Application of LIBS in Detection of Escherichia coli in Sausages
1 Introduction
Since the early beginning of lasers, Laser Induced Breakdown Spectroscopy has been extensively studied[1]. Based on atomic emission spectroscopy for the analysis of the constituents and their concentrations of samples, LIBS has been applied in diverse and extensive fields, such as material analysis, environmental monitoring, industrial production control, biomedicine, botany and so on[2]. In this chapter, a new approach to identification of E.coli in sausages by means of Laser induced breakdown spectroscopy is proposed.
E.coli is naturally found in the intestine of all warm-blooded animals, including humans. It is a Gram negative rod-shaped bacterium which is classed in enterbacteriaceae. Most of the E.coli strains are innocuous, but partially strains of E.coli are virulent and in relation to human diseases. It is generally contains five virulent categories: enterotoxigenic E.coli (ETEC), enterohemorrhagic E.coli (EHEC), enteroinvasive E.coli (EIEC) and enteroadhesive E.coli (EAEC). The presence of E.coli in food becomes accepted as food has been contaminated. The contaminated food would immediately dangerous to human health if they are eaten by humans. E.coli have the ability to survive for short periods outside the body which makes it be the hygienic bacteria index of fecal contamination in food[3]. As a received hygienic monitoring indicator, its detective methods are very important and there are many detective methods have been invented. For example, the traditional methods include Multiple fermentation (MTF) and Membrane filter (MF)[3], other technique such as polymerase chain reaction(PCR), high perfomance liquid chromatography (HPLC), high performance liquid chromatography(HPLC) and so on. However, some disadvantages exist, such as complexity, spending a long time or poor specificity. So a better method is necessary to be found.
In recent years, Laser Induced Breakdown Spectroscopy has been successfully applied in bacterial identification and has shown promising results. For example, Matthieu Baudelet[4] once used LIBS technique to distinguish different kinds of bacterium species successfully, he came to a conclusion that high sensitivity for mineral trace detections, larger intensity from molecular bands and precise kinetic study are among benefits using short pulses. Jonathan Diedrich[5,7] made a study of E.coli O157:H7, a kind of toxic bacteria and compared to three nonpathogenic E.coli strains using LIBS. In our laboratory, teacher Mingyin Yao[6] has gave a pilot study of identification of E.coli by Laser Induced Breakdown Spectroscopy. In her work, spectral intensity and variety can be precisely determined by LIBS, the samples can be characterized by their profile of spectral intensity and varieties of trace mineral elements. Though the above researches only study the cultured bacteria in the lab, enormous support and theoretical guidance for the direct identification of E.coli in food have been provided. In this work, an attempt has been made to identify E.coli in sausages directly by LIBS. We observe and analyze the spectral line of the samples.
2 Materials and Methods
2.1 Setup
Fig.1 shows the schematic map of the experimental setup of LIBS. In our experiment, a 10HZ Q-switched ND:YAGns laser (BeamTech, Nimma-200, China) was employed as a laser source, which generates laser pulses with a pulse duration of 8ns and a maximum pulse energy of 200mJ at 1064nm, 100mJ at 532nm and 50mJ at 355nm. In this work, we choose 1064nm as the fundamental wavelength. The laser pulse is focused on the surface of a sample through a mirror with 45 degree and a convergent lens with 30mm diameter and 200mm focal length. In order to have a different ablation spot for each laser shot, the samples were placed into an automatic controlled rotating stage.
The plasma emission is injected into the 400um core diameter, 2m length spectrometer fiber via another lens with a collection angle of approximately 45 degrees. A eight-channel spectrometer (2048-USB2-RM, Avantes B.V.) connected the fiber and equipped with a 2048 pixel ICCD detector which can automatically subtract the dark current from the measured spectral data and provided a resolution of 0.09nm at 200 to 317nm, 0.07nm at 315 to 417nm, 0.06nm at 415-499nm, 0.08nm at 497-565nm, 0.08nm at 563-673nm, 0.12nm at 671-750nm, 0.13nm at 748-931nm, and 0.11nm at 929-1050nm respectively in the wavelength range from 200nm to 1100nm. The AvaSpec spectrometer was controlled by personal computer (PC) running manufacturer-provided software. Throughout the whole experimental process, the integration time, delay time, repetition rate, laser pulse energy and the number of accumulated pulses and spectra were fixed at 2ms, 1.28us, 5HZ, 150mj , 10 pulses and 20 spectra, respectively.
2.2 Sample
The sausages were bought from the market. Preparing for two pieces of equal length sausages. E.coli was grown on a trypticasesoy agar growth medium for 24h at 37℃ under aeration[6]; impact by aspiration of 20 ml solution of bacteria on a filter paper and also impact by aspiration of 20ml solution of bacteria on one piece of sausage; washing of the charged filter paper and the sausage by 20ml distilled water with the same aspiration process; Under the same experimental conditions, spectra of a sausage, a filter paper charged with E.coli, a sausage charged with E.coli were collected and analyzed.
2.3 Detection Principle
E.coli is a Gram negative rod-shaped bacterium, it has a property that in which divalent cations maintain the cohesion of proteins in its outer membrane. So some trace mineral elements in sausage charged with E.coli would contribute to the characterization of E.coli, since they participate in the metabolism of the bacterium and influence its structure. Thus less mineral elements would be contained in sausage charged with E.coli than in pure sausage sample or E.coli sample. And this difference would be reflected on the spectra. Therefore, we can characterize the samples by identifying and analyzing the spectroscopy line.
3 Results and Discussions
Fig.2(a) shows the spectra of sausage at 580-660nm wavelength range. Mineral atomic spectroscopy lines, such as the KⅠ (578.24nm), FeⅠ (585.51nm), NaⅠ (588.9964nm), and CaⅠ (610.27nm) can be found.
Fig.2(b) shows spectra of E.coli at 580nm-660nm wavelength range. We can observe mineral atomic lines like FeⅠ(564.67nm, 579.39nm, 630.15nm), CaⅠ(612.22nm), NaⅠ(616.07nm, 674.31nm) and so on.
Fig.3(b) shows spectra of sausage which was charged with E.coli. As the figure shows, we can just observe the spectroscopy line of Fe.Fig.3 shows the spectra of sausage (a), E.coli (b), sausage charged with E.coli (c) at the wavelength range of 750-900nm. From these three figures, we can observe the spectra of FeⅠ(766.15nm), KⅠ(819.27nm, 890.40nm), NaⅠ(818.3255nm), CaⅠ(831.25nm), FeⅡ(875.99nm) in figure (a) and FeⅠ(868.13nm), NaⅠ(818.33nm), KⅠ(892.33nm), MgⅠ(846.88nm), CaⅠ(769.79nm) in figure (b) and FeⅠ(766.43nm), KⅠ(786.52nm), MgⅠ(899.73nm), CaⅠ(831.25nm) in figure (c). Compared figure (a) and (b) with figure (c), the intensity of spectra in the first two was more than in figure (c), and there are more mineral elements could be detected in figure(a) and (b) than in figure (c).
In summary, the spectra of sausage, E.coli and sausage charged with E.coli were different obviously from the figures. First, the spectral intensity in E.coli was far higher than sausage charged with E.coli sample. Second, in E.coli and sausage the elements spectrum lines could be detected more and more obvious than in sausage that charged with E.coli. For example, we can observe the spectral line of Na Ⅰ (588.99nm) or Na Ⅰ (616.07nm, 674.31nm) in figure (a) and (b), but in sausage charged with E.coli, none of Na element was found.
Likewise, compared with Fig.3 (b), less mineral elements such as K, Mg, Na could be observed in Fig.3 (c).
This was because the property of Gram negative bacterium, just as has been said before. Some trace mineral elements have been contributed to the characterization of E.coli to maintain the metabolism of the bacterium and influence its structure[6].
4 Conclusion
The spectra of sausage, E.coli and sausage charged with E.coli can be identified and analyzed by LIBS. Their profile of spectral intensity and variety of trace mineral elements can be used for characterizing the samples and such spectral intensity and variety can be precisely determined by LIBS. The results fit well with the property of Gram negative bacterium and offer some valuable information about how to use LIBS to detect E.coli in food. We can judge whether the sausage has been infected with E.coli by study the spectrum of the different samples. Of course, more and more progress should be created in using LIBS technique to detecting E.coli in food. At present, food safety problems catch everybody’s sight more and more. It is, thus that interest in food products safety testing research that remains strong. Thus, more and more further studies are under way in our laboratory to let LIBS to be a more effective and practical detecting and identifying technique in food detection field.
Acknowledgment
This work was supported by a grant from the freedom foundation of Jiangxi Agricultural University (No.2943).Authors are most grateful to them.
References:
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