Research Journal of Biotechnology

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Research Journal of Biotechnology

Screening of high-yield Lovastatin strain of Penicillium spp. and optimization of its fermentation conditions

Zheng Jieshi and Jiang Donghua

To screen for the strains suitable for industrial manufacture, 163 Penicillium spp. were isolated from various products. The yield of Lovastatin was determined by thin layer chromatography (TLC) combined with analytical-high performance liquid chromatography (HPLC) and a strain which produces high yield of Lovastatin was screened from 163 strains. Studies on 18S rDNA sequence and colony characteristics such as colony size, color, surface morphology, aerial mycelium, substrate mycelium and edge features showed that this strain belongs to Penicillium oxalicum. By analyzing the influence of carbon source, nitrogen source, fermentation temperature, initial pH and so on to explore the optimal fermentation conditions, the results were as follows: carbon source glycerol, nitrogen source peptone, fermentation temperature 28℃, shaking speed 180 rpm, initial pH 5.0, inoculum amount 7%. Then the same culture medium and fermentation condition were applied to amplified fermentation in a stirred tank bioreactor. After 7 days’ incubation, content of Lovastatin reached 153.2 mg/L.

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Effects of Sires Genotyping for κ-Casein on B Allele Frequency in Romanian Brown Cattle

- Neamt Radu Ionel, Ilie Daniela Elena and Cziszter Ludovic Toma

The CSN3*B allele of bovine is associated with higher milk protein content influencing significantly the cheese yields. Currently it is being considered as a potential selection trait for several cattle breeds. The aim of the current study was to increase the frequency of the favourable B allele for CSN3 in Romanian Brown herd rearing for milk production, using marker - assisted selection (MAS). The trial was conducted on 153 Brown cattle. The study began in the year 2006 when entire herd was genotyped. The genotyping for CSN3 was done by using PCR-RFLP and PCR-HRM assays. Only sires with BB and AB genotypes were further used for artificial insemination schemes. An increase by 5.8% of CSN3*B frequency was calculated as a result of applied selection in Romanian Brown investigated herd and ranged between 62.12% in 2007 to 67.92% in 2016 respectively. The use of marker - assisted selection in Brown breed cattle has been proven to be an efficient tool for improving the herd genetic structure for the CSN3 locus.

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Purification and production of antiserum against pepper mild mottle virus isolated from Saudi Arabia

Al-Wabli Afaf S., Khattab Eman A.H. and Farag Azza G.

The virus was identified on the basis of host range, symptomatology, stability in crude extracts, modes of transmission, serological reaction, inclusion bodies and electron microscopy. The virus was purified from infected plants using the purified procedure. The UV-absorption spectrum had a maximum at 260 nm and a minimum at 245 nm. The ratios of Amax/ Amin and A260/ A280 were 1.19 and 1.25 respectively. The yield of purified pepper mild mottle virus (PMMoV) preparation was about 5mg/100g of infected tissue collected on the basis of an extinction coefficient 3.18 and polyclonal antibodies raised against PMMoV had a maximum titer of 1: 1024 from bleeding taken 12 days after the last injection. The validity of antiserum production was determined by tissue blot immuno binding assay (TBIA) and dot blot immuno binding assay (DBIA).

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Analysis of Molecular Assortment in Sugarcane Varieties using RAPD and ISSR markers

Jomhe Farsangi Forough, Thorat Avinash and Devarumath Rachayya

Molecular assortment within 55 sugarcane cultivars/clones including associated genera was studied using RAPD and ISSR markers. The molecular diversity study with 15 RAPD markers generated total 153 alleles of 146 (95.4%) and were found polymorphic. The PA varied from 7-14 amplified with an average of 10.5 alleles. The PIC varied from 0.31-0.61 with an average of 0.46 found and Rp varied from 2.55-10.29 with an average 6.42. Similarly, 15 ISSR markers generated total 138 alleles of 123 (89.1%) were found polymorphic. The PA varied from 3-11 amplified with an average of 7 alleles. PIC varied from 0.17-0.52 with an average of 0.34 and the Rp found varied from 1.31-6.85 with an average of 4.08. RAPD marker showed more polymorphism than ISSR on selected sugarcane genotypes which together would be used to ascertain the selection of more genetically diverse sugarcane from the groups and utilized for crop improvement program.

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Formate co- feeding improved Candida antarctica Lipase B activity in Pichia pastoris

Charumathi Jayachandran, Balakumaran Palanisamy Athiyaman and Meenakshisundaram Sankaranarayanan

Addition of non - repressive carbon sources together with methanol can minimize the excess energy demand induced by recombinant protein expression. The present study investigated formate co - feeding along with methanol to enhance the cellular energy level and recombinant Candida antarctica Lipase B (CALB) secretion. The methylotrophic yeast P. pastoris efficiently metabolizes formate to promote ATP formation. P. pastoris expressing CALB gene (GSCALB) was used in this study. Formate co-feeding (0.1%) in GSCALB strain increased intracellular ATP levels and CALB activity by 23%. The formate co- utilization improved methanol uptake rate which in turn enhanced NADH levels and NADH/NAD+ ratio. The augment in adenylate energy charge value in formate supplemented culture indicates the progress in energy state of cell. Therefore, formate can act as an extra energy source like sorbitol in P. pastoris to improve recombinant CALB production. Moreover, formate supplementation as a non-repressible carbon source in the medium tends to be economically cheaper alternative and enables P. pastoris to act as a biocatalyst for production of industrial products through cofactor regeneration.

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Characterization of a milk-clotting enzyme produced by Bacillus mojavensis P47M strain isolated from Algerian dairy farm soil

Kahlouche Amal and Nouani Abdelouahab

A strain P47M which produces a milk-clotting enzyme isolated from soil of Algerian dairy factory was identified as Bacillus mojavensis based on morphology and internal transcription spacer sequence. Phylogenetic and sequencing analysis of 16S rDNA gene showed 100% identity of the tested strain with the B. mojavensis in the database (GenBank KY694978.1). This strain produced a good active extracellular protease which is purified by partial purification with fractionation with ammonium sulfate (40-80% saturation) followed by molecular exclusion chromatography which revealed the presence of a single active peak of 31 kDa. Monomeric band of 29 kDa was revealed by SDS-PAGE. The optimal activity was observed at pH 6,0, 70°C and 0,05 M of CaCl2. The clotting enzyme proved to be a metalloprotease by EDTA inhibition. Furthermore, according to the ratio MCA /PA (Milk clotting enzyme/proteolitic activity) of the order of 164 and its distinctive properties, P47M protease might be a useful contribution to the cheese industry.

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Phytochemical screening and in vitro antioxidant activity of Parkia timoriana (DC.) Merr.

Papitha Ruthiran and Chinnadurai Immanuel Selvaraj

Parkia timoriana is a traditional medicinal plant. The aim of this study was to estimate the antioxidant potential of P.timoriana and also to analyse its phytochemical composition by qualitative and quantitative methods. The results of FT-IR and GC-MS analysis showed that the phytochemicals present in the extract are rich in carboxyl (C=O) and alcohol (O-H) functional groups. Qualitative phytochemical screening showed that the plant is rich in tannin and phenol content with minor portion of flavonoid content. Phenol, tannin and flavonoid content in the extract were quantified by comparison with known standards. The antioxidant potential analysis was studied using DPPH and FRAP method indicating that methanol extract of P.timoriana contains highest antioxidant potential. In this study we conclude that P.timoriana contains significant antioxidant activity and contains valuable phytochemicals with anticipated bioactivity.

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Phytochemical analysis of Quercus infectoria galls extracts using FTIR, LC-MS and MS/MS analysis

Amira Raudhah Abdullah, Hermizi Hapidin and Hasmah Abdullah

The present study was carried out to characterise and compare types of phytoconstituents present in crude and aqueous extract of Quercus infectoria (QI) using FTIR, LC-MS followed by confirming using MS/MS analysis. QI gall was purchased from local market and grinded to obtain the crude in powdered form. The aqueous extract produced by refluxing in water bath at 50°C for 24 hours was filtered and concentrated using rotary evaporator followed by freeze-drying. The crude and aqueous extract was scanned at wavelength 400 to 3400 nm using Tensor-II-FTIR spectrometer. LC-MS analysis utilised Agilent-1290-Infinity-system coupled to Agilent-6520-Accurate-Mass-Q-TOF mass-spectrometer with dual ESI. Detected constituents matched with METLIN_AM_PCDL-N-130328.cdb database. Compounds with >95 DB score were selected to be confirmed with MS/MS analysis. The FTIR spectrum confirmed presence of several similar functional groups in both crude and aqueous extract. LC-MS screening results indicated similar phytoconstituents present in both crude and aqueous QI extract including 1,2,3,6-Tetrakis-O-galloyl-ß-D-glucose (6.86% and 4.36%), 1,2,3,4,6-Pentakis-O-galloyl-ß-D-glucose (8.11% and 3.36%), gallic acid (1.61% and 2.62%), digallate (1.8% and 2.38%) and gallotannin (1-O,2-O,6-O-Digalloyl-β-D-glucose) (2.64% and 2.16%) respectively. MS/MS analysis confirmed presence of all targeted constituents in aqueous QI extract. In conclusion, LC-MS and MS/MS results show that all major constituents analysed were from polyphenolic derivatives primarily gallic acid and hydrolysable tannins.

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Metabolic Engineering for Tocotrienol Accumulation in Perilla [Perilla frutescens (L.) Britton]

Tariq Hossain H.M.M., Lee Young-Sang, Lee Joo-Young and Kim Yong-Ho

Tocotrienols are the major form of Vitamin E in the seed of most monocot plants. They have been reported to exert a protective effect against cancer, diabetes and cardiovascular and neurological diseases. And it has been known that homogentisate geranylgeranyl transferase (HGGT) is a key enzyme for tocotrienol biosynthesis. Perilla (Perilla frutescens L. Britton) which has been cultivated as a fresh leafy vegetable as well as an oil crop in Asian countries, produces tocopherols, while tocotrienols do not accumulate in leaf tissues. To develop a transgenic perilla plant producing tocotrienol in leaf, the OsHGGT gene cloned from rice was introduced to a wild type perilla through Agrobacterium-mediated transformation. The T-DNA integration in plants was verified by PCR and the results of northern hybridization analysis showed there is the constitutive expression of OsHGGT gene in transgenic perilla. Also, α-tocotrienol identified by GC-Mass spectrometry was accumulated in leaves of transgenic Perilla, whereas the content of tocopherols was not reduced. α-Tocopherol was the predominant tocochromanol in both transformants and wild type plant. The average content of α-tocotrienol was 4.7 μg/g DW (ranges from 2.5 to 8.6 μg/g DW) in leaf tissues of transgenic plants. These results indicate that introducing the OsHGGT gene was useful for the accumulation of tocotrienols in leaf of perilla. The transgenic perillia with enhanced tocotrienol may provide a significant nutritional benefit to human health.

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Quantitative and qualitative evaluation of three commercial probiotic products from India

Chavali Kavyasudha, Mohandass Ramya and Madasamy Parani

Studies from other countries have shown that many commercial probiotics did not contain the organisms that were listed in the label or they contained species that were not listed – some of them were even pathogenic. There are no regulatory guidelines governing the production and sale of probiotics in India and thus it is very important to bring the regulatory guidelines. Therefore, a pilot study was conducted to evaluate the microbial contents of the probiotics in terms of quality and quantity. Commercial probiotics were purchased from local drug stores. As per the label, all the three probiotics should contain Clostridium butyricum, Streptococcus faecalis and Bacillus mesentricus. In addition, BIFILAC® and ViBact® should contain Lactobacillus sporogenes while Pre-Pro® should contain Lactobacillus acidophilus. In analysis, S. faecalis and C. butyricum were present in all the three probiotics but in case of Pre-Pro®, CFU/sachet was several fold less than what was claimed in the label. Both BIFILAC® and ViBact® contained L. sporogenes in expected quantity but according to latest nomenclature it should be renamed as B. coagulans. Bacillus mesentricus was absent in all the three products and L. acidophilus was absent in Pre-Pro®. All the three products contained B. subtilis which was not claimed in the label. Fortunately, we did not find any pathogenic bacteria in any of the probiotic products analyzed. Our study clearly demonstrates mislabeling of the organisms, absence of claimed organisms, presence of unclaimed organisms and significantly less number of certain claimed organisms in the probiotics tested.

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Antimycobacterial activity of potential plant metabolites with emphasis on management of drug resistant Mycobacterium tuberculosis strains

Rajiniraja Muniyan and Jayaraman Gurunathan

Tuberculosis (TB) is one of the major health problems worldwide. Extensive efforts are in progress to eradicate TB using new drugs with new modes of action and with lesser side effects. The major challenge in this effort is the management of multi-drug resistant Mycobacterium tuberculosis strains. Nature has provided wider scope for the discovery of new lead molecules. Therefore, the purpose of this review is to underline the importance of plant derived drugs and to consolidate the literature pertaining to phytochemicals that have been proven to have antimycobacterial property. Owing to the vast literature, the inclusive criteria for selecting the phytochemicals for reviewing are the following: (a) the minimum inhibitory concentration (MIC) to be below 50 µg/ml and (b) capacity to manage the multi-drug resistant (MDR) and extensive drug resistant (XDR) strains. Though most of the phytochemicals discussed here have potential antimycobacterial property, the most promising candidates could be licarin A, dihydroguaiaretic acid, ursolic acid, oleanolic acid and plumbagin since these compounds were reported to inhibit wide variety of resistant strains as well as broad biological activity. We opine that these compounds or their respective analogs may eventually develop into effective drug candidate(s) against drug-resistant M. tuberculosis strains.

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