DNA extraction and PCR optimization for DNA barcode analysis of commercially-grown coffee varieties in Nepal

The isolation of high-quality genomic DNA is an essential criterion for further molecular analysis. Coffea genus is well known for its high amount of polyphenols, polysaccharides, and other secondary metabolites that degrades the quality of the DNA isolation needed for further down streaming processes. The present work was carried out to obtain a simple and efficient DNA isolation protocol generating high-quality amplification for barcoding. The protocol involves modifying the CTAB extraction, incorporating the use of polyvinylpyrrolidone and β -mercaptoethanol yielding quality DNA with a ratio (A260/280) between 1.8–2.0 indicating low contamination. The PCR conditions were optimized for high amplification based on the optimal concentration of MgCl2 (3 mM), primer (0.5 µM), Taq polymerase (0.2 U), 50–60 ng of DNA template, and cycle conditions as initial denaturation of 94○C for 4 min followed by 35 cycles of denaturation at 94○C for 50 sec, annealing (respective of barcodes) for 50 sec and extension at 72○C for 80 sec, followed by a final extension at 72○C for 7 min. The optimal conditions produced highly amplified reproducible data. Thus, the optimized method proposed enabled a simple DNA extraction and PCR amplification for Coffea genus and may serve as an efficient tool for further molecular analysis

Genetic diversity analysis of commercial Arabica coffee in Nepal using Molecular markers

Coffee is an established plant for its flavor and has high commercial use. In Nepal, the popularity of coffee is increasing for its high economic value. However, its diversity and the status of its genetic mapping have not been studied in Nepal. In the present study, the genetic diversity of 28 coffee accessions was assessed by using twenty-four SSR markers with the aim of studying the variation of coffee in accord with the genetic markers from a molecular approach. With the use of DNA extraction and marker selection for its amplification using PCR tools, a total of 81 loci from SSR were identified. Of all SSR 63.22% showed for mean polymorphism. The mean polymorphic information content of SSR was 0.38, which showed low genetic diversity of SSR markers among Coffea genotypes.  On the basis of the SSR marker, the unweighted pair group method with arithmetic mean (UPGMA) dendrogram constructed showed a similar group of distribution among 28 accessions, which was further supported by a principle coordinate analysis scatter plot. The phylogenetic relationships among the accessions were assessed by SSR marker, which also showed low diversity in coffee genotypes. Our study demonstrated the use of SSR markers in diversity analysis as the data were informative and highly reproducible for evaluating relationships among coffee cultivars in Nepal. The use of more markers systems and a high genotype pool would have been beneficial in accessing more accurately. Regardless, the information from the phylogenetic relationship study could be useful for breeding, varietal improvement, and for conservation programs

Purification and Characterization of a Noble Thermostable Alpha-amylase from Anoxybacillus tengchongensis RA1-2-1 Isolated from Geothermal Spring of Nepal

A thermophilic amylolytic strain, Anoxybacillus tengchongensis RA1-2-1 was isolated from geothermal spring of Rasuwagadi district of Nepal. The BLAST alignment of the 16s rRNA sequence revealed 99.3% similarity with the type strain Anoxybacillus tengchongensis T-11. The morphological, physiological and biochemical properties were similar to the type strain. The enzyme from the strain was purified to 40-fold purification by DEAE-cellulose ion exchange chromatography. The Km value of the enzyme was 0.68±0.05 mg/ml. The optimum pH and temperature were 7.0 and 70 °C. SDS-PAGE analysis showed a single band at 69 kDa. The half-life of the enzyme at 70°C and 80°C were 85.01min and 51.96 min respectively. TLC analysis of the hydrolysis product showed that the enzyme is maltogenic amylase. The calcium independent enzyme was completely inhibited by Hg2+ but showed inhibitory effect in the range of 100 %-30 % in the presence of other salts at 1-10 mM concentrations.</jats:p

Molecular Identification and Antioxidant Activity Determination among Coffee Varieties Cultivated in Nepal

Coffee is the most popular beverage containing numerous phytochemical components that have antioxidant activity capable of scavenging free radicals. Antioxidant and phenolic contents have considerable benefits for human health. The aim of this study was the molecular identification of 9 coffee samples from the Nepal Agricultural Research Council, Lalitpur, Nepal, and the determination of the antioxidant activity and total phenolic content of green and roasted coffee beans. Molecular identification was performed using ITS-specific PCR followed by sequencing and phylogenetic tree construction using the maximum parsimony method. The DPPH assay was used to determine the antioxidant activity, and the Folin–Ciocalteu (F-C) assay was used to determine the total phenolic content. All the samples belonged to the taxa Coffea arabica. The antioxidant activity in roasted beans varied from 2.49 to 4.62 AAE mg/g and from 1.4 to 3.9 AAE mg/g in green beans. The total phenolic content varied from 2.58 to 3.38 GAE mg/g and from 4.16 to 5.36 GAE mg/g for the roasted beans and green beans, respectively. The data revealed that the highest antioxidant content (4.62 AAE mg/g) was found in roasted coffee and that the highest phenolic content (5.36 GAE mg/g) was found in green coffee. The study concludes that roasting increases the antioxidant activity but decreases the phenolic content of coffee

Screening and Identification of Thermotolerant and Osmotolerant Bacillus amyloliquefaciens BKHE Isolated from Kinema of Eastern Nepal for Alkaline Protease Production

Alkaline protease is one of the most important industrial enzymes which are excessively used in the detergent industry, food industry, feed industry, pharmaceutical industry, leather industry, etc. 60% of the produced alkaline protease is consumed by the detergent industry alone. In the present study, bacterial isolates that can produce alkaline protease for purpose of bio-detergent were screened among the isolates isolated from kinema (an alkaline fermented food of eastern Nepal). Selected bacterial isolates were further screened for hemolysis activity and the production of other hydrolytic enzymes. Four bacterial isolates selected were tested for their capacity to produce alkaline protease in five different fermentation mediums. Isolate BKHE produces a high amount of alkaline protease (0.4705 ± 0.035 U/mL/min) in fermentation medium M2 (sucrose, 11 g/L; yeast extract, 5 g/L; and KNO3, 5.2 g/l, pH 9). The selected isolate was identified as Bacillus amyloliquefaciens BKHE based on 16S rRNA sequencing and phenotypic features. This bacterial strain was also found to be thermotolerant (confluent growth at 50°C) and salt tolerant up to 10% NaCl concentration. With its versatile ability, bacterial isolate or purified enzymes have potential applications in the food and detergent industry

Selection and Characterization of Potential Baker’s Yeast from Indigenous Resources of Nepal

The study aims to isolate the yeast strains that could be used effectively as baker’s yeast and compare them with the commercial baker’s yeast available in the market of Nepal. A total of 10 samples including locally available sources like fruits, Murcha, and a local tree “Dar” were collected from different localities of Bhaktapur, Kavre, and Syangja districts of Nepal, respectively. Following enrichment and fermentation of the samples, 26 yeast strains were isolated using selective medium Wallerstein Laboratory Nutrient Agar. From the differential tests which included morphological and microscopic observation and physiological and biochemical characterization such as nitrate reduction and lactose utilization tests, 8 strains were selected as possible Saccharomyces strain. The selected strains were further assessed for their efficient leavening ability by tests such as ethanol tolerance, osmotolerance, invertase test, and stress exclusion test. The three most potent strains ENG, MUR3B, and SUG1 isolated from grape, Murcha, and sugarcane, respectively, were used in the fermentation and baking of dough. These strains also carried a possibility of being used as industrial baker’s yeast