FTIR Spectroscopy Tracks Changes in Bacterial Spores Under Heat and Pressure

Lynn M. Savage

Bacterial spores — protective shells that help certain bacteria resist environmental stress — are challenging to the food industry. Typically, raising the temperature high enough eventually will inactivate spores, but often this adversely affects the nutrition and sensory properties of the foodstuffs on which they can be found.

Thermal processing, or pasteurization, of food can be aided by adding high pressure to the technique; in fact, many low-acid foods, such as eggs and potatoes, often are treated with pressure-assisted thermal processing methods. However, little is known about exactly how bacterial spores are inactivated by these methods, and knowing the mechanism of action would be important for the food-processing industry.

Researchers used Fourier transform infrared spectroscopy and multivariate analysis to characterize spores of various bacterial species and to discriminate among them after they had been treated with high heat, or high heat and high pressure. Multiple spectra acquired from each species readily clustered, indicating species. Reprinted with permission of the American Chemical Society.

Now a group of researchers from Ohio State University department of food science and technology in Columbus has applied a Fourier transform infrared (FTIR) spectroscopy technique to elucidating the chemical changes that occur in the spores of five strains of bacteria. Led by assistant professor Luis Rodriguez-Saona, the investigators found that the technique, combined with multivariate analysis, can help identify specific strains of bacteria. Furthermore, they believe that the method may be used one day to screen spores that are resistant to pressure-assisted thermal processing altogether.

The researchers analyzed 10-μl samples that contained spores of Clostridium tyrobutyricum, Bacillus sphaericus or one of three strains of B. amyloliquefaciens in suspension. They processed some samples at 121 °C at ambient pressure and others at the same temperature but with an increase in pressure to 700 MPa. The remainder were untreated controls.

They placed each treated or untreated sample onto a crystal in an attenuated total reflectance device made by Pike Technologies of Madison, Wis. As reported in the Oct. 31 issue of Journal of Agricultural and Food Chemistry, drying the spore suspensions on the crystals created a uniform film that enabled the scientists to collect high-quality spectra with distinct spectral features. They measured the spectra using an FTIR spectrometer made by Digilab Inc. of Canton, Mass. (part of a line of devices now manufactured by Varian Inc. of Palo Alto, Calif.).

According to researcher Anand Subramanian, they chose FTIR spectroscopy because it is simple, sensitive and cost-effective in the long run. “In microbial analysis, the limiting step is the plating method, which can take several days to provide quantitative results. FTIR enables rapid analysis and is capable of providing quantitative as well as biochemical information,” he said.

The researchers found that the major spectral bands that helped discriminate among the bacterial species were at 1626 cm^–1, which is asso-ciated with the amide I band of β-pleated sheets of secondary proteins, and at 1281, 1378, 1440 and 1616 cm^–1, the bands associated with dipicolinic acid (DPA), a major constituent of the spores.

Importantly, they found also that not only is DPA released rapidly during inactivation of the spores via pressure-assisted thermal processing, but also that there is a correlation between levels of the calcium chelate of DPA and the spores’ resistance to the treatment.

The investigators plan to elucidate further the biochemical changes that occur in spores during inactivation.

Their research plans include using FTIR to investigate changes in bacterial spores under various combinations of pressure and temperature and examining the response of spores to processing when they are present in food.

Contact: Luis E. Rodriguez-Saona, Ohio State University; e-mail: rodriguez-saona.1@osu.edu.