Rapid Detection,Application,And Antibiotic Resirtance Of Bacteria In Food

Bacteria in food not only include strains that cause disease, but also include lactic acid bacteria(LAB) that are good for food production and human health. Foodborne pathogen is one of the key factors affecting food safety, therefore, rapid detection of foodborne pathogens is critically important for ensuring food safety. However, traditional methods for foodborne pathogens detection were very time consuming. Surface-enhanced Raman spectroscopy(SERS) was widely used in microorganism researches due to its simplicity, high sensitivity, and capability for nondestructive detection. In this study, SERS combined with mapping technique and chemometric methods were used to rapidly detect and identify foodborne pathogens. Lactic acid bacteria(LAB) are widely used in food production since they can improve the flavor, preservation and nutrition value of food. However, application of LAB with antibiotic resistance has raised some safety issues since they may transfer their resistance to pathogenic bacteria in human gastrointestinal. Rapid identification of LAB with antibiotic resistance is predominant for eliminating the potential safety threat in food industry. This study investigated the application of LAB in food fermentation and developed a SERS-based method for the rapid discrimination of antibiotic susceptibility of LAB.Contents of this research and the main results were as follows:(1) A simple, rapid and label-free SERS based mapping method was developed for the rapid detection and discrimination of Salmonella enterica and Escherichia coli on silver(Ag) dendrites. Sample preparations were first optimized to maximize sensitivity. The mapping technique was then used to scan through the bacterial cells situated in the surface of Ag dendrites. The intrinsic and distinct SERS signals of bacterial cells were used as the basis for label-free detection and discrimination. Results showed that the developed method was able to detect single bacterial cells situated in the silver dendrites with a limit of detection(LOD) as low as 104 CFU/m L, which was two orders of magnitude lower than that for the normal SERS method under the same experimental condition. The time needed for collecting a 225 points map was approximately 24 min. Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica subsp enterica BAA1045(SE1045) and Escherichia coli BL21 from a mixture sample using principle component analysis(PCA).(2) A novel method was developed to rapidly concentrate, detect and differentiate bacteria in skimmed milk using SERS mapping on 4-mercaptophenylboronic acid(4-MPBA) functionalized Ag dendrites. 4-MPBA functionalized Ag dendritic SERS substrate was used to capture bacterial cells and enhance their SERS signal. Salmonella, a significantly important food pathogen was used as the representative strain to optimize and evaluate the developed method. The capture efficiency for SE1045 was 84.92 ± 3.25% at 106 CFU/m L and as high as 99.65 ± 3.58% at 103 CFU/m L. Four different strains, two gram-negative and two gram-positive could be clearly distinguished by their SERS spectra using PCA. A mapping technique was utilized to automatically collect 400 spectra over an area of 60 ?m × 60 ?m to construct a visual image for a sensitive and statistically reliable detection of SE1045 within 30 min. Using this method, we were able to detect as low as 103 CFU/m L SE1045 in 50 m M NH4HCO3 solution and 102 CFU/m L cells in both 1% casein and skimmed milk. Our results demonstrate the feasibility of using SERS mapping method coupled with 4-MPBA functionalized Ag dendrites for rapid and sensitive bacteria detection in complex liquid samples.(3) Malolactic fermentation(MLF) properties of different Oenococcus oeni(O. oeni) strains were evaluated and malolactic enzyme(MLE), the key enzyme for MLF was purified and characterized. The MLF properties of 23 O. oeni strains isolated in China was first evaluated in synthetic wine media with increasing selective pressure, such as low p H and high ethanol concentration. And the selected strains were further evaluated in newly alcohol-fermented Cabernet Sauvignon wine. Finally, O. oeni CS-4b and O. oeni ME-5b, which exhibited better MLF capability than the commerical starter O. oeni 31 MBR, were selected as promising MLF starters. MLE was purified from O. oeni SD-2a through protamine sulfate precipitation, anion exchange chromatography and gel ltration chromatography. The MLE was purified by 43-fold with a yield of 0.42 % and possessed a specific activity of 419.2 U/mg. The purified enzyme showed a nominal molecular mass of 59 k Da and a theoretical p I of 4.76 and exhibited a maximum enzyme activity at 35 °C and p H 6.0. Under the conditions of temperature 30 °C and p H 6.0, the Km and Vmax of MLE on L-malic acid were 12.5×10-3 M and 43.86 ?mol/(min × mg), respecitively.(4) SERS was used to characterize the biochemical profiles of Lactococcus lactis(L. lactis) responding to antibiotics. L. lactis exposed to antibiotics was mixed with 50-nm gold nanoparticles for subsequent SERS measurements. The SERS spectra analyzed by PCA showed no significant change after 30 min of antibiotic treatment, whereas distinct changes were clearly observed after 60 and 90 min of antibiotic treatments. Different antibiotics induced different spectral changes, and these changes revealed the detailed biochemical information of cellular responses. This study demonstrates that the developed SERS method not only senses the changes in the bacterial cell wall, but also reveals the detail information of the changed biochemical profile, which help us to understand how LAB respond to antibiotics, as well as to set a base for the detection of antibiotic susceptibility of bacteria by SERS.(5) SERS was used to discriminate the susceptibility of LAB to antibiotics targeting bacterial cell wall. Lactobacillus delbrueckii subsp. bulgaricus(Lb. bulgaricus) ATCC11842 was used to represent LAB strains that were widely used in food industry. Penicillin G, ampicillin and vancomycin were used to treat Lb. bulgaricus ATCC11842 at different toxic levels for 30, 60 and 90 min, respectively. SERS spectra of Lb. bulgaricus after each treatment were collected to characterize the responses of Lb. bulgaricus. The collected spectra were analyzed by PCA and partial least squares regression(PLSR). Results suggested that all three antibiotics induced significant peak changes in LAB, and the spectral changes induced by each antibiotic treatment were significantly different. Moreover, the antibiotic induced spectral changes in Lb. bulgaricus had good correlation with its proliferation ability and could be potentially used as a base for rapid quantification of antibiotic susceptibility of bacteria. The developed method could detect the reaction of LAB to antibiotics treatments within 3 h. Therefore, the developed SERS method could be used to rapidly discriminate and quantify the antibiotic susceptibility of LAB.