Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.

Extraction of oligosaccharides and phenolic compounds by roasting pretreatment and enzymatic hydrolysis from spent coffee ground

Spent coffee ground (SCG) is the waste generated during the preparation of instant coffee and is the sourceof industrially valuable organic compounds. In this article, SCG was pretreated by roasting at 150°C for 30minutes and heated with water at 90°C for extracting carbohydrates and phenolic compounds, after which1.0% (w/w) β-mannanase was applied for the hydrolysis of pretreated SCG. SCG is characterized in terms ofits total sugar content by the anthrone–sulfuric assay and phenolic compounds by Folin−Ciocalteu’s procedure.In this study, the total sugar increased by 14.79% (w/w) by the roasting process, and subsequently enzymatichydrolysis increased the total sugar yield up to 17.43% (w/w) compared to the untreated SCG, i.e., 10.24%(w/w). The reducing sugar was estimated by the dinitrosalicylic acid method and the end product increased to106.10 (mg Glucose/g) from the initial content 5.32 (mg Glucose/g raw SCG). The total phenolic compoundincreased to 291.86 (mg Gallic acid/g lyophilized material), which was a 6.39-fold increase compared to thenative SCG (45.68 mg Gallic acid/g). These results point to the valuable compounds present in SCG, can beenhanced by combining the roasting pretreatment and enzymatic hydrolysis, and can be utilized in the foodand biotech industries.

Purification and physicochemical characterisation of Aspergillus niger USM F4 β-mannanase

Aims@#This present study focused on purification of fungal β-mannanase produced by Aspergillus niger USM F4 and also physicochemical characterisation of the purified enzyme.@*Methodology and results@#The purified β-mannanase with a molecular mass of ~47.4 kDa was demonstrated on SDSPAGE gel. The enzyme signified a purification degree of 4-fold, with final specific activity of 196.42 U/mg. It reached an optimum catalytic activity at pH 4.0 and 60 °C. The thermal stability of the enzyme was up to 70 °C and maintained the 50% activity after 30 min at 80 °C. Meanwhile, the pH stability was in the range of pH 3.0-9.0 and a 30 min half-life at pH 10.0. All chemical substances manifested an inhibitory effect on purified β-mannanase, with SDS (28.16 ± 0.05% residual activity) as the strongest inhibitor, followed by cupric ion (Cu2+) (49.51 ± 0.09% residual activity). As a whole, the enzyme displayed a substrate specificity in the order of locust bean gum (LBG) > carboxymethylcellulose > soluble starch > xylan from oat spelt > α-cellulose. Its preference for LBG has generated the Km and Vmax values of 0.20 mg/mL and 9.82 U/mL, respectively.@*Conclusion, significance and impact of study@#The outcomes of our study offer potential for use at industrial scales, particularly in the oligosaccharides production that involve acid-related activity, wide-ranging temperature and pH stability.

Dietary β-mannanase decreases cloacal temperature of broiler chickens under hot conditions without affecting growth performance / El consumo de β-mananasa disminuye la temperatura cloacal de pollos de engorde bajo condiciones de calor sin afectar su rendimiento de crecimiento / O consumo de β-mananase diminui a temperatura da cloaca do frango de corte em condições de calor sem afetar seu desempenho do crescimento

Abstract Background: High amounts of nonstarch polysaccharides in the diet may increase the amounts of fermentative materials in the hindgut, leading to an increase in fermentative heat production. Dietary β-mannanase is reported to decrease antinutritional effects of β-mannans, such as the potential increase of body heat; however, its efficacy on broiler chickens raised under hot climatic conditions has not been investigated. Objective: To investigate the effects of dietary β-mannanase on growth performance, cloacal temperature, relative lymphoid organ weight, and blood characteristics of broiler chickens raised under hot climatic conditions. Methods: A total of 1,701 1-day-old Ross 308 broiler chickens were randomly allotted to one of three dietary treatments with nine replicates. A basal diet was prepared and added with β-mannanase at 0.05 or 0.10% inclusion levels. The experiment was conducted for 30 days. Average room temperature was 28.8 ± 1.74 ˚C and average relative humidity (RH) was 76.1 ± 11.49% during the experiment. Results: Growth performance of broiler chickens raised under hot climatic conditions was not affected by β-mannanase inclusion. Cloacal temperature decreased at the end of experiment (linear, p<0.05) with increasing inclusion levels of dietary β-mannanase. Increasing inclusion levels of β-mannanase tended to increase (linear, p=0.076) the relative weight of thymus, but had no effects on the relative weight of spleen and bursa of Fabricius. Blood characteristics were not influenced by dietary β-mannanase. Conclusion: Increasing inclusion levels of β-mannanase decrease cloacal temperature; however, it does not directly influence growth performance nor alleviates the heat stress of broiler chickens raised under hot climatic conditions.

Resumen Antecedentes: Altas cantidades de carbohidratos no almidonosos en la dieta pueden aumentar la cantidad de materiales fermentativos en el intestino posterior, aumentando la producción de calor fermentativo. La β-mananasa dietaria disminuye los efectos antinutricionales de los β-mananos, tales como el potencial incremento de la producción calorica; Sin embargo, no se ha investigado su eficacia en pollos de engorde criados bajo condiciones de calor ambiental. Objetivo: Investigar los efectos de la β-mananasa en la dieta sobre el crecimiento, la temperatura cloacal, el peso relativo de los órganos linfoides y las características sanguíneas de pollos de engorde criados bajo condiciones climáticas calientes. Métodos: Un total de 1.701 pollos de engorde de 1 día de edad (Ross 308) fueron asignados al azar a uno de tres tratamientos dietarios con nueve repeticiones. A una dieta basal se le adicionó β-mananasa en niveles de inclusión de 0,05 o 0,10%. El experimento duró 30 días. La temperatura ambiente durante el experimento fue de 28,8 ± 1,74 ˚C y la humedad relativa de 76,1 ± 11,49%. Resultados: La inclusión de β-mananasa no afecto el rendimiento de los pollos. La temperatura cloacal medida al final del experimento disminuyó (lineal, p<0,05) con niveles de inclusión dietarios crecientes de β-mananasa. Niveles incrementales de β-mananasa tendieron a aumentar (lineal, p=0,076) el peso relativo del timo, pero no hubo efecto sobre el peso del bazo o la bursa de Fabricio. La β-mananasa no influenció las características de la sangre. Conclusión: Niveles incrementales de β-mananasa disminuyen la temperatura cloacal, aunque afectan el crecimiento ni alivian el estrés térmico del pollo de engorde criado bajo condiciones climáticas calientes.

Resumo Antecedentes: Altas quantidades de polissacáridos não amiláceos na dieta podem aumentar as quantidades de materiais fermentativos no intestino posterior, levando a um aumento na produção de calor fermentativo. A β-mananase dietética é relatada para diminuir os efeitos antinutricionais da β-manana, tal como o possível aumento da produção de calor; Entretanto, não tem sido investigada a eficácia de la em frangos de corte criados em condições climáticas quentes. Objetivo: Investigar os efeitos da β-mananase na dieta sobre o desempenho do crescimento, a temperatura cloacal, o peso relativo dos órgãos linfóides e as características de sanguíneas de frangos de corte criados em condições climáticas quentes. Métodos: Um total de 1.701 frangos de corte de um dia de idade (Ross 308) foram distribuídos aleatoriamente em um dos três tratamentos dietéticos com nove repetições. A dieta basal foi preparada e a β-mananase foi adicionada à dieta basal com níveis de inclusão de 0,05 ou 0,10%. O experimento foi conduzido durante 30 dias. A temperatura ambiente média foi de 28,8 temperatura ahumidade relativa (HR) média foi de 76,1 ± 11,49% durante o experimento. Resultados: O desempenho de crescimento dos frangos de corte criados em condições climáticas quentes não foi afetado pelos níveis de inclusão de β-mananase. A temperatura cloacal medida no final do experimento foi diminuída (linear, p<0,05) com níveis crescentes de inclusão de β-mananase na dieta. O aumento dos níveis de inclusão da β-mananase tendeu a aumentar (linear, p=0,076) o peso relativo do timo, mas não teve efeitos sobre o peso relativo do baço e da bursa de Fabricius. As características do sangue não foram influenciadas pela β-mananase dietética. Conclusão: Os níveis crescentes de inclusão de β-mananase diminuem a temperatura cloacal; entretanto, isso não influencia diretamente o desempenho do crescimento e alivia o estresse térmico dos frangos de corte criados em condições climáticas quentes.

Production of β-Mannanase by Penicillium italicum Subjected to Different Growth Conditions

Aims: The process parameters affecting enzyme production were optimized to ascertain the best optimal conditions for β-mannanase production by Penicillium italicum in solid state fermentation. Study Design: Four stages of experimental processes were designed for this study. The first experiment, samples were withdrawn after 24, 48, 72, 96, 120, 144,168 and 192 h incubation. In second experiment, the fermentation media were incubated at different temperatures. In third experiment, the effect of different pH values on β-mannanase production was evaluated, while the fourth experiment described the supplementation of surfactants in mineral salt solution for β-mannanase production. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: β-mannanase production was conducted using Locust Bean Gum (LBG) as the sole carbon source; moisten with mineral salt solution, and enzyme activity determined by dinitrosalicylic acid method, while protein content was determined by Lowry method. Results: Maximum enzyme activity (146.389 U/ml) was observed after 72 h of incubation. Different surfactants were supplemented in the basal medium, and Sodium Dodecyl Sulfate (SDS) was observed to give the highest β-mannanase activity of 53.335 U/ml. Initial pH of the culture medium was optimized and a pH of 6.0 was found to support maximum enzyme activity (173.241 U/mg protein). The optimum incubation temperature was achieved at 35°C. Conclusion: The results obtained provide information on optimal process parameters that might improve the yield of β-mannanase by P. italicum for better fish feed formulation, especially in the larval stages of fish fingerlings when the enzyme system is not efficient.

Establishment of fingerprint of Astragali Radix polysaccharides based on endo-1,4-β-mannanase hydrolysis and identification of Astragali Radix of different germplasm resources / 中国中药杂志

The polysaccharides of different germplasm resources of Astragalus membranaceus var. mongholicus〓(cultured Astragalus Radix (RA) and natural RA) and A. membranaceus (MJ) (cultured RA and natural RA) were studied by using the optimal enzymatic conditions of endo-1,4-β-mannanase. Saccharide fingerprints were obtained for the identification and evaluation of the germplasm resources of RA by Fluorophore-assisted Carbohydrate Electrophoresis (FACE). The data were analyzed by principal component analysis to obtain the difference between RA of different germplasm resources. The results showed that trisaccharide, tetrasaccharide and pentasaccharide of endo-1,4-β-mannanase hydrolyzate could be used as the differential fragments to distinguish MG (cultured RA and natural RA); the pentasaccharide and hexasaccharide can be used as differentially expressed carbohydrate fragments that distinguish MJ (cultured RA and natural RA); the trisaccharide and tetrasaccharide can be used as the differential fragments to distinguish the cultured MG and cultured MJ. Studies have shown that polysaccharide products degraded by endo-1,4-β-mannanase can well distinguish RA species (MG and MJ), growth mode (cultured RA and natural RA). This study laid the foundation for the quality evaluation of Astragalus medicinal herbs and screening of active oligosaccharides.

Comparison of Signal Peptides for Two Hemicellulase Secretory Expression / 中国生物工程杂志

Xylanase and mannanase are two hemicellulases,which are widely used in many fields.To improve the expression of thermostable xylanase DSB and thermostable mannanase ManA in Pichia pastoris,three endogenous signal peptides of Pichia pastoris(Scw1 1,Dse4 and Exg1) were chosed.Their capability to mediate the secretion of DSB and ManA with that of the Saccharomyces cerevisiae α-factor were compared.In shake-flask cultivation,three endogenous signal peptides and α-factor efficiently mediated the secretion of DSB and ManA,but the secretion efficiency has obvious difference.As for DSB,the expression efficiency of α-factor was much higher than three endogenous signal peptides.But as for ManA,the expression efficiency of Dse4 was equal to α-factor and much higher than Scw11 and Exg1.Therefore,α-factoris the most efficient signal peptide for DSB expression and Dse4 or α-factor are the most efficient signal peptide for ManA expression in Pichia pastoris X33.Moreover,the intracellular activities of DSB and ManA by α-factor are higher than Scw1 1,Dse4 and Exg1,and the intracellular activity of ManA was higher than DSB (the molecular weight of ManA was larger than DSB).Thus,when ManA were expressed in Pichia pastoris,different signal peptide,such as Dse4,could be used for improving the secretion efficiency.A basis for identifying more available signal peptides and screening for the optimal signal peptide for the target protein was provided.

Screening and Molecular Identification of New Microbial Strains for Production of Enzymes of Biotechnological Interest

ABSTRACT: This research focused on isolation, identification and characterization of new strains of fungi and bacteria, which were able to produce extracellular xylanase, mannanase, pectinase and α-amylase. Fungi isolates were identified on the basis of analyses of 18S gene sequencing and internal transcribed spacer region. The closest phylogenetic neighbors according to 18S gene sequence and ITS region data for the two isolates M1 and SE were Aspergillus fumigatus and Aspergillus sydowii, respectively. I4 was identified as Bacillus mojavensis on the basis of the 16S rRNA gene sequencing and biochemical properties. The enzyme production was evaluated by cultivating the isolated microorganisms in liquid-state bioprocess using wheat bran as carbon source. Two fungi (M1, and SE) and one bacterium (I4) strains were found to be xylanase producer, and several were proven to be outstanding producers of microbial xylanase. The strains producing xylanase secreted variable amounts of starch-debranching enzymes and produced low level β-mannan-degrading enzyme systems. The bacterium strain was found to be capable of producing pectinolytic enzymes on wheat bran at high level. Some of the strains have good potential for use as sources of important industrial enzymes.

Identification of Suitable Condition for Mannanase Production by Bacillus sp. GA2(1).

Aims: The effects of 1% (w/v) supplementation of additional 5 agricultural wastes, corn cob, bagasses, coffee residues, soybean meal, and copra meal for mannanase production by Bacillus sp.GA2(1) were studied. Hence, partial characterization of mannanase was determined. Methodology: The 1%(v/v) overnight cultured of Bacillus sp. GA2(1) was transferred into the basal medium and shaken at 150 rpm for 18 h at 37ºC. The additional of 5 AWs, corn cob, bagasses, coffee residues, soybean meal, and copra meal for the mannanase production were investigated. The cell suspension was centrifuged, and the crude mannanases were collected and stored at – 20ºC for enzyme assay. The mannanase activities were measured by the dinitrosalicylic acid method. The optimal pH of mannanase were studied by measuring enzyme activity at pH 3-10 using 50 mM of following buffers; citrate (pH 3.0-6.0), phosphate (pH 6.0-8.0), and glycine-NaOH (pH 8.0-10.0). The optimal temperature was measured at 30-80ºC. Under standard assay conditions, locust bean gum was used as substrate to determine the optimal pH and temperature of the reaction. Thermostability was determined by preincubating the enzyme at different temperatures (30-80ºC) for 1 h. The residual mannanase activities were measured under standard condition. Results: Among bagasses, coffee residues, soybean meal, corn cob and copra meal, the coffee residues was the most effective carbon source, the maximum yield of mannanase activity was 0.26 U/ml. The optimal temperature and pH for mannanase activity was pH 6.0 and 50ºC of 0.44 and 0.35 U/ml, respectively. The stability of enzyme was determined at 30-80ºC for 60 min. The results revealed that mannanase retained more than 96% of remaining activity after incubation of 60 min at 50ºC. Conclusion: The maximum mannanase production was found when the medium was supplemented with coffee residues. Crude mannanase showed the highest activities of 0.44 U/ml at pH 6.0 and of 0.35 U/ml at 50ºC. The mannanase from Bacillus sp. GA2(1) retained more than 90% of theirs activities at 30-60ºC after preincubated for 60 min and then rapidly decreased.

Characterization and identification of mannanolytic actinomycete Nonomuraea sp. ID06-379

Aims: The study focused on screening, identification and characterization of mannanolytic actinomycetes isolated from soil and leaf litter samples obtained from several sites in Indonesia. Methodology and results: A total of 337 isolates of actinomycetes isolated from soil and leaf litter samples collected from various areas in Indonesia were screened for their mannanolytic activity. Mannanase activity was analysed using locus bean gum (LBG) as the substrate. The strain ID06-0379 displayed significant mannanase activity. The strain ID06- 0379 was analysed for its mannanase activity by determining the rate of enzyme production when cultured in the presence of palm kernel cake (PKC) as a substrate. The highest mannanase activity from ID06-0379 was 4.40 U/mL at 5% PKC concentration at 5 days incubation. Chemotaxonomic and phenotypic characterisation of mannanolytic actinomycete was done and the strain ID06-0379 contained meso-diaminopimelic acid, and madurose was the diagnostic sugar in whole cell sugar. The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, and hydroxy-phosphatidylethanolamine. The predominant menaquinone of strain ID06-0379 was MK-9(H4). The major cellular fatty acids were C16:0 (31.47%), cis9-C16:1 (15.23%) and iso-C16:0 (10.84%), and the G+C content of the DNA was 71.7 mol%. Phylogenetic analysis based on the 16S rDNA sequences revealed that strain ID06- 379 was closely related to species of Nonomuraea jabiensis A4036T with 99% nucleotide similarity. Conclusion, significance and impact study: The results from this study revealed that the mannanolytic actinomycete strain ID06-379 belongs to the genus Nonomuraea that closely related to N. jabiensis A4036T . Mannanase production using agricultural waste such as palm kernel cake may contribute to the development and utilisation of biomass bioconversion processes. Keywords: Indonesian actinomycetes, mannanase enzyme, locus bean gum, palm kernel cake, Nonomuraea sp. ID06- 0379.

Screening of Bacterial Strains of Agricultural Waste Origin for Βeta-mannanase Production and Optimization of Process Parameters.

Aims: This study was carried out to screen bacterial strains of agricultural wastes origin for β-mannanase production and optimization of culture conditions. Study Design: The first experiment, bacterial strains were screened for β-mannanase production. In the second experiment, the best incubation time was determined. In the third experiment, different agricultural wastes were screened. In the fourth experiment, different nitrogen sources were screened. In the fifth and sixth experiments described the effect of different pH values and incubation temperatures on β-mannanase production. The best moisture content was determined in the seventh experiment, while in experiment eight; effect of different inoculum concentrations was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure, Nigeria between September 2011 and March 2012. Methodology: Bacterial strains were screened and β-mannanase production from optimization studies was conducted on Locust Bean Gum. Enzyme activity was determined by dinitrosalicylic acid method. Results: Out of the sixteen bacterial strains screened, Klebsiella edwardsii designated 1A was selected as the most potent in producing enzyme of high activity and it was therefore selected for further studies. Pineapple peels were found to be the most effective carbon source with a highest β-mannanase activity of 8.533±0.08U/ml. Ammonium nitrate (NH4NO3) was obtained to be the best nitrogen source out of all the nitrogen sources screened. The best moisture content was obtained at 1:11 (ratio of substrate to salt solution). Inoculum concentration of 1.0% (v/v) yielded highest β-mannanase activity of 15.833±0.01U/ml. Addition of simple carbon sources to medium containing LBG caused a catabolic repression of β-mannanase synthesis. Conclusion: The optimal culture conditions obtained from this study will help to standardize the requirements for optimum β-mannanase production using cheaper substrates.

Kinetic Properties of Purified β-Mannanase from Penicillium italicum.

Aim: The study aimed at purification and characterization of β-mannanase from Penicillium italicum. Study Design: The first experiment, β-mannanase from Penicillium italicum was produced in basal medium supplemented with Locust Bean Gum (LBG). The second described the purification of crude β-mannanase, while the third experiment dealt with characterization and kinetic studies of purified β-mannanase from Penicillium italicum. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between July and August 2012. Methodology: β-mannanase from Penicillium italicum was produced in basal medium supplemented with LBG. The enzyme was purified by ammonium sulphate precipitation, ion exchange chromatography (DEAE-Sephadex A-50) and gel filtration (Sephadex G-150). The purified enzyme was characterized to determine its optimal conditions by standard assay procedures. The kinetic parameters of the purified enzyme were determined by Lineweaver-Bulk plot. Results: Fractionation of ammonium sulphate precipitated β-mannanase from Penicillium italicum on sephadex A-50 produced one major activity peak. Further fractionation of partially purified enzyme from ion exchange on Sephadex G-150 yielded one activity peak. A pH of 5.0 was optimum for purified enzyme activity and relatively stable between 40 to 100 min of incubation at this pH. The optimum temperature was 70ºC and 100% thermostable for 40 min after which a slight decline in activity was observed. The apparent Km for the hydrolysis of LBG from Lineweaver-Bulk plot was approximately 0.26 mg/mL, while the Vmax was 0.12 μmol/min/mL. The incubation of salts and organic compounds at 10 mM and 40 mM caused inhibition of enzyme activity. At 20 mM, enzyme activity was enhanced by FeSO4.7H2O, SDS and ZnSO4. 7H2O, while others caused inhibition of enzyme activity. The incubation of enzyme with CaCl2 and FeSO4.7H2O at 60 mM enhanced enzyme activity, while others caused inhibition. Conclusion: The result obtained from this study revealed that purified β-mannanase is active over a wide pH and temperature, and its stability implies that the enzyme will be useful during industrial processes where extreme conditions are required.

Isolation and Screening of Mannanase Producing Bacteria from Agricultural Wastes.

Aim: The work focused on the isolation and screening of mannanase-producing bacteria associated with selected agricultural wastes. Study Design: The first experiment, mannanase-producing bacteria were screened for mannanase production on Locust Bean Gum (LBG) agar medium and total bacterial count was determined. In the second experiment, the isolated bacteria were further screened for mannanase production in submerged state fermentation. Place and Duration of Study: Microbiology Research Laboratory Federal University of Technology, Akure and Postgraduate Research Laboratory, Obafemi Awolowo University Ile-Ife, Nigeria between September 2011 and March 2012. Methodology: The associated bacterial isolates were isolated on agar medium containing LBG and counted by standard microbiological methods. Quantitatively, mannanase production was conducted in mineral salt medium into which copra meal had been incorporated as the sole carbon source and enzyme activity was determined by dinitrosalicylic acid method. Results: The highest bacteria counts were recorded in compost from wood dust with 5.5×1011 cfu/g, while cassava peels had the least of 1.02×106 cfu/g. In this study, 23 bacterial isolates showed positive results with clear zone around the cultures. Bacterial isolate 1A showed the highest ratio of clear zone to colony, while the lowest was observed in isolate 4B. In liquid broth, all the 23 isolates displayed mannanase activity between 0.28 to13.89 U/ml for static and 0.56 to 13.43 U/ml for shaken condition, with the highest mannanase activity observed with isolate IA for both culture conditions. In the comparative study between static and shaken conditions, it was revealed that shaken cultures exhibited better yield than static cultures. According to the morphological and biochemical studies, the isolate 1A was primarily identified as the Klebsiella edwardsii. Conclusion: In this investigation, bacterial isolates evaluated for mannanase production from agricultural wastes elaborated considerable mannanase activity and this could be applied in feed and prebiotic.

Screening and Identification of Mannanase- Producing Fungi Isolated from Selected Agricultural Wastes.

Aim: The study evaluated potential performance of different fungal isolates from agricultural by-products for mannanase production. Study Design: The first experiment, fungal isolates were screened for mannanase production on agar medium containing Locust Bean Gum (LBG) and total fungal count was conducted. In the second experiment, the fungal isolates were further screened for mannanase production in submerged state fermentation. Place and Duration of Study: Microbiology Research Laboratory Federal University of Technology, Akure and Postgraduate Research Laboratory, Obafemi Awolowo University Ile-Ife, Nigeria between September 2011 and March 2012. Methodology: The fungal isolates associated with some agricultural wastes were isolated on LBG containing agar medium by plate assay techniques and counted by standard microbiological methods. Mannanase production was conducted in submerged state fermentation (shaken & static) into which copra meal had been supplemented as the sole carbon source and enzyme activity was determined by dinitrosalicylic acid method. Results: In this study, 11 fungal isolates showed positive results with clear zone around their cultures. Fungal isolate 5A showed the highest activity ratio of 1.8, while the least was observed in isolate 9A12 with activity ratio of 0.64. The highest fungal counts were recorded in fermented coconut with 7.4×102 sfu/g, while cocoa pod and groundnut shell had no fungal growth. In terms of percentage occurrence of fungal isolates from selected agrowastes, it was revealed that Rhizopus japonicus had the highest occurrence of 66.67%, while the same value of 8.33% was observed for Aspergillus fumigatus, A. glaucus, R. stolonifer and Trichosporonoides oedocephalis. In fermentation broth, all the 11 isolates displayed mannanase activity ranging from 0.370 to 21.667 U/ml for static and 0.278 to 3.982 U/ml for shaken condition, with the highest mannanase activity observed with isolate 5A for both culture conditions. According to the cultural characters and microscopic morphology, the isolate 5A being the highest mannanase producer was identified as the Aspergillus fumigatus. Conclusion: In this study, fungal isolates screened and evaluated for mannanase production from agricultural by-products elaborated considerable mannanase activity and this could be exploited for prebiotic preparation.

Fermenation Products of β-Mannanase Producing Bacteria Inhibit Selected Poultry Borne Pathogens.

Aim: The study evaluated the inhibitory effect of fermentation products of β-mannanaseproducing bacteria on selected poultry borne pathogens. Study Design: The first experiment, bacterial isolates previously confirmed positive for mannanase by plate assay technique were further screened for mannanase production in submerged state fermentation. In the second experiment, inhibitory effect of fermentation products of mannanase-producing bacteria on selected poultry pathogens was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: Bacterial isolates from agricultural wastes previously confirmed positive for mannanase activity by plate assay were further screened for their potential performance under submerged state fermentation and enzyme activity determined by dinitrosalicylic acid method. The inhibitory action of β-mannanase-producing bacteria was determined by supplementation of supernatant and plating method. Results: Isolate 1A showed highest mannanase activity (13.430 U/ml), displayed broad inhibition to selected poultry borne pathogens; Klebsiella oxytoca, Shigella alkalescens, Escherichia coli, Salmonella typhii, Staphylococcus aureus and Streptococcus sp. Apart from isolate 1A, fermentation products of other isolates generated from the mannolytic action of β-mannanase on mannan containing substrate displayed different percentage inhibition on selected poultry borne pathogens. Conclusion: The results suggested that fermentation products from β-mannanaseproducing bacteria might possess antibacterial properties which could be applied in poultry farms.

Characterization of Solid State Fermentation Culture Conditions for Growth and Mananase Production by Aspergillus niger USM F4 on Rice Husk in Tray System.

Aim: The study evaluated various fermentation conditions for the production of mannanase. Place and Duration of Study: Industrial Biotechnology Research Laboratory (IBRL), School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia between May 2009 and September 2010. Methodology: Solid substrate fermentation was carried out in a shallow aluminum tray system (16 cm x 16 cm x 5 cm) for maximum mannanase production by Aspergillus niger USM F4 using rice husk as a substrate. Results: The maximum mannanase activity of 119.91 U/g substrate was achieved on the 6 days of cultivation when the optimized physical parameters were used (substrate thickness of 1.6 cm or equivalent to 80 g of 0.75 mm rice husk, moisture content to substrate ratio of 1:1 (w/v), cultivation temperature at room temperature (28±2ºC), inoculum size of 6x106 spores/ml and in static condition (no mixing during the fermentation process). The results showed an increment of about 30.79% of mannanase activity after the optimization (119.91 U/g substrate) compared to before optimization (91.68 U/g substrate). Conclusion: The results obtained from this study revealed that rice husk can be used as a substrate for mannanase production in solid state fermentation process.

Armazenamento e qualidade de sementes de tomate enriquecidas com micronutrientes e reguladores de crescimento / Storage and quality of tomato seeds enriched with micronutrients and growth regulators

O estudo foi desenvolvido na Universidade Federal de Lavras, em Lavras, MG, com o objetivo de avaliar o efeito da aplicação de micronutrientes e reguladores de crescimento na germinação, no vigor, na atividade de algumas enzimas e no teor de proteínas totais em sementes de tomate, durante o armazenamento. As sementes foram tratadas com os produtos Starter®, Cellerate® e Stimulate® nas dosagens correspondentes a 0 por cento, 50 por cento, 100 por cento, 150 por cento e 200 por cento da dose recomendada pelo fabricante, utilizando a técnica de peliculização. As avaliações foram realizadas aos zero, 6 e 12 meses de armazenamento, pelos seguintes parâmetros: porcentagem de germinação, porcentagem e índice de velocidade de emergência, atividade das enzimas endo-â-mananase e esterase, teor de proteínas totais e sanidade. Foi utilizado o delineamento inteiramente casualizado, em esquema fatorial 3 x 5 x 3 ( 3 produtos x 5 doses x 3 épocas de armazenamento), com quatro repetições de 50 sementes por tratamento. Concluiu-se que a combinação dos reguladores de crescimento contidos no produto Stimulate® promove aumento na velocidade de emergência das plântulas de tomate quando aplicados na dose recomendada e na pré-semeadura; a atividade da enzima esterase aumenta com o período de armazenamento das sementes de tomate, indicando aumento no processo de deterioração; o revestimento enriquecido com os micronutrientes e reguladores de crescimento contidos nos produtos Starter®, Cellerate® e Stimulate® e o armazenamento interferem na atividade da enzima endo-â-mananase em sementes de tomate; há um aumento no teor de proteínas totais em sementes de tomate com o aumento do período de armazenamento.

The objective of this study was to evaluate the effect of the application of micronutrients and growth regulators on the germination, vigor, activity of some enzymes, and on the total protein contents in tomato seeds during the storage. The seeds were treated with the chemicals Starter®, Cellerate®, and Stimulate® at the dosages corresponding to 0 percent, 50 percent, 100 percent, 150 percent, and 200 percent of the dose recommended by the manufacturer, utilizing the film-coating technique. The evaluations were performed at 0, 6, and 12 months of storage by the following parameters: percentage of germination, percentage and emergency velocity rate, activity of enzymes endo-â-mannanase and esterase, total protein content, and health. The completely randomized design in a 3 x 5 x 3 factorial structure was utilized, namely, three chemicals, five doses, and three storage periods with four replicates of 50 seeds per treatment. Coating with the chemicals based on micronutrients and growth regulators and storage interfere on the activity of enzyme endo-â-mannanase in the tomatoes and seeds; the activity of enzyme esterase increases with the storage of tomato seeds, pointing to an increase in the deterioration process; there is an increase in the total protein content in tomato seeds throughout the storage period; chemicals based on growth regulators promote an increase in emergency velocity rate of the tomato seedlings when applied at pre-sowing at the recommended dose.

Evaluation of the activities of concentrated crude mannan-degrading enzymes produced by Aspergillus niger

The mannan-degrading enzymes produced by Aspergillus niger were concentrated and the activities were evaluated. The optimum pH for -mannanase, endoglucanase and -galactosidase were obtained at pH 3.5 while pH optimum for -mannosidase was occurred at pH 3.0. The -mannanase, endoglucanase, -mannosidase and -galactosidase was stable at pH 3.5 to 7, pH 3.5 to 6.5, pH 4 to 7 and pH 3.5 to 5.0, respectively. The enzymes obtained in this study were characterized and defined as acidic proteins. The -mannanases from A. niger had two optimum temperatures (at 50 °C and 60 °C) and its half-life was 6 h and 4 h at 60 °C and 70 °C, respectively. The -mannosidase, endoglucanase and -galactosidase displayed optimal activity at 70 °C, 60 °C and 50 – 60 °C, respectively. The -mannosidase had half-life of 1.5 h at 70 °C, while -galactosidase had a half-life of 2.5 h at 60 °C and endoglucanase had a half-life of 6 h at 60 °C and 45 min at 70 °C.

Purification and Characterization of ?-mannanase atMAN47 from Armillariella tabescens / 微生物学通报

Armillariella tabescens EJLY2098 was induced to produce ?-mannanase with konjac fine flour (Amorphopallus rivieri) as single carbon source. This induced enzyme was then purified using DEAE ion exchange chromatography and named atMAN47. Zymologic analysis showed that the molecular weight of this ?-mannanase was approximately 47 kD. The enzyme was stable when pH ranged from 5.0 to 6.5 and could be activated by Na+ and Ba2+. With an optimal temperature of 50?C. Action mode analysis of TLC revealed that the enzyme belonged to the endo-?-mannanase family. Being a meta-acid endo-?-mannanase, it was suitable to be applied to feed industry with a promising future as an enzyme preparation.

Identification of Chaetomium sp. CQ31 and Optimization of Xylanase Production in Solid State Fermentation / 微生物学通报

A fungus CQ31 isolated from soil samples was identified as Chaetomium sp.. This strain produced effectively xylanases utilizing several liguocellulosic materials in the solid-state fermentation (SSF), and corn straw was the best carbon source. The results of single-factor-experiment showed that the corn straw as the carbon source, tryptone as the nitrogen source, initial moisture content of 80% and initial pH 9.0 were the optimal conditions for xylanase production. Under the optimized conditions, it produced xylanase which was 4897 U/g dry substrate while mannanase was 803 U/g dry substrate after 7 days of cultivation. Therefore, xylanase and mannanase production by Chaetomium sp. CQ31 in SSF possess potential for commercial applications.