Initial Starch Research Articles

Production of 2,5‐furandicarboxylic acid (FDCA) from starch, glucose, or high‐fructose corn syrup: techno‐economic analysis

AbstractThis paper presents a techno‐economic analysis of a low‐cost and high‐efficiency technology for the production of 2,5‐furandicarboxylic acid (FDCA) from starch, glucose, or high‐fructose corn syrup (HFCS). With the design presented here, it is viable to convert starch to glucose through enzymatic hydrolysis followed by catalytic dehydration of glucose with niobium phosphate, an innovative low‐cost catalyst, to produce 5‐hydroxymethyl furfural (HMF). The HMF produced is then converted into FDCA via air oxidation over a cobalt‐manganese mixed oxide catalyst. Three variations of this design are assessed: Scenario 1 starts with starch, and Scenarios 2 and 3 start directly with glucose or HFCS, without the initial starch hydrolysis step. The minimum selling price (MSP) and discounted payback period (PBP) were calculated to investigate the feasibility of the scenarios. A sensitivity analysis was also performed for the key cost drivers, selling price of FDCA, and spent catalysts. All scenarios were feasible; however, the HFCS to FDCA process was the most profitable (MSP 1802 US$/t and a PBP below 5 years). Above all, the feasibility of this technology is mostly affected by variations in recovered catalysts and FDCA selling prices. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd.

Efficient butanol production using corn-starch and waste Pichia pastoris semi-solid mixture as the substrate

Corn starch is the traditional substrate for butanol production by Clostridium acetobutylicum. However, the production suffers the problems of high substrate price and low sugar utilization yield. Waste P.pastoris semi-solid enriches with carbohydrate/protein and could be potentially used as an alternative substrate. In this study, NaOH was used to treat the waste semi-solid into suspensions. The fermentations were implemented by largely reducing initial starch content, and then adding the suspensions into broth after fermentations entered solventogenesis phase. Using the mixed substrate, final butanol concentrations in a 7 L anaerobic fermentor could reach and stabilize around 9.5–10.5 g/L, which was equivalent to that using 15% pure corn-starch; total sugar utilization yield largely increased from 50% to more than 90%, as disaccharides/trisaccharides were effectively utilized; 53% carbohydrate in the semi-solid was effectively digested which was beneficial for waste amount reduction; 57% expensive corn-starch could be saved.

Dynamic Modeling of Carbon Metabolism During the Dormant Period Accurately Predicts the Changes in Frost Hardiness in Walnut Trees Juglans regia L.

The leafless period is often considered as inactive, although trees have to actively modulate their metabolism through the cold acclimation/deacclimation processes, to cope with frost exposure during winter and to restore growth ability in spring. Carbon metabolism is a key component of these processes through the osmotic control of extracellular ice formation and the trophic control of bud growth. The influence of temperature on the inter-conversion between starch and soluble carbohydrate has been evidenced for years, but we are currently missing an operational tool to predict starch vs. soluble carbohydrate contents during this period, which should allow to better predict frost hardiness. For this purpose, we exposed 1-year-old branches of Juglans regia to constant temperature for one to 3 weeks and measured the changes in carbohydrate composition at three periods (autumn, winter, and spring). As expected, the temperature significantly affected the changes in carbohydrate composition, but the water content and the sampling period were also relevant. Higher starch hydrolysis was observed at low temperature (<5°C) for all sampling periods. Starch hydrolysis was also observed at warm temperature, but in autumn only. These data were used to compare three modeling approaches simulating the changes in carbohydrate composition through enzymatic analogy. The most empirical and the most mechanistic approach did not succeed to simulate external observations (Root Mean Standard Error of Prediction (RMSEP) > 30 mg.g DM−1, Efficiency (Eff) <0), whereas the intermediate model was more efficient (RMSEP = 15.19 mg.g DM−1, Eff = 0.205 and 16.61 mg.g DM−1, Eff = 0.366, for GFS (Glucose + Fructose + Sucrose) and starch, respectively). The accuracy of the model was further improved when using field data for calibration (RMSEP = 5.86 mg.g DM−1, Eff = 0.962; RMSEP = 10.56 mg.g DM−1, Eff = 0.752, for GFS and starch, respectively). This study provided an operative tool to simulate carbohydrate dynamics over leafless period that could predict frost hardiness with approx. 3.4°C accuracy with temperature, water content and initial starch and soluble carbohydrate measurements. It should now be tested under various meteorological conditions and biological systems.

A new kinetic model for growth based on simultaneous substrate and biomass limitation in solid-state fermentation using agar spheres as the model substrate

Substrate limitation is a major concern in solid-state fermentation, especially for bacteria, as they are not capable of penetrating into the substrate particles. The use of a model substrate, such as agar spheres, makes it possible to study the intra-particle transfer phenomena in solid-state fermentation, reduce the system complexity, and determine the cell dry weight (CDW). A semi-mechanistic model was used to predict the growth of Pseudomonas aeruginosa on agar spheres that contain starch as the only carbon source. The model was developed by introducing a new simultaneous substrate- and biomass-limited growth kinetic equation. The parameters of the growth kinetic were estimated by fitting the experimental data of both the CDW and cumulative CO2 production on agar spheres containing glucose. Statistical analysis (F-test) showed that the new growth kinetic equation was significantly better than the conventional substrate-limited growth kinetics. The model predictions were validated by the experimental data of growth, starch concentration and cumulative CO2 production (CCP) of Pseudomonas aeruginosa growing on agar spheres containing starch in aerated small packed-bed bioreactors. The model confirmed that at high initial starch concentration, free glucose and O2 were not the limiting factors, and bacterial growth is limited by other factors that are related to the amount of biomass in the biofilm.

Enhanced production of exopolysaccharides using industrial grade starch as sole carbon source

Industrial grade soluble corn starch was used directly and effectively as the fermentation substrate for microbial exopolysaccharides production. Bacillus subtilis mutant strain NJ308 grew with untreated starch raw material as the sole carbon source. The real-time PCR results demonstrated that up-regulated genes encoding N-acetylglucosaminyltransferase, mannosyltransferase, and N-acetylglucosamine-1-phosphate uridyltransferase were the key elements of B. subtilis mutant strain NJ308 for exopolysaccharides production from industrial grade starch. Subsequently, the culture conditions for B. subtilis NJ308 were optimized using Plackett-Burman design and central composite design methods, and the related key genes in the synthesis pathway of exopolysaccharides from the starch raw material were analyzed by real-time PCR. The maximum exopolysaccharides titration (3.41g/L) was obtained when the initial starch concentration was 45g/L. This corresponds to volumetric productivity values of 71.04mg/Lh.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5–7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Pickering emulsions stabilized by media-milled starch particles

A simple, organic solvent-free media-milling process was applied to modify the structure and morphology of maize starch granules. During the milling process, the sizes of starch granules reduced dramatically while their crystalline structure was largely damaged as verified by differential scanning calorimetry and X-ray diffraction. The surface hydrophobicity of milled starch was also characterized by contact angle measurement. These media-milled starch suspensions with different starch concentrations (i.e., 2.4%, 7.2% and 10.0%) were used as stabilizers to form Pickering emulsions. The droplet sizes of the Pickering emulsions prepared in this study ranged from approximately 10μm to 900μm depending on the milling time and the initial starch concentration. The droplet sizes decreased with prolonged milling time and increased initial starch concentration. Milled starch particles of around 100nm anchored at the emulsion interfaces were observed by cryo-scanning electron microscopy. Although some emulsions creamed immediately after homogenization, most emulsions exhibited stability against coalescence during the long-term storage of 6months. The emulsions also exhibited benign stability against a wide range of pH, ionic strength and temperature conditions. This research provides a new green approach to form food-grade emulsions to deliver active food ingredients.

Process development of starch hydrolysis using mixing characteristics of Taylor vortices.

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor-Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.

Bioethanol production from potato starch by a novel vertical mass-flow type bioreactor with a co-cultured-cell strategy

The purpose of this study was to develop a vertical mass-flow type bioreactor (VMFB) consisting of both aerobic and anaerobic zones to produce ethanol by Simultaneous Saccharification and Fermentation (SSF). Potato starch and microorganisms of Aspergillus awamori, Rhizopus japonicus, and Zymomonas mobilis were used. The highest ethanol concentration with 6.0 % (w/v) initial starch were 2.99g/L by traditional two-stage Separate Hydrolysis and Fermentation (SHF, co-suspension culture, flask, 48 h), 17.71g/L (SHF, co-immobilization culture, flask, 84h), and 21.68g/L (SSF, co-cultured in VMFB, 27 h), respectively. In the repeated-batch process (3 cycles) using the VMFB, stable ethanol production was 95.97g/L after 72h. Increase of the starch concentration from 6.0% to 12.0% (w/v) resulted in higher ethanol production of 60.18g/L after 51h. Another type of starch was tested, and 27.91g/L of ethanol was obtained from 6.0% (w/v) cassava starch. The results showed that the performance of VMFB cannot only enhance the ethanol yield, but also has great benefit for high fermentation efficiency and less cost.

Cassava roots: perspectives of a traditional staple for bio-solvents production

Sidi Seibou T.M., Rychtera M., Melzoch K. (2015): Cassava roots: perspectives of a traditional staple for Clostridium acetobutylicum DSM 1731. The purpose of these tests was to determine the initial starch concentration which could be used as a starting point for the fermentation in laboratory bioreactors. In this case, we used two culture media: the first one made with cassava starch (M1), and the second one with mashed fresh cassava (M2). The culture media were supplemented with organic and inorganic nutrients. Fermentation in M1 medium resulted in the production of 17.8 g/l solvents, i.e. 12.3 g/l butanol, 3.7 g/l acetone, and 1.8 g/l ethanol, while in M2 it provided 16.2 g/l solvents representing approximately 11.4 g/l of butanol, 3.0 g/l of acetone, and 1.8 g/l of ethanol. The optimum production of butanol in M1 was 12.3 g/l in 96 h of fermentation, which is a low yield of 25% and 0.13 g/l/h productivity. Although the fermentation in medium M2 was also low (with 11.4 g/l butanol optimally), it allowed to save on the nutrient sup -

The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids.

The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P < 0.05) increased following both gastric and duodenal digestion. This was also in correlation with the antioxidant assays quantifying the water-soluble antioxidant capacity of the extracts. The lipophilic Oxygen Radical Absorbance Capacity assay did not indicate a statistically significant change in the values during any of the digestion phases. Statistically significant (P < 0.05) reductions in the anthocyanin contents were observed during the digestion phases in contrast to the carotenoid contents. With the exception of the cumin seed extract, none of the spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities.

Non-Carbohydrate Components on the Surface of Starch Granules According to the Hardness of Wheat

An attempt was made to demonstrate the usefulness of pre-knowledge of the quality and quantity analysis of non-carbohydrate components of starch surface, such as proteins and lipids, in starch isolated from wheat with varying degrees of hardness, for receiving glucose hydrolysates. The chemical composition of starches isolated from wheat grains of different hardness was examined. The amount of protein fraction (surface proteins, puroindolins and internal proteins), total and surface lipids, apparent amylose, phosphorous and pentosans contents were evaluated. It was found that starches, depending on the hardness of wheat grain, have a different chemical composition. A clear influence of wheat hardness on lipid and proteins content on the surface of starch grains and on apparent amylose was proved. It was showed that the presence of polar lipids (glyco and phospholipids) on the surface of starch was connected with their role in binding of surface proteins fraction (friabilins) to starch grains. There was a clear positive correlation between the amount of lipid fraction on starch surface and the amount of protein fraction, depending on wheat hardness. Starches from soft wheat varieties were characterized by higher amounts of proteins and lipids on their surface. In addition, it was determined that basing on the knowledge of mechanical properties of wheat grains, namely its hardness and initial starch chemical content analysis, one may predict its further directions of processing, including evaluation, whether the starch from a given wheat variety would be appropriate for obtaining hydrolysates with good physicochemical properties.

Effects of wheat and rye bread structure on mastication process and bolus properties

Chemical composition, baking process and structure of breads influence their degradation in digestion leading to different postprandial responses. Rye bread has a very different structure as compared to wheat bread, and rye breads are known to induce lower postprandial insulin responses than wheat bread. The aim of this study was to find out potential differences in mastication and initial starch hydrolysis rate of rye and wheat breads. Three rye breads (wholemeal rye, endosperm rye and endosperm rye with gluten) and wheat bread were masticated by fifteen participants and the process was monitored using electromyography. The particle size distribution and initial in vitro starch hydrolysis of the bread boluses were analysed. Specific volume correlated negatively and closed porosity of breads correlated positively with work required for mastication. When compared to wheat bread, wholemeal rye bread required more work for mastication process (p=0.004). Rye breads were degraded to smaller particles than wheat bread during mastication. There was a trend (p=0.098) towards slower in vitro starch hydrolysis rate in rye bread boluses than in wheat bread boluses. The results indicate that the digestion process of rye breads differs from that of wheat bread already in the early phase of digestion. This may be one reason behind the unique postprandial responses reported for rye breads.

Preparation of Oxidized Starch Using Environment Friendly Chlorine Dioxide as Oxidant

Abstract In the present work, the oxidized corn starch was prepared by using chlorine dioxide as novel oxidant. It was characterized by X-ray diffraction, scanning electron microscope (SEM), and transmission electron microscope (TEM). The main factors that affect the oxidation process were studied, and the oxidation degree of oxidized starch was determined by the carboxyl content and viscosity of the product. The results show that carboxyl groups are introduced into the starch chains successfully, and the oxidation reaction mainly carries out at the amorphous area. Compared with native starch, the light transmittance and anti-retrogradation of oxidized starch increase, whereas the viscosity reduces. The best oxidized starch can be obtained at the sodium chlorate dosage of 3%, hydrochloric acid 4%, and sulfuric acid 3% (all based on the initial starch weight), while the oxidation reaction is conducted at 50°C for 5 h.

Experimental studies and kinetic modeling of cyclodextrin glucanotransferase production from starch with the use ofBacillussp. DSM 2523

Cyclodextrin glucanotransferase (CGTase) production from starch with the use of Bacillus sp. DSM 2523 was studied. The specific growth rate (µ) considering the initial starch concentrations (S0) ranging from 0.2% to 2% w/v, was determined and a maximum value for µ of 0.1151/h was found at S0 = 0.5% w/v. The maximum specific CGTase production rate (qP) at S0 = 0.2% w/v, was 1.1105/h with considerable fluctuations in enzyme production. By performing a repeated batch experiment using dextrin as feeding substrate, the cell's ability to maintain its responses for an extended period of time was examined. The trend toward a decrease in the µ value and qP with an increase in the substrate concentration was evident. The experimental data showing the inhibitory function of starch on the growth and enzyme production were best predicted using Haldane model. On basis of the cell maintenance concept the observed dependency of the growth yield (Y) on the starch concentration was applied and the relationship between maintenance energy and µ was evaluated. At concentrations of starch below 0.5% w/v, the constant maintenance energy can be obtained. The variation of maintenance energy dependency on µ was assessed in the inhibited culture system (S0 ≥ 0.5% w/v).

Preparation of water‐soluble oxidized starch with high carbonyl content by sodium hypochlorite

Water‐soluble oxidized starch with high carbonyl content (DOCO = 0.40) was prepared using a one‐step method with sodium hypochlorite (NaClO) as the primary oxidant and under the mediation of sodium bromide (NaBr) and hydroxylamine hydrochloride (NH2OH · HCl). The influences of pH, initial starch concentration, and the amount of NaBr and NH2OH · HCl on the carbonyl and carboxyl content were discussed. The molar ratio of carbonyl to carboxyl groups can be adjusted from 0.8 to 2.2 by controlling the dropping speed of NaClO. The formation of carbonyl and carboxyl groups on the oxidized starch was confirmed by Fourier‐transform infrared (FT‐IR) and 13C NMR spectroscopy. The molecular weight of the oxidized starch determined by SEC revealed modest degradation. Besides, a good solubility of the oxidized starch in cold water was also proved based on the study of its solubility and swelling properties.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function.

Ethanol fermentation from food processing waste

This study focuses on the use of restaurant waste for production of ethanol. Food wastes (corn, potatoes, and pasta) were converted to ethanol in a two‐step process: a two‐part enzymatic digestion of starch using α‐amylase and glucoamylase and then fermentation of the resulting sugars to ethanol using yeast. Because of the low initial composition of starch in the food waste, low ethanol concentrations were achieved: at best 8 mg/mL ethanol (0.8% by mass). Ethanol concentration increased with increasing enzyme dosage levels. Calculations were conducted to evaluate whether waste heat from restaurant waste could be used to drive flash vaporization to purify ethanol. If the solution produced by fermenting food waste is flashed at a temperature of 99.7°C, 77% of the ethanol is recovered in a vapor stream with 1.14 mol % ethanol (2.87 mass %). Waste heat could provide over a third of the energy for this vaporization process. If 4 mol % ethanol could be produced in the fermentation step by increasing the initial starch content in the waste solution and improving the fermentation process, then a single flash at 98.9°C will recover nearly 99% of the ethanol, giving a mass concentration of ethanol of 10.3%, which is similar to that achieved in industrial grain fermentation. © 2012 American Institute of Chemical Engineers Environ Prog, 32: 1280–1283, 2013

Interaction of cationic starch derivatives and cellulose fibres in the wet end and its correlation to paper strength with a statistical evaluation

AbstractThe adsorption onto a softwood pulp of cationic modified starches with a degree of substitution (DS) ranging from 0.015 to 0.130 and native potato starch was investigated using a method based on size exclusion chromatography‐multi angle laser light scattering (SEC‐MALLS). This technique provides information about the amount and molecular composition of the adsorbed starch product such as the AP–AM‐ratio, the average MW and molar mass distribution. Factors influencing adsorption, like the electrolyte content in the suspension and the initial starch polysaccharide concentration, were investigated. Furthermore, hand sheets were made and the resulting mechanical paper strength was tested. Results were analysed statistically using analysis of variance. Representative results were discussed in detail. In general, low‐substituted cationic starch (DS 0.030) exhibited a higher degree of adsorption and resulted in high paper strength when there was a low electrolyte content in the starch–pulp‐suspension. However, high amounts of electrolytes impeded electrostatic interaction of the charged groups of the starch polysaccharides and cellulose fibre due to partial screening and therefore strongly limited adsorption. This resulted in lowered mechanical paper strength. The water quality of the suspensions had little impact on the polyelectrolyte adsorption of the highly substituted cationic starch (DS 0.100). However, paper strength was markedly higher in the case of starch adsorption where there was a high ionic content and when both starch polysaccharides–AP and AM– were adsorbed. The initial starch concentration determined starch adsorption both quantitatively and qualitatively and subsequently paper strength as well. The relation between adsorption behaviour and paper strength was discussed, particularly in the case of 6% initial starch concentration. In deionised water (DW), the low‐substituted starch (DS 0.03) adsorbed onto the pulp to a greater extent with an AP–AM‐ratio similar to that of the initial starch. Moreover, this achieved the best strength properties in contrast to 36 dGH, where less starch adsorbed and that being almost exclusively AP. In water with 36 dGH, the highly substituted starch (DS 0.100) with both the AP and AM was absorbed and this resulted in higher paper strength. In contrast, AM was almost exclusively adsorbed in DW. Increasing the initial starch concentration increased the amount adsorbed while subsequently changing the AP–AM‐ratio towards increased AM adsorption and decreased AP adsorption in the case of DS 0.100 in DW. Exclusive adsorption of AM markedly lowered the development of the paper strength.

Hydrolysis and fermentation of sweetpotatoes for production of fermentable sugars and ethanol

Liquefaction, saccharification, and fermentation of FTA-94 industrial sweetpotatoes (ISPs) were examined using α-amylase and glucoamylase for the production of ethanol. Starch degradation and sugars produced over time were examined for (1) α-amylase (Liquozyme SC) at different loading rates (0.045, 0.45, and 4.5%KNU-S/g dry ISP) during liquefaction; and (2) three glucoamylases (Spirizyme Fuel, Spirizyme Plus Tech, and Spirizyme Ultra) at different loading rates (0.5, 1.0, and 5.0AGU/g dry ISP) during saccharification. The majority of starch, 47.7 and 65.4% of dry matter, was converted during liquefaction of flour and fresh sweetpotato preparations, respectively, with the addition of 0.45KNU-S/g dry ISP of Liquozyme SC after 2h (66.4 and 80.1% initial starch in dry matter, respectively). The enzymes used during saccharification increased starch breakdown, but was more effective in conversion of short chain carbohydrates to fermentable sugars. The addition of 5.0AGU/g of Spirizyme Ultra after 48h produced 795.4 and 685.3mg glucose/g starch with flour and fresh preparations, respectively. Yeast fermentation on hydrolyzed starch was examined over time with and without the addition of salt nutrients. Yeast converted all fermentable sugar (e.g. glucose, fructose, maltose) and produced 62.6 and 33.6g ethanol/L of hydrolysate for flour (25% w/v, substrate loading) and fresh (12.5% w/v, substrate loading) ISP, respectively, after 48h without salt addition.