The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy

Previous studies dealing with bacterial identification by means of Raman spectroscopy have demonstrated that micro-Raman is a suitable technique for single-cell microbial identification. Raman spectra yield fingerprint-like information about all chemical components within one cell, and combined with multivariate methods, differentiation down to species or even strain level is possible. Many microorganisms may accumulate high amounts of polyhydroxyalkanoates (PHA) as carbon and energy storage materials within the cell and the Raman bands of PHA might impede the identification and differentiation of cells. To date, the identification by means of Raman spectroscopy have never been tested on bacteria which had accumulated PHA. Therefore, the aim of this study is to investigate the effect of intracellular polymer accumulation on the bacterial identification rate. Combining fluorescence imaging and Raman spectroscopy, we identified polyhydroxybutyrate (PHB) as a storage polymer accumulating in the investigated cells. The amount of energy storage material present within the cells was dependent on the physiological status of the microorganisms and strongly influenced the identification results. Bacteria in the stationary phase formed granules of crystalline PHB, which obstructed the Raman spectroscopic identification of bacterial species. The Raman spectra of bacteria in the exponential phase were dominated by signals from the storage material. However, the bands from proteins, lipids, and nucleic acids were not completely obscured by signals from PHB. Cells growing under either oxic or anoxic conditions could also be differentiated, suggesting that changes in Raman spectra can be interpreted as an indicator of different metabolic pathways. Although the presence of PHB induced severe changes in the Raman spectra, our results suggest that Raman spectroscopy can be successfully used for identification as long as the bacteria are not in the stationary phase.     

Raman spectroscopic identification of single bacterial cells at different stages of their lifecycle

Early, rapid, and reliable bacterial identification is of great importance in natural environments and in medical situations. Numerous studies have shown that Raman spectroscopy can be used to differentiate between different bacteria under controlled laboratory conditions. However, individual bacteria within a population exhibit macromolecular and metabolic heterogeneity over their lifetime. Therefore it is important to be able to identify and classify specific bacteria at different time points of the growth cycle. In this study, four species of bacteria were used to explore the capability of confocal Raman spectroscopy as a tool for the identification of (and discrimination between) diverse bacterial species at various growth time points. The results show that bacterial cells from different growth time points (as well as from a random growth phase) can be discriminated among the four species using principal component analysis (PCA). The results also show that bacteria selected from different growth phases can be classified with the help of a prediction model based on principal component and linear discriminant analysis (PC-LDA). These findings demonstrate that Raman spectroscopy with the application of a PC-LDA model rooted in chemotaxonomic analysis has potential for rapid sensing of microbial cells in environmental and clinical studies.     

Assessment of two isolation techniques for bacteria in milk towards their compatibility with Raman spectroscopy

The identification of single microorganism in food samples by conventional plating techniques or molecular genetic methods requires a time consuming enrichment step. Raman spectroscopy in combination with a suitable extraction method however offers the possibility to rapidly identify bacteria on a single cell level. Here we evaluate the two well-known bacteria extraction methods from milk: "buoyant density centrifugation" and "enzymatic milk clearing" towards their recovery efficiency and their compatibility with Raman spectroscopy for a rapid identification of microorganisms in milk. The achieved recovery yields are slightly better compared to those which are already applied for food investigations, where a loss of one order of magnitude is usually reached. For example, buoyant density centrifugation allows collecting up to 35% of the milk-spiked microorganisms. To prove the suitability of the isolation techniques for use in combination with the spectroscopic approach, a small Raman database has been created by recording Raman spectra of well-known contaminants in dairy products. Two subspecies of Escherichia coli and three different Pseudomonas species, which were inoculated to UHT (ultra-high-temperature processed) milk and afterwards extracted by the two techniques mentioned above, were analysed. At a first glance, grave spectral artefacts caused by the matrix itself or especially by the extraction techniques were not obvious. But via chemometric analysis, it could be shown that these factors noticeably influence the identification rates: while the samples prepared via milk clearing did not provide sufficient identification results, buoyant density centrifugation allows an identification of the investigated species with an overall accuracy of 91% in combination with linear discriminant analysis.     

Simultaneous isolation and label‐free identification of bacteria using contactless dielectrophoresis and Raman spectroscopy

The traditional bacterial identification method of growing colonies on agar plates can take several days to weeks to complete depending on the growth rate of the bacteria. Successfully decreasing this analysis time requires cell isolation followed by identification. One way to decrease analysis time is by combining dielectrophoresis (DEP), a common technique used for cell sorting and isolation, and Raman spectroscopy for cell identification. DEP‐Raman devices have been used for bacterial analysis, however, these devices have a number of drawbacks including sample heating, cell‐to‐electrode proximity that limits throughput and separation efficiency, electrode fouling, or inability to address sample debris. Presented here is a contactless DEP‐Raman device to simultaneously isolate and identify particles from a mixed sample while avoiding common drawbacks associated with other DEP designs. Using the device, a mixed sample of bacteria and 3 μm polystyrene spheres were isolated from each other and a Raman spectrum of the trapped bacteria was acquired, indicating the potential for cDEP‐Raman devices to decrease the analysis time of bacteria.     

Rapid identification of closely related muscle foods by vibrational spectroscopy and machine learning

Muscle foods are an integral part of the human diet and during the last few decades consumption of poultry products in particular has increased significantly. It is important for consumers, retailers and food regulatory bodies that these products are of a consistently high quality, authentic, and have not been subjected to adulteration by any lower-grade material either by accident or for economic gain. A variety of methods have been developed for the identification and authentication of muscle foods. However, none of these are rapid or non-invasive, all are time-consuming and difficulties have been encountered in discriminating between the commercially important avian species. Whilst previous attempts have been made to discriminate between muscle foods using infrared spectroscopy, these have had limited success, in particular regarding the closely related poultry species, chicken and turkey. Moreover, this study includes novel data since no attempts have been made to discriminate between both the species and the distinct muscle groups within these species, and this is the first application of Raman spectroscopy to the study of muscle foods. Samples of pre-packed meat and poultry were acquired and FT-IR and Raman measurements taken directly from the meat surface. Qualitative interpretation of FT-IR and Raman spectra at the species and muscle group levels were possible using discriminant function analysis. Genetic algorithms were used to elucidate meaningful interpretation of FT-IR results in (bio)chemical terms and we show that specific wavenumbers, and therefore chemical species, were discriminatory for each type (species and muscle) of poultry sample. We believe that this approach would aid food regulatory bodies in the rapid identification of meat and poultry products and shows particular potential for rapid assessment of food adulteration.     

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.     

Molecular methods for identification and detection of bacterial food pathogens

The polymerase chain reaction (PCR) shortens conventional microbiological methods for the detection of food pathogens either by replacing the conventional biochemical and serological identification or by its direct use on pre-enrichment media or food products. PCR allows fast and highly reliable identification of bacterial taxa, particularly phenotypically atypical bacterial strains. For reliablity, PCR primers and reaction conditions must be thoroughly optimized and evaluated, appropriate sample preparations must be developed, and a stringent laboratory protocol must be followed. Positive control systems are used to monitor possible inhibition of the reaction and negative controls are needed to monitor for contamination. The most recent developments involve messenger RNA-based (mRNA-based) detection of viable bacterial pathogens and real-time PCR quantitation of pathogens.     

Covalent Reactions on Chemical Vapor Deposition Grown Graphene Studied by Surface‐Enhanced Raman Spectroscopy

Graphene is a material of unmatched properties and eminent potential in disciplines ranging from physics, to chemistry, to biology. Its advancement to applications with a specific function requires rational design and fine tuning of its properties, and covalent introduction of various substituents answers this requirement. We challenged the obstacle of non‐trivial and harsh procedures for covalent functionalization of pristine graphene and developed a protocol for mild nucleophilic introduction of organic groups in the gas phase. The painstaking analysis problem of monolayered materials was addressed by using surface‐enhanced Raman spectroscopy, which allowed us to monitor and characterize in detail the surface composition. These deliverables provide a toolbox for reactivity of fluorinated graphene under mild reaction conditions, providing structural freedom of the species to‐be‐grafted to the single‐layer graphene.     

Raman spectroscopic study of Lactarius spores (Russulales, Fungi)

Fungi are important organisms in ecosystems, in industrial and pharmaceutical production and are valuable food sources as well. Classical identification is often time-consuming and specialistic. In this study, Raman spectroscopy is applied to the analysis of fungal spores of Lactarius, an economically and ecologically important genus of Basidiomycota. Raman spectra of spores of Lactarius controversus Pers.: Fr., Lactarius lacunarum (Romagn.) ex Hora, Lactarius quieticolor Romagn. and Lactarius quietus (Fr.: Fr.) Fr. are reported for the first time. The spectra of these species show large similarity. These spectra are studied and compared with the Raman spectra of reference substances known to occur in macrofungi, including saccharides, lipids and some minor compounds that may serve as specific biomarkers (adenine, ergosterol and glycine). Most Raman bands could be attributed to specific components. In agreement with the biological role of fungal spores, high amounts of lipids were observed, the main fatty acid being oleate. In addition to different types of lipids and phospholipids, the polysaccharides chitin and amylopectin could be detected as well. The presence of trehalose is not equivocally shown, due to overlapping bands. Raman band positions are reported for the observed bands of the different species and reference products.     

Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions

Microbial contamination is not only a medical problem, but also plays a large role in pharmaceutical clean room production and food processing technology. Therefore many techniques were developed to achieve differentiation and identification of microorganisms. Among these methods vibrational spectroscopic techniques (IR, Raman and SERS) are useful tools because of their rapidity and sensitivity. Recently we have shown that micro-Raman spectroscopy in combination with a support vector machine is an extremely capable approach for a fast and reliable, non-destructive online identification of single bacteria belonging to different genera. In order to simulate different environmental conditions we analyzed in this contribution different Staphylococcus strains with varying cultivation conditions in order to evaluate our method with a reliable dataset. First, micro-Raman spectra of the bulk material and single bacterial cells that were grown under the same conditions were recorded and used separately for a distinct chemotaxonomic classification of the strains. Furthermore Raman spectra were recorded from single bacterial cells that were cultured under various conditions to study the influence of cultivation on the discrimination ability. This dataset was analyzed both with a hierarchical cluster analysis (HCA) and a support vector machine (SVM).     

UV Raman spectroscopic study on the synthesis mechanism and assembly of molecular sieves

In the past decade, UV Raman spectroscopy has become a powerful technique for the characterization of the synthesis mechanism and assembly of molecular sieves. Ultraviolet excitation avoids fluorescence that plagues visible Raman spectroscopy and concurrently enhances the Raman signal because of the short wavelength of excitation and the resonance Raman effect. The advances of UV Raman spectroscopy, UV resonance Raman spectroscopy and in situ UV Raman spectroscopy and their applications to the characterization of zeolite assembly mechanisms are provided in this tutorial review. Using UV Raman spectroscopy, the synthesis mechanism of zeolites, including the identification of primary units, assembly through key intermediates, transition metal species, and the roles of the organic templates in framework formation have been elucidated, and are discussed herein.     

表面增强拉曼光谱在食品安全分析中的应用

拉曼光谱技术具有样品用量少、快速高效、无损分析等特点,表面增强拉曼光谱克服了常规拉曼光谱灵敏度低的缺点,可以获得更多物质结构信息,在现场快速筛查、检测和鉴别农兽残、限用或禁用添加剂分析检测中具有广阔的应用前景。本文综述了表面增强拉曼光谱在食品中农药残留、兽药残留和限/禁用添加剂检测中的研究进展,并展望了其发展前景。     

Single Step Fabrication of Chitosan Nanocrystals Using <Emphasis Type="Italic">Penaeus semisulcatus</Emphasis>: Potential as New Insecticides,Antimicrobials and Plant Growth Promoters

In this study, chitosan nanoparticles (CH-NPs) were synthesized using Penaeus semisulcatus shrimp shells and characterized using UV–Vis and FT-IR spectroscopy, as well as XRD and HR-TEM analyses. CH-NPs were investigated for growth inhibition properties against selected species of bacterial and fungal pathogens, showing performances higher or comparable over positive controls, respectively. Furthermore, CH-NPs were tested on three important mosquito vectors, achieving LC₅₀ from 12.27 to 14.62 µg/ml. In addition, CH-NPs were evaluated using in vitro plant tissue culture by rooting gel method, to enhance the vegetative growth of the medicinal plant species Sphaeranthus indicus. With the simple technique presented here, large-scale industrial production of CH-NPs is possible. They can be used to develop pesticides highly effective against mosquito vectors of high medical and veterinary importance, as well as for plant tissue culture and food packaging applications.     

Evaluation of an accurate calibration and spectral standardization procedure for Raman spectroscopy

Due to modern developments Raman spectroscopy has evolved into a fast vibrational technique. Detailed fingerprints in combination with non-destructivity and minimal sample preparation has allowed the construction of reference libraries in a variety of research fields. Long-term stability and comparability are important characteristics when developing reference libraries. In addition, small shifts in highly similar spectra of different samples may limit the full potential of Raman spectroscopy. Since libraries often contain a large number of different and/or highly similar spectra, it is important that each data point in all the spectra corresponds to the exact Raman wavenumber. This is often not the case, due to shifts in optical pathway and/or shifts in laser wavelength. This paper describes a complete calibration protocol (wavelength and intensity) and evaluates the procedure for both short and long term stability, by means of 60 randomly selected measurement sessions spread over a period of nine months. A two-step standardization procedure is proposed to deal with spectral shifts.     

表面增强拉曼光谱:应用和发展

表面增强拉曼光谱技术(Surface-enhanced Raman spectroscopy,SERS)是一种具有超高灵敏度的指纹光谱技术,目前已广泛应用于表面科学、材料科学、生物医学、药物分析、食品安全、环境检测等领域,是一种极具潜力的痕量分析技术。 本文对SERS技术及相关的针尖增强拉曼光谱(Tip-enhanced Raman spectroscopy,TERS),壳层隔绝纳米粒子增强拉曼光谱(Shell-isolated nanoparticle-enhanced Raman spectroscopy,SHINERS)技术的发展及应用进行了综合评述,并探讨了其未来的研究热点及发展方向。     

DNA microarray technology used for studying foodborne pathogens and microbial habitats: minireview

Microarray analysis is an emerging technology that has the potential to become a leading trend in bacterial identification in food and feed improvement. The technology uses fluorescent-labeled probes amplified from bacterial samples that are then hybridized to thousands of DNA sequences immobilized on chemically modified glass slides. The whole gene or open reading frame(s) is represented by a polymerase chain reaction fragment of double-strand DNA, approximately 1000 base pair (bp) or 20-70 bp single-strand oligonucleotides. The technology can be used to identity bacteria and to study gene expression in complex microbial populations, such as those found in food and gastrointestinal tracts. Data generated by microarray analysis can be potentially used to improve the safety of our food supply as well as ensure the efficiency of animal feed conversion to human food, e.g., in meat and milk production by ruminants. This minireview addresses the use of microarray technology in bacterial identification and gene expression in different microbial systems and in habitats containing mixed populations of bacteria.     

Analytical methods for monitoring contaminants in food—An industrial perspective

Incoming legislation on the registration, evaluation, authorisation and restriction of chemical substances places responsibility on the chemical industry, including downstream users of chemicals, to provide appropriate safety information with which to improve the protection of human health and the environment through the better and earlier identification of the intrinsic properties of chemical substances. Food consumption is only one of several potential exposure routes, but if industrial chemicals enter the food chain, the diet may be a significant pathway of human exposure. Consequently strong measures are taken to protect the integrity of the human food chain and these are constantly revised to address perceived chemical safety threats. In order to understand the risk presented by the possible presence of a chemical residue in food, knowledge is required of its toxicology and of the level of exposure. Reliable exposure assessment requires robust analytical methodology. Existing standards for the validation and performance evaluation of methods have led to improved analytical capability and better inter-laboratory agreement of results. However, increasing the availability of robust, cost-effective methodology should be the benchmark for future developments in the field of food chemical residue analysis. Chromatography meets the needs of target analyses well and largely provides the selectivity of measurement needed to assess compliance with food regulatory limits. However, to keep pace with the increased need for expanded analytical capability – faster throughput, more analytes per sample – chromatographic separation capability still needs to grow. In this respect, orthogonal separation techniques and multi-dimensional chromatography are key tools for the future.     

A review on solid phase microextraction-high performance liquid chromatography as a novel tool for the analysis of toxic metal ions

This paper reviews the practical applications of solid phase microextraction-High performance liquid chromatography in the analysis of toxic metal species as these are important contaminants and are carcinogenic. Their determination in formulations, in feed and food, and in complex environmental matrices (e.g., waste water and industrial effluents) often requires analytical methods capable of high efficiency, unique selectivity, and high sensitivity. Solid phase microextraction (SPME) requires low solvent consumption and is quick in use. SPME is used for extraction and online desorption of analytes with the mobile phase of HPLC and subsequent detection by UV, ICP-MS or ESI-MS as detectors. Different SPME-HPLC methods are summarized in this article to demonstrate the usefulness of this technique for metallic species of As, Cr, Pb, Hg and Se.     

Development of nanofibrillated cellulose coated with gold nanoparticles for measurement of melamine by SERS

The objective of this study was to develop nanofibrillated cellulose (NFC)-based substrate for rapid detection of melamine in milk by surface-enhanced Raman spectroscopy (SERS). NFC were served as a highly porous platform to load with gold nanoparticles (AuNPs), which can be used as a flexible SERS substrate with nanoscale roughness to generate strong electromagnetic field in SERS measurement. The NFC/AuNP substrate was characterized by UV–Vis spectroscopy and electron microscopy. Milk samples contaminated by different concentrations of melamine were measured by SERS coupled with NFC/AuNP substrate. The spectral data analysis was conducted by multivariate statistical analysis [i.e. partial least squares (PLS)]. Satisfactory PLS result for quantification of melamine in milk was obtained (R = 0.9464). The detection limit for melamine extracted from liquid milk by SERS is 1 ppm, which meets the World Health Organization’s requirement of melamine in liquid milk. These results demonstrate that NFC/AuNP substrate has improved homogeneity and can be used in SERS analysis for food safety applications.     

In situ Raman studies during sulfidation, and operando Raman-GC during ammoxidation reaction using nickel-containing catalysts: a valuable tool to identify the transformations of catalytic species

Ni-containing catalysts are investigated under reaction conditions for two different cases, during sulfidation, with Ni-Mo based catalysts, and during ammoxidation reaction, with the Ni-Nb catalysts. It is shown how Raman spectroscopy can follow some of the transformations of these catalysts upon different treatments. For the NiMo/Al(2)O(3)-SiO(2) system it was possible to identify some of the sulfided Mo species formed during the sulfidation of the oxide precursors, while for the bulk Ni-Nb oxide catalysts the simultaneous reaction-Raman results strongly suggest that the incipient interaction between niobium and nickel oxides at low Nb/Ni atomic ratios is directly related to catalytic activity, and that a larger size well-defined NiNb(2)O(6) mixed oxide phase is not active for this reaction. Moreover, the promotion by niobium doping appears to be limited to a moderate niobium loading. It was found that in situ and operando Raman are valuable techniques that allowed the identification of active Mo-S and Ni-Nb species under reaction conditions, and that are not stable under air atmospheres.