern (XRD) of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
C.K. Reddy et al. / Food Chemistry 155 (2014) 38–44 Table 4 Thermal properties of S1, S2 and S3: transition temperatures (T0; TP; TC), enthalpy of gelatinization (DHgel), peak height index (PHI) and gelatinization range (R).# Parameter
Type S1
T0 (°C) TP (°C) TC (°C) DHgel (J/g) PHI (J/g C) R (°C)
53.073 ± 1.471b 93.66 ± 2.094c 149.603 ± 1.316a 284.14 ± 5.65c 6.51 ± 0.17b 96.606 ± 1.272a
S2
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resistant starch III of elephant foot yam has improved functional properties resulting from the enzymatic hydrolysis, thermal and retrogradation processes, and can potentially used as a substitute of native starches used currently in the food industry.
S3 b
54.993 ± 0.655 98.25 ± 0.84b 150.443 ± 2.157a 316.37 ± 3.815b 7.123 ± 0.07a 97.703 ± 0.401a
58.233 ± 0.773a 103.21 ± 0.773a 152.103 ± 3.282a 329.143 ± 3.3a 7.027 ± 0.047a 98.106 ± 0.15a
T0, onset temperature; TP, peak temperature; TC, conclusion temperature; DHgel, enthalpy of gelatinization; PHI, peak height index ((Dgel/(TP T0)); R, gelatinization range (TC T0); S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
Fig. 4. Differential scanning calorimetry (DSC) thermograms of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
endothermic enthalpy of S3 (329.1 J/g) was higher, and directly proportional to the amount of RS, the interaction between amylose–amylose molecules and the content of crystallinity. The enthalpy of S3 was significantly different from S2 (316.3 J/g) and S1 (284.1 J/g). The complete crystallinity of the samples depends on the enthalpy of gelatinization and is an indicator of the loss of molecular order within the granule that occurs on gelatinization (Tester & Morrison, 1990). The difference in R values between the samples suggests a transformation in the crystalline regions of the starch granules. 4. Conclusion Results revealed that the enzymatic hydrolysis of elephant foot yam starch by pullulanase followed by thermal and retrogradation process increases the contents of amylose and the formation of resistant starch with enhanced water solubility, absorption capacity and swelling power properties. The resistant starch III of elephant foot yam when compared with the native starch proved to have a better thermal stability with high crystallinity along with the potential to reduce the viscosity, gel forming ability and hardness of the gel. Reduced viscosity and hardness of the gel is inversely proportional to the amount of resistant starch. The inclusion of many tuber and root starches in food applications is very low due to its poor functional properties. This study has shown that
Acknowledgement This project is financially supported by the Department of Food Science and Technology, Pondicherry Central University, Puducherry, India. References Amani, N. G., Buleon, A., Kamenan, A., & Colonna, P. (2004). Variability in starch physicochemical and functional properties of yam (Dioscorea sp.) cultivated in Ivory Coast. Journal of the Science of Food and Agriculture, 84(15), 2085–2096. Anderson, R. A., Conway, H. F., Pfeifer, V. F., & Griffin, E. (1969). Gelatinisation of corn grits by roll and extrusion cooking. Cereal Science Today, 14, 4–7. Annison, G., & Topping, D. L. (1994). Nutritional role of resistant starch: Chemical structure vs. physiological function. Annual Review of Nutrition, 14, 297–320. Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46(Suppl. 2), 33–50. Gao, Q. Y., Li, S. L., Jian, H. L., & Liang, S. Z. (2011). Preparation and properties of resistant starch from corn starch with enzymes. African Journal of Biotechnology, 10(7), 1186–1193. Gelencser, T., Juhasz, R., Hodsagi, M., Gergely, S., & Salgo, A. (2008). Comparative study of native and resistant starches. Acta Alimentaria, 37(2), 255–270. Govindasamy, S., Campanella, O. H., & Oates, C. G. (1996). High moisture twin-screw extrusion of sago starch: 1. Influence on granule morphology and structure. Carbohydrate Polymers, 30, 215–286. Gunaratne, A., & Hoover, R. (2002). Effect of heat-moisture treatment on the structure and physicochemical properties of tuber and root starches. Carbohydrate Polymers, 49, 425–437. Hoover, R. (2001). Composition, molecular structure, and physicochemical properties of tuber and root starches: A review. Carbohydrate Polymers, 45(3), 253–267. Kaur, L., Singh, J., McCarthy, O. J., & Singh, H. (2007). Physico-chemical, rheological and structural properties of fractionated potato starches. Journal of Food Engineering, 82(3), 383–394. Leong, Y. H., Karim, A. A., & Norziah, M. H. (2007). Effect of pullulanase debranching of sago (Metroxylon sagu) starch at subgelatinization temperature on the yield of resistant starch. Starch-Starke, 59(1), 21–32. Liu, Q., Donner, E., Yin, Y., Huang, R. L., & Fan, M. Z. (2006). The physicochemical properties and in vitro digestibility of selected cereals, tubers and legumes grown in China. Food Chemistry, 99, 470–477. Miao, M., Jiang, B., & Zhang, T. (2009). Effect of pullulanase debranching and recrystallization on structure and digestibility of waxy maize starch. Carbohydrate Polymers, 76(2), 214–221. Nara, S., & Komiya, T. T. (1983). Studies on the relationship between water saturated state and crystallinity by the diffraction method for moistened potato starch. Starch/Starke, 35, 407–410. Nattapulwat, N., Purkkao, N., & Suwithayapan, O. (2009). Preparation and application of carboxymethyl yam (Dioscorea esculenta) starch. AAPS PharmSciTech, 10(1), 193–198. Okaka, J. C., Okorie, P. A., & Ozo, O. N. (1991). Quality evaluation of sun-dried yam chips. Tropical Science, 30, 265–267. Polesi, L. F., & Sarmento, S. B. S. (2011). Structural and physicochemical characterization of RS prepared using hydrolysis and heat treatments of chickpea starch. Starch/Starke, 63, 226–235. Raja, M. K. C., & Sindhu, P. (2000). Properties of steam-treated arrowroot (Maranta arundinacea) starch. Starch-Starke, 52(12), 471–476. Ratnayake, W. S., & Jackson, D. S. (2007). A new insight into the gelatinization process of native starches. Carbohydrate Polymers, 67(4), 511–529. Ravi, V., Ravichandran, C. S., & Suja, G. (2009). Growth and productivity of elephant foot yam (Amorphophallus paeonifolius) (Dennst.Nicolson): An overview. Journal of Root crops, 35, 131–142. Reddy, C. K., Suriya, M., & Haripriya, S. (2013). Physico-chemical and functional properties of resistant starch prepared from red kidney beans (Phaseolus vulgaris L.) starch by enzymatic method. Carbohydrate Polymers, 95(1), 220–226. Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant starch – A review. Comprehensive Reviews in Food Science and Food Safety, 5, 1–17. Shin, S. I., Byun, J., Park, K. H., & Moon, T. W. (2004). Effect of partial acid hydrolysis and heat-moisture treatment on formation of resistant tuber starch. Cereal Chemistry, 81, 194–198. Singh, N., & Kaur, L. (2004). Morphological, thermal, rheological and retrogradation properties of potato starch fractions varying in granule size. Journal of the Science of Food and Agriculture, 84(10), 1241–1252. Tester, R. F., & Morrison, W. R. (1990). Swelling and gelatinization of cereal starches. Cereal Chemistry, 67, 558–563. Thompson, D. B. (2000). Strategies for the manufacture of resistant starch. Trends in Food Science & Technology, 11(7), 245–253.
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Preparation and characterization of resistant starch III from elephant foot yam (Amorphophallus paeonifolius) starch Chagam Koteswara Reddy, Sundaramoorthy Haripriya ⇑, A. Noor Mohamed, M. Suriya Department of Food Science and Technology, Pondicherry Central University, Puducherry 605 014, India
a r t i c l e
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Article history: Received 1 July 2013 Received in revised form 26 November 2013 Accepted 11 January 2014 Available online 23 January 2014 Keywords: Elephant foot yam starch Enzymatic hydrolysis Physico-chemical properties Pullulanase Resistant starch
a b s t r a c t The purpose of this study was to assess the properties of resistant starch (RS) III prepared from elephant foot yam starch using pullulanase enzyme. Native and gelatinized starches were subjected to enzymatic hydrolysis (pullulanase, 40 U/g per 10 h), autoclaved (121 °C/30 min), stored under refrigeration (4 °C/24 h) and then lyophilized. After preparation of resistant starch III, the morphological, physical, chemical and functional properties were assessed. The enzymatic and retrogradation process increased the yield of resistant starch III from starch with a concomitant increase increase in its water absorption capacity and water solubility index. A decrease in swelling power was observed due to the hydrolysis and thermal process. Te reduced pasting properties and hardness of resistant starch III were associated with the disintegration of starch granules due to the thermal process. The viscosity was found to be inversely proportional to the RS content in the sample. The thermal properties of RS increased due to retrogradation and recrystallization (P < 0.05). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction In the food processing industries, starch contributes to important characteristics including thickening, gelling, consistency and shelf stability in a diverse range of diverse applications. Potato, rice and wheat starch have been exclusively employed for this purpose in transforming the microstructure and functionality of products in the food industries (Wheatley, Liping, & Bofu, 1996). Though starch contributes significantly to the quality and consistency of commercial products, it has a high glycemic index which often makes it unfavourable in terms of its effects to the consumer. In order to meet the growing demands of the consumers for functional foods, carbohydrates which can act as functional ingredients and have a beneficial effect to human health are favoured over carbohydrates with a high glycemic index. Resistant starch (RS) is one of the naturally occurring carbohydrate which is defined as the sum of starch and starch degradation products which cannot be digested in the small intestine of humans and when reached in the large intestine, it undergoes fermentation by the commencal Abbreviations: DHgel, enthalpy of gelatinization; DSC, differential scanning calorimetry; PHI, peak height index ; R, gelatinization range; RC, relative crystallinity; RS, resistant starch; SEM, scanning electron microscopy; SP, swelling power; T0, onset temperature; TC, conclusion temperature; TP, peak temperature; TPA, texture profile analyzer; WAC, water absorption capacity; WSI, water solubility index; XRD, X-ray diffractometer. ⇑ Corresponding author. Tel.: +91 9443701906; fax: +91 413 2654621. E-mail address: [email protected] (S. Haripriya). 0308-8146/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2014.01.023
intestinal microorganisms production, resulting in the production of short chain fatty acids (Annison & Topping, 1994). These short chain fatty acids can be partially absorbed in the small intestine and be a source of energy to the mucosal cells or can support the growth and metabolism of the colonic microbiota, with the undigested mass being excreted in the stool (Xue, Newman, & Newman, 1996). Besides its vital physiological role as a functional ingredient in lowering the risk of diet-related diseases, RS when compared with traditional insoluble fibres also has many favourable features for thea food industry. More specifically, RS is a natural white colour powder with bland taste and has acceptable appearance and texture (Sajilata, Singhal, & Kulkarni, 2006). RS is classified on the basis of its botanical source and processing methods (Englyst, Kingman, & Cummings 1992). RS can be classified in four types including RS1, physically inaccessible starch; RS2, native starch granules; RS3, retrograded starch and RS4, chemically modified starch (Sajilata et al., 2006). Sources of cereal grains, roots, tubers and legumes produce resistant starch through the process of cyclic heating, autoclaving and extrusion methods. Gelatinization and cooling processes which are generally referred to as annealing procedures, are the common methods used to enhance the formation of RS3 (retrograded starch) (Thompson, 2000). Ample studies are available on the RS of the cereals, pulses, and tubers, especially cassava and potato starch. One such starch source which is not explored commercially is Amorphophallus paeonifolius, which is also known as elephant foot yam, an herbaceous, perennial C3 crop. It is a tropical tuber which has originated
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from the south eastern Asian region and is extensively used in Indian cuisines (Ravi, Ravichandran, & Suja, 2009). Though several researchers have studied the flour and starch obtained from yam tubers in order to find new food applications (Okaka, Okorie, & Ozo, 1991) a limited number of studies on starch and resistant starch fr A. paeonifolius are available. Taking into account the need for RS3, as this would constitute a functional food ingredient, the study was designed to elucidate the preparation of starch and resistant starch (RS3) from elephant foot yam. The objectives of the study were to prepare RS3 retrograded starch from isolated starch of elephant foot yam (A. paeonifolius) and characterise its physico-chemical and functional properties.
2. Materials and methods 2.1. Materials The tuber of elephant foot yam (A. paeonifolius) was purchased from a local market. The resistant starch assay kit was purchased from Megazyme International (Ireland), whereas the enzymes used in the study included pullulanase from Bacillus acidopullulyticus (Promozyme 400L) and heat stable a-amylase from Bacillus licheniformis (Termamyl 120L), obtained from Sigma (USA).
2.2. Isolation of starch The starch was isolated from elephant foot yam (A. paeonifolius) according to the method of Amani, Buleon, Kamenan, and Colonna (2004). The tuber was peeled, cut into small pieces and immediately suspended in 0.1% (w/v) sodium metabisulphite solution. Then, the sample was homogenised with warring blender and suspended in a large amount of 4% NaCl. The slurry was filtered through a 100 lm sieve and the filtrate was centrifuged at 2660g for 15 min. This procedure was repeated for four times and the recovered white coloured starch was then oven dried at 48 h at 45 °C.
2.3. Preparation of resistant starch 2.3.1. Enzymatic hydrolysis of elephant foot yam starch Enzymatic hydrolysis of elephant foot yam starch was performed by using the procedure used by Polesi and Sarmento (2011) with a slight modification. The elephant foot yam starch (10% w/w db) was suspended in sodium acetate buffer (0.1 M and pH 5.3) and mixed with pullulanase enzyme (40 U/g dry starch), and the mixture incubated in a shaking water bath at 60 °C for 10 h. The sample was heated in a boiling water bath for 10 min to inactivate the enzyme. Starch gelatinization, prior to adding the enzyme, was performed by boiling the sample in a water bath for 10 min.
2.3.2. Preparation of resistant starch Starch samples, namely elephant foot yam starch (S1), native hydrolyzed by enzyme (S2) and gelatinized hydrolyzed by enzyme (S3) in suspensions (10% w/w db) were autoclaved at 121 °C for 30 min, cooled and kept at 4 °C for 24 h. The samples (S1, S2 and S3) were then lyophilized.
2.3.3. Determination of resistant starch content The amount of RS in the samples was analysed by using a Megazyme resistant starch assay kit, which is based on the Association of Official Analytical Chemists (AOAC) methods (2002.02).
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2.4. Scanning electron microscopy (SEM) The morphological characteristics of S1, S2 and S3 were evaluated using scanning electron microscope (HITACHI Model S-3000H) with a magnification 500 to 1500. The powdered samples were sprinkled on double-sided stick tape placed on aluminium stubs and were covered with a gold–palladium layer. 2.5. Physicochemical characteristics 2.5.1. Chemical composition The moisture content of S1 was recorded in terms of weight loss after heating at 130 ± 2 °C for 2 h using 2 g of sample. The amount of ash, protein, and fat were analysed according to AACC methods 08–01, 46–13 and 30–25, respectively. The amount of starch was analysed using the AOAC method 8.020. 2.5.2. Amylose content The total amylose content of the starch samples (S1, S2and S3) was analsyed according to the method described by Williams, Kuzina, and Hlynka (1970). Starch samples (20 mg) were added to 10 mL of 0.5 N KOH and the resultant suspension was thoroughly mixed. Subsequently, the dispersed sample was transferred into a 100 mL volumetric flask and diluted to the mark with distilled water. An aliquot (10 mL) was pipetted into a 50 mL volumetric flask and 5 mL of 0.1 N HCl were added followed by 0.5 mL of iodine reagent, and the solution made up to 50 mL with water; the absorbance was then measured at 625 nm. The measurement of amylose was determined from a standard curve developed using amylose as the standard. 2.5.3. Water absorption capacity (WAC) and water solubility index (WSI) The WAC and WSI of the samples (S1, S2 and S3) were analysed according to the procedure described by Anderson, Conway, Pfeifer, and Griffin (1969). Briefly, a sample of 0.5 g was mixed with 6 mL of distilled water, and centrifuged. The supernatant was heated at 30 °C with continuously stirring for 30 min in a water bath. The suspension was placed in a petridish and dried at 105 °C for 4 h to obtain the dry solids weight, and the wet sediment was weighed. The WSI and WAC were determined as: WSI = (weight of dry solids in supernatant/weight of dry sample) 100; WAC = weight of wet sediment/(weight of the dry sample-weight of the dry solids). 2.5.4. Swelling power (SP) The swelling power of the samples (S1, S2and S3) was anasyed using the method described by Nattapulwat, Purkkao, and Suwithayapan (2009). A sample (0.2 g) was dispersed in water (20 mL) to form a suspension. The suspension was heated to 85 °C in a water bath for 30 min with vigorous shaking every 5 min. The starch gel was then centrifuged at 2200 rpm for 15 min. The weight of the sediment was used to calculate the swelling power. The supernatant was dried and weighed to measure the amount of dissolved starch in the supernatant. The swelling power was determined as: swelling power = weight of sediment/(weight of dry starch weight of dissolved starch). 2.5.5. Pasting properties The viscoamylographic property of the samples (S1, S2 and S3) were performed with a Rapid Visco Analyser (RVA starch master 2, Newport Scientific, Warriewood, NSW, Australia) using 2 g of sample in 25 mL of water. The following parameters: paste temperature, peak viscosity, breakdown viscosity, final viscosity and setback viscosity were measured from the viscoamylographs.
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2.5.6. Textural characteristics The textural properties of RVA gels were studied using the method Kaur, Singh, McCarthy, and Singh (2007) using a texture profile analyzer (HDP/BS blade of texture analyzer (TA) TA–HD plus, Stable Micro Systems, Surrey). After 24 h incubation at 4 °C the gels formed from the RVA analysis were examined by the texture analyzer using probe No. 5. From the texture profile curve, hardness, cohesiveness, gumminess, adhesiveness, springiness, chewiness and stringiness were calculated. 2.5.7. XRD and relative crystallinity (RC) The X-ray patterns of the samples (S1, S2 and S3) were studied with a X-ray diffractometer (Shimadzu XRD 7000) according to the procedure described by Zobel (1964). The RC of the starches was quantitatively estimated following the method of Nara and Komiya (1983) using a software (Origin version 8, Microcal Inc., Northampton, MA, USA). The graphs were plotted between 2h of 4° to 30° and smoothed with the ‘‘Adjacent Averaging’’ tool. 2.5.8. Thermal properties The thermal properties of the samples (S1, S2 and S3) were examined using the method described by Gao, Li, Jian, and Liang (2011) based on differential scanning colorimetry (TA-Q20 DSC). The DSC curve which showed the onset (T0), peak (Tp), final (Tf), gelatinization temperatures and enthalpy (DH) were analysed using Universal Analysis 2000 3.9A software. 2.6. Statistical analysis The experiments were conducted in triplicate. The obtained data were submitted to one way ANOVA and Duncan’s Multiple Range Test (DMRT) (P 6 0.05) to analyse the significance of difference between the mean values of the samples using SPSS 18 software (SPSS Institute Inc., Cary, NC, USA). 3. Results and discussion 3.1. Morphological characteristics The SEM micrographs (Fig. 1) of S1 when compared to S2 and S3 differed significantly. SEM studies revealed that the elephant foot yam starch (S1) granules are round, elliptical and polygonal in shape with smooth surfaces, with no obvious effects or signs of damage on the surface. However, the granular structure of samples was destroyed after being cooked, debranched and dried. S2 and S3 showed a cohesive structure, leading to the loss of granular appearance and to a irregular shape, which is the consequence of gelatinization temperature where the coupled starch granules forms sponge like structure within the inner region of the retrograded starch (Ratnayake & Jackson, 2007). 3.2. Chemical composition The proximate composition in terms of moisture, ash, protein and fat in the isolated elephant foot yam starch (S1) was 8.5%, 2.3%, 0.9% and 0.2% respectively. The moisture content of S1 (8.5%) was comparatively lower when compared with cassava starch (10.2%) and arrow root starch (9.82%). Moisture content plays a significant role for flow and other mechanical properties (Raja & Sindhu, 2000). The moisture content of a starch powder is dependent upon the extraction procedure and drying process along with surrounding atmosphere humidity. The starch and amylose content in the extracted starch were 87.7% and 24.2%, respectively, on a dry weight basis. These parameters were compa-
rable with the proximate composition, and the starch and amylose content of other yam varieties (Wang, Yu, Liu, & Chen, 2008). 3.3. Physicochemical properties The amylose, RS content, SP, WAC and WSI of S1, S2 and S3 are shown in Table 1. The amylose content of S1 (24.21%) was found to be significantly lower than S2 (46.78%) and S3 (50.18%). Furthermore, S3 (50.18%) was found to be significantly greater than S2 (46.78%). The RS content of S3 (36.27%) was significantly higher when compared with S2 (30.67%) and S1 (17.98%) owing to the increased amylose content in samples S2 and S3 which are the resultant retrograded starch of elephant foot yam starch upon the treatment with pullulanase enzyme before and after gelatinization, respectively. The considerable increase of RS content in S3 followed by S2 would be attributed to the effect of pullulanase enzyme on debranching the a-(1–6) linkage of amylopectin (Leong, Karim, & Norziah, 2007); the latter is converted into small chain linear polysaccharides like amylose molecules, which form strong gel network through retrogradation (Polesi and Sarmento, 2011). The RS formed in S2 and S3 were subjected to retrogradation which ensures the conversion of RS2–RS3. The swelling power measures the tenacity of the bonds in the crystalline portion of the starch granule, showing the ease with which the starch will cook. Granules with greater crystalline areas along with stronger bonds in the crystalline regions swell less in cold water as well as when heated. The gels formed from these crystalline regions are weak and have greater tendency towards retrogradation because of the bonds. The swelling power of elephant foot yam starch at 85 °C (8.14%) was significantly differed from the swelling power of S2 at 85 °C (5.6%) and that of S3 at 85 °C (5.4%). The swelling power ascertains the magnitude of interaction among the starch chain within the regions of crystalline and amorphous nature. The distribution of molecular weight, degree of debranching, length of branches and the conformation of the molecules along with the ratio of amylose and amylopectin places a very important influence on the extent of water and starch interaction (Hoover, 2001). The observation of the decreased SP in S2 and S3, irrespective to the increase in temperature, could be attributed to the effect of gelatinization and the autoclaving process, which would have occurred during the preparation of S2 and S3. The properties of WAC and WSI of all starch samples were greatly dependent on the source, content of amylose/amylopectin, extraction procedure and thermal stability. WAC is a phenomenon linked to the wet heat treatment of starch samples. In this study, the gelatinization induced by heating and autoclaving significantly increased the WAC in S3 (6.04%) when compared to S1 (3.59%) and S2 (5.46%). The WAC of all the starch samples was comparable with other studies of RS from chick pea starch (Polesi and Sarmento, 2011). An increased water activity in retrograded starch was noted which is the result of a change in molecular structure or any other mechanisms leading to an easier mobility of the starch components, where leaching of starch was also noticed (Govindasamy, Campanella, & Oates, 1996). The parameter WSI is used as an indicator of the destruction of starch components. The WSI of S3 (13.89%) and S2 (12.06%) was significantly different from S1 (2.57%). The S2 and S3 have undergone an hydrolytic process when compared to S1. The enzymatic hydrolysis contributed to the increased water solubility in S2 and S3. 3.4. Pasting properties The pasting properties of all samples were determined using a rapid visco analyzer. The pasting property plays a vital role in the application of starch and resistant starch in food industries and these parameters are depended on the source, amount of
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Fig. 1. Scanning electron micrographs (SEM) of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
Table 1 Amylose, resistant starch, swelling power, water absorption capacity, water solubility index and relative crystallinity of S1, S2 and S3.# Parameter
Amylose (%) RS (%) SP (%) WAC (%) WSI (%) RC (%)
Type S1
S2
S3
24.21 ± 0.983c 17.98 ± 0.382c 8.14 ± 0.201a 3.59 ± 0.176c 2.57 ± 0.110b 19.37 ± 0.633c
46.78 ± 1.454b 30.67 ± 1.632b 5.67 ± 0.108b 5.46 ± 0.415a 12.06 ± 1.010a 25.12 ± 0.949b
50.18 ± 1.171a 36.27 ± 1.167a 5.48 ± 0.168b 6.04 ± 0.185a 13.89 ± 1.249a 27.84 ± 1.201a
RS, resistant starch; SP, swelling power; WAC, water absorption capacity; WSI, water solubility index; RC, relative crystallinity; S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
starch, and the interaction between molecules and testing conditions (Liu, Donner, Yin, Huang, & Fan, 2006). In the presence of water, the starch suspension was subjected to a thermal process, where the starch granules swell and thus the viscosity increases progressively. The viscoamylographs of S1, S2 and S3 are shown in Fig. 2 and Table 2. The pasting properties of S1 showed a significant difference with S2 and S3 (P < 0.05) which represents the typical pasting behaviour of native starch (Miao, Jiang, & Zhang, 2009). The pasting temperature was not detectable for S3 and the low pasting temperature for S2 (61.2 °C) could be due to the destruction of starch granules when subjected to autoclave at 121 °C during RS preparation which is also comparable with other studies like the RS from chickpea and red kidney bean starch (Polesi and Sarmento, 2011; Reddy, Suriya, & Haripriya, 2013). S2 and S3 show a lower amylographic pattern for parameters including peak viscosity, hold viscosity, final viscosity, break down and set back when compared with S1. This attribute could be a result of the
Fig. 2. Typical RVA starch pasting curves for of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
enzymatic hydrolysis process which improves the levels of short linear chain molecules and RS. The formation of starch gel ability was reduced in S2 and S3 when compared with S1 due to ithets autoclave process (Polesi and Sarmento, 2011; Gelenscer et al., 2008). 3.5. Textural properties After 24 h incubation at 4 °C, the retrograded starch pastes (RVA gels) of S1, S2 and S3 were examined with a texture profile analyzer and the obtained data are shown in Table 3. Higher peak viscosity and hardness was observed in S1 when compared with S2 and S3 (P < 0.05), which may be due to the presence of larger size starch granules and lower levels of amylose. The reduced hardness
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Table 2 Pasting properties of S1, S2 and S3: pasting temperature (°C), peak time (min), peak viscosity (cP), hold viscosity (cP), final viscosity (cP), break down (cP) and set back (cP).# Parameter
Pasting temp (°C) Peak time (min) Peak viscosity (cP) Hold viscosity (cP) Final viscosity (cP) Breakdown (cP) Setback (cP)
Table 3 Textural properties of S1, S2 and S3: hardness, cohesiveness, adhesiveness, gumminess, springiness, chewiness and stringiness.# Parameter
Type
Type S1
S1
S2
S3
89 ± 0.7a 4.42 ± 0.5c 1320.0 ± 31.320a 1248.33 ± 56.888a 1819.33 ± 30.171a 122.33 ± 4.725a 626.0 ± 7.151a
61.2 ± 0.5b 7.10 ± 0.33a 435.0 ± 55.865b 418.66 ± 29.022b 598.33 ± 60.351b 16.33 ± 1.527b 131.66 ± 3.511b
nd 5.30 ± 0.61b 238.0 ± 16.370c 228.0 ± 11.532c 275.0 ± 13.258c 13.33 ± 1.527c 42.0 ± 3.0c
S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch; nd, not detected. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
and the formation of gel ability of S2 and S3 is due to the partial hydrolysis of starch granules and also the destruction of granules by enzymatic hydrolysis and the autoclave process. The results elicited significant difference between all samples (S1, S2 and S3) with respect to the texture parameters including hardness, cohesiveness, adhesiveness, gumminess, springiness, chewiness and stringiness. The difference in the textural properties of all sample gels were influenced by rigidity in gelatinized starch, amylose content as well as interaction between the dispensed and continuous phase of the gel, which in turn is dependent on the amylose and amylopectin structure (Yamin, Lee, Pollak, & White, 1999). 3.6. X-ray diffraction The X-ray diffraction patterns of samples (S1, S2 and S3) are shown in Fig. 3. S1 gave an intermediate intensity peak at diffraction angles of 2h = 17.92° and a strong peak at 2h = 23.05°, with a weak peak at 14.7° showing an intermediate diffraction pattern A- and B-type (C-type). The S2 and S3 exhibited peaks at 16.85° and 16.95°, respectively, indicating a B-type crystallinity due to the retrogradation at low temperature. Similar crystalline patterns were also observed in wheat, corn and sago starch (Leong et al., 2007) when treated with pullulanase, and were autoclaved and
Hardness (N) Cohesiveness Adhesiveness (Ns) Gumminess (N) Springiness (s) Chewiness (Ns) Stringiness
0.506 ± 0.051a 0.323 ± 0.041b 13.04 ± 0.632b 0.17 ± 0.01a 1.14 ± 0.026a 0.127 ± 0.092a 5.973 ± 0.935a
S2 0.113 ± 0.005b 0.523 ± 0.035a 3.963 ± 0.241a 0.046 ± 0.011b 0.876 ± 0.096b 0.04 ± 0.01a 6.17 ± 0.238a
S3 0.093 ± 0.005b 0.536 ± 0.025a 4.04 ± 0.07a 0.046 ± 0.005b 0.903 ± 0.06b 0.0366 ± 0.005a 5.476 ± 0.434a
S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch; nd, not detected. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
retrograded. The type of crystallinity of samples is influenced by the chain length of amylopectin, growth temperature and fatty acids content (Gunaratne & Hoover, 2002). S3 gave a single broad peak when compared with S2 due to the recrystallization with retrogradation. The relative crystallinity of S1 (19.37%) and S2 (25.12%) were lower when compared with that of S3 (27.84%). The highest crystallinity of S3 could be due to the increased RS content, which could have been enhanced by gelatinization, enzymatic treatment and retrogradation (Table 1).
3.7. Thermal properties Generally, the endothermic transition of all types of starch samples are influenced by the interactions between amylose–amylose, amylose–amylopectin and the amylose–lipid contents (Shin, Byun, Park, & Moon, 2004). However in S1 there were no considerable levels of lipid present. The thermal properties of S1, S2 and S3 were influenced by the granule shape, amylopectin chain length and crystalline regions (Singh & Kaur, 2004). Thermal analyses of S1, S2 and S3 were performed by DSC and the data are shown in Table 4 and Fig. 4. The DSC profiles of the samples showed a low peak temperature for S1 and S2 when compared with S3, which is attributed to the gelatinization and retrogradation process. The
Fig. 3. X-ray diffraction pattern (XRD) of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
C.K. Reddy et al. / Food Chemistry 155 (2014) 38–44 Table 4 Thermal properties of S1, S2 and S3: transition temperatures (T0; TP; TC), enthalpy of gelatinization (DHgel), peak height index (PHI) and gelatinization range (R).# Parameter
Type S1
T0 (°C) TP (°C) TC (°C) DHgel (J/g) PHI (J/g C) R (°C)
53.073 ± 1.471b 93.66 ± 2.094c 149.603 ± 1.316a 284.14 ± 5.65c 6.51 ± 0.17b 96.606 ± 1.272a
S2
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resistant starch III of elephant foot yam has improved functional properties resulting from the enzymatic hydrolysis, thermal and retrogradation processes, and can potentially used as a substitute of native starches used currently in the food industry.
S3 b
54.993 ± 0.655 98.25 ± 0.84b 150.443 ± 2.157a 316.37 ± 3.815b 7.123 ± 0.07a 97.703 ± 0.401a
58.233 ± 0.773a 103.21 ± 0.773a 152.103 ± 3.282a 329.143 ± 3.3a 7.027 ± 0.047a 98.106 ± 0.15a
T0, onset temperature; TP, peak temperature; TC, conclusion temperature; DHgel, enthalpy of gelatinization; PHI, peak height index ((Dgel/(TP T0)); R, gelatinization range (TC T0); S1, elephant foot yam starch; S2, retrograded enzyme hydrolyzed native starch; S3, retrograded enzyme hydrolyzed gelatinized starch. # All data were means of triplicates. Values with the same superscripts in a row did not differ significantly (P < 0.05) by DMRT.
Fig. 4. Differential scanning calorimetry (DSC) thermograms of S1 (elephant foot yam starch), S2 (retrograded enzyme hydrolyzed native starch) and S3 (retrograded enzyme hydrolyzed gelatinized starch).
endothermic enthalpy of S3 (329.1 J/g) was higher, and directly proportional to the amount of RS, the interaction between amylose–amylose molecules and the content of crystallinity. The enthalpy of S3 was significantly different from S2 (316.3 J/g) and S1 (284.1 J/g). The complete crystallinity of the samples depends on the enthalpy of gelatinization and is an indicator of the loss of molecular order within the granule that occurs on gelatinization (Tester & Morrison, 1990). The difference in R values between the samples suggests a transformation in the crystalline regions of the starch granules. 4. Conclusion Results revealed that the enzymatic hydrolysis of elephant foot yam starch by pullulanase followed by thermal and retrogradation process increases the contents of amylose and the formation of resistant starch with enhanced water solubility, absorption capacity and swelling power properties. The resistant starch III of elephant foot yam when compared with the native starch proved to have a better thermal stability with high crystallinity along with the potential to reduce the viscosity, gel forming ability and hardness of the gel. Reduced viscosity and hardness of the gel is inversely proportional to the amount of resistant starch. The inclusion of many tuber and root starches in food applications is very low due to its poor functional properties. This study has shown that
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