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Pattern of Enzyme Hydrolysis in Raw Sago Starch: Effects of Processing History
Abstract
Hydrolysis patterns of five batches of sago starch were studied by using Novo Nordisk and Sigma α-amylases and glucoamylases. Native sago starch was a poor substrate to the enzymes and the hydrolysis patterns were surface erosion, pitting and crevassing. After incubation with pH 3·5 acetate buffer at 60 °C for 2 h, the hydrolysis pattern was different: a single deep round hole developed regardless of the batch or enzyme(s) used. This step also significantly increased the degree of hydrolysis. Granule size distribution results indicated that at about 67% hydrolysis, treated granule residues were the same mean size as native granules while untreated granule residues had two major size populations. DSC results suggested that amorphous regions of the untreated granule were preferentially hydrolysed, however, upon pretreatment regions within the granule were more uniform towards enzymes' action.
... Sago starch granules have a broad size range, between 10 and 50 µm in diameter with an average granule diameter of 32 µm (Wang and others 1995). Sago starch granules are generally bigger than those of rice (3 to 10 µm), corn (5 to 20 µm), wheat ...
... Sago starch digestibility in the granular state has been studied by several researchers (Monma and others 1989; Haska and Ohta 1991; Gorinstein and others 1994; Wang and others 1995; Oates 1997). Haska and Ohta (1992) found that sago starch is resistant to enzymes compared to cereal starches. ...
... Combination of α-amylase and glucoamylase could more effectively hydrolyze the raw sago, rice, corn, and potato starches (Monma and others 1989). Starch pretreatment at lower pH and heating below gelatinization temperature were found to significantly increase the degree of hydrolysis (Haska and Ohta 1991; Wang and others 1995). Before treatment , amorphous regions of the granules were preferentially hydrolyzed . ...
The common industrial starches are typically derived from cereals (corn, wheat, rice, sorghum), tubers (potato, sweet potato), roots (cassava), and legumes (mung bean, green pea). Sago (Metroxylon sagu Rottb.) starch is perhaps the only example of commercial starch derived from another source, the stem of palm (sago palm). Sago palm has the ability to thrive in the harsh swampy peat environment of certain areas. It is estimated that there are about 2 million ha of natural sago palm forests and about 0.14 million ha of planted sago palm at present, out of a total swamp area of about 20 million ha in Asia and the Pacific Region, most of which are under- or nonutilized. Growing in a suitable environment with organized farming practices, sago palm could have a yield potential of up to 25 tons of starch per hectare per year. Sago starch yield per unit area could be about 3 to 4 times higher than that of rice, corn, or wheat, and about 17 times higher than that of cassava. Compared to the common industrial starches, however, sago starch has been somewhat neglected and relatively less attention has been devoted to the sago palm and its starch. Nevertheless, a number of studies have been published covering various aspects of sago starch such as molecular structure, physicochemical and functional properties, chemical/physical modifications, and quality issues. This article is intended to piece together the accumulated knowledge and highlight some pertinent information related to sago palm and sago starch studies.
... However, the raw sago starch exists as large granules with compact crystalline structure. As a result, the enzyme reaction rate and yield of products from raw sago starch was reported to be too low for industrial application (Wang et al., 1995). Sakano et al. (1986) and Takao et al. (1986) reported that bioconversion of sago starch was limited by the resistance of the raw granule to enzymatic hydrolysis. ...
... Degree of hydrolysis was low when the enzyme concentrations were less than 100 units/mL. Similar results were reported by Wang et al. (1995Wang et al. ( , 1996Wang et al. ( , 1997 and Govindasamy et al. (1995) using commercial α-amylase and glucoamylase. ...
The native sago starch exists as a compact crystalline structure and is not efficiently hydrolyzed by Raw Starch Degrading Enzyme (RSDE). In order to enhance its hydrolysability, the starch was treated with acid and heated below its gelatinization temperature, thus increasing the accessibility of the sago starch granule to enzymatic attack. Results showed that treatment of sago starch with acid at pH 2.0 and temperature 65°C for 2 hours greatly enhanced its conversion rate to glucose from 53.3% to 71.9%. It is clearly shown that high yield of glucose is produced during hydrolysis of acid-treated sago starch using the Raw Starch Degrading Enzyme from Acremonium sp. The difference between the acid-treated and untreated sago starch in this study could be due to the differences on the surface of the sago starch granule which may influence the accessibility and diffusion of enzyme into the starch during hydrolysis.
... However, the raw sago starch exists as large granules with compact crystalline structure. As a result, the enzyme reaction rate and yield of products from raw sago starch was reported to be too low for industrial application (Wang et al., 1995). Sakano et al. (1986) and Takao et al. (1986) reported that bioconversion of sago starch was limited by the resistance of the raw granule to enzymatic hydrolysis. ...
... Degree of hydrolysis was low when the enzyme concentrations were less than 100 units/mL. Similar results were reported by Wang et al. (1995Wang et al. ( , 1996Wang et al. ( , 1997 and Govindasamy et al. (1995) using commercial ±-amylase and glucoamylase. ...
The native sago starch exists as a compact crystalline structure and is not efficiently hydrolyzed by Raw Starch Degrading Enzyme (RSDE). In order to enhance its hydrolysability, the starch was treated with acid and heated below its gelatinization temperature, thus increasing the accessibility of the sago starch granule to enzymatic attack. Results showed that treatment of sago starch with acid at pH 2.0 and temperature 65 o C for 2 hours greatly enhanced its conversion rate to glucose from 53.3% to 71.9%. It is clearly shown that high yield of glucose is produced during hydrolysis of acid-treated sago starch using the Raw Starch Degrading Enzyme from Acremonium sp. The difference between the acid-treated and untreated sago starch in this study could be due to the differences on the surface of the sago starch granule which may influence the accessibility and diffusion of enzyme into the starch during hydrolysis.
... In hydrolysis, starch is converted into fermentable sugars, which is carried out in three stages: gelatinization, liquefaction, and saccharification [16]. In gelatinization, heating in the presence of water enhances chemical reactivity toward hydrolytic enzymes due to converting the starch structure from semicrystalline to amorphous conformation [17,18]. The liquefaction step usually occurs the partial starch hydrolysis by adding α-amylase at a temperature around 86-90 °C; gelatinization can occur at higher temperatures than liquefaction or is generally carried out simultaneously with liquefaction [5,7]. ...
The biorefinery using wasted sweet potatoes is an attractive way to replace fossil fuels and integrate other products. This work aimed to produce sweet potato ethanol using conventional commercial amylolytic enzymes usually applied at 60–90 °C, but at a low temperature (28–42 °C). Additionally, simultaneous hydrolysis and fermentation were performed. Enzyme concentration in the hydrolysis, temperature, time, and concentration of the potassium metabisulfite solution in the fermentation yield was also studied through central composite design (CCD). Moreover, the effect of alpha-amylase addition before and after (before cooling) the pretreatment of sweet potato was evaluated. The sweet potato was characterized by moisture and total reducing sugars. When the enzymes are added simultaneously, the optimal conditions are 35 °C and 25.1 h for a minimum yield of 75 %. When alpha-amylase is added just after heating, the yield is 79.7 % in only 22 h of incubation, achieving a gain of 8.6 h. Thus, this work shows that it is possible to use enzymes generally used at higher temperatures at lower temperatures, which can help reduce the energy costs of sweet potato ethanol production.
... The degree of hydrolysis (DH) was defined by the following equation (Wang et al. 1995): ...
In most tropical countries, carbohydrate-based agricultural products occur in large quantities. Wider utilization of local starch crops will offer various economic and ecological advantages. Local starch crops can be a catalyst for rural industrial development and eventually open up new markets. The potential glucose yield from the typical Indonesian starches (edible canna, arrowroot, sago, and sweet potato) using lower dosage of cold-starch hydrolyzing enzyme preparation Stargen™ 002 was compared with that of corn and potato. The glucose equivalent yield reached 88.4 g/L and 86.3 g/L after 24 h of hydrolysis when 40% (w/v) raw sago and sweet potato starches were used, respectively, as compared to corn starch (89.6 g/L). While arrowroot and edible canna gave much lower amounts (53.3 g/L and 39.7 g/L, respectively). This study demonstrates that sago and sweet potato starches may provide interesting alternatives to corn starch in a cold hydrolysis conversion process of starch into glucose.
... Only α-amylase, dextrose amylase, and a few amylases have been found to act directly on raw starches, and they are called raw amylases. Hydrolases include exo-amylase and endo-amylase; the hydrolytic performance of exo-amylase is better than that by endo-amylase [16]. Although pullulanase or isoamylase alone cannot hydrolyze starch, they can accelerate hydrolysis when they work synergistically with glucose amylase. ...
As the second largest production material, starch has important value in textile, food, chemical and other fields. The shortcomings of natural starch can be solved, and its properties can be improved by modifying its structure, developing original properties, or introducing new functions, making it more suitable for certain application requirements. At present, the methods of starch modification mainly include chemical, physical, and enzymatic modification. In comparison with the two traditional modification methods (chemical and physical modification) mentioned above, enzymatic modification has the advantages of mild conditions, high substrate selectivity, and high product safety, and it is the most ideal green modification method. In this paper, we present an overview of the modified starch by enzymatic structure design. The modification process and mechanism for granule starch and gelatinized starch are summarized. Further, the difficulties encountered in starch modification by enzymatic modification were also analyzed. These analyses could pave a way for understanding and broadening the preparation and applications of modified starch, and provide theoretical references for the utilization of amylase in starch modification.
... Compared to corn and tuber starches, hydrolysis of a-amylase on sago starch granule produces a different type of pore structure. Instead of having well-distributed pores or honeycomb-like-structure, sago starch ends up with single deep round cavity structure as a result of enzyme hydrolysis (Wang, Powell, and Oates 1995;Cui and Oates 1999;Yang et al. 2010). According to Cui and Oates (1999), this deep round cavity is formed from the complete digestion of the granule's inner part. ...
Starch is a complex carbohydrate formed by the repeating units of glucose structure connected by the alpha-glycosidic linkages. Starch is classified according to their derivatives such as cereals,legumes, tubers, palms, fruits, and stems. For decades, native starch has been widely utilized in various applications such as a thickener, stabilizer, binder, and coating agent. However, starches need to be modified to enhance their properties and to make them more functional in a wide range of applications. Porous starch is a modified starch product which has attracted interest of late. It consists of abundant pores that are distributed on the granule surface without compromis�ing the integrity of its granular structure. Porous starch can be produced either by enzymatic, chemical, and physical methods or a combination thereof. The type of starch and selection of the modification method highly influence the formation of pore structure. By carefully choosing a suit�able starch and modification method, the desired morphology of porous starch can be produced and applied accordingly for its intended application. Innovations and technologies related to starch modification methods have evolved over the years in terms of the structure, properties and
modification effects of different starch varieties. Therefore, this article reviews recent modification methods in developing porous starch from various origins.
... Furthermore, the surface is smooth with no cracks or holes. Wang et al. [16] suggests that the presence of holes or cracks in the surface may increase the granule's susceptibility to enzymatic attack. Therefore, it is possible to suggest that cassava starch has its susceptibility to enzymatic attack reduced due to the absence of these holes and cracks on its surface. ...
Barley malt was used as a source of amylases for the hydrolysis of cassava starch to produce reducing sugars for the alcoholic fermentation. Two routes of hydrolysis were evaluated in this work. One using milled barley malt and the other using the enzyme extract of this grain. The first one evaluated three concentrations of milled barley malt: 5, 10 and 15% (w/w) and there was no significant difference between the values of reducing sugars obtained as a function of the three concentrations. Three concentrations were also tested for barley malt extract: 0.5, 1.0 and 1.5 mL of extract. The higher content of reducing sugars was found for the 0.5 mL concentration of extract. The barley malt extract was more efficient in the enzymatic hydrolysis of cassava starch due to a better contact of the enzymes with substrate. The alcoholic fermentation of the wort obtained with 0.5 mL yielded an ethanol content of 7.74 ± 3.19 g/L with an efficiency of 88.6%. DOI: http://dx.doi.org/10.17807/orbital.v13i3.1525
... Comparatively, starch granules eroded with a rougher surface with more pinholes formed during germination were observed in HHP-pretreated GBR grains (Fig. 4), illustrating that the high pressure processing prior to soaking made the morphology of starch granules more easily influenced during germination. As a result, the highly eroded surface led to the higher values of in vitro digestibility, because porous structure facilitated the accessibility of hydrolytic enzymes to starch by altering the pattern of hydrolysis behavior from surface to internal erosion (Wang, Powell, & Oates, 1995). ...
This investigation aims to evaluate the effects of high hydrostatic pressure (HHP) applied prior to germination on functionality and quality of wholegrain-germinated brown rice (GBR). Wholegrain brown rice (WBR) were firstly stressed by HHP treatments (50-350 MPa/20 min), and then incubated at 37 oC to obtain GBR grains after a 2-day soaking period. Gama-aminobutyric acid contents significantly depended on the pressure applied, showing 25% increment in 50 MPa-stressed grains compared to the control. HHP shock led to significantly improved in vitro starch digestibility, which was related to the transformation of crystalline starch granules into amorphous form as consistently revealed by scanning electron microscope imaging and Fourier-transform infrared spectroscopy. HHP-pretreated samples achieved markedly enhanced storability by influencing kinetic curves of lipid hydrolysis and oxidation. These results suggested that metabolic response to HHP during germination could significantly improve functional and quality characteristics of WBR products.
... Nevertheless, AM, CGTase and BE treated starches offered great resistance to enzymatic hydrolysis. Similar effects were reported when corn starch was treated with AMG and CGTase for 24 h [13,14], although opposite results have been observed with AM treated starch subjected to longer production time that intensified changes in the crystalline areas [21]. ...
Studies on porous starch have been directed to explore different industrial applications as bio-adsorbents of a variety of compounds. However, the analysis of starch digestibility is essential for food application. The objective of this study was to determine the impact of porous structure on in vitro starch digestibility. Porous starches were obtained using a range of concentrations of amyloglucosidase (AMG), α-amylase (AM), cyclodextrin-glycosyltransferase (CGTase) or branching enzyme (BE). Porous starches exhibited major content of digestible starch (DS) that increased with the intensity of the enzymatic treatment, and very low amount of resistant starch (RS). Porous starches behaved differently during in vitro hydrolysis depending on their enzymatic treatment. AMG was the unique treatment that increased the digestive amylolysis and estimated glycemic index, whereas AM, CGTase and BE reduced them. A significant relationship was found between the pore size and the severity of the amylolysis, suggesting that a specific pore size is required for the accessibility of the digestive amylase. Therefore, pore size in the starch surface was a limiting factor for digestion of starch granules.
... Conversion of starch to glucose, maltose and dextrin, involves gelatinization, liquefaction and sacharification process [4], which is achieved by either acid hydrolysis or enzymatic process, but dilute acid and high temperature corroded the equipment and it causes undesirable products limited yield and was costly. The use of enzyme has more advantages [5], but high temperature liquid phase enzymatic hydrolysis is used widely. ...
Conversion of raw starch by amylase is important in a way that some of the products could be used as industrial raw materials for value added products. This will reduce wastage and improve economic gain. This study was performed to isolate raw starch digesting microorganism from soil. This begins with the sample collection screened for amylase producers by observing the halo zone appeared around the colonies. Microorganism was characterized by saline wet mount and LPCB staining technique was Aspergillus carbonarius S-CSR-0002, gave the amylase yield (880 U/ml) in submerged fermentation process. Amylase from Aspergillus carbonarius S-CSR-0002, a fungus isolated from soil contaminated with canteen kitchen waste showed an ability to degrade tapioca, corn, arrow root, rice starches. Tapioca has the highest degree of hydrolysis followed by corn, rice, arrow root, consecutively. The crude amylase preparation had temperature and pH optimal activities at 30 °C and 7.0 respectively, optimal stabilities at 30 °C and 7.5 respectively. The optimum substrate concentration was 5%. The highest adsorption of crude enzyme was found with rice starch followed by arrow root, corn and tapioca. Crude enzyme was partially purified by 20-100% ammonium sulphate precipitation followed by dialysis, in which maximum amylolytic activity shown by 20% fraction was 1850 U/ml.
... Hence, diverging part of sago crop to ethanol production does not only help in reducing the fuel price but also maintaining the price of sago derived products [10]. Furthermore, it will also provide agricultural sustainability for sago palm since it is fully utilized not only for food, but also energy consumption [11]. ...
Two stage processes prior to ethanol production has been proposed which is hydrolysis and fermentation. Commercial enzymes were used in this two steps hydrolysis; α-amylase (liquefaction step) and glucoamylase (saccharification step). Optimization was carried out in both stages; hydrolysis and fermentation. Three parameters are involved in optimization of % dextrose equivalent (DE); sago starch concentration (20% (w/v), 30% (w/v), 40% (w/v)), glucoamylase enzyme (52 U/g, 78 U/g, 104 U/g) and time during saccharification (1, 2, 3 hours). Three parameters are involved in optimization of ethanol; agitation (100 rpm, 150 rpm, 200 rpm), inoculums (1% (v/v), 3% (v/v), 5% (v/v)) from constant initial stock of 2.5x10 6 cells/mL and pH (4, 5, 6). Both optimization studies were carried out using Box-Behnken design. The experiment showed that the optimum parameters for hydrolysis study was identified to be glucoamylase (75.87 U/g), substrate concentration (28.49% (w/v)), and time (2 hours) which produced 62.15 g/L glucose as the fermentation substrate. For ethanol fermentation study, it was identified that the optimum parameters that produced 29.25 g/L were 167 rpm agitation, 3.43% (v/v) inoculums and pH 5.
... Samples were incubated at 35°C with constant shaking. Degree of hydrolysis was determined as outlined by Wang et al. (1995). ...
Two strains of Vibrio parahaemolyticus, ATCC 17802 Kanagawa negative (K-) and ATCC 27519, Kanagawa positive (K+) were used in this study. The effect of temperature on the production of hemolysin was studied in trypticase soy broth (TSB) with 3 % NaCl. Maximum hemolysin productions by both strains were obtained at 35°C with little or no production of hemolysin at 4 °, 10° and 42°C. The K+ strain consistently produced higher titers of hemolytic activity than its K- counterpart. Hemolysin production at lower temperatures (4° and 10°C) could only be detected after 10 or 12 h in TSB with 3 % NaCl. At 20 o, 25°, 30° and 35°C in this medium, production of hemolysin could be detected after 6 or 8 h of incubation. At these same temperatures, both K+ and K- strains produced comparable cell numbers by 12 h; however, the greatest hemolysin production occurred at 35 °C. This indicates that temperature, rather than cell numbers, determines the total hemolysin produced during a controlled incubation time period.
... Samples were incubated at 35°C with constant shaking. Degree of hydrolysis was determined as outlined by Wang et al. (1995). ...
The studies at Suwan Farm during 1997-98 and 1998-99 were set up to show exp.2 with the application of neem extract integrated with cotton resistant variety lines as the insect control method to give better yields than those obtained in exp.1 using resistant plant as principal control alone. AP1 and AP2, the mutant lines possessing antibiosis mechanism against some major insect pests of cotton and SR60, the recommended variety were used in the test. It was found that the average amount of jassids on the mutants and SR60 in exp.1 did not significantly differ from those in exp.2 while there are significant differences between the numbers of bollworm and cotton plant bug on each variety/line of both tests. The comparison of average seed yields also revealed highly significant increase of seed weight in AP1 and AP2 in exp.2 whereas the increase in SR60 was not that high. Concerning fiber quality parameters, fiber length of all variety/line in both experiments were in medium group with their uniformities and micronaires to be very high and desirable respectively.
... The number and the position of the subsites are unique for each type of amylase (Meagher et al., 1989). The hydrolysis occurs layer by layer with an attacked layer of granule being completely hydrolyzed (Wang et al., 1995). Besides that, a structural support which is known as the specialized starch granule-binding domains has been identified to exist in some amylases and glucoamylases. ...
Native granular starches (corn, cassava, mung bean, and sago) were hydrolyzed using a mixture of alpha-amylase and glucoamylase at 35°C for 24h. Hydrolyzed starches were analyzed for the degree of hydrolysis and for physicochemical and functional properties. Corn starch showed the highest degree of hydrolysis, as evidenced by the presence of distinct pores penetrating deep into the granules. Enzymatic erosion occurred mainly at the surface for cassava, whereas isolated porous structures were observed in hydrolyzed mung bean and sago starch. The amylose content was significantly lower in all starches except for sago starch. The powder X-ray diffraction of all starches showed no significant changes after hydrolysis, but hydrolyzed starches showed a more crystalline nature. The action of enzymes caused significant changes in some pasting properties and in the swelling/solubility of starches. Evidently, enzymes were able to hydrolyze granular starches to a variable degree at sub-gelatinization temperature, and produced a relatively high degree of conversion.
... According to Chew and Shim (1993), microscopic examination revealed a large number of starch granules to be trapped within the lignocellulosic matrix of sago hampas [22]. e sago starch granules were either pear or cigar shaped and had a generally smooth outer surface with some shallow indentations whereas the size distribution was in a narrow range of 10-50 m with a mean size of 32 m [23]. ...
Lower concentration of glucose was often obtained from enzymatic hydrolysis process of agricultural residue due to complexity of the biomass structure and properties. High substrate load feed into the hydrolysis system might solve this problem but has several other drawbacks such as low rate of reaction. In the present study, we have attempted to enhance glucose recovery from agricultural waste, namely, "sago hampas," through three cycles of enzymatic hydrolysis process. The substrate load at 7% (w/v) was seen to be suitable for the hydrolysis process with respect to the gelatinization reaction as well as sufficient mixture of the suspension for saccharification process. However, this study was focused on hydrolyzing starch of sago hampas, and thus to enhance concentration of glucose from 7% substrate load would be impossible. Thus, an alternative method termed as cycles I, II, and III which involved reusing the hydrolysate for subsequent enzymatic hydrolysis process was introduced. Greater improvement of glucose concentration (138.45 g/L) and better conversion yield (52.72%) were achieved with the completion of three cycles of hydrolysis. In comparison, cycle I and cycle II had glucose concentration of 27.79 g/L and 73.00 g/L, respectively. The glucose obtained was subsequently tested as substrate for bioethanol production using commercial baker's yeast. The fermentation process produced 40.30 g/L of ethanol after 16 h, which was equivalent to 93.29% of theoretical yield based on total glucose existing in fermentation media.
... The degree of hydrolysis (DH) was calculated by the following equation: DH (%) = (H 1 / H 0 ) 9 100, where H 1 was reducing sugar produced by enzyme hydrolysis, and H 0 was reducing sugar produced by acid hydrolysis. Acid hydrolysis was carried out by treating raw starch with 1 mol l À1 HCl at 100°C for 2 h (Wang et al. 1995). ...
The aims were to isolate a raw starch–degrading α-amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.
A 1539 complete open reading frame of a starch–degrading α-amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α-amylase (BaqA) has no starch-binding domain, and together with a few putative α-amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch.
A novel raw starch–degrading B. aquimaris MKSC 6.2 α-amylase BaqA is proposed to be a member of new GH13 subfamily.
This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.
... In view of energy costs and effective utilization of natural resources, direct enzymatic hydrolysis of starch below gelatinization temperature is desirable. In recent years, starch can be hydrolyzed by ␣-amylase together with glucoamylase to obtain porous starch as the final product (Kimura & Robyt, 1995;O'Brien & Wang, 2008;Sarikaya, Higasa, Adachi, & Mikami, 2000;Uthumporn, Zaidul, & Karim, 2010;Wang, Powell, & Oates, 1995;Yamada, Hisamatsu, Teranishi, Hasegawa, & Hayashi, 1995;Yan & Zhengbiao, 2010;Yao & Yao, 2002). Further modification can increase the efficiency of native starch hydrolysis (Luo et al., 2008;Shariffa, Karim, Fazilah, & Zaidul, 2010). ...
The structural changes of cassava starch granules were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and differential scanning calorimeter after starch granules were hydrolyzed by a mixture of α-amylase and glucoamylase. The surface of starch granules was porous after hydrolysis treatment. Enzymatic erosion occurred mainly at the surface for cassava starch. The BET-specific surface area of hydrolyzed cassava starch improved 10.7 times compared with that of native starch. The powder XRD intensity of hydrolyzed starch was higher than that of native starch. The crystallinity in the hydrolyzed cassava starch increased due to hydrolysis. Compound enzymes could hydrolyze cassava starch granules at sub-gelatinization temperature, and could produce porous starch.
... The degree of hydrolysis (DH) was defined by the following equation: DH (%) ¼ (H 1 /H 0 ) Â 100, where H 1 was reducing sugar produced by enzyme hydrolysis, and H 0 was reducing sugar produced by acid hydrolysis. Acid hydrolysis was carried out by treating raw starch with 1 M HCl at 1008C for 2 h [7]. ...
Characteristics of raw starch degrading a-amylase from Bacillus aquimaris MKSC 6.2 associated with soft coral Sinularia sp. Partially purified a-amylase from Bacillus aquimaris MKSC 6.2, a bacterium isolated from a soft coral Sinularia sp., Merak Kecil Island, West Java, Indonesia, showed an ability to degrade raw corn, rice, sago, cassava, and potato starches with adsorption percentage in the range of 65–93%. Corn has the highest degree of hydrolysis followed by cassaca, sago potato and rice, consecutively. The end products of starch hydrolysis were a mixture of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and small amount of glucose.
... As reported, a-amylase and glucoamylase may accommodate the non-reducing end of substrate chain in their active centre and catalyze hydrolysis reaction, in order to the cleavage of glycosidic bonds and formation of glucoses. Moreover, the starch hydrolysis occurs granule by granule with an attacked granule being completely hydrolyzed (Oates & Powell, 1996; Wang, Powell, & Oates, 1995 ). As observed in the measurement of SEM, the enzymes initially erodes selected zones of granule surface, resulting in the formation of pits which will become canals. ...
Microwave heat-moisture treated Canna edulis Ker starch has been applied to study structural changes in vitro using porcine a-amylase, pancreatin and amyloglucosidase. The structures at different digestion stages were characterized using scanning electron microscopy (SEM), polarized optical microscopy (POM), small angle X-ray scattering (SAXS), X-ray diffraction (XRD), Fourier transforms infrared (FT-IR) spectroscopy and solid state (13)C nuclear magnetic resonance (NMR) spectroscopy. The increase in molecular order was observed with increasing digestion time and reflected in higher scattering intensity measured by SAXS, higher crystallinity observed by XRD, the increase of the double helix order obtained by solid state (13)C NMR spectroscopy, and corresponding changes in the measurement of FT-IR, in favour of increasing resistant starch content. SAXS reveals a single peak around 0.6335 nm(-1), suggesting that the enzyme may erode the special site of granule surface and catalyze whole hydrolysis reaction through pitted canals. In the process of digestion, amorphous region of starch granule is susceptible to the attack of enzyme. Moreover, the result also demonstrates that the resistance to enzymatic digestion may mainly depend on specific structure in the treated starches. (C) 2009 Elsevier Ltd. All rights reserved.
Over the past few decades, the adoption of enzymatic modification with partial replacement of physical and chemical methods has been trending due to enhanced yield with safe and healthier starch. In the enzymatic modification process, exposure of starch is employed to numerous enzymes (primarily hydratases), causing to accelerate the production of highly functional derivatives. The resultant process mainly involves the depolymerization of starch into oligosaccharides or the change of starch by shifting glycosidic linkages and residues. This chapter provides a comprehensive understanding of the enzymatic modification of starch, various enzymes used in the process, and, thereby, characteristics changes in terms of morphology, crystallinity, gelatinization, and rheological property. Furthermore, the significance and application of enzymatic modification of starch production used for food products, packaging, pharmaceuticals, and cosmetics are briefly discussed to highlight the enhanced characteristics of this modification.KeywordsModified starchEnzymatic modificationEnzymatic hydrolysisEnzymesFunctionalityα-amylaseAmyloglucosidase
Starch is omnipresent in plant material and is the most important polysaccharide as well as storage polymer. Starch undergoes various transformations during food processing. Heating of starch in presence of water results in gelatinization which is accompanied by granule breakdown, loss of ordered structure, and loss of optical birefringence. Low temperature favors the re-association of disrupted chains of amylose and amylopectin in a process known as retrogradation. These changes are very much affected by the nature of starch and the amylose to amylopectin ratio. Therefore, it is important to study or monitor the various changes under the influence of various treatments to expand its commercial value. Starch possesses substantial nutritional, pharmaceutical, and industrial importance owing to its distinctive physical, chemical, and functional characteristics. Starch owns varied functional properties and applications in the food industry. However, the native starches lack desired functional characteristics for anticipated applications in the food industry. To increase its commercial significance, the functional properties of starch can be upgraded under the stimulus of several physical, chemical, and enzymatic techniques. Alterations in the structure of starch molecules initiated by various treatments are characterized as modifications. The starch structure is sensitive to high temperature, very high and low pH, high pressure, osmotic pressure, electric field, plasma, sonic waves, microwaves, irradiation, ozone, mechanical stress, various chemicals, and enzymes. These alterations may employ either desirable or undesirable changes in the structure and functionality of starch. Therefore, an appropriate selection of modification techniques concerning cost economics, environmental factors, and efficiency is required to attain targeted improvements in functional characteristics. A practical alternative to chemical and heat-induced alterations is non-thermal treatments that are clean, environmental friendly, more efficient, free from toxic residues, and are sustainable. These techniques are of most interest to the food scientist in the present times. Also, the structural transformations of starch caused by various methods are important to visualize the effectiveness of the process and can be studied by a range of available methods based on different principles such as microscopic, spectroscopic, differential scanning calorimetry, rheological, X-ray diffractions and chromatographic methods, etc. With the scientific and technological interventions, newer techniques for starch modification are being introduced to achieve the targeted characteristics in a simple and more practical approach.
Starch-sugar homeostasis and starch molecular configuration regulates the dynamics of starch digestibility which result in sweet sensory perception and eliciting glycemic response, which has been measured in vitro as inherent glycemic potential (IGP). The objective of the research was to understand the key determinants of IGP as well as sweetness in different Pearl millet (PM) genotypes. To understand the intricate balance between starch and sugar, total starch content (TSC) and total soluble sugars (TSS) were evaluated. Higher concentrations of TSC (67.8%), TSS (2.75%), glucose (0.78%) and sucrose (1.68%) were found in Jafarabadi Bajra. Considering the role of compact molecular configuration of starch towards digestibility, X-ray powder diffraction (XRD) analysis was performed. A-type crystallinity with crystallinity degree (CD %) ranged from 53.53-62.63% among different genotypes, where the least CD % (53.53%) was found in Jafarabadi Bajra. In vitro starch hydrolyzation kinetics carried out to determine IGP, revealed a maximum of 77.05% IGP with minimum 1.42% resistant starch (RS) in Jafarabadi Bajra. Overall our results suggest higher sweet sensory perception of Jafarabadi Bajra which is contributed by the matrix composition with least molecular compactness of starch. Also, the interdependence among starch quality parameters; CD%, IGP, RS and amylose has also been discussed.
Starch is a complex carbohydrate formed by the repeating units of glucose structure connected by the alpha-glycosidic linkages. Starch is classified according to their derivatives such as cereals, legumes, tubers, palms, fruits, and stems. For decades, native starch has been widely utilized in various applications such as a thickener, stabilizer, binder, and coating agent. However, starches need to be modified to enhance their properties and to make them more functional in a wide range of applications. Porous starch is a modified starch product which has attracted interest of late. It consists of abundant pores that are distributed on the granule surface without compromising the integrity of its granular structure. Porous starch can be produced either by enzymatic, chemical, and physical methods or a combination thereof. The type of starch and selection of the modification method highly influence the formation of pore structure. By carefully choosing a suitable starch and modification method, the desired morphology of porous starch can be produced and applied accordingly for its intended application. Innovations and technologies related to starch modification methods have evolved over the years in terms of the structure, properties and modification effects of different starch varieties. Therefore, this article reviews recent modification methods in developing porous starch from various origins.
Maize starch is an abundant renewable biopolymer that can be processed into sugars and ethanol, or other fermented chemicals. The objectives of this study were to examine how partial swelling of starch granules enhanced the starch saccharification process, characterize the indigestible residues, and determine the optimal conditions for glucose production. Normal maize starch (2.0% in 50 mM citrate butter) was partially swollen by heating at 62, 65, and 70 °C for 30 min, and the preswollen starch hydrolyzed by addition of a granular starch hydrolyzing enzyme (GSHE). After preswelling at 70 °C, enzyme kinetics study of the release of glucose showed a 54% reduction in the Michaelis-Menten constant (Km), suggesting that preswelling increased the complexing of GSHE with the starch granules. Preswelling the starch at 62, 65, and 70 °C followed by digestion with 1.0% GSHE (starch basis) at 62 °C for 24 h in 50 mM citrate buffer (pH 4.5) resulted in 61.3, 76.0, and 94.5% conversion to glucose. The indigestible fraction, compared to the untreated starch, had a lower A-type crystallinity and probably less amylose-lipid complex, was more highly branched, contained a higher proportion of short chains, and showed a higher gelatinization temperature but lower enthalpy of gelatinization. The indigestible fraction, after preswelling at 100 °C in citrate buffer, was converted practically quantitatively to glucose. Partial swelling of granules offers great potentials in converting starches to sugars by GSHE.
This study developed an energy efficient procedure to produce size-controlled starch nanoparticles (SNPs) via precipitation. A 0.06 g/ml aqueous starch suspension was solubilized in a solvents system including NaOH and urea at temperature of 35 °C. Applying only 3 minutes ultrasonic treatment leads to formation of SNPs in a narrow size range of 50-70 nm with production yield of 92±5%. TEM and FESEM revealed formation of uniform short nanofibers. Unlike starch/water suspension, the nanoparticles/water suspension remained stable for 48 hours.
This paper reviews reported studies on the hydrolysis of starch especially sago via acid and enzyme. The review begins with overview of sago palm and the starch industry, followed by process of extracting the starch from sago pith. Physicochemical properties of sago starch were tabulated for better understanding of hydrolysis process. Factors or process condition influencing hydrolysis process is discussed based on results from previous researches. Advantages and disadvantages of each hydrolysis is also discussed. Generally, there are very few researches dedicated on sago starch as compared to other starches. It can be concluded that, enzyme hydrolysis gives higher yield at milder process conditions. However, the reaction rate of enzyme hydrolysis is still low compared to acid hydrolysis.
Enzyme technology has many potential applications in the baking industry because carbohydrate-active enzymes specifically react with carbohydrate components, such as starch, in complex food systems. Amylolytic enzymes are added to starch-based foods, such as baking products, to retain moisture more efficiently and to increase softness, freshness, and shelf life. The major reactions used to modify the structure of food starch include: (1) hydrolysis of α-1, 4 or α-1, 6 glycosidic linkages, (2) disproportionation by the transfer of glucan moieties, and (3) branching by formation of α-1, 6 glycosidic linkage. The catalytic reaction of a single enzyme or a mixture of more than two enzymes has been applied, generating novel starches, with chemical changes in the starch structure, in which the changes of molecular mass, branch chain length distribution, and the ratio of amylose to amylopectin may occur. These developments of enzyme technology highlight the potential to create various structured-starches for the food and baking industry.
Heat pretreatment was investigated as a way to improve the α-amylase-catalyzed hydrolysis of granular corn starch at high concentration (45%, w/w). Native and preheated starches were hydrolyzed in the granular state for 1h at 40°C using a commercially available α-amylase. The initial dextrose equivalent (DE) value of the native granular starch (8.0%) increased significantly (to 12.6%) after pretreatment at 60°C for 15min. Microscopic analysis of the starch granules indicated that heat pretreatment increased the number and size of the pores and pinholes on the granule surface, which facilitated the adsorption and subsequent penetration of enzyme molecules. Furthermore, the amylose present in amorphous regions leached rapidly when corn starch was incubated at 60°C, suggesting weaker interactions within the granules. The DE value of the hydrolysate showed a linear dependence on the amount of amylose that leached during the course of heat pretreatment (R² =0.9771). Mobilization of amylose chains within the granule, caused by the heat pretreatment, may have allowed the enzyme to make greater use of amylose, which is its primary substrate during starch granule hydrolysis. In addition, a 35% reduction in the K m value showed that heat pretreatment increased the affinity of α-amylase for the starch granules. This can be attributed to the mobilization of amylose chains within the granule and expansion of the surface pinholes. These effects demonstrate that proper heat pretreatment of granular starch before amylolysis has great potential in industry.
We have previously reported that re-dispersible amidinium chitin nanofibers are obtained from an amidinated chitin by CO2 gas bubbling with ultrasonic treatment in water. On the other hand, amylose is precisely synthesized by phosphorylase-catalyzed enzymatic polymerization. In this study, grafting of amylose on the amidinium chitin nanofibers was investigated by the phosphorylase-catalyzed enzymatic polymerization to produce amylose-grafted chitin nanofiber materials. Depending on the reaction conditions, the reaction mixtures turned into hydrogels. The hydrogels were constructed via the formation of double helixes from a part of amylose graft chains closely present among the nanofibers. Microstructures, which were hierarchically constructed by lyophilization of the hydrogels, were changed from network to porous morphologies in accordance with the molecular weights of amylose graft chains. Most of the amylose graft chains with higher molecular weights, which did not participate in double helixes, formed amorphous membranes in the nanofiber networks by lyophilization, to construct porous structures.
Abstract This article surveys methods for the enzymatic conversion of starch, involving hydrolases and nonhydrolyzing enzymes, as well as the role of microorganisms producing such enzymes. The sources of the most common enzymes are listed. These starch conversions are also presented in relation to their applications in the food, pharmaceutical, pulp, textile, and other branches of industry. Some sections are devoted to the fermentation of starch to ethanol and other products, and to the production of cyclodextrins, along with the properties of these products. Light is also shed on the enzymes involved in the digestion of starch in human and animal organisms. Enzymatic processes acting on starch are useful in structural studies of the substrates and in understanding the characteristics of digesting enzymes. One section presents the application of enzymes to these problems. The information that is included covers the period from the early 19th century up to 2009.
This study investigates the compression and mechanical properties of directly compressible pregelatinized sago starches in comparison with Spress® B820 and Avicel® PH 101. The sago starch is pregelatinized at 65 °C with different pregelatinization times of 15, 30, 45, and 60 min, creating samples PS1, PS2, PS3, and PS4, respectively. Compressibility of the powders is analyzed by Heckel and Kawakita equations. The compressibility of sago starch is found to be lower than that of its pregelatinized forms, and the compressibility increases with an increase in the pregelatinization time. Avicel® PH 101 is the most compressible among the powders evaluated, followed by PS4, Spress® B820, PS3, PS2, PS1, and sago starch. As for mechanical properties, Avicel® PH 101 is found to have the highest radial tensile strength and the hardest compacts, indicating that it has the highest compactibility, followed by Spress® B820, PS4, PS3, PS2, PS1, and sago starch.
Starch has very important physicochemical properties that depend on its botanical source and structural characteristics. Peruvian carrot starch displays special characteristics, which make it appropriate for industrial application in many processed foods. In this work, starch from two Peruvian carrot varieties, Amarela de Carandaí (AC) and Amarela de Senador Amaral (ASA), were isolated and their physicochemical and structural properties were determined. Starch from the ASA variety exhibited granules with a greater average diameter than those from the AC variety and more than double the number of granules with size >20 µm, when compared to the other variety. Starches from both varieties, observed in a Scanning Electron Microscope, showed smooth granule surface with circular and polyhedral shapes for large and small granules, respectively. Molecular size distribution, intrinsic viscosity and degree of cristallinity of starches from both varieties were similar; however, amylose content was higher for the starch from the AC variety. Higher values of viscosity, swelling power and gelatinization temperatures were observed for the starch from the ASA variety, which could be related to the lower amylose content and the higher proportion of large granules exhibited by this starch.
Starch has very important physicochemical properties that depend on its botanical source and structural characteristics. Peruvian carrot starch displays special characteristics, which make it appropriate for industrial application in many processed foods. In this work, starch from two Peruvian carrot varieties, Amarela de Carandaí (AC) and Amarela de Senador Amaral (ASA), were isolated and their physicochemical and structural properties were determined. Starch from the ASA variety exhibited granules with a greater average diameter than those from the AC variety and more than double the number of granules with size >20 µm, when compared to the other variety. Starches from both varieties, observed in a Scanning Electron Microscope, showed smooth granule surface with circular and polyhedral shapes for large and small granules, respectively. Molecular size distribution, intrinsic viscosity and degree of cristallinity of starches from both varieties were similar; however, amylose content was higher for the starch from the AC variety. Higher values of viscosity, swelling power and gelatinization temperatures were observed for the starch from the ASA variety, which could be related to the lower amylose content and the higher proportion of large granules exhibited by this starch.
The influence of processing conditions in a combined gelatinisation and liquefaction extrusion system on subsequent saccharification was examined. Extrusion conditions investigated were barrel temperature (70–130 °C), screw speed (70–190 rpm), enzyme concentration (0–1%) and feed moisture content (28.5–50.5%). Feed moisture, barrel temperature and enzyme concentration had significant effects on saccharification, which ranged from 32 to 98% (dextrose equivalent = 83–98) after 8 h reaction with 0.50 AGU/ml amyloglucosidase (AMG). Die pressure was negatively correlated with saccharification (r = −0.75), implying that products formed from a high viscous mass were poorly saccharified. Greater extent of saccharification was achieved with lower SME input. Saccharification in the extruder was initiated by adding AMG during the extrusion processing. Both initial and subsequent saccharification were dependent on the AMG concentration, moisture content and the stage of AMG addition. In this mixed-enzyme system, synergistic effects of both enzymes (α-amylase and amyloglucosidase) were evident. High-performance size-exclusion chromatography profiles showed that incorporating AMG led to loss of G6 and G2 in the oligosaccharide spectra but with formation of G5.
Simultaneous pregelatinization and preliquefaction of sago starch with a thermostable α-amylase was carried out in a Brabender single screw extruder. Response surface methodology was employed to study the effects of processing conditions; feed moisture content (21–38%), enzyme concentrations (1.48–6.52%) and mass temperatures in the compression and die zones (70.5–97.5 °C), on the properties of the extrudates. Twenty runs were performed based on a multifactorial composite rotatable design. Changes in the dextrose equivalent (DE), water solubility index (WSI), water absorption index (WAI), degree of gelatinization (DG) and high performance size exclusion chromatography (HPSEC) profiles were assessed. From the HPSEC profiles, the degree of degradation (DGR) and oligosaccharide content were calculated. Feed moisture content and enzyme concentration were found to be the most significant variables affecting most of the measured physicochemical properties. Hydrolysis of starch granules was the fundamental reaction occurring during the extrusion process as indicated by the higher DE, WSI, oligosaccharide content and correspondingly lower WAI, DG and DGR. Product spectra from HPSEC showed that the amylopectin (Ap) was preferentially degraded and amylose (Am) was apparently protected. The predominant oligosaccharide species were G3, G5 and G6.
As part of an ongoing study to improve the cassava starch manufacturing process, a potential improvement to the dewatering stage was explored. Two types of starch dewatering were compared, a pressure filter and a conventional centrifuge. Performance with respect to the dewatering efficiency of the starch slurry, implied by the filtration rate and percentage of dry solids in circulation, was measured for a pressure filter and a conventional centrifuge. For the pressure filter, effect of different filter cloths, feed time and pressing time were evaluated. At all filtering conditions, the pressure filter provided improved dewatering efficiency. The filtration rate significantly increased from 162 to 226 g m-2 s-1 and starch loss, to the circulation, notably decreased from 15 to 0.15%. Improvement in processing-efficiency did not sacrifice starch quality. Granule morphology and functional properties, such as paste viscosity, water adsorption, and solubility characteristics remained unchanged. One notable exception was that chemical compounds and microorganisms appeared to be more readily absorbed to the granule surface. The pressure filter not only improved dewatering efficiency but also minimized production cost due to a lower starch cake moisture, which requires less energy consumption for subsequent drying.
Sulphur dioxide addition, usually during the centrifugal or extraction stage of starch processing, is believed to improve starch extraction and is a common practice in Thailand. Examination of the effect of sulphur dioxide addition during commercial scale processing revealed that in addition to an obvious bleaching effect, inclusion levels, such that the final product contained 180 mg sulphur dioxide/g starch, also altered functional properties. Changes were thought to result from granule stabilization and to be acting at the level of amylopectin, this fraction was very much less stable to physical disruption when no sulphur dioxide was present. Increased granule stabilization was expressed by an increase in gelatinisation temperature of about 2°C, a decrease in swelling at lower temperatures and paste viscosity. Entry and possibly exit of material into the granule was also influenced by the presence of sulphur dioxide – enzyme, acid and water were all apparently restricted in their access to the granule. In the presence of sulphur dioxide enzyme hydrolysis was both lower and of a pattern that indicated only surface activity, whereas granules that did not contain sulphur dioxide, enzyme activity occurred from within the granule, resulting in breakdown, fragmentation and increased hydrolysis (from 44.8 to 53.5%). Lower water absorption, granule swelling and limited acid hydrolysis all characterized starch samples containing sulphur dioxide. The influence of sulphur dioxide was thought to be at the level of granule structure and could not be accounted for by differences in proximate composition or microbial activity, both starch were identical in all respect except amount of associated sulphur dioxide.
Successive extraction of sago pith with cold water, hot water, dimethylsulfoxide, and 5% NaOH yielded 48.9% cold-water-soluble starch, 11.5% hot-water-soluble starch, 21.9% DMSO-soluble starch, and 7.1% alkali-soluble starch, which contain 5.4%, 7.0%, 3.6%, and 16.2% nonstarch polysaccharides, such as β-glucan and hemicelluloses, respectively. The total pure starch accounted for 83.9% of the dried sago pith. Cold- and hot-water-soluble starches contained noticeable amounts of amylose, whereas DMSO- and alkali-soluble starches predominated in amylopectin. All the four starch fractions also contained trace quantities of ash (0.05%-0.4%), crude protein (0.1%-0.15%), and lipids (0.1%-0.16%). The isolated straches were further analyzed by UV, FT-IR, and 13 C-NMR spectroscopies as well as thermal analysis, and the results are reported.
The raw starch-digesting alpha amylase of Bacillus sp. IMD 434 was purified to homogeneity and displayed substantial hydrolysis of raw starch but did not adsorb onto the insoluble substrates, corn, rice, wheat or potato starch, at any of the pH values examined. The degree of hydrolysis ranged from 10% hydrolysis of potato starch to 32% hydrolysis of corn starch after 24 h. α- and β-Cyclodextrins (CDs) inhibited raw starch digestion but did not affect hydrolysis of soluble starch. In the presence of 10 mM α-CD or β-CD, hydrolysis of raw corn starch by the amylase decreased by 88 and 97%, respectively. The enzyme did adsorb onto α-CD Sepharose 6B, suggesting that an affinity site may be present on this non-raw starch-adsorbing amylase. After incubation with Pronase E, alpha amylase (434a Mr 69,200) was hydrolysed into two components, a large enzymatically active component, EA 434b (Mr 56,200) and a small inactive peptide, IA 434c (Mr 13,000). EA 434b, although active on soluble starch, was incapable of hydrolysing raw starch, unable to adsorb onto raw starch and lost its ability to adsorb onto α-CD Sepharose 6B. Conversely, IA 434c was inactive on soluble and raw starch, did not adsorb onto raw starch but did adsorb onto α-CD Sepharose 6B and a range of linear maltooligosaccharide Sepharose 6B matrices. Thus, the alpha amylase simultaneously lost the ability to hydrolyse raw starch and adsorb onto α-CD Sepharose 6B when IA 434c was removed by proteolysis. The ability of the raw starch-digesting alpha amylase to adsorb onto α-CD Sepharose 6B was then exploited successfully in the development of a one-step purification for the amylase using CD affinity chromatography.
Native sago starch was incubated at 60°C with lysophosphatidylcholine, monomyristin, monopalmitin, and monostearin. Differential scanning calorimetry peaks centred at 100–120°C indicated formation of amylose–lipid complexes. Among the four lipids, lysophosphatidylcholine showed the highest complexing ability, while that of the monoglycerides decreased with the increasing chain length. Part of the amylose leached during the incubation, and the amount of leached material decreased in the presence of lipids. Starch–lipid samples were subjected to enzyme hydrolysis by porcine pancreatic α-amylase. The bioavailability of native and freshly gelatinised sago starch was decreased in the presence of lipids, while retrograded starch–lipid samples showed higher digestibility than starch control. ©
Tapioca starch was annealed at 60°C for 90 min followed by hydrolysis with α-amylase at 60°C at various lengths of time (30, 60 and 120 min) to obtain high-crystalline starches. The reaction products were subjected to spray drying to obtain annealed–enzymatically hydrolyzed–spray dried tapioca starch (SANET) in the form of spherical agglomerated granules. The properties of SANET were compared with those of annealed–spray dried tapioca starch without enzymatic treatment (SANT) and native–spray dried tapioca starch (SNT). Scanning electron micrographs of the starch samples were used to study the morphological changes and to suggest the mode of enzyme attack during hydrolysis. The á-amylase preferentially attacked the interior of the starch granules, leaving a deep round hole on the starch granule surface. It was found by X-ray diffraction that both annealing and amylolysis did not alter the A type diffraction pattern. The% relative crystallinity of SANET was raised with increasing hydrolysis time and with decreasing amylose content. High performance size exclusion chromatography (HPSEC) demonstrated the decrease of the degree of polymerization (DP) of the amylose fraction of SANET after prolonged hydrolysis. For the utilization of SANET as tablet filler, it was directly compressed by a tablet compression machine at 4 kN to obtain tablets. The increased relative crystallinity of starch resulted in increased crushing strength and disintegration time, but in a decreased tablet friability.
Acid and enzyme hydrolyses followed by ball milling were applied to fracture cassava starch granules. Microscopic and chromatographic evidence suggested different mechanisms of the two hydrolyses. Using the enzyme process, granules with a sponge-like structure and shells with the interior hydrolysed were produced. Amylose and amylopectin were subjected equally to multiple attacks by enzymes, with no significant change in granule crystallinity. The hydrolysed residues could not be effectively broken down by ball milling, although the crystallinity was destroyed. In contrast, the acid treatment caused superficial external corrosion, mainly at the amorphous lamellae, ie the branch points of amylopectin. Acid-lintnerised starch granules were mostly of Degree of polymerization, DP 10–15 and exhibited increased crystallinity and brittleness, making them more susceptible to breakdown upon milling. Ball milling, although destroying some degree of crystallinity, could effectively reduce the size of acid-hydrolysed starch, with no further degradation of amylodextrin molecules. By a combination of lintnerisation and ball milling, smaller particle starch (3–8 µm compared with 3–30 µm for native starch) could be produced. It is clear that removal of the amorphous phase prior to milling is critical for effective rupture of the granules. Copyright © 2003 Society of Chemical Industry
Edible canna (Canna edulis Ker) as an alternative starch source was evaluated on the basis of genetic characteristics, agronomic traits and starch properties. Four canna varieties indigenous to Thailand were examined including Thai-green, Japanese-green, Thai-purple and Chinese-purple and compared with cassava (Manihot esculenta Crantz). Using the Random Amplified Polymorphic DNA (RAPD) technique employing ten 10-base primers, four primers implied that at least three types of canna including Thai-green, Japanese-green and Thai/Chinese-purple existed and corresponded to plant characteristics as identified by flower, stem, leaf and rhizome colors. Despite genetic diversification, starch properties were not variable. All four varieties produced 30.4–38.4 tonne/ha of rhizomes with starch content about 13% (wet basis). Starch yields of canna (4.1–4.9 tonnes/ha) were comparatively lower than cassava (6.5 tonnes/ha). The starches were characterized by giant granules (10–80 μm), and compared with cassava starch pastes had a higher peak viscosity (930–1060 BU for canna starches and 815 BU for cassava starch), occurring at a higher temperature. Pastes of canna starch were more stable and when cooled, viscosity increased to 1800 BU. Gelatinized pastes of canna starches also rapidly formed good gels on cooling. It is evident that edible canna provides starches with very attractive properties and totally different from cassava and is the greatest promise for the new base starch to be employed complementarily with cassava starch.
Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting
the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were
determined. The highest kefiran concentration of 0.85g/l was obtained at the initial pH of 5.5, temperature of 30°C, starch
concentration of 4% and mixed-enzymes with activity of 100U/g-starch. The use of a mixture of α-amylase and glucoamylase
could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40
gave the highest kefiran production rate of 11.83mg/l/h. This study showed that sago starch could serve as a low-cost substrate
for kefiran production.
Poly(lactic acid) (PLA) composites consisting of PLA, rice starch (RS) (0–50 wt%) and epoxidised natural rubber (ENR50) were compounded by a twin-screw extruder and compression moulded into dumbbell specimens. Tensile tests were performed to characterize the mechanical properties of the PLA/RS composites. Morphological studies were done on the tensile fractured surface of the specimens by using scanning electron microscopy (SEM). Twenty weight percent of RS achieved a good balance of strength and stiffness. Beyond 20 wt% loading of RS, the tensile strength and elongation at break of PLA decreased drastically. This may be attributed to the agglomeration of RS, which could then act as stress concentrator. The incorporation of ENR50 increased the tensile strength and elongation at break of the PLA/RS composites remarkably, owing to the elastomeric behaviour and compatibilisation effects of ENR50. Interestingly, the morphology of PLA/RS composites transformed to a more ductile one with the addition of ENR. The kinetics of water absorption of the PLA/RS composites conforms to Fick's law. The Mm and D values are dependent on the RS and ENR concentrations. The tensile properties of the PLA/RS composites deteriorated after water absorption. The retention-ability and recoverability of the PLA/RS composites are relatively low, attributed to the hydrolysis of PLA, degradation of the PLA–RS interface and leaching of the RS particles. In addition, the tensile properties of PLA/RS composites decreased drastically upon exposure to enzymatic degradation. Extensive pinhole and surface erosion on the PLA/RS composites indicate high degree of hydrolysis. Whilst the addition of ENR leads to some improvements in tensile properties, nevertheless, it enhanced the biodegradability of the PLA/RS composites when exposed to water and α-amylase enzymatic treatments.
Gelatinized sago starch was stored for different times and under different temperature conditions for the investigation of retrogradation. DSC was used to monitor the thermal properties of retrograded starch. Bioavailability of starch samples was determined by porcine pancreatic α-amylase at 37 °C. Amylose retrograded rapidly at 5 °C, whereas amylopectin recrystallization was enhanced by sequential storage at 5 °C followed by 30 °C. Increased extent of retrogradation (high melting enthalpy values and melting temperature) caused reduced enzyme susceptibility of sago starch and sago products at 37 °C.
Starch constitutes a major component of foods and also a raw material for use in the production of industrial products. The constituent macromolecules of starch are packaged in a highly ordered and compact manner, resulting in inert, insoluble granules. Food processing destroys starch structure, thereby releasing the component molecules, which are subsequently made available for hydrolysis or serve a functional role in the food system. The application of starch as a raw material usually requires the prior disruption of the inert granule structure, which involves additional processing steps. The hydrolysis of native granules has ramifications at all levels of the food processing chain, from postharvest losses to nutritional consequences of the ingested food. Technologists have not been able to take advantage of, or control, this process because the body of the information that is available on starch granule structure and the behaviour of hydrolysing enzymes is still incomplete. In this review, I have highlighted some of the more recent advances in this field, with the view to opening up the way for more efficient native starch hydrolysis.
Sago starch annealed at varying temperatures, time intervals and pH was used to study granule hydrolysis by a glucoamylase (AMG) and α-amylase (Termamyl) mixture. Differential scanning calorimetry (DSC) indicated that there was a relationship between the extent of annealing and starch granule hydrolysis. Enthalpy of gelatinisation of annealed starch granules remained unchanged, suggesting that no gelatinisation had occurred. The degree of hydrolysis increased and the granule degradation pattern altered—from surface erosion to preferential digging of the internal regions of the granule. Sections of the hydrolysed granule residues revealed that enzymes attacked from one point on sufficiently annealed granules, and that after extensive hydrolysis, only an empty shell remained.
A wide variety of microorganisms produce, and in most cases secrete extracellularly, amylases having different specificities and some rather interesting properties (Fogarty & Kelly, 1980; Fogarty, 1983). Gram-positive bacteria, and particularly the genus Bacillus, are prolific producers of amylases, although very few are found among Gram-negative bacteria. A wide range of moulds produce amylases, particularly the genus Aspergillus. In recent years interest and awareness in the amylolytic activities of yeasts has generated a number of studies of these systems. It is not the purpose of this review to provide an exhaustive or detailed treatise of microbial amylases. Other publications have dealt with the subject area extensively (Fogarty & Kelly, 1979, 1980; Fogarty, 1983) and this work has as its aim an update of developments which have taken place in the area in recent years.
The raw starch-binding a-amylase of C. butyricum, an acidogenic anaerobe found in mesophilic methane sludge, was purified by chromatography on a column of Bio-Rex 70 after the enzyme had been liberated from potato starch granules. The molecular weight of the purified enzyme was determined to be 89,000 by sodium dodecyl sulfate (SDS) disc electrophoresis. The iodine reaction completely disappeared at 9% hydrolysis of soluble starch by the enzyme, and the successive formation of maltooligosaccharides proceeded at pH 5.0 and 37°C. Decreases in the levels of maltopentaose and maltotetraose then took place as well as the formation of glucose, there being eventually almost 36% hydrolysis of the substrate.
Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/l and the ethanol yield, Yp/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.
Raw sago starch digesting amylase was obtained from Penicillium brunneum No. 24. with strong ability to digest sago starch granules. The crude enzyme from this strain contains CMC‐ase and avicelase. The specific activity of the enzyme did not increase proportionally with purification. We tried combination of our purified enzyme with other hydrolytic enzymes as a means of improving the hydrolysis of sago starch granules.
Addition of cellulase at the initial stage of the hydrolyzation process resulted in an increase in the ability of raw starch digesting amylase to digest sago starch granules. Adding 10 unit/g starch of cellulase. followed of our purified raw starch digesting amylase in small portion at various time intervals was found effective in the hydrolysis of untreated sago starch granules. The treatment resulted in a convertion rate of untreated sago starch granules to glucose near to complete after 120h enzymes reaction, and was also effective in reducing the reaction time of hydrolysis of treated sago starch granules. This process showed that mainly glucose was produced.
An amylolytic yeast strain, isolated from Thai Loogpang Lauw and identified as Lipomyces starkeyi HN-606, was found to produce a novel yeast α-amylase characteristically showing raw-starch digestibility almost to the same extent as the commercial amylolytic Aspergillus, but with a lower culture temperature of 15°C rather than 25°C as optimal for growth. The yeast α-amylase (MW 56,000) was adsorbed onto raw corn-starch and could digest it to form glucose, maltose and maltotriose. α-Cyclodextrin at less than 10 mM completely inhibited the raw-starch adsorption and digestion with a Kl value of 0.38 mM, but could hardly inhibit gelatinized-starch hydrolysis.
The study deals with comparison of the susceptibility to fungal glucoamylase and salivary α-amylase of starch granules from navane, panivaragu, black pepper and black gram. The crude glucoamylase was purified by fractionation and column chromatography to give two pure fractions of which one was used. The rate of amylolysis was followed by estimating the amount of glucose and maltose released. The type and extent of damage of the starch granules were observed by scanning electron microscopy which revealed characteristic degradation patterns in navane and panivaragu, whereas black gram granules were resistant to the attack. Very small-sized black pepper starch granules did not exhibit any obvious signs of amylolytic attack.
Raster-Elektronenmikroskopie von enzym-behandelten Stärkekörnern.
Die Untersuchung befaßt sich mit der Angreifbarkeit von Stärkekörnern aus Navane, Panivaragu, schwarzem Pfeffer und Mungobohnen durch Pilz-Glucoamylase und Speichel-α-Amylase. Die rohe Glucoamylase wurde durch Fraktionierung und Sälen-chromatographie gereinigt; dabei ergaben sich zwei reine Fraktionen, von denen eine verwendet wurde. Die Geschwindigkeit der Amylolyse wurde durch Bestimmung der freigemachten Glucose und Maltose verfolgt. Die Art und das Ausmaß des Angriffs auf die Stärke wurden durch Raster-Elektronenmikroskopie beobachtet, wobei charakteristische Abbauerscheinungen in Navane und Panivaragu festgestellt wurden, denen gegenüber sich die Stärkekörner der Mungobohne als widerstandsfähig erwiesen. Sehr kleinkörnige Stärkekörner des schwarzen Pfeffers zeigten keine eindeutigen Merkmale eines amylolytischen Angriffs.
Several microorganisms have been found to produce raw starch digesting amylase. We have isolated Penicillium brunneum from sago palm tree at a sago processing site, which was used as a source of starch digesting amylase. All the raw starch digesting enzymes were effective for cereal starches, but root starches and sago starch were resistant to the enzyme reaction.
Treatment of sago starch by heating to temperature below gelatinization temperature at lower pHs resulted in an increase in the ability of enzyme to digest sago starch granules. Heating to 60°C at pH 2.0 resulted in a conversion rate of sago starch granules to glucose near to the conversion rate of raw corn starch to glucose. At higher concentration, the degree of hydrolysis of treated sago starch granules was about 275% as compared to that of untreated sago starch granules.
Addition of the enzyme in large amount or small portion at various time intervals was found effective in the hydrolysis of treated sago starch granules.
Glucoamylase and α‐amylase of Chalara paradoxa were separated by hydrophobic column chromatography using butyl‐Toyopearl 650M. The α‐amylase showed the highest activity at pH 5.5 and 45°C, and was stable in the pH range of 5.5–6.5 and at temperatures lower than 40°C. The glucoamylase showed the highest activity at pH 5.0 and 45°C, and was stable in the pH range of 4.0–7.5 and at temperatures lower than 45°C. The molecular mass of the α‐amylase and glucoamylase estimated by SDS polyacrylamide gel electrophoresis was 80,000 and 68,000, respectively. Both glucoamylase and α‐amylase could digest more effectively raw rice starch and raw corn starch than raw sago starch and raw potato starch. 2% raw rice starch in 10 ml solution was digested by more than 90% by 100 units of each amylase. When these amylases were used combined, raw corn starch was more effectively digested than they were used singly. This cooperative action in raw corn starch digestion was also observed when. C. paradoxa α‐amylase and R. niveus glucoamylase were combined.
The disruption of molecular oders which occur during the gelatinisation of starch granules has been studied by isolating dried samples from maize, waxy maize, wheat, potatoe, and tapioca starches after defined thermal pre-treatments. Residual molecular and crystalline order was quantified by 13C-c.p.-m.a.s.-n.m.r. spectroscopy and powder X-ray diffraction, respectively, and the results compared with residual gelatinisation enthalpy determined by d.s.c. For native starches, molecular (double-helical) order was significantly greater than crystalline order. Molecular and crystalline order were both found to correlated with the residual enthalpy of gelatinisation following thermal pre-treatment, indicating that both levels of structure are disrupted concurrently during gelatinisation. From the data obtained, predicted enthalpy values for the disruption of fully ordered crystalline analogues of the starches studied were calculated, and compared with values for essentially fully ordered and crystalline model material. This comparison suggests that the enthalpy of gelatinisation primarily reflects the loss of molecular (doube-helical) order.
Eighty-eight amylolytic Bacillus strains representing 18 species were examined for the ability to hydrolyse starch granules. Only strains of Bacillus stearothermophilus and Bacillus amylolyticus secreted amylases which showed high activity towards corn starch. The amylase from B. stearothermophilus NCA26 hydrolysed corn and wheat starch granules efficiently but had low activity on potato starch granules when the digestions were performed at 40°C; however, when the temperature was increased to 60°C, hydrolysis of potato starch granules was more effective and 45% conversion to glucose was achieved in 12 h.
Apparent molecular weight profiles of native and hydrolysed sago starch were investigated. Components of sago starch were characterized by high-performance size-exclusion chromatography (HPSEC) and the relative peak areas of amylose and amylopectin calculated as 27·2 and 61·8%, respectively. Techniques to disperse molecularly the components of sago starch with minimal degradation were developed. Storage of samples at −20°C prior to analysis resulted in depolymerization. Polymer integrity was, however, maintained if samples were stored at 40°C in the presence of a suitable antimicrobial agent. The hydrolysis pattern of Termamyl 120L on sago starch was monitored using kinetic, HPSEC and scanning electron microscopy (SEM) studies. Kinetic studies showed that both Km and Vmax were temperature dependent; Km at 90°C was lower than that at 40°C and the activity of 1 ml of Termamyl 120L solution on solubilized sago starch at 90°C, pH 6·0 and 30 ppm of Ca2+ was determined and found to hydrolyse an average of 7716 μequivalent glycosidic bonds of the starch per minute. Product spectra from HPSEC showed that the amylolysis was dependent on temperature, enzyme concentration, chain length and nature of substrate. SEM studies appeared to suggest that the enzyme tunnelled into the granular interior and then hydrolysed from within, along concentric rings.
The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.
The neocuproine test as described by Brown (4) has been modified to include the range of 5 to 125 μg of glucose. The replacement of the tartrate chelating agent by glycine reduces the value of the blank and decreases the relative standard error of the conventional oxidation procedure by a factor of approximately 6 at low substrate concentrations and by a factor of 2 at high substrate concentrations.
The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.
A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting alpha-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30 degrees C. The raw-potato-starch-digesting alpha-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying alpha-amylase.
Kinetic expressions that represent the synergistic action of α-amylase and glucoamylase on the hydrolysis of native starch granules are proposed. They are simultaneous differential equations consisting of an equation representing the α-amylase action and another one representing the glucoamylase action. α-amylase splits randomly the substrate molecules on the surface of the granules in order to supply new nonreducing end groups to glucoamylase. But, most of these split molecules remain on the surface of the granules. Glucoamylase forms soluble sugar successively from a molecule on the surface, in other words, peels the molecule from the surface and reveals new glucoside bonds on the next layer of the granule, which are reacted by α-amylase.
Cereal starches and proteins
- Evers