Inactivation of Bacillus cereus Spores by High Hydrostatic Pressure at Different Temperatures

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1 599 Journal of Food Protection, Vol. 66, No. 4, 2003, Pages Copyright q, International Association for Food Protection Inactivation of Bacillus cereus Spores by High Hydrostatic Pressure at Different Temperatures SANGSUK OH* AND MYOUNG-JOO MOON Department of Food and Nutrition, Ewha Womans University, 11-1, Daehyun-dong, Sudaemoon-ku, Seoul, , South Korea MS : Received 12 April 2002/Accepted 18 October 2002 ABSTRACT The effect of ph on the initiation of germination and on the inactivation of Bacillus cereus (KCTC 1012) spores during high hydrostatic pressure processing (HPP) with pressures of 0.1 to 600 MPa at different temperatures was investigated. Two different high-pressure treatments were adopted to evaluate the effect of ph on the inactivation of B. cereus on sporulation medium and in suspension medium. Inactivation of B. cereus spores with HPP treatment was affected more by sporulation medium ph than by suspension medium ph. B. cereus spores obtained through sporulation at ph 6.0 showed more resistance to inactivation by HPP at 20, 40, and 608C than did those obtained through sporulation at phs of 7.0 and 8.0. Constituents of B. cereus spores obtained through sporulation at ph 6.0 may undergo electrochemical charge changes comparable to those for spores obtained through sporulation at ph 7.0. The initiation of B. cereus spore germination was more sensitive to pressure around 300 MPa at 208C. Increasing processing temperatures during HPP enhanced the effect of sporulation medium ph (i.e., environmental ph) on the inactivation of B. cereus spores. High hydrostatic pressure processing (HPP) treatment of food products can be used for nonthermal food processing because of its food preservation capability and its potential to achieve interesting functional effects (2, 9, 12). HPP s effect is instantaneous and uniform throughout a mass of food regardless of the food s size, shape, or composition (23). Bacillus cereus has been detected in diverse foods such as milk, vegetables, spices, rice, and sauces. Its pathogenicity is related to the production of two toxins, a thermostable emetic enterotoxin and a thermosensitive diarrhoegenic enterotoxin (8, 20). In general, a population of.10 5 bacteria per gram causes intoxication. The occurrence of viable spores in food products is a common phenomenon; it is therefore important to understand the germination and growth of the microorganism in food products, taking food characteristics and environmental conditions into consideration. Different microorganisms have different levels of resistance to HPP. Vegetative cells of bacteria and yeast are inactivated at pressures of ca. 300 to 400 MPa at ambient temperatures. Bacterial spores, however, can survive higher pressures (2). The inactivation of bacterial spores through HPP, unlike the inactivation of vegetative bacteria, occurs in two steps: (i) high pressures cause spore germination, and (ii) high temperatures inactivate the germinated spores (3, 5, 7, 22). The synergistic effect of high pressure and high temperature inactivates the spores. The resistance of bacterial spores to physicochemical agents is genetically determined, but it is also in uenced by many environmental factors, such as sporulation temperature, ph, and water ac- * Author for correspondence. Tel: ; Fax: ; ssoh71@mm.ewha.ac.kr. tivity. The effect of sporulation temperature is one of the strongest produced by any of the environmental factors (6, 17, 18, 21, 22). Spores can be inactivated by a medium s ph (13, 14). The objective of this study was to investigate the effects of HPP at different temperatures on the initiation of germination and on the inactivation of B. cereus spores obtained through sporulation on medium at phs of 6.0, 7.0, and 8.0 and suspended in McIlvaine citrate phosphate buffer at phs of 4.5, 6.0, 7.0, and 8.0. MATERIALS AND METHODS Preparation of spores. A colony isolated from B. cereus (KCTC 1012) on a nutrient agar (Difco Laboratories, Detroit, Mich.) was inoculated into nutrient broth (Difco) and incubated at 308C. The culture (0.03 ml) was then inoculated onto nutrient agar plates (30 ml) supplemented with MnSO 4 (3 mg/liter) and incubated at 308C (11). The ph of the medium was adjusted to 6.0, 7.0, or 8.0 with the addition of McIlvaine buffers (0.1 M citric acid and 0.2 M sodium phosphate) (4). Sporulation was assessed by microscopic examination. Spores were harvested when 80 to 90% sporulation was obtained after 2 to 3 days at 308C. Spores were collected by ooding the surface of the culture with sterile distilled water. The plate was then scraped with a sterile bent glass rod. After harvesting, spores were washed three times by centrifugation (10, 11) and resuspended in distilled water (16). The resulting spore suspension contained no visible vegetative cells and was stored at 48C. HPP. Spores obtained by sporulation on medium at phs of 6.0, 7.0, and 8.0 were diluted in McIlvaine buffer (4) (suspension buffer, ph 7.0) to a concentration of 10 8 to 10 9 spores per ml, and spores obtained by sporulation on medium at ph 7.0 were diluted in McIlvaine buffer (suspension buffer, ph 4.5, 6.0, 7.0, and 8.0) to a concentration of 10 8 to 10 9 spores per ml. Prepared diluted spore suspensions (3 ml each) were then aseptically trans-

2 600 OH AND MOON J. Food Prot., Vol. 66, No. 4 ferred to sterile polyethylene pouches (Stomacher, Seward Medical) and heat sealed after air was expelled. The sample pouches containing spores were processed with high pressure by using an isostatic pressing system (MFP-7000, Mitsubishi Heavy Ind., Japan) with a cylindrical pressure chamber (height, 0.26 m; diameter, 0.06 m). The samples were subjected to pressures ranging from 0.1 to 600 MPa for 15 min at 20, 40, and 608C. The pressureprocessed sample pouches were immediately immersed in cold water after treatment for the enumeration of surviving spores. For each experiment, an untreated sample was used as a control to determine the initial number of spores. The spores in the processed and unprocessed samples were enumerated by the method of Raso et al. (17). The samples were serially diluted in sterile 0.1% peptone (Difco) water, plated on a nutrient agar (Difco), and incubated at 308C for 36 to 48 h. Each experiment was repeated three times, and average counts were obtained. Initiation of spore germination. The extent of the germination of B. cereus spores after pressure treatment was determined by the method of Raso et al. (17). The samples (1 ml) were introduced into screw-cap test tubes containing 9 ml of the Mc- Ilvaine citrate phosphate buffer and immersed for 30 min in a water bath at 658C to kill the vegetative cells. The heat-resistant spores that survived (ungerminated spores) were then counted by the pour plating method. The viability of the intact spores of B. cereus was found to be unaffected by heating at 658C for 30 min (10). A spore count reduction was expressed as the log count for an untreated sample (control, N 0 ) minus the log count for a treated sample (N). Statistical analysis. Experiments were carried out in duplicate, and three samples per experiment were analyzed. Signi cant differences (at the 0.05 level) in spore reduction logarithms due to treatments were determined by analysis of variance and Duncan s multiple-range test (SAS Institute Inc., Cary, N.C.). RESULTS AND DISCUSSION Effect of nutrient agar ph on sporulation time for B. cereus. Attempts to produce satisfactory spore crops at ph 4.5 were unsuccessful, in part because of growth suppression. The sporulation times for 80 to 90% of the initial vegetative cells were 60 (90% sporulation), 36 (90% sporulation), and 48 h (80% sporulation) at phs of 6.0, 7.0, and 8.0, respectively. Slight ph changes were observed throughout the sporulation period. The most rapid sporulation rate was observed at ph 7.0. These results are similar to those of Mazas et al. (11), who reported that maximum spore yield is obtained at a nearly neutral ph. Lethal effect of HPP at different temperatures on spores suspended in buffer at different ph values. Two different HPP treatments were employed to evaluate the effect of ph on the inactivation of B. cereus spores. HPP has been used with suspended spores in a buffer at different phs for previous experiments demonstrating the effect of ph on the inactivation of spore-forming microorganisms. Data on the in uence of HPP at different temperatures on the inactivation of B. cereus spores obtained by sporulation at phs of 6.0, 7.0, and 8.0 are shown in Figure 1. B. cereus spores treated with high pressure at 208C were not signi - cantly different with regard to their resistance to inactivation by HPP at the ph values tested. B. cereus spores obtained by sporulation at ph 6.0 and pressure treated ($300 FIGURE 1. Inactivation of B. cereus (KCTC 1012) spores obtained through sporulation at ph 6.0 ( ), ph 7.0 ( ), and ph 8.0 ( ) at various pressures for 15 min at (a) 20, (b) 40, and (c) 608C (average log N/N 0 6 standard deviation). MPa) at 40 and 608C appeared to be more resistant to inactivation by HPP than spores obtained through sporulation at phs of 7.0 and 8.0. The extent of the reduction of spores obtained through sporulation at ph 6.0 with pressure treatment ($300 MPa) was signi cantly different (a ) from that of the reduction of spores obtained through sporulation at ph 7.0 or 8.0. For B. cereus spores obtained through sporulation at phs of 6.0 and 7.0,.1.5-log differences in levels of resistance to inactivation by HPP at 300, 450, and 600 MPa were observed when spores were treated with high pressure at 40 and 608C. When the processing temperature increased from 20 to 408C, the effect of ph on the inactivation of B. cereus spores was signi cantly increased. The magnitude of the effect of ph on the inactivation of B. cereus spores was apparently greatest when pressure treatment was carried out at 408C. The effects of ph at 608C appear to be less extensive than those at 408C,

3 J. Food Prot., Vol. 66, No. 4 INACTIVATION OF B. CEREUS SPORES BY HPP 601 FIGURE 2. In uence of the ph of the suspension buffer (M, ph 4.5;, ph 6.0;, ph 7.0;, ph 8.0) on the inactivation of B. cereus (KCTC 1012) spores at various pressures for 15 min at (a) 20, (b) 40, and (c) 608C (average log N/N 0 6 standard deviation). probably due in part to the increased impact of temperature. Furthermore, spores showing higher levels of resistance took longer to sporulate on medium at ph 6.0 than at ph 7.0 or 8.0. No optimum inactivation pressure was observed; an increase in pressure resulted in more extensive inactivation of spores at all phs studied. To investigate the effect of suspension buffer ph on spore inactivation by HPP, the spores obtained by sporulation at ph 7.0 were suspended in buffers of phs 4.5, 6.0, 7.0, and 8.0. Spores suspended in a buffer of ph 4.5 were substantially more resistant to inactivation than those suspended in buffers of phs 6.0, 7.0, and 8.0 at all temperatures when treatment with 300, 450, and 600 MPa was used (Fig. 2). No signi cant differences in the levels of inactivation of B. cereus spores suspended in buffers of phs 6.0, 7.0, and 8.0 were observed. There was, however, a clear difference between the magnitude of the inactivation of B. cereus spores suspended in a buffer of ph 4.5 and that of the inactivation of spores suspended in a buffer of ph 7.0 at 408C. No differences in the levels of inactivation of B. cereus spores at phs of 6.0, 7.0, and 8.0 were found. An increase in pressure resulted in more extensive inactivation at all phs tested except ph 4.5. B. cereus spores appear to be more sensitive to pressure treatments of 300 and 450 MPa at a neutral ph than at ph 4.5. This nding is in agreement with results reported by Sale et al. (22), who found the inactivation of bacterial spores by HPP was more extensive when the buffer ph was nearly neutral. However, Roberts and Hoover (19) reported increased sensitivity to HPP at lower phs for B. coagulans spores. When the levels of inactivation of B. cereus spores on sporulation medium (Fig. 1) and in suspension buffer (Fig. 2) at phs of 6.0, 7.0, and 8.0 are compared, it is apparent that the in uence of ph on spores resistance to inactivation is more extensive for sporulation medium than for suspension buffer. This nding is possibly due to changes in electrochemical charges of spores structural components. Movahedi and Waites (14) reported that heat shock proteins formed when B. subtilis cells were treated at 488C. These investigators suggested that heat shock proteins increase spores heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth. Enhanced resistance of spores to HPP at ph 6.0 at 408C (Fig. 2) may have been due to changes in electrochemical charges of structural components of spores during their formation. The differences in the inactivation resistance levels of B. cereus spores obtained at ph 4.5 and those obtained at ph 7.0 may indicate the importance of electrical charges of spore structural components such as Ca 21 and dipicolinate. In addition, the sporulation rate was slower at ph 6.0 than at phs of 7.0 and 8.0, as discussed above. This nding could be related to changes in the electrochemical charges of the components brought about by environmental ph. Initiation of germination of B. cereus spores by HPP at different ph values. Data on the in uence of HPP on the initiation of the germination of B. cereus spores obtained through sporulation at phs of 6.0, 7.0, and 8.0 are presented in Figure 3. After 15 min of pressurization, HPP had no signi cant effect on the initiation of the germination of B. cereus spores at 208C (Fig. 3). Regardless of ph, the initiation of the germination of B. cereus spores appeared to be more affected by pressure at ca. 300 MPa at 208C, a nding similar to that of Raso et al. (16), who reported that the optimum pressure for the initiation of germination of spores was ca. 250 MPa. These results suggest that the initiation of germination of B. cereus spores was sensitive to pressure at,300 MPa. Nakayama et al. (15) demonstrated that spores of various Bacillus spp. were inactivated more ef ciently at moderate pressures (200 to 500 MPa) than at a high pressure (.500 MPa). These investigators indicated that a moderate pressure range could induce spores to germinate and subsequently lose their resistance to high pressure, assuming that germination was less ef cient at a high

4 602 OH AND MOON J. Food Prot., Vol. 66, No. 4 FIGURE 3. Initiation of germination of B. cereus (KCTC 1012) spores obtained through sporulation at ph 6.0 ( ), ph 7.0 ( ), and ph 8.0 ( ) at various pressures for 15 min at (a) 20, (b) 40, and (c) 608C (average log N/N 0 6 standard deviation). pressure. A relatively well de ned optimum pressure of 300 MPa was identi ed for the germination of the four spore suspensions of B. cereus at different phs. An optimum pressure of ca. 250 MPa was also identi ed by Sale et al. (22), who worked with spores of various species of Bacillus and Clostridium. An increase in treatment pressure at 40 or 608C resulted in more extensive inactivation at all phs. As shown in Figure 3b and 3c, spores at ph 6.0 clearly show more extensive resistance to high-pressure treatments (.300 MPa) than do those at ph 7.0. B. cereus spores suspended in buffer at ph 4.5 exhibited substantially higher levels of resistance to the initiation of germination by HPP than did those suspended at phs of 6.0, 7.0, and 8.0 at the same pressures and temperatures FIGURE 4. In uence of the ph of the suspension buffer (M, ph 4.5;, ph 6.0;, ph 7.0;, ph 8.0) on the initiation of the germination of B. cereus (KCTC 1012) spores at various pressures for 15 min at (a) 20, (b) 40, and (c) 608C (average log N/ N 0 6 standard deviation). (Fig. 4). Spore surface electrical charges may have some effect on resistance to the inactivation of spores at high pressure. It is, however, not clear whether there was any difference in the HPP resistance levels of B. cereus spores suspended at phs of 6.0, 7.0, and 8.0. On the basis of the results presented in Figures 3 and 4, it can be assumed that sporulation medium phs of 6.0, 7.0, and 8.0 may have affected changes in electrochemical charges of spore structural components, which may have contributed to the resistance of spores to HPP. Environmental conditions during sporulation can also in uence the pressure resistance of bacterial spores. The in uence of ph on bacterial spore germination at ambient pressure is well known (17, 19, 24). Our results show the

5 J. Food Prot., Vol. 66, No. 4 INACTIVATION OF B. CEREUS SPORES BY HPP 603 effects of the ph of the sporulation medium and the ph of the suspension buffer on the initiation of germination and on the inactivation of bacterial spores by HPP, even though the exact mechanism of spore injury and loss of dormancy resulting from HPP is not known. Raso et al. (17) reported that pressure-induced germination of bacterial spores depends on the ph of the suspension buffer. Many food preservation processes take advantage of the fact that bacterial spores cannot germinate ef ciently at low phs (1). Results obtained by other researchers indicate that the inactivation of bacterial spores by HPP, unlike the inactivation of vegetative bacteria, occurs in two stages (3, 5, 7, 22). First, pressure induces the germination of spores, and then the pressure inactivates the germinated spores. With pressure, temperature produced a synergistic effect on spore inactivation. The results of this study con rm this pattern of inactivation: under all treatment conditions, more spores were germinated and subsequently inactivated at pressures of $300 MPa. An increase in processing temperatures during HPP could enhance the effect of sporulation medium ph (i.e., environmental ph) on the inactivation of B. cereus spores. ACKNOWLEDGMENTS We thank Pulmuone Co., Ltd., for allowing us to use the MFP-7000 systems. We are indebted to Dr. N. R. Reddy for his critical review of this manuscript. This work was supported by the Intramural Research Grant of Ewha Womans University. REFERENCES 1. Blocher, J. C., and F. F. Busta Bacterial spore resistance to acid. Food Technol. 11: Cheftel, J. C High-pressure, microbial inactivation and food preservation. A review. Int. Food Sci. Technol. 1: Clouston, J. G., and P. A. Wills Kinetics of initiation of germination of Bacillus pumilus spores by hydrostatic pressure. J. Bacteriol. 103: Dawson, R. M. C., P. A. Elliot, W. H. 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