赖氨酸修饰对梯棱羊肚菌黑色素结构和理化特性的影响

    Effects of lysine modification on the structural and physicochemical properties of melanin from Morchella importuna

    • 摘要: 为了进一步提高梯棱羊肚菌黑色素的提取率及溶解性,该研究采用单因素、Plackett-Burman试验、响应面试验对纤维素酶-超声波协同提取梯棱羊肚菌黑色素的提取工艺进行优化。通过赖氨酸修饰,并对修饰前后的梯棱羊肚菌黑色素进行结构表征、理化性质及稳定性分析。结果表明,在NaOH浓度为1.54 mol/L,纤维素酶添加量为20 mg/g,纤维素酶酶解时间为78.6 min,料液比为1:30,酶解温度为40 ℃,超声时间为80 min条件下提取的梯棱羊肚菌黑色素最优。未修饰的梯棱羊肚菌黑色素不溶于水,色价值为480.24,修饰后的黑色素溶解度为1 016 g/L,色价值为1 771.18,比未修饰的黑色素在溶解性、色价值方面均有所提高。此外,在不同的温度、光照、pH条件下,修饰前后的梯棱羊肚菌黑色素均比较稳定。以上研究结果为梯棱羊肚菌黑色素的高效提取及其产品的开发利用奠定了理论基础。

       

      Abstract: Melanin is a natural ingredient produced widely in edible fungi with excellent bioactivity. The edible fungi can provide a rich source of melanin. Morchella importuna (MIM), a precious edible and medicinal fungus, has been domesticated in China in recent years. However, the extraction of melanin from MIM has been rarely reported. Besides, the hydrophobicity of melanin can limit its applications. Hence, it is a high demand to efficiently extract and improve the water solubility by modification. Alkali-extraction and acid-precipitation with ultrasound-assisted approach can often be used to extract melanin. Furthermore, it has not yet been reported to extract melanin from MIM using cellulase and ultrasonic. In cellulase extraction, the cellulase can be used to degrade cellulose components in the microbial cell wall, in order to increase the extraction rate. In ultrasonic extraction, ultrasonic vibration can be used to make the microbial tissue structure form a cavity combine with its thermal effect. The better extraction of melanin can be obtained, in terms of the short extraction time and high extraction rate. It is expected to combine the cellulase and ultrasonic extraction for the active ingredients of natural products. In this study, the extraction process was optimized by single-factor, Plackett-Burman and response surface test, in order to improve the extraction rate of the melanin from MIM. The solubility of MIM was improved by modification with lysine. The structural and physicochemical properties of MIM were evaluated before and after modification, together with solubility and stability, using UV-visible absorption, FTIR spectra, scanning electronic microscope (SEM). The results showed that the highest extraction rate of MIM was achieved under the conditions of NaOH solution concentration of 1.54 mol/L, cellulase addition of 20 mg/g, cellulase digestion time of 78.6 min, material-liquid ratio of 1:30, digestion temperature of 40 °C, and ultrasound time of 80 min. The absorbance of modification was compared with four amino acids, in order to determine the optimal amino acid and mass ratio. The water solubility was significantly improved after lysine modification. The color values of MIM and lysine-modified Morchella importuna melanin (L-MIM) were 480.24 and 1 771.18, respectively. The solubility and color value of L-MIM were higher than those of MIM. The maximum absorption peaks of MIM and L-MIM were 230 and 219 nm, respectively. The absorption of MIM and L-MIM showed a decreasing trend with the increasing wavelength. It was also found that the MIM and L- MIM had no special absorption peaks at 260 and 280 nm, indicating less nucleic acids and proteins without impurity peaks. The FTIR spectra demonstrated the typical indole structure of melanin. SEM tests revealed that there were the smooth surface and uniform cubic crystal structure in MIM samples, whereas, the L-MIM samples were in the form of large lumps, with the raised surfaces that adhered to each other. The reason was that the particle adhesion was susceptible to moisture absorption in the L-MIM samples. In addition, both MIM and L-MIM were relatively stable under the temperature from 20 to 100 ℃, in the dark and natural light, while both were basically stable, when pH value from 8.0 to 12.0. It was found that Fe3+ had a significant impact on both MIM and L-MIM stability. These findings can provide the clear directions for their applications in functional foods.

       

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