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20102
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Subject: Dairy Science × Author: KAU × End date: 2019 × Start date: 2010 ×
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    ThesisItemOpen Access
    Detergent potential of enzymes of dairy microflora and their effect on the shelf life of milk products
    (Department of Dairy Science, College of Veterinary and Animal Sciences, Mannuthy, 2010) Beena, A K; KAU; Geevarghese, P I
    A study was conducted to assess the detergent potential of a spoilage protease enzyme obtained from the microflora of dairy plant environment. An attempt was also made to study the impact of selected enzyme producers on the shelf life of curd (dahi) and sterilised skim milk. A total of 71 bacterial isolates obtained from dairy environment were screened for their ability to produce spoilage enzymes like proteases lipases and lecithinases. Based on the spoilage potential, Pseudomonas aeruginosa (P12) isolated from pasteurised milk and Bacillus cereus (S4) isolated from sterilized skim milk were selected for further work. The influence of spoilage enzymes on selected physico-chemical characteristics of curd (dahi) and sterilized skim milk was evaluated by preparing the products from milk precultured with isolate P12 and S4. In general, proteolysis of milk was found to have an adverse effect on the quality of products. The stimulatory effect of proteolytic products of P12 and S4 on curd starters was evident from the higher values of acidity, firmness and syneresis in treated curd. The spoilage enzymes adversely affected the overall quality and shelf life of curd. In treated sterilised milk, tyrosine and NPN values were highly elevated. A linear correlation was found to exist between off-flavour and proteolysis. Curd and sterilised skim milk prepared from milk precultured with proteolytic organism were significantly different from that of control. The possibility of exploiting an alkaline protease from spoilage organism in dairy plant sanitation was also looked into. Environmental conditions for the production of alkaline protease by a psychrotrophic strain of Bacillus cereus (S4) was optimised in whey based medium. The protease used in this trial preferred an alkaline medium to remain stable. The enzyme was found to be stable over a wide temperature range of -10°C to 80°C and a pH range of 7.0 to 12.0. The metal ions Ca++, Mg++, Zn++ and Hg++ enhanced the enzyme activity. Lack of inhibition by Hg++ suggested lack of disulphide bonds in the active site of enzyme. Significant inhibition of activity by serine inhibitors indicated an essential serine residue in the active site of enzyme. The deleterious effect of EDTA on enzyme activity showed the supportive role of divalent cations. Marked residual activity on treatment with β-mercaptoethanol indicated the absence of cysteine residue for the enzyme. Enhancement of protease activity in the presence of surfactants and stability in the presence of H2O2 signified its potential to be used as detergent additive. Qualitative assessment of cleaning efficiency of inbuilt formulation substantiated the superiority of enzyme based formulations. Ammonium sulphate fractionation, dialysis and gel filtation using seralose 4B and Seralose 6B were effective in purifying the protease preparation by 141.31 fold. The purified protease was found to be a homogenous preparation of molecular weight of 50.5 kDa as determined by SDS PAGE.
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    ThesisItemOpen Access
    Development and evaluation of different vaccines against duck pasteurellosis
    (Department of Dairy Science, College of Veterinary and Animal Sciences, Mannuthy, 2010) Jesto, George; KAU; Krishnan Nair
    This study was undertaken to develop biofilm vaccines against duck pasteurellosis using oil, saponin and aluminium hydroxide as adjuvants and to experimentally evaluate their immunogenicity in ducks. Identity of P. multocida serotype A: 1 (DP1) used for study was confirmed by biochemical tests and by PM-PCR and pathogenicity was established in Swiss albino mice before vaccine production. The LD50 (11 w) of the DP1 isolate determined was 10 CFU / bird and MDT was 23.75 h in 11 week old ducklings when a high dose of 3 X 10 6 CFU of P. multocida per bird was given. At 11 Weeks age MDT gradually increased as the dose of inoculum decreased. In 21 week old ducks, the LD50 (21w) of the isolate DP1 was estimated to be 3 ×108 CFU of P. multocida and it showed that the Kuttanad duck had decreased susceptibility to pasteurellosis with age. On light microscopic studies planktonic cells appeared to be Gram negative coccobacillary, while biofilm cells were Gram negative and pleomorphic. Electron microscopic studies revealed that P. multocida could form well differentiated classic biofilm and 0.32 per cent TSB media supplemented 0.5 per cent chitin seemed to be excellent medium for biofilm formation by P. multocida. Four different vaccines viz. OV, OBV, SV and SAV were prepared and all of them were found to be sterile and safe. The oil adjuvanted vaccines (OV and OBV) offered better protection compared to saponin and aluminium hydroxide adjuvanted vaccine groups (SV and SAV) following primary vaccination, up to seven weeks. The modified oil adjuvanted vaccine prepared was not only found to be homogenous, but also more efficient in stimulating a humoral immune response and hence may be recommended. The oil adjuvanation gave better protection than saponin and aluminium hydroxide adjuvanation. The SAV gave better protection than SV which might be due to the presence of aluminium hydroxide which potentiated the immunostimulating ability of saponin. The combined vaccine (SAV) although was found to be better than single vaccine (SV) they cannot be used as a substitute to oil adjuvanted vaccines. The booster vaccination was found to have added advantageous effect on protection and is a must, to prevent losses. Pasteurella biofilms although found to be weak in inducing a primary immune response had the potency to evoke a more powerful secondary response compared to planktonic cells. Vaccination done at six weeks age followed by booster vaccination at 16 weeks age seemed to be a better modification of existing schedule and may be recommended. In histopathological studies, lymphoid hyperplasia was observed in spleen in survived control birds and in SV and SAV vaccine groups that did not survive challenge test, which indicated the persistence of Pasteurella organisms through mild infection in them following experimental challenge. Lymphoid depletion was observed in caecal tonsil in experimental pasteurellosis as in spleen. As the survived vaccinated birds following challenge test showed normal intact caecal tonsil, the course of disease and lesions might be less prominent in vaccinated birds during infection process. The well developed bursa observed in OV and OBV birds that survived challenge test indicated that the humoral immune response was well induced in them compared to other groups. The designed primers E1 and E2 amplified the gene E and hence, this pair of primers could be used for the production of amplified Gene E sequences for further studies on recombinant ghost system. In conclusion, 0.32 per cent TSB media supplemented 0.5 per cent chitin seemed to be an excellent medium that support classical biofilm formation by P. multocida. Booster vaccination definitely had added advantageous effect on protection. Immunization at 6 weeks of age with OV followed by booster vaccination at 16 weeks age with OBV seemed to be a better modification of existing schedule and may be recommended. In histopathological studies, the lesions were less prominent in vaccinated birds than control birds which indicated that the vaccines were effective.