Vinod K. Kansal

Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme.

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

Therapeutic Effect of Probiotic Dahi on Plasma, Aortic, and Hepatic Lipid Profile of Hypercholesterolemic Rats

This study examined the effects of probiotic dahi prepared by Lactobacillus plantarum Lp9 and dahi culture in buffalo milk on lowering cholesterol in rats fed a hypercholesterolemic basal diet. Male Wistar rats were divided into 3 groups and fed with probiotic dahi, dahi, or buffalo milk for 120 days. Following the consumption of supplements (probiotic dahi, dahi or buffalo milk), the animals were fed a basal hypercholesterolemic diet. Plasma total cholesterol and triglycerides (TAGs) were decreased by 35% and 72% in rats fed with probiotic dahi group, while cholesterol levels increased by 70% and TAGs increased by 97% in buffalo milk and 59% in dahi fed groups. Supplementation of probiotic dahi further lowered plasma low-density lipoprotein (LDL) + very-low-density lipoprotein (VLDL)- cholesterol by 59%, while it elevated plasma high-density lipoprotein (HDL)-cholesterol by 116%. As a result, atherogenic index, the ratio of HDL to LDL + VLDL was markedly improved. Deposition of cholesterol and TAGs in liver and aorta were significantly reduced in rats fed with probiotic dahi. These observations suggest that probiotic dahi may have therapeutic potential to decrease plasma, hepatic and aortic lipid profile, and attenuate diet-induced hypercholesterolemia.

Age-related decline in macrophage and lymphocyte functions in mice and its alleviation by treatment with probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum

This study evaluated the effects of probiotic Dahi administration in ageing mice on macrophage and lymphocyte functions. Probiotic Dahi were prepared by co-culturing in buffalo milk (3% fat) Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lc. lactis ssp. lactis biovar diacetylactis NCDC-60) along with Lactobacillus acidophilus LaVK2 (La-Dahi) or combined Lb. acidophilus and Bifidobacterium bifidum BbVK3 (LaBb-Dahi). Four groups of 12 mo old mice were fed for four months, with the supplements (5 g/day) of buffalo milk (3% fat), Dahi, La-Dahi and LaBb-Dahi, respectively, in addition to basal diet, and a fifth group that received no supplements served as control. The immune functions of young mice (4 mo old) were also compared with those of ageing adult mice (16 mo old). The production of nitric oxide and cytokines IL-6 and TNF-α declined and that of immunosuppressive prostaglandin E2 (PGE2) increased by stimulated peritoneal and splenic macrophages in ageing mice, compared with their young counterparts. The proliferation of stimulated splenocytes diminished and the production of IL-2 decreased and that of IL-6 and TNF-α enhanced in ageing compared with young mice. Feeding ageing mice with La-Dahi or LaBb-Dahi improved peritoneal macrophage functions stimulating nitric oxide and IL-6 and diminishing PGE2 production. Feeding La-Dahi or LaBb-Dahi also improved lymphocyte functions stimulating their proliferation and production of IL-2 in ageing mice. To conclude, the probiotic La-Dahi and LaBb-Dahi are effective in reversing age related decline in immune functions in mice.

Heterogeneity of cationic amino acid transport systems in mouse mammary gland and their regulation by lactogenic hormones.

The mechanism of cationic amino acid transport in lactating mouse mammary gland was investigated. Two Na(+)-independent systems of arginine transport were discriminated on the basis of their sensitivity to leucine. The leucine-sensitive uptake of arginine (Km 0.4 mM) was through a broad specificity system that interacted with both cationic and neutral amino acids, and was inhibited by preloading mammary tissue with neutral amino acids. The leucine-insensitive uptake was identified as the y+ system (Km 0.76 mM). Preloading mammary tissue with cationic amino acids increased the uptake of arginine by the y+ system. Decreasing the pH of the external medium to 6.0 suppressed the y+ system-mediated uptake by approximately 25%, whereas the broad specificity system remained unaffected. Lactogenic hormones upregulated the y+ system-mediated uptake of arginine in pregnant mouse mammary tissue cultured in vitro, although the broad specificity system remained unaffected. The y+ system-mediated uptake increased 2-fold with insulin alone and 4-fold with the combination of insulin, cortisol and prolactin.

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum modulates immunoglobulin levels and cytokines expression in whey proteins sensitised mice.

BACKGROUND Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. This study investigated whether orally administrated probiotics could suppress sensitisation in whey proteins (WP)-induced allergy mouse model. Two types of probiotic Dahi were prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3. Mice were fed with probiotic Dahi (La-Dahi and LaBb-Dahi) from 7 days before sensitisation with WP, respectively, in addition to milk protein-free basal diet, and control group received no supplements. RESULTS Feeding of probiotic Dahi suppressed the elevation of whey proteins-specific IgE and IgG response of WP-sensitised mice. In addition, sIgA levels were significantly (P < 0.001) increased in intestinal fluid collected from mice fed with La-Dahi. Production of T helper (Th)-1 cell-specific cytokines, i.e. interferon-γ (IFN-γ), interleukin (IL)-12, and IL-10 increased, while Th2-specific cytokines, i.e. IL-4 decreased in the supernatant of cultured splenocytes collected from mice fed with probiotic Dahi as compared to the other groups. Moreover, the splenic mRNA levels of IFN-γ, interleukin-10 were found to be significantly increased, while that of IL-4 decreased significantly in La-Dahi groups, as compared to control groups. CONCLUSION Results of the present study indicate that probiotic Dahi skewed Th2-specific immune response towards Th1-specific response and suppressed IgE in serum. Collectively, this study shows the potential use of probiotics intervention in reducing the allergic response to whey proteins in mice.

Effect of thermal processing of cow and buffalo milk on the allergenic response to caseins and whey proteins in mice.

BACKGROUND Heat treatment is the most common method for reducing pathogen load, but it remains controversial in reducing the incidence of hyperimmune reactions. The aim of this study was to compare the allergenicity of caseins (CSN) and whey proteins (WP) of thermally processed cow and buffalo milk in a mouse model. Swiss albino mice were sensitised by intraperitoneal injections (administered in three doses at weekly intervals) of CSN or WP from cow or buffalo milk for the evaluation of humoral response and splenocyte stimulation index. RESULTS After 3 weeks of intraperitoneal stimulation of mice with milk proteins, the sterilised milk protein group displayed significantly lowered (P ≤ 0.05) serum IgG and IgE levels, while considerably increased cow milk protein-specific responses (IgE) were shown by proteins of pasteurised milk compared with those of raw milk. The stimulation index of splenocytes induced by CSN or WP of boiled and sterilised milk was also lower (P ≤ 0.05) than that of raw milk of both cow and buffalo. CONCLUSION The experiment showed that boiling and sterilisation of cow and buffalo milk clearly affect the allergenicity by decreasing the humoral and cell-mediated responses in mice. All results indicated that CSN and WP of sterilised milk are less allergenic than those of raw milk in mice.

Exploring the ameliorative potential of probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum on dextran sodium sulphate induced colitis in mice.

Conventional medical therapies for ulcerative colitis (UC) are still limited due to the adverse side effects like dose-dependent diarrhoea and insufficient potency to keep in remission for long-term periods. So, new alternatives that provide more effective and safe therapies for ulcerative colitis are constantly being sought. In the present study, probiotic LaBb Dahi was selected for investigation of its therapeutic effect on DSS-induced colitis model in mice. LaBb Dahi was prepared by co-culturing Dahi culture of Lactococci along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 in buffalo milk. Four groups of mice (12 each) were fed for 17 d with buffalo milk (normal control), buffalo milk plus DSS (Colitis control), Dahi plus DSS, and LaBb Dahi plus DSS, respectively, with basal diet. The disease activity scores, weight loss, organ weight, colon length, myeloperoxidase (MPO) and β-glucoronidase activity was assessed, and the histopathological picture of the colon of mice was studied. All colitis control mice evidenced significant increase in MPO, β-glucoronidase activity and showed high disease activity scores along with histological damage to colonic tissue. Feeding with LaBb Dahi offered significant reduction in MPO activity, β-glucoronidase activity and improved disease activity scores. We found significant decline in length of colon, organ weight and body weight in colitis induced controls which were improved significantly by feeding LaBb Dahi. The present study suggests that LaBb Dahi can be used as a potential nutraceutical intervention to combat UC related changes and may offer effective adjunctive treatment for management of UC.

Biochemical characterization of buffalo ( Bubalus bubalis ) milk lysozyme

Lysozyme, a low-molecular weight basic protein, is an important component of the antibacterial system in milk. Lysozyme activity is higher in buffalo milk (60±3·9×10 −3 units/ml) than in bovine milk (29·1±1·5×10 −3 units/ml). Buffalo colostrum contains five-times more lysozyme activity than mature milk (Priyadarshini & Kansal, 2002a). Lysozyme activity in buffalo milk is not influenced by the parity of animal or stage of lactation, but it increases during extreme weather (winter and summer). Lysozyme in buffalo milk is more stable than in cow milk during storage and heat treatment. A sharp increase in milk lysozyme has been observed in buffaloes with sub-clinical mastitis (Priyadarshini & Kansal, 2002a).

Characteristics of transport systems of L-alanine in mouse mammary gland and their regulation by lactogenic hormones: evidence for two broad spectrum systems.

The characteristics of the transport systems of L-alanine in lactating mouse mammary gland and their regulation by lactogenic hormones have been studied. L-alanine uptake was mediated by three Na(+)-dependent and one Na(+)-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na(+)-dependent uptake exhibited the usual characteristics of system A. Cl- dependency has been established for system A. The other two Na(+)-dependent systems, which we have named BCl(-)-dependent and BCl(-)-independent, are described for the first time. These are systems with broad specificity and were distinguished on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. The Na(+)-independent route was identified as system L, which operates independent of Cl-. The A, L and BCl(-)-independent transport systems were upregulated in pregnant mouse mammary tissue cultured in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin). Insulin alone also upregulated systems A and L to some extent in pregnant mouse mammary tissue. BCl(-)-dependent activity was not detected in pregnant mouse mammary tissue and was not induced by lactogenic hormones in vitro.

Mechanism of glycine transport in mouse mammary tissue.

The mechanism of glycine transport in lactating mouse mammary gland was investigated. Three Na+-dependent systems of glycine transport, distinguished on the basis of their ionic requirement and sensitivity to 2-(methylamino)isobutyric acid (MeAIB), were A (Na+-dependent, MeAIB-sensitive); (Na++Cl-)-dependent, MeAIB-insensitive; and Na+-dependent, Cl--independent, MeAIB-insensitive. These systems were further distinguished on the basis of inhibition analysis and sensitivity to pH of the extracellular medium and preloading mammary tissue with amino acids. The uptake of glycine via the A system (Km 0.53 mM) was inhibited by preloading mammary tissue with alanine, while glycine uptake mediated by the (Na++Cl-)-dependent, MeAIB-insensitive system (Km 0.47 mM) was downregulated by preloading mammary tissue with all amino acids (alanine, sarcosine and histidine) tested. Treatment of mammary tissue with N-ethylmaleimide inhibited the uptake of glycine via both these systems. Decreasing the pH of the extracellular medium inhibited the uptake of glycine via the A system but not the (Na++Cl-)-dependent, MeAIB-insensitive system. On the basis of ionic requirement, system A appears to comprise two components, one dependent on Na+ plus Cl- and the other on Na+ alone. Insulin upregulated the A system-mediated uptake of glycine in pregnant mouse mammary tissue cultured in vitro, while the (Na++Cl-)-dependent, MeAIB-insensitive system remained unaffected.