Title Page
Contents
ABSTRACT 9
1. Introduction 11
2. Materials and Methods 15
2.1. Materials and chemicals 15
2.2. Lactic acid bacteria strains 16
2.3. Preparation of samples 17
2.3.1. Manufacture of pistachio milk 17
2.3.2. Fermentation of pistachio milk 19
2.3.3. Preparation of supernatants from pistachio milk and fermented pistachio milk 19
2.4. Proximate composition analysis 20
2.5. Measurements of acidification kinetics parameters 22
2.6. Gas chromatography (GC) analysis 22
2.7. Cell culture and treatments 23
2.8. Cytotoxicity assay 23
2.9. Cell morphologic observation 24
2.10. Annexin V/propidium iodide assay for determination of apoptosis 24
2.11. Western blot analysis for determination of protein expression level of microtubule- and apoptosis-related markers 25
2.12. Immunofluorescence microscopy for evaluation of microtubules and apoptosis 26
2.13. Statistical analysis 27
3. Results 28
3.1. Proximate composition of pistachio, pistachio milk, and bovine milk 28
3.2. Changes of pH and acidification kinetics of fermented pistachio milks 30
3.3. Concentrations of SCFAs and lactate in pistachio milk and fermented pistachio milk 33
3.4. Cytotoxicity and morphological changes in human colon carcinoma cells (Caco-2 cells) treated with fermented pistachio milk, acetate, and lactate 35
3.5. Determination of apoptosis in Caco-2 cells treated with fermented pistachio milk, acetate, and lactate 38
3.6. Evaluation of α-tubulin protein expression in Caco-2 cells treated with fermented pistachio milk, acetate, and lactate 41
3.7. Determination of apoptosis through activated caspase-3 and disruption of α-tubulin in Caco-2 cells treated with fermented pistachio milk and acetate 43
4. Discussion 45
5. Conclusions 52
References 53
Abstract (in Korean) 65
Table 1. The conditions for dry matter and ash content measurements in pistachio, PM, and bovine milk 21
Table 2. Proximate composition of pistachio, PM, and bovine milk 29
Table 3. Acidification kinetic parameters of fermented PM groups 32
Figure 1. The process for the production of pistachio milk (PM). 18
Figure 2. Changes in pH of fermented PMs depending on fermentation time. C = PM fermented with Streptococcus thermophilus and Lactobacillus bulgaricus; C-La = PM... 31
Figure 3. The concentrations of SCFAs ((A) acetate, (B) propionate, and (C) butyrate), and (D) lactate in PMs and fermented PMs with or without 4% inulin as measured by... 34
Figure 4. Effects of fermented PM, acetate, and lactate on cell viability and morphology in Caco-2 cells. The cell viability treated with (A) fermented PM, (C) acetate, and (E)... 36
Figure 5. Effects of fermented PM (fPM), acetate, and lactate on apoptosis in Caco-2 cells. Flow cytometry scatter plots of propidium iodide (y-axis) versus annexin V-... 39
Figure 6. Effect of fermented PM, acetate, and lactate on microtubule protein expression in Caco-2 cells. Protein expression of α-tubulin treated with (A) fermented PM, (B)... 42
Figure 7. Effects of fermented PM and acetate on apoptosis markers, α-tubulin disruption, and cell nuclei damage in Caco-2 cells. (A) Protein expression of caspase-3... 44