【作者】 武浩；

【导师】 朱拓；

【作者基本信息】 江南大学 ， 光学工程， 2009， 硕士

【摘要】 益生菌是一类对人体有益的细菌,服用含有益生菌的食品,可以帮助人体维持肠道菌丛的平衡,保持人体的健康。益生菌的应用,取得了很好的医疗和保健效果,帮助人们获得了经济和社会效益,促进了人类自身健康的发展。为了更好地应用益生菌造福人类,需要从多方面研究益生菌。国外已有关于细菌荧光光谱的报道,多集中在低浓度溶液下的荧光光谱,并结合主成分分析方法对不同菌种的光谱进行分类识别。国内尚未开展有关益生菌荧光光谱的研究,本文将荧光分析技术与导数光谱、偏振光谱、小波分析、神经网络等方法结合,研究益生菌的荧光光谱,填补了这一部分的空白。将荧光分析法用于益生菌的研究,从一个新的角度研究益生菌的特性,揭示益生菌的特征,为益生菌的研究提供更多的参考价值。本学位论文分别研究了植物乳杆菌、嗜热链球菌、干酪乳杆菌、嗜酸乳杆菌、保加利亚乳杆菌和变异链球菌的荧光光谱,其中运用二维荧光光谱和三维荧光光谱技术对植物乳杆菌溶液的荧光进行表征,并定量检测了不同浓度植物乳杆菌溶液的荧光峰值强度,同时运用荧光偏振技术对植物乳杆菌进行了研究;对比分析了干酪乳杆菌和嗜热链球菌的荧光发射光谱以及二阶导数荧光光谱特征,给出了两者之间的差异;结合径向基函数神经网络算法对嗜酸乳杆菌、保加利亚乳杆菌和变异链球菌的光谱进行了识别。本文所做的研究为在益生菌领域中运用荧光分析技术定性、定量的研究提供了参考。最后对光镊在益生菌荧光光谱中的应用作了介绍。鉴于目前实验条件限制,此部分实验将在下一步工作中实现。通过对植物乳杆菌的吸收和荧光光谱特性进行分析、研究发现:植物乳杆菌对紫外波段的光有明显的吸收,峰值位于215nm,在280nm附近有一个对吸收光谱产生扰动的肩峰。通过对植物乳杆菌的荧光光谱检测,发现该菌种在吸收紫外光后发射荧光。在300nm—650nm波段范围内,植物乳杆菌菌液的荧光光谱有四个峰值,分别位于338nm,387nm,465nm,538nm附近,这四个峰值分别对应细胞中主要荧光物质的荧光峰值,提供了细胞内部的相关信息。对荧光强度和菌液浓度关系的进一步研究,给出了峰值强度随浓度变化的规律,并根据其变化规律进行数据拟合,给出相应的函数表达式。此外我们还对植物乳杆菌溶液的激发光谱以及300nm-430nm波段的静态偏振光谱作了研究,实验结果显示前两个峰值的偏振度分别为0.4485和0.0921,第一个峰值体现了一定的偏振性,但是第二个峰值的偏振度很低,文中运用不同荧光团中能量转移机制作了分析,解释第二个峰值的偏振度小的原因。通过对干酪乳杆菌和嗜热链球菌荧光光谱以及导数光谱的研究发现:两种菌的荧光光谱相似,从光谱上很难直接进行区分。导数光谱能够减少干扰,增强特征光谱精细结构的分辨能力,区分光谱的细微变化,使得两种菌荧光光谱间的区别明显。实验结果显示了在285nm以及340nm波长的光激发下,两种菌的导数光谱有明显差异。可以用来区分这两种益生菌。采用小波变换和径向基函数神经网络方法对实验数据进行处理,进行三种菌的识别。小波变换对数据压缩,既减少了数据量,保持了光谱的峰值特征,又节约了神经网络的处理时间。应用神经网络方法对压缩后的数据处理,进行光谱的预测和识别,可以实现计算机化和自动化,数据处理的结果显示此方法对益生菌的识别准确。最后对光镊的原理和应用作了介绍,简述了光镊技术在益生菌单细胞研究中的应用,并用光镊捕获嗜酸乳杆菌的细胞,实现细胞的捕获和移动。此外我们还对光镊在单细胞荧光中的应用作了展望,将光镊与荧光分析法结合,可以固定细胞,减少细胞运动对实验的影响,而且光镊产生的力不会伤害细胞,能够在自然的状态下研究单个细胞,从而得到单个生命个体的组成和细胞内部生化变化的信息。本论文的研究丰富了荧光光谱技术的应用领域,并结合三维技术、偏振技术、导数荧光、小波分析、径向基函数神经网络算法等技术,使得荧光分析技术在益生菌研究领域中有了方法上的改进和提高,为进一步对益生菌的研究,提供了依据;为荧光光谱技术益生菌研究领域的广泛应用提供了有意义的参考。更多还原

【Abstract】 The probiotics are very beneficial to human health, which can be taken as food additive, and maintain the balance of intestinal flora. It is the application of probiotics to obtain good medical effects, health effects, and achieve economic, social and ecological benefits. In order to benefit mankind by the application of probiotics, further studies need to be done. There are a lot of reports about the fluorescence of bacteria abroad, which are about the fluorescence spectra of bacteria with low concentration, and identifying bacteria by the method of principal component analysis. But there are no reports about the fluorescence of probiotics at home, and the studies of this paper fill in the blank. The combination of fluorescence analysis and the study of probiotics is a new perspective of studying the characteristics of probiotics, and provide more reference value about the research of probiotics.We used physical method to study the fluorescence spectra of Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, Latobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus mutans, and provided reference for the application of fluorescence in the qualitative and quantitative investigation of probiotics. The fluorescence of Lactobacillus plantarum solution were characterized by two- dimensional fluorescence spectroscopy and three-dimensional fluorescence spectroscopy, and the fluorescence intensities of different concentrations of Lactobacillus plantarum solution were quantitative detected, while the fluorescence polarization technology was used to study Lactobacillus plantarum. Comparing and analyzing the zero-order derivative fluorescence spectra of Lactobacillus casei and Streptococcus thermophilus, as well as the spectral characteristics of the second-order derivative fluorescence spectra, and giving the differences between the two strains. Combination of radial basis function (RBF) neural network algorithm, we identified the spectra of Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus mutans. Introducing the application of optical tweezers in studying fluorescence spectrometry of probiotics, and the experiments of this part will be achieved in the next step as the result of the current experimental conditions.The absorption spectra and fluorescence spectra of Lactobacillus Plantarum are measured and studied by spectral analysis technique, and the results will be applied to the study and classification of probiotic bacteria. The results show that Lactobacillus Plantarum absorbs ultraviolet rays and emits obvious fluorescence in the range of 300nm and 650nm. There are four peaks in this range, and the locations are 338nm, 387nm, 465nm and 535nm respectively, these correspond to the major organic material in cells: aromatic amino, nucleotide, NADH, FAD, FMN and so on. The studies of the spectra show relevant information in cells about Lactobacillus Plantarum. The relations between fluorescence intensities and the concentrations are also studied and the change law of intensity with increasing concentration is found. The functions of data fitting based on the law are shown in this article. The relationship between the intensity change of two peaks (em1 and em2) and concentration is non-linear, but the other two (em3 and em4) are linear. The studies of Lactobacillus Plantarum provide reference for the research on the application of fluorescence analysis in the field of bacteria, and also provide experimental data for realizing identification of strains via fluorescence spectra. With the frontal experiment, we studied the excitation spectra and polarization spectroscopy in the 300nm-430nm range of Lactobacillus plantarum solution. The experimental results show that the polarization degrees of the two peaks are 0.4485 and 0.0921 respectively. The first peak embodies a certain polarization, but the polarization degree of the second is very low. The mechanism of energy transfer between different fluorophore was used to explain the reason of the small polarization degree of the second peak.From the research, we found that the fluorescence spectra of Lactobacillus casei-BDI are similar to Streptococcus thermophilus. It is difficult to identify the two strains from the spectra directly. Second-order spectra can reduce the interference and increase the resolving power. According to the results, with the excitation wavelengths of 285nm and 340nm, there are significant differences between the Second-order spectra.Using wavelet transform and RBF neural network method to process experimental data and identify three different probiotics. The data were compressed by wavelet transform, which reduced the amount of data and maintained the spectral characteristics of the peak, but also reduced the neural network processing time. Neural network was used to process compressed data, and identify the spectra, which can achieve computerization and automation, and the results showed that this method of identification of probiotics is accurate.Finally, the principle of optical tweezers was introduced and the applications in probiotics study of single-cell were also introduced briefly. Capturing Lactobacillus acidophilus cells by optical tweezers, and achieving cell captured and moved. The combination of optical tweezers and fluorescence analysis can reduce cell movement and Brownian motion interference and improve signal-to-noise ratio. Studying the single cell in the condition closing to the natural physiological state can obtain the information of single life’s components and real-time and biochemical changes information in groups under the average individual.In this paper, the research has enriched the fluorescence spectroscopy application areas. Combined with three-dimensional technology, polarization, derivative fluorescence, wavelet analysis, radial basis function neural network algorithm, the applications of fluorescence analysis in studies of probiotics are improved and enhanced. Providing a basis for further research on probiotics and a meaningful reference for the wide applications of fluorescence analysis technology更多还原