PhD

Investigation on “Evaluation and utilisation of edible lichen Parmotrema
tinctorum (Nyl.) Hale for food preservation” was carried out in the Department of
Post Harvest Technology, College of Horticulture ,Vellanikkara during 2014-2017.
The main objectives of the study were to evaluate the biochemical constituents,
proximate composition, antimicrobial activity, feasibility for food preservation and
to study the toxicological effect of the lichen Parmotrema tinctorum.
The lichen samples were collected from Chembra, Meppadi, Moolankavu and
Ambalavayal areas in Wayanad district, and they were identified as Parmotrema
tinctorum by colour spot tests. The samples read K-, C+, KC+ and Pd- for the lichen
Parmotrema tinctorum. The habitat of lichen was found to be the shady places of the
evergreen forests at 736m -2100m above MSL. Parmotrema tinctorum is found to be
corticolous (growing on the surface of trees) in habit. Thallus of the lichen is foliose,
loosely attached, lobes irregular, margins entire, upper surface grey, smooth, shining;
lower surface black and marginal area brown.
Proximate analysis of Parmotrema tinctorum revealed a high content of total
protein (15.70 %), crude fibre (14.16%), ash (10.50%) and total phenols (322
mg/100g). Parmotrema tinctorum also contained total carbohydrate (20.03 g/100g),
crude fat (1.28%), ascorbic acid (4.66 mg/100g) and total free amino acids (8.25
mg/g). High content of calcium, magnesium, potassium and iron were found in the
mineral analysis of Parmotrema tinctorum. Methanol, ethyl acetate and acetone
extracts of Parmotrema tinctorum were analysed for antioxidant activity by DPPH
and ABTS assays, and the highest scavenging action was detected in the methanol
extract against the DPPH free radicals (IC50-1.47 mg/ml) and the ABTS radicals
(IC50-1.27 mg/ml).
Preliminary phytochemical screening of Parmotrema tinctorum revealed
maximum phytochemicals in methanol extract viz. carbohydrates, phenols,
flavonoids, tannins, terpenoids, fixed oils and coumarins. The TLC profiling of lichen
extracts (hexane, methanol and acetone) showed maximum compounds in acetone
extract, and the spots indicated the presence of phenols and terpenoids. A range of
volatile compounds were observed when the lichen extracts (methanol, hexane,
acetone, chloroform and ethanol) were subjected to GC-MS analysis.Volatile
compounds with antimicrobial properties identified were orcinol, methyl orsellinate,
atraric acid, atranorin, methyl haematommate, glyceryl trilaurate, lauric acid vinyl
ester and gamma-sitosterol.
In vitro testing of antimicrobial activity of acetone, ethanol and chloroform
extracts of Parmotrema tinctorum using disc and well diffusion methods revealed
their inhibitory action against the selected food spoilage organisms. Ethanol extract
(EE) of Parmotrema tinctorum produced maximum inhibition of Aspergillus niger,
while chloroform extract (CE) produced maximum inhibition of Aspergillus oryzae.
The growth of both yeast species, Saccharomyces cerevisiae and Zygosaccharomyces
bailii, was found to be inhibited maximum by the ethanol extract followed by the
chloroform extract of the lichen Parmotrema tinctorum. In both disc diffusion and
well diffusion methods, the growth of Bacillus subtilis and Staphylococcus aureus
were remarkably inhibited by the acetone extract (AE) followed by chloroform
extract (CE) forming zones of inhibition at all the concentrations tested.
Feasibility of utilizing Parmotrema tinctorum for food preservation was
evaluated by adding in powder and in ethanol extract form in two processed products
viz. lime pickle and tomato sauce. In lime pickle, bacterial count was least in
treatments T8 and T7 (added with 0.3% and 0.2% ethanol extracts respectively).
Product preserved with 250 ppm sodium benzoate (T2) revealed least fungal count
(0.6x103 CFU/g), which was on par with that containing 0.3% ethanol extract
(1.0x103 CFU/g). Lowest yeast count was observed in T8, followed by T2 (0.3 x103
CFU/g). The shelf life of lime pickle treated with T2 (product preserved with 250
ppm sodium benzoate) was estimated to be six months, while that of T8 (product
treated with 0.3% ethanol extract) was found to be five months. Unpasteurised tomato
sauce in which 0.1% ethanol extract was added had significantly lower bacterial
count (5.2X106 CFU/g). The tomato sauce preserved with 750 ppm sodium benzoate
recorded lowest fungal and yeast count. The products added with 0.05% and 0.1%
ethanol extract of lichen also recorded lower fungal and yeast counts. Microbial
analysis of products showed the relevance of ethanol extract as an alternative to
sodium benzoate in preventing the microbial spoilage of foods. Sensory analysis
revealed that lichen extract added products were acceptable for consumption.
Acute oral toxicity study of the ethanol extract of Parmotrema tinctorum
conducted in Wistar rats revealed the absence of clinical signs of toxicity and
mortalities. There were no treatment related changes in body weight and gross
pathological changes in the test animals. Single dosing of ethanol extract of
Parmotrema tinctorum upto a dose of 2000 mg/kg body weight orally was found to
be safe in Wistar rats.