Physicochemical characteristics and effects of nanopowdered eggshell on ovariectomy-induced osteoporosis in rats and its application into yogurt

국문 초록 (Abstract)

국문초록

난각나노분말의 이화학적 특성과 난소절제 흰쥐에서
항-골다공증 효과 규명 및 요구르트에 적용 연구

Mohammad Al Mijan
식품공학과
세종대학교 대학원

나노기술은 최근에 빠르게 성장하고 있으며, 특히 식품 및 의약분야에서 가까운 미래에 전도유망한 기술로 각광 받을 것으로 기대되고 있다. 화장품, 의약품 분야뿐만 아니라, 음료산업을 비롯한 식품산업에서도 다양한 생리활성물질의 생 이용율과 기능성을 향상시키기 위한 노력이 지속적으로 진행되고 있으며, 나노기술은 이에 따른 극복방안으로 떠오르고 있다. 본 연구는 3단계로 진행되었으며, 우선 난각나노분말의 이화학적특성을 분석하고, 동물실험으로 난소절제흰쥐에서 항-골다공증 효과를 규명하였으며, 마지막으로 이를 요구르트에 첨가하여 기능성 식품소재의 가능성을 검증하였다.
첫 번째 실험에서는 전계 방출형 주사전자현미경 (FE-SEM), 투과전자현미경 (TEM), dynamic vapor sorption (DVS) analyzer, X-선 회절 분석기 (XRD) and fourier transform infrared spectroscopy (FT-IR)를 사용하여 이화학적 특성을 규명하였다. SEM과 TEM을 이용하여 입자의 크기와 형태를 분석한 결과 구형의 난각나노분말 입자가 응집되어 균일하게 분산되어있는 모습을 관찰하였다. 수분흡수 분석에서는 난각나노분말 (NPES)이 일반난각분말 보다 높은 흡수성을 나타내었으며, XRD 분석을 통하여 난각나노분말과 난각일반분말에 칼사이트를 포함하고 있는 것을 확인하였다. 난각나노분말과 난각일반분말의 화학적 조성은 염화칼슘 유무에 따른 큰 연관성이 있음이 나타났다. 이와 같은 결과에서 NPES가 식품 또는 의료용 소재로써, 인체에 무해하며 체내에 적응이 용이할 것으로 판단되었다.
두 번째 실험에서는 난소절제된 흰쥐를 대상으로 나노난각분말의 항-골다공증 효과에 대한 연구를 하였다. 암컷 흰쥐 7주령을 대상으로 난소절제수술을 실시한 뒤 4개의 그룹을 다음과 같이 나누었다. 모의 수술을 실행한 정상 대조군 (sham), 난소를 적출한 골다골증 유발 대조군 (OVX), 난각나노분말 (5g/kg body weight), 난각일반분말 (5g/kg body weight) 그룹으로 나누어 시료를 60일 동안 경구투여 하였다. 골밀도 측정 결과, 대조군과 비교하여 난각나노분말그룹의 경우 6.6 %, 난각일반분말그룹에서는 2.2 % 증가하였다. 난각나노분말 의 골소주는 대조군보다 12.4 %, 섬유주 수는 7.5 % 높았으나, 섬유주의 분리는 대조군 보다 14.5 % 낮았다. 혈청 분석 결과 난각나노분말에서 osteocalcin 농도는 대조군보다 22.4% 높았으며, 정상 대조군과는 차이가 없었다. 요 분석결과, 대조군과 비교하여 난각나노분말의 deoxypyridinoline 농도는 43.7 %, N-teleopeptide of type I collagen 농도는 87% 낮았다. 위에서와 같은 결과를 통해서 난각나노분말은 난소절제 흰쥐의 골다공증에 유의적인 예방효과가 있는 것으로 나타났다.
세 번째 실험에서는 난각나노분말 (0.15, 0.30, 0.45 %)을 첨가한 요구르트의 5 °C 에서 16일 동안 저장 품질변화와 안정성을 측정하였다. pH측정결과 난각나노분말 요구르트의 pH는4.31 - 4.66 이었으며, 대조군의 pH는 4.15 - 4.44이었다. 이는 나노난각분말 첨가 요구르트가 대조군과 비교하였을 때 비교적 안정한 것으로 나타났다. 난각나노분말의 유산균수는 저장기간 동안 6.56×108에서 8.56×108 CFU/ml으로 다소 증가하였으며, 대조군에 비해 낮았으나 유의적이지 않았다. 색도측정 결과 나노난각분말의 L, a, b 값은 유의적인 차이를 보이지 않았다 (p>0.05). 묘사분석을 통한 나노난각분말 첨가한 요구르트의 관능검사 결과 신 맛 (sourness)은 대조군에 비해 유의적으로 낮았으며, 떫은 맛 (astringency)은 유의적으로 높았다. 난각나노분말의 향과 맛을 나타내는Calcite값은 Control 값과 비교 하였을 때 나노난각분말 첨가 요구르트가 높았다. 그러나 전체적인 기호도 평가에서 나노난각분말 첨가 요구르트와 대조군에서 유의적 차이가 관찰되지 않았다 (p>0.05). 위와 같은 결과를 바탕으로 난각나노분말을 요구르트에 첨가할 경우 0.15 0.30 % 농도가 적합한 것으로 사료된다.
결론적으로, 난각나노분말은 난소절제 흰쥐의 골다공증모델에서 뛰어난 생체 적합성과 항-골다공증효과를 나타냈다. 또한 요구르트에 적용하였을 때, 요구르트의 품질의 영향을 주지 않았으며, 저장기간 동안 안정성은 향상되었다. 그러므로, 난각나노분말은 폐경기 이후 골다공증과 골 관련 질환에 치료효과가 있을 것으로 기대되며, 난각의 나노분말화는 난각의 생체이용률을 증가시켜, 난각나노분말은 식품에 적용하기 적합한 기능성소재로 사료된다.

다국어 초록 (Multilingual Abstract)

Abstract

Physicochemical Characteristics and Effects of Nanopowdered Eggshell on Ovariectomy-Induced Osteoporosis in Rats and its Application into Yogurt


Mohammad Al Mijan
Department of Food Science and Technology
Sejong University

Nanotechnology is among the few fast growing and promising technologies that are receiving a great deal of attention for potential application in food and pharmaceutical industries. Application of nanotechnology to enhance the bioavailability and functionality of different nutritional elements in food and beverages has already become familiar. A growing interest is being focused upon the application of nanotechnology in food research with a view to improve the bioavailability of nutritional ingredients. Hence, this study has investigated the physicochemical characteristics and antiosteoporotic effects of nanopowdered eggshell (NPES) in the ovariectomized rats and the possibility of its application into yogurt.
Part I of the study characterized the physicochemical properties of NPES using a field emission scanning electron microscope (FE-SEM), a transmission electron microscope (TEM), a particle size analyzer, a dynamic vapor sorption (DVS) analyzer, an X-ray diffractometer (XRD) and a fourier transform infrared spectroscopy (FT-IR). Homogeneous, aggregated and spherical shaped crystals of NPES particles were observed through SEM and TEM. The average particle sizes of NPES and powdered eggshell (PES) were 202.3±28.9 nm and 96.31±79.37µm, respectively. Zeta potentials of nanopowdered eggshell (-15.41 mV) and powdered eggshell (-22.66 mV) suggesting that both of the particles were moderately stable in colloidal system. Moisture sorption analyses indicated a higher water adsorption capacity of NPES than the PES. XRD analyses confirmed the presence of calcite in both NPES and PES. The chemical composition of the NPES and PES particles were strongly associated with the presence of calcium carbonate from the FT-IR spectra. The physicochemical properties determined in this study indicate that NPES particles possessed improved biocompatibility without any phase change. Hence, eggshell nanopowder could be utilized as a functional component in nutraceutical foods.
In part II, the preventive effects of NPES on postmenopausal osteoporosis were investigated in ovariectomized rat model. Seven-week old female rats were ovariectomized and assigned to 4 groups: sham operated (sham), ovariectomized rats treated with vehicle (OVX), ovariectomized rats treated with NPES (5g/kg body wt), and PES (5g/kg body wt). The rats fed with NPES and PES exhibited 6.6% and 2.2% greater bone mineral density (BMD) than the OVX rats. Femoral microstructure manifested that the NPES group had a remarkably greater trabecular area than PES and OVX groups. Investigation of the trabecular bone in NPES ingested rats revealed a 12.4% higher bone volume (BV/TV), 7.5% higher trabecular number (Tb.N), and 14.5% lower trabecular separation (Tb.Sp) compared to OVX rats. Serum analysis demonstrated that NPES fed rats showed a significantly higher (22.4%) osteocalcin level than the OVX rats and the concentration of osteocalcin in NPES fed rats resembled that of sham rats. Analysis of urine in NPES supplemented rats revealed a 43.7% lower deoxypyridinoline (DPD) and 87% lower N-teleopeptide of type I collagen (NTX) than OVX rats. Collectively, these results indicated that NPES had significant pharmaceutical effects in preventing the ovariectomy induced severe bone loss in rats which evidenced that this ingredient could be an alternative treatment for postmenopausal osteoporosis.
In part III, yogurt samples supplemented with NPES (0.15-0.45%) were examined for their quality and stability during 16 d storage at 5°C. The pH values in NPES supplemented yogurts ranged from 4.31 to 4.66 and the pH value in the control yogurt ranged from 4.15 to 4.44 which implied a substantial effect of NPES to improve the yogurt stability. The population of lactic acid bacteria (LAB) in NPES-added yogurts was little lower than that in control yogurt and were ranged from 6.56X108 to 8.56×108 cfu/mL. Color analysis showed that the L*, a* and b* values were not significantly altered by the addition of NPES. Sensory evaluation revealed that NPES-added yogurts showed a notably lower sourness score and a higher astringency score than the control one. Calcite score was found somewhat higher in NPES supplemented yogurt compared to the control one. However, overall acceptability did not differ significantly between the control and the NPES supplemented yogurts. Based on the results above, it is worth suggesting that the concentration (0.15% to 0.3%) of NPES can be used to formulate NPES supplemented yogurt.
To conclude, nanogrinding did not make any phase change in the chemical composition while it improved the physicochemical properties of eggshell powder. A powerful antiosteoporotic effect of eggshell nanopowder has been demonstrated in the ovariectomized osteoporotic rat models. Due to the addition of NPES, the quality of yogurts did not deteriorate considerably while it rather improved the yogurt stability. Therefore, NPES has the potential to treat postmenopausal osteoporosis and other bone related diseases and can be considered as a functional material for food application.

Key words: Nanopowdered eggshell, physicochemical properties, postmenopausal osteoporosis, ovariectomized rats, nanopowdered eggshell-supplemented yogurt.

목차 (Table of Contents)

• Table of contents
• Abstract 1
• Part 1: Physicochemical properties of nanopowdered eggshell 5
• 1. Introduction 5
• 2. Materials and methods 7
• 2.1 Materials 7
• 2.2 Physicochemical analysis 7
• 2.2.1 Electron microscopy 7
• 2.2.2 Particle size distribution 8
• 2.2.3 Zeta potential 8
• 2.2.4 Moisture sorption analysis 8
• 2.2.5 X-ray diffraction 9
• 2.2.6 Fourier transform infrared spectroscopy 9
• 3. Results and discussion 10
• 3.1 Particle morphology 10
• 3.2 Particle size distribution 14
• 3.3 Zeta potential 16
• 3.4 Moisture sorption analysis 18
• 3.5 X-ray diffraction 20
• 3.6 Fourier transform infrared spectroscopy 22
• 4. Conclusion 24
• Part II: Effect of nanopowdered eggshell on postmenopausal osteoporosis: a rat study 25
• 1. Introduction 25
• 2. Materials and methods 28
• 2.1 Animal care and treatments 28
• 2.2 Sample collection and storage 29
• 2.3 Bone analyses 29
• 2.3.1 Bone Ca and P measurement 29
• 2.3.2 Bone microstructure assessment 30
• 2.4 Biochemical assays 31
• 2.4.1 Alkaline phosphatase, Ca and P in serum 31
• 2.4.2 Creatinine, Ca and P in urine 31
• 2.4.3 Ca and P in liver, kidney and feces 31
• 2.4.4 Serum osteocalcin 32
• 2.4.5 Urinary deoxypyridinoline 32
• 2.4.6 Urinary N-telopeptides of type I collagen 32
• 2.5 Statistical analysis 32
• 3. Results and discussion 34
• 3.1 Body weights 34
• 3.2 Analyses of bone 36
• 3.2.1 Femoral calcium and phosphorous contents and the wet weight and length
• of femur 36
• 3.2.2 Bone mineral density 38
• 3.2.3 Bone µCT evaluation 40
• 3.3 Biochemical assays 43
• 3.3.1 Calcium and phosphorous content in serum, urine and feces 43
• 3.3.2 Osteocalcin and alkaline phosphatase in serum 46
• 3.3.3 Deoxypyridinoline, N-teleopeptide of type I collagen in urine 49
• 3.3.4 Creatinine level in urine 52
• 4. Conclusion 54
• Part III: Physicochemical and sensorial properties of nanopowdered eggshell- supplemented yogurt 55
• 1. Introduction 55
• 2. Materials and methods 58
• 2.1 Materials 58
• 2.2 Preparation of nanopowdered and powdered eggshell supplemented yogurts 58
• 2.3 pH 59
• 2.4 Viscosity 59
• 2.5 Lactic acid bacteria (LAB) 59
• 2.6 Color 60
• 2.7 Sensory analysis 60
• 2.8 Statistical analysis 60
• 3. Results and discussion 61
• 3.1 pH 61
• 3.2 Viscosity 63
• 3.3 Lactic acid bacteria 65
• 3.4 Color analysis 67
• 3.5 Sensory evaluation 69
• 4. Conclusion 73
• References 74