Arthrographis curvata and Rhodosporidium babjevae as New Potential Fungal Lipase Producers for Biotechnological Applications

Abstract Fungi have always attracted a lot of attention as they are able to produce a vast repertoire of enzymes that find a broad spectrum of uses in biotechnological and industrial fields. Undoubtedly, one of the most promising biocatalysts is the lipase, which has been widely used for the biotransformation of a number of commercial products due to its high stability, high catalytic efficiency, versatility and selectivity, making it one of the most attractive and best-studied enzymes. In this study we report the isolation and molecular identification of new lipase-producing fungi from different environmental samples from Morocco. The production and activity of extracellular lipases, at different parameters, was evaluated using the Rhodamine B agar, submerged fermentation and biochemical methods. Two fungal strains Arthrographis curvata and Rhodosporidium babjevae, were isolated and found to produce large amounts of lipases. The optimal activity of the extracellular lipase was detected at 40°C and pH 9.0 for A. curvata and at 40 °C and pH 8.0 for R. babjevae. This study add new information at the growing list of fungal species producing lipases with improved physicochemical proprieties which could constitute a new line of research for further studies and to be exploited for industrial or bioremediation purposes.

Proteolytic and lipolytic potential of Pseudomonas spp. from goat and bovine raw milk / Potencial proteolítico e lipolítico de espécies de Pseudomonas do leite cru caprino e bovino

Pseudomonas, the main genus of gram-negative microorganisms isolated from milk, is psychrotrophic, biofilm-forming, and thermo-resistant deteriorating enzyme producers. The aim of this study was to quantify Pseudomonas spp. in goat's and cow's milk produced in the Paraná state, Brazil, to evaluate the deteriorating activity of the isolates at mesophilic and psychrotrophic conditions and to identify, at the species level, the isolates with alkaline metalloprotease (aprX gene) production potential. Microbiological, biochemical and molecular methods were used for isolating, confirming and identifying of isolates. The mean counts were 1.6 (±6.3)x104 and 0.89(±3)x102 CFU/mL for goat and bovine milk samples, respectively, immediately after milking. Of the Pseudomonas colonies isolated from goat milk (n=60), 91.7% showed proteolytic potential when incubated at 35°C/48 h and 80% at 7°C/10 days, and lipolytic potential was observed in 95% of the isolates incubated in mesophilic and 78.3% at refrigeration conditions. From the isolates of bovine milk (n=20), 35% showed proteolytic activity only when incubated at 35°C/48 h, and lipolytic potential was observed in 25% of the isolates incubated at 7°C/10d and 35°C/48h. It was observed that 83.3% and 25% of the isolates genetically confirmed as Pseudomonas spp. of goat and bovine milk showed the potential for alkaline metalloprotease production, with the species P. azotoformans, P. koreensis, P. gessardii, P. monteilii and P. lurida being the most frequent in goat milk and P. aeruginosa the only species identified in cow milk.(AU)

Pseudomonas é o principal gênero de micro-organismos Gram negativos isolados do leite, são psicrotróficos, formadores de biofilmes e produtores de enzimas deteriorantes termodúricas. O objetivo do presente trabalho foi quantificar Pseudomonas spp. no leite de cabras e vacas produzido no estado do Paraná, Brasil, avaliar a atividade deteriorante em temperatura mesofílica e psicrotrófica e identificar, em nível de espécie, os isolados com potencial de produção de metaloprotease alcalina (geneaprX). Foram utilizados métodos microbiológicos, bioquímicos e moleculares para isolamento, confirmação e identificação dos isolados. As contagens médias foram de 1,6 (±6,3) x 104 e 0,9 (±3) x 102 UFC/mL para as amostras de leite caprino e bovino, respectivamente. Dos isolados de Pseudomonas do leite de cabra (n=60), 91,7% demonstraram potencial proteolítico quando incubadas a 35°C/48h e 80% a 7°C/10dias e lipolíticos em 95% dos isolados incubados em mesofilia e em 78,3% dos incubados em temperatura de refrigeração. Dos isolados do leite bovino (n=20), foi verificada atividade proteolítica de 35% apenas quando incubadas a 35°C/48h e lipolítica em 25% dos isolados incubados a 7°C/10d e 35°C/48h. Foi observado que 83,3% e 25% dos isolados confirmados geneticamente como Pseudomonas spp. do leite caprino e bovino, respectivamente, apresentaram o potencial de produção de metaloprotease alcalina, sendo as espécies P. azotoformans, P. koreensis, P. gessardii, P. monteilii e P. lurida as mais frequentes no leite de cabras e P. aeruginosa a única identificada do leite de vacas.(AU)

Evaluation of alkali and thermotolerant lipase from an indigenous isolated Bacillus strain for detergent formulation

Background: Lipases are used in detergent industries to minimise the use of phosphate-based chemicals in detergent formulations. The use of lipase in household laundry reduces environmental pollution and enhances the ability of detergent to remove tough oil or grease stains. Results: A lipase-producing indigenous Bacillus subtilis strain [accession no. KT985358] was isolated from the foothills of Trikuta mountain in Jammu and Kashmir, India. The lipase (BSK-L) produced by this strain expressed alkali and thermotolerance. Lipase has an optimal activity at pH 8.0 and temperature 37°C, whereas it is stable at pH 6.0­9.0 and showed active lipolytic activity at temperatures 30 to 60°C. Furthermore, lipase activity was found to be stimulated in the presence of the metal ions Mn2+, K+, Zn2+, Fe2+ and Ca2+. This lipase was resistant to surfactants, oxidising agents and commercial detergents, suggesting it as a potential candidate for detergent formulation. BSK-L displayed noticeable capability to remove oil stains when used in different washing solutions containing buffer, lipase and commercial detergent. The maximum olive oil removal percentage obtained was 68% when the optimum detergent concentration (Fena) was 0.3%. The oil removal percentage from olive oil-soiled cotton fabric increased with 40 U/mL of lipase. Conclusions: This BSK-L enzyme has the potential for removing oil stains by developing a pre-soaked solution for detergent formulation and was compatible with surfactants, oxidising agents and commercial detergents.

Use Of Biological Parameters To De-Escalate Chemoradiation In Hpv Associated Squamous Cell Carcinoma Of Different Subsites

Human papillomavirus infection is now a well-established cause of many common cancers like cervical, other anogenital, and head and neck cancers. The mortality and morbidity rate associated with these cancers constitute a major burden especially for the underdeveloped and developing countries of the world, where they are more common. Traditionally, all these subsites are being treated with different chemoradiation protocols with variable results. Toxicities associated with the standard high dose chemoradiation protocols form a major obstacle in the completion of treatment for these patients and often affects the outcome negatively. Personal experience and published reports and reviews suggests that HPV associated squamous cell cancers are a distinct biological sub group of cancer which can be treated safely with reduced intensity of chemoradiation. The establishment of a similar de-intensified chemoradiation protocol for all HPV associated squamous cell carcinoma will certainly improve the quality of life of such patients.

Effects Of Natural Products On Body Weight And Biochemical Parameters In Healthy Rats

The present study was undertaken to evaluate effect of natural products i.e. Anthocephalus indicus; KADAM, roots, Hibiscus rosa sinensis roots, Tinospora cordifolia stem and Cassia tora seeds in normal healthy rats. In this study ethanol extract of above mentioned medicinal plants had macerated with aqueous gum acacia (2%, w/v) suspension and fed orally (500 mg/kg bw p.o.) to male adult healthy normal rats of Charles Foster strain for 30 days. Results of this study showing that alcoholic extracts caused no any signicant reduction in blood glucose, total cholesterol, triglyceride, phospholipids, free fatty acid, lipid peroxide and no signicant increased in post heparin lipolytic activity, but on the other hand as per pre-existing data and my published studies in diabetic patients and diabetic experimental animals showed that extracts exerting all above effects signicantly. That's why it is very clear here if healthy person will take natural products, it never cause hypoglycemia, hypolipidemia and under weight. Natural products also not cause any signicant change on hepato-specic parameters. Thus from this study we conclude that, natural products are safe, non toxic and free from side effects, in comparison to synthetic drugs.

Experimental Validation Of Anti-Diabetic And Antioxidant Potential Of T. Cordifolia Stems (M.): An Indigenous Medicinal Plant

This case control study had been carried out to evaluate antidiabetic and antioxidant activities of Tinospora cordifolia (T. cordifolia; family: Menispermaceae) against streptozotocin induced diabetes in experimental rats to scientifically validate its use against diabetes. Ethanolic extract of T. cordifolia stem extract and standard drug (glibenclamide) macerated with aqueous gum acacia (2%, w/v) suspension and fed orally to streptozotocin induced male adult diabetic rats of Charles Foster strain for 30 days. Biochemical parameters in normal, diabetic control, standard (600µg/kg bw p.o.) and treated (500 mg/kg bw p.o.) animals group were determined and compared. Treatment of streptozotocin induced diabetic rats with ethanolic extract caused significant (p<0.001) reduction in blood glucose, total cholesterol, triglyceride, phospholipids, free fatty acid, lipid peroxide and significant increased (p<0.001) post heparin lipolytic activity. Furthermore, the stem extract (100-400 µg) when tested for its antioxidant activity in vitro, shown significant (p<0.001) inhibit the generation of super oxide anions in enzymic system a, in enzymic system b, non enzymic system and hydroxyl radicals in enzymic system and non-enzymic system. The results of the present study demonstrated antidiabetic antidyslipidemic and anti oxidant activities of T. cordifolia stem extract which could help in prevention of diabetic- dyslipidemia and related complications.

Characterization and lipolytic activity of lactic acid bacteria isolated from Thai fermented meat.

Twenty six strains of lactic acid bacteria (LAB) were isolated from Thai fermented meat products, including 7 samples of Nham (fermented pork), 7 Sai-krog-prieo (fermented sausage) and one Mum (fermented beef). The isolates were identified as Lactobacillus pentosus (5 isolates), Lactobacillus sp. (11 isolates), Pediococcus pentosaceus (5 isolates) and each isolate of Pediococcus lolii, Leuconostoc fallax, Weissella thailandensis, W. cibaria, and W. paramesenteroides based on their phenotypic characteristics and 16S rRNA gene sequence similarities (99.8-100 %). The isolates were primary screened for their lipolytic activity on agar plates. The rodshaped isolates showed 0.031±0.030-0.938±0.127 U/ml of lipase activity in broth supplemented with Tween 20, Tween 40, Tween 60 or Tween 80. The isolate SS50-1 identified as Lactobacillus pentosus showed the highest activity in Tween 80 (0.938±0.127 U/ml). The coccal isolates showed lipase activity ranged from 0.029±0.006- 1.090±0.033 U/ml when Tween 20, Tween 40, Tween 60 or Tween 80 was used as a substrate. The isolate SS48-4 identified as Pediococcus lolii showed the highest activity in Tween 80 (1.090±0.033 U/ml).

Identification and characterization of lipolytic bacteria from Thai fermented foods

Aims: Gram-positive spore forming rod-shaped bacteria from Thai fermented foods were isolated, identified and screened for lipolytic activity. Methodology and results: Bacterial strains were isolated from Thai fermented foods by the standard dilution technique using Tryptic soy agar. Seven isolates which belong to the genus Bacillus and one isolate which belongs to Paenibacillus were characterized based on their phenotypic characteristics and 16S rRNA gene sequence analyses. Isolates NM8-1 (Group 1), PR9-2 (Group 2), PR11-1 (Group 3), KM15-1 and SS49-4 (Group 4), SS48-5 (Group 5) and SS503 (Group 6), were closely related to Bacillus methylotropicus, B. pumilus, B. flexus, B. cereus, B. subtilis and B. anthracis, based on 99.90-100% similarities, respectively. Isolate NM45-3 (Group 7) was closely related to Paenibacillus pasadenensis based on 99.55% similarity. All the isolates were susceptible to amikacin, chloramphenicol, clindamycin, erythromycin, gentamicin, imipenem, kanamycin, levofoxacin, novobiocin, streptomycin, tetracycline and vancomycin. Their lipase production in nutrient broth with Tween 20, Tween 40, Tween 60 or Tween 80 ranged from 0.014±0.129 - 3.231±0.087 U/mL. Bacillus subtilis SS48-5 exhibited highest lipase production when cultured with Tween 80 at pH 7.5 for 24 h. The optimum lipase production of this strain was at 40 °C after incubated for 30 h. Conclusion, significance and impact of study: The characterization and evaluation of the lipolytic activity of Bacillus and Paenibacillus strains isolated from Thai fermented foods will be useful for the species diversity, food fermentation and the lipase production.

Lipolytic Activity and Molecular Identification of Pseudomonas aeruginosa and Lysinibacillus sphaericus Isolated from Domestic Oil Rich Wastewater.

Microbial lipases have been heightened in bioremediation and various industries. Place and Duration of Study: Department of Microbiology, Ekiti State University, Ado-Ekiti, Ekiti State, Nigeria, between September 2010 and August 2011. To identify the lipolytic enzyme producing microbial strains in domestic oil rich wastewater, the 16S rRNA gene was amplified and sequenced. The sequences were used to identify the strains by comparing with related sequences in database using BLAST analysis. The enzyme activity was quantified by HPLC analysis. All the lipolytic bacteria showed appreciable growth rates in the wastewater (between 0.67 and 1.67 mg/day) within 5 days. The most effective lipolytic bacteria isolates in the oil-rich wastewater were two species of the genus Pseudomonas and one of Bacillus. Comparing the weights on the first day to the twelfth day values when lipolytic organisms were grown in palm oil, some appreciable increases in weight difference were recorded in some isolates: 28.3%, 7.84%, 4.44% and 6.98% for Pseudomonas, Staphylococcus, Bacillus and Klebsiella, respectively. The weight increase of each of the microbial cells in palm oil culture was usually lesser than what was obtained in the oil-rich wastewater culture. Two isolates showed high similar sequence (99%) to that of Pseudomonas aeruginosa and Lysinibacillus sphaericus, respectively. From palm oil, Lysinibacillus sp. produced various forms of fatty acids in the medium, including myristic acid (2.61%), palmitic acid (6.22%), stearic acid (5.18%) and arachidic (3.66%). These strains are versatile in utilizing the limited nutrient and had the ability to grow appreciably in the toxic condition (soap solution), suggesting that they may serve as candidates in treating dietary oil-rich wastewater.

Lipolytic Enzymes Involved in the Virulence of Human Pathogenic Fungi

Pathogenic microbes secrete various enzymes with lipolytic activities to facilitate their survival within the host. Lipolytic enzymes include extracellular lipases and phospholipases, and several lines of evidence have suggested that these enzymes contribute to the virulence of pathogenic fungi. Candida albicans and Cryptococcus neoformans are the most commonly isolated human fungal pathogens, and several biochemical and molecular approaches have identified their extracellular lipolytic enzymes. The role of lipases and phospholipases in the virulence of C. albicans has been extensively studied, and these enzymes have been shown to contribute to C. albicans morphological transition, colonization, cytotoxicity, and penetration to the host. While not much is known about the lipases in C. neoformans, the roles of phospholipases in the dissemination of fungal cells in the host and in signaling pathways have been described. Lipolytic enzymes may also influence the survival of the lipophilic cutaneous pathogenic yeast Malassezia species within the host, and an unusually high number of lipase-coding genes may complement the lipid dependency of this fungus. This review briefly describes the current understanding of the lipolytic enzymes in major human fungal pathogens, namely C. albicans, C. neoformans, and Malassezia spp.

Evaluation of the induction of lipolytic enzymes froma Pseudomona aeruginosa isolated from african palm fruit (Eleasis guimeensis) / Evaluación de la inducción de enzimas lipolíticas a partir de una Pseudomona aeruginosa aislada del fruto de palma africana (Eleasis guineensis)

Background: Extracellular lipases are found in the culture broth when the fermentation is at the end of the exponential phase. Lipases can be induced easily since they are produced by the presence of oily sources or other materials as surfactants, fatty acids, some esters, glycerol and biliary salts. Objective: The aim of this work is to study the effect of carbon source concentration and the use of inductors on biomass production, and the lipolytic activity of a bacterium isolated from mature palm oil fruits. Methods: The yield biomass/substrate was evaluated with glucose as carbon source at different concentrations (3, 5, 7, 10, 15 y 20 g/L) by dry weight and OD (600 nm). Lipolytic activity was evaluated by spectrophotometric assay using p-nitrofenilpalmitate at 37°C for 15 min. Results: Gram negative microorganisms with lipolytic activity isolated from palm fruit were identified as Pseudomona aeruginosa. The growth of the bacteria was inhibited when glucose was used at concentrations greater than 5%. The production of lipase was induced by using three inducers (Palm oil, Tween 20 and palm oil:Tween 20 mixture), at three different induction times (0, 11 and 18 hours of fermentation). The highest activity (3,81 µmoles/mL*min) was observed when the palm oil:Tween 20 mixture was added at 11 hours of fermentation. The kinetic of p-nitrophenylpalmitate hydrolysis using the supernatant of a culture induced with palm oil:Tween 20 mixture at 11 hours showed the production of p-nitrophenol beyond 300 minutes, with the greatest hydrolysis rate during the first 7 minutes. Conclusions: The growth of P. aeruginosa was not affected by using glucose as carbon source at concentrations of 3% and 5%. There was a basal level of lipase production without inducer, and greater lipolytic activity was achieved with the addition of inducers.

Hypolipidemic activity of Hibiscus rosa sinensis root in rats.

The hypolipidemic activity of Hibiscus rosa sinensis (family Malvaceae) root extract was studied on triton and cholesterol-rich high fat diet (HFD) induced models of hyperlipidemia in rats. In triton WR-1339-induced hyperlipidemia, feeding with root extract (500 mg/kg body wt/day p.o.) exerted lipid-lowering effect, as assessed by reversal of plasma levels of total cholesterol (TC), phospholipids (PL) and triglycerides (TG) and reactivation of post-heparin lipolytic activity (PHLA) of plasma. The other model was fed with cholesterol-rich HFD and root extract (500 mg/kg body wt/ day p.o.) simultaneously for 30 days. This also caused lowering of lipid levels in plasma and liver homogenate and reactivation of plasma PHLA and hepatic total lipoprotein lipase activity. The hypolipidemic activity of Hibiscus rosa sinensis root was compared with a standard drug guggulipid (200 mg/kg body wt/day p.o.), a known lipid- lowering agent in both models. Histopathological findings in rat liver supported the protective role of H. rosa sinensis root extract in preventing cholesterol-rich HFD-induced hepatic steatosis.

Las dietas cetogénicas: fundamentos y eficacia para la pérdida de peso

El consenso internacional es que los carbohidratos son la base de la pirámide alimenticia de una dieta saludable, de tal forma que la mayoría de los especialistas de hoy en día piensan que la mejor forma de perder peso es mediante una reducción en el número total de calorías ingeridas diariamente, principalmente las que vienen en forma de grasa. No obstante, esta revisión intentará demostrar que las dietas cetogénicas son desde un punto de vista fisiológico, bioquímico y práctico, un camino mucho más efectivo para perder peso. Además se argumenta cómo este tipo de dietas proporciona ventajas metabólicas como son la capacidad de preservar la masa muscular, reducir el apetito, tener una eficiencia metabólica más baja, producir una activación de la termogénesis y favorecer una mayor pérdida de grasa incluso en presencia de una mayor cantidad de calorías.

Ketogenic diets and weight loss: basis and effectiveness. The international consensus is that carbohydrates are the basis of the food pyramid of a healthy diet. Today’s specialists believe that the best way to lose weight is by cutting down on calories, essentialy in the form of fat. However, this paper will clarify that ketogenic diets are, from a physiological, biochemicale and practical point of view, a much more effective way of losing weight, since such diets provide metabolic advantages such as the capacity to preserve muscle mass, reduce appetite, to have a lower metabolic efficiency, produce a metabolic activation of thermogenesis and favour a greater fat loss even with a greater number of calories.

Effects of Portulaca Oleracea L. Extract on Lipolysis and Hormone Sensitive Lipase (HSL) Gene Expression in 3T3-L1 Adipocytes

This study was performed to investigate the lipolytic effects of Portulaca oleracea L. extract in 3T3-L1 adipocytes. The Portulaca oleracea L. was extracted with extrusion method using twin-screw extruder under 58~60 rpm screw speed, 4~5 kg/hr feed rate, 140degrees C extrusion temperature. The lipolytic action of Portulaca oleracea L. extract was estimated by measuring the amount of glycerol and free fatty acids (FFA) released from 3T3-L1 adipocytes and by measuring the cellular lipid content in 3T3-L1 adipocytes. The hormone sensitive lipase (HSL) mRNA level was analyzed using quantitative real-time PCR. The Portulaca oleracea L. extract at 1 to 100 microgram/ml suppressed lipid accumulation. The release of glycerol and FFA into the medium, and the mRNA level of HSL were significantly increased by the addition of Portulaca oleracea L. extract at dose-dependent manner. In conclusion, the Portulaca oleracea L. extract was suggested to have the lipolytic effect through release of lipolytic products (FFA and glycerol) of triacylglyceride to the culture medium and suppression of lipid accumulation via up-regulation of HSL gene expression in 3T3-L1 adipocytes.

Effects of endurance training under hyperoxia on carbohydrate and fat metabolism in rats / 体力科学

The effects of 6 weeks (5 days/week) of endurance training under hyperoxia (60% O<SUB>2</SUB> plus 40% N<SUB>2</SUB>) on carbohydrate and fat metabolism were studied in 42 male rats. The rats were divided into four groups ; normoxia control (NC, n=8), hyperoxia control (HC, n=9), normoxia training (NT, n=12), and hyperoxia training (HT, n=13) . NT and HT groups were made to run on a treadmill in a metabolic chamber at a speed of 20 m/min for 30 min. The metabolic chamber was perfused with hyperoxic gas. VCO<SUB>2</SUB> values at rest (HC) and during exercise (HT) under hyperoxia were significantly lower (p<0.01) than VCO<SUB>2</SUB> values at rest (NC) and during exercise (NT) under normoxia, respectively. These results appear to indicate that a decreased respiratory exchange ratio was induced by hyperoxia. The results showed that at 15 min after the last training there were no differences between NT and HT in the glycogen or triglyceride content of the liver, heart, m, gastrocnemius, and m. soleus. However, blood glucose at 15 min in NT (109±13 mg/d<I>l</I>) was significantly lower (p<0.05) than the corresponding value at 15 min in HT (133±11 mg/d<I>l</I>) and at 48 h after the last training in NT (149±7 mg/d<I>l</I>) . The glycogen content of the liver in HC (36.4±2.6 mg/g wet wt) was significantly higher (p<0.05) than the corresponding value in NC (26.1±1.9 mg/g wet wt) . In the HT group, the triglyceride content of the liver at 48 h was lower (p<0.01) than the corresponding value at 15 min. However, the triglyceride content of the heart at 48 h in HT was significantly higher (p<0.05) than the value at 15 min. Basal lipolysis in HC was significantly higher than the corresponding values in NC, NT and HT, but there were no differences among the groups in norepinephrine-induced lipolysis. These results indicate that endurance training under hyperoxia might alter the content of tissue glycogen and triglyceride as a result of decreased carbohydrate consumption and increased fat utilization during fasting and/or exercise.