Showing papers on "Amylase published in 2015"
TL;DR: It is proposed that plant foods containing high quantities of starch were essential for the evolution of the human phenotype during the Pleistocene and the auxiliary role copy number variation in the salivary amylase genes may have played in increasing the importance of starch in human evolution following the origins of cooking.
Abstract: We propose that plant foods containing high quantities of starch were essential for the evolution of the human phenotype during the Pleistocene. Although previous studies have highlighted a stone tool-mediated shift from primarily plant-based to primarily meat-based diets as critical in the development of the brain and other human traits, we argue that digestible carbohydrates were also necessary to accommodate the increased metabolic demands of a growing brain. Furthermore, we acknowledge the adaptive role cooking played in improving the digestibility and palatability of key carbohydrates. We provide evidence that cooked starch, a source of preformed glucose, greatly increased energy availability to human tissues with high glucose demands, such as the brain, red blood cells, and the developing fetus. We also highlight the auxiliary role copy number variation in the salivary amylase genes may have played in increasing the importance of starch in human evolution following the origins of cooking. Sa...
174 citations
TL;DR: Kinetic analyses of α-amylase hydrolysis of maize starch in the presence of cellulose showed that the inhibition is of a mixed type, suggesting that cellulose in the diet can potentially attenuate starch Hydrolysis.
171 citations
TL;DR: The results of the present study indicate that, fraction 2 of A. caudatum and fraction 4 of C. argentea, rich in flavonoids, are effective α- amylase and α- glucosidase inhibitors, which may be helpful to reduce the postprandial glucose levels.
Abstract: Objective: The objective of the present study was to provide an in-vitro evidence for the potential inhibitory activity of extracts and fractions of Adiantum caudatum Linn. and Celosia argentea Linn. on α-amylase and α-glucosidase enzymes . Materials and Methods: The plant extracts were prepared, first with cold maceration (70% v/v ethanol) and then by Soxhlation techniques (95% v/v ethanol). Subsequently, the combined extracts were subjected for fractionation. Different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/ml) of extract and fractions were subjected to α-amylase and α-glucosidase inhibitory assay. The absorbance was measured at 540 and 405 nm using multiplate reader and the percentage of α- amylase and α- glucosidase inhibitory activity and IC 50 values of extract and fractions were calculated. Results: Fraction 2 of A. caudatum and fraction 4 of C. argentea has shown highest α-amylase and α-glucosidase inhibitory potential with IC 50 values of 0.241, 0.211 and 0.294, 0.249 mg/ml, respectively, which was comparable with acarbose (0.125 and 0.93 mg/ml). Whereas, extracts and remaining fractions of both the plants have shown lesser activity. Conclusion: The results of the present study indicate that, fraction 2 of A. caudatum, rich in triterpenoids and phenolics and fraction 4 of C. argentea, rich in flavonoids, are effective α- amylase and α- glucosidase inhibitors, which may be helpful to reduce the postprandial glucose levels. Hence, further studies may throw light on the antidiabetic potential of A. caudatum and C. argentea, especially in the management of type 2 diabetes.
161 citations
TL;DR: The results allow targeted design of starch digestion experiments through a thorough understanding of the contributions of α-amylase and amyloglucosidase to digestion rates.
113 citations
TL;DR: In this paper, the starch structural factors which control the rate of action of alpha-amylase are reviewed, and it is concluded that local starch molecular density has the major influence on amylase digestion kinetics, and that density sufficient to either limit enzyme binding and/or slow down catalysis can be achieved by either crystallization or dense amorphous packing.
Abstract: To aid in the design of starch-containing foods with slow and/or incomplete digestion in the upper gastrointestinal tract, the starch structural factors which control the rate of action of alpha-amylase are reviewed. It is concluded that local starch molecular density has the major influence on amylase digestion kinetics, and that density sufficient to either limit enzyme binding and/or slow down catalysis can be achieved by either crystallization or dense amorphous packing.
111 citations
TL;DR: The fluorescence data revealed that the green tea extract bound with α-amylase to form a new complex with static quenching mechanism, which could be used to manipulate starch digestion for potential health benefits.
107 citations
TL;DR: It is shown that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures used and that qPCR data in other studies have been further compromised by systematic miscalibration.
Abstract: The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations.
99 citations
TL;DR: A pathway for starch utilization by E. rectale may be conserved among other starch‐degrading Clostridium cluster XIVa organisms in the human gut and three solute‐binding proteins display a range of glycan‐binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6‐branched glycans are described.
Abstract: Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane-associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut.
97 citations
TL;DR: It could be stated that productive medium supplemented with 2% potato starchy waste as a low price substrate could be more favorable than basal medium containing 1% starch for amylases production in submerged fermentation, as it increased α, β and γ amylase activity by 1.98, 7.69 and 12-fold than that produced in basal medium (control), respectively.
95 citations
TL;DR: An enzymatic-colorimetric starch assay developed in 1997 that had advantages in ease of sample handling and accuracy compared to other methods was considered and was further modified to improve utilization of laboratory resources and reduce time required for the assay.
Abstract: Starch, glycogen, maltooligosaccharides, and other α-1,4- and α-1,6-linked glucose carbohydrates, exclusive of resistant starch, are collectively termed "dietary starch". This nutritionally important fraction is increasingly measured for use in diet formulation for animals as it can have positive or negative effects on animal performance and health by affecting energy supply, glycemic index, and formation of fermentation products by gut microbes. AOAC Method 920.40 that was used for measuring dietary starch in animal feeds was invalidated due to discontinued production of a required enzyme. As a replacement, an enzymatic-colorimetric starch assay developed in 1997 that had advantages in ease of sample handling and accuracy compared to other methods was considered. The assay was further modified to improve utilization of laboratory resources and reduce time required for the assay. The assay is quasi-empirical: glucose is the analyte detected, but its release is determined by run conditions and specification of enzymes. The modified assay was tested in an AOAC collaborative study to evaluate its accuracy and reliability for determination of dietary starch in animal feedstuffs and pet foods. In the assay, samples are incubated in screw cap tubes with thermostable α-amylase in pH 5.0 sodium acetate buffer for 1 h at 100°C with periodic mixing to gelatinize and partially hydrolyze α-glucan. Amyloglucosidase is added, and the reaction mixture is incubated at 50°C for 2 h and mixed once. After subsequent addition of water, mixing, clarification, and dilution as needed, free + enzymatically released glucose are measured. Values from a separate determination of free glucose are subtracted to give values for enzymatically released glucose. Dietary starch equals enzymatically released glucose multiplied by 162/180 (or 0.9) divided by the weight of the as received sample. Fifteen laboratories that represented feed company, regulatory, research, and commercial feed testing laboratories analyzed 10 homogenous test materials representing animal feedstuffs and pet foods in duplicate using the dietary starch assay. The test samples ranged from 1 to 70% in dietary starch content and included moist canned dog food, alfalfa pellets, distillers grains, ground corn grain, poultry feed, low starch horse feed, dry dog kibbles, complete dairy cattle feed, soybean meal, and corn silage. The average within-laboratory repeatability SD (sr) for percentage dietary starch in the test samples was 0.49 with a range of 0.03 to 1.56, and among-laboratory repeatability SDs (sR) averaged 0.96 with a range of 0.09 to 2.69. The HorRat averaged 2.0 for all test samples and 1.9 for test samples containing greater than 2% dietary starch. The HorRat results are comparable to those found for AOAC Method 996.11, which measures starch in cereal products. It is recommended that the dietary starch method be accepted for Official First Action status.
92 citations
TL;DR: It is proposed that the slightly longer branch lengths of amylopectin which leads to the formation of more stable B-type double helical structures compared to their A-type counterparts is the major parameter, with other factors such as granule size, surface pores and interior channels having secondary roles, in determining the rate of enzymatic hydrolysis of rice starch granules.
TL;DR: An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB) was constructed using different plasmids containing the amyl enzyme gene of Panibacillus sp.
Abstract: Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer. An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB), was constructed using different plasmids containing the amylase gene of Panibacillus sp. and PHB synthesis genes from Ralstonia eutropha. This engineered strain can utilize starch as the sole carbon source. The maximum PHB production (1.24 g/L) was obtained with 2% (w/v) starch in M9 media containing 0.15% (w/v) yeast extract and 10 mM glycine betaine. The engineered E. coli SKB99 strain can accumulate intracellular PHB up to 57.4% of cell dry mass.
TL;DR: The strongest stimulative effects on amylase were shown by both synbiotics, on lipase synbiotic Bi²tos + Lactococcus lactis subsp.
TL;DR: This is the first report of direct ethanol production from natural starchy substrates (without any pre‐treatment or commercial enzyme addition) using industrial yeast strains co‐secreting both a glucoamylase and α‐amyl enzyme.
Abstract: The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase. Biotechnol. Bioeng. 2015;112: 1751–1760. © 2015 Wiley Periodicals, Inc.
TL;DR: In this article, the ability of fresh tropical fruits to inhibit starch-hydrolyzing enzymes was determined by using high-performance liquid chromatography coupled with diode array detector.
TL;DR: In this article, the molecular structures and functional properties of high-amylose starches from a rice transgenic line and commercial maize were investigated and compared and compared with those obtained from normal rice and maize starches.
TL;DR: Overall, germinated rice flour showed appropriate functionality for being used as raw ingredient in gluten free breadmaking, and significant changes on hydration and pasting properties of brown rice flour were found during germination.
Abstract: The effect of germination time on physicochemical characteristics of brown rice flour and its effect on gluten free bread qualities have been investigated. Germination was carried out at 28 °C and 100 % RH for 12, 24 and 48 h; brown rice and soaked brown rice was also analyzed. Significant changes on hydration and pasting properties of brown rice flour were found during germination. The starch degradation by enzyme activity could be evidenced with the decrease in viscosity and water binding capacity (WBC). No significant effect in specific volume, humidity and water activity of the gluten free bread was found as germination time increase, but a significant softness of the crumb was obtained. However, at 48 h of germination, the intense action of α amylase could result in excessive liquefaction and dextrinisation, causing inferior bread quality. Overall, germinated rice flour showed appropriate functionality for being used as raw ingredient in gluten free breadmaking.
TL;DR: In this paper, the authors evaluated the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease, using in vitro batch digestion with simulated oral, gastric, and duodenal phases.
Abstract: cope
Resistance of proteins to gastrointestinal digestion may play a role in determining immune-mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood.
Methods and results
Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS-PAGE and immunoblotting using monoclonal antibodies specific for celiac-toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases.
Conclusion
The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten-starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices.
TL;DR: In this article, the effects of dietary inulin on growth performance, diet utilization, survival rate, carcass composition and digestive enzymes activities (amylase, lipase and protease) of carp (Cyprinus carpio) fry were investigated.
Abstract: This study was conducted to investigate the effects of dietary inulin on growth performance, diet utilization, survival rate, carcass composition and digestive enzymes activities (amylase, lipase and protease) of carp (Cyprinus carpio) fry (0.55 ± 0.02 g). After acclimation, fish were allocated into 9 tanks (40 fish per tank) and triplicate fish groups were fed, control diet (0 g) or diets containing 5 g and 10 g inulin kg−1 for 7 weeks. No significant effect on growth performance and diet utilization of fish fed inulin compared with the control group was observed. However, supplementation of inulin significantly increased survival rate and carcass lipid content, while carcass protein content significantly decreased. Dietary inulin had no significant effects on digestive lipase, protease and amylase activities.
TL;DR: The digestion behaviour showed that enzyme treated starch particle had a low digestion rate, which would help designing highly branched nano-particles as a potential delivery carrier for functional components.
TL;DR: Results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.
Abstract: A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.
TL;DR: Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2-independent, which may have potential applications in starch-to-ethanol conversion process.
Abstract: Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0–5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0–9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process.
TL;DR: The results suggest that the maltogenic amylase could play a powerful role in inhibiting the staling of baked products even at limited starch hydrolysis.
Abstract: Maltogenic α-amylase is widely used as an antistaling agent in bakery foods. The objective of this study was to determine the degree of hydrolysis (DH) and starch structure after maltogenic amylase treatments in relation to its retrogradation. Waxy maize starch was cooked and hydrolyzed to different degrees by a maltogenic amylase. High-performance anion-exchange chromatography and size exclusion chromatography were used to determine saccharides formed and the molecular weight (Mw) distributions of the residual starch structure, respectively. Chain length (CL) distributions of debranched starch samples were further related to amylopectin (AP) retrogradation. Differential scanning calorimetry (DSC) results showed the complete inhibition of retrogradation when starches were hydrolyzed to >20% DH. Mw and CL distributions of residual AP structure indicated that with an increase in %DH, a higher proportion of unit chains with degree of polymerization (DP) ≤9 and a lower proportion of unit chains with DP ≥17 were formed. A higher proportion of short outer AP chains that cannot participate in the formation of double helices supports the decrease in and eventual inhibition of retrogradation observed with the increase in %DH. These results suggest that the maltogenic amylase could play a powerful role in inhibiting the staling of baked products even at limited starch hydrolysis.
TL;DR: An evolutionary approach is presented by considering salivary amylase in the animal kingdom with special focus on mammalians divided into the three main nutritional types carnivores, herbivores, and omnivores; it is hypothesized that nutritional habits affect expression of enzymes in the saliva of animals.
TL;DR: The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-SacCharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study.
Abstract: During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.
TL;DR: The evidence from this study suggests that Cys alleviates salt-induced growth inhibition and suppresses oxidative damage in germinating barley seeds by modulating cellular redox status due to its own antioxidant property.
Abstract: This research was an attempt to study the effects of exogenous cysteine (Cys) on early seedling growth, amylase activity, enzymatic and non-enzymatic antioxidant systems, reactive oxygen species (ROS), oxidative stress parameters and DNA damage of germinating barley seeds under salt stress (125 mmol l−1 NaCl). Salt stress markedly reduced root elongation and coleoptile growth; these changes could be alleviated by Cys application. Amylase activity exhibited results parallel to early seedling growth. The observed salinity-mediated reduction in amylase activity was markedly impeded by Cys. Isozyme profile confirmed the mitigating effect of Cys on salt-induced amylase inhibition. ROS production was significantly elevated under salt stress. Changes in lipid peroxidation level and DNA damage coincided with high levels of ROS. However, Cys significantly reduced salt-induced ROS production and mitigated oxidative damage to membranes and genetic material. This mitigating effect of Cys was related to low level of ROS as a conclusion its own antioxidant properties rather than its effect on enzymatic and non-enzymatic antioxidants. We found that although Cys application resulted in different effects on antioxidant system in root and coleoptiles, it had partial reducing effect as compared to stressed seedlings in both organs (especially roots). These results were supported by the isozymes activity profiles. Despite the partial reduction determined in antioxidant system in Cys-applied seedlings, low ROS levels revealed that Cys decreases the need for activation of antioxidant system by acting as a ROS scavenger. The evidence from this study suggests that Cys alleviates salt-induced growth inhibition and suppresses oxidative damage in germinating barley seeds by modulating cellular redox status due to its own antioxidant property.
TL;DR: Exercise may not only lead to the anticipated increase in amylase and protein secretion, but also to an increase in salivary flow rate and MUC5B secretion.
TL;DR: Findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.
TL;DR: Results from this experiment show that corn-soy diets having phytase and supplemented with amylase and xylanase led to increased growth performance, AMEn, and starch digestibility in broilers.
TL;DR: In this article, the orange oil-in-water (O/W) emulsion stabilized by Octenylsuccinic anhydride (OSA) modified waxy maize starch (OS-starch) was investigated.