Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum improves phagocytic potential of macrophages in aged mice.

The present study investigated the effect of Dahi containing potential probiotic bacterial strains on macrophage functions in ageing mice. Probiotic Dahi was prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis biovar diacetylactis) along with Lactobacillus acidophilus LaVK2 (La-Dahi) or combined L. acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 (LaBb-Dahi) in buffalo milk. The effect of ageing on phagocytic function was evaluated on 4 mo, 12 mo and 16 mo old mice. The effect of probiotic Dahi was evaluated for macrophage functions in ageing mice (12 mo old) fed La-Dahi or LaBb-Dahi supplements for 4 months. The production of extracellular superoxide and H2O2 declined in peritoneal macrophages but enhanced in splenic macrophages, while intracellular superoxide declined in both peritoneal and splenic macrophages with ageing in mice. A decline in phagocytic activity of peritoneal macrophages was also observed in aged mice. Supplementation of diet with La-Dahi or LaBb-Dahi for 4 months improved production of reactive oxygen species and phagocytic and adherence indices of peritoneal macrophages in aged mice. These results suggest that oral administration of La-Dahi or LaBb-Dahi has potential to improve immune functions in ageing individuals.

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum modulates the formation of aberrant crypt foci, mucin-depleted foci, and cell proliferation on 1,2-dimethylhydrazine-induced colorectal carcinogenesis in Wistar rats.

Aberrant crypt foci (ACF) and mucin-depleted foci (MDF) are pre-neoplastic lesions identified in the colon of carcinogen-treated rodents and in humans at high risk for colon cancer. The present study was carried out to divulge the protective potential of the probiotic Dahi containing Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 alone or in combination with piroxicam (PXC) on the development of early biomarkers of colorectal carcinogenesis in male Wistar rats administered 1,2-dimethylhydrazine (DMH). DMH was injected subcutaneously at the rate of 40 mg/kg body weight per animal twice a week for 2 weeks. A total of 120 male Wistar rats were randomly allocated to five groups, each group having 24 animals. The rats were fed with buffalo milk or probiotic supplement (20 grams) alone or as an adjunct with PXC in addition to a basal diet ad libitum for 32 weeks. Group I was offered buffalo milk (BM) and served as the control group. Group II was administered DMH along with BM and served as the DMH-control group; group III was administered BM-DMH-PXC, in which besides administering BM-DMH, PXC was also offered. Group IV was offered probiotic LaBb Dahi and DMH, and group V was offered both probiotic LaBb Dahi and PXC along with DMH. The rats were euthanized at the 8(th), 16(th), and 32(nd) week of the experiment and examined for development of ACF, aberrant crypts per ACF (AC/ACF), mucin-depleted foci (MDF), large MDF, and proliferating cell nuclear antigen (PCNA) labeling index. Administration of DMH in rats induced pre-neoplastic lesions (ACF and MDF) and increased the PCNA index in colorectal tissue. A significant (p<0.05) reduction in the number of ACF, AC/ACF, MDF, large MDF, and PCNA labeling index were observed in the probiotic LaBb Dahi group compared with the DMH control group. Feeding rats with LaBb Dahi or treatment with PXC diminished the initiation and progression of DMH-induced pre-neoplastic lesions and the PCNA index, and treatment with LaBb Dahi and PXC combined was significantly more effective. The dietary intervention of probiotics and PXC significantly protects against the development of CRC in the rat-DMH model. These observations suggest that probiotic LaBb Dahi alone or as an adjunct with PXC may have anti-neoplastic and anti-proliferative activities. Moreover, probiotic LaBb Dahi possesses the medicinal properties to prevent colorectal carcinogenesis.

Modulation of expression of Programmed Death-1 by administration of probiotic Dahi in DMH-induced colorectal carcinogenesis in rats.

UNLABELLED: Interaction of probiotic bacteria with the host immune system elicits beneficial immune modulating effects. Although, there are many published studies on interaction of probiotics with immune system focusing on activation of immune system by bacterial cell wall through the engagement of Toll-like receptor family; very few studies have focused on molecules involved in the T-cell activation, and not much work has been executed to study the correlation of probiotics and programmed death-1 in colorectal carcinogenesis in animal models. Hence, the present study was carried out to assess the effect of probiotic Dahi on expression of programmed death (PD-1) in colorectum of 1, 2-dimethylhydrazine treated Wistar rats. METHODS: DMH was injected subcutaneously at the rate of 40 mg/kg body weight per animal twice a week for 2 weeks. A total of 168 male Wistar rats were randomly allocated to seven groups, each group having twenty-four animals. The rats were euthanized at the 8th, 16th and 32nd week of the experiment and examined for the expression of PD-1 in colorectal tissues by immunohistochemical staining. RESULTS: Expression of PD-1 was observed in colorectal tissues of normal and DMH-treated rats. Feeding rats with probiotic Dahi or the treatment with piroxicam decreased the expression of PD-1 in DMH-induced colorectal mucosa, and the combined treatment with probiotic Dahi and piroxicam was significantly more effective in reducing the expression of PD-1. CONCLUSION: PD-1 expressed independent of carcinogen administration in normal colonic mucosa and may play a role in modulation of immune response in DMH-induced colorectal carcinogenesis. The present study suggests that probiotic Dahi can be used as an effective chemopreventive agent in the management of colorectal cancer.

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 ß-glucoronidase activity was assessed, and the histopathological picture of the colon of mice was studied. All colitis control mice evidenced significant increase in MPO, ß-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, ß-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.

Non-fat milk solids attenuate hypercholesterolemic effect of conjugated linoleic acid.

The present study instigates the notion that non-fat milk has a beneficial effect on hypercholesterolemia caused by dietary conjugated linoleic acid (CLA). Two groups of mice (6 per group) were fed synthetic CLA (predominantly a mixture of t10, c12 and c9, t11 isomers) at 1 % level in the diet with or without skim milk powder (SMP). Another group (control) was fed a similar diet without CLA and SMP. The study was conducted for 60 days (d). Daily feed intake and weekly body weight were recorded. Plasma lipid profile was determined monthly. At the conclusion of experiment, animals were sacrificed and liver tissue was removed to assay cholesterol and triacylglycerols (TAG). Following 60 day feeding, the plasma total cholesterol content increased significantly in CLA group (218 mg/dl) than in control group (167 mg/dl), while in CLA + SMP group (fed non-fat milk solids along with CLA), it was similar to that in control group. The fractions of high density lipoproteins (HDL) and low density lipoprotein + very low density lipoprotein (LDL + VLDL) cholesterol as well as triacylglycerols (TAG) in plasma increased in CLA group, but in CLA + SMP group it remained similar to the control levels. Thus, it is concluded that dietary CLA increases plasma cholesterol and TAG contents in mice on normocholesterolemic diet and the inclusion of non-fat milk solids reverses this effect.

Effects of cow ghee (clarified butter oil) & soybean oil on carcinogen-metabolizing enzymes in rats.

BACKGROUND & OBJECTIVES: Our previous study showed that cow ghee relative to soybean oil had a protective effect against carcinogen induced mammary cancer in rats. The objective of this study was to elucidate its biochemical mechanism. METHODS: Two groups of 21 day old rats (20 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). Five animals from each group were sacrificed at 0 day and at 5, 21 and 44 wk for analysis of phase I and phase II pathways enzymes of carcinogen metabolism. RESULTS: Dietary cow ghee relative to soybean oil decreased the activities of cytochrome P450 (CYP) enzymes, CYP1A1, CYP1A2, CYP1B1 and CYP2B1, responsible for activation of carcinogen in liver. Carcinogen detoxification activities of uridinediphospho-glucuronosyl transferase (UDPGT) and quinone reductase (QR) in liver, and γ-glutamyltranspeptidase (GGTP) and QR in mammary tissue were significantly higher in cow ghee fed rats than in soybean oil fed rats. The hepatic GGTP activity decreased on soybean oil diet; while in cow ghee group it remained unaffected. INTERPRETATION & CONCLUSIONS: Our findings show that dietary cow ghee compared to soybean oil downregulates the enzyme activities responsible for carcinogen activation in liver and upregulates carcinogen detoxification activities in liver and mammary tissues.

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkers of ageing in mice.

The potential benefiting effects of probiotic Dahi on age-inflicted accumulation of oxidation products, antioxidant enzymes and expression of biomarkers of ageing were evaluated in mice. Probiotic Dahi were prepared by co-culturing in buffalo milk (3% fat) 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 (La-Dahi) or combined L. acidophilus and Bifidobacterium bifidum BbVK3 (LaBb-Dahi). Four groups of 12 months old mice (6 each) were fed for 4 months supplements (5 g/day) of buffalo milk (3% fat), Dahi, La-Dahi and LaBb-Dahi, respectively, with basal diet. The activities of catalase (CAT) and glutathione peroxidase (GPx) declined and the contents of oxidation products, thiobarbituric acid reactive substances (TBARS) and protein carbonyls, increased in red blood corpuscles (RBCs), liver, kidney and heart tissues and superoxide dismutase (SOD) activity increased in RBCs and hepatic tissues during ageing of mice. Feeding ageing mice with La-Dahi or LaBb-Dahi increased CAT activity in all the four tissues, and GPx activity in RBCs and hepatic tissue, and a significant decline in TBARS in plasma, kidney and hepatic tissues and protein carbonyls in plasma. Feeding mice with probiotic Dahi also reversed age related decline in expression of biomarkers of ageing, peroxisome proliferators activated receptor-α, senescence marker protein-30 (SMP-30) and klotho in hepatic and kidney tissues. The present study suggests that probiotic Dahi containing selected strains of bacteria can be used as a potential nutraceutical intervention to combat oxidative stress and molecular alterations associated with ageing.

Immunoprotective Effect of Probiotic Dahi Containing Lactobacillus acidophilus and Bifidobacterium bifidum on Dextran Sodium Sulfate-Induced Ulcerative Colitis in Mice.

In the present study, probiotic Dahi (LaBb Dahi) containing Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 was selected as a probiotic therapy to investigate its protective effect on dextran sodium sulfate (DSS)-induced ulcerative colitis model in mice that mimics the picture in human. LaBb Dahi was 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 L. acidophilus LaVK2 and B. bifidum BbVK3 in buffalo milk (3% fat). Four groups of swiss albino male mice (12 each) were fed buffalo milk (3% fat), buffalo milk (3% fat) plus DSS, Dahi plus DSS, and LaBb Dahi plus DSS, respectively, for 17 days with basal diet. The myeloperoxidase (MPO) activity, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon (IFN-γ) were assessed as inflammatory markers, and the histopathological picture of the colon of mice was studied. DSS-induced colitis appeared to induce significant increase in MPO activity, levels of TNF-α, IL-6 and IFN-γ. Feeding with LaBb Dahi offered significant reduction in MPO activity, levels of TNF-α, IL-6 and IFN-γ when compared to either buffalo milk group or group III (Dahi). The present study suggests that LaBb probiotic Dahi can be used to combat DSS-induced biochemical and histological changes and to achieve more effective treatment for ulcerative colitis.

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.

Study on cow ghee versus soybean oil on 7,12-dimethylbenz(a)-anthracene induced mammary carcinogenesis & expression of cyclooxygenase-2 & peroxisome proliferators activated receptor-γ in rats.

BACKGROUND & OBJECTIVES: Breast cancer is a leading cause of cancer death in women; dietary fat is the one of the factors that influences its incidence. In the present study we investigated the effect of feeding cow ghee versus soybean oil on 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary cancer in rat and expression of cyclooxygenase-2 and peroxisome proliferators activated receptor-γ (PPAR-γ) in mammary gland. METHODS: Two groups of 21 day old female rats (30 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). The animals were given DMBA (30 mg/kg body weight) through oral intubation after 5 wk feeding. Another two groups (8 each) fed similarly but not given DMBA served as control for the gene expression study. RESULTS: In DMBA treated groups, the animal fed soybean oil had higher tumour incidence (65.4%), tumour weight (6.18 g) and tumour volume (6285 mm3) compared to those fed cow ghee (26.6%, 1.67 g, 1925 mm3, respectively). Tumour latency period was 23 wk on soybean oil compared to 27 wk on cow ghee. Histological analysis of tumours showed that the progression of carcinogenesis was more rapid on soybean oil than on cow ghee. The expression of cyclooxygenase-2 was observed only in DMBA treated rats and it was significantly less on cow ghee than on soybean oil. The expression of PPAR-γ was significantly more on cow ghee than on soybean oil. INTERPRETATION & CONCLUSIONS: Our results show that dietary cow ghee opposed to soybean oil attenuates mammary carcinogenesis induced by DMBA; and the effect is mediated by decreased expression of cyclooxygenase-2 and increased expression of PPAR-γ in the former group.

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.