黑木耳黑色素对细菌群体感应调控行为的抑制及其抗氧化活性的研究
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摘要
黑木耳(Auricularia auricula)以“黑”而出名,但有关黑木耳子实体中黑色素的研究目前却鲜见报道。黑色素是一类结构复杂多样的多聚芳香族化合物的总称,它不仅是一类光保护剂、抗辐射剂、鳌合剂、生物抗氧化剂和免疫促进剂,而且还可用作生物半导体材料和光电传递材料,黑色素还具有抗蛇毒、抗癌、抑制艾滋病毒复制、治疗帕金森症等作用,因此黑色素在医药、化妆品、食品、电子等方面有着广泛的应用前景。目前,黑色素的生产主要依靠化学合成,而源自动植物的天然色素由于受到季节、气候及地理区域限制等,不能满足实际需求。黑木耳是我国珍贵的药食两用菌,其黑色素安全性极高,应用范围很广,开展黑木耳色素的提取纯化及其生物活性研究,特别是对群体感应调控行为的抑制作用及抗氧化活性研究,无论是对黑木耳的高附加值综合利用和深加工开发,还是对黑色素的深入研究都具有十分重要的理论意义和实用价值。主要研究结果如下：
1.采用超声波辅助的碱提酸沉法提取黑木耳子实体黑色素,并采用强酸水解、有机溶剂洗涤和反复沉淀的方法得到了纯化的黑木耳黑色素。对纯化后的黑木耳黑色素通过化学测试、紫外光谱分析、红外光谱分析、电子顺磁共振波谱分析和元素分析进行了鉴定。结果表明,黑木耳黑色素不溶于水和常见的有机溶剂,但在1.5mol/L NaOH溶液中完全溶解；黑木耳黑色素的最大吸收波长在210nm,黑木耳黑色素的吸光度的对数对波长作图斜率为-0.0028；红外光谱中在3287.6cm-1附近和1619.4cm-1附近各有一个强的吸收带；电子顺磁共振波谱为典型的单线一次微商波谱,无超精细结构,g=2.0042；黑色素的S：N为0.08。
2.采用报告平板检测法和紫色杆菌素提取法研究了黑木耳黑色素对紫色杆菌(Chromobacterium violaceum CV026)群体感应系统的抑制作用。结果表明,黑木耳黑色素可以有效抑制C.violaceum CV026紫色杆菌素的产生,其抑制作用与黑色素浓度呈明显的量效关系,但是对C.violaceum CV026的生长无明显的抑制。
3.铜绿假单胞菌(Pseudomonas aeruginosa PAO1)浮游现象已经被公认是群体感应调控的行为。我们采用双层平板法研究了黑木耳黑色素对铜绿假单胞菌浮游现象的影响。结果表明,黑木耳黑色素在低于P. aeruginosa PAO1最低抑菌浓度下,可以有效抑制P. aerugionsa PAO1的浮游现象。随着浓度的增大,抑制率从21.06%到94.25%。
4.通过定性和定量两种方法检测了黑木耳黑色素对大肠杆菌(Escherichia coli K-12、.铜绿假单胞菌(P.aeruginosa PAO1)以及荧光假单胞菌(P.fluorescens P-3)形成生物膜能力的影响。结果表明,黑木耳黑色素在低于最低抑菌浓度下(sub-MIC),可以明显抑制3株细菌生物膜的形成,其抑制作用与黑色素浓度呈现明显的量效关系。当黑木耳黑色素浓度达到80μg/mL时,其对E. coli K-12、P. aeruginosa PAO1和P.fluorescens P-3生物膜形成的抑制率分别达到73.78%、53.01%和55.58%。
5.研究了黑木耳黑色素结合防腐剂山梨酸钾、ε-多聚赖氨酸和Nisin同时使用对E. coli K-12、P. aeruginosa PAO1以及P.fluorescens P-3生物膜的影响,结果表明,在低于最低抑菌浓度下,黑木耳黑色素可显著降低防腐剂对3株供试菌的最低生物膜消除浓度。采用激光共聚焦显微镜观察证实,80μg/mL的黑木耳黑色素对大肠杆菌、铜绿假单胞菌和荧光假单胞菌成熟生物膜有不同程度的破坏作用,其破坏率分别达到41.99%、36.28%和40.25%。
6.通过DPPH自由基、超氧自由基以及羟自由基的清除试验发现黑木耳黑色素具有清除体外自由基的活性,其活性在一定浓度范围内随着浓度的增大而增强,对羟基自由基清除活性尤为明显。
综上所述,采用超声波辅助的碱提酸沉法提取并采用强酸水解、有机溶剂洗涤和反复沉淀纯化的黑木耳黑色素是一种真黑色素(Eumelanins),具有典型的天然真黑色素溶解性、紫外光谱、红外光谱、电子顺磁共振波谱特征。纯化的黑木耳黑色素对细菌群体感应调控行为(紫色杆菌色素产生、铜绿假单胞菌浮游现象以及细菌生物膜形成)有显著抑制作用并对体外DPPH自由基、超氧自由基以及羟自由基有明显清除效果。因此,黑木耳黑色素有作为天然的群体感应抑制剂和抗氧化剂的潜力,有望应用于食品和医药等工业领域。
Auricularia auricula is well known for black, but there is sparse for the characterization and biological activity studies on A. auricula melanin. Melains are not only a kind of photoprotectants, chelators, bio-antioxidants and immuno-stimulators but also a kind of amorphous organic semiconductors, photoconductors and electrical conductors. They have many bio-activities such as anti-venom, antitumor, inhibiting HIV replication and protection against Parkinson disease. So they have a vast range of prospects for making use in functional food industry, cosmetic, medicine, pharmacy, bioelectronics and other fields. At present, the production of melanin is mainly rely on the synthesis, and natural melanin that is got from plants and animals can not meet the actual demand because of seasonal, climatic and geographical constraints. A. auricula is a precious edible fungus both for medicine and food. A. auricula has widely application and high security. To study the extraction, purification and biological activity of A. auricula melanin is of great importance in theoretical and practical. It can improve its added-value and deep processing, and it is also useful for further investigations of melanin. The main results were as follows:
1. A. auricula melanin was extracted with NaOH and HCl. Purified melanin was obtained by using strong acid hydrolysis and washing by organic solvents. It was characterized by chemical tests, ultraviolet-visible (UV) absorption spectrum, infrared (IR) spectroscopy, electron paramagnetic resonance (EPR) and elemental analysis. The melanin was insoluble in both water and organic solvents, and dissolved in1.5mol/L NaOH. The overall characteristic absorption peak was showed at210nm, and the log of optical density of the melanin solution when plotted against wavelength produces a linear curve with negative slopes of-0.0028. The IR spectrum of the melanin exhibited broad absorption band at3287.6cm-1and1619.4cm-1. The EPR spectrum was a typical single first derivative spectrum, and the melanin did not have hyperfine structure. The peak at2.0042(G value) of the pigment indicated it is melanin. The S:N of the melanin is0.08.
2. The biological methods of reporter agar diffusion assay and extraction of violacein were used to determine the capacity of QSI for A. auricula melanin. The results showed that A. auricula melanin did not inhibit the growth of C. violaceum CV026, but it can inhibit the production of violacein. Inhibition with the concentration of the melanin showed significant dose-effect relationship.
3. The swarming motility in P. aeruginosa PAO1was recognized as QS regulation of behavior. It was screened using agar diffusion method. The results showed that A. auricula melanin reduced swarming motility in P. aerugionsa PAO1at sub-MIC. As the concentration increases, the inhibition rate was from21.06%to94.25%.
4. The influence on biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin was checked. The results of qualitative and quantitative methods showed that the A. auricula melanin can inhibit biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3at sub-MIC. Furthermore, we studied the inhibition of biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin at different concentrations and found that inhibition with the concentration of the melanin showed significant dose-effect relationship. The percentage inhibition of biofilm formation by A. auricula melanin at a concentration of80μg/mL were73.78%,53.01%and55.58%, respectively.
5. The influence on mature biofilm of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin with potassium sorbate, ε-polylysine and Nisin was checked. Sub-MIC of A. auricula melanin can reduce the minimum biofilm bacterium elimination concentration (MBEC) of the preservative to E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3significantly. The confocal laser scanning microscopy (CLSM) images showed that the80μg/mL of A. auricula melanin can damage the mature biofilm of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3. The failure rate were41.99%,36.28%and40.25%, respectively.
6. Using DPPH, superoxide and hydroxyl radical scavenging assay, the A. auricula melanin exhibited a moderate antioxidant activity. It is clearly demonstrated that free radical scavenging increases with increasing melanin concentration. A. auricula melanin had strong scavenging activity on hydroxyl radical.
Therefore, A. auricula melanin that was extracted with NaOH and HC1and purified by using strong acid hydrolysis and organic solvent was eumelanins. It had the typical characteristics of the natural melanin (including solubility, UV spectroscopy, IR spectroscopy, EPR). Purified A. auricula melanin can inhibit the production of violacein of C, violaceum CV026, reduce swarming motility in P. aerugionsa PAO1and inhibit biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3. A. auricula melanin also can scavenge DPPH, superoxide and hydroxyl. A. auricula melanin can be used as a natural QSI and antioxidant in food and pharmaceutical industries.
1.采用超声波辅助的碱提酸沉法提取黑木耳子实体黑色素,并采用强酸水解、有机溶剂洗涤和反复沉淀的方法得到了纯化的黑木耳黑色素。对纯化后的黑木耳黑色素通过化学测试、紫外光谱分析、红外光谱分析、电子顺磁共振波谱分析和元素分析进行了鉴定。结果表明,黑木耳黑色素不溶于水和常见的有机溶剂,但在1.5mol/L NaOH溶液中完全溶解；黑木耳黑色素的最大吸收波长在210nm,黑木耳黑色素的吸光度的对数对波长作图斜率为-0.0028；红外光谱中在3287.6cm-1附近和1619.4cm-1附近各有一个强的吸收带；电子顺磁共振波谱为典型的单线一次微商波谱,无超精细结构,g=2.0042；黑色素的S：N为0.08。
2.采用报告平板检测法和紫色杆菌素提取法研究了黑木耳黑色素对紫色杆菌(Chromobacterium violaceum CV026)群体感应系统的抑制作用。结果表明,黑木耳黑色素可以有效抑制C.violaceum CV026紫色杆菌素的产生,其抑制作用与黑色素浓度呈明显的量效关系,但是对C.violaceum CV026的生长无明显的抑制。
3.铜绿假单胞菌(Pseudomonas aeruginosa PAO1)浮游现象已经被公认是群体感应调控的行为。我们采用双层平板法研究了黑木耳黑色素对铜绿假单胞菌浮游现象的影响。结果表明,黑木耳黑色素在低于P. aeruginosa PAO1最低抑菌浓度下,可以有效抑制P. aerugionsa PAO1的浮游现象。随着浓度的增大,抑制率从21.06%到94.25%。
4.通过定性和定量两种方法检测了黑木耳黑色素对大肠杆菌(Escherichia coli K-12、.铜绿假单胞菌(P.aeruginosa PAO1)以及荧光假单胞菌(P.fluorescens P-3)形成生物膜能力的影响。结果表明,黑木耳黑色素在低于最低抑菌浓度下(sub-MIC),可以明显抑制3株细菌生物膜的形成,其抑制作用与黑色素浓度呈现明显的量效关系。当黑木耳黑色素浓度达到80μg/mL时,其对E. coli K-12、P. aeruginosa PAO1和P.fluorescens P-3生物膜形成的抑制率分别达到73.78%、53.01%和55.58%。
5.研究了黑木耳黑色素结合防腐剂山梨酸钾、ε-多聚赖氨酸和Nisin同时使用对E. coli K-12、P. aeruginosa PAO1以及P.fluorescens P-3生物膜的影响,结果表明,在低于最低抑菌浓度下,黑木耳黑色素可显著降低防腐剂对3株供试菌的最低生物膜消除浓度。采用激光共聚焦显微镜观察证实,80μg/mL的黑木耳黑色素对大肠杆菌、铜绿假单胞菌和荧光假单胞菌成熟生物膜有不同程度的破坏作用,其破坏率分别达到41.99%、36.28%和40.25%。
6.通过DPPH自由基、超氧自由基以及羟自由基的清除试验发现黑木耳黑色素具有清除体外自由基的活性,其活性在一定浓度范围内随着浓度的增大而增强,对羟基自由基清除活性尤为明显。
综上所述,采用超声波辅助的碱提酸沉法提取并采用强酸水解、有机溶剂洗涤和反复沉淀纯化的黑木耳黑色素是一种真黑色素(Eumelanins),具有典型的天然真黑色素溶解性、紫外光谱、红外光谱、电子顺磁共振波谱特征。纯化的黑木耳黑色素对细菌群体感应调控行为(紫色杆菌色素产生、铜绿假单胞菌浮游现象以及细菌生物膜形成)有显著抑制作用并对体外DPPH自由基、超氧自由基以及羟自由基有明显清除效果。因此,黑木耳黑色素有作为天然的群体感应抑制剂和抗氧化剂的潜力,有望应用于食品和医药等工业领域。
Auricularia auricula is well known for black, but there is sparse for the characterization and biological activity studies on A. auricula melanin. Melains are not only a kind of photoprotectants, chelators, bio-antioxidants and immuno-stimulators but also a kind of amorphous organic semiconductors, photoconductors and electrical conductors. They have many bio-activities such as anti-venom, antitumor, inhibiting HIV replication and protection against Parkinson disease. So they have a vast range of prospects for making use in functional food industry, cosmetic, medicine, pharmacy, bioelectronics and other fields. At present, the production of melanin is mainly rely on the synthesis, and natural melanin that is got from plants and animals can not meet the actual demand because of seasonal, climatic and geographical constraints. A. auricula is a precious edible fungus both for medicine and food. A. auricula has widely application and high security. To study the extraction, purification and biological activity of A. auricula melanin is of great importance in theoretical and practical. It can improve its added-value and deep processing, and it is also useful for further investigations of melanin. The main results were as follows:
1. A. auricula melanin was extracted with NaOH and HCl. Purified melanin was obtained by using strong acid hydrolysis and washing by organic solvents. It was characterized by chemical tests, ultraviolet-visible (UV) absorption spectrum, infrared (IR) spectroscopy, electron paramagnetic resonance (EPR) and elemental analysis. The melanin was insoluble in both water and organic solvents, and dissolved in1.5mol/L NaOH. The overall characteristic absorption peak was showed at210nm, and the log of optical density of the melanin solution when plotted against wavelength produces a linear curve with negative slopes of-0.0028. The IR spectrum of the melanin exhibited broad absorption band at3287.6cm-1and1619.4cm-1. The EPR spectrum was a typical single first derivative spectrum, and the melanin did not have hyperfine structure. The peak at2.0042(G value) of the pigment indicated it is melanin. The S:N of the melanin is0.08.
2. The biological methods of reporter agar diffusion assay and extraction of violacein were used to determine the capacity of QSI for A. auricula melanin. The results showed that A. auricula melanin did not inhibit the growth of C. violaceum CV026, but it can inhibit the production of violacein. Inhibition with the concentration of the melanin showed significant dose-effect relationship.
3. The swarming motility in P. aeruginosa PAO1was recognized as QS regulation of behavior. It was screened using agar diffusion method. The results showed that A. auricula melanin reduced swarming motility in P. aerugionsa PAO1at sub-MIC. As the concentration increases, the inhibition rate was from21.06%to94.25%.
4. The influence on biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin was checked. The results of qualitative and quantitative methods showed that the A. auricula melanin can inhibit biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3at sub-MIC. Furthermore, we studied the inhibition of biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin at different concentrations and found that inhibition with the concentration of the melanin showed significant dose-effect relationship. The percentage inhibition of biofilm formation by A. auricula melanin at a concentration of80μg/mL were73.78%,53.01%and55.58%, respectively.
5. The influence on mature biofilm of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin with potassium sorbate, ε-polylysine and Nisin was checked. Sub-MIC of A. auricula melanin can reduce the minimum biofilm bacterium elimination concentration (MBEC) of the preservative to E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3significantly. The confocal laser scanning microscopy (CLSM) images showed that the80μg/mL of A. auricula melanin can damage the mature biofilm of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3. The failure rate were41.99%,36.28%and40.25%, respectively.
6. Using DPPH, superoxide and hydroxyl radical scavenging assay, the A. auricula melanin exhibited a moderate antioxidant activity. It is clearly demonstrated that free radical scavenging increases with increasing melanin concentration. A. auricula melanin had strong scavenging activity on hydroxyl radical.
Therefore, A. auricula melanin that was extracted with NaOH and HC1and purified by using strong acid hydrolysis and organic solvent was eumelanins. It had the typical characteristics of the natural melanin (including solubility, UV spectroscopy, IR spectroscopy, EPR). Purified A. auricula melanin can inhibit the production of violacein of C, violaceum CV026, reduce swarming motility in P. aerugionsa PAO1and inhibit biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3. A. auricula melanin also can scavenge DPPH, superoxide and hydroxyl. A. auricula melanin can be used as a natural QSI and antioxidant in food and pharmaceutical industries.
引文
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