光谱法研究甲醇和盐酸对短梗霉黑色素的分离效果

应用紫外和红外光谱对甲醇和盐酸分离的短梗霉黑色素进行了分析。分析结果表明:甲醇和盐酸法分离的黑色素在紫外图谱215 nm处都有最大吸收峰,而甲醇法分离的黑色素其紫外图谱在260、280 nm处无吸收峰表明此法分离的黑色素不含核酸及蛋白质;红外图谱中,在3340 cm-1、1637 cm-1处有很强的吸收峰表现为黑色素的典型特征;同时在对照中发现盐酸多次处理后的黑色素显示较少的结构信息,说明盐酸沉降法有可能破坏黑色素的结构。由此选用甲醇作为沉淀剂有利于黑色素的纯化及确保其结构信息的完整,为进一步分析短梗霉黑色素的结构表征奠定了基础。     

短梗霉黑色素的分离纯化及结构的初步分析

采用热碱提取、水煮酸沉法从短梗霉发酵液中提取得到黑色素粗品,再经DMSO萃取、酸性甲醇(pH=2)沉淀得到不含多糖和蛋白的短梗霉黑色素。此黑色素不溶于水及常规有机溶剂,可溶于碱性溶液和DMSO;离子交换色谱分析表明黑色素组分均一,出峰时间26±0.5 m in;紫外光谱谱图最大吸收峰为215 nm左右,未见蛋白(280 nm)与核酸(260 nm)的特征吸收峰;红外光谱谱图具有黑色素3μm和6μm的特征吸收峰,并含大量的羟基、氨基,与核磁共振和液质联机谱图结合分析推出短梗霉黑色素可能含有酚羟基、羧基和吲哚等官能团,主要结构骨架为5,6-二羟基吲哚-羧酸和多巴醌,推断该黑色素为酪氨酸酶控制合成的真黑素。     

二价阳离子对短梗霉多糖发酵的影响

就二价阳离子对短梗霉多糖和黑色素的影响进行了分析和研究。结果表明 ,二价阳离子对短梗霉多糖的合成和黑色素的形成均有较大的影响。通过对培养基中二价阳离子含量和种类的控制不仅可以抑制细胞黑色素的形成 ,而且还保持了很高的多糖发酵水平 ,在 30L生物反应器中短梗霉多糖的产量和转化率分别达到了 59 8g/L和 61 5%。     

毛木耳黑色素提取条件优化及体外抗氧化活性研究

为实现毛木耳黑色素的高效提取,并对其抗氧化性进行评价。以黑色素的提取率为指标,实验采用Box-Behnken响应面法对超声波辅助提取毛木耳黑色素的提取条件进行优化,并对毛木耳黑色素进行红外光谱与体外化学和细胞抗氧化活性进行分析。结果表明,超声波辅助提取毛木耳黑色素的最优条件为:NaOH浓度0.34 mol/L,料液比1∶30,超声时间52 min,超声功率250 W,超声温度70℃;最优条件下的黑色素提取率可达9.38%±0.15%。红外光谱分析结果表明,超声波组与非超声波组提取的黑色素主要吸收峰一致,官能团未发生变化。体外抗氧化实验结果表明,毛木耳黑色素对2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸自由基(ABTS)、1,1-二苯基-2-三硝基苯肼自由基(DPPH)与羟基自由基具有良好清除活性,且对H_2O_2诱导的L02细胞损伤具有明显的保护作用。本研究明确了毛木耳黑色素超声波辅助提取条件及其体外抗氧化活性,为毛木耳黑色素功能产品的开发和运用提供理论依据。     

出芽短梗霉新菌株RM1603产普鲁兰多糖条件优化及多糖分析

目的:出芽短梗霉RM1603是一株高产胞外多糖新菌株,通过优化其产糖条件,鉴定其多糖结构,为进一步开发利用RM1603产胞外多糖奠定理论基础。方法:以RM1603为出发菌株,在单因素分析确定最佳氮源与无机盐的基础上,利用正交试验探究RM1603最佳发酵产糖条件;薄层层析及红外光谱分析确定胞外多糖产物结构。结果:出芽短梗霉菌RM1603的初始多糖产量为28.91g/L,发酵条件优化后产糖量达到65.213g/L,提高了约2.3倍;结构分析表明RM1603的多糖产物为普鲁兰多糖。结论:出芽短梗霉RM1603是一株具有较大产糖优势,极具开发潜力的高产普鲁兰糖新菌株;奠定了开发利用RM1603生产普鲁兰多糖的理论基础。     

应用FTIR和NMR研究短梗霉多糖分子结构

短梗霉多糖是出芽短梗霉产生的一种胞外多糖,具有极好的成膜、成纤维、阻气、粘接、易加工、无毒性等特性,是微生物多糖中最令人瞩目的多糖之一．本研究应用ＦＴＩＲ和ＮＭＲ技术对由出芽短梗霉胞外产生的短梗霉多糖进行了分析．短梗霉多糖的红外光谱（４０００～４００ｃｍ－１）,具有明显的多糖特征吸收峰,证明多糖是由α－Ｄ－吡喃葡萄糖残基组成．应用先进的一维和二维核磁共振技术,在绝对温度３４３Ｋ下获得短梗霉多糖１Ｈ－ＮＭＲ谱和１３Ｃ－ＮＭＲ谱,确证短梗霉多糖的结构单元是α－１,４麦芽三糖,归属了短梗霉多糖的１Ｈ和１３Ｃ的全部化学位移     

聚苹果酸生产菌出芽短梗霉CCTCC M2012223的全基因组测序及序列分析

【目的】解析出芽短梗霉CCTCC M2012223的基因组序列信息,分析其代谢产物聚苹果酸、黑色素、普鲁兰多糖合成相关基因,为深入研究遗传多样性和代谢工程改造提供序列背景信息。【方法】使用Illumina Hi Seq高通量测序平台对出芽短梗霉CCTCC M2012223菌株进行全基因组测序,并对测序数据进行序列拼接,基因预测与功能注释,COG/GO聚类分析,比较基因组学分析等。下载其他5株出芽短梗霉基因组序列,比较分析6株菌的种内同源基因、全基因组进化以及代谢产物合成相关基因。【结果】出芽短梗霉CCTCC M2012223基因组序列全长30756831 bp,GC含量47.49%,编码9452个基因。比较基因组分析表明出芽短梗霉CCTCC M2012223的基因组组装长度最长,6株菌的同源基因数达到7092个,普鲁兰多糖和聚苹果酸合成相关基因的蛋白序列有很高的保守性。出芽短梗霉CCTCC M2012223和Aureobasidium pullulans var.melanogenum亲缘关系最近,而这2株菌的黑色素合成相关基因的蛋白序列有一些插入和突变。【结论】本研究解析了出芽短梗霉CCTCC M2012223的基因组序列信息,获得黑色素、普鲁兰多糖和聚苹果酸合成相关基因,为后续的代谢机制解析和改造提供相关依据。     

出芽短梗霉产聚苹果酸发酵条件的优化

【背景】出芽短梗霉可发酵葡萄糖生成聚苹果酸,但存在转化率和转化效率低等瓶颈,阻碍其实现商业化生产。【目的】通过优化发酵培养条件,提高出芽短梗霉的聚苹果酸产量、糖酸转化率和生产强度。【方法】采用单因素试验优化适宜出芽短梗霉BK-10菌株产生聚苹果酸的培养条件,通过Plackett-Burman法对培养基组分筛选显著性影响因素,并对其培养基中无机盐进行正交试验优化,最后进行5 L发酵罐验证。【结果】最优培养基配方和培养条件:100 g/L葡萄糖,1.5 g/L尿素,0.20 g/L KH_2PO_4,0.20 g/L ZnSO_4,0.05 g/L MgSO_4,0.75 g/L KCl,30 g/L CaCO_3,0.01%吐温-80,发酵温度26°C,250 mL摇瓶装液量50 mL。【结论】通过优化,聚苹果酸的糖酸转化率达到0.71 g/g,生产强度达到0.89 g/(L·h),较优化前分别提高了18.33%和71.15%,为发酵葡萄糖合成聚苹果酸进而生产L-苹果酸工艺的工业化生产奠定经济性基础。     

二价阳离子短梗霉多糖发酵的影响

就二价阳离子对短梗霉多糖和黑色素的影响进行了分析和研究。结果表明,二价阳离子对短梗霉多糖的合成和黑色素的形成均有较大的影响。通过对培养基中二价阳离子含量和种类的控制不仅可以抑制细胞黑色素的形成,而且还保持了很高的多糖发酵水平,在３０Ｌ生物反应器中短梗霉多糖的产量和转化率分别达到了５９．８ｇ／Ｌ和６１．５％。     

大鲵皮肤色素提取工艺及抗氧化研究

为优化大鲵皮肤黑色素的提取工艺条件,探讨大鲵皮肤黑色素组成成分及体外抗氧化活性,采用酶法和碱溶酸沉法提取大鲵皮肤黑色素,以氢氧化钠浓度、液料比、提取温度为影响色素提取率因素,优化黑色素提取工艺条件,用紫外-可见光谱仪、红外光谱仪和超高效液相质谱仪测定黑色素的光谱特性,测定其抗氧化性。结果表明:大鲵皮肤黑色素最佳提取工艺条件为氢氧化钠浓度1.5 mol/L、液料比1∶15、提取温度45℃,黑色素提取率达0.65%。大鲵皮肤黑色素的紫外最大吸收波长为214 nm,由真黑色素和脱黑色素两种色素组成,其对超氧阴离子自由基的清除率为30.51%,对羟基自由基的清除率为54.17%。大鲵皮肤黑色素具有一定的体外抗氧化能力。     

Study of photochemical and surface-active melanins of some representatives of black fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < lambda < 400 nm) and in the blue-violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm2. The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Extraction and Identification of the Pigment in the Adductor Muscle Scar of Pacific Oyster Crassostrea gigas

In this study, UV (ultraviolet) and IR (infrared radiation) spectral analysis were integrated to identify the pigment in the adductor muscle scar of the Pacific oyster Crassostrea gigas. The pigment was extracted from the adductor muscle scars of cleaned oyster shells that were pulverized, hydrolyzed in hot hydrochloric acid, purified with diethyl ether, and dissolved in 0.01 mL/L NaOH. The maximum absorption of the pigment in the UV absorption spectrum within the range of 190–500 nm was observed between 210–220 nm. The UV absorbance decreased with increasing wavelength which was consistent with the UV spectral absorption characteristics of melanin. In addition, Fourier transform infrared spectroscopy scanning revealed characteristic absorption peaks that emerged near 3440 cm^-1 and 1630 cm^-1, which was consistent with infrared scanning features of eumelanin (a type of melanin). This study has demonstrated for the first time that the pigment in the adductor muscle scar of the Pacific oyster is melanin, hinting that the adductor muscle could be another organ pigmenting the mollusc shell with melanin other than mantle.     

一品红苞片花色素的分离及初步鉴定

用紫外-可见光分光光度计、高效液相色谱（HPLC）和质谱（MS）技术对一品红（Euphorbia pulcherrima）红色苞片中的花色素提取液进行了初步鉴定.一品红花色素的甲醇溶液分别在270、340和520 nm处有3个吸收峰;在440 nm吸光度与可见光最大吸收波长520 nm吸光度的比值为0.29;花色素的甲醇溶液中加入AlCl3后发生红移,再加入HCl后发生蓝移;色素溶液在紫外光下无荧光;色素样品经液相色谱分离后在270 nm检测有5个比较明显的吸收峰;质谱中得到595、611、381、571和589等对应的分子离子峰;花色素酸解液高效液相色谱图谱和鼠李糖、葡萄糖的出峰时间一致.由这些结果可推断一品红花色素样品中主要含有5种组分：矢车菊花色素芸香苷、飞燕草花色素芸香苷、飞燕草花色素苯甲酰基葡糖苷、矢车菊花色素苯甲酰基葡糖苷和一种未知成分.     

Characterisation of Bacillus thuringiensis mutant highly producing melanin pigment and active against potato tuber moth

The bacteria Bacillus thuringiensis mutant is highly producing melanin pigment with increased ultra violet resistance and insecticidal activity against the potato tuber moth Phthorimaea operculella (Zeller). The results showed that the high decrease of crystal protein formation rate ranged from 100% (B.t.EMS-M2 and B.t.EMS-M6) to 91.82% (B.t.EMS-M9). The EMS–UV-induced mutants (B.t.EMS–UV-2h-1, B.t. EMS–UV-2h-2, B.t.EMS–UV-2h-3, B.t.EMS–UV-2h-5, B.t.EMS–UV-4h-1, B.t.EMS–UV-4h-3 and B.t.EMS–UV-6h-2) showed 100% decrease in the crystal protein formation. Results also showed that the growth rate of B. thuringiensis isolates was detected by measuring the light absorption of culture broth (BP media at pH 8) at the wavelength of 600 nm. The absorbance values of the standard melanin were 2.055 and 0.134 at wavelengths of 226.5 and 602 nm, respectively. This means that the maximum absorbance at wavelength was 226.5 nm, this result is similar to that of the synthetic melanin which has the absorbance of 226 nm. Our experiments detected that the pigment extracted from the mutant isolate B.t.EMS-M3 (EMS-induced mutant) gave the maximum value of absorbance (2.615) at wavelength of 227.5 nm that was similar to standard melanin which gave absorbance value about 2.055 at a wavelength of 226.5 nm. This may be due to the genetic alterations that happened to the mutant isolates due to the mutation by EMS or/and UV irradiation.     

Separation of the bovine colostrum M-1 glycoprotein into two components

1. Two glycoproteins were isolated from the M-1 acid glycoprotein fraction of bovine colostrum. 2. The lighter glycoprotein had a molecular weight of 7200, contained about 28.4% of carbohydrate, and had an absorption maximum at 275nm. The heavier glycoprotein had a molecular weight of 12000, contained 39.0% of carbohydrate, and had no absorption maxima in the 240-300nm. range of the spectrum. 3. The carbohydrate moiety of both glycoproteins was removable from the polypeptide moiety under the conditions of the beta-elimination reaction. 4. Periodate oxidation experiments showed that sialic acid was linked to galactose in both proteins.     

The Photochemical and Surface-Active Properties of Melanins Isolated from Some Black Fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < < 400 nm) and in the blue–violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm². The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

一品红苞片花色素的分离及初步鉴定

用紫外-可见光分光光度计、高效液相色谱(HPLC)和质谱(MS)技术对一品红(Euphorbia pulcherrima)红色苞片中的花色素提取液进行了初步鉴定。一品红花色素的甲醇溶液分别在270、340 和520 nm 处有 3 个吸收峰; 在 440 nm 吸光度与可见光最大吸收波长 520 nm 吸光度的比值 为0.29; 花色素的甲醇溶液中加入AlCl3后发生红移, 再加入HCl后发生蓝移; 色素溶液在紫外光下无荧光; 色素样品经液相色谱分离后在 270 nm检测有 5 个比较明显的吸收峰; 质谱中得到 595、611、381、571 和 589 等对应的分子离子峰; 花色素酸解液高效液相色谱图谱和鼠李糖、葡萄糖的出峰时间一致。由这些结果可推断一品红花色素样品中主要含有 5 种组分: 矢车菊花色素芸香苷、飞燕草花色素芸香苷、飞燕草花色素苯甲酰基葡糖苷、矢车菊花色素苯甲酰基葡糖苷和一种未知成分。