Glucoamylase immobilized on montmorillonite: Synthesis, characterization and starch hydrolysis activity in a fixed bed reactor

Glucoamylase from Aspergillus Niger was immobilized on montmorillonite clay (K-10) by two procedures, adsorption and covalent binding. The immobilized enzymes were characterized using XRD, surface area measurements and 27Al MAS NMR and the activity of the immobilized enzymes for starch hydrolysis was tested in a fixed bed reactor (FBR). XRD shows that enzyme intercalates into the inter-lamellar space of the clay matrix with a layer expansion up to 2.25 nm. Covalently bound glucoamylase demonstrates a sharp decrease in surface area and pore volume that suggests binding of the enzyme at the pore entrance. NMR studies reveal the involvement of octahedral and tetrahedral Al during immobilization. The performance characteristics in FBR were evaluated. Effectiveness factor (η) for FBR is greater than unity demonstrating that activity of enzyme is more than that of the free enzyme. The Michaelis constant (Km) for covalently bound glucoamylase was lower than that for free enzyme, i.e., the affinity for substrate improves upon immobilization. This shows that diffusional effects are completely eliminated in the FBR. Both immobilized systems showed almost 100% initial activity after 96 h of continuous operation. Covalent binding demonstrated better operational stability.

Fundamental studies of sourdoughs fermented with Weissella cibaria and Lactobacillus plantarum: influence on baking characteristics, sensory profiles and in vitro starch digestibility of gluten-free breads

The application of sourdough can improve texture, structure, nutritional value, staling rate and shelf life of wheat and gluten-free breads. These quality improvements are associated with the formation of organic acids, exopolysaccharides (EPS), aroma or antifungal compounds. Initially, the suitability of two lactic acid bacteria strains to serve as sourdough starters for buckwheat, oat, quinoa, sorghum and flours was investigated. Wheat flour was chosen as a reference. The obligate heterofermentative lactic acid bacterium (LAB) Weissella cibaria MG1 (Wc) formed the EPS dextran (a α-1,6-glucan) from sucrose in situ with a molecular size of 106 to 107 kDa. EPS formation in all breads was analysed using size exclusion chromatography and highest amounts were formed in buckwheat (4 g/ kg) and quinoa sourdough (3 g/ kg). The facultative heterofermentative Lactobacillus plantarum FST1.7 (Lp) was identified as strong acidifier and was chosen due to its ubiquitous presence in gluten-free as well as wheat sourdoughs (Vogelmann et al. 2009). Both Wc and Lp, showed highest total titratable acids in buckwheat (16.8 ml; 26.0 ml), teff (16.2 ml; 24.5 ml) and quinoa sourdoughs (26.4 ml; 35.3 ml) correlating with higher amounts of fermentable sugars and higher buffering capacities. Sourdough incorporation reduced the crumb hardness after five days of storage in buckwheat (Wc -111%), teff (Wc -39%) and wheat (Wc -206%; Lp -118%) sourdough breads. The rate of staling (N/ day) was reduced in buckwheat (Ctrl 8 N; Wc 3 N; Lp 6 N), teff (Ctrl 13 N; Wc 9 N; Lp 10 N) and wheat (Ctrl 5 N; Wc 1 N; Lp 2 N) sourdough breads. Bread dough softening upon Wc and Lp sourdough incorporation accounted for increased crumb porosity in buckwheat (+10.4%; +4.7), teff (+8.1%; +8.3%) and wheat sourdough breads (+8.7%; +6.4%). Weissella cibaria MG1 sourdough improved the aroma quality of wheat bread but had no impact on aroma of gluten-free breads. Microbial shelf life however, was not prolonged in any of the breads regardless of the starter culture used. Due to the high prevalence of insulin-dependent diabetes mellitus particular amongst coeliac patients, glycaemic control is of great (Berti et al. 2004). The in vitro starch digestibility of gluten-free breads with and without sourdough addition was analysed to predict the GI (pGI). Sourdough can decrease starch hydrolysis in vitro, due to formation of resistant starch and organic acids. Predicted GI of gluten-free control breads were significantly lower than for the reference white wheat bread (GI=100). Starch granule size was investigated with scanning electron microscopy and was significantly smaller in quinoa flour (<2 μm). This resulted in higher enzymatic susceptibility and hence higher pGI for quinoa bread (95). Lowest hydrolysis indexes for sorghum and teff control breads (72 and 74, respectively) correlate with higher gelatinisation peak temperatures (69°C and 71°C, respectively). Levels of resistant starch were not increased by addition of Weissella cibaria MG1 (weak acidifier) or Lactobacillus plantarum FST1.7 (strong acidifier). The pGI was significantly decreased for both wheat sourdough breads (Wc 85; Lp 76). Lactic acid can promote starch interactions with gluten hence decreasing starch susceptibility (Östman et al. 2002). For most gluten-free breads, the pGI was increased upon sourdough addition. Only sorghum and teff Lp sourdough breads (69 and 68, respectively) had significantly decreased pGI. Results suggest that the increase of starch hydrolysis in gluten-free breads was related to mechanism other than presence of organic acids and formation of resistant starch.

Effects of processing methods on amaranth starch digestibility and predicted glycemic index

Amaranth has attracted a great deal of interest in recent decades due to its valuable nutritional, functional, and agricultural characteristics. Amaranth seeds can be cooked, popped, roasted, flaked, or extruded for consumption. This study compared the in vitro starch digestibility of processed amaranth seeds to that of white bread. Raw seeds yielded rapidly digestible starch content (RDS) of 30.7% db and predicted glycemic index (pGI) of 87.2, the lowest among the studied products. Cooked, extruded, and popped amaranth seeds had starch digestibility similar to that of white bread (92.4, 91.2, and 101.3, respectively), while flaked and roasted seeds generated a slightly increased glycemic response (106.0 and 105.8, respectively). Cooking and extrusion did not alter the RDS contents of the seeds. No significant differences were observed among popped, flaked, and roasted RDS contents (38.0%,46.3%, and 42.9%, respectively), which were all lower than RDS content of bread (51.1%). Amaranth seed is a high glycemic food most likely because of its small starch granule size, low resistant starch content (< 1%), and tendency to completely lose its crystalline and granular starch structure during those heat treatments.

Cassava bagasse cellulose nanofibrils reinforced thermoplastic cassava starch

Cellulose cassava bagasse nanofibrils (CBN) were directly extracted from a by-product of the cassava starch (CS) industry, viz. the cassava bagasse (CB), The morphological structure of the ensuing nanoparticles was investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), presence of other components such as sugars by high performance liquid chromatography (HPLC), thermogravimetric analysis (TGA), and X-ray diffraction (XRD) experiments. The resulting nanofibrils display a relatively low crystallinity and were found to be around 2-11 nm thick and 360-1700 nm long. These nanofibrils were used as reinforcing nanoparticles in a thermoplastic cassava starch matrix plasticized using either glycerol or a mixture of glycerol/sorbitol (1:1) as plasticizer. Nanocomposite films were prepared by a melting process. The reinforcing effect of the filler evaluated by dynamical mechanical tests (DMA) and tensile tests was found to depend on the nature of the plasticizer employed. Thus, for the glycerol-plasticized matrix-based composites, it was limited especially due to additional plasticization by sugars originating from starch hydrolysis during the acid extraction. This effect was evidenced by the reduction of glass vitreous temperature of starch after the incorporation of nanofibrils in TPSG and by the increase of elongation at break in tensile test. On the other hand, for glycerol/sorbitol plasticized nanocomposites the transcrystallization of amylopectin in nanofibrils surface hindered good performances of CBN as reinforcing agent for thermoplastic cassava starch. The incorporation of cassava bagasse cellulose nanofibrils in the thermoplastic starch matrices has resulted in a decrease of its hydrophilic character especially for glycerol plasticized sample. (C) 2009 Elsevier Ltd. All rights reserved.

Direct fermentation of potato starch in wastewater to lactic acid by Rhizopus oryzae

The fungal species of Rhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30degreesC was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%similar to85.5% associated with 1.5similar to2.0 g/L fungal biomass produced in 36 h of fermentation.

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87-0.97 g/g starch associated with 1.5-2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.

Simultaneous saccharification and fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of simultaneous saccharification and fermentation (SSF) for lactic acid production by fungal species of Rhizopus arrhizus 36017 and Rhizopus oryzae 2062 was studied with respect to growth pH, temperature and substrate. Both R. arrhizus 36017 and R. oryzae 2062 had a capacity to carry out a single stage SSF process for lactic acid production from potato starch wastewater. The kinetic characteristics, termed as starch hydrolysis, accumulation of reducing sugars, lactic acid production and fungal biomass formation, were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.85-0.92 g/g associated with 1.5-3.5 g/l fungal biomass produced in 36-48 h fermentation. R. arrhizus 36017 had a higher capacity to produce lactic acid, while R. oryzae 2062 produced more fungal biomass under similar conditions. (c) 2005 Elsevier B.V. All rights reserved.

Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. 27Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N2 adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. Km was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.

Immobilization of Diastase α-amylase on to synthetic and natural polymers

Several natural and synthetic supports have been assessed for their efficiency for enzyme immobilization. Synthetic polymer materials are prepared by chemical polymerization using various monomers. As a kind of important carrier, synthetic polymer materials exhibit the advantages of good mechanical rigidity, high specific surface area, inertness to microbial attack, easy to change their surface characteristics, and their potential for bringing specific functional group according to actual needs. Hence, they have been widely investigated and used for enzyme immobilization. When it comes to the natural polymer materials, much attention has been paid to cellulose and other natural polymer materials owing to their wide range of sources, easy modification, nontoxic, and pollution-free, with a possibility of introducing wide variety of functional groups and good biocompatible properties. In this work report the use of synthetic polymer, polypyrrole and its derivatives and natural polymers coconut fiber and sugarcane bagasse as supports for Diastase α- amylase immobilization. An attempt was also made to functionalize both synthetic and natural polymers using Amino-propyl triethoxysilane. Supports and their immobilized forms were characterized via FT-IR, TG, SEM, XRD, BET and EDS techniques. Immobilization parameters were also optimized so as to prepare stable immobilized biocatalyst for starch hydrolysis.

Study of some physical properties of biodegradable films based on blends of gelatin and poly(vinyl alcohol) using a response-surface methodology

The aim of this work was to study the effect of the hydrolysis degree (HD) and the concentration (C(PVA)) Of two types of poly(vinyl alcohol) (PVA) and of the type (glycerol and sorbitol) and the concentration (C(P)) of plasticizers on some physical properties of biodegradable films based on blends of gelatin and PVA Using a response-surface methodology. The films were prepared with a film forming solutions (FFS) with 2 g of macromolecules (gelatin+PVA)/100 g de FFS. The responses analyzed were the mechanical properties, the solubility, the moisture Content. the color difference and the opacity. The linear model was statistically significant and predictive for puncture force and deformation. elongation at break, solubility in water, Moisture content and opacity. The CPVA affected strongly the elongation at break of the films. The interaction of the HD and the C(P) affected this property. Moreover. the puncture force was affected slightly by the C(PVA). Concerning the Solubility in water, the reduction of the HD increased it and this effect was greater for high CPVA Values. In general. the most important effect observed in the physical properties of the films was that of the plasticizer type and concentration. The PVA hydrolysis degree and concentration have an important effect only for the elongation at break, puncture deformation and solubility in water. (C) 2008 Elsevier B.V. All rights reserved.

The floral nectary of Hymenaea stigonocarpa (Fabaceae, caesalpinioideae): Structural aspects during floral development

Background and Aims Considering that few studies on nectary anatomy and ultrastructure are available for chiropterophilous flowers and the importance of Hymenaea stigonocarpa in natural 'cerrado' communities, the present study sought to analyse the structure and cellular modifications that take place within its nectaries during the different stages of floral development, with special emphasis on plastid dynamics.Methods For the structural and ultrastructural studies the nectary was processed as per usual techniques and studied under light, scanning and transmission electron microscopy. Histochemical tests were employed to identify the main metabolites on nectary tissue and secretion samples.Key Results The floral nectary consists of the inner epidermis of the hypanthium and vascularized parenchyma. Some evidence indicates that the nectar release occurs via the stomata. The high populations of mitochondria, and their juxtaposition with amyloplasts, seem to be related to energy needs for starch hydrolysis. Among the alterations observed during the secretory phase, the reduction in the plastid stromatic density and starch grain size are highlighted. When the secretory stage begins, the plastid envelope disappears and a new membrane is formed, enclosing this region and giving rise to new vacuoles. After the secretory stage, cellular structures named 'extrastomatic bodies' were observed and seem to be related to the nectar resorption.Conclusions Starch hydrolysis contributes to nectar formation, in addition to the photosynthates derived directly from the phloem. In these nectaries, the secretion is an energy-requiring process. During the secretion stage, some plastids show starch grain hydrolysis and membrane rupture, and it was observed that the region previously occupied by this organelle continued to be reasonably well defined, and gave rise to new vacuoles. The extrastomatic bodies appear to be related to the resorption of uncollected nectar.

Avaliação da farinha de mandioca e do fubá de milho como substratos para a obtenção de bebida fermento-destilada

A farinha de mandioca e o fubá de milho foram avaliados como matérias-primas alternativas na obtenção de uma bebida fermento-destilada, visando gerar informações úteis à aplicação industrial. Os substratos foram caracterizados e comparou-se a eficiência da mosturação, o perfil de açúcares no mosto, bem como as curvas de fermentação dos substratos. Os resultados demonstraram que o conteúdo de amido foi de 78,1 % para o fubá de milho e 92,7 % para a farinha de mandioca. Quanto ao rendimento da mosturação foi de 57, 4 % para o milho e 66,4 % para a mandioca, sendo que o perfil de açúcares no mosto demonstrou que 95 % dos açúcares presentes no mosto de mandioca foi glicose e o restante pequenas porcentagens de dextrinas e maltose. Já o perfil do mosto de milho apresentou cerca de 85 % de glicose , 10 % de dextrinas e cerca de 4 % de maltose. Para o processo fermentativo, observou -se que o consumo de açúcares no mosto de mandioca foi mais rápido que no mosto de milho.

Production of CGTase by a bacillus Alkalophilic CGII strain isolated from wastewater of a manioc flour industry

GCTase production by a new strain of Bacillus alkalophillc CGII isolated from Brazilian wastewater of manioc flour industry was examined. The growth medium used was composed by 1.5% starch, 1.5% nitrogen and 1% Na 2CO3. Higher activity was obtained with starch, maltodextrin and galactose. When glucose was added to the medium, no enzyme production was observed. High enzyme activity and growth were reached when aeration was increased (88.6 U/mL). The enzyme characterization showed an optimum pH and temperature 8.0 and 55°C for starch hydrolyses, respectively. Mg+ and Ca++ showed small activation; however, Hg + and Cu+ showed a strong enzyme inhibition.

Production and application of amylases of Rhizopus oryzae and Rhizopus microsporus var. oligosporus from industrial waste in acquisition of glucose

Amylases from Rhizopus oryzae and Rhizopus microsporus var. oligosporus were obtained using agro-industrial wastes as substrates in submerged batch cultures. The enzymatic complex was partially characterised for use in the production of glucose syrup. Type II wheat flour proved better than cassava bagasse as sole carbon source for amylase production. The optimum fermentation condition for both microorganisms was 96 hours at 30°C and the amylase thus produced was used for starch hydrolysis. The product of the enzymatic hydrolysis indicated that the enzyme obtained was glucoamylase, only glucose as final product was attained for both microorganisms. R. oligosporus was of greater interest than R. oryzae for amylase production, taking into account enzyme activity, cultivation time, thermal stability and pH range. Glucose syrup was produced using concentrated enzyme and 100 g L-1 starch in a 4 hours reaction at 50°C. The bioprocess studied can contribute to fungus glucoamylase production and application. © 2013 Institute of Chemistry, Slovak Academy of Sciences.

Isolamento de linhagens microbianas termofílicas amilolíticas, produção, caracterização e aplicação das amilases na hidrólise do amido de mandioca

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)