Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid.
A study was conducted to determine the effect of sugarcane maturation on the contents of chlorophyll, tannin, and polyphenol oxidase (PPO) activity and on color change of sugarcane juice. The maturation period of the cane studied was between 3 and 10 months after planting. Different parts of the cane, namely, the top, middle, and bottom portions, were analyzed. Results obtained indicated that there were significant (P < 0.01) decreases in total chlorophyll a and b and tannin contents during maturity followed by slower rates of decrease of both parameters at the end of maturity stages. There were no significant differences (P > 0.05) in chlorophyll and tannin contents between the middle and bottom portions. On the other hand, the top portion of the stem had a significantly (P < 0.01) lower concentration of chlorophyll and a significantly (P < 0.01) higher content of tannin. PPO activity of sugarcane juice was determined using chlorogenic acid as a substrate. There was a highly significant difference (P < 0.01) in PPO activity of cane juice during maturity. PPO activity was high at the early development stage, decreased during maturation, and then remained relatively constant at the end of maturity. PPO activity was higher when chlorogenic acid was used as substrate. There were also significant differences (P < 0.01) in juice color (L*, a*, b* values) from different portions at different maturity stages. At the early stages, the color of extracted juice was dark, and then the juice turned to yellowish green during maturity. The decrease in green color or the increase in the yellow color could be associated with the decline in chlorophyll. The overall color change (DeltaE) at maturity indicated that the color of the middle and bottom portions was lower than that of the top portion.
The effects of different drying methods (freeze drying, vacuum oven drying, and shade drying) on the phytochemical constituents associated with the antioxidant activities of Z. officinale var. rubrum Theilade were evaluated to determine the optimal drying process for these rhizomes. Total flavonoid content (TFC), total phenolic content (TPC), and polyphenol oxidase (PPO) activity were measured using the spectrophotometric method. Individual phenolic acids and flavonoids, 6- and 8-gingerol and shogaol were identified by ultra-high performance liquid chromatography method. Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used for the evaluation of antioxidant activities. The highest reduction in moisture content was observed after freeze drying (82.97%), followed by vacuum oven drying (80.43%) and shade drying (72.65%). The highest TPC, TFC, and 6- and 8-shogaol contents were observed in samples dried by the vacuum oven drying method compared to other drying methods. The highest content of 6- and 8-gingerol was observed after freeze drying, followed by vacuum oven drying and shade drying methods. Fresh samples had the highest PPO activity and lowest content of flavonoid and phenolic acid compounds compared to dried samples. Rhizomes dried by the vacuum oven drying method represent the highest DPPH (52.9%) and FRAP activities (566.5 μM of Fe (II)/g DM), followed by freeze drying (48.3% and 527.1 μM of Fe (II)/g DM, respectively) and shade drying methods (37.64% and 471.8 μM of Fe (II)/g DM, respectively) with IC50 values of 27.2, 29.1, and 34.8 μg/mL, respectively. Negative and significant correlations were observed between PPO and antioxidant activity of rhizomes. Vacuum oven dried rhizomes can be utilized as an ingredient for the development of value-added food products as they contain high contents of phytochemicals with valuable antioxidant potential.
Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cubense, especially by tropical race 4 (Foc TR4), is threatening the global banana industry. Musa acuminata Pahang, a wild diploid banana that displays strong resistance to Foc TR4, holds great potential to understand the underlying resistance mechanisms. Microscopic examination reports that, in a wounding inoculation system, the Foc TR4 infection processes in roots of Pahang (resistant) and a triploid cultivar Brazilian (susceptible) were similar by 7 days post inoculation (dpi), but significant differences were observed in corms of both genotypes at 14 dpi. We compare transcriptomic responses in the corms of Pahang and Brazilian, and show that Pahang exhibited constitutive defense responses before Foc TR4 infection and inducible defense responses prior to Brazilian at the initial Foc TR4 infection stage. Most key enzymatic genes in the phenylalanine metabolism pathway were up-regulated in Brazilian, suggesting that lignin and phytotoxin may be triggered during later stages of Foc TR4 infection. This study unravels a few potential resistance candidate genes whose expression patterns were assessed by RT-qPCR assay and improves our understanding the defense mechanisms of Pahang response to Foc TR4.
Freshly prepared, hand-pressed strawberry fruit juice was exposed to ultraviolet radiation (254 nm) at room temperature (25 ℃ ± 1 ℃) for 15, 30 and 60 min with 0 min serving as control. Results revealed decrease in pH, total soluble solids and titratable acidity, while colour parameters (L*, a* and b* values) and clarity of juice (% transmittance) increased significantly. All the results corresponded to exposure time to ultraviolet radiation. Bioactive compounds (total phenolics, ascorbic acid and anthocyanins) decreased along with a recorded reduction in polyphenol oxidase enzyme and 1,1-diphenyl-2-picryl hydrazyl radical scavenging activities, which were again dependent on exposure time. Results on the microbial studies showed significant reduction by 2-log cycles in aerobic plate count as well as in total yeast and mould counts. Though negative results were observed for certain parameters, this is the first time it was endeavoured to demonstrate the impact of ultraviolet radiation radiation on freshly prepared, hand-pressed strawberries juice.
Composition, physicochemical properties and enzyme inactivation kinetics of coconut water were compared between immature (IMC), mature (MC) and overly-mature coconuts (OMC). Among the samples studied, pH, turbidity and mineral contents for OMC water was the highest, whereas water volume, titratable acidity, total soluble solids and total phenolics content for OMC water were the lowest. Maturity was found to affect sugar contents. Sucrose content was found to increase with maturity, and the reverse trend was observed for fructose and glucose. Enzyme activity assessment showed that polyphenol oxidase (PPO) in all samples was more heat resistant than peroxidase (POD). Compared to IMC and MC, PPO and POD from OMC water showed the lowest thermal resistance, with D83.3°C=243.9s (z=27.9°C), and D83.3°C=129.9s (z=19.5°C), respectively.
Trichoderma is a genus of soil-borne fungus with an abundance of reports of its economic importance in the agriculture industry. Thus, the correct identification of Trichoderma species is necessary for its commercial purposes. Globally, Trichoderma species are routinely identified from micro-morphological descriptions which can be tedious and prone to errors. Thus, we emphasize that the accurate identification of Trichoderma strains requires a three-pronged approach i.e. based on its morphological characteristics, multilocus gene sequences of the rDNA [internal transcribed spacer (ITS) 1 and 2 regions], translation elongation factor 1-α (TEF-1α), Calmodulin (CAL) and its lignocellulolytic activities. We used this approach to identify a total of 53 Trichoderma strains which were isolated from a wet paddy field located at Tuaran, Sabah, Malaysia. The 53 strains were positively identified as belonging to three Trichoderma species, namely T. asperellum (43 strains), T. harzianum (9 strains), and T. reesei (one strain) on the basis of its morphological characteristics and multilocus gene sequences. Phylogenetic trees constructed based on the UPGMA method of the ITS 1 and 2 regions of the rDNA, TEF-1α and CAL revealed three distinct groups with the T. asperellum, T. harzianum and T. reesei strains placed under the section of Trichoderma, Pachybasium and Longibrachiatum, respectively. In addition, the lignocellulolytic activities of the isolates were measured based on the diameters of the halo zones produced when degrading cellulose, lignin, and starch, respectively. This diagnostic assay can be used to identify Trichoderma as it produces polyphenol oxidase when Tannic Acid Media is used for the lignin test, endoglucanases when Jensen media is used for cellulose, and it hydrolyzes starch to glucose when the modified Melin-Nokrans media is used for the starch test. Accurate identification of Trichoderma species is needed as these strains can potentially be used as a biocontrol agent to prevent diseases and to increase yield in agriculture crops.
One of the major steps in the innate immune response of shrimp includes the activation of serine proteinases of the pro-phenoloxidase pathway by the prophenoloxidase activation enzyme (PPAF). In this study, the cDNA encoding a serine proteinase homologue (SPH) with prophenoloxidase activating activity of Penaeus monodon (PmPPAF) was cloned and characterized. PmPPAF cDNA consists of 1444 nucleotides encoding a protein with 394 amino acid residues. The estimated molecular weight of PmPPAF is 43.5 kDa with an isoelectric point of 5.19. PmPPAF consists of a signal peptide, a CLIP domain and a carboxyl-terminal trypsin-like serine protease domain. It is highly similar to the masquerade-like protein 2A (61% similarity) of the crayfish Pacifastacus leniusculus, other serine proteases (42.9-67% identity) of P. monodon, and the PPAF of the crab (61% similarity). Unlike other SPH of P. monodon, which express mainly in the hemocytes, PmPPAF transcripts were detected in the hemocytes, eyestalk, hypodermis, gill, swimming leg and brain. Similar to the crab PPAF, PmPPAF transcript level is high in shrimp at the premolt stages and PmPPAF expression is up-regulated in shrimp infected with white spot syndrome virus (WSSV). Gene silencing of PmPPAF decreased expression of a prophenoloxidase-like gene and injection of Anti-PmPPAF antibody causes a decrease in PO activity. Taken together, these results provided evidence that PmPPAF is a serine proteinase homologue, and is involved in the pro-PO activation pathway of the shrimp innate immune system.