Research Journal of Biotechnology

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Vol. 14(11) November 2019

Keratin-wastes valorization for production of hydrolysates with high antioxidant potential

Sharma Shikha, Singh Satbir, Gupta Sahil and Bajaj Bijender Kumar

Page No: 1-8

Abstract: Keratin rich wastes produced from diverse sources could be easily valorized by the application of robust microbial proteases. Bacillus licheniformis K-3 (BLK-3) was previously characterized for its enormous hydrolytic potential and current study presents the production of a thermostable and pH tolerant keratinase from Bacillus licheniformis K-3 by optimization of process variables using response surface methodology. Optimal combination of four cultural variables (% w/v) viz. wool 0.5, molasses 2.0, mustard cake 0.5 and phosphates 0.5 resulted in 1.96-fold increment in keratinase production. BLK-3 keratinase successfully hydrolyzed both α and β-keratin wastes and the hydrolysate demonstrated excellent antioxidant potential.

Out of hydrolysates generated by catalytic action of keratinase on different keratinous wastes i.e. wool, hooves and feather, the one produced from wool exhibited the highest antioxidant potential. The potential capability of Bacillus licheniformis K-3 for possessing tremendous hydrolytic potential coupled with and its process-apt secretome could serve a boon for different industrial production processes. Thus, a newly isolated Bacillus licheniformis K-3 exhibits excellent keratinolytic activity and the hydrolysate generated from various keratin wastes has high antioxidant potential.

The genetic variability of two Egyptian chicken breeds based on mitochondrial DNA D-loop region patterns

Yacoub Haitham A. and Badwy Moataz I.

Page No: 9-14

Abstract:We analyzed the genetic variability of Egyptian chicken breeds through mitochondrial D-loop region patterns. The current results found that eleven nucleotide sequences (ACTACGGGAAC) were observed only in all individuals of both of Egyptian chicken breed at the beginning of d-loop region of mitochondrial DNA. The results showed that the D-loop region of mitochondrial DNA has reliable polymorphisms as single nucleotide polymorphisms (SNPs) in the two Egyptian traditional chicken breeds. The phylogenetic tree revealed that both Fayoumi and Dandarawi shared same cluster and they are close to domesticated chicken species such as Gallus gallus spadiceus and Gallus gallus bankiva respectively with tiny distance in case of the wild type of Gallus lafayetii.

TA total number of eleven haplotypes observed in both of Egyptian chicken breed and nine polymorphic site were found in Egyptian chicken. The estimated haplotype diversity was 1.00±0.063 (Fayoumi breed) and 0.857±0.10 (Dandarawi breed) whereas the nucleotide diversity was 0.00398±0.0017 for Fayoumi and 0.00313±0.0015 in case of Dandarawi.

High-Frequency direct regeneration of Woodfordia fruticosa (Linn.)Kurz, A valuable ayurvedic herb

Grover Neha, Mishra Preeti and Patni Vidya

Page No: 15-21

Abstract:The present study deals with high frequency micropropagation studies of an ayurvedic medicinal plant, Woodfordia fruticosa (Linn.) Kurz. Dhatki also known as the Fire Flame bush due to its beautiful red flowers. It has danger to be rare in near future due to exploitation for ayurvedic medicines. Shoot tip explants were inoculated on solid MS medium supplemented with different concentrations of cytokinins like BAP and Kinetin and auxins alone and in combinations. Synergistic effects of cytokinins and auxins were studied on the direct regeneration through shoot tip.

Most significant results were achieved in MS medium fortified with BAP (2.0 µM) with 52 shoots per explant. ½ MS medium supplemented with IBA (5.0µM) and 3% sucrose resulted in maximum number of roots. The complete plantlets were hardened and transferred to the green house after two weeks with survival rate of 70%.

Value addition of cottonseed meal for feed industries by degossypolization employing papain and solvent

Kumar Manoj, Mageshwaran V., Saxena Sujata, D’ Souza Charlene, Ashtaputre Nandita, Patil Sharmila, Mahapatra Archana, Patil P.G. and Tak Yamini

Page No: 22-29

Abstract: The current study was carried out to improve the utilization of cottonseed meal (CSM) as a feed to monogastric animals. Papain enzyme was considered as one of the green channels for removing the gossypol from the CSM. The cottonseed meal was evaluated for the free and total gossypol for assessing the effect of enzyme for its improvement. A Box-Behnken design (BBD) with three-level, three-factor, under response surface methodology (RSM) was performed to standardize different parameters such as concentrations of papain (0.5–1.5%), extraction time (60–180 min) and extraction temperatures (40–60 ºC). From the response surface model, a polynomial equation was developed and was provided a set of optimal conditions for getting the minimum level of free and total gossypol in CSM.

Following are the predicted optimal conditions from the model: enzyme concentration = 1.48%, temperature = 46.38°C and extraction time = 178.2 min. Under the optimal conditions, CSM obtained through papain treatment had free gossypol content (FGC) (0.514 ± 0.020) and total gossypol content (TGC) (0.977 ± 0.032). Results indicate the suitability of papain for removing the gossypol from the CSM for improving its utilization to broader extent.

Evolutionary variations in parrotfishes via molecular markers

Saad Yasser M., Shaikh-Omar Abdulkader M. and Gharbawi Waleed Y.

Page No: 30-38

Abstract:Developing informative DNA markers facilitate understanding of the evolution of parrotfish biological resources. Therefore, we present a comparative analysis among a group of tmo-4C4 gene fragment sequences identified in some parrotfish species (Scarus russelii, S. ghobban, S. ferrugineus, S. fuscopurpureus, S. psittacus and Chlorurus sordidus) and other tmo-4C4 gene sequences (obtained from NCBI). Nucleotide composition, sequence conservation, single nucleotide polymorphism and nucleotide diversity were calculated in all estimated DNA sequences. The evaluated fishes were clustered into two groups (Scaridae and Labridae). The GC and GC3 contents in Scaridae were higher than in Labridae. Averages of genetic distances were 0.046 and 0.095 within Scaridae and Labridae respectively. Analysis of nucleotide variations was used to reconstruct phylogenetic relations among evaluated fish species.

Maximum Likelihood and Neighbor-Joining methods on the resulting DNA sequences produced parallel topologies that confirm the monophyly of each evaluated fish family. S. psittacus is distantly related from both S. russelii and C. sordidus. The low distance was observed between S. psittacus and S. fuscopurpureus. The tmo-4C4 gene system provides a reliable solution for understanding parrotfish species biodiversity and evolution. This study provides a guide for future investigations within these aquatic biological resources in the Red Sea.

Optimization of bacterial biomass production for bioaugmentation of the hydrocarbons contaminated sites

Olga Muter, Galina Makarenkova, Dagnija Vecstaudza, Karlis Shvirksts, Mara Grube and Uldis Kalnenieks

Page No: 39-51

Abstract:Bacteria with hydrocarbon (HC)-degrading activity coexist and dominate in oil-polluted areas in consortia. Investigation of cooperation between different microbial species is vital for assessing the potential efficiency of various environmental biotechnologies. In this study, the metabolic activity of eight phenotypically distinct isolates with HC-degrading activity was tested. Pairwise comparison of the 16S rRNA gene sequences of the soil isolates revealed that 5 of these showed high similarity with Stenotrophomonas and 3 with Pseudomonas genus. A hierarchical cluster analysis based on the Fourier Transform Infrared Spectroscopy, has demonstrated the grouping of one Pseudomonas and two Stenotrophomonas isolates within one cluster while the rest (5 isolates) formed another cluster. The response of bacteria to 1% diesel oil addition against background of molasses concentrations ranging from 0.05 % to 3.00 % has revealed that molasses concentrations below 1 % are likely to be more efficient for microbial activity when grown with diesel oil.

Comparative testing of bacterial activity in a Bushnell Haas broth supplemented with six nitrogen sources showed ammonium salts as a preferential nitrogen source for HC degradation. That was proved using 2,6-dichlorophenol indophenol (DCPIP) indicator. Serial batch experiments with a stepwise increase of diesel oil concentration from 1% to 3% and up to 5% showed that step 2 with 3 % diesel oil represented the highest biomass concentration and the highest phenotypical diversity of colonies among the tested variants. These results provide additional tools towards optimization of bacterial biomass production for its use in bioaugmentation of the HC-contaminated sites.

Biotyping and Phylogenetic analysis of bacterial isolates prevalent in a local hospital environment

Padhan Monalisa and Pattnaik Smaranika

Page No: 52-61

Abstract:This study aims at isolation and identification of bacterial strains residing in the ambulatory environment of a local hospital (VSSIMSAR, Burla, Odisha). The side wall of female medicine ward was selected as the investigation site and bacterial strains were isolated using the Medical Microbiological diagnostic tools. The bacterial colonies developed on agar plates were observed as various morpho types. The results obtained from biotyping experiments added the differential biochemical characteristic features. In addition, the 16s r DNA phylogenetic analysis had inferred about prevalence of members of Enterobacteriaceae family, Pseudomonadaceae family and Corynebacteriaceae family.

Further variation in ‘Antibiogram’ pattern was observed. The possible reason behind this phenomenon could be due to occurrence of ‘non-specific horizontal gene transfer’ among the bacterial strains inhabiting the supposed environment.

Drought and UV-B radiation effect on growth and antioxidant parameters in Triticum aestivum L.

Amal Algarawi, Ibrahim A. Arif and Mona S. AlWhibi

Page No: 62-70

Abstract: Crop plants are affected by different environmental changes which naturally affect their growth. In this study, physiological responses from the exposure of wheat plant were assessed as one of the most important food crops in the world for drought and exposure to UVB and their effect together. The response of plants to different pressures was different from individual stresses. In this study, nerves affected by drought stress and / or UV-B were treated for 4 days of treatment. They were evaluated in terms of roots, dry and dry weights, root height, height and leaf area, relative water content and electrolyte conductivity.

In general, double-stress (UV-B + D) appears to be less effective than any of the individual treatments and the stress effect of the drought is stronger than UV-B radiation. We also observed a strong increase in oxidative stress that indicates hydrogen peroxide (H2O2) and Malondialdehyde (MDA) in drought or UVB than cumulative stress in addition to antioxidant enzyme activities such as superoxide dismutase SOD and increased glutathione concentration more in wheat plants that have been subjected to cumulative pressures of UVB and drought more than individual pressures. According to our findings, it can be concluded that joint dehydration and UV-B provide resistance to more than one stress.

Proteomic insight into the Effect of Pigment Production by Fusarium Chlamydosporum: A SWATH based Quantitative Proteomics

Tessy Anu Thomas, Suguna S.R. and Sharmila Tirumale

Page No: 71-78

Abstract: Since many industrial microbiological products result from secondary metabolism, researchers have sought to explain the role of secondary metabolites in the survival of the organism. Fusarium chlamydosporum normally a saprophyte is known to produce a red pigment which still remains unidentified. The mechanism and functional pathway for production of secondary metabolites can be known by profiling the fungal proteome at the time of pigment production i.e. during the late idiophase or early tropophase period of the growth curve. Proteomics will contribute to our understanding of the gene function and metabolic pathways of complex organisms.

In this study, a relatively new approach of quantitative proteome profiling was performed. After successful extraction of the proteins from the protoplast (cell wall of the fungus was removed with suitable solvents in order to reduce the complexity of proteins), protein extracts were identified and sequenced by Liquid Chromatography coupled with Mass Spectrometry and Triple-Time of Flight (LC-MS/MS-Triple TOF). Homologous fungal sequences were derived by searching the available data bases with the help of Sequential Window Acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Our analysis identified a total of 1228 proteins. A stringent analysis led to the identification of 139 proteins and 775 distinct peptides with 99% confidence. Out of the identified 139 proteins, 45 proteins were found to be significantly regulated wherein 24 were up regulated and 21 were down regulated.

In vitro production of anacardic acid from suspension cultures of Anacardium occidentale L.

Sija S.L.

Page No: 79-87

Abstract: Cashew (Anacardium occidentale L.) is an economically and medicinally important plantation crop which is the main source of the major phenolic compounds such as anacardic acid, cardol and cardanol. Plant tissue culture has been used as an alternative method for enhancing the production of secondary metabolites. This study reports in vitro production of anacardic acid from A. occidentale. In vitro callus cultures of Anacardium were initiated from cotyledon explant on Murashige and Skoog’s (MS) medium supplemented with various concentrations of plant growth hormones. Maximum callus induction and proliferation was noted at 25 mg/l 2,4-D + 25 mg/l BAP.

Suspension cultures were established by transferring friable callus to the above-mentioned medium composition without solidifying agents. Cultures were continuously agitated on a rotary shaker (110 rpm) under dark condition for anacardic acid production under in vitro condition until the end of culture cycle (10 days). Quantification of anacardic acid in cell suspension culture was done using High Performance Thin Layer Chromatography (HPTLC). The maximum production of anacardic acid (234.8 µg/g dry weight) was observed on 8th day of cell suspension culture of A.occidentale.

Isolation and characterization of extracellular β-D-fructan fructanohydrolase producing Trichoderma asperellum RSBR08

G. Babul Reddy, Sukumar Karuganti, Muralitharan G., Swarnabala G., N. Venkata Raju and Anjani Devi Chintagunta

Page No: 88-93

Abstract:From the past few decades, several drawbacks of sucrose usage have been noticed because of which fructose and fructo-oligosaccharides have drawn much focus. Inulin contains a linear polymer of fructose units which is abundant in plant tubers and roots. Inulinases possess an industrial applicability in the production of ultra-high fructose syrups, fructo-oligosaccharides and inulo-oligosaccharides. In the current research, potent β-D-fructan fructanohydrolase producing fungal strain was isolated from agricultural lands of Bowrempet village, Telangana. High inulinase producing RSBR08 was identified as Trichoderma asperellum by phenotypic and molecular identification.

DNA sequence obtained was deposited in NCBI, Genbank and accession number was assigned as MH782167. Harvesting time optimization studies were carried out and high yield of inulinase was observed at 96 h (48.5 U/ml) followed by 120 h (41.2 U/ml) and 72 h (36.1 U/ml). Hydrolysis of fructose units and exo-inulinase production was assessed by thin layer chromatography which showed high fructose units liberation at 96 h. Molecular weight of the exo-inulinase was tested by using SDS–PAGE which showed 75 kD with standard molecular weight markers. This research study reveals the effective utilization of exo-inulinase producing Trichoderma asperellum RSBR08 in the industrial applications.

Studies on Effective Production of Pullulan from corn cob by Aureobasidium pullulans and Rheological Behavior of Pullulan Fermentation Broth

Farjana Begum A., Harivardhinie R., Kalaiezhil P., Nithya R. and Ashok Kumar A.

Page No: 94-100

Abstract:Pullulan is a well-known extra cellular polysaccharide produced by yeast-like fungus Aureobasidium pullulans (A. pullulans). Pullulan has a wide range of commercial and industrial applications due to its material properties. Considering the economics of pullulan production, efforts have been put forward to enhance the production from agro waste. In this research, a new fermentation strategy to obtain pullulan using a sustainable raw material as the single carbon source was analyzed. Corn cob dilute-acid hydrolysates were obtained and used as a carbon source for a fermentative medium formulation in order to achieve biopolymer biosynthesis. The dilute-acid hydrolysate with 36.82 g/L total sugars content assured the fermentative conditions for microbial growth and pullulan bioproduction. The Pullulan structure was analyzed by FTIR analysis. The yield of Pullulan produced after the 120 hr in liquid medium based on corn cob hydrolysate was 34.1% w/w which is comparable to the results reported in the literature.

The rheological properties of the pullulan fermentation broth based on Corn cob hydrolysates were also studied. The relationship between shear rate/shear stress and shear rate/apparent viscosity showed a Non-Newtonian behaviour of the fermentation broth. Our findings offer valuable perspectives regarding the use of corn cob agro-waste resources for the formulation of a fermentation medium for pullulan production by an economical biotechnological approach.

Multiple shoot formation and plant regeneration of a commercially useful tropical plant Citrus aurantifolia (Swingle)

Bhalerao S.R. and Kumre K.R.

Page No: 101-107

Abstract:Citrus is one of the important fruit crops of the world. It occupies a place of prime importance among the major fruit crops of India, ranking third after mango and banana. A rapid protocol is developed for in vitro regeneration of seedless lime (Citrus aurantifolia Swingle) using nodal segment as explant.

The efficacy of different hormones for shoot proliferation on nodal segment was investigated on Murashige and Skoog (MS) medium. BAP individually 1mg/l (4.4±1.74) and in interaction with kinetin (0.25mg/lBAP+0.5mg/l kinetin) showed the maximum sprouting (3.1±0.44) of buds which was on par with the other treatments. The maximum multiple shooting (6.0±2.55) was observed on MS media containing 1mg/l BAP+0.5mg/l kinetin. In vitro raised shoots were cultured on root induction medium. Rooting was initiated after 30 days of shoot inoculation. Maximum rooting (46.66%) was obtained on MS medium supplemented with 2.5mg/l IAA. The plantlets with well-formed rooting system were successfully hardened and acclimatized.

Sorbitol and mannitol effect on chrysanthemum growth in vitro

Malyarovskaya Valentina, Samarina Lidiia, Choudhary Ravish, Koninskaya Natalia, Rakhmangulov Ruslan, Gvasaliya Maya, Platonova Natalia, Klemeshova Kristina and Trautvein Kristina

Page No: 108-113

Abstract:Sorbitol and mannitol are polyols which can be useful for germplasm conservation of Chrysanthemum cultivars in vitro. The aim of this study was to evaluate the effect of sorbitol and mannitol on the morphological and physiological parameters of the three commercial cultivars of Chrysanthemum x morifolium in vitro. A decrease in the shoot length, plant biomass, relative water content and increase of proline were observed as effect of sorbitol and mannitol. The best plant performance with no plant necrosis was observed with Variant III (20 g l-1 sucrose + 50 g l-1 sorbitol).

On the other hand, the greatest negative effect on the growth and development of chrysanthemum plants was observed in variant IV (80 g l-1mannitol + 20 g l-1 sucrose) where two chrysanthemum cultivars did not survive. In all variants, the plant biomass was inversely related to the proline content and is directly related to the relative water content. Our study shown that mannitol and sorbitol can be an alternative source of carbohydrates like sucrose in a nutrient medium to support the growth of chrysanthemum at least for 6 months. The results will be useful for slow growth conservation of chrysanthemum germplasm in vitro.

Antimicrobial Activity of Phaseolus vulgaris L. Cultivar Baspa Trypsin Inhibitor Protein

Thakur Yamini, Sharma Ankita, Parmar Subhash Chand, Sharma Satish K. and Nath Amarjit Kaur

Page No: 114-126

Abstract:Antimicrobial activity of partially purified trypsin inhibitor from seed flour of bean (Phaseolus vulgaris L.) cultivar Baspa was determined. Maximum per cent (88.12 %) inhibition of growth was against fungal strain Cercospora punicae in presence of 300 µg ml -1 inhibitor protein. Inhibition in growth was 84.20 %, 81.00 % and 79.41 % against Fusarium oxysporum, Colletotricum gloeosporioides and Alternaria solani, respectively. The inhibitor protein was highly effective against bacterial strain Xanthomonas campestris and less effective against Ralstonia solanacearum.

The zone of growth inhibition was 8, 10, 11, 16 and 21 mm in Xanthomonas campestris , 6, 8, 12, 13 and 16 mm in Agrobacterium tumefaciens and 4, 8, 16, 17 and 19mm in Ralstonia solanacearum in presence of 26, 52, 104, 208 and 416 µg/ml of inhibitor protein loaded into the well. No bacterial growth was observed when clear zone was streaked in control media indicating death of bacterial cells. The studies indicated partially purified inhibitor protein to be effective against plant pathogenic bacterial and fungal strains with varying efficiencies. Inhibitor proteins produced from natural products emerge as potential antimicrobial agents. It can be applied in agricultural sector for development of transgenic resistant to plant diseases.

Microstructure of Fermented Catfish (Clarias sp.) Sausages infected by Listeria monocytogenes

Happy Nursyam and Asep Awaludin Prihanto

Page No: 127-129

Abstract:The effect of several lactic acid bacteria was studied on the fermented African Catfish (Clarias sp) sausages infected by Listeria monocytogenes. Pediococcus acidilactici and Lactobacillus casei were used as starter of fermented sausage. The aim of this study was to investigate the microstructure of the infected fermented sausage. It was noted that myosin filaments which existed in all sausage were affected by the breakdown of tissue cells as a result of chopping the fish flesh. The formation of elongated fibrils indigenous sausage was presumably caused by lactic acid bacteria which was lower than fermented sausage using lactic acid bacteria starter.

The structure of Pediococcus acidilactici sausage starter has cavities looks and there are granules around it. Meanwhile, Lactobacillus casei starter was dominated by wider and compact swollen myofibril. Pediococcus acidilactici-fermented sausage had rougher swollen myofibril than that of combination of Pediococcus acidilactici and Lactobacillus casei starter-fermented sausage. In conclusion, the combination of Pediococcus acidilactici and Lactobacillus casei gave a compact microstructure in fermented catfish sausage.

Applications of Lipases

Joshi Swapnil S. and Jobanputra Arpana H.

Page No: 130-138

Abstract:Lipases are water soluble enzymes that catalyze the hydrolysis of triacylglycerols to liberate glycerol and free fatty acids. Many organisms like fungi, bacteria, yeast, animals etc. produce lipases; but lipases produced by bacteria and fungi are of commercial interest because of ease of their production, recovery and cheaper production costs as compared to other sources. Lipases along with other enzymes have wide range of applications because of their versatility like tolerance to wide pH and temperature range.

Other enzymes dominated the industry but in the last two decades industrial interest has shifted towards lipase production. Today many bacterial and fungal species like Bacillus, Pseudomonas and Aspergillius are exploited on industrial scale for the lipase production. Today lipases are used in industries such as food, cosmetics, detergent, pharmaceutical, leather etc.