Suppression of Fusarium graminearium growth by differently structured starch types

We investigated the growth behavior and amylolytic enzymes of Fusarium graminearum cultivated on different types of native starch granules including barley (A‐type crystalline polymorph), potato and Curcuma zedoaria (B‐type crystalline polymorph), cassava (C‐type crystalline polymorph), and high AM maize (A + Vh‐type crystalline polymorphs). F. graminearum grew poorly on B‐type starches and the accumulation of biomass was similar to that obtained for fungi cultivated under carbohydrate starvation conditions. In comparison, three‐ to fivefold higher accumulation of fungal biomass was observed for growth on the A‐, C‐ and A + Vh‐type starches. Fungal glucoamylase and α‐amylase activity increased over time in the presence of native starch granules. Interestingly, resistant B‐type starches induced the highest amylolytic activity indicating that F. graminearum interacts with B‐type granules although only limited degradation occur. Starch degradation products maltose and malto‐oligosacharides was found to increase glucoamylase and α‐amylase activity, whereas glucose acted as a catabolite repressor.     

Effect of Organic Nitrogen Sources on Raw Starch‐Digesting Glucoamylase Production of Rhizopus sp. MKU 40

In a liquid cultivation of Rhizopus sp. MKU 40, supplementation of the medium with 1.5% (w/v) organic nitrogen sources (neopeptone, casein from milk, and meat extract) had a slightly positive effect on glucoamylase (GA) (EC 3.2.1.3) activity compared with the medium lacking organic nitrogen sources. The addition of organic nitrogen sources induced production of protease. Supplementation of the medium with 1.5% (w/v) organic nitrogen sources resulted in an acid and neutral protease activity of 11 — 25 U/mL and 12 — 20 U/mL, respectively. The co‐existence of GA‐I [a highly raw starch‐digesting glucoamylase (RSDG)] and protease in the same medium leads to the production of Ga‐II (an extremely weak RSDG) from GA‐I. As a result the RSDG activity in the medium decreases. Raw starch adsorption rates of a medium without organic nitrogen sources were 100%, because the medium contained only GA‐I. In contrast, the media supplemented with organic nitrogen sources had low starch adsorption rates because the media contained both GA‐I and GA‐II. The results presented in this paper indicate that supplementation of the culture medium of Rhizopus strains with organic nitrogen sources negatively affects GA‐I production.     

Cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits

Both culture‐dependent method and molecular technique were firstly used to simultaneously investigate the cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits. The results showed that both cultivable bacterial diversity and amylase production were obviously different. The species of nine bacteria from Deshan, nine from Baisha and six from Wuling Daqus were identified. The total bacterial strains of 17, 15 and 14, and 9, 16 and 10 could produce α‐amylase and glucoamylase, respectively, from the Daqus, and the enzyme yields were different. Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens not only were dominant bacteria in the Daqus, but also possessed high activities of α‐amylase and glucoamylase. By comparison, Bacillus cereus and Bacillus oleronius were found to be another predominant bacterial species and good producers of α‐amylase and glucoamylase in Deshan and Wuling Daqus, respectively.     

Daqu— A Traditional Chinese Liquor Fermentation Starter

Chinese liquor is one of the world's oldest distilled alcoholic beverages, and it is typically obtained with the use of Daqu fermentation starters. Daqu is a saccharifying and fermenting agent, having a significant impact on the flavour of the product. Daqu can be categorized according to maximum incubation temperatures (high, medium and low) and flavour (sauce, strong, light and miscellaneous). Most Daqu are prepared by solid‐state fermentation from wheat, barley and/or peas with ingredient formulation, grinding and mixing, shaping, incubation and maturation. Although there is a wealth of artisanal experience in the production of a range of different types of Daqu, the scientific knowledge base—including the microbiota, their enzymes and their metabolic activities—needs further development. Daqu as a specific alcoholic starter is compared with other Asian amylolytic fermentation starters in terms of microbial diversity and function. Filamentous fungi (Rhizopus, Rhizomucor, Aspergillus and other genera), yeasts (Saccharomyces, Candida, Hansenula and other genera) and bacteria (acetic acid bacteria, lactic acid bacteria and Bacillus spp.), are considered to be the functional microbiota, responsible for the formation of a range of lytic enzymes, formation of substrates for alcoholic fermentation and formation of flavour compounds. However, the knowledge about the microbiota composition and their function is still fragmentary information, so further research is required to establish the functionality and growth kinetics of the microbiota in diverse types of Daqu.     

Raw Starch Digestion by α-Amylase and Glucoamylase from Chalara paradoxa

Glucoamylase and α-amylase of Chalara paradoxa were separated by hydrophobic column chromatography using butyl-Toyopearl 650M. The α-amylase showed the highest activity at pH 5.5 and 45°C, and was stable in the pH range of 5.5–6.5 and at temperatures lower than 40°C. The glucoamylase showed the highest activity at pH 5.0 and 45°C, and was stable in the pH range of 4.0–7.5 and at temperatures lower than 45°C. The molecular mass of the α-amylase and glucoamylase estimated by SDS polyacrylamide gel electrophoresis was 80,000 and 68,000, respectively. Both glucoamylase and α-amylase could digest more effectively raw rice starch and raw corn starch than raw sago starch and raw potato starch. 2% raw rice starch in 10 ml solution was digested by more than 90% by 100 units of each amylase. When these amylases were used combined, raw corn starch was more effectively digested than they were used singly. This cooperative action in raw corn starch digestion was also observed when. C. paradoxa α-amylase and R. niveus glucoamylase were combined.     

Enhanced production of α‐amylase from Bacillus subtilis subsp. spizizenii in solid state fermentation by response surface methodology and its evaluation in the hydrolysis of raw potato starch

This is an attempt to lower the cost of starch hydrolysis by the discovery of new generation α‐amylase. A natural isolate of Bacillus subtilis subsp. spizizenii was capable of producing appreciable amounts of raw potato starch digesting α‐amylase in solid state fermentation of wheat bran. The enzyme productivity has been substantially enhanced by supplementing various nutrients and statistically studying their interactions by response surface methodology. A central composite design for amylase production system elucidated a wheat bran‐based medium supplemented with soybean meal, threonine, and B‐complex vitamins predicting a yield of 521 391 U/g dry solids. The enzyme preparation could effectively digest 5–15% suspension of insoluble potato starch in 6 h revealing the dextrose equivalent of 32–44. The supplementation of a glucoamylase preparation, thereafter, brought about complete saccharification. The yield achieved in the statistically optimized amylase system may be one of the best to date and its capability in directly liquefying raw potato starch granules makes this study novel.     

Effect of bioaugmentation on biochemical characterisation and microbial communities in Daqu using Bacillus,Saccharomycopsis and Absidia

Daqu, a saccharifying and fermenting agent for the production of Chinese vinegar and liquor, is manufactured through a spontaneous solid-state fermentation process (SSF). To investigate the influence of bioaugmentation with native microorganisms on Daqu SSF process, physicochemical properties, microbial biomass, volatile compounds and microbial communities were analysed in laboratory-scale Daqu. The results showed that the amylase activity of Daqu enhanced 6.35% ± 0.74% by inoculating Bacillus amyloliquefaciens, Saccharomycopsis fibuligera and Absidia corymbifera. Obviously higher microbial richness was found in the Daqu with bioaugmentation, although the microbial community structure remained relatively stable. Moreover, a total of twenty-two volatile compounds were detected in the mature Daqu. Slightly increase in the content of alcohols was found in the enhanced group by fungal inoculation, including ethanol (46.58 ± 1.36 mg kg⁻¹), isobutanol (0.19 ± 0.04 mg kg⁻¹) and isoamyl alcohol (1.55 ± 0.13 mg kg⁻¹). This study demonstrated that bioaugmentation had a positive effect on the amylase activity, main volatile compounds and microbial community richness.     

Amylolytic Hydrolysis of Native Starch Granules Affected by Granule Surface Area

ABSTRACT: Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, α‐amylase from Bacillus subtilis, glucoamylase I (GA‐I) and II (GA‐II) from Aspergillus niger, and β‐amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver–Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.     

Kinetics of enzymic hydrolysis of cassava flour starch — optimization and modelling

This study was conducted to model the kinetics of cassava flour hydrolysis by Miles Taka-Therm L–170 alpha amylase and Diazyme L–200 glucoamylase to produce glucose syrup. Maximum starch concentration was 33% due to a controlled process of flour gelatinization by gradual temperature increase, and parallel starch hydrolysis by thermostable alpha amylase activity, preventing excess viscosity. The time of hydrolysis was two and half hours of alpha amylase activity and 36 h of glucoamylase activity with the final yield of 90–93% of glucose. Exponential hyperbolic models were obtained to predict the kinetic of hydrolysis both amylase and glucoamylase, with a generalized correlation coefficient >0.94.     

A comparison of the hydrolysis of sweet potato starch with β-amylase and infrared radiation allows prediction of reducing sugar production

The apparent reaction rate constants (k) of the reaction that generates reducing sugar (presented as mg maltose equivalents) from raw sweet potato starch by using β‐amylase were determined by varying enzyme concentration, starch concentration and pH. The Arrhenius plot of the k‐value reached a peak at approximately 86 °C; the activation energy and frequency factor were also determined. Moreover, the amount of reducing sugar produced in sweet potato heated by infrared irradiation was estimated by using the values of k and the kinetic parameters. The calculated amounts of reducing sugar produced generally agreed with the experimental values. These results indicate that the amount of reducing sugar produced in heat‐treated sweet potato can be predicted by the methodology described in this study.     

1H NMR‐based metabolomics approach for understanding the fermentation behaviour of Bacillus licheniformis

Bacillus licheniformis has been found to be one of the persistent dominant microorganisms in Daqu, which is a traditional fermentation starter, and it has been used to intensify certain strains. To understand the impact of B. licheniformis on Daqu, the fermentation behaviour of B. licheniformis was studied using ¹H NMR‐based non‐targeted analysis and principal component analysis. During the fermentation, 53 compounds were identified. Among them, seven compounds were largely consumed and 17 metabolites were largely accumulated. The macromolecular starch and cellulose were degraded by B. licheniformis, and then utilized to produce acetic acid, lactic acid, propionic acid, succinate acid, etc. Principal component analysis was carried out to study the variations of analytes during the fermentation. Samples collected at each time point could be clearly discriminated and the biomarkers of each time point were defined. A variety of biochemical compounds (such as acetate, ethanol, lactate, pyruvate, malate, maltose and sucrose) were changed during the fermentation of B. licheniformis. The results are useful to reveal how and why B. licheniformis becomes dominant in Daqu, and to reveal its impact on the aroma formation of Daqu and its derived products. Copyright © 2015 The Institute of Brewing & Distilling     

Isolation of a Recombinant Bacillus sp. TS‐23 α‐Amylase by Adsorption‐Elution on Raw Starch

A Bacillus sp. TS‐23 α‐amylase produced by recombinant Escherichia coli was adsorbed onto raw starch and the adsorbed enzyme was eluted with maltose or maltodextrin in 50 mM Tris/HCl buffer (pH 8.5). The adsorption‐elution procedure resulted in a yield of 53% α‐amylase activity and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) analysis showed that the eluted α‐amylase had a molecular mass of approximately 64 kDa. Raw starch could be used repeatedly in the adsorption‐ elution cycle with good reproducibility. Scanning electron microscopy of the isolated corn starch exhibited a smooth appearance of the granules before adsorption and only a small change in appearance after three adsorption‐elution cycles. These results suggest that the raw starch adsorption‐elution technique has a great potential in the isolation of Bacillus sp. TS‐23 α‐amylase from the culture broth of recombinant E. coli.     

Evaluation of potential side activities of commercial enzyme preparations used in winemaking

Twenty‐one commercial enzyme preparations used in winemaking were characterised for the α‐L‐rhamnosidase, α‐L‐arabinosidase, β‐D‐xylosidase, ?β‐D‐galactosidase, β‐D‐glucosidase, esterase, protease, cinnamoyl esterase and laccase activities. A new rapid fluorimetric method to assay esterase activity was developed. Enzyme preparations differed for the level of each enzyme activity assayed rather than for the type of enzymatic activity detected. High levels of protease, glycosidase, esterase and cinnamoyl esterase activity were found among enzyme preparations for different technological applications. A drastic reduction in the level of cinnamoyl esterase was observed in commercial grape juice, pH 3.6, total acidity 5.3 g L^?1_, sugar 170 g L^?1. Protease activity was only weakly reduced, from 10 to 20%, in commercial grape juice. β‐glucosidase activity levels are reduced in the presence of increasing concentration of glucose but are still present at the higher glucose concentration (100 g L^?1). The extent of the reduction observed was dependent on the enzymatic preparations tested.     

Properties of Residual Starches of Sugary-2 and Sugary-2 Opaque-2 Maize Following Amylase Hydrolysis

Properties of residual starches of sugary-2 opaque-2 and sugary-2 maize starch granules hydrolyzed with glucoamylases were investigated. A crude and two crystalline glucoamylases were used. The amylopectin fractions of both starches hydrolyzed easier than that of amylose with all enzymes. Residual starches hydrolyzed by the crude glucoamylase accumulated low-molecular weight materials, which was not observed in residual starches attacked by crystalline glucoamylase. It was suggested that in the crude enzyme the contaminating α-amylase caused the accumulation of the minified fraction. It is also suggested that the crystalline region of sugary-2 opaque-2 starch may consist of a mixture of A-type and B-type patterns. Evidence for this was from observation of the changes in X-ray diffraction patterns of residual starch following amylase and acid hydrolysis.     

The influence of proteolytic and cytolytic enzymes on starch degradation during mashing

The objective of this research was to investigate the impact of proteolysis and cytolysis on starch degradation over the course of the mashing process. A proteolytic enzyme (Neutrase 0.8L, Novozymes) and a number of cytolytic enzymes (barley β‐glucanase from Megazyme, Shearzyme 500L and Ultraflo Max from Novozymes) were used to test their efficiency on starch degradation during mashing. The proteolytic and cytolytic enzymes had positive effects on the levels of starch‐degrading enzymes and starch solubilization during mashing, resulting in higher levels of wort sugar compared with the control, indicating that proteins and residual non‐starchy polysaccharides limited the digestibility of starch during mashing. Moreover, the proteolytic enzyme showed a significantly greater improvement than the cytolytic enzymes, yielding a 57% increase in β‐amylase, a 173% increase in limit dextrinase, rapid starch solubilization during mashing and a higher percentage of fermentable sugars in resultant wort. The increases in limit dextrinase and β‐amylase promoted by the protease suggest that sufficient proteinaceous inhibitor existed in the wort during mashing to inhibit their activities, leading to the unavailability of β‐amylase and especially limiting dextrinase. Furthermore, Ultraflo Max, which contained a β‐glucanase–xylanase mixture, showed a greater improvement than either the individual β‐glucanase or the xylanase on starch hydrolysis. These findings suggest that β‐glucanase is the major enzyme responsible for the degradation of cell walls, and that the complete hydrolysis of the residual cell walls depends on the synergistic effect of β‐glucanase and xylanase. The results suggest that brewers should adjust the degradation of the cell walls and correct the degree of protein modification in order to obtain the desired wort composition. Copyright © 2014 The Institute of Brewing & Distilling     

Characterization and detection of toxoflavin‐producing Burkholderia in rice straws and Daqu for Chinese Maotai‐flavour liquor brewing

Rice straws are widely used in Daqu (starter) preparation for Maotai‐flavour liquor brewing to provide adequate thermal insulation, water retention and aeration. As the straws have direct contact with the Daqu during its preparation, the microorganisms carried by the straws can be transferred to the Daqu. In this study, yellow pigment‐producing strains were isolated from the rice straws using a dilution plate method. Most of these strains were characterized as toxoflavin‐producing Burkholderia spp. by 16S rRNA sequencing, toxA gene‐specific PCR and toxoflavin detection. The PCR assays revealed the existence of Burkholderia in the Daqu samples. The toxoflavin content in a number of Daqu samples was determined using HPLC. The highest observed content of toxoflavin in the Daqu was >8 mg/kg, close to its LD₅₀. Although no toxoflavin was detected in the distilled liquor, the safety problem regarding the use of rice straws in Daqu preparation is still noteworthy, since Burkholderia and toxoflavin can be transferred and can accumulate during the brewing process. The results can contribute to a further assessment of the contamination risk in liquor brewing caused by Burkholderia and to safer production in this traditional process. Copyright © 2015 The Institute of Brewing & Distilling     

Kinetics of Granular Starch Hydrolysis in Corn Dry‐Grind Process

A promising modification to the dry grind process is the use of granular starch hydrolyzing enzymes (GSHE) for corn fractionation and ethanol production. Modeling the kinetics of granular starch hydrolysis in the corn dry‐grind process is challenging given the heterogeneity of the substrate and the combined activities of α‐amylase and glucoamylase. By measuring reducing sugars, the hydrolysis progress in dry‐grind corn slurry treated with a commercial GSHE was monitored during 3 h of reaction. Progress curves consisted initially of a rapid product formation phase (<15 min), followed by a longer range kinetics characterized by relatively slower product formation. The initial product from the accelerated kinetics was proportional to the adsorbed GSHE concentration, after accounting for the contribution of dissolved starch hydrolysis. The long range kinetics was modeled by an empirical equation that yielded two parameters: a which stood for the asymptotic limit of the product concentration, and ln (b) which was the time required to reach a product concentration of a/2. In response to increasing GSHE concentration, a increased and ln (b) decreased towards a saturation limit. In response to decreasing corn particle size (geometric mean diameter), a increased and ln (b) decreased linearly. These behaviors indicated both parameters were controlled by the concentration of accessible substrates in the starch granules. The findings have implications on the optimal usage of GSHE in dry‐grind processes.     

Characteristics of Pleurotus sp. TD36 and its ability to reduce wood extractives in pretreatment for pulping

Lipophilic extractives of wood cause pitch deposition during pulp and paper production, leading to serious economic losses and environmental pollution. Natural seasoning of wood material before pulping can reduce wood extractives, but there is no control of the pulp yield and quality. Pretreatment of the wood chips with selected fungi is regarded as better controlled seasoning with effective removal of problematic extractives. Commercially available fungal preparations are effective mostly for softwood, but not quite suitable for Acacia mangium and hybrid hardwood—the main pulpwood of Vietnam. In this paper, a fungal isolate TD36 was identified as white-rot basidiomycete Pleurotus sp. TD36 based on the biological characteristics and phylogenetic analysis. Having high activity of ligninolytic enzymes, amylase and protease, Pleurotus sp. TD36 was able to degrade lignin-like compounds (guaiacol, Remazol Brilliant Blue), starch and casein. Degradation of wood extractives was evaluated by laccase and sterol esterase assays of the fungus in shake flask cultures and in pretreatment of Acacia wood chips. Favorable conditions for wood pretreatment were selected, including: inoculum size, 1% v/w; wood moisture, 60% v/w; temperature, 28–30 °C and 30 days. Under such conditions, Pleurotus sp. TD36 removed about 68?±?2.75% of wood acetone extractives soluble in chloroform. Of that, about 73–89% of fatty acids and fatty alcohols, 76% of free sterols and 63% of sterol ester, were removed. Fungal pretreatment did not significantly affect the cellulose content of wood and Kraft pulp yield.

     

Genetic Engineering of white Shochu-Koji to achieve Higher Levels of Acid-Stable α-Amylase and Glucoamylase and other Properties when used for Shochu Making on a Laboratory Scale

The genes for acid-stable α-amylase and glucoamylase were cloned from white shochu-koji, Aspergillus kawachii. Both genes were used to transform the parent strain of white shochu-koji which carried a dominant selective marker gene, amdS, that originated from A. oryzae. Three lines of transformants were identified that secreted about 6-fold more acid-stable α-amylase activity and about 7-fold more glucoamylase activity than the parent strain in liquid culture. In solid culture, all three transformants had 2-fold higher acid-stable α-amylase activity and 2.4-fold higher glucoamylase activity than the parent strain. When koji was prepared on a laboratory scale, acid-stable α-amylase activity was 5.7-fold higher and glucoamylase activity was 3.8-fold higher than when the parent strain was used. Shochu was produced with a koji ratio of 33% or 10% using one line of transformants on a laboratory scale. Even with a koji ratio of 10%, the weight of the mash obtained with the transformant decreased to almost the same extent as with a koji ratio of 33% and the parent strain. Levels of flavour compounds in shochu produced with koji of the transformant were higher than in the shochu prepared with koji of the parent strain. In particular, levels of isoamyl acetate and β-phenethyl acetate were as high as 12.9 mg/litre and 3.8 mg/litre, respectively.