Raw Starch Research Articles

Starch saccharification and fermentation of uncooked sweet potato roots for fuel ethanol production

An energy-saving ethanol fermentation technology was developed using uncooked fresh sweet potato as raw material. A mutant strain of Aspergillus niger isolated from mildewed sweet potato was used to produce abundant raw starch saccharification enzymes for treating uncooked sweet potato storage roots. The viscosity of the fermentation paste of uncooked sweet potato roots was lower than that of the cooked roots. The ethanol fermentation was carried out by Zymomonas mobilis, and 14.4g of ethanol (87.2% of the theoretical yield) was produced from 100g of fresh sweet potato storage roots. Based on this method, an energy-saving, high efficient and environment-friendly technology can be developed for large-scale production of fuel ethanol from sweet potato roots.

1H NMR spectroscopy for the in vitro understanding of the glycaemic index

The glycaemic index (GI) characterises foods by using the incremental area under the glycaemic response curve relative to the same amount of oral glucose. Its ability to differentiate between curves of different shapes, the peak response and other aspects of the glycaemic response is contentious. The present pilot study aimed to explore the possibility of using 1H NMR spectroscopy to better understand in vivo digestion characteristics as reflected in the glycaemic response of carbohydrate-rich foods; such an approach might be an adjunct to the in vivo GI test. The glycaemic response of two types of raw wheat flour (2005 from Griffith NSW, Chara, Row 10, Plot 6:181 and store-bought Colese Plain Flour) and a cooked store-bought flour was tested and compared with results recorded during the in vitro enzymatic digestion of the wheat flour samples by glucoamylase from Aspergillus niger (EC 3.2.1.3) as monitored by 1H NMR spectroscopy. Comparing the digestion time courses of raw and cooked wheat starch recorded in vitro strongly suggests that the initial rate of glucose release in vitro correlates with the glycaemic spike in vivo. During the in vitro time courses, approximately four times as much glucose was released from cooked starch samples than from raw starch samples in 90 min. Monitoring enzymatic digestion of heterogeneous mixtures (food) by 1H NMR spectroscopy showcases the effectiveness of the technique in measuring glucose release and its potential use as the basis of an in vitro method for a better understanding of the GI.

The Nature of Raw Starch Digestion

The Nature of Raw Starch Digestion

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

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.

Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans

A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650 bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723 U mg−1 protein with a molecular mass of 59 kDa. It is optimally active at pH 5.0 and 80 °C with t1/2 values of 283, 184 and 56 min at 70, 80 and 90 °C, respectively. The activation energy required for its temperature deactivation is 84.96 kJ mol−1. Ca2+ strongly inhibits Gt-amy at 10 mM concentration, and inhibition kinetics with Ca2+ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca2+ binding site in the active centre in a mixed-type inhibition manner. The Km and kcat of the Gt-amy are 0.315 mg mL−1 and 2.62 × 103 s−1, respectively. Gt-amy is Ca2+-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures.

The <i>in vitro</i> caecal fermentation of different starch sources in rabbits

The objective of our study was to extend the knowledge of microbial fermentation of starch in the rabbit caecum using the in vitro gas production technique. Different sources of starch (wheat and maize grain, raw and cooked potato) and different starch isolates (wheat, maize and potato starch) were incubated with inoculum, prepared from rabbit caecum content. The gas production parameters such as total potential gas production (parameter “B”), gas production till 10 h of incubation (Gas10), maximum fermentation rate (MFR) and time of maximum fermentation rate (TMFR) were estimated with Gompertz model and were significantly affected by the substrata. Differences between maize grain and maize starch were small: MFR and TMFR were not different, while Gas10 of maize starch was higher than from maize grain (5 and 3 ml/kg DM, respectively; P < 0.05). MFR of wheat starch was higher than that of wheat grain (3.1 and 2.4 ml/h, respectively; P < 0.05), but reached maximum rate later (TMFR: 12.4 and 10.9 h, respectively; P < 0.05) and had lower Gas10 (6 and 9 ml/kg DM, respectively; P < 0.05). The fermentation of cooked potato reached the TMFR after 8.5 h of incubation and had the highest MFR (2.9 ml/h) and Gas10 (16 ml/kg DM) of all substrata. The MFRs of raw potato and potato starch were similar (2.1 and 2.3 ml/h, respectively), although TMFR and Gas10 of raw potato were shorter and higher than those of potato starch (TMFR of 10.7 and 17.2 h and Gas10 of 10 and 1 ml/kg DM, respectively; P < 0.05).

Xanthan gum production by Xanthomonas campestris pv. campestris 8004 using cassava starch as carbon source

Cassava starch is a main renewable bio-resource with low price and mass production in Guangxi, China. It was used as carbon source in growing Xanthomonas campestris pv. campestris 8004 (Xcc 8004) for xanthan gum production in this study. The xanthan gum yield of gelatinized cassava starch was higher than that of raw cassava starch; the maximal reducing sugar and amylase activity reached 14.30 g/L and 1.19U/ml, respectively. The maximal xanthan gum yield of 16.95 g/L was achieved with 30 g/L cassava starch as carbon source. The concentration of residual sugar was 6.82 g/L at the end of fermentation, and the conversion efficiency of sugar reached 73.12%. Thus, Xcc 8004 should be used as a competitive strain for xanthan gum production using cassava starch in industrial applications. Keywords: Xanthan gum, cassava starch, Xanthomonas campestris pv. campestris 8004, amylase activity

Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

Engineering yeasts for raw starch conversion

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10% of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future.

Synthesis of Starch-Graft-Polyacrylamide by Salt Solution Media Polymerization

Starch-graft-polyacrylamide (St-g-PAM) was prepared using a salt media polymerization technique, and characterized by FITR. The type of starch and the synthesis method of different system were studied through the mass of product, the percentage of grafting (PG) and the monomer conversion (MC), and the difference polymerization system. The results showed that the pretreated starch and the salt solution system, respectively, were more advantageous than the raw starch and the aqueous solution system. The intrinsic viscosity, transmittance, product loss, penetration and apparent viscosity of St-g-PAM by salt solution polymerization were 12.49 dL•g-1, 18.6%, 0.56%, 53.96, 18.40 Pa•S, respectively. The method could get bigger branched-chain molecular than the others with high yield, high monomer conversion, high the percentage of grafting, low product losses and easy post-processing

Physical Characterisation of Millet Starch during Roasting

The gelatinization and liquefaction properties of roasted millet starch were investigated in this study. Results showed that the particle morphology of roasted millet starch greatly changed as compared with control, and its solubility and swelling degree increased with temperature increment, in which the former increased rapidly above 70°C and was basically stable after 80°C, while the latter was overall lower than the raw millet starch's. The viscosity of millet starch paste increased with decreasing temperature, but that of the roasted starch increased more slowly than that of the raw. There was little effect of pH value on viscosity of roasted millet starch. Compared with the raw, the roasted millet starch would be firstly reached scheduled DE value under the same conditions, which could reduce liquefaction time in the course of production.

Optimization of the Preparation Technology of Resistant Starch of Cowpea Using Response Surface Methodology

Response surface methodology was used to optimize the preparation technology of resistant starch (RS) production by raw cowpea bean starch. In the first optimization step, single factor experiments designed was used to evaluate the influence of RS yield. The RS yield were influenced significantly by some factors of preparation RS, including the starch concentration, autoclaving time, pullulanase dosage and enzymolysis temperature. The others in the investigation scope had no significant influence on the RS production. In the last step, four main factors were further optimized using Box-Behnken designs and response surface analysis. The optimized conditions in the process of preparation RS were starch concentration as 29%, autoclaving time as 38min, pullulanase dosage as 4.0PUN/g, enzymolysis temperature as 60°C. In our optimal conditions, rather good RS yield was 23.52±0.15% and repeatability of the preparation process was good which was valuable in further production.

Preparation and Characterization of Enzymatically-Treated Granular Cassava Starch and Poly(butylene adipate-co-terephthalate) Blends

Raw cassava starch was treated with α-amylase and amyloglucosidase in aqueous solution under annealing condition to obtain starch granules with rough and porous surfaces. Many different pits and pores formed by the activity of the enzymes on the surface granules and were observed by scanning electron microscopy (SEM). The obtained starch granules with rough surfaces were mechanically blended with poly(butylenes adipate-co-terephthalate)(PBAT) at different ratios by using a single screw extruder. The results showed that the samples comprised of enzymatically treated starch blends had higher elongation than those comprised of untreated starch blends. At 10% starch content, the treated starch/PBAT blend had about 37.55% more elongation than the untreated starch/PBAT blend. This resulted in the improved compatibility of the starch granules and PBAT matrix in the blends as confirmed by SEM.

Salt-independent thermophilic α-amylase from Bacillus megaterium VUMB109: An efficacy testing for preparation of maltooligosaccharides

An amylase (est. Mw 150kDa) was purified 27.39-folds from the culture broth of Bacillus megaterium VUMB109. The purified enzyme was not inhibited by p-chloromercuro benzoate and iodoacetamide (10mM), it rapidly decolorized the blue color of starch–iodine complex and produced α-anomeric products from starch hydrolysis, thus, it is an endo-attacking α-amylase. The enzymatic activity was not affected by any metal ion and EDTA, therefore, it is not in the class of metalloenzyme. The purified α-amylase showed higher affinity (Km=1.5μM; Vmax/Km=0.38 and Kcat/Km=2.5×106) to starch than other tested substrates like amylose, amylopectin and glycogen. Maltooligomer mixture with high proportion of maltopentaose (G5) and maltotriose (G3) was produced during hydrolysis of starch, amylopectin and amylose. It exhibited high degree of hydrolysis on raw potato starch than wheat, rice and corn starches. Thus the studied α-amylase could be exploited as a useful catalyst in the bioprocessing of maltooligomer mixture as food supplement for baby and aged people.

Influence of different carbohydrate composition in barley varieties on Salmonella Typhimurium var. Copenhagen colonisation in a “Trojan” challenge model in pigs

Based on previously performed in vitro studies, which showed that hulless barley varieties could reduce large intestinal Salmonella Typhimurium var. Copenhagen proliferation in pigs, two in vivo experiments were conducted to prove these observations. In Experiment (Exp.) 1, 126 weaning piglets were randomly allocated into pens of seven animals each and fed one of six experimental diets. Three diets contained (75% as-fed) one of three hulless barley varieties with β-glucan (BG) contents ranging from 5 to 11% and amylose from 5 to 40%, and two diets contained a low BG and amylose hulless barley supplemented with isolated barley BG or raw potato starch. A hulled barley diet served as a control. Two piglets per pen (“Trojan” pigs) were orally infected with Salmonella Typhimurium var. Copenhagen (ST). The remaining five pigs per pen were designated “Contact” pigs. The ST shedding was determined over one week after infection. On day 6, the two Trojans and two random Contacts from each pen were euthanised and intestinal contents and mesenteric lymph nodes cultured for ST. Intestinal volatile fatty acids and microbial composition were determined. In Exp. 2, 126 piglets were assigned to one of three diets based on hulled or hulless barleys. The timeline, infection, sampling and analyses were similar as in Exp. 1 except samples were taken from four Contact pigs. Hulless barley varieties with high BG and amylose tended to decrease ST persistence in Exp. 1. Clostridia from cluster I in the colon were reduced with high amylose hulless barley or diets supplemented with potato starch (p < 0.05), whereas other microbial groups were not. Propionate increased (p < 0.05) and acetate decreased (p < 0.05) with hulless barley inclusion. Exp. 2 revealed a reduced ST shedding and reduced number of clostridia for high BG hulless barley as compared to common hulled barley and a low BG variety (p < 0.05). In conclusion, high BG hulless barley do not prevent ST colonisation but might help to reduce transmission in pigs, likely by supporting an intestinal environment limiting growth of this zoopathogen.

Preferential and rapid degradation of raw rice starch by an α-amylase of glycoside hydrolase subfamily GH13_37

The α-amylase (AmyP) from a marine metagenomic library belongs to the recently classified glycoside hydrolase subfamily GH13_37. The degradation abilities of AmyP on a broad range of raw starch granules were examined at 40 °C and pH 7.5. It was found that AmyP is a raw starch-degrading enzyme, exhibiting a unique and remarkable ability to preferentially and very rapidly digest raw rice starch. The specific activity of raw rice starch was reached 118.5 ± 0.6 Umg(-1), which was much higher than that of other raw starches. The final hydrolysis degrees were obtained in 4 h for 1 % raw rice starch and 1 h for 8 % concentration, indicating a very rapid speed of hydrolysis. The presence of a starch residue resistant was the main limiting factor for complete hydrolysis, although end product inhibition also existed, especially at high starch concentrations. AmyP randomly attacks unique or susceptible sites on raw rice starch granules, and releases glucose, maltose, and maltotriose as end products. This is the first biochemical characterization of the raw starch-degrading ability of an α-amylase of family GH13_37. The specific ability towards raw rice starch has never been described before, and this makes AmyP a promising candidate for use as a novel enzyme in rice starch processing.

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75% of the theoretical maximum yield.

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5g/l of ethanol from 200g/l of raw corn starch after 120h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.

Flaking as a corn preparation technique for dry-grind ethanol production using raw starch hydrolysis

A 23 full-factorial study was designed to study the effect of corn preparation methods (flaking and grinding) on dry-grind ethanol performance using raw starch hydrolysis (RSH) process. Moisture content (15, 22%), flaker roller gapsetting (0.508 mm, 1.016 mm), and grinding were studied. Fifteen hundred g of corn samples were cracked, roller pressed, and were either ground further or retained, along with control ground corn. A bimodal size distribution was observed for ground corn, regardless of flaking. Moisture at 22% resulted in bigger-sized flakes with d50 between ∼1.3 and 1.8 mm, compared to ∼138–169 μm for ground corn. Not all ground corn resulted in higher ethanol concentration in fermentation beer; the ethanol levels in beer did not reflect the starch hydrolysis trend that favored ground corn. In a related study, the beer ethanol concentration did not show a clear trend with rollermill gapsetting while fermenting the flakes produced at 0.203, 0.305, 0.406, and 0.508 mm gapsettings. Generally, flakes from corn at 22% moisture resulted in higher ethanol content in beer. Rollermill flaking was found comparable to hammermill grinding for dry-grind corn ethanol via raw starch hydrolysis and yeast fermentation.

Characterization of morphological forms of Aspergillus carbonarius and the effect of inoculum size on raw starch digesting amylase (RSDA) production

The effect of spore inoculum level and mycelia morphology on raw starch digesting amylase production by Aspergillus carbonarius in submerged culture was investigated. Shake flask cultures were inoculated directly with spore concentration range of 104 to 109spores/ml. Morphology was differentiated into, pellets, clumps, branched and free filaments. Results on the percentage morphological occurrences revealed that for all tested inoculum a gradual change in morphology was observed as innoculum level increased from 104 to 109spores/ml though pellets predominated. A decrease in the mean main and total hyphal length, mean hyphal growth unit and marginal increase in mean number of tips/mycelium was observed in the mycelia trees as inoculum levels increased from 104 to 109spores/ml. In the aggregated forms, there was a decrease in the mean area, perimeter, roughness and equivalent diameter while mean compactness increased as inoculum increased from 104 to 109 spores/ml. Irrespective of the predominating form a change in morphology was observed. RSDA production appeared to be related to spore inoculum level. Maximum yield was observed at fermentation inoculated with 104 and 105spores/ml where pellets were most dominant. The impact of spore inoculum size on the resulting mycelia morphology was marginal but had a significant effect on RSDA production. Key words: Aspergillus carbonarius, raw starch digesting amylase, fungal morphology and inoculum.