Cholesterol Reducing Effects of Lactobacillus Spp

cholesterol Reducing Effects of Lactobacillus SppCHOLESTEROL cut EFFECTS OF Lactobacillus spp. ISOLATED FROM BREAST MILK OF LACTATION MOTHER.SUHANIS NADIA BINTI SALLEHCHAPTER 1 submissionBackground of the studyCholesterol is the precursor of primary bile salts form in the liver and shop as conjugated bile salts in gall bladder to be release in digestive tract. (Corzo Gilliland, 1999). lipoid and cholesterol rich food intake act as the primary(prenominal) factor in increasing of heart disease (Anandharaj Sivasankari, 2014). Thus, it is important to shrivel up cholesterol as prevention to cardiovascular disease. (Yildiz et. al, 2011). Even though pharmaceutical agent or therapy exists for hypercholesterolemia treatments, they ar expensive and may provoke side effect. (Schuster, 2004).Due to the reason, non pharmaceutical approaches which yield cholesterol reduction were examined and probiotics ar one of several approaches that drive been used (Anandharaj Sivasankari, 20 14).Problem statement1.3 query Objective1.3.1 General objectiveTo investigate the cholesterol reducing dimension of Lactobacillus spp. assignd from teat milk of lactation m another(prenominal) .1.3.2Specific objective1.3.2.1 To isolate Lactobacillus spp. from mamilla milk of lactation mother.1.3.2.2 To identify Lactobacillus spp. isolated from thorax milk of lactation mother.1.3.2.3 To determine cholesterol reducing property of Lactobacillus spp. isolated from breast milk of lactation mother.Research surmise1.4.1 Study hypothesis at that place is significant difference between Lactobacillus spp. isolated from breast milk of divers(prenominal) lactation mother on its cholesterol reducing property.1.4.2 Null hypothesisThere is no significant difference between Lactobacillus spp. isolated from breast milk of lactation mother on its cholesterol reducing property.1.5 reach and limitation of the studyThis study focusing on identification of bacterium and its properties and inclu des molecular technique. The scope of this study involves both phe nonypic and genotypic characterization. about limitations arise in this study. The cholesterol reduction assay which exit be done in vitro to mimic the in vivo mechanism may not be totally similar with in vivo environment.1.6 Significant of study chest of drawers milk is a possible source of Lactobacillus strains but there are only few studies done on isolation of probiotic from humans milk. (Anandharaj Sivasankari , 2014 Martin et al., 2004). The reliability of cholesterol reduction by using probiotics for hypercholesterolemia treatments have gain increase of interest. (Jones et al. 2004 Lim et al. 2004). Even so, the findings are more on lactic acid bacteria strains among Western origin subject (Yildiz et al. 2011). Different bequeath may be obtained from other population subject and this study may enhance the finding of probiotic strains that capable in cholesterol assimilation.CHAPTER 2 books REVIEWCHAPTER 3METHODOLOGYProposed methodology (Descriptive)Ethical issuesStudy arenaThe study get out be conducted in the final year look laboratory of microbiology section at UiTM Puncak Alam. Most of the experiments volition be conducted at the laboratory except for sequencing which lead be send away. Sample assembly is done outside the study area and will be store in the laboratory storage section.Sample collectionBreast milk sample will be collected from volunteers in sterile container. forward to collection, the breast is clean with sterile water and apply with chlorexidine to remove other normal flora. The sample will be store on spyglass until delivery to the laboratory. The sample will then stored in -80oC if not direct use or for further use. For storage, the sample will previously remove into several small vials to avoid multiple freeze and thaw.Isolation of Lactobacillus spp.1ml of breast milk sample is transfer into 9ml of sterile saline (0.85% sodium chloride). The turn off samples will be plate on Man Ragosa Sharpe, MRS medium. The plate is continue at 37oC for 24 to 48 hour in anaerobic condition. denomination of Lactobacillus spp.Isolated Lactobacillus spp. will be confirm based on yield on MRS medium, colony morphology, Gram staining, and catalase reaction. The isolated colony will be proceed with subculture to obtain pure culture. Further species identification will be performed by carbohydrate fermentation pattern using API 50 CHL bear witness strip. The result is analyze using API LABTM PLUS software. MRS broth medium containing 20% glycerol is use to preserve the pure cultures and store at -80oC.PCR involution of 16S rDNA and sequencingModified method of Smoker and Barnum (1988) will be conducted for DNA isolation. The 16S rDNA will then amplify in thermocycler by 30 cycles which include denaturation at 940C for 30s, annealing at 56oC for 30s and elongation at 720C. The PCR result will be separated on gel electrophoresis to check for t he purity and of the amplicon. The amplified rDNA will be purified with PCR purification kit and send for sequencing.Phylogenetic analysisCholesterol reducing assayStatistical analysisThe data will be collected in triplicate and will be analysed by SPSS software. entropy will be expressed as mean and standard deviation. iodine way ANOVA test with significance level p Proposed methodology (Flow chart)5.0 Expected outcomesFor isolation and identification of Lactobacillus spp., various species may be identified. Generally, they are expected to be catalase negative, gram positive, rods or cocci. Results on API 50 CHL will confirm the specific species. In PCR amplification, there will be bearing of bands of specific base pair for Lactobacillus spp. after the amplicons separated on the agarose gel. As for cholesterol reducing assay, there will be species which show cholesterol reducing property and the species with the most significant reduction of cholesterol will be identified.6.0 Fin ancial implications7.0 Ganntt chart Work designing 2014 (September December)Work plan 2015 (March July)Work plan 2015 (March June)