Ashok Kumar Mohanty

Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

Background Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt. Methods and Findings An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus. Conclusion The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.

Bovine chymosin: Production by rDNA technology and application in cheese manufacture

Bovine chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preprochymosin and secreted as prochymosin which is autocatalytically activated to chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106. The gene encoding chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture.

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis).

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line.

Background The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. Methodology Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. Principal Findings The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. Conclusions We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ~60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).

In vitro phagocytic activity of milk neutrophils during lactation cycle in Murrah buffaloes of different parity

Milk samples were collected from 34 lactating Murrah buffaloes on days 0, 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270 and day 300 after calving. Milk somatic cell counts (SCC) were highest in multiparous buffaloes. Milk SCC were significantly lower in buffaloes of third and fourth parity during early lactation and than increased significantly (p < 0.01) by the end of lactation. Milk neutrophils were significantly lower in all the buffaloes during early lactation, but increased significantly (p < 0.01) afterwards. Milk lymphocytes were significantly higher during early lactation, but decreased significantly (p < 0.01) by the end of lactation. Phagocytic activity (PA) was highest in day 1 colostrum and then decreased significantly (p < 0.01) by the fourth milking in buffaloes of second, third and fourth parity. Phagocytic index (PI) was also highest in colostrums of primiparous buffaloes. Irrespective of parity, maximum PA and PI was observed during mid lactation. In terms of in vitro phagocytic activity, early lactation is the most critical period followed by late and mid lactation.

Effect of supplementation of vitamin E, copper and zinc on the in vitro phagocytic activity and lymphocyte proliferation index of peripartum Sahiwal (Bos indicus) cows

To study the effect of vitamin E (VE), copper (Cu) and zinc (Zn) supplementation on the in vitro phagocytic activity (PA) and lymphocyte proliferation response (LPR) of blood neutrophils and lymphocytes, thirty Sahiwal pregnant cows (six in each group) in their late gestation at 30 days before the expected date of calving were selected from the NDRI experimental herd and supplemented with various micronutrients from 30 days before calving to 45 days after calving. Cows were supplemented individually with VE (1000 IU/cow/day), Cu (20 ppm/cow/day) and Zn (80 ppm/cow/day) and also with a combination of VE, Cu and Zn to study cumulative effect of all micronutrients. One group without any supplementation acted as a control. Blood neutrophils and lymphocytes were isolated and studied for their PA and LPR. Supplementation of micronutrients like VE, Cu, Zn and a combination of all these nutrients significantly (p < 0.01) increased the PA of experimental cows as compared to control (unsupplemented) cows during the pre-partum period. During post-partum, all the micronutrients (VE, Cu, Zn and their combination) showed a significant (p < 0.01) increase in the PA of experimental cows as compared to control cows. Of all the groups, significant (p < 0.01) and maximum PA was observed in the combination group followed by Zn-supplemented group during both the pre- and post-partum period. A significant (p < 0.01) increase in LPR of B lymphocytes was observed in combination-supplemented group during the pre-partum period and during both the pre- and post-partum period in the Cu-supplemented group.

Early Pregnancy Diagnosis in Bovines: Current Status and Future Directions

An early and accurate diagnosis of reproductive dysfunctions or aberrations is crucial to better reproductive management in livestock. High reproductive efficiency is a prerequisite for high life-time production in dairy animals. Early pregnancy diagnosis is key to shorten the calving interval through early identification of open animals and their timely treatment and rebreeding so as to maintain a postpartum barren interval close to 60 days. A buffalo, the most important dairy animal in the Indian subcontinent, is known for problems related to high calving interval, late puberty, and high incidence of anestrus. Lack of reliable cow-side early pregnancy diagnosis methods further aggravates the situation. Several methods of pregnancy diagnosis are being practiced in bovine species, yet none qualifies as the ideal pregnancy diagnosis method due to the inherent limitations of sensitivity, accuracy, specificity, speed, and ease of performing the test. The advancement of molecular techniques like proteomics and their applications in animal research has given a new hope to look for pregnancy biomarker molecules in these animals. This review attempts to examine common pregnancy diagnosis methods available for dairy animals, while assessing the usefulness of the modern technologies in detecting novel pregnancy markers and designing future strategies for research in this area.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Genome-wide analysis of the heat stress response in Zebu (Sahiwal) cattle.

Environmental-induced hyperthermia compromises animal production with drastic economic consequences to global animal agriculture and jeopardizes animal welfare. Heat stress is a major stressor that occurs as a result of an imbalance between heat production within the body and its dissipation and it affects animals at cellular, molecular and ecological levels. The molecular mechanism underlying the physiology of heat stress in the cattle remains undefined. The present study sought to evaluate mRNA expression profiles in the cattle blood in response to heat stress. In this study we report the genes that were differentially expressed in response to heat stress using global scale genome expression technology (Microarray). Four Sahiwal heifers were exposed to 42°C with 90% humidity for 4h followed by normothermia. Gene expression changes include activation of heat shock transcription factor 1 (HSF1), increased expression of heat shock proteins (HSP) and decreased expression and synthesis of other proteins, immune system activation via extracellular secretion of HSP. A cDNA microarray analysis found 140 transcripts to be up-regulated and 77 down-regulated in the cattle blood after heat treatment (P<0.05). But still a comprehensive explanation for the direction of fold change and the specific genes involved in response to acute heat stress still remains to be explored. These findings may provide insights into the underlying mechanism of physiology of heat stress in cattle. Understanding the biology and mechanisms of heat stress is critical to developing approaches to ameliorate current production issues for improving animal performance and agriculture economics.