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In recent years, the use of microwave radiation has become popular in the food industry for thawing, drying, and baking foods, as well as for the inactivation of microorganisms in foods (21, 31, 32). In particular, microbial destruction by microwave radiation has great potential in the pasteurization of foods (31). Its short heating and exposure time is less destructive to food than longer conventional heating (15).

There have been many studies on the use of microwaves for the reduction of microorganisms in various foods, including turkey, beef, corn-soy milk, chicken, frozen foods, and potatoes (1, 5, 8, 12, 14, 28, 36). All of these works have led to the conclusion that microwave radiation extends food preservation by reducing microbial cells in food. Microwave heating is known to inactivate many microorganisms, such as Escherichia coli, Streptococcus faecalis, Clostridium perfringens, Staphylococcus aureus, Salmonella, and Listeria spp. (2, 4, 5, 9, 10, 14, 15, 19, 20, 23, 24, 30). Bacterial and mold spores, as well as the bacteriophage PL-1, which is specific to Lactobacillus casei, have also been reported to be sensitive to microwave radiation (20-22).

Despite many studies on microbial destruction by microwave radiation, the mechanism of destruction is not fully understood. It is generally thought that the destruction of microorganisms is mainly due to a thermal effect of microwave exposure (16, 37, 38). However, another argument has also been proposed to explain microbial inactivation by microwaves. Several researchers have attempted to ascertain if such radiation has a nonthermal effect on microorganisms (7, 10, 27, 34). The destruction of microorganisms by microwave at temperatures lower than the thermal destruction point has been observed (11, 13, 22, 24, 27). In particular, microwave-stressed cells of S. aureus exhibited a greater metabolic imbalance than conventionally heated cells (27). Morozov and Petin found that hypertonic solutions (1.0%) of sodium chloride were less effective in protecting cells against heat damage during microwave heating than during thermal heating (29). This study examined the mechanism of microbial cell inactivation by microwave heating along with the differences in the effects on gram-positive and -negative bacteria.

Bacterial culture and microwave treatment. E. coli wild-type strain K-12 (3) was obtained from the Korean Collection for Type Cultures, and Bacillus subtilis KM107 (24) was obtained from the stock culture in our laboratory. E. coli was grown in Luria broth (1% Bacto Tryptone, 0.5% yeast extract, 1% NaCl) (33), and B. subtilis was grown in nutrient medium (0.3% Bacto beef extract, 0.5% Bacto Peptone) (35). The bacteria were cultured in 500 ml of the liquid medium at 37°C for 15 h on a rotary shaker (150 rpm). Cells were harvested by centrifugation and washed twice with a sterile 0.9% NaCl solution. The cell pellets were resuspended in a 0.9% NaCl solution at a cell concentration of 10⁹ to 10¹⁰ CFU/ml, which was used for the microwave radiation.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Changes in the temperature of bacterial cell suspensions relative to microwave exposure time. A bacterial cell suspension in 0.9% NaCl was exposed to microwave radiation at 600 W, and its temperature changes were monitored.

Measurements of viable cell counts and nucleic acid and protein amounts. The microwave-radiated cell suspensions were serially diluted with a sterile 0.9% NaCl solution and spread on Luria-Bertani agar (E. coli) or nutrient agar (B. subtilis) plates. The plates were incubated at 37°C for 24 h, and cells were enumerated. Cell density was measured at 600 nm using a spectrophotometer (CE393; Cecil Instruments, Cambridge, United Kingdom). The amount of protein released from the microwave-treated cells was measured at 595 nm by the method of Bradford (6). Bovine serum albumin was used as the standard protein. The nucleic acid content of the supernatants was directly measured at 260 nm using a UV spectrophotometer (U200; Hitachi Co., Tokyo, Japan). All the experiments were carried out in triplicate.

Electron microscopy. After the cells were treated by microwave radiation, the shape of the cells was examined by electron microscopy. The cells were fixed at 24°C for 60 min with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany), dehydrated with a serial concentration of ethanol, and then dried on a critical point dryer (HCP-2; Hitachi Co.). The dried cell samples were coated with gold (26), and examined using a scanning electron microscope (S-4100; Hitachi Co.). For transmission electron microscopy, dehydrated cells were embedded in a medium type LR white resin (Sigma Chemical Co., St. Louis, Mo.), which was polymerized at 60°C for 24 h (26). The polymerized samples were sliced with an ultramicrotome and observed using a transmission electron microscope (Hitachi Co.).

Inactivation of bacterial cells by microwave radiation. The inactivation patterns of the microwave-radiated cells were investigated using cell suspensions (10⁹ to 10¹⁰ CFU/ml) of E. coli, a gram-negative bacterial strain, and B. subtilis, a gram-positive strain (Fig. 2). The viable counts in both cell suspensions were found to dramatically diminish relative to an increase in the microwave heating temperatures. Treatment up to 80°C resulted in an approximate 5-log reduction of the viable count in both strains compared to the initial counts. The highest reduction ratio in the viable counts was observed when the temperature was increased from 50 to 60°C, which was a ca. 3-log reduction in E. coli organisms (from 1.1 × 10⁸ to 2.5 × 10⁵ CFU/ml) and a ca. 2-log reduction in B. subtilis organisms (from 3.3 × 10⁶ to 1.6 × 10⁴ CFU/ml). When the microwave heating temperature exceeded 60°C, the amount by which viable counts were reduced dramatically decreased. When the temperature was increased from to 60 to 80°C, the viable counts were reduced only by factors of 10 in the E. coli and 3 in the B. subtilis cell suspensions. Therefore, it is assumed that microwave heating for microbial inactivation is highly efficient up to a temperature of 60°C, yet not as effective at higher temperatures.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Changes in viable count and cell density in E. coli (left panel) and B. subtilis (right panel) cell suspensions relative to the temperature produced by microwave radiation. The temperature of 20°C shown on the x axis represents the temperature of cell suspensions before microwave treatment. , viable cell count; , cell density.

Leakage of cell materials caused by microwave processing. Another general indication of heat damage to microorganisms is the leakage of nucleic acid and protein from cells. Microwave-injured cells have often been reported to release ninhydrin-positive material, purines, and pyrimidines into a suspension (23). Nucleic acid and its related compounds, such as pyrimidines and purines, are well known to absorb UV light at a wavelength of 260 nm. The presence of these materials in a suspension indicates damage to the cell at the membrane level. Furthermore, similarly injured cells are also known to release intracellular proteins into a suspension.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Nucleic acid and protein amounts released into the cell suspension from microwave-radiated bacterial cells relative to the temperature produced by microwave radiation. After the E. coli () and B. subtilis () cell suspensions were heated to the temperature shown on the x axis, the amounts of nucleic acid and protein in the supernatant of the cell suspension were measured.

Effect of microwave radiation on the surface structure of bacterial cells. The opposite release patterns for the release of nucleic acid and protein in two bacterial strains prompted us to examine the surface structure of microwave-radiated cells (Fig. 4). The untreated cells and cells heated up to 70°C were examined using a scanning electron microscope, and the shapes of their surface structures were compared. It was found that untreated E. coli cells had a smooth surface, while most of the microwave-radiated cells exhibited severe destruction. The surfaces of the microwave-heated cells were damaged and had become rough and swollen. However, all the B. subtilis cells exhibited the same smooth surface. Whether the cells were microwave heated or not, no damage to their surface structures was observed. This result suggests that the microwave-radiated cells remained unlysed in suspension, although they were inactivated by the radiation. Furthermore, the damage to the surface structure of E. coli cells may not, therefore, be the main reason for inactivation by microwave heating.

View larger version (143K): [in this window] [in a new window]   FIG. 4.   Scanning electron microphotograph of untreated and microwave-radiated (up to 70°C) bacterial cells.

Sensitivity of microwave-heated cells to SDS. In order to investigate the sensitivity of microwave-injured cells to lysis by sodium dodecyl sulfate (SDS), microwave-heated cells in 0.9% NaCl were incubated at 37°C with shaking (150 rpm) in the presence of 0.1% SDS, and the cell density was monitored at 600 nm (Fig. 5). For E. coli, the density of the microwave-heated cell suspension was dramatically reduced within an hour of incubation in the presence of SDS, but it did not decrease significantly in the absence of SDS. In the case of the untreated cell suspension, no significant reduction in the cell density was observed during 4 h of incubation, regardless of the presence of SDS. These results support the conclusion that most of the cells inactivated by microwave radiation remain unlysed in a cell suspension in the absence of SDS. In addition, they are also highly sensitive to lysis by SDS.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Sensitivity of untreated and microwave-radiated bacterial cells to SDS. E. coli and B. subtilis cell suspensions in 0.9% NaCl, both untreated and microwave heated up to 70°C, were incubated at 37°C with shaking (150 rpm). Changes in cell densities were then monitored at 600 nm, both with and without the presence of 0.1% SDS. , untreated cells in the absence of SDS; , untreated cells in the presence of SDS; , microwave-radiated cells in the absence of SDS; , microwave-radiated cells in the presence of SDS.

Effect of microwave radiation on the intracellular components of cells. To investigate the effect of microwave heating on intracellular components, cells that were microwave heated up to 70°C were examined using a transmission electron microscope (Fig. 6). When microwave heated, both types of bacteria showed several dark spots in their cytoplasm. However, no dark spots were observed in the untreated cells, suggesting that the dark spots were the result of microwave heating. Heat treatment has been known to cause protein denaturation and aggregation in cytoplasm as well as to induce heat shock proteins (39, 40). Therefore, the dark spots are thought to be aggregated proteins caused by microwave heating. The two bacterial strains showed similar results for protein aggregation regardless of their cell wall structure, which suggests that protein aggregation may participate somehow in microbial inactivation caused by microwave heating. Further studies on the induction of heat shock proteins are in progress to elucidate whether microwave heating induces heat shock proteins in bacterial cells.

View larger version (122K): [in this window] [in a new window]   FIG. 6.   Transmission electron microphotograph of untreated and microwave-radiated (up to 70°C) bacterial cells.