Physicochemical Properties of Marine and Terrestrial Proteins and Their Association with Myotube Atrophy Attenuation

다국어 초록 (Multilingual Abstract)

Sarcopenia, characterized by the progressive loss of skeletal muscle mass and function with aging and physical inactivity, has become a major health concern. Protein intake is essential for maintaining muscle protein synthesis and preventing age-related muscle loss; however, the comparative effectiveness of different protein sources remains unclear.
This study examined the physicochemical properties of proteins from eight sources—marine (Pyropia yezoensis [P. yezoensis], Paralichthys olivaceus [P. olivaceus], Thunnus orientalis [T. orientalis], Haliotis discus [H. discus], and Crassostrea gigas [C. gigas]) and terrestrial (Glycine max (L.) Merr. [G. max], Gallus gallus [G. gallus] and Bos taurus [B. taurus])—and their associations with muscle function improvement and sarcopenia prevention. Protein hydrolysates were prepared through enzymatic hydrolysis with Alcalase and ethanol precipitation, followed by comprehensive characterization including amino acid composition, molecular weight distribution, secondary structure (FT-IR), ζ-potential, surface hydrophobicity, solubility, oil-holding capacity, emulsifying stability, and antioxidant capacity. Muscle differentiation and muscle atrophy prevention were evaluated using C2C12 myotube cells exposed to dexamethasone-induced atrophy. Statistical analysis (t-test and correlation analysis) was performed to compare muscle functional parameters: myotube coverage, fusion index, total nuclei, and myotube diameter. Lipidomics analysis was conducted to investigate lipid composition changes during muscle differentiation. Marine protein hydrolysates demonstrated significantly higher myotube coverage and fusion index compared to Terrestrial proteins (p<0.05), indicating superior muscle-promoting effects. Specific amino acids (tyrosine, phenylalanine, arginine, glutamic acid), peptide molecular weight ranges (700–899 Da), and secondary structural features (β-turn) showed strong correlations with muscle functional indices. Lipidomics analysis revealed that marine protein treatment induced favorable changes in structural lipids, including phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC), as well as the signaling lipid lysophosphatidic acid (LPA), thereby promoting cell membrane fluidity and myoblast fusion. These findings demonstrate that marine-derived proteins are more effective for sarcopenia prevention and muscle function improvement compared to terrestrial proteins, primarily through advantageous modulation of lipid metabolism and cellular signaling pathways.
This study scientifically demonstrated that, compared with terrestrial proteins, marine-derived proteins more effectively prevent muscle atrophy and improve muscle function by markedly increasing myotube coverage and fusion index in C2C12 cells and by regulating the expression of membrane structural lipids (PE, PG, LPC) and the signaling lipid LPA. These findings provide important evidence to guide the selection and development of protein-based foods targeting age-related muscle loss.

국문 초록 (Abstract)

근감소증은 노화와 신체활동 부족에 의해 진행성으로 나타나는 골격근 질량 및 기능 감소를 특징으로 하는 주요 건강 문제이다. 단백질 섭취는 근단백질 합성 유지와 노화 관련 근육 손실 예방에 필수적이지만, 다양한 단백질 원천 간의 효과 비교는 명확하지 않다.
본 연구는 8가지 수산물 및 농축산 단백질 원료(김, 콩, 광어, 닭, 참치, 소, 전복, 굴)의 물리화학적 특성과 근육 기능 개선 및 근위축 예방과의 연관성을 평가하였다. Alcalase 효소 가수분해 및 에탄올 침전을 통해 단백질 가수분해물을 제조하고, 아미노산 조성, 분자량 분포, 입자 크기, 2차 구조(FT-IR), 제타 전위, 표면 소수성, 용해도, 유분 보유력, 유화 안정성 및 항산화능을 종합적으로 분석하였다. C2C12 근관세포를 이용한 Dexamethasone 유도 위축 모델에서 근육 분화 및 근위축 예방 효과를 평가하고, Myotube coverage, Fusion index, Total nuclei, Myotube diameter 등의 근육 기능 지표를 통계적으로 비교하였다. 또한, 근육 분화 과정에서의 지질 조성 변화를 리피도믹스 분석으로 탐색하였다. 수산물 단백질 가수분해물은 농축산물 단백질에 비해 Myotube coverage 및 Fusion index에서 유의하게 높은 효과를 보여 근육 촉진에 우수함을 나타냈다 (p<0.05). 특정 아미노산 (Tyrosine, Phenylalanine, Arginine, Glutamic acid), 펩타이드 분자량 범위 (700–899 Da) 및 2차 구조 (β-turn)가 근육 기능과 강한 상관관계를 보였다. 리피도믹스 결과, 수산물 단백질 처리군은 세포막 유동성 및 근모세포 융합을 촉진하는 Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Lysophosphatidylcholine (LPC)와 같은 구조 지질 계열과 신호 지질 계열인 Lysophosphatidic acid (LPA)의 발현을 증가시키는 양상을 보였다.
본 연구는 수산 유래 단백질이 농축산 단백질보다 C2C12 근관세포에서 Myotube coverage와 Fusion index를 보다 유의미하게 증가시키고, 동시에 PE, PG, LPC 등 막 구조 지질과 LPA와 같은 신호 지질의 발현을 조절함으로써 근위축 예방과 근섬유 형성·융합 및 근육 기능 개선에 더 우수한 효과를 나타냄을 과학적으로 증명하였다. 이는 노화 관련 근육 감소 해결을 위한 단백질 식품 선택과 개발에 중요한 근거자료를 제공한다.

목차 (Table of Contents)

• I. INTRODUCTION 1
• II. MATERIALS AND METHODS 5
• 2-1. Materials 5
• 2-2. Preparaion of protein hydrolysates 5
• 2-3. Protein hydrolysate analysis 6
• 2.3.1 Measurement of protein content 6
• 2.3.2 Measurement of moisture content 6
• 2.3.3 Measurement of ash content 6
• 2.3.4 Color properties 7
• 2.3.5 Amino acid composition analysis 7
• 2.3.6 Molecular weight measurement 7
• 2.3.7 Fourier transform infrared spectroscopy (FT-IR) 8
• 2-4. Surface characteristics 8
• 2.4.1 Dynamic light scattering (DLS) & Scanning electron microscopy (SEM) measurements 8
• 2.4.2 ζ- potential 9
• 2.4.3 Surface hydrophobicity 9
• 2-5. Functional properties 9
• 2.5.1 Solubility 9
• 2.5.2 Oil holding capacity (OHC) 10
• 2.5.3 DPPH radical scavenging activity 10
• 2.5.4 Emulsifying capacity 10
• 2-6. Cell experiment 11
• 2.6.1 Cell culture and myotube differentiation 11
• 2.6.2 Immunofluorescence staining 11
• 2-7. Lipidomics 12
• 2.7.1 Lipidomics analysis procedure 12
• 2.7.2 Lipidomics data analysis 13
• 2-8. Statistical analysis 14
• 2.8.1 Independent two-sample t.test 14
• 2.8.2 Correlation test 16
• III. RESULTS 18
• 3-1. Proximate composition 18
• 3-2. Color 21
• 3-3. DLS & SEM 23
• 3-4. Amino acid composition of protein hydrolysates 26
• 3-5. Molecular weight distribution 28
• 3-6. FT-IR 31
• 3-7. ζ- potential 33
• 3-8. Surface hydrophobicity 34
• 3-9. Protein solubility 36
• 3-10. OHC 37
• 3-11. DPPH radical scavenging activity 39
• 3-12. Emulsifying stability 41
• 3-13. The protective effect of protein hydrolysates against dexamethasone-induced muscle atrophy 44
• 3-14. Independent two-sample t-test analysis 47
• 3-15. Correlation analysis between muscle function indicators and physicochemical properties 50
• 3-16. Lipidomics 56
• IV. DISCUSSION 60
• V. CONCLUSIONS 64
• VI. REFFERENCES 66