Characterization of Melanoidins based on Amadori and Heyns Rearrangement Products by Racemization in Maillard Reaction Scheme

국문 초록 (Abstract)

본 연구에서는 환원당인 포도당, 과당과 L형, D형 아미노산에 의해 형성되는 아마도리 화합물과 헤인스화합물의 비효소적 갈변 과정을 통해 형성되는 멜라노이딘의 특성을 규명하였다. 따라서 본 연구는 (1) 모델 마일라드 시스템을 통한 당의 엔올화와 아미노산의 라세미화에서의 pH의 효과, (2) 투석 과정을 통해 형성된 멜라노이딘의 항산화 효과에서의 pH의 효과, (3) 모델 시스템을 통해 형성된 멜라노이딘의 색 형성에서의 pH의 효과, (4) FT-IR과 XRD를 통한 멜라노이딘의 구조적 특성 평가, (5) 모델 마일라드 시스템에서의 furfural compounds의 형성 평가를 연구하였으며 그 결과는 다음과 같다.
1. pH 증가에 따라 당의 엔올화 반응은 포도당 시스템에서 더 쉽게 나타났으며 아미노산의 라세미화는 L형의 아미노산이 D형으로 전환되는 비율이 큰 것으로 나타났다. 또한 pH 증가에 따라 멜라노이딘의 형성은 증가하였으며 이성질체에 따른 멜라노이딘 형성의 차이는 없었다.
2. 멜라노이딘의 항산화 효과는 chelating activities를 제외하고 투석 후 급격히 증가하였으며 Glc(Fru)/Gly system으로부터 형성된멜라노이딘은 pH 7.0에서 효과가 가장 크게 나타난 반면 Glc(Fru)/L(D)-lysine 으로부터 형성된 멜라노이딘은 pH 증가에 따라 항산화 효과가 감소하는 것으로 나타났다. 또한, 이성질체에 따른 멜라노이딘의 항산화 효과의 차이는 없었다.
3. 멜라노이딘의 extinction coefficients는 pH 7.0에서 이성질체 간의 차이가 적은 것으로 나타났으며 색 형성 평가 결과 멜라노이딘의 주된 색소는 파랑색으로 나타났고 pH 증가에 따라 노란색이 증가하는 것으로 나타났다. 또한, 이성질체에 따른 멜라노이딘의 색 형성의 차이는 없었다.
4. FT-IR 결과, 멜라노이딘의 화학구조는 상당량의 OH, NH, CH2, CH3, amide I, amide II 그리고 amide III groups이 존재하며 aromatic furanic compound의 결합도 존재하는 것으로 나타났다. 또한 XRD 결과, 멜라노이딘의 주된 결정상은 10 ~ 21°(2θ) 범위에서 존재하며 pH 증가에 따라 멜라노이딘의 결정상의 강도는 증가하였으며 이성질체의 차이는 없었다.
5. 모델 마일라드 시스템을 통해 형성된 furfural compounds의 평가 결과, 산성조건에서는 hydroymethylfurfura1 (HMF)의 형성이 증가하였으며 염기성조건에서는 furfuryl alcohol (FFA)와 2-furaldehyde (F)가 형성되는 것으로 나타났다.
이상의 결과, 아마도리 화합물과 헤인스 화합물의 비효소적 갈변 과정을 통해 형성되는 멜라노이딘은 pH 변화에 따라 그 특성이 다르게 나타나며 특히 저분자량의 멜라노이딘의 경우 chelating activity가 더 우수하였다. 또한 마일라드 반응의 연구에 있어서 당의 엔올화와 아미노산의 라세미화 반응은 또 하나의 중요한 영향 요인으로 여겨지며 멜라노이딘의 기능적 측면에서 제과제빵과 장류산업에 적용이 가능할 것으로 사려된다.

다국어 초록 (Multilingual Abstract)

The objective of this study was to investigate the characterization of melanoidins due to the Maillard reactions, which based on enolization and racemization of glucose (or fructose) with D (or L) forms of amino acid while heating as a function of pH.
In the present study, remarkable enolization of sugars was observed in the course of the Maillard reaction. The degree of sugar enolization was increased as the pH increased, which was especially higher in the fructose system than in the glucose system. On the other hand, enolization of sugars with amino acid during heating was higher in the glucose system than in the fructose system. In addition, the racemization of amino acid was higher in glucose-based system. The formation of isomer was easier with the L-amino acid than the D-amino acid. The relative amounts of isomers in fructose with lysine were increased with the increase of pH, regardless of enantiomer form. While glucose with D or L lysine systems were not affected by the pH. Development of browning during heating was more significant (p<0.05) in the sugar-amino acid model system especially with fructose compared to glucose solution alone, which was affected by the pH. The L- and D- isomers showed different absorption with similar shape in the UV-visible spectra. Every peak has a stable absorbance in the range of 260 to 320 nm, which is the characteristic of melanoidins.
In addition, antioxidative activities of melanoidins were different before and after dialysis. Antioxidative activities of melanoidins after dialysis were significantly (p<0.05) higher than those before dialysis, except for ferrous ion chelating activities. At pH 7, antioxidative activities of melanoidins from glucose with the glysine system were greater compared to those at pH 4 and pH 10. Moreover, the difference in the extinction coefficients of melanoidins with amino acids enantiomers became less significant at pH 7.
On the other hand, antioxidative activities of melanoidins with lysine were decreased as the pH increased, regardless of the type of sugar. In addition, melanoidins that formed with D-isomers had similar antioxidative activities to those of L-isomers. Most of the melanoidins were shown as blue-green locus at the wavelength of 475 nm. The color of melanoidins was grouped within a narrow band from the blue-green spectrum to the direction of yellow-red locus as the pH increased. Especially, the yellowness increased in the glucose-based system with the increase in pH. Furthermore, the difference became less significant with amino acids enantiomer. The structure of melanoidins was composed of OH, NH, CH2, CH3, amide I, amide II and amide III groups. Particularly, the structure of melanoidins formed from the glucose-based system had a stronger union than that of the fructose-based system. The chemical composition of melanoidins was also observed in the band of aromatic furanic or conjugated compounds. The crystallinity of melanoidins products was formed in the 10-21° (2θ) range. As the pH increased, the intensity of the crystallinity of melanoidins increased as well. Similar crystallinity of melanoidins was formed from D or L -isomer with a different intensity. In order to quantify the furfural compounds as the indicator of the advanced Maillard reaction, hydroymethylfurfura1 (HMF) was formed in the acidic condition, while furfuryl alcohol (FFA) and 2-furaldehyde (F) were formed in the basic condition. The results of the present study indicate that the pH affected the enolization and racemization during the Maillard reaction, which caused the different characteristic of melanoidin. In addition, the results of this study can apply this functional aspect of melanoidin, particularly in the industries of bakery and fermented soy sauce. In the future, enolization and racemization must be considered in the kinetic study of Maillard reactions.

목차 (Table of Contents)

• I. Introduction = 1
• II. Background = 2
• 1. Chemistry of the Maillard reaction = 3
• 2. Factors affecting the rate of Maillard reaction = 23
• 3. The Lobry de Bruyn-Alberda van Ekenstein (LdB-AvE) transformation = 26
• 4. Mechanisms of the formation of free D-amino acids = 33
• III. Materials and methods = 40
• 1. Chemicals = 40
• 2. Preparation of Maillard reaction products (MRPs) = 41
• 3. Effect of pH on enolization and racemization reactions of glucose and fructose with amino acid enantiomers on heating and formation of melanoidins as a result of the Maillard reaction = 41
• 3.1. Determination of sugars in MRPs = 41
• 3.2. Derivatization of amino acids with 1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide (FDAA) in MRPs = 42
• 3.3. Determination of amino acids in MRPs = 42
• 3.4. Measurement of browning = 43
• 3.5. Wavelength spectra of melanoidins = 43
• 4. The antioxidant activity of melanoidins from glucose and fructose/amino acids enantiomers Maillard reaction before and after dialysis = 44
• 4.1. Dialysis = 44
• 4.2. Total ferric ions (Fe3+) reduction capability = 44
• 4.3. Ferrous (Fe2+) metal ions chelating activity = 44
• 4.4. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity = 45
• 4.5. Antioxidant capacity by ferric reducing/antioxidant power (FRAP) assay = 45
• 4.6. 2,2´-azinobis(3-ethylbenothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization assay = 46
• 5. Effect of pH on the color development of melanoidins from glucose and fructose/amino acids enantiomers Maillard reaction = 46
• 5.1. Spectrophotometric analysis = 46
• 5.2. Color measurements by spectrophotometer = 47
• 5.3. Color measurements by colorimeter = 47
• 6. Fourier transform-infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) characterization of melanoidins from the glucose and fructose/amino acids enantiomers Maillard reaction = 48
• 6.1. FT-IR analysis = 48
• 6.2. X-ray diffraction analysis = 49
• 7. Quantification of the furfural compounds, as indicator of the advanced Maillard reaction = 49
• 7.1. Determination of furfural compounds = 49
• 8. Statistical analysis = 49
• IV. Results and Discussion = 50
• 1. Effect of pH on enolization and racemization reactions of glucose and fructose with amino acid enantiomers on heating and formation of melanoidins as a result of the Maillard reaction = 50
• 1.1. Effect of pH on the loss and enolization of sugar in MRPs = 50
• 1.2. Effect of pH on the loss and racemization of amino acids in MRPs = 53
• 1.3. Browning and formation of melanoidins = 58
• 2. The antioxidant activity of melanoidins from glucose and fructose/amino acids enantiomers Maillard reaction before and after dialysis = 62
• 2.1. Total ferric ions (Fe3+) reduction capability using the potassium ferricyanide reduction method = 62
• 2.2. Ferrous (Fe2+) metal ions chelating activity = 65
• 2.3. DPPH radical scavenging activity = 68
• 2.4. Antioxidant capacity by ferric reducing/antioxidant power (FRAP) assay = 71
• 2.5. ABTS radical scavenging activity = 74
• 3. Effect of pH on the color development of melanoidins from glucose and fructose/amino acids enantiomers Maillard reaction = 77
• 3.1. Extinction coefficient = 77
• 3.2. Color measurements by spectrophotometer = 79
• 3.3. Color measurements by colorimeter = 82
• 4. FT-IR and XRD characterization of melanoidins from the glucose and fructose/amino acids enantiomers Maillard reaction = 89
• 4.1. FT-IR analysis of melanoidins = 90
• 4.2. X-ray diffraction pattern of melanoidins = 93
• 5. Quantification of the furfural compounds, as indicator of the advanced Maillard reaction = 95
• V. Conclusion = 103
• VI. References = 105
• 국문초록 = 118
• Acknowledgements = 119