| Lactobacillus rhamnosus is an important probiotics,which has the physiological function like keeping the balance of intestinal microflora,inhibiting the growth of harmful bacteria and eliminating allergic reactions.At present,there are many kinds of dairy products containing Lactobacillus rhamnosus,but due to the wide variety of equipment conditions and technical levels,the quality of the products is uneven.Many products have quality problems such as the number of live bacteria not meeting the standards and the species identification is inconsistent.Therefore,the establishment of rapid,sensitive and accurate quantitative detection method of Lactobacillus rhamnosus is of great significance for evaluating the quality of probiotics and screening ideal strains.This paper has carried out the following work on the rapid quantitative detection of Lactobacillus rhamnosus.(1)This thesis established a detection method of Lactobacillus rhamnosus immunomagnetic bead through preparing immunomagnetic beads by recombinant SpaA polyclonal antibody,and amplifying electrical signals by HRP.The results showed that the standard working curve was established under the conditions of optimized antibody concentration,magnetic bead dosage,immune time and temperature.The linear range was 2.56 ? 103-2.56? 107CFU mL-1,and the limit of detection was 22 CFU mL-1.At the same time,we also considered the specificity,repeatability and stability of this method,the results showed that the method had no cross reaction with Lactobacillus casei?Lactobacillus paracasei?Lactobacillus plantarum?Lactobacillus fermentum?Lactobacillus bulgaricus and Streptococcus thermophilus,indicating that the good specificity of the sensor.The RSD of 5 samples were lower than 5%in repeated experiments.In the stability experiment,7 days later,it was found that the current value decreased by 2.07%compared with the beginning,and decreased by 5.07%and 9.91%,respectively,14 and 21 days later.The whole detection process showed a good stability.In Milk simulated sample,the recovery rate was between 91.74%-108.67%,and the RSD was between 2.72%-5.16%,indicating that the sensor could accurately detect the rat effective Lactobacillus rhamnosus,and the whole detection process was no longer than 3 hours.(2)In this thesis,blue fluorescent carbon quantum dots were synthesized by microwave method using citric acid as the carbon source ethylenediamine as surface passivator.The surface morphology and fluorescence properties were studied.The synthesized carbon quantum dots were labeled with a SpaA antibody by covalent cross-linking method,and the fluorescence intensity of the Lactobacillus rhamnosus was specifically measured by a fluorescence spectrophotometer to quantitatively detection.The results show that the synthesized carbon quantum dots have uniform size and good dispersion.The optimal excitation wavelength is 350 nm,which is not affected by K+and Na+.The fluorescence intensity between pH4-11 is relatively stable and suitable for microbial detection.A standard working curve was established under optimized optimal working conditions with a linear range of 5×104-5×108 CFU mL-1 and the limit of detection was 103 CFU mL-1.The specificity of the method was also considered and compared with colony immunoblotting and flow cytometry.The results showed that the method did not cross-react with Bm01,LYO and BG.LV108 and CRL1505 had certain positive signals but significantly lower than the fluorescence intensity of LGG.The results of colony immunoblot experiments were basically consistent,indicating that the method has good specificity.In the flow cytometry experiment,the CDs were compared with the traditional dye FITC,and the results were basically the same,but the CDs have photobleaching resistance,low cytotoxicity,good biocompatibility,low cost and easy operation,which provides a novel and green tool for microbial detection.(3)In order to screen the specific recognition component of cheap Lactobacillus rhamnosus,this study used Lactobacillus rhamnosus GG as the host strain to isolate Lactobacillus rhamnosus phage from fecal samples and named it LRPYZU021.Transmission electron microscopy showed that the phage form of LRPYZU021 was long-tailed phage.The cleavage spectrum test showed that phage LRPYZU021 could only lyse its host LGG and could not lyse other five kinds of Lactobacillus rhamnosus,reflecting the excellent specificity of the phage.The basic biological characteristics of phage were studied.The results showed that phage LRPYZU021 had good stability at pH 6.0 to 11.0 and 50 0C and below,the tolerance was better,and the optimal phage was multiplicatively infected.The one-step growth curve and the determination of the antibacterial ability provide a new materials for further utilizing the Lactobacillus rhamnosus phage as a specific component for detection.(4)The carbon quantum dots were labeled with Lactobacillus rhamnosus phage LRPYZU021,and the efficiency of labeling and the activity of the labeled phage during storage and the change of fluorescence intensity were investigated.The results showed that the phage survival rate of CDs-labeled phage was 82.42±4.72%,and the phage labeling at 108 PFU mL-1 showed strong fluorescence,which showed good labeling efficiency,and the phage activity during storage was not significantly reduced within 5 days,and the fluorescence intensity remained stable during storage.The labeled phage-specific adsorption of Lactobacillus rhamnosus was measured by fluorescence spectrophotometer to determine the fluorescence intensity,thereby detecting the Lactobacillus rhamnosus,and the specificity of the method and flow cytometry were also considered.Compared.The results showed that the fluorescence intensity was not obvious when the bacterial concentration was below 107 CFU mL-1,which may be due to the low efficiency of CDs binding to phage and the phage-induced cell lysis may hinder the interaction between quantum dots and phage;the specificity indicates that the method Bm01,LYO,BG,LV108 and CRL1505 have no cross-reaction,indicating that the phage has better specificity than the antibody,and provides a novel green and specific method for microbial detection. |
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