Enterotoxin B Solution Conformation And Environmental Effect On Growth For Staphylococcus Aurus

Staphylococcus aureus is one of the major human and animal pathogens that produce a wide range of toxins and a group of enzymes contributing to their ability to cause diseases. Staphylococcal enterotoxins (SEs), a family of ten major serological types of heat stable enterotoxins, are a key cause of staphylococcal food poisoning and gastroenteritis in human. Staphylococcal food poisoning is the second most common food poisoning events caused by bacterial pathogens. In this paper, Staphylococcus aureus S6 producing staphylococcal enterotoxin B was studied. On the basis of purified SEB, aggregation structure, the molecule chain morphology and secondary structural parameters of SEB were investigated with the advanced techniques, such as X-ray diffraction, scan electron microscopy, atomic force microscopy, fourier transform infrared spectrum, laser raman spectrum, circular dichroism spectrometer and fluorescence spectrum. The characteristic conformation and possible unfolding process of SEB in dilute solution and different conditions were emphatically studied. Furthermore, the inhibition of Staphylococcus aureus by lactic acid bacteria and their metabolizing substance was studied. The inhibiting mechanism of nisin on Staphylococcus aureus was also studied from the point of microcalorimetry. The main results from this study are as follows:1. The isolation and purification of SEBStaphylococcal enterotoxin B was obtained in the relative yield of 26.8% by the method of cellophane covered agar plate and electrophoretic homogeneity from the culture of Staphylococcus aureus. The major purification procedure involved ion-exchange chromatography with CM-cellulose and gel filtration with Sephadex G-200. The molecular weight of purified SEB was determined as 2.8 × 10~4 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the characteristic absorbance peak on UV spectrum was 276.6nm, which is consistent with that in the reported literature.2. Structure characterization of SEBThe aggregation structure, the molecule chain morphology and secondary structural parameters of SEB were studied by modern analytic techniques, such as X-ray diffraction,scan electron microscopy, atomic force microscopy > fourier transform infrared spectrum, and laser raman spectrum.The results showed that there was the formation of crystallizing field in SEB with crystallinity of 37.8%. The SEB molecule was conglomerated with the shape of sheet and plate. The molecular chain conformation of SEB was observed directly by AFM, and its length, width and height of molecular chain was 1500nm, 20~40nm and 0.6nm respectively. The secondary structural unit of SEB was mainly composed of P -sheets and a -helixs with few random coils. The side chain conformation of C—C—S — S —C—C was trans-twist-trans.3. The conformation and unfolding of SEB in the dilute solutionThe secondary structure of SEB derived from the far-UV circular dichroic spectra were composed of 22.6% a -helixs,40.4% P -sheets,19.2% turns and 17.7% random coils in water solution at the concentration of 0.02mol/L (25 °C). Within the range of 50°C and 121 "C, the content of a -helixs gradually descended from 22.6% at 25 °C to 9.6% at 95 °C with the increase of temperature, and a -helixs conformation of SEB completely disappeared at 121 °C. In opposition to the change of a -helixs content in SEB, the content of 3-sheets remained comparatively stable, with the exception at 75 °C the content of P -sheets was 8.5%. The content of P -sheets was up to 39.9% at 121 "C (more than 30.2% at other temperatures). The results showed that the change of temperature almost could not induce unfolding of SEB, and P -sheet was the major unit to maintain the structural stability and microbio-activity of SEB. The Laser Raman Spectrum of SEB obtained by heat treatment at 121 °C indicated that the characteristic vibration of a -helixs and S-S linkage was disappeared, but still remained vibrating band of P -sheets at 1672cm"1. These results proved that SEB was a heat stable proteins, and provided theoretical foundation for this property. There have been no reports on similar results previously.The effect of SDS, urea and nisin on the conformation of SEB was also studied by circular dichroism spectrometer and fluorescence spectrum. Though the content of a -helixs in the secondary structure of SEB decreased, the content of P -sheets and random coils increased, the major conformation was still P -sheets after the treatment of SDS and nisin. This means that SDS and nisin did not make SEB deactivated. On the other hand, the content of a -helixs in (he secondary structure of SEB remarkably increased, while P -sheets and random coils disappeared, and turned to P -turn after the treatment of urea. This means that urea did unfolding action on SEB, and confirms the reported prediction that urea treatment was not suitable for the detection of heat-denatured enterotoxins in thethermally processed foods.It was indicated in fluorescence spectrum experiments that the tryptophane residue in SEB turned to hydrophobic environment and fluorescence quantum yield and relative intensity decreased with the increase of urea concentrations (0~6mol/L). On the other hand, the tryptophane residue in SEB turned to hydrophilic environment and the relative intensity of SEB increased with the concentration increase of SDS and nisin. In addition, 30% ethanol and 30% glycerol made the intrinsic fluorescence intensity of SEB an increase of 29.9% and 461.0% respectively, but the maximum emission wavelength remained unchanged, which showed the same effect as SDS did. SEB showed faintly effect on the fluorescence spectra of nisin with little increasing of relative intensity, and the maximum emission wavelength of nisin remained unchanged.4. Preparation of the antibody against staphylococcal entrotoxin B andestablishment of indirect competition ELISAfor SEB detectionThe immune protocol of little dose and long period was adopted to immune Balb/c mouse with injecting SEB by multi dots and multi times. When the mouse serum titer was appropriate, the Balb/c mouse was injected with a non-antibody-secreting myeloma cell P2/0 to induce ascites formation, which was then extracted for the preparation of antibody. It had a potential application when the titer of antibody measured by indirect ELIS A achieved l:105.An indirect competition ELISA for detecting SEB was established by homemade SEB and anti-SEB antibody in this study. The detection limit of indirect ELISA assay established in this study was lng/ml with a detection concentration range of 1—10Jng/mL and correlation coefficient r=0.9951.The recovery of artificially contaminated sample was 94.5%.5.The inhibitory effects of lactic acid bacteria and their metabolicsubstances on Staphylococcus aureusThe growth of staphylococcus aureus was inhibited by a low pH> the occurring of anti-microbial substances and action of nutritional competition among microorganisms when Staphylococcus aureaus, Lactobacillus bulgaricus and Streptococcus thermophilus were grown by a commixture culture. Lactic acid, acetic acid and H2O2 showed inhibitory effect on the growth of staphylococcus aureus with positive correlation to their concentrations. The inhibition of lactic acid and acetic acid on Staphylococcus aureus was stronger than that of inorganic acid (hydrochloric acid), and they both showed obviousinhibitions on the growth of Staphylococcus aureus at pH 3.5. The inhibition of nisin on the growth of Staphylococcus aureus was correlative with its concentration, and the correlation could be expressed as y=0.8025x+5.12, R2=0.9939.6. Effect of nisin on the growth of Staphylococcus aureus by amicrocalorimetric methodA novel microcalorimetric technique was applied to evaluate the biological effect of nisin on the growth of Staphylococcus aureus. The physiological, biochemical and metabolic characteristics of Staphylococcus aureus changed in the presence of nisin. The results showed that the growth rate constant of Staphylococcus aureus decreased and the generation time prolonged with the increasing of nisin concentration. The relationship between the growth rate constant (k) and the concentration of nisin (c) is k=0.0377-3.988xl0"4c, with a correlation coefficient of -0.9971. Half inhibitory concentration of nisin on the growth of Staphylococcus aureus was 48.05IU/mL, the critical inhibitory concentration was 94.53IU/mL. The growth rate constant, the generation time, the half inhibitory concentration and the critical inhibitory concentration obtained by microcalorimetric method which could be used as a quantitative index for evaluating the inhibitory effect of nisin on microorganisms, which could be also applied for evaluating the inhibitory effect of other bacteriocins. There have been no reports on similar results previously.7. Aspects on the ecology of Staphylcococcus aureus in yoghurtThe initial inoculating amount of Staphylococcus aureus in raw milk was investigated on its existent ability in yoghurt. No Staphylcococcus aureus and its products thermonuclease and staphylococcal enterotoxin B were detected in yoghurt during the fermentation and storage when its initial inoculation in raw milk were 1.8 X 10 cfu/mL and 1.8 X102 cfu/mL. When the initial inoculation in raw milk were 8.5 X103, 2.7 X104, 2.7 X105 and 1.8 X 106cfu/mL, the amount of Staphylcococcus aureus increased gradually during the process of fermentation, while it decreased during the stage of storage, and at the same time the thermonuclease was positive and the content of SEB were 0> 6.5, 38.5 an 77.9ng/mL respectively. This means that the growth of Staphylcococcus aureus and the production of enterotoxins were not effectively inhibited by the low pH and metabolic substances when the raw milk was contaminated with large amount of the bacteria. There was a hazard to cause staphylococcal food poisoning if these foods were consumed.