Enzymatic preparation of fructooligosaccharides-rich burdock syrup with enhanced antioxidative properties

Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

Abstract The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30 °C, 6% (v/v), inoculum size and 150 rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Enhancing inulinase yield by irradiation mutation associated with optimization of culture conditions

A new inulinase-producing strain was isolated from rhizosphere soils of Jerusalem artichoke collected from Shihezi (Xinjiang, China) using Jerusalem artichoke power (JAP) as sole carbon source. It was identified as an Aspergillus niger strain by analysis of 16S rRNA. To improve inulinase production, this fungus was subjected to mutagenesis induced by ⁶⁰Co γ-irradiation. A genetically stable mutant (designated E12) was obtained and it showed 2.7-fold higher inulinase activity (128 U/mL) than the parental strain in the supernatant of a submerged culture. Sequential methodology was used to optimize the inulinase production of stain E12. A screening trial was first performed using Plackett-Burman design and variables with statistically significant effects on inulinase bio-production were identified. These significant factors were further optimized by central composite design experiments and response surface methodology. Finally, it was found that the maximum inulinase production (185 U/mL) could be achieved under the optimized conditions namely pH 7.0, yeast extract concentration of 5.0 g/L, JAP concentration of 66.5 g/L, peptone concentration of 29.1 g/L, solution volume of 49.4 mL in 250-mL shake flasks, agitation speed of 180 rpm, and fermentation time of 60 h. The yield of inulinase under optimized culture conditions was approximately 1.4-fold of that obtained by using basal culture medium. These findings are of significance for the potential industrial application of the mutant E12.

New isolate for enhancement production of microbial inulinase / Novo isolado para reforço de produção microbiana inulinase

The optimization of growth conditions for the production of inulinase by Penicillium funiculosum cells were studied as well as the continuous production of the enzyme using immobilized cells. The highest amount of enzyme (163.5U/mL) was obtained when the producing cells were incubated for 96 hours at 27oC and 200 rpm in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen sources respectively. However, when the cells of the tested microorganism were adsorbed on different carriers, especially linen fibers, their production ability was also successfully maintained, to different extends, for seven successive batches. Moreover, commercially pure inulin is very expensive in only small quantities, this fermentation medium was later substituted by a crude inulin solution obtained from Jerusalem artichoke tubers (Helianthus tuberosus). The crude inulin juice was able to sustain inulinase production during the second batch cultivation of the P. funiculosum, immobilized by their adsorption on linen fibers, in a satisfactory level of about 122U/mL. Moreover, the use of the previously mentioned crude inulin preparation was also compared to the use of either complete or minimal media, composed solely of 1% pure inulin. The method, adopted in this study for inulinase production, is simple, economic, time saving, non-toxic to the microorganism and the loaded linen pads are reusable.

A otimização das condições de crescimento para a produção de inulinase por células de Penicillium funiculosum foram estudados, bem como a produção contínua da enzima utilizando células imobilizadas. A maior quantidade de enzima (163.5U / mL) foi obtida quando as células produtoras foram incubadas durante 96 horas a 27 ° C e 200 rpm num meio de fermentação contendo ambos inulina e peptona como fontes de carbono e nitrogênio, respectivamente. No entanto, quando as células do microorganismo testado foram adsorvidas em diferentes suportes, especialmente fibras de linho, a sua capacidade de produção foi também mantida com sucesso, por diferentes extensões, e por sete lotes sucessivos. Por outro lado, a inulina comercialmente pura é muito dispendiosa em apenas pequenas quantidades. Este meio de fermentação foi depois substituído por uma solução de inulina bruta obtida a partir de tubérculos de alcachofra-girassol (Helianthus tuberosus). A inulina bruta foi capaz de sustentar a produção de inulinase durante o segundo lote de cultura de P. funiculosum, imobilizado pela sua adsorção nas fibras de linho, em um nível satisfatório de aproximadamente 122U / mL. Além disso, a utilização da preparação de inulina bruta anteriormente mencionada foi também comparada com o uso de meios completos ou mínimos, compostos unicamente de 1% de inulina pura. O método, adotada neste estudo para produção da enzima, é simples, de baixo custo e com economia de tempo. Além disso, não apresenta toxicidade para o microorganismo e os suportes de linho são reutilizáveis.

Production of Inulinase by Free and Immobilized Cells of Penicillium funiculosum p.36

The aim of this work was to optimize the growth conditions and continuous production of the enzyme using free and immobilized cells of inulinase by Penicillium funiculosum. The highest yield of enzyme (163.5U/mL) was obtained when the culture was incubated at 27oC and 200 rpm for 96h in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen source, respectively. When the cells of the P. funiculosum were immobilized on different carriers, especially linen fibers, their production ability was successfully maintained for seven successive batches. When the fermentation was carried out using inulin juice prepared from Jerusalem artichoke tubers (in place of pure inulin), inulinase production could be sustained till the second cultivation batch of the P. funiculosum immobilized on linen fibers, yielding 122 U/mL enzyme. Results proved the feasibility of using crude inulin juice as a simple and economic carbon source for the production of inulinase.

Immobilization and characterization of inulinase from Ulocladium atrum on nonwoven fabrics.

Ulocladium atrum inulinase was immobilized on different composite membranes composed of chitosan/nonwoven fabrics. Km values of free and immobilized U. atrum inulinase on different composite membranes were calculated. The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester nonwoven fabric coated with 3% chitosan solution (PPNWF3), but optimum pH was 5 for immobilized U. atrum inulinase on polyester and polypropylene nonwoven fabrics coated with 1% chitosan solution. The enzyme had optimum temperature at 40°C for immobilized enzyme on each of polyester and polypropylene composite membranes coated with 1% chitosan, while it was 50°C for free and immobilized enzyme on polypropylene nonwoven fabric coated with 3% chitosan solution. Free U. atrum inulinase was stable at 40°C but thermal stability of the immobilized enzyme was detected up to 60°C. Reusability of immobilized enzyme was from 38 to 42 cycles of reuse; after this, the immobilized enzyme lost its activity completely. In conclusion, immobilized U. atrum inulinase was considerably more stable than the free enzyme, and could be stored for extended periods.

Isolation of inulinase producing bacteria from sugarcane soil.

Microbial inulinases have a great potential for industrial use in the production of fructose from inulin. Optimization of the growth parameters of the microbes is essential to obtain inulinase in sufficient quantity. The present work aimed to obtain inulinase from natural strains isolated from sugarcane fields and to optimize its growth parameters. In a total of ten isolates, four organisms Bacillus subtilis, Lactobacillus casei, Pseudomonas aeruginosa, and Achromobacter sp. were identified as efficient inulinase producers. The optimum temperature and pH of these organisms were found to be 40°C and pH 5 respectively. Inulin was observed to be the suitable carbon source for these organisms. The molecular weight of this enzyme was estimated at 45 KDa using SDS-PAGE. On thin layer chromatography inulin hydrolysis showed mono and disaccharides as the main end products. The highest enzyme activity was obtained from Achromobacter spp. of 333 U/L at 22nd hour which showed exo-inulinase type.

Partial characterization of an inulinase produced by Aspergillus japonicus URM5633

Enzymes obtained by fermentation processes offer a number of advantages and have been widely researched and used throughout the world. This study aimed to partially characterise an inulinase produced from palm and cassava peel. The enzyme was produced via the solid-state fermentation of Aspergillus japonicus URM5633. The optimal temperatures were 50ºC and 55ºC, and the optimal pH values were 5.2 and 3.4 for inulinase fermentatively produced from palm and cassava peel, respectively. The thermostability measurements for inulinase produced in palm showed that the relative activity remained below 100% until 30 minutes of stability for all temperatures, but reached 106.8% at a temperature of 50ºC after 60 minutes. Inulinase from the crude extract of cassava peel was pH stable and only decreased to 55% of the maximal activity over the course of the assay, suggesting that this enzyme can be used in inulinase production and can be utilized in food industries.

Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611

Background: Inulinase is a versatile enzyme from glycoside hydrolase family which targets the beta-2, 1 linkage of fructopolymers. In the present study, the effect of medium composition and culture conditions on inulinase production by Aspergillus niger ATCC 20611 was investigated in shake-flasks. Results: The highest extracellular inulinase (3199 U/ ml) was obtained in the presence of 25 percent (w/v) sucrose, 0.5 percent (w/v) meat extract, 1.5 percent (w/v) NaNO3 and 2.5 mM (v/v) Zn2+, at initial pH of 6.5, temperature 35ºC and 6 percent (v/v) of spores suspension in the agitation speed of 100 rpm. Surfactants showed an inhibitory effect on enzyme production. The optimum temperature for inulinase activity was found to be 50ºC. TLC analysis showed the presence of both exo- and endo-inulinase. Conclusion: Sucrose, Zn2+, and aeration were found to be the most effective elements in inulinase production by A. niger ATCC 20611. TLC analysis also showed that the crude enzyme contained both endo and exo-inulinases. The strain is suggested as a potential candidate for industrial enzymatic production of fructose from inulin.

Production of inulinase from Kluyveromyces marxianus using Dahlia tuber extract

Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL-1) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL-1). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL-1) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C- sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL-1) and peptone (13.8 nkat mL-1). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production.

Effects of culture conditions on the production of inulinase by Kluyveromyces marxianus

The present study was conducted to investigate the influence of initial sucrose concentration, pH and aeration rate on biomass and inulinase production by Kluyveromyces marxianus var. bulgaricus in a stirred batch reactor. Maximum inulinase activity (15.29 UmL-1) was obtained at a sucrose concentration of 10 g L-1, pH 5.0 and aeration rate of 1 vvm. The 20 g L-1 sucrose concentration was suitable for cell growth; however, enzymatic activity at this concentration was inhibited due to catabolic repression. The increase in aeration rate caused a reduction in enzyme activity with no relevant biomass increase.

O estudo foi conduzido para investigar a influência da concentração inicial da sacarose, a taxa da aeração e do pH na biomassa e na produção da inulinase pela Kluyveromyces marxianus var. bulgaricus em um reator em batelada. A máxima atividade de inulinase, 15.29 UmL-1, foi obtida na concentração de 10 g L-1 de sacarose, no pH 5.0 e na taxa da aeração de 1 vvm. A concentração de sacarose de 20g L-1 foi apropriada para o crescimento celular, porém nesta concentração a atividade enzimática foi inibida, devido a repressão catabólica. O aumento na taxa da aeração propiciou redução da atividade enzimática, ao mesmo tempo em que não houve aumento considerável do biomassa.

Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

Aspergillus niger - 245, a strain isolated from soil samples showed good β-fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I) was between 4.0 - 4.5 and for invertase activity (S) between 2.5 and 5.0. The optimum temperature was 60O.C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The Km value was 1.44 and 5.0, respectively, for I and S and Vm value 10.48 and 30.55, respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10-3 M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism, acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained, although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrups.

Aspergillus niger - 245, isolado do solo mostrou boa atividade de b-frutosidase meio formulado com extrato de tubérculos de dahlia. A enzima foi purificada por precipitação em sulfato de amônia e percolada em colunas de DEAE-Sephadex A-50 e CM-celulose, produzindo um único pico em todas as fases de purificação e mantendo a relação I/S entre 0,32 a 0,39. O pH ótimo para a atividade de inulinase (I) foi encontrado entre 4,0 - 4.5 e para a atividade de invertase (S) em 2,5 e 5,0. A temperatura ótima foi de 60O.C para ambas as atividades e nenhuma perda foi observada quando mantida nesta temperatura por 30 min. Os valores de Km foram de 1,44 e 5,0, respectivamente, para I e S e os valores de Vm de 10,48 e 30,55, respectivamente. A atividade I foi fortemente inibida por Hg2+, Ag+ e 2 x 10-3 M de glicose, mas não por frutose na mesma concentração. A enzima mostrou um mecanismo de exo-ação, atuando sobre a inulina de diferentes origens. Em condições de ensaio foi obtida hidrólise total de frutanas, apesar de ter mostrado maior atividade sobre a inulina de chicória que sobre as de alcachofra de Jesrusalém e dahlia, nos primeiros 30 minutos de reação. Os resultados obtidos sugerem que a enzima apresenta bom potencial para aplicações industriais na preparação de xaropes de frutose.

Studies on the Inulinase Protective Agent of the Aspergillus niger U?-2 / 微生物学通报

The thermostability of the inulinase was studied in this resea rc h. Some alcoholic materials and thickening agent could enhance the thermostabli lity of the inulinase. Using glycerol、xanthic pastern and though orthogonal ex periments of three elements and three levels, a satisfying protective agent, whi ch included glycerin(6%), xanthan gum(0.6%) and CaCl_2 (100mmol/mL) and ha d a significant effect on the enhancement of the inulinase thermostability, was acquired.