Raw Starch Research Articles

Metabolomic and transcriptomic responses induced in the livers of pigs by the long-term intake of resistant starch.

The present study investigated metabolomic and transcriptomic responses in the livers of pigs to evaluate the effects of resistant starch on the body's metabolism at the extraintestinal level. Thirty-six Duroc× Landrace × Large White growing barrows (70 d of age) were randomly allocated to either the corn starch (CS) group or the raw potato starch (RPS) group with a randomized complete block design; each group consisted of 6 replicates (pens), with 3 pigs per pen. Pigs in the CS group were offered a corn-soybean-based diet, whereas pigs in the RPS group were put on a diet in which 230 (growing) or 280 g/kg (finishing) purified CS was replaced with purified RPS during a 100-d trial. The livers of pigs were collected for metabolome and gene expression analysis. Gas chromatography-mass spectrometry analysis showed that compared with the CS diet, the RPS diet decreased ( < 0.05) cholesterol and palmitic acid as well as increased ( < 0.05) 3-hydroxybutyric acid, which indicated the reduction of adipose weight and fatty acid biosynthesis and the elevation of fatty acid β-oxidation. In addition, 2-ketoglutaric acid and glucose-6-phosphate were increased (< 0.05) although pyruvic acid was decreased ( < 0.05) in the RPS group, indicating the upregulated capacity of glucose phosphorylation and glycolysis. Microarray analysis showed that the mRNA expression of (), (), and () were downregulated ( < 0.05) whereas (), (), and () were upregulated ( < 0.05) in the RPS diet, indicating a decrease in fatty acid intake and synthesis and an increase in fatty acid oxidation and glycerophospholipid synthesis. The results demonstrated that the long-term consumption of RPS could modulate hepatic lipid metabolism by decreasing fatty acid synthesis as well as increasing lipid oxidation and glycerophospholipid synthesis.

Glucoamylase from the Predacious Fungus Arthrobotrys conoides: a Cationic Enzyme with High Debranching Activity and Raw Starch Digestibility

The extracellular amylolytic activity elaborated by the nematophagous fungus Arthrobotrys conoides was found to resolve into 2 amylolytic peaks when fractionated on Sephadex G-100 column. Around 80% of the eluted glucoamylase activity was attributed to peak I (GA A). GA A being cationic in nature was purified about 70-fold with 57% yield by negative chromatography on DEAE Sephadex at pH 7.0. The enzyme was stable over a broad pH range of 4.8–9.0. KM for the linear polysaccharide amylose was 0.34 mg/mL. Enzyme showed high affinity for the branched polysaccharides as the KM values for amylopectin, glycogen and starch were 0.056, 0.062 and 0.065 mg/mL, respectively. The enzyme clearly demonstrated raw starch digestibility. Probable involvement of Trp and His residues in enzyme catalysis was elucidated using group-specific reagents.

Screening and Selection of Medium Components for Cyclodextrin Glucanotransferase Production by New Alkaliphile Microbacterium terrae KNR 9 Using Plackett-Burman Design.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) production using new alkaliphile Microbacterium terrae KNR 9 was investigated by submerged fermentation. Statistical screening for components belonging to different categories, namely, soluble and raw starches as carbon sources, complex organic and inorganic nitrogen sources, minerals, a buffering agent, and a surfactant, has been carried out for CGTase production using Plackett-Burman factorial design. To screen out k (19), number of variables, k + 1 (20), number of experiments, were performed. Among the fourteen components screened, four components, namely, soluble starch, corn flour, yeast extract, and K2HPO4, were identified as significant with reference to their concentration effect and corresponding p value. Although soluble starch showed highest significance, comparable significance was also observed with corn flour and hence it was selected as a sole carbon source along with yeast extract and K2HPO4 for further media optimization studies. Using screened components, CGTase production was increased to 45% and 87% at shake flask level and laboratory scale fermenter, respectively, as compared to basal media.

Acha ( Digitaria exilis ) Malt as a Source of Enzyme for Bio-Ethanol Production from Starchy Materials

Three acha varieties were malted and their enzyme activities were compared with that of barley malt. During the malting, the maximum amylase activity was achieved on day 3 for white and black acha but in the case of brown acha, the maximum activity was obtained on day 4. Under the optima malting conditions, all the three acha varieties gave higher amylase activity than barley malt. Of the three varieties of acha, white acha gave the highest glucoamylase activity (50 U/ml) and the least was exhibited by barley malt (26 U/ml). Application of malted acha as a source of enzyme for simultaneous hydrolysis and ethanol fermentation of two substrates (cassava and corn flour) was investigated under various conditions. Cassava flour yielded higher ethanol than corn flour under all the conditions investigated. Comparison of acha malt with koji enzymes for simultaneous hydrolysis and ethanol fermentation under various conditions showed that acha malt enzymes are superior to koji enzymes (microbial enzymes) under the three conditions investigated. Furthermore, regardless of the substrate used, gelatinization of starch improved ethanol productivity and yields. Incubating a mixture of raw starch and acha malt at 50 o C for 30 minutes before inoculation of yeast cells improved the ethanol production but the final ethanol concentration was not as high as the value obtained with gelatinized starch. These results have demonstrated that acha malt has great potential application in brewery and ethanol production industries. Keywords: Acha malt, Koji enzymes, Bio-ethanol, Cassava flour, Corn flour

Effect of Interacting Organic Co-solutes with Enzyme Substrate Complex on the Hydrolysis of Raw Soluble Starch with α-amylase: Theory and Experimentation

Effect of Interacting Organic Co-solutes with Enzyme Substrate Complex on the Hydrolysis of Raw Soluble Starch with α-amylase: Theory and Experimentation

The Effect of Additives and Temperature on the Velocity of Hydrolysis of Raw Starch with Human Salivary α - amylase

The Effect of Additives and Temperature on the Velocity of Hydrolysis of Raw Starch with Human Salivary α - amylase

Physicochemical properties and digestibility of eleven Vietnamese rice starches with varying amylose contents.

The physicochemical properties of eleven Vietnamese rice starches with apparent amylose contents ranging from 0.2% to 28.4% were investigated to identify the interplaying factors contributing to the in vitro digestibility of starch: in uncooked, cooked in excess water and under limited water conditions. The amylose content of rice starches governed the physicochemical properties of starch such as swelling power, pasting and gelatinization of starch which strongly correlated with raw starch digestibility. Amylopectin predominantly contributed to the crystallinity of starches both in short range and long range orders (observed by FT-IR and X-ray diffraction respectively). This study found that cooking destructively changed the molecular structure of starch that lead to an increase in the rate of starch digestion. Cooking in excess water resulted in a higher rate of starch digestion at least by 2 fold in all examined starches, suggesting that under these cooking conditions, factors that would normally limit the rate of digestion were eliminated. Cooking in limited water that was similar to domestic cooking conditions, seemed to allow rice starch granules to rearrange and interact together and limited the rate of digestion compared to cooking in excess water. It can be concluded that the physicochemical properties of starch can predict the digestibility of raw starches only but not cooked starches. The digestion of rice starch cooked in limited water cannot be predicted by just studying the uncooked starches or cooking in excess water.

Overcoming hydrolysis of raw corn starch under industrial conditions with Bacillus licheniformis ATCC 9945a α-amylase.

α-Amylase from Bacillus licheniformis ATCC 9945a (BliAmy) was proven to be very efficient in hydrolysis of granular starch below the temperature of gelatinization. By applying two-stage feeding strategy to achieve high-cell-density cultivation of Escherichia coli and extracellular production of BliAmy, total of 250.5 U/mL (i.e. 0.7 g/L) of enzyme was obtained. Thermostability of amylase was exploited to simplify purification. The hydrolysis of concentrated raw starch was optimized using response surface methodology. Regardless of raw starch concentration tested (20, 25, 30 %), BliAmy was very effective, achieving the final hydrolysis degree of 91 % for the hydrolysis of 30 % starch suspension after 24 h. The major A-type crystalline structure and amorphous domains of the starch granule were degraded at the same rates, while amylose-lipid complexes were not degraded. BliAmy presents interesting performances on highly concentrated solid starch and could be of value for starch-consuming industries while response surface methodology (RSM) could be efficiently applied for the optimization of the hydrolysis.

Evaluation of a recombinant insect-derived amylase performance in simultaneous saccharification and fermentation process with industrial yeasts.

Starch is the dominant feedstock consumed for the bioethanol production, accounting for 60 % of its global production. Considering the significant contribution of bioethanol to the global fuel market, any improvement in its major operating technologies is economically very attractive. It was estimated that up to 40 % of the final ethanol unit price is derived from the energy input required for the substrate pre-treatment. Application of raw starch hydrolyzing enzymes (RSHE), combined with operation of the process according to a simultaneous saccharification and fermentation (SSF) strategy, constitutes the most promising solutions to the current technologies limitations. In this study, we expressed the novel RSHE derived from an insect in Saccharomyces cerevisiae strain dedicated for the protein overexpression. Afterwards, the enzyme performance was assessed in SSF process conducted by industrial ethanologenic or thermotolerant yeast species. Comparison of the insect-derived RSHE preparation with commercially available amylolytic RSH preparation was conducted. Our results demonstrate that the recombinant alpha-amylase from rice weevil can be efficiently expressed and secreted with its native signal peptide in S. cerevisiae INVSc-pYES2-Amy1 expression system (accounting for nearly 72 % of the strain’s secretome). Application of the recombinant enzyme-based preparation in SSF process secured sufficient amylolytic activity for the yeast cell propagation and ethanol formation from raw starch. (Oligo)saccharide profiles generated by the compared preparations differed with respect to homogeneity of the sugar mixtures. Concomitantly, as demonstrated by a kinetic model developed in this study, the kinetic parameters describing activity of the compared preparations were different.

Granule Size and Distribution of Raw and Germinated Oat Starch in Solid State and Ethanol Solution

The morphology and granular properties of oat starch during germination were investigated by scanning electron microscopy and X-ray powder diffractometer. The granule size distribution of oat starch was studied in various concentrations in ethanol using a laser particle-size analyzer. The starch granule shape before germination remained almost the same after germination, as shown by low-magnification (×1000) scanning electron microscopy. However, surface fissures at high-magnification (×5000) were found on the sample at the end of germination. After soaking, the relative crystallinity of the samples significantly reduced, by following increased and then decreased, reaching a maximum crystallinity of 16% at the end of germination. In various ethanol concentrations, the size of starch granules produced in different germination phase slightly varied, but this difference was not easy to detect in the scanning electron microscope ×1000 images. Overall, the particle size of oat starch generally decreased and the specific surface area increased significantly during oat germination.

Synthesis of large-ring cyclodextrin from tapioca starch by amylomaltase and complex formation with vitamin E acetate for solubility enhancement

Recombinant amylomaltase from Corynebacterium glutamicum (CgAM) was used to synthesize large-ring cyclodextrins (LR-CDs) from tapioca starch. A mixture of LR-CDs (CD22–CD54) was obtained from both raw and soluble tapioca starch, but with different ratio of main products (CD25–CD30). Almost three-fold higher amount of products was formed indicating the preference for raw tapioca starch. The optimal condition was to incubate 1.0% (w/v) starch with 0.05U/ml CgAM in buffer pH 6.0 at 30°C for 18h. Total LR-CDs yield was 60.5mg/g starch. LR-CDs were resistant to hydrolysis by Rhizopus sp. glucoamylase but completely hydrolyzed by CgAM at high enzyme concentration. Solid inclusion complex between LR-CDs mixture and vitamin E acetate was formed, as analyzed by FTIR. For complex preparation, the 1:10mol ratio of LR-CDs to vitamin E was chosen because maximum amount of the vitamin was bound to LR-CDs and high complex yield was obtained. Aqueous solubility of vitamin E acetate in complex form increased 800-fold while the antioxidant activity decreased about 30%. This is the first work to demonstrate the use of tapioca starch in LR-CDs synthesis by CgAM and the complex formation between LR-CDs and vitamin E acetate as solubility enhancement process of vitamin.

Cover: Journal of Basic Microbiology. 11/2015

Journal of Basic MicrobiologyVolume 55, Issue 11 Cover PictureFree Access Cover: Journal of Basic Microbiology. 11/2015 First published: 26 October 2015 https://doi.org/10.1002/jobm.201570111AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Bacillus licheniformis α-amylase is active on starch, e.g. jackfruit seeds raw starch (background image). The enzyme structure was revealed from B. licheniformis AS08E and B. licheniformis NH1. The superimposed structure reveals metal binding domains and catalytic sites containing the substrate analog acarbose. (Photo and image: J.K. Roy, Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur (Assam), India) Volume55, Issue11November 2015 RelatedInformation

Simultaneous liquefaction, saccharification and fermentation at very high gravity of rice at pilot scale for potable ethanol production and distillers dried grains composition

In this work, a simultaneous liquefaction, saccharification, and fermentation (SLSF) process at very high gravity (VHG) of broken rice for potable ethanol production was developed at pilot scale. The SLSF–VHG process was performed in a unique fermenter, at 30°C. Rice flour (RF) was dissolved in tap water to reach 311.5g/l dry matter and then the mixture was simultaneousely liquefied, saccharified, and fermented. Thanks to the addition of a raw starch hydrolyzing enzyme containing a mixture of alpha-amylase and gluco-amylase (Stargen 002 at 991.8 GAU/kg RF), gluco-amylase (Amigase Mega L at 0.035% w/w), protease (Fermgen at 600 SAPU/kg RF), active dry yeast Saccharomyces cerevisiae (Red Ethanol at 3.5×107cells/ml), KH2PO4 (4.8mM), and urea (16.0mM). Under these conditions, the SLSF–VHG process finished after 120h and achieved an ethanol content of 17.6% v/v corresponding to 86.3% of the theoretical ethanol yield. We scaled up this SLSF process at very high gravity at pilot scale (25l) and achieved an ethanol content of 17.0% v/v corresponding to a yield of 83.2% of the theoretical ethanol yield. Rice-based distillers dried grains (DDG) was produced from the whole stillage of SLSF process at very high gravity by being plate-filtered and dried. The obtained DDG had high contents of crude protein (47.5%) and fibers (15.8%). Our results suggest, the SLSF under VHG condition of broken rice as well as the recovery of protein-rich DDG could have a great potential for the ethanol and animal feeding industry in Vietnam.

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases.

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol

The major bottlenecks in achieving competitive bioethanol fuel are the high cost of feedstock, energy and enzymes employed in pretreatment prior to fermentation. Lignocellulosic biomass has been proposed as an alternative feedstock, but because of its complexity, economic viability is yet to be realized. Therefore, research around non-conventional feedstocks and deployment of bioconversion approaches that downsize the cost of energy and enzymes is justified. In this study, a non-conventional feedstock, inedible wild cassava was used for bioethanol production. Bioconversion of raw starch from the wild cassava to bioethanol at low temperature was investigated using both a co-culture of Aspergillus sp. and Saccharomyces cerevisiae, and a monoculture of the later with enzyme preparation from the former. A newly isolated strain of Aspergillus sp. MZA-3 produced raw starch-degrading enzyme which displayed highest activity of 3.3 U/mL towards raw starch from wild cassava at 50°C, pH 5.5. A co-culture of MZA-3 and S. cerevisiae; and a monoculture of S. cerevisiae and MZA-3 enzyme (both supplemented with glucoamylase) resulted into bioethanol yield (percentage of the theoretical yield) of 91 and 95 at efficiency (percentage) of 84 and 96, respectively. Direct bioconversion of raw starch to bioethanol was achieved at 30°C through the co-culture approach. This could be attractive since it may significantly downsize energy expenses.

Production of pullulan from raw potato starch hydrolysates by a new strain of Auerobasidium pullulans

In the present study, hydrolysis of potato starch with marine cold-adapted α-amylase and pullulan production from the hydrolysates by a new strain of Auerobasidium pullulans isolated from sea mud were conducted. The hydrolysis conditions were optimized as follows: reaction time 2h, pH 6.5, temperature 20°C, and α-amylase amount 12U/g. Under these optimum hydrolysis conditions, the DE value of the potato starch hydrolysates reached to 49.56. The potato starch hydrolysates consist of glucose, maltose, isomaltose, maltotriose, and trace of other maltooligosaccharides with degree of polymerization ranged 4–7. The maximum production of pullulan at 96h from the hydrolysate of potato starch was 36.17g/L, which was higher than those obtained from glucose (22.07g/L, p<0.05) and sucrose (31.42g/L, p<0.05). Analysis of the high performance liquid chromatography of the hydrolysates of the pullulan product with pullulanase indicated that the main composition is maltotriose, thus confirming the pullulan structure of this pullulan product.

Engineering Yarrowia lipolytica to produce biodiesel from raw starch.

BackgroundIn the last year, the worldwide concern about the abuse of fossil fuels and the seeking for alternatives sources to produce energy have found microbial oils has potential candidates for diesel substitutes. Yarrowia lipolytica has emerged as a paradigm organism for the production of bio-lipids in white biotechnology. It accumulates high amounts of lipids from glucose as sole carbon sources. Nonetheless, to lower the cost of microbial oil production and rival plant-based fuels, the use of raw and waste materials as fermentation substrate is required. Starch is one of the most abundant carbohydrates in nature and it is constituted by glucose monomers. Y. lipolytica lacks the capacity to breakdown this polymer and thus expensive enzymatic and/or physical pre-treatments are needed.ResultsIn this work, we express heterologous alpha-amylase and glucoamylase enzymes in Y. lipolytica. The modified strains were able to produce and secrete high amounts of active form of both proteins in the culture media. These strains were able to grow on starch as sole carbon source and produce certain amount of lipids. Thereafter, we expressed both enzymes in an engineered strain able to overaccumulate lipids. This strain was able to produce up to 21 % of DCW as fatty acids from soluble starch, 5.7 times more than the modified strain in the wild-type background. Media optimization to increase the C/N ratio to 90 increased total lipid content up to 27 % of DCW. We also tested these strains in industrial raw starch as a proof of concept of the feasibility of the consolidated bioprocess. Lipid production from raw starch was further enhanced by the expression of a second copy of each enzyme. Finally, we determined in silico that the properties of a biodiesel produced by this strain from raw starch would fit the established standards.ConclusionsIn this work, we performed a strain engineering approach to obtain a consolidated bioprocess to directly produce biolipids from raw starch. Additionally, we proved that lipid production from starch can be enhanced by both metabolic engineering and culture condition optimization, setting up the basis for further studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0335-7) contains supplementary material, which is available to authorized users.

THE IMPORTANCE OF DIETARY CARBOHYDRATE IN HUMAN EVOLUTION.

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. Salivary amylases are largely ineffective on raw crystalline starch, but cooking substantially increases both their energy-yielding potential and glycemia. Although uncertainties remain regarding the antiquity of cooking and the origins of salivary amylase gene copy number variation, the hypothesis we present makes a testable prediction that these events are correlated.

Responses in colonic microbial community and gene expression of pigs to a long-term high resistant starch diet.

Intake of raw potato starch (RPS) has been associated with various intestinal health benefits, but knowledge of its mechanism in a long-term is limited. The aim of this study was to investigate the effects of long-term intake of RPS on microbial composition, genes expression profiles in the colon of pigs. Thirty-six Duroc × Landrace × Large White growing barrows were randomly allocated to corn starch (CS) and RPS groups with a randomized block design. Each group consisted of six replicates (pens), with three pigs per pen. Pigs in the CS group were offered a corn/soybean-based diet, while pigs in the RPS group were put on a diet in which 230 g/kg (growing period) or 280 g/kg (finishing period) purified CS was replaced with purified RPS during a 100-day trial. Real-time PCR assay showed that RPS significantly decreased the number of total bacteria in the colonic digesta. MiSeq sequencing of the V3-V4 region of the 16S rRNA genes showed that RPS significantly decreased the relative abundance of Clostridium, Treponema, Oscillospira, Phascolarctobacterium, RC9 gut group, and S24-7-related operational taxonomic units (OTUs), and increased the relative abundance of Turicibacter, Blautia, Ruminococcus, Coprococcus, Marvinbryantia, and Ruminococcus bromii-related OTUs in colonic digesta and mucosa. Analysis of the colonic transcriptome profiles revealed that the RPS diet changed the colonic expression profile of the host genes mainly involved in immune response pathways. RPS significantly increased proinflammartory cytokine IL-1β gene expression and suppressed genes involved in lysosome. Our findings suggest that long-term intake of high resistant starch (RS) diet may result in both positive and negative roles in gut health.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases.

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.