The fungus Rhizopus oryzae 1009 was obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India. The fungus was foremost grown on Potato dextroglucose agar angles at 30oC for seven yearss. This civilization was transferred one time a month to a fresh angle. All the civilizations were stored at -2oC to -6oC and pureness was tested before utilizing.
IDENTIFICATION OF FUGI BY MICROSCOPY ( Aneja et al. , 2001 )
Scotch tape readying for analyzing morphology of Fungis
This is a rapid technique for fixing a impermanent microscopic saddle horse of a fungus without upseting the agreement of a fungous morphology.
Fungus settlement on agar home base,
A strip of clear cello tape10 centimeter,
Lactophenol cotton blue,
Microscopic slide and
Cover faux pas.
1. In a clean slide, a bead of lactophenol cotton blue was placed in the centre of the slide.
2. The crystalline adhesive tape was held with gluey side down, between pollex and index
of the each manus and pressed steadfastly.
3. The centre of the gluey side of the tape was pressed steadfastly on to the surface of the fungus
settlement, where monogenesis was seeable.
4. The tape was gently pulled off from the settlement and placed on the bead of lactophenol
5. The drawn-out terminals of the tape were folded over the terminals of the slide.
6. The morphology of the Fungi was observed under 10X.
Picture of Fungis
Whenever required, the fungus was cultured on Potato dextroglucose agar home bases, incubated at 30oC and exposed to endorse visible radiation to excite monogenesis. The fungus was allowed to turn for seven yearss for the formation of spores. Spores were harvested by deluging the civilization plates with sterile distilled H2O. A concluding spore suspension ( 1 x 106 spore/ml ) was prepared and used for inoculating in the agitation stock. For experimentation, the fungous spores in the angle were suspended in sterilised H2O maintained at 4oC. For storage, the spores were placed in 20 % glycerin solution at -80oC. Pelletized seed was cultured on the medium with murphy dextrose stock. In footings of accomplishing pellet signifier, the spore solution was inoculated in a 250 milliliter Erlenmeyer flask, incorporating 50 milliliter of seed medium with a spore concentration of 1 ten 106 spores per milliliter and cultured at 27oC on a orbital shaker bath set at 200 revolutions per minute for one twenty-four hours. The civilization temperature was fixed at 27oC.
Fermentation medium ( Chen et al. , 2001 )
The agitation medium contains 20 g/L of glucose, 10 g/L of peptone, 1 g/L of barm infusion, 5 g/L of ammonium sulfate, 1 g/L of K H phosphate dibasic, 1 g/L of Mg sulfate, 0.1 g/L of Ca chloride and 1 g/L of Na chloride. After vaccination, the Fungi was grown in the agitation stock for an extra two yearss in a shaking brooder set at 28oC with agitation of 200 revolutions per minute, the pH of the medium was maintained between 3-5 throughout the agitation. The sterilised medium was inoculated with 10 % v/v of newly prepared inoculant. At the terminal of coveted incubation period the mycelia was harvested by filtration.
The biomass was recovered from the agitation medium by filtration ( No.1 Whatmann ) and washed with distilled H2O until clear filtrate was obtained. The mycelium was so treated with 1M Na hydrated oxide ( 1:30g/V ) and the mixture was autoclaved at 121oC for 15 proceedingss. The mixture was later filtered ( No.1 Whatmann ) to sediment the base indissoluble stuffs ( AIF ) and washed with distilled H2O and ethyl alcohol. The washed stuff was further extracted with 10 % acetic acid solution ( 1:40g/ml ) and refluxed at 65oC for 6 hours. The ensuing slurry was isolated by filtration ( No.1Whatmann ) giving an acid indissoluble precipitate ( incorporating chitin ) and acid soluble supernatant. The chitin was eventually washed with distilled H2O, 95 % ethyl alcohol and propanone later.It was so air dried.
Lactic acid extraction
From the filtrate of agitation stock, lactic acid was extracted. In the filtrate, lactic acid was present either in the signifier of salts or esters which was isolated and separated. The filtrate were acidified with 15 beads of concentrated sulfuric acid and boiled for one hr. Then it was filtered and to the filtrate 20 milliliter of diethyl quintessence was added and shaken for 15 proceedingss in a separating funnel. Ether bed contains lactic acid that was extracted in a beaker and the bed incorporating indissoluble substance was discarded. Then ether was allowed to vaporize. After vaporization the coloured substance of lactic acid was removed utilizing wood coal. It was so heated for 5 to 10 proceedingss and filtered ( No.1 Whatmann ) . Lactic acid was eventually obtained colorless.
Determination of growing curve, extractible chitin and lactic acid ( Shimhara, et al. , 1998 )
The growing of extractible chitin and lactic acid of Rhizopus oryzae NCIM 1009 were determined by culturing Fungis in the agitation medium. This was done by inoculating 30 milliliter of spore inoculant in 270 milliliter of agitation medium taken in seven 500 milliliters Erlenmeyer flask. Each incubation flask was incubated at different periods of clip 24, 48, 72, 96, 120, 144 and 168 hours in a rotary shaker. At the terminal of each incubation period mycelia were harvested from all of the seven flasks and the corresponding biomass were dried. Three replicate civilizations were done for this procedure.
Chitin word picture
Two mgs of fungous chitin was dried nightlong at 60oC and exhaustively assorted with 100mg of KBr to bring forth 0.5 millimeters thick phonograph record. Spectrum was recorded utilizing JASCO FTIR 410 in the Pharmaceutical analysis research lab, College of Pharmacy, KMCH, Coimbatore.
Acute toxicity surveies
Swiss albino mice of either sex ( 40-80g ) maintained under standard research lab conditions. A sum of five animate beings were used which received a individual unwritten dosage of ( 2000mg/kg organic structure weight ) of chitin obtained from Rhizopus oryzae NCIM 1009. Animals were kept fasted nightlong prior to the disposal of the stray fungous chitin. Food was withheld for farther 3-4 hour. Animals were observed separately at least one time during the first 24 hour ( with particular attending for first 24 hour ) and day-to-day thereafter for a period of 14 yearss. Mortality, if any, was determined over a period of 2 hebdomads ( OECD, 2000 ) .
PROCEDURE FOR PARACETAMOL INDUCED HEPATOTOXICITY
Animals were randomized and divided in to five groups ( I-V ) of six animate beings in each group.
Group I served as Normal Control and fed orally with Normal Saline 5 ml/kg organic structure weight daily for 14 yearss.
Group II served as Negative Control and rats were likewise treated as Group I.
Group III and IV animate beings were treated with assorted doses of infusion for 14 yearss.
On 14th twenty-four hours, paracetamol suspension was given by unwritten path at a dosage of 750 mg/kg organic structure weight to all group of rats except the rats in Group I.
The infusions were administered by unwritten forced feedings 1h before paracetamol disposal.
Group V rats were treated with standard drug silymarin 25 mg/kg organic structure weight.
The blood samples ( volume of blood is 2-5 milliliter ) were collected from all animate beings by cardiac puncture after mercy killing with diethyl quintessence ( 5 milliliter ) .
The blood samples were used for the appraisal of assorted biochemical parametric quantities including SGOT, SGPT, SALP, Bilirubin and Total Protein.
Object of the survey is:
Co-production of two economically and pharmaceutically of import merchandises such as lactic acid and chitin from Rhizopus oryzae NCIM 1009.
Determination of best seed civilization status, influence of agitating velocity and pH on lactic acid agitation.
Word picture of lactic acid by IR spectrometry and Optical rotatary scattering.
Word picture of chitin by IR spectrometry.
To analyze the hepatoprotective activity of fungous chitin.
To analyze the antimicrobic activity of lactic acid isolated from Fungis.