Isolation and Characterization of Alkaline Pectinase Productive Bacillus tropicus from Fruit and Vegetable Waste Dump Soil

Abstract Alkaline pectinase is the utmost significant industrial enzyme of the bioscouring process. By considering bio scouring of cotton, 30 microbial isolates from fruit and vegetable waste rich dump soil of Solang Valley and Vasishta (Manali, Himachal Pradesh, India) were isolated and screened for the alkaline pectinase production in the current research work. Only four isolates P3, P16, P21, and P27 were capable to produce extracellular alkaline pectinase at pH 9. Further by applying submerged fermentation, the alkaline pectinase production was quantitatively screened. The most efficient isolate was P3 identified as Bacillus tropicus, based on morphological, biochemical, and molecular characterization. Molecular characteristics confirmed by 16S rDNA sequence analysis. The nucleotide sequence of the isolate was novel with a 97% similarity index and submitted to the GenBank with accession number MK332379. The Bacillus strain selected was active at broad pH range from 8-10.5 and a temperature range from 25-50 oC. Optimum pH and temperature observed were 9 and 37 oC respectively and can be suitably used for the bio scouring process for the pretreatment of the fabrics.

Elaboration of mangaba vinegar by semi-solid fermentation combined with enzymatic activity: chemical characterization and sensory evaluation / Elaboração de vinagre de mangaba por fermentação semi-sólida com ação enzimática: caracterização química e avaliação sensorial

ABSTRACT: The objective of this study was to produce vinegar from mangaba pulp using semi-solid alcoholic fermentation combined with the enzymatic activity of pectinase and to investigate the chemical composition and sensory characteristics of the final product. was evaluated for volatile acidity and the reduced dry extract was evaluated for ashes, alcohol content, sulfates, pH, total phenolic compounds, total carotenoids, color parameters, yield, productivity, and sensory analysis. Average and standard deviation was used for descriptive statistics. Principal component analysis (PCA) was applied to all variables except total carotenoid content. Physicochemical characterization of the raw and alcoholically fermented pulp was also carried out. The main results showed that, in the vinegar, the reduced dry extract, volatile acidity, pH, and ashes were 44.3±1.5 (g/L), 4.4±0.1 (% w/v), 3.1±0.0, and 3.0±0.41 (g/L), respectively. The total phenolic compound content and total carotenoid content for the mangaba vinegar were 19.2±8.20. mg/100 g and 2.6±0.6. mg/100 g, respectively. The conversion yield from ethanol to acetic acid was 90%. PCA showed that pH and volatile acidity had a strong influence on the product, and there was a strong positive correlation between color and aroma. The final product met all legal requirements, showing that it is possible to produce mangaba vinegar with antioxidant potential for consumers. In the sensory evaluation, it was favored by the tasters, demonstrating potential economic value in the Cerrado fruit.

RESUMO: Objetivou-se produzir vinagre, a partir da polpa de mangaba por fermentação alcoólica semi-sólida com ação enzimática através da pectinase, investigar a composição química e avaliação sensorial do produto final. O vinagre foi avaliado através da acidez volátil, extrato seco reduzido, cinzas, teor alcoólico, sulfatos, pH, compostos fenólicos totais, carotenoides totais, parâmetros de cor, rendimento, produtividade e análise sensorial. Os dados foram submetidos a estatística descritiva com média e desvio padrão. Foi aplicado a análise de componentes principais (ACP) para todas as variáveis, exceto para análise de carotenoides totais. Também foi realizada a caracterização físico-química da polpa e fermentado alcoólico. Os principais resultados mostraram que, no vinagre, extrato seco reduzido, acidez volátil, pH e cinzas foram, respectivamente, 44,3±1,5 (g/L), 4,4±0,1 (% m/v), 3,1±0,0, 3,0±0,41 (g/L). Os compostos fenólicos totais e carotenoides totais apresentaram valores de 19,2±8,20 (mg/100 g) e 2,6±0,6 (mg/100 g), respectivamente. O rendimento de conversão de etanol a ácido acético foi de 90%. ACP foi aplicada nas variáveis físico-químicas do vinagre no qual os parâmetros de cor, pH e acidez volátil apresentaram forte influência no produto e, para os atributos da análise sensorial, cor e aroma apresentaram uma forte correlação positiva entre si. O produto final atendeu a todos os quesitos legais, demonstrando ser possível a produção de vinagre de mangaba com potencial antioxidante. Na avaliação sensorial teve boa aceitação pelos provadores, valorizando o uso deste fruto do Cerrado.

Purification and biochemical characterization of pectinase produced by Aspergillus fumigatus isolated from soil of decomposing plant materials

This study isolated, screened, and identified a pectinase-producing fungus from a decomposing plant material. Italso cultured the isolated fungus under optimized conditions to obtain crude pectinase enzyme as well as purifiedand investigated the biochemical characteristics of the purified enzyme. The fungal strain was isolated on pectinasescreening agar medium containing 1% pectin and obtained a clear zone. It was identified as Aspergillus fumigatus andcultivated for enzyme production using banana, plantain, and orange peels as the solid substrate. Under optimizedconditions, a maximum of 3.52 U/ml pectinase activity was obtained at 65% moisture content after 144 h (6 days)of incubation period on orange peel, 1.5 ml inoculum, and 3% salt content. A. fumigatus pectinase was purified4.45-fold and a yield of 26.16% with a specific activity of 38.88 U/mg. The molecular weight determined on sodiumdodecyl sulfate (SDS-PAGE) was 31.6 kDa. The pectinase exhibited maximum activity at 60°C, optimum pH of 5.0,and stability at 40–50°C. The enzyme showed a preference for polygalacturonic acid as its primary substrate with aKM 3.08 mg/ml and Vmax of 1.61 U/ml. The enzyme was activated by 0.5 mM Na+, K+, and 1–5% toluene. The enzymeactivity was inhibited by metal cations; 20% ethanol, 4.0 mM SDS, and L-cysteine. The obtained results showed thatA. fumigatus pectinase could be a candidate for potential industrial and biotechnological application

Assessment of pectinase-producing fungi isolated from soil and the use of orange waste as a substrate for pectinase production

Pectinases are important enzymes not only for their potential applications in different industries such animal feed, agricultural, textile, beverage, food processing, oil extraction, etc. Ten fungal species were isolated from the soil and screened for production of pectinase enzyme by using the pectin agar medium. Pectinolytic enzymes synthesis were attained at a temperature of 30 °C and activities were determined after a seven-days culture of Aspergillus sp. 391 and Aspergillus sp. 031, in a basic medium containing 2% citrus pectin and as the sole carbon source. The extract enzymatic showed an optimum activity for exo-polygalacturonase (PG) and pectin lyase (PNL) against galacturonic acid and pectin at pH 4.5 and 5.5, respectively. There were variations in PG and PNL enzymes levels produced in culture filtrates obtained of Aspergillus sp. 391 with addition of citrus waste (2.0 and 4.0 % w/v) to the medium. Maximum activity for PNL activity was observed in the medium containing 5% pectin or 4% citrus waste, as sole carbon source, after 7 days of growth. The results showed that the isolate Aspergillus sp. 391 is a promising for pectinolytic enzymes production at the industrial level.(AU)

Process optimization for pectinase production by locally isolated fungal strain using submerged fermentation / Otimização de processos para produção de pectinase por cepa fúngica localmente isolada usando fermentação submersa

The present study deals with the isolation screening and optimization of fungal strain for pectinase production. The fungal strains were isolated from different sources, including soil, fruits etc. Qualitative screening was performed on the basis of the pectin hydrolysis zone. While, quantitative screening was carried out employing submerged fermentation. Among all the strains the strains showing highest pectinolytic potential were selected identified and assigned the code Aspergillus niger ABT-5.The influence of different fermentation media on pectinase production was evaluated. The M5 medium containing 10g wheat bran, nutrient medium containing (g/l) of (NH4)2SO4 6.0, K2HPO4 6.0, KH2PO4 6.0, MgSO4.7H2O 0.1 gave the highest pectinase production. The other important physico chemical parameters including incubation period, temperature, and volume of media, size of inoculum, carbon and nitrogen sources were also optimized for pectinase production. The highest pectinase production (15.5U/ml) was obtained at 72h of incubation, pH 6, temperature 30°C, volume of media 50ml. Fructose and urea were designated as best carbon and nitrogen sources subsequently.

O presente estudo trata da triagem de isolamento e otimização da cepa fúngica para produção de pectinase. As cepas fúngicas foram isoladas de diferentes fontes, incluindo solo, frutas, etc. A triagem qualitativa foi realizada com base na zona de hidrólise da pectina. Enquanto, a triagem quantitativa foi realizada utilizando fermentação submersa. Entre todas as cepas, as cepas que apresentaram maior potencial pectinolítico foram selecionadas e atribuídas ao código Aspergillus niger ABT-5. Avaliou-se a influência de diferentes meios de fermentação na produção de pectinase. O meio M5 contendo 10g de farelo de trigo, meio nutriente contendo (g / l) de (NH4)2SO4 6.0, K2HPO4 6.0, KH2PO4 6.0, MgSO4.7H2O 0.1, proporcionou a maior produção de pectinase. Os outros parâmetros físico-químicos importantes, incluindo período de incubação, temperatura e volume dos meios, tamanho do inóculo, fontes de carbono e nitrogênio também foram otimizados para a produção de pectinase. A maior produção de pectinase (15,5U / ml) foi obtida às 72h de incubação, pH 6, temperatura 30 ºC, volume dos meios 50ml. A frutose e a ureia foram designadas como melhores fontes de carbono e nitrogênio posteriormente.

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.

Production and Partial Characterization of Pectinase from Snail (Archachatina marginata) Gut Isolates.

Aim: To identify isolates for industrial production of pectinase. Methodology: The isolates were screened for pectinolytic activity using pectin as substrate. Enzyme activity was expressed as mg of glucose equivalent released per ml of crude enzyme solution per second. The kinetic parameters of the enzyme were determined to obtain the optimum pH, temperature, Km and Vmax. Pectinase produced from Bacillus subtillis was immobilized on chitosan and its activity was compared with that of free enzyme. Results: Pectinase from Bacillus subtilis was screened to have the highest enzymatic activity among bacterial isolates while pectinase from Aspergillus niger had the highest enzymatic activity among fungal isolates. High yield of pectinase enzyme was obtained from B. subtilis after 24hrs with activity of 4.01×10-4 mg/ml/sec while high yield of pectinase was obtained from A. niger on the 5th day with activity of 2.07×10-4 mg/ml/sec. The optimum pH for pectinase produced from B. subtilis and A. niger were 8 and 6, respectively. The optimum temperature for pectinase from B. subtilis and A. niger were at 50ºC and 40ºC, respectively. The Vmax and Km of pectinase from B. subtilis and A. niger were 16.88×10-4 (mg/ml/sec) and 10 (mg/ml); 9.29×10-4 (mg/ml/sec) and 30 (mg/ml), respectively. Optimum temperature and pH of immobilized pectinase were 70ºC and 4.0, respectively. Residual activity of immobilized enzyme was 92% after storage at 4ºC for 14 days. Conclusion: This study revealed that Bacillus subtilis from snail gut may be considered as a good candidate for industrial production of pectinase.

Study on Extraction Technology of Polysaccharides from Gossypium herbaceum L. by Pectinase Hydrolysis / 中国药房

OBJECTIVE:To optimize the extraction technology of polysaccharides from Gossypium herbaceum L. by pectinase hydrolysis. METHODS:Polysaccharides were extracted from G. herbaceum L. by pectinase hydrolysis combined with traditional hot water extraction. With the extraction rate of polysaccharides as the evaluated index,single factor test and orthogonal design were applied to investigate the effects of 4 factors including enzymolysis temperature,enzymolysis time,the amount of enzyme and pH value on polysaccharides extraction rate,and verification tests were conducted. RESULTS:The optimal extraction technolo-gy was as follows as enzymolysis temperature of 40 ℃,enzymolysis time of 150 min,enzyme accounting for 2.0%,pH value for enzymolysis of 4.6. Under the above conditions,the average extraction rate of polysaccharides from G. herbaceum L. was 2.474%(RSD=3.34%,n=5). CONCLUSIONS:Pectinase hydrolysis is an effective method to extract polysaccharides from G. herbace-um L.. The optimal extraction technology is reasonable and feasible.

Multistep mutagenic strain improvement in Aspergillus Carbonarius to enhance pectinase production potential.

Pectinase is one of the most important commercially synthesised enzyme having its application in several industrial sectors like food and beverages, fruit clarifications etc. A.carbonarius has the capacity to produce Exo-pectinase 50 U/ml by submerged fermentation process as per the previous study. The present study describes the improvement of previously identified fungal strain Aspergillus carbonarius for enhancement of pectinase production by inducing mutations using physical and chemical mutagens. Aiming to increase the potentiality in pectinase production, the parental strain was treated for three times with four mutagens - UV irradiation, Colchicine, Hydrogen peroxide and Ethidium bromide to obtain mutants. Mutants were selected based on higher enzyme activity, improved growth rates and varied morphology with increased pectinase production. All the surviving mutants were assessed quantitatively after first mutagenic treatment. The stability of the best mutants was tested by repeating the exposures for two times to obtain 3rd generation mutants. These mutants were tested quantitatively to assess the pectinase production. Of all the best mutants E8 showed maximum activity producing 65U/ml pectinase enzyme compared to wild and sister mutants. The wild strain of A. carbonarius is a low pectinase producing organism as per literature. This strain was successfully mutated to increase the productivity rate to 1.8 fold in comparison to wild strain. This overproduction and strain stability may be due to repeated mutagenic treatments.

Ação da enzima eectinase na extração do suco de jabtiicaba / Pectiase enzyme action in jabuticaba?s juice extraction

A enzima pectinase destaca-se pelo potencial de extração de sólidos solúveis presentes nas cascas de frutas. No caso da casca de jabuticaba, este fator é muito interessante, pois esta é rica em nutrientes e compostos que contribuem para o aumento da doçura e da cor. Assim, o objetivo deste trabalho foi realizar um estudo para avaliação da influência da adição da enzima pectinase na extração do suco de jabuticaba (Myrcia jabuticaba cauliflora Berg). Os efeitos foram observados por meio de análises físico-químicas (acidez, açúcares redutores e não redutores, anidrido sulfuroso livre e total, sólidos solúveis totais, concentração de polifenóis e antocianinas, pH e cinzas) e sensorial (teste de escala hedônica e escala de atributos) dos sucos elaborados com (T2) e sem a adição da enzima (T1). Os resultados mostraram diferença significativa entre as amostras, principalmente em relação à concentração de antocianinas e cinzas, ambos maiores em T2. Além disso, T2 apresentou maior rendimento (± 5%), coloração mais intensa e um suco mais límpido. Os resultados da análise sensorial mostraram diferença significativa no teste de escala hedônica e no atributo sabor, onde ambos apresentaram as médias de 5 em T1 e 6 em T2, o que corresponde respectivamente a ?não gostei, nem desgostei? e ?gostei ligeiramente?. Assim, comprovou-se que com a adição da enzima pectinase, melhorou o aspecto visual e sensorial, além da maior extração de composto de grande interesse para a saúde como as antocianinas e os minerais.

Pectinase enzyme stands out because of its soluble solid extraction potential found in fruit peels. In the case of jabuticaba peel, this factor is really interesting, because it is rich in nutrients and compounds that contribute to increase sweetness and color. Thus, the objective of this study was to evaluate the influence of pectinase enzyme in jabuticaba juice extraction (Myrcia jabuticaba cauliflora Berg). The effects were observed through physical and chemical analysis (acidity, reducing and non-reducing sugars, free and total sulfurous anhydride, total soluble solids, concentration of polyphenols and anthocyanins, pH and ashes) and sensory (hedonic scale test and range of attributes) from juices prepared with (T2) and with no enzyme addition (T1). The results have shown a meaningful difference among the samples, mainly in regarding anthocyanin concentration, and ashes, both higher in T2. Besides this, T2 had a bigger yield (± 5%), more intense coloring and clearer juice. The sensory analysis results showed meaningful difference in the hedonic scale test and in the flavor attribute where both presented the average of 5 in T1 and 6 in T2, respectively corresponding to ?I didn?t like it nor dislike it.? and ?I slightly liked it.?. Therefore, it was proven that with the addition of pectinase enzyme, the visual and sensory aspect improved, besides the bigger compound extraction which can be of interest for health as well as for anthocyanins and minerals.

Preparation and In-vitro characterization of ethylcellulose coated pectin alginate microspheres of 5-fluorouracil for colon targeting.

The main objective of the present study was to prepare and evaluate the colon-specific pectin alginate microspheres of 5-fluorouracil (5-FU) for the treatment of colon cancer. Calcium alginate beads were prepared by extruding 5-FU loaded alginate solution to calcium chloride solution and gelled spheres were formed instantaneously by ionotropic gelation reaction using different ratios of 5- FU and alginate, alginate and calcium chloride, stirring speeds (500-1500 rpm) and reaction time. The core beads were coated with ethyl cellulose to prevent drug release in the stomach and provide controlled dissolution of enteric coat in the small intestine and maximum drug release in the colon. Morphology and surface characteristics of the formulation were determined by scanning electron microscopy. In vitro drug release studies were performed in conditions simulating stomach to colon transit in the presence and absence of pectinase enzyme. No significant release was observed at acidic pH, however, when it reached the intestinal pH where ethyl cellulose starts to dissolve, drug release was observed. Also, release of drug was found to be higher in presence of pectinase enzyme. The DSC and FT-IR studies were also indicates there were no interactions between the drug and the polymers used.

Aplicação da enzima pectinase na vinificação / Application of pectinase enzyme on vinification

As enzimas são substâncias encontradas natural ou artificialmente, capazes de governar e estimular processos químicos. Seu uso no processo de vinificação promove um aumento nos teores dos compostos fenólicos no vinho. O objetivo do presente estudo foi verificar o efeito da enzima pectinase na vinificação da uva niágara rosada. As uvas, safra 2006, foram coletadas e encaminhadas para o laboratório de Bioquímica da Universidade Paranaense, Campus Toledo, dividida em quatro lotes, e sulfitadas com 50,0 mg/L de metabissulfito de sódio. Após, foram realizadas as correções de açúcar para 23,0 ºBrix, seguidos da adição de nutrientes na proporção de 0,2 g/L de fosfato de amônio dibásico por kg de uva e inoculados com a levedura Saccharomyces Cerevisiae seca e ativada (30 mg/kg de uva). O tratamento 1 foi tomado como controle. Ao segundo foram adicionados 2,5 g da enzima pectinase (Ultrazym AFPL da Novozymes); ao terceiro 5,0 g, e ao quarto foram adicionados 7,5 g/100 kg de uva. As microvinificações foram realizadas em triplicata, sendo acompanhadas pelo ºBrix. Finalizada a fermentação alcoólica, o vinho foi decantado, pasteurizado e mantido sob refrigeração. Foram realizadas análises de pH (a 20º C), acidez total (meq/L), acidez volátil (meq/L), SO2 livre (g/L), SO2 total (g/L) e densidade (g/cm³), polifenóis totais, antocianinas, taninos e índices de cor. A enzima pectinase favoreceu a difusão dos compostos fenólicos da película para o vinho. A concentração de 5,0 g de enzima por 100 kg de uva foi a que se mostrou mais adequada.

Enzymes are substances naturally or artificially found which are able to govern and stimulate chemical processes. Its use in the vinification process promotes the increase of the levels of phenolic compounds in wine. The objective of this study was to verify the effects of the enzyme pectinase in the vinification of the Niagara pink grape. The grapes, harvested in 2006, were collected and taken to the laboratory of Biochemist of the Universidade Paranaense, Campus Toledo, divided into four sets, and sulfited with 50.0 mg/L of metabissulfito of sodium. Then, corrections of sugar to 23.0 ºBrix followed by the addition of nutrients in the ratio of 0,2 g/L of dibasic ammonium phosphate per inoculated kg of grape and dry and activated Saccharomyces Cerevisiae yeast (30 mg/kg of grape) were carried out. Treatment 1 was considered control. To the second, 2.5 g of the enzyme pectinase (Ultrazym®, AFP-L, Novozymes do Brasil) were added; 5.0 g to the third, and 7.5 g/100 kg to the fourth. The microvinification had been carried through in third copy, being followed by ºBrix. The wine was decanted, pasteurized and kept under refrigeration after the alcoholic fermentation. Analyses of pH (20º C), total acidity (meq/L), volatile acidity (meq/L), free SO2 (g/L), total SO2 (g/L) and density (g/cm³), total, anthocyanins, polyphenols, tanines, and color indexes were carried out. The pectinase enzyme favored the diffusion of skin phenolic composites to the wine. The concentration of 5.0g enzyme per 100 kg of grape presented to be more suitable.

Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media

A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of beta-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing beta-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong beta-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

Clinical study on patients with gastrolithiasis treated by pectinase / 中国普通外科杂志

Objective To determine the pathological mechanism of gastrolithiasis formation in vitro and the effects of pectinase on gastrolithiasis. Methods The test in vitro was divided randomly into two groups:(1) Haws and persimmons(25g respectively) were chewed and put into two clean containers filled with fresh gastric juice.(2) Wine containing 30% alcohol(50ml) was added to the container. Each group included 4 collections.All two groups were placed in 37℃ calorstat box to observe the course and time of gastrolith formation. After gastrolith formation, 2g pectinase was added to the container and observed the decomposition of pectinase to gastrolith and the time was recorded. The effects of pectinase on 54 patients with gastrolithiasis were also investigated. Results The mean time of haws juice coagulation and persimmons was 23 minutes and 25 minutes respectively in the group without wine; but in the group with wine, the mean time of haws juice coagulation and persimmons was 14 minutes and 19 minutes respectively. After adding pectinase, the mean time of clot initiated decomposition was about 17 minutes in both groups, but complete decomposition needed 34 minutes. All the 54 patients with gastrolithiasis who received pectinase were cured.The gastrolith disappeared 6 hours after taking pectinase confirmed by barium meal examination. Conclusions Haws and/or persimmons mixed with gastric juice could induce gastrolith formation. The effects of pectinase on patients with haws or persimmons gastrolithiasis are good, this therapeutic procedure is simple, and the gastrolith resolves quickly.Pectinase may be widely used in the treatment of gastrolithiasis.