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The fungus Rhizopus oryzae 1009 was obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India. The fungus was first grown on Potato dextrose agar slants at 30oC for seven days. This culture was transferred once a month to a fresh slant. All the cultures were stored at -2oC to -6oC and purity was tested before using.
IDENTIFICATION OF FUGI BY MICROSCOPY (Aneja et al., 2001)
Scotch tape preparation for studying morphology of fungi
This is a rapid technique for preparing a temporary microscopic mount of a fungus without disturbing the arrangement of a fungal morphology.
Fungus colony on agar plate,
A strip of clear cello tape10 cm,
Lactophenol cotton blue,
Microscopic slide and
1. In a clean slide, a drop of lactophenol cotton blue was placed in the center of the slide.
2. The transparent adhesive tape was held with sticky side down, between thumb and forefinger
of the each hand and pressed firmly.
3. The center of the sticky side of the tape was pressed firmly on to the surface of the fungus
colony, where sporulation was visible.
4. The tape was gently pulled away from the colony and placed on the drop of lactophenol
5. The extended ends of the tape were folded over the ends of the slide.
6. The morphology of the fungi was observed under 10X.
Picture of fungi
Whenever required, the fungus was cultured on Potato dextrose agar plates, incubated at 30oC and exposed to back light to stimulate sporulation. The fungus was allowed to grow for seven days for the formation of spores. Spores were harvested by flooding the culture plates with sterile distilled water. A final spore suspension (1 x 106 spore/ml) was prepared and used for inoculating in the fermentation broth. For experimentation, the fungal spores in the slant were suspended in sterilized water maintained at 4oC. For storage, the spores were placed in 20% glycerol solution at -80oC. Pelletized seed was cultured on the medium with potato dextrose broth. In terms of achieving pellet form, the spore solution was inoculated in a 250 ml Erlenmeyer flask, containing 50 ml of seed medium with a spore concentration of 1 x 106 spores per ml and cultured at 27oC on a orbital shaker bath set at 200 rpm for one day. The culture temperature was fixed at 27oC.
Fermentation medium (Chen et al., 2001)
The fermentation medium contains 20 g/L of glucose, 10 g/L of peptone, 1 g/L of yeast extract, 5 g/L of ammonium sulphate, 1 g/L of potassium hydrogen phosphate dibasic, 1 g/L of magnesium sulphate, 0.1 g/L of calcium chloride and 1 g/L of sodium chloride. After inoculation, the fungi was grown in the fermentation broth for an additional two days in a shaking incubator set at 28oC with agitation of 200 rpm, the pH of the medium was maintained between 3-5 throughout the fermentation. The sterilized medium was inoculated with 10% v/v of freshly prepared inoculum. At the end of desired incubation period the mycelia was harvested by filtration.
The biomass was recovered from the fermentation medium by filtration (No.1 Whatmann) and washed with distilled water until clear filtrate was obtained. The mycelium was then treated with 1M sodium hydroxide (1:30g/V) and the mixture was autoclaved at 121oC for 15 minutes. The mixture was subsequently filtered (No.1 Whatmann) to sediment the alkali insoluble materials (AIF) and washed with distilled water and ethanol. The washed material was further extracted with 10%acetic acid solution (1:40g/ml) and refluxed at 65oC for 6 hours. The resulting slurry was isolated by filtration (No.1Whatmann) yielding an acid insoluble precipitate (containing chitin) and acid soluble supernatant. The chitin was finally washed with distilled water, 95% ethanol and acetone subsequently .It was then air dried.
Lactic acid extraction
From the filtrate of fermentation broth, lactic acid was extracted. In the filtrate, lactic acid was present either in the form of salts or esters which was isolated and separated. The filtrate were acidified with 15 drops of concentrated sulphuric acid and boiled for one hour. Then it was filtered and to the filtrate 20 ml of diethyl ether was added and shaken for 15 minutes in a separating funnel. Ether layer contains lactic acid that was extracted in a beaker and the layer containing insoluble substance was discarded. Then ether was allowed to evaporate. After evaporation the colored substance of lactic acid was removed using charcoal. It was then heated for 5 to 10 minutes and filtered (No.1 Whatmann). Lactic acid was finally obtained colorless.
Determination of growth curve, extractable chitin and lactic acid (Shimhara, et al., 1998)
The growth of extractable chitin and lactic acid of Rhizopus oryzae NCIM 1009 were determined by culturing fungi in the fermentation medium. This was done by inoculating 30 ml of spore inoculum in 270 ml of fermentation medium taken in seven 500 ml Erlenmeyer flask. Each incubation flask was incubated at different periods of time 24, 48, 72, 96, 120, 144 and 168 hours in a rotary shaker. At the end of each incubation period mycelia were harvested from all of the seven flasks and the corresponding biomass were dried. Three replicate cultures were done for this process.
Two milligrams of fungal chitin was dried overnight at 60oC and thoroughly mixed with 100mg of KBr to produce 0.5 mm thick discs. Spectrum was recorded using JASCO FTIR 410 in the Pharmaceutical analysis laboratory, College of Pharmacy, KMCH, Coimbatore.
Acute toxicity studies
Swiss albino mice of either sex (40-80g) maintained under standard laboratory conditions. A total of five animals were used which received a single oral dose of (2000mg/kg body weight) of chitin obtained from Rhizopus oryzae NCIM 1009. Animals were kept fasted overnight prior to the administration of the isolated fungal chitin. Food was withheld for further 3-4 hrs. Animals were observed individually at least once during the first 24 hrs (with special attention for first 24 hrs) and daily thereafter for a period of 14 days. Mortality, if any, was determined over a period of 2 weeks (OECD, 2000).
PROCEDURE FOR PARACETAMOL INDUCED HEPATOTOXICITY
Animals were randomized and divided in to five groups (I-V) of six animals in each group.
Group I served as Normal Control and fed orally with Normal Saline 5 ml/kg body weight daily for 14 days.
Group II served as Negative Control and rats were similarly treated as Group I.
Group III and IV animals were treated with various doses of extract for 14 days.
On 14th day, paracetamol suspension was given by oral route at a dose of 750 mg/kg body weight to all group of rats except the rats in Group I.
The extracts were administered by oral gavages 1h before paracetamol administration.
Group V rats were treated with standard drug silymarin 25 mg/kg body weight.
The blood samples (volume of blood is 2-5 ml) were collected from all animals by cardiac puncture after euthanasia with diethyl ether ( 5 ml).
The blood samples were used for the estimation of various biochemical parameters including SGOT, SGPT, SALP, Bilirubin and Total Protein.
Object of the study is:
Co-production of two economically and pharmaceutically important products such as lactic acid and chitin from Rhizopus oryzae NCIM 1009.
Determination of best seed culture condition, influence of shaking speed and pH on lactic acid fermentation.
Characterization of lactic acid by IR spectroscopy and Optical rotatary dispersion.
Characterization of chitin by IR spectroscopy.
To study the hepatoprotective activity of fungal chitin.
To study the antimicrobial activity of lactic acid isolated from fungi.