International Research Symposium of UWU-2018

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Anti-oxidant Activities of Bioactive Compounds Extracted from Pterygoplichthys pardalis (Scavenger Fish) Harvested at Digana, Central Province, Sri Lanka
(Uva Wellassa University of Sri Lanka, 2018) Perera, B.B.A.N.S.; Aruppala, A.L.Y.H.; Abeyrathne, E.D.N.S.
Pterygoplichthys pardalis (Scavenger fish) survive by competing with native biota. This species is an omnivore whichthreat to endemic fish species and inland aquaculture industry. However, these fishes contain compounds which autolysis proteins under low temperatures. Objective of this study was to check the difference in the water soluble proteins which can be used as bioactive compounds separated from scavenger fish after slaughtering stored at 4 °C for 24 hrs. Female fish (n = 3) were collected from local reservoir and slaughtered in the field. Slaughtered fish which stored at 4 °C were separated in to 4 main components as flesh, GI tract, mucus and other gonads in 0, 3, 6, 9, 12 and 24 hrs of storing. Separated parts were homogenized with distilled water (1:4) and centrifuged to collect the supernatant. Level of separation was observed using SDS-PAGE gel electrophoresis. Then samples were lyophilized and used for further analysis. Antioxidant activity was measured using TBARS inhibitory assay and DPPH scavenging activities. SDSPAGE images confirmed that there were no differences in the extracted compounds after 03 hrs of slaughtering. According to the TBARS assay, three extractions from flesh, mucus and other organs had stronger antioxidant properties compared to the control (p < 0.05). While DPPH scavenging results showed over 75% activity (other organs-91.26±8.28%, flesh- 87.07±4.49%, GI-86.20±3.94%, mucus- 75.20±4.09%) but no difference was observed among the extracted compounds (p > 0.05). Concluding water extracted in 0-3 hrs after slaughtering of female scavenger fish showed strong antioxidant activities and this can be used as natural anti-oxidant agent in food industry.
Antibacterial Activities of Endophytic Fungi of Cyperus iria Collected from Matale Distric
(Uva Wellassa University of Sri Lanka, 2018) Jayasundara, J.M.N.M.; Ratnaweera, P.B.; de Silva, E.D.
Antibiotic resistance of bacteria has become an ongoing severe human health concern which requires extensive research priority. Endophytic fungi of Cyperaceae family plants are considered as a potential source for isolating bioactive compounds. Hence, the objective of the current study was to isolate endophytic fungi of Cyperus iria and investigate the antibacterial activities of the crude fungal extracts. Healthy C. iria plants were collected from Weragama in Matale district and endophytic fungi were isolated from the surface sterilized roots and aerial parts using five types of media (SYP, YPD, ME, PDA and MEA) enriched with antibiotics. Each pure fungal culture was sub cultured in ten PDA dishes, incubated close to sporulation, extracted into ethyl acetate, filtered and resulting crude extracts were obtained. The crude extracts were tested for antibacterial activity using agar disc diffusion assay against four bacteria, Staphylococcus aureus (ATCC 25928), Bacillus cereus (ATCC 11718), Pseudomonas aeruginosa (ATCC 9027) and Escherichia coli (ATCC 35218) at 400 ug disc-1 concentration where Gentamycin (10 ug disc-1) and methanol (10 IA disc') were used as the positive and negative controls, respectively. Total 34 morphologically distinct putative endophytic fungi, 23 from aerial parts and 11 from roots, were isolated. Thirteen fungal extracts exhibited antibacterial activity against S. aureus, 24 against B. cereus, 12 against P. aeruginosa and one against E. coli. Among all, 29 fungi were active against at least one bacterium tested while five fungi were inactive to all. Activity of three extracts against B. cereus, 12 against P. aeruginosa, and one against E. coli was similar to the activity of the positive control. Fifteen extracts against B. cereus, 27 against P. aeruginosa, 33 against E. coli and 21 against S. aureus showed significant (p < 0.05) antibacterial activities compared to the negative control. In conclusion, C. iria from Matale harbors a lot of endophytic fungi, where several are capable of producing bioactive secondary metabolites with selective antibacterial properties.
Antifungal Activity of Bacillus amyloliquefaciens Ethyl Acetate Extract and Fractions Against the Fungus Khuskia oryzae
(Uva Wellassa University of Sri Lanka, 2018) Lohanathen, G.P.; Jayasundara, J.M.N.M.; Ratnaweera, P.B.; de Silva, E.D.
Fungal infections are common among plants and animals which result in economic losses. Finding new antifungal agents from alternative sources may help to solve the above issue. It was observed that, in culture Bacillus amyloliquefaciens isolated from a contamination shows antifungal activity against the fungus Khuskia oryzae. Therefore, the objective of the current study is to determine the antifungal activity of the crude ethyl acetate extract and the fractions of crude extract of B. amyloliquefaciens against the fungus K oryzae. B. amyloliquefaciens was grown on Luria-Bertani Agar (LBA), extracted into ethyl acetate after an incubation period of three days and the antifungal activity of the crude extract was tested against K. oryzae at 400 lig disc-1 using agar disc diffusion method. Crude extract of 1.5 g was first fractionated by Kupchan solvent-solvent partitioning scheme, sequentially using hexane, methanol/water (9:1); chloroform, methanol/water (6:4) and ethyl acetate, water. Antifungal activity of the three fractions hexane, chloroform and ethyl acetate was determined and the chloroform fraction was active against K. oryzae. The active fraction was further purified using Sephadex LH2O size exclusion chromatography using methanol as the eluent. Fractions were combined according to the thin layer chromatography (TLC) profiles and the antifungal activity was tested for the combined fractions (A-F). Flucanozole and methanol was used as the positive and negative controls respectively. Fraction C (32.8 mg) resulted from size exclusion chromatography of the chloroform fraction exhibited 18 mm radius inhibition zone against K. oryzae while none of the other fractions showed any activity. Activity of fraction C was similar (p > 0.05) to the activity of the positive control. However, TLC profile of the fraction C showed the presence of more than one compound. Thus, further purification of fraction C is necessary in order to isolate the active compound/s which may lead to a potential antifungal agent.
Antioxidant Activity of Selected Ten Underutilized Fruit Species
(Uva Wellassa University of Sri Lanka, 2018) Dilani, V.J.; Singhalage, I.D.
The study was conducted to assess the antioxidant potential of ten underutilized fruit species namely Phyllanthus emblica L,Flacourtia sp.,Elaeocarpus serratus L., Phyllanthus acidus (L.) Skeels , Averrhoa carambola L., Averrhoa bilimbi L., Cynometra cauliflora L., Morus sp., Spondias sp. and Manilkara zapota (L.) P.Royen grown in Sri Lanka. Fruit extracts were prepared by centrifuging (4500 rpm) finely ground fresh fruit samples (20 g) with distilled water (40 ml) for 90 minutes. The extraction was repeated twice and the supernatants were used for analysis. Total Ascorbic Acid Content (TAsC) was determined using an iodomatric titration technique, calculated as mol per gram of fresh weight. Antioxidant potential was determined using DPPH• and ABTS•+ radical scavenging assays. The DPPH• Radical Scavenging Activity (RSA) of the extracts was expressed as IC50 values. The ABTS•+ activity (RSA) was calculated as percentage of discoloration. This experiment was conducted by following complete randomized design with 3 replicates. Ascorbic acid was used as a positive control and distilled water was used as a negative control. The TAsC varied among the species from 0.125x10-3 ± 0.000025 mol g- of fresh fruit of C. cauliflora L. to 4.608 x 10-3 ± 0.0001665 mol g - of fresh fruit of P. emblica L. The TAsC and antioxidant activity (DPPH• and ABTS•+ assays) were found in order to, C. cauliflora L.< Spondias sp.
Antioxidant Activity of the Crude Extract of Ulva lactuca (Sea Lettuce) Collected from the South Coast of Sri Lanka
(Uva Wellassa University of Sri Lanka, 2018) Rumeshani, K.L.G.; Aruppala, A.L.Y.H.; Abeyrathne, E.D.N.S.
Ulva lactuca is one of the famous edible seaweeds around the world. It is a rich source of many nutrients and bioactive compounds. Solvent extraction method is commonly used in extracting bioactive compounds in seaweeds which may be nonfood grade. The objective of this study was to develop a simple, food grade extraction method to extract bioactive compounds from Ulva lactuca and identify the antioxidant activity of the crude water soluble extract and compare with a commonly used solvent extraction method. Four types of seaweed treatments (05 g) namely Fresh, Air-dried (AD), Oven-dried (OD) and Freeze-dried (FD) were used for the water extraction using three different ratios as 1:10, 1:20 and 1:30 and Airdried sample using Methanol as the control. The crude extracts derived from different extraction methods were used for analyzing the antioxidant activity by Thiobarbituric Reactive Substance (TSARS) assay and 2,2-dipheny1-1- picrylhydrazyl (DPPH) free radical scavenging assay. All the trials were done in triplicates. Data were analyzed using the Minitab 18. In TBARS assay, methanol extracts showed the lowest TEARS value (-1.10±0.08) and AD (1:30) and OD (1:10, 1:20, 1:30) showed no significant difference (p > 0.05) compared to antioxidant activity of the methanol extracts. Almost all the samples showed antioxidant activity except Fresh (1:10, 1:20) and AD (1:10). However, In DPPH scavenging assay, DPPH scavenging activity of Fresh (1:10, 1:20, 1:30), AD (1:10, 1:20, 1:30), OD (1:10. 1:30) and FD (1:30) showed no significant difference (p > 0.05) compared to that of methanol extracts. AD (1:10) showed the highest DPPH scavenging activity (89.54±4.56%) which is higher than methanol (87.75±2.87) and Ascorbic acid (85.73±0.19). Therefore it can be concluded that considering the time of production, simplicity, toxicity and cost; water extraction of Fresh (1:10) or AD (1:10) can be used as a best extraction for producing antioxidant agents in food industry.
Antioxidant and Metal Chelation Activities of Fish Protein Hydrolysates Produced from (Scomber japonicus) Pacific Mackerel Canned Fish Processing Fin Wastage
(Uva Wellassa University of Sri Lanka, 2018) Ediriweera, T.K.; Aruppala, A.L.Y.H.; Abeyrathne, E.D.N.S.
Pacific chub mackerel (Scomber japonicus) is a salience fish species which highly utilized in canned fish processing. In production, around 30% of raw fishes are discarded as wastes which lead to economic losses and environmental pollution. Hence, production of Fish Protein Hydrolysates (FPH) utilizing fish wastes, which contains bioactive compounds may be an ideal remedy. In this study Scomber japonicus canned fish processing fin wastage was collected and blended. Aqueous extracts of Fish Protein Concentrates (FPC) were produced with 04 different ratios as sample: distilled water, 1:1, 1:2, 1:3 and 1:4. Crude extraction was observed using 10% SDS-PAGE. Extracted FPCs were hydrolyzed using Papain, Pepsin, Trypsin and Protease enzymes (1:100) under 370C with their optimum pH conditions for 0, 3, 6, 9, 12 and 24 hours followed by heat inactivation at 1000C for 15 minutes. Hydrolyzed samples were lyophilized and observed for antioxidant activities by TBARS and DPPH scavenging assay and metal chelation activity by Fe (II) chelating activity. According to the observations there was no significant difference between the 04 ratios in yield (p > 0.05). So 1:1 ratio was selected with periods as Papain-24 h, Pepsin3 h, Trypsin-3 h, Protease-0 h for further experiments. According to the results obtained from TBARS assay, none of the FPHs showed antioxidant properties (p < 0.05), instead all showed high oxidative activity. However DPPH scavenging assay showed significance difference among the treatments (p < 0.05). Results obtained by Fe (II) chelation activity analysis revealed that the produced FPHs show Fe(II) releasing activity instead of chelation (1.84, 13.99, 16.48, 1.84%,), while FPHs produced according to standard protocol showed a slight chelating activity (0.73%). This concludes the FPHs produced using aqueous extracts of Scomber japonicus do not contain strong antioxidant activity and they have iron releasing properties. Keywords
Biological Activities of Polysaccharides Extracted from Vernonia cinerea and Vernonia zeylanica
(Uva Wellassa University of Sri Lanka, 2018) Wickramasinghe, W.M.C.N.K.; Henagamage, A.P.; Alakolanga, A.G.A.W.
Many members of the genus Vernonia are shown to have biological activity which contained important chemical compounds. In recent years, there has been a growing interest in polysaccharides obtained from higher plants that may have biological activities. This study was focused to investigate the antioxidant, antimicrobial and protease inhibition activity of crude polysaccharides extracted from Vernonia cinerea and Vernonia zeylanica. Plant materials were collected from Ankumbura forest area in Kandy district, Sri Lanka. Polysaccharides were extracted from the powdered above ground plant materials by hot water and alkali extraction methods and were fractionated seperately. FT-1R analysis was performed using potassium bromide powder for the purified polysaccharide fractions in the frequency range of 400 to 4000 cm-1.Total carbohydrate analysis and antimicrobial activity were performed using phenol sulfuric acid method and agar well diffusion method against Staphylococcus aureus and Cladosporium cladosporoides respectively. Antioxidant activity was determined by DPPH radical scavenging activity assay and OH scavenging activity assay. The highest total carbohydrate content (9.1 mg ml-') was recorded from NaOH extraction of V.zeylanica. The FTIR analysis confirmed the presence of polysaccharides, which displayed bands characteristic to O-H, C-O- C, and C-O groups. NaOH extraction of Polysaccharide from V.zeylanica displayed the highest antibacterial activity against Stapphylococcus aureus. NaOH extraction of V. zeylanica showed the highest significant scavenging abilities (p < 0.05) on hydroxyl radicals (IC50, 4.832 mg ml-1) whereas the highest significant scavenging abilities (p < 0.05) on DPPH radicals (IC50, 9.594 mg ml-1) was shown by the hot water extraction. Significant difference was not observed in all fractions for protease inhibition activity. Thus, this study reveals that polysaccharides extracted from V. zeylanica have significant biological activities.
Comparison of Antioxidant Activity of Hydrolysate Products of Crude Collagen Extracted from Chicken Egg Shell Membrane Using Different Methods
(Uva Wellassa University of Sri Lanka, 2018) Dissanayake, K.S.M.; Aruppala, A.L.Y.H.; Abeyrathne, E.D.N.S.
Collagen is a highly valuable protein used in food industry. Egg shell membrane is a safe source for collagen. Extraction of collagen from chicken membrane and producing its hydrolysates were carried out using different methods. Objective of this study was to extract collagen from chicken egg membrane with simple and non-toxic method followed by hydrolysis to find out the functional properties of the hydrolysates. Shell membrane was separated by manual peeling by adding 0.5 M Acetic acid, 0.5 M Citric acid followed with extraction of collagen with pepsin digestion. pH of the extracted collagen was adjusted to 6.5 and hydrolyzed using protease with different time combinations (0, 3, 6, 9, 12, 24 hours) at 37 °C followed by heat inactivation at 100 °C for 15 min. Best hydrolysates were selected by 10% SOS-PAGE by visual observations. Selected hydrolysates were subjected to antioxidant activity by Thiobarbituric Acid Reactive Substances (TBARS) method and Diphenyl-l-picryhydrazyl (DPPH) radical scavenging activity. The highest collagen yield was observed from citric acid (0.15 g 1 0g-1) extraction than acetic acid (0.08 glOg-1) treatment (P > 0.05). SOS-PAGE did not show bands even 0 hours, so, 0 hour hydrolysates were selected for antioxidant testing. In DPPH analysis, citric acid extraction shows higher scavenging activity (96.79%) than acetic acid (72.37%) (P < 0.05).flowever in TBARS method also did not show significant difference among the treatments (P > 0.05) and it showed 0.00 mg 1.1level of Melonaldehyde. This concludes that collagen hydrolysates showed good antioxidant activity with citric acid extraction than acetic acid extraction comparing with the ascorbic acid as positive control which can be used as a natural antioxidant in food industry.
Control of Colletotrichum gloeosporioides L. Caused Anthracnose Using isolated Yeast Species
(Uva Wellassa University of Sri Lanka, 2018) Ranasinghe, R.A.W.U.; Prematilake, M.M.S.N
Anthracnose is one of the most widely spread post harvest diseases which is mainly caused by the fungus Colletotrichum gloeosporioides L. for a vast variety of fruits around the world. To control this damage various commercialized fungicides are being used. But due to harmful effects of these on consumers, scientists have now turned towards novel bio controlling methods. In this study the antagonistic activity of four types of wild yeasts species were tested against C. gloeosporioides as yeast has become one of the promising alternative to chemical fungicides. Four types of yeasts (Y162, Y234, Y342 and Y467) were isolated from surfaces of leaves of Carica papaya L (papaya), Psidium guajava L (Guava) and Cocos nucifera L (Coconut) water and C. gloeosporioides was isolated from infected papaya and banana tissues obtained from Badulla area. Yeast isolates were identified using colony, morphological characteristics and C. gloeosporioides isolate was also identified using colony, morphological characteristics through slide cultures. Four types of yeast isolates were tested for antagonistic activity against C. gloeosporioides using dual culture method. A commercialized fungicide (Fucanazole) was used as the positive control. Antagonistic activity was tested by calculating percentage of inhibition of colony radial growth (PIRG %). All the yeast isolates showed antagonistic activity against C. gloeosporioides according to the analysis (One way ANOVA, P = 0.03), but the yeast isolate Y162 showed the highest mean PIRG % of 57.33%. Interestingly, yeast isolate Y162 showed a higher mean PIRG % when compared to the positive control (29.67%). So the results of the current study revealed that the yeast isolate Y162 has the best antagonistic activity against C. gloeosporioides. Therefore, further studies are required to identify the yeast Y162 and its mechanism of inhibition, which would lead to the production of a commercial biological control agent for Anthracnose caused by C. gloeosporioides. Keywords
Degradation of Cellulose and Pectin in Organic Wastes by Developed Fungal-Bacterial Biofilms
(Uva Wellassa University of Sri Lanka, 2018) Wickramage, K.C.; Singhalage, I.D.; Seneviratne, G.
An organic waste consists of cellulose and pectin which are resistant to rapid degradation due to their complexity. The objective of this study was to find out the most efficient fungal-bacterial biofilm/s (FBB/s) for the organic waste degradation. Pectinolytic and cellulolytic activity of isolates were tested by standard plate assays and best strains were used to develop 25 FBBs. The best FBBs were selected based on the physical attachment of bacterial cells to fungal filaments (light microscopic observations), and those were symbolized as F3B1, F5B1, F2B2, F3B2, F3B3, F4B3, F2B5 and F3B5. Coffee silver skin, barely skin and rice husk were the organic wastes used. Developed FBBs were inoculated separately to 10 g of above wastes in petri dishes and were incubated for 40 days. The control was maintained without FBBs inoculation. Three replicates were maintained for each treatment and the experiment was arranged in a completely randomized design. The mass reduction, Fourier Transform Infrared Spectroscopy (FTIR) and sugar accumulation of samples were analyzed within five day intervals. Data were analyzed by ANOVA. Results revealed that in the 40th day, the sugar production was highest in barley husk treated by F3B5. In coffee silver skin, the sugar accumulation was similar under all FBBs. In rice husk, F3B1 showed the highest sugar level in 15th day, but F4B3 dominated on the 25th day. The mean weights of the samples decreased with time, but after 25th day they came into a plateau with having 0.1-0.3 % weight loss percentage. According to FTIR data, all FBBs except F3B3 showed the degradation of barely husk. F2B5 was the best in terms of weight loss during the last five days of incubation. F4B3, F3B2 and F5B3 were the best biofilms in terms of weight loss at the end of 10th, 15th and 20th days, respectively. It can be concluded that F3B3 and F3B2 were the best FBBs for degradation of all three types of organic wastes. Keywords
Detecting Mislabelling of Packaged Frozen Seafood Products in Sri Lanka: A DNA Barcoding Approach
(Uva Wellassa University of Sri Lanka, 2018) Perera, A.G.D.M.; Amarakoon, A.A.D.G.U.; Herath, D.R.; Senevirathna, J.D.M.; Liyanage, N.P.P.
Seafood trade has conquered a vast market in global, as well as the local context. Substitution of high value species with those of low cost has become an emerging problem for the expanding market, since some of the products are visually unrecognizable and morphologically indistinguishable. The objective of this study was to assess the suitability of DNA barcoding as a method for species identification of packaged seafood to detect any mislabelling. Eight packaged frozen seafood product samples including finfish, crab, shrimp and cuttlefish were obtained from randomly selected supermarkets and subjected to DNA extraction by standard phenol chloroform DNA extraction protocol. Mitochondrial cytochrome oxidase subunit I gene (COI) was amplified in those samples with appropriate primers. Then successful five PCR products of tuna (T1 & T2), sailfish (SF1 & SF2) and crab (Cl) were selected for sequencing to build DNA barcodes. The prepared DNA sequences were compared with the Barcode of Life Database (BOLD) system for species identification. Close related sequences of each sample were downloaded from NCBI Genbank and phylogenetic trees were constructed using Maximum Parsimony, Maximum Likelihood and Neighbor Joining methods. Samples were identified as follows, T1 - Thunnus albacares (100%) and T2 Thunnus alalunga (99.85%), SF1 and SF2 - Istiophorus platypterus (100%) and Cl - Portunus pelagicus (97.71%). Identical tree topologies were resulted from three methods and three major clades were revealed in the phylogenetic tree as tuna, sailfish and crab groups. All selected five samples were matched (100%) showing that correct labelling had been done. This study concluded that DNA barcoding is a feasible, efficient and reproducible method for detection of mislabelling of packaged frozen seafood. However, the packaged frozen seafood trade has not reached high extent in Sri Lanka yet, hence substitution of seafood products does not occur frequently.
Determination of Genetic Purity of the Yellow Dwarf Coconut Seedlings Rejected from Nurseries Using SSR Markers
(Uva Wellassa University of Sri Lanka, 2018) Wijewickrama, W.L.H.; Meegahakumbura, M.K.; Attanayaka, D.P.S.T.G.; Alwis, L.M.H.R.
Hybrids coconut cultivars usually produce 40% higher yields over commonly grown Sri Lankan Talls. At present about 10,000 yellow dwarf coconut seedlings are rejected yearly from nurseries, as there is not true to type hybrids based on yellow color petiole as a visible marker for hybrid seedlings. The ambiguity of this phenotypic marker for selection often results in considerable wastage of true hybrid seedlings from the nurseries widening the gap between the demand and the supply of hybrid planting material. In the current study, microsatellite (SSR) marker-based approach was used to test the true hybridity of seedlings raised in the nursery. One hundred rejected seedlings were screened with 2 SSR primers, namely CAC 68 and CAC 23 which exhibited potential to distinguish parental varieties, Sri Lanka Tall (SLT), Sri Lanka Yellow Dwarf (SLYD), and resulting hybrids. The results of the study revealed that on average 36% of the rejected plants were true hybrids which are suitable for planting. The percentages of parental types, the true contaminants were 62% comprising with 6% Tall types and 56% SLYD types. As a result the current visible marker used to select off type seedlings from the coconut nursery is inaccurate resulting a loss of 36 true hybrids to the industry for every 100 seedlings rejected. The two SSR markers can be used to confirm the hybridity of seedlings derived from SLT X SLYD crosses reducing the loss by authenticated plants from the nurseries.
Effect of Methanolic Extracts of Emilia sonchifolia (Lilac tassel flower), Ageratum conyzoides (Billy goat weed) and Mikania micrantha (Bitter vine) on Protease Enzyme Inhibition
(Uva Wellassa University of Sri Lanka, 2018) Nisrina, M.N.; Alakolanga, A.G.A.W.; Premetilake, M.M.S.N.; Wijesekara, K.B.
According to previous studies, methanolic extracts of Emilia sonchifolia, Ageratum conyzoides and Mikania micrantha have shown significant antimicrobial activity against Staphylococcus aureus. But their mode of action on the microorganisms is still unknown. It is expected that these three plants may have acted as protease inhibitors in the respective microorganism. So in the present study, 40 g of shade dried leaves of each plants, E sonchifolia, A. conyzoides and M. micrantha were extracted using methanol and subsequently subjected to solvent-solvent partitioning using hexane, chloroform and ethyl acetate. Those fractions were evaporated to obtain concentrated fractions. Then these concentrated fractions were used to prepare four concentration gradients, such as: 250 𝜇g ml-1, 500 𝜇g m1-1, 750 𝜇g m1-1 and 1000 𝜇g m1-1 and tested against protease enzyme. The protease enzyme assay was carried out based on Kunitz method, using casein as the substrate. According to the results obtained, highest inhibitory percentage was shown by E. sonchifolia. Even though statistical output has shown a significant difference of inhibition percentage among the concentration gradient of the plant fractions used (p value = 0.000), the range of the values are narrowed from 83.8% and 93.5% only for all three plants. So the results do not confirm the protease inhibitory activity of the extracts of the selected plants. Protease inhibitors control the action of proteases that are vital for the growth and development of the organism. Therefore, the reason for the antimicrobial activity of the methanolic extracts of these plants may not be due to protease inhibitory reaction but due to some other reasons.
Effect of Solvent Type and Extraction Time on Yield and Purity of Lotus (Nelumbo nucifera)Leaf Wax
(Uva Wellassa University of Sri Lanka, 2018) Prasadika, H.K.A.E.; Liyanage, N.P.P.; Pitawala, H.M.J.C.; Senevirathna, J.D.M.
This study was conducted with the main objective of extracting lotus leaf wax using a simple methodology and to analyze the wax yield and purity of the extracted wax corresponding to different time treatments. Past studies have found that lotus plant leaf wax contains a mixture of aliphatic compounds mainly nonacosanol and nonacosanediols. Fresh, cleaned lotus leaves with 1 cm2 in surface area were exposed to three organic solvents (methanol, acetone and chloroform) and time taken for the presence of light green colour (due to the extraction of chlorophyll) in the medium was recorded to find out the most efficient organic solvent. Further, contact angle measurements of water drops placed on each of the leaf samples treated with different solvents were calculated to find out the efficiency of wax extraction. Based on the results obtained from this study leaf samples with surface area of 72.41 cm2 were exposed to chloroform by changing the dipping time duration ranging from two seconds to 30 minutes with time intervals of two seconds for the first five treatments ,15 seconds for the next three and five minutes for the last seven treatments. Extracted wax was subjected to the FTIR analysis to find out the purity of the wax. According to the results it was revealed that green colour was appeared in methanol within the first five minutes and there was no color change in the chloroform and acetone for about 30 minutes. Moreover, least change of the contact angle was shown by the leaf sample which was treated with acetone and it reveals that wax extraction was not done in an efficient manner. Chloroform is the best solvent to extract lotus leaf wax among three organic solvents used. The highest mean yield gives out by the time treatment with the dipping time of 20 minutes. And it shows that the purity decreases with the increase of the dipping time duration. Keywords
Effects of Different Cooking Methods on Antioxidant Activity of Selected Underutilized Tuber Crops of Sri Lanka
(Uva Wellassa University of Sri Lanka, 2018) Perera, P.P.M.; Wijesekara, K.B.
This study was carried out to investigate the effect of cooking on antioxidant activity of five underutilized tuber crops of Sri Lanka namely, Dioscorea alata (raja ala), D.esculenta (kukulala), Amorphophallus paeoniifolius (kidaran), D. alata (kondol), D. alata (hingurala) The two cooking methods tested in the study were frying and boiling. Selected tubers were peeled, cut into strips of 0.5 W x 4 L x 0.5 H cm separately. Then the strips were cooked separately either by boiling in water until soft or fried in coconut oil until golden brown and crispy for 15 minutes. Uncooked strips from each tuber variety served as controls. Methanolic extracts were prepared for boiled, fried and fresh tuber strips by shaking (780 rpm) shade dried finely powdered samples (20 g) with 30 ml of methanol. Total ascorbic acid content of each extract was measured by Iodometric titration method and total antioxidant activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assays. Each experiment was conducted as 3 replicates. Results indicated that total ascorbic acid content of boiled yam extracts were significantly lower (p < 0.05) than the control and fried tubers. Raw yam extracts of all five varieties had higher ascorbic acid and antioxidant contents than cooked tuber extracts. The DPPH and ABTS radical scavenging capacity of boiled yam extracts were significantly lower (p < 0.05) than fried tubers. Amongst five varieties, highest radical scavenging capacity was shown by Amorphophallus paeoniifolius raw yam extract and lowest value was obtained for D. alata (hingurala) boiled yam extract (p < 0.05). IC50 values (concentration for 50% inhibition of DPPH) and ABTS.discoloration percentage of each variety was 85.2±2.7 µg m1-1, 86±1% and 910.8±11.8 𝜇g m1-1, 9.57±1.89% respectively. Antioxidant activities of all cooked tubers are lower than the raw tubers. For all tubers boiling in water had lower antioxidant activity.
Enhancement of Cellulolytic Activity through Biofilm Action for Bioethanol Production
(Uva Wellassa University of Sri Lanka, 2018) Jayathilaka, M.G.L.W.; Henagamage, A.P.; Peries, C.M.; Seneviratne, G.
Cellulosic biomass is a biopolymer with great potential for bioconversion to valueadded products. However, efficient degradation of cellulose is a problem in many industries including bioethanol production. Although a variety of microorganisms are capable of degrading cellulose, few of them produce significant quantities of enzyme fractions which hydrolyze cellulose to simple sugars. Extensive studies on bio-degradation by cellulolytic mixed microbial cultures would be beneficial in cellulosic biofuel production. Thus, this study was focused to evaluate the efficiency of cellulolytic activity of mono and mixed microbial cultures. Microbial isolations were carried out using soil samples obtained from a land at Kuliyapitiya, in Kurunegala district, Sri Lanka. They were streaked on Cellulose-Congo red Agar medium to screen for potential cellulolytic microorganisms. The selected microorganisms were inoculated on Carboxy Methyl Cellulose Agar medium to screen the most effective cellulolytic fungi and bacteria. Fungal-bacterial biofilms (FBB) were developed from the selected cellulolytic fungi and bacteria using Combine Carbon Broth. The efficiency of cellulolytic activity of the selected microbial combinations was evaluated using the production of reducing sugar through 3,5-Dinitro Salicylic acid after treating with cellulose powder. Two fungal (F1 and F2) and three bacterial isolates (B1, B2 and B3) were selected as the best cellulolytic microorganisms. Out of the selected cellulolytic microorganism, F2 and B I showed the significantly highest cellulolytic activities (P < 0.05). This mean reducing sugar level (113.90 ppm) was observed with the F2B1 combination after twenty three days of incubation. In addition, F2, B1 and B2 mono cultures showed significantly higher yield of reducing sugar than that of the other mono and mixed cultures, except F2B1. Thus, the selected FBB combination can be used to enhance the hydrolysis efficiency of cellulose for bioethanol production.
Extraction of Crude Bone Collagen from Yellowfin Tuna (Thunnus albacares) and Determination of Anti-oxidative Activity of Its Hydrolysates
(Uva Wellassa University of Sri Lanka, 2018) Wijekoon, W.M.M.P.; Aruppala, A.L.Y.H.; Kariyawasam, M.G.T.R.; Abeyrathne, E.D.N.S.
Fish bones are significant part of fish processing by-product and rich source of collagen proteins. Utilization of yellowfin tuna bones are important economically as well as environmentally. Objective of this research was to extract crude collagen from yellowfin tuna bones and to identify the anti-oxidative activities of its hydrolysates which can be a potential natural anti-oxidative agent in food industry. Acid-pepsin soluble collagens were extracted from fresh yellowfin tuna bones. As with the pre-treatment process EDTA and citric acid were tested to decalcify. Extracted collagens from two treatments were subjected to the hydrolysis using protease enzyme with different time combinations (0, 3, 6, 9, 12, 24 h) at 37 °C followed with heat inactivation at 100 °C for 15 minutes. Antioxidant activity of the best hydrolysates were evaluated using thiobarbituric acid reactive substances (TBARS) assay and 2,2-dipheny1-1-picrylhydrazyl (DPPH) free radical scavenging activity method. All treatments were replicated (n = 3). Resulting extracts with citric acid treatment (1.23±0.05%) showed higher yield compared to the EDTA treatment (0.62±0.18%) (p < 0.05). Both treatments showed similar band patterns with 08% SDS-PAGE gel electrophoresis confirming the extracted collagen are same. Hydrolysates produced after incubating for 3 hours at 37 °C followed with heat inactivation was selected as the best (p < 0.05). The results showed that collagen hydrolysate of yellowfin tuna bones inhibited lipid oxidation in oil emulsion system and also control free radicals (DPPH). TBARS results of EDTA and citric acid treatment showed no significance difference with the control(p > 0.05). EDTA (86.14±1.88%) and citric acid (87.92±7.72%) treatments showed DPPH free radical scavenging activity compared with ascorbic acid (89.10±0.64%). These results suggest that hydrolysates produced from yellowfin tuna bones with citric acid can be used as a potential natural antioxidant agent in food industry.
Genome Analysis on Drought Resistance of Hevea brasiliensis
(Uva Wellassa University of Sri Lanka, 2018) Rajapaksha, R.L.P.N.D.; Weerasinghe, A.R.; Attanayaka, D.P.S.T.G.; Wijesinghe, C.R.; Alwis, L.M.H.R.
Hevea brasiliensis (para rubber tree) plays an important role in the economy of Sri Lanka. Potential drought stress conditions due to climatic changes will have a severe effect on the yield and the survival of the rubber tree. Understanding the underlying genetic basis of drought tolerance through identification and systematic analyses of the candidate genes associated with drought tolerance of Hevea will help rubber breeding by marker assisted selection and transgenic improvement. This study was undertaken to generate information about the genes related to drought tolerance in Hevea. Biologically validated eighteen Arabidopsis thaliana genes with known functional pathways were used as query sequences to find orthologous Hevea genes from the ASM165405v1 genome assembly using the BLASTP program of the BLAST tool. Query coverage higher than 50%, bit score higher than 80 and E-value lower than 1 x10-5° were taken as cut off criteria for the search. Nine Hevea orthologous genes were identified and they represented six functional groups involved with both physiological and molecular adaptation to drought. Highest number of candidate genes identified encodes transcription factors. Systematic analyses of the identified genes related to drought tolerance suggest that transcription regulation, phospholipid metabolism, growth control, detoxification signaling, osmolyte biosynthesis and signal transduction pathways play important roles in drought tolerance in Hevea. Identification and analysis of conserved regions was conducted for the identified three transcription factors using the MEME and InterPro tools respectively. Three domains were identified which shared Gene Ontology terms related to drought tolerance. The results of the research not only enrich information about the genes related to drought tolerance, but also provide new insights into understanding the drought tolerance mechanisms in rubber tree.
Molecular mapping of leaf rust resistance in C14.16/Aus91433 RIL population
(Uva Wellassa University of Sri Lanka, 2018) Pakeerathan, K.; Bariana, H.S.; Bansal, U.K.
Leaf rust, caused by P. triticina (Pt) is one of the most wide-spread diseases of wheat. This pathogen was introduced into Australia by first migrant settlers. Due to its wide range of adaptation, rapid evolution of P. triticina (Pt) pathotypes in Australia has defeated many leaf rust resistance genes. Deployment of genetically diverse sources of resistance in wheat cultivars with the help of tightly linked molecular markers is the most rapid and eco-friendly means of leaf rust control. Seedling-resistant and adult plant leaf rust susceptible cultivar Aus91433 was crossed with the selection C16.14 and the recombinant inbred line population was developed. The C16.14/Aus91433 RIL population was screened with Pt pathotype 104-2,3,6,(7),12 at the seedling stage in greenhouse. Resistant RILs and Aus91433 expressed IT 12C and susceptible RILs and C16.14 exhibited IT 3+. Monogenic segregation for leaf rust response was observed. The resistance locus was temporarily named LrPak. Genomic DNA extracted from the resistance RILs and susceptible RILs were processed with high throughput 90K SNP Array-based BSA to detect the chromosomal location of the resistance. The 90K SNP Array identified association of 32 SNPs from chromosome 2B with LrPak. These SNPs spanned across 6.0 cM to 27.0 cM interval of the 90K consensus map. Based on the parental polymorphic screening, final linkage map of 17.4 cM showing marker order and position of the gene was drawn. KASP_80930 and KASP_51150 flanked LrPak 2.1 cM and 2.5 cM proximally and distally, respectively. Identified closely linked molecular markers in this study will be used for marker assisted back crossing of LrPak into modern wheat varieties to create diversity for leaf rust resistance.
Phytochemical Screening and Antioxidant Activities of Selected Tropical Underutilized Fruits
(Uva Wellassa University of Sri Lanka, 2018) Chathurangi, R.P.D.D.; Wathsara, H.P.T.; Samarakoon, K.W.; Ranasinghe, P.; Dissanayake, P.K.
Fruits are rich sources of bioactive compounds as majority of them have antioxidant properties. The objectives of this study were to screen particular phytochemicals and to determine the antioxidant properties of selected fruit species, namely Cynometra cauliflora, Psidium cattleianum, Annona squamosa and Diospyros discolor. Phytochemical screening was done using colourimetric qualitative analysis to screen phenol, flavonoid, tannin, alkaloid and saponin. Cynometra cauliflora seed and Psidium cattleianum fruit extracts were positive for all the tested phytochemicals. Antioxidant properties of fruit extracts were determined by in vitro antioxidant assays using 96 well micro plates. Total Phenolic Content, Total Flavonoid Content, Ferric Reducing Antioxidant Power and Oxygen Radical Absorbance Capacity were determined by using standard procedures. All values of antioxidant properties were significantly different for the tested fruits (p < 0.05). The highest total phenolic content value was recorded for Cynometra cauliflora seed (306±1.64 mg of Gallic Acid Equivalents per g of extract) while the lowest value was recorded for Annona squamosa (19.17±4.78 Gallic Acid Equivalents per g of extract). The highest total flavonoid content value was shown by Psidium cattleianum (4.76±0.19 Quercetin Equivalents per g of extract) while the lowest value was shown by Diospyros discolor (0.35±0.02 Quercetin Equivalants g-1 of extract). The highest ferric reducing antioxidant power value was shown by Cynometra cauliflora seed (685.38±1.63 Trolox Equivalents per g of extract) while the lowest value was shown by Cynometra cauliflora pericarp (30.74±1.3 Trolox Equivalents per g of extract). The highest oxygen radical absorbance capacity value was recorded for Psidium cattleianum (549.79±6.91 Trolox Equivalents per g of extract) and the lowest value was recorded by Annona squamosa (33.57±0.31 Trolox Equivalents per g of extract). Overall results of the study revealed Cynometra cauliflora seed, Cynometra cauliflora pericarp, Psidium cattleianum, Annona squamosa and Diospyros discolor extracts posses antioxidant properties and in general Cynometra cauliflora seed and Psidium cattleianum have high antioxidant properties.

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Browsing International Research Symposium of UWU-2018 by Subject "Biotechnology"

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