CHOLESTEROL REDUCING EFFECTS OF Lactobacillus spp. ISOLATED FROM BREAST MILK OF LACTATION MOTHER.

• SUHANIS NADIA BINTI SALLEH

CHAPTER 1

INTRODUCTION

1. Background of the study

Cholesterol is the precursor of primary bile salts formed in the liver and store as conjugated bile salts in gall bladder to be release in digestive tract. (Corzo & Gilliland, 1999). Lipid and cholesterol rich food intake act as the main factor in increasing of heart disease (Anandharaj & Sivasankari, 2014). Thus, it is important to reduce cholesterol as prevention to cardiovascular disease. (Yildiz et. al, 2011). Even though pharmaceutical agent or therapy exists for hypercholesterolemia treatments, they are expensive and may produce side effect. (Schuster, 2004).

Due to the reason, non pharmaceutical approaches which yield cholesterol reduction were examined and probiotics are one of several approaches that have been used (Anandharaj & Sivasankari, 2014).

2. Problem statement

1.3 Research Objective

1.3.1 General objective

To investigate the cholesterol reducing property of Lactobacillus spp. isolated from breast milk of lactation mother .

1.3.2Specific objective

1.3.2.1 To isolate Lactobacillus spp. from breast milk of lactation mother.

1.3.2.2 To identify Lactobacillus spp. isolated from breast milk of lactation mother.

1.3.2.3 To determine cholesterol reducing property of Lactobacillus spp. isolated from breast milk of lactation mother.

4. Research hypothesis

1.4.1 Study hypothesis

There is significant difference between Lactobacillus spp. isolated from breast milk of different lactation mother on its cholesterol reducing property.

1.4.2 Null hypothesis

There is no significant difference between Lactobacillus spp. isolated from breast milk of lactation mother on its cholesterol reducing property.

1.5 Scope and limitation of the study

This study focusing on identification of bacteria and its properties and includes molecular technique. The scope of this study involves both phenotypic and genotypic characterization. Some limitations arise in this study. The cholesterol reduction assay which will be done in vitro to mimic the in vivo mechanism may not be totally similar with in vivo environment.

1.6 Significant of study

Breast milk is a possible source of Lactobacillus strains but there are only few studies done on isolation of probiotic from human’s 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 result may be obtained from other population subject and this study may enhance the finding of probiotic strains that capable in cholesterol assimilation.

CHAPTER 2

LITERATURE REVIEW

CHAPTER 3

METHODOLOGY

1. Proposed methodology (Descriptive)
   1. Ethical issues
   2. Study area

The study will be conducted in the final year research laboratory of microbiology section at UiTM Puncak Alam. Most of the experiments will be conducted at the laboratory except for sequencing which will be send away. Sample collection is done outside the study area and will be store in the laboratory storage section.

3. Sample collection

Breast milk sample will be collected from volunteers in sterile container. Prior to collection, the breast is clean with sterile water and apply with chlorexidine to remove other normal flora. The sample will be store on ice until delivery to the laboratory. The sample will then stored in -80^oC if not directly use or for further use. For storage, the sample will previously transfer into several small vials to avoid multiple freeze and thaw.

4. Isolation of Lactobacillus spp.

1ml of breast milk sample is transfer into 9ml of sterile saline (0.85% sodium chloride). The dilute samples will be plate on Man Ragosa Sharpe, MRS medium. The plate is incubate at 37^oC for 24 to 48 hour in anaerobic condition.

5. Identification of Lactobacillus spp.

Isolated Lactobacillus spp. will be confirm based on growth 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 test strip. The result is analyze using API LAB^TM PLUS software. MRS broth medium containing 20% glycerol is use to preserve the pure cultures and store at -80^oC.

6. PCR amplification of 16S rDNA and sequencing

Modified 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 56^oC for 30s and elongation at 72⁰C. The PCR result will be separated on gel electrophoresis to check for the purity and of the amplicon. The amplified rDNA will be purified with PCR purification kit and send for sequencing.

7. Phylogenetic analysis
8. Cholesterol reducing assay

9. Statistical analysis

The data will be collected in triplicate and will be analysed by SPSS software. Data will be expressed as mean and standard deviation. One way ANOVA test with significance level p < 0.05 will be used to determine the significant difference between means.

2. Proposed methodology (Flow chart)

5.0 Expected outcomes

For 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 presence 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 Financial implications

7.0 Ganntt chart

Work plan 2014 (September – December)

Work plan 2015 (March – July)

Work plan 2015 (March – June)