Spores of foodborne pathogens such as Clostridium botulinum,
Clostridium perfringens and Bacillus cereus are widely distributed in nature.
Presence of those spores in food products, particularly C. botulinum spores in
vacuum packed, ready-to-eat low-acid products, is a great safety concern. The
research here described is a first effort towards understanding the role of the
spore coat proteins in the inactivation of bacterial spore using high pressure
processing. This study proposes a coat protein solubilization methodology
using non-ionic detergents minimizing protein damage and compatible with
spectroscopy methods. The methodology developed here was compared with
approaches proposed in the literature with respect to protein yield, protein
fractions identified, amino acid composition and suitability with spectroscopy
techniques for the further analysis of coat proteins.
Bacillus subtilis ATCC 6633 spore coat proteins were solubilized
(n=3) using octyl-β-D-glucopyranoside (OGP) at room temperature and
urea/sodium dodecyl sulphate (UDS) at 37C and 70C. Analysis of variance
(ANOVA) showed no significant (95% confidence) differences between the
three repetitions of the three spore coat protein solubilization methods. Protein
yield was significantly larger (95% confidence) when using UDS at 70C as
compared to UDS at 37C. OGP gave the lowest protein yield but allowed circular dichroism (CD) analysis of the spore coat protein solution with
minimum blank signal. SDS-PAGE revealed that the UDS-70C coat protein
solutions consisted of five major and six minor proteins ranging 6 to 65 kD
while the OGP solution appear to consist of four major and nine minor bands
in the same mw range. Amino acid analysis of the protein extracted by the
OGP method was conducted using reverse phase HPLC (RP-HPLC) and
compared with published information. The OGP spore coat protein solution
showed a higher proportion of aspartate, glutamate, alanine and tyrosine.
Pressure, heat and time effects were studied on spore coat proteins
obtained from untreated and pressure-treated B. subtilis ATCC 6633 spores.
Pressure treatments of spores, and of extracted spore coat protein solutions, at
50 kpsi (345 mPa) and 85 kpsi (586 mPa) for 10 and 30 min at constant 85C
along with appropriate heat- and pressure-only controls and untreated sample,
were used to study the effect of pressure, heat and time on spore coat proteins.
Both spore coat protein solubilization procedures showed a significant
reduction in protein yield for pressure-only, heat-only and pressure/heat treated
spores when compared with untreated spores. When OGP-solubilized proteins
from untreated spores were pressure treated, SDS-PAGE profile showed an
increasing overall band intensity with increasing pressure and time. In the case
of protein solution obtained from pressure-treated spores the electrophoretic
pattern showed the loss of higher molecular weight proteins.
The significance of this study is that for the first time we have observed
extensive changes on spore coat proteins caused by pressure, as well as heat
treatments. Future studies will examine what is the probable physiological role
of the proteins damaged by these physical treatments. An advantage of the
protein solubilization here developed will allow the application of
spectroscopy techniques to characterize changes in spore coat proteins. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27290 |
| Date | 08 November 2002 |
| Creators | Gandhi, Kalpesh K. |
| Contributors | Torres, J. Antonio |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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