Effects of nanopowderized oyster shell on antiosteoporosis and bone growth and its application : 나노굴껍질분말의 이화학적 특성과 흰쥐에서 항-골다공증과 키 성장의 효과와 유제품에 적용 연구

다국어 초록 (Multilingual Abstract)

Nanotechnology is receiving a great deal of attention regarding its potential application in the food and other industries. This technology enhances the bioavailability and functionality of various food components. The current growing interest in nanotechnology focuses on its application to food research, with a view to increasing the solubility and bioavailability of nutritional food ingredients. This study has investigated the physicochemical properties, bioavailability, and toxicity of nanopowderized oyster shell (NPOS) and zinc-activated nanopowderized oyster shell (Zn-NPOS). It also examined the effects of NPOS and Zn-NPOS on postmenopausal osteoporosis and bone growth in rats, and the possible application to dairy products.
In the first part of this study, the physicochemical characteristics, bioavailability, and toxicity of NPOS and Zn-POS were examined by particle size analysis, scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), fourier transform infrared spectroscopy (FT-IR). The resulting data demonstrated that the surfaces of powderized oyster shell (POS), NPOS and Zn-NPOS were spherical in shape, and their particle sizes were 180 μm, 257 nm and 389 nm, respectively. The XRD and FT-IR analyses confirmed that calcium carbonate was major component in POS, NPOS and Zn-NPOS. Additionally, both the solubility and bioavailability of Zn-NPOS were higher than those of NPOS and POS. Cytotoxic studies showed that no toxicity of NPOS and Zn-NPOS was observed for four days. Overall, the physicochemical properties, bioavailability, and toxicology investigations indicated that NPOS and Zn-NPOS particles improved biocompatibility without any phase changes.
In the second part of this study, the preventative effects of NPOS and Zn-NPOS on postmenopausal osteoporosis were investigated in ovariectomized rats. Seven-week old female rats were ovariectomized and assigned to five groups: sham operated (sham); ovariectomized rats treated with vehicles (OVX); ovariectomized rats treated with POS (20 mg/kg body wt); ovariectomized rats treated with NPOS (20 mg/kg body wt); and ovariectomized rats treated with Zn-NPOS (20 mg/kg body wt). The analysis of femur bones indicated that the treatment with Zn-NPOS resulted in a greater extent of recovery from ovariectomy-induced bone loss compared to the NPOS and POS treated groups. In addition, treatment with Zn-NPOS resulted in greater bone strength and superior trabecular architecture in rats than that treated with NPOS and POS. Moreover, Zn-NPOS showed a greater efficiency in increasing bone formation markers, as well as reducing bone resorption markers. Overall, these results indicated that both NPOS and Zn-NPOS treatments could potentially prevent ovariectomy-induced severe bone loss in rats.
In the third part of this study, the effects of NPOS and Zn-NPOS on bone growth were examined in rats. Three-week old male rats were divided into four groups, based on the following treatments administered for seven weeks: control (non-treated sample); treated group 1 (treated with POS); treated group 2 (treated with NPOS); and treated group 3 (treated with Zn-NPOS). The rats fed with Zn-NPOS significantly increased in length, from 0.43 mm to 0.62 mm (44.19%). Treatments with NPOS and Zn-NPOS resulted in greater bone strength and superior trabecular architecture in rats than that with POS treatment. In addition, the serum IGF-1 levels of the Zn-NPOS groups appeared to be greater than those of the control groups. This part of the study indicated that NPOS and Zn-NPOS treatment groups enhanced bone calcium metabolism, resulting in a growth in rat length.
In the fourth part of this study, milk samples, supplemented with dispersible oyster shell powders (0.5–2.0%, v/v), were examined for physicochemical and sensory properties at 4C during a 16 day storage period. Oyster shell powder was added to distilled water (10%, w/v) and stirred at 800 rpm for 2 h, and then 0.5% polyglycerol monostearate (PGMS) was added and stirred for 24 h. The pH values of all the milk samples ranged from 6.62 to 6.88 during the storage period, and the dispersibility of NPOS- and Zn-NPOS-added milk were more stable than that of the POS-added milk at low concentrations (0.5 and 1.0%, v/v) during storage. The sensory properties revealed that the dispersibility score in appearance increased significantly at higher concentrations of POS-, NPOS- and Zn-NPOS-added milk (1.5 and 2.0%, v/v; p<0.05) during the storage. Therefore, the dispersible NPOS or Zn-NPOS can be supplemented into milk without any adverse effect.
In the fifth part of this study, the characteristics of tablet milk supplemented with NPOS or Zn-NPOS were investigated. The data showed that the hardness was not significantly different between the control sample and NPOS- or Zn-NPOS- added tablet milk samples (p>0.05). The sensory properties showed that the appearance, flavor, taste, texture, and overall acceptability of all the samples were similar to the control samples. Finally, it was concluded that the quality of the tablet milk samples, supplemented with nanosized oyster shell powders, was similar to the control sample.
In summary, nanopowderized oyster shell was not significantly different from POS in terms of chemical composition, purity, crystallinity and toxicology. The results showed that NPOS and Zn-NPOS had greater calcium dispersibility and bioavailability. In addition, NPOS and Zn-NPOS treatments had significant antiosteoporotic effects and increased bone growth. The quality of milk and tablet milk, supplemented with NPOS or Zn-NPOS, did not change considerably sensory property but was stable physically. In conclusion, both NPOS and Zn-NPOS have the potential to treat postmenopausal osteoporosis and improve bone growth. Thus, they could be considered as functional ingredients in food applications.

목차 (Table of Contents)

• Literature review 1
• Chapter I: Evaluation of Physicochemical properties, bioavailability and toxicity of nanopowderized oyster shell 43
• 1. Abstract 43
• 2. Introduction 45
• 3. Materials and Methods 48
• 3.1 Materials 48
• 3.2 Manufacture of nanopowderized and Zn-activated nano-powderized oyster shell 48
• 3.3 Size distribution 49
• 3.3.1 Electron microscopy 49
• 3.3.2 Particle size distribution 50
• 3.4 Physicochemical analysis 51
• 3.4.1 Mineral 51
• 3.4.2 Zeta potential 51
• 3.4.3 Moisture sorption analysis 52
• 3.4.4 X-ray diffraction 52
• 3.4.5 Fourier transform infrared spectroscopy 53
• 3.5 Absorption properties 53
• 3.5.1 Acidic solubility 53
• 3.5.2 Bioavailability 54
• 3.6 Cytotoxicity 55
• 3.7 Statistical analysis 56
• 4. Results and Discussion 57
• 4.1 Size distribution 57
• 4.1.1 Electron microscopy 57
• 4.1.2 Particle size distribution 60
• 4.2 Physicochemical analysis 62
• 4.2.1 Mineral 62
• 4.2.2 Zeta potential 62
• 4.2.3 Moisture sorption analysis 64
• 4.2.4 X-ray diffraction 68
• 4.2.5 Fourier transform infrared spectroscopy 71
• 4.3 Absorption properties 73
• 4.3.1 Acidic solubility 73
• 4.3.2 Bioavailability 74
• 4.4 Cytotoxicity 79
• 5. Conclusion 82
• 6. References 83
• Chapter II: Effect of nanopowderized oyster shell on postmenopausal osteoporosis in rats 91
• 1. Abstract 91
• 2. Introduction 93
• 3. Materials and Methods 96
• 3.1 Animal treatments 96
• 3.2 Bone analysis 99
• 3.2.1 Bone calcium and phosphorus measurement 99
• 3.2.2 Bone microstructure assessment 99
• 3.3 Biochemical assays 100
• 3.3.1 Alkaline phosphatase, calcium and phosphorus in serum 101
• 3.3.2 Creatinine, calcium and phosphorus in urine 102
• 3.3.3 Calcium and phosphorus in liver, kidney and feces 102
• 3.3.4 Serum osteocalcin 102
• 3.3.5 Urinary deoxypyridinoline 103
• 3.3.6 Urinary N-telopeptides of type I collagen 103
• 3.4 Statistical analysis 104
• 4. Results and Discussion 105
• 4.1 Body weights 105
• 4.2 Bone analysis 105
• 4.2.1 Femoral calcium, zinc and phosphorous contents 105
• 4.2.2 Bone mineral density 108
• 4.2.3 Bone stiffness 110
• 4.2.4 Bone µCT evaluation 112
• 4.3 Biochemical assays 118
• 4.3.1 Calcium, zinc and phosphorous content in serum, feces, liver and kidney 118
• 4.3.2 Alkaline phosphatase and creatinine in serum 121
• 4.3.3 Osteocalcin in serum and deoxypyridinoline and N-teleopeptide of type I collagen in urine 123
• 5. Conclusion 128
• 6. References 129
• Chapter III: Promoting effects of nanopowderized oyster shell on bone growth in rats 133
• 1. Abstract 133
• 2. Introduction 135
• 3. Materials and Methods 138
• 3.1 Animal treatments 138
• 3.2 Bone growth factor 141
• 3.2.1 Plasma insulin-like growth factor-1 141
• 3.3 Bone analysis 141
• 3.3.1 Bone calcium and phosphorus measurement 141
• 3.3.2 Measurements of femur lengths 142
• 3.3.3 Bone microstructure assessment 142
• 3.4 Concentration of calcium and phosphorus 143
• 3.4.1 Serum 143
• 3.4.2 Liver, kidney and fecal 144
• 3.5 Statistical analysis 144
• 4. Results and Discussion 145
• 4.1 Body weights 145
• 4.2 Bone growth factor 145
• 4.2.1 Plasma insulin-like growth factor-1 145
• 4.3 Analysis of bone 148
• 4.3.1 The femoral calcium, zinc and phosphorous contents 148
• 4.3.2 Bone growth 151
• 4.3.3 Bone mineral density 153
• 4.3.4 Bone µCT evaluation 155
• 4.4 Biochemical assays 161
• 4.4.1 Calcium, zinc and phosphorous content in the liver and kidney, fecal and serum 161
• 5. Conclusion 164
• 6. References 165
• Chapter IV: Physicochemical and sensory properties of milk supplemented with dispersible nanopowderized oyster shell during storage 173
• 1. Abstract 173
• 2. Introduction 175
• 3. Materials and Methods 179
• 3.1 Optimization of dispersible nanopowderized oyster shell 179
• 3.2 Manufacture of dispersible nanopowderized oyster shell-supplemented milk 179
• 3.3 Physicochemical properties of dispersible nanopowderized oyster shell-supplemented milk 180
• 3.3.1 pH 180
• 3.3.2 Mineral 180
• 3.3.3 Color measurement 181
• 3.4 Sensory analysis 181
• 3.5 Statistical analysis 182
• 4. Results and Discussion 183
• 4.1 Optimization of dispersible nanopowderized oyster shell 183
• 4.2 Physicochemical properties of dispersible nanopowderized oyster shell-supplemented milk 189
• 4.2.1 pH 189
• 4.2.2 Amount of calcium 192
• 4.2.3 Dispersible nanopowderized oyster shell in milk 195
• 4.2.4 Color 198
• 4.3 Sensory evaluation 201
• 5. Conclusion 205
• 6. References 206
• Chapter V: Characteristics of tablet milk supplemented with nanopowderized oyster shell 213
• 1. Abstract 213
• 2. Introduction 215
• 3. Materials and Methods 218
• 3.1 Manufacture of tablet milk 218
• 3.2 Physicochemical properties of nanopwderized oyster shell-supplemendted tablet milk 221
• 3.2.1 pH 221
• 3.2.2 Mineral 221
• 3.2.3 Moisture sorption 222
• 3.2.4 Texture profile analysis 223
• 3.2.5 Color measurement 223
• 3.3 Sensory analysis 224
• 3.4 Statistical analysis 225
• 4. Results and Discussion 226
• 4.1 Shape 226
• 4.2 Physicochemical properties of nanopwderized oyster shell-supplemendted tablet milk 228
• 4.2.1 pH 228
• 4.2.2 Mineral 228
• 4.2.3 Moisture sorption 231
• 4.2.4 Texture profile analysis 233
• 4.2.5 Color 235
• 4.3 Sensory evaluation 237
• 5. Conclusion 240
• 6. References 241