THERMAL RESISTANCE OF BACILLUS CEREUS SPORES AS AFFECTED BY ADDITIVES IN THE RECOVERY MEDIUM

ISAAC GONZALEZI, MERCEDES LOPEZ~, MARGARITA WAS, JOSEFA GONZkEZI and ANA BERNARD01.3

'Dpto. de ffigiene y Tecnoiogia de Ios Alimentos Universidad de Ledn, Spain

2Area Tecnologia de 10s Alimentos Universidad de Vigo, Spain

Received for Publication November 25, 1996 Accepted for Publication January 22, 1997

ABSTRACT

The effects of the addition of starch, glucose, sodium chloride, sodium citrate, monopotassium phosphate and disodium phosphate to the recovery medium on apparent heat resistance of Bacillus cereus spores (ATCC 4342, 7004 and 9818) were investigated. Sodium citrate, monopotassium and disodiurn phosphate at concentrations of 0. I% were efective inhibitory agents for heat injured B. cereus spores especially for strain 9818, although only monopotassium and disodium phosphate caused a signifcant reduction ($0.05) in D-values obtained for strain 9818. Sodium chloride also had a marked efect on the recovery of heat injured spores. Concentration as low as 0.5% caused a significant reduction in the recovery rates for strains 981 8 and 7004. In all cases, increasing the salt levels from 0.5 to 4% resulted in a progressive decrease in spore recovery. D-values gradually decreased as the salt content increased, although the concentrations which produced statistically significant differences (p< 0.05) varied among strains. The addition of starch at 0. I % resulted in a significant increase in the counts for strains 9818 and 7004. In contrast, glucose (0. I %), did not significantly mod& the counts obtained. Neither of these compound aflected decimal reduction times. No statistical signlficance (PO. 05) diferences were detected among z-values for the spores of the three strains recovered in the presence of dflerent additives assayed. z- Values rangedfiom 6.67 to 8.32, with a mean value of 7.56 f 0.46C.

'Correspondence to: Dr. h a Bemardo, Dpto. de Higiene y TecnologIa de 10s Alimentos, Facultad de Veterinaria, Universidad de Le6n, Campus de Vegazanash, 24071-Le611, Spain. Phone: 34 87 291 182; Fax: 34 87 291284.

Journal of Food Safety 17 (1997) 1-12. AN Rights Reserved. OCopyright 1997 by Food d; Nutrition Press, Inc., Trumbull, CT06611 1

2 I . GONZALEZ ETAL.

INTRODUCTION

Bacillus cereus causes food-borne illness and is frequently isolated from pasteurized dairy products. It is also occasionally isolated from processed foods (Franklin 1970; Bradshaw et al. 1975; Mostert et al. 1979). Despite the important role of B. cereus in heat processed foods, data on its thermal resistance characteristics are very scarce, Furthermore, the influence of environmental factors on its heat resistance is practically unknown. Our group studied the effects of some recovery conditions on parameters determining thermal resistance of this organism such as recovery medium and temperature (Gonziilez et al. 1995) and pH of the recovery medium (God lez et ul. 1996). No information is available on the effects of components normally present in foods (starch, glucose, sodium chloride) and certain stabilizing additives (phosphates and citrates) could have on the behavior of heated B. cereus spores. These compounds are used extensively in the food industry and may affect the recovery of surviving heat damaged spores.

For other sporeformers there is sufficient information on the beneficial effect of starch in recovery media on the recovery of heated spores. Some authors have reported that heat resistance is greater in media containing starch (Cook and Gilbert 1968; Mallidis and Scholefield 1986). Thus, this compound may promote recovery of thermally injured B. cereus spores.

It is also well established that the presence of sodium chloride in the recovery medium influences heat resistance of spores. However, the effect exerted by salt seems to vary among the species. Thus, while for B. stearothermophilus and Clostridium sporogenes, most authors agree that as the concentration of salt increases both the recovery rates and the D-values drop (Jarvis et al. 1976; Labbe 1979; Chumey and Adams 1980; Feeherry et al. 1987; Hutton et ul. 1991; L6pez et al. 1994); for B. subtilis and B. pumilus spores, the recovery is better when the medium contained this ingredient (Briggs and Yazdany 1970).

The aim of this work was to determine the influence of several compounds added to the recovery medium (starch, glucose, sodium chloride, phosphates and citrates) upon recoverability and D and z-values of three B. cereus strains.

MATERIALS AND METHODS

Microorganisms and Spore Preparation

Three strains of B. cereus (ATCC 4342,7004 and 98 18) were used in this study. Spores were produced in nutrient agar (NA, Difco, Detroit, MI) supplemented with 1 ppm Mn" at 32C as described by Gondlez et ul. (1 995). Stock suspensions were stored in McIlvaine buffer -citric aciddisodium hydrogen phosphate- (Mckenzie and Dawson 1969) pH 7.0 at 0-5C for hrther use.

HEAT RESISTANCE OF B. CEREUS 3

Thermal Treatments

Thermal treatments were carried out in a thermoresistometer TR-SC (Condbn et a/. 1989) over a wide range of temperatures (92-1 14C). Buffer McIlvaine (400 mL) pH 7.0 was used as a heating menstruum and was inoculated with 0.2-1 mL of spore suspensions of each strain. Intervals between viability determinations ranged from 0.5 s to 2 min, depending on the heat resistance of the strain and treatment temperature.

Recovery Conditions

Aliquots of 0.1-0.5 mL of the heated spore suspensions were sampled at different treatment times and were directly placed on petri dishes poured with nutrient agar. Parallel counts were also made on NA containing 0.1% (w/v) of glucose (Difco, Detroit, MI), starch (Difco, Detroit, MI), monopotassium phosphate (Panreac, Montplet & Esteban SA, Barcelona, Spain), disodium phosphate (Panreac, Montplet & Esteban SA, Barcelona, Spain) or sodium citrate (Panreac, Montplet & Esteban SA, Barcelona, Spain). The media in experiments performed with monopotassium phosphate and disodium phosphate were adjusted to pH 6.8 with 1N NaOH (Panreac, Montplet & Esteban SA, Barcelona, Spain) and 1N HCI (Panreac, Montplet & Esteban SA, Barcelona, Spain), respectively.

Finally, sodium chloride was tested by adding to the plating medium concentrations of 0.5, 1,2,3 and 4% (wh) of NaCl (Panreac, Montplet & Esteban SA, Barcelona, Spain).

Plates were incubated at 30C for 20-24 h, but plates containing sodium chloride were incubated for up to 3 days. Longer incubation time did not result in a significant increase in the number of CFU/plate.

Colonies of strains ATCC 4342 and 7004 were counted using the method described by Cond6n et ul. (1987). Strain 9818, due to its overspread, required suitable dilutions to determine the counts. Spore level recovery was determined as described by Gonzhlez et ul. (1 995).

D and Z-values

D-values were calculated as the negative reciprocals of the slopes of the regression lines plotted with the straight portion values of the survival curves (log population versus time). Strain 7004 exhibited tails at all temperatures tested. Decimal reduction times were obtained from the first portion of their survival curves. D-values were determined in triplicate at 1OOC. Student’s “t” test (Steel and Torrie 1960) was used to check the significance on the slope value. z-Values were determined as the negative reciprocals of the lines of regression of thermal death time curves (log D-values versus temperature). In order to compare z-values, the homogeneity test was used, calculated as described by Steel and Torrie (1960).

4 I. GONZALEZ ETAL

RESULTS AND DISCUSSION

Effect of Starch and Glucose on Heat Resistance

The results presented in Table 1 show that the three strains tested showed increase in recovery percentages when starch was added to the recovery medium, although with strain 4342, it was not statistically significant (p0.05). The addition of glucose did not significantly modify the recovery efficiency obtained with any of the strains studied.

'The average D,, values and z-values obtained in different assayed conditions are summarized in Table 2. Neither decimal reduction times nor z-values were affected by the addition of these compounds to the recovery medium (p>0.05).

The positive effect of starch on the recovery has already been reported for other sporeformers, such as Clostridium botulinum (Olsen and Scott 1950; Sugiyama 1951) and Bacillus steurothermophilus (Cook and Gilbert 1968; Labbe 1979;

TABLE 1. EFFECT OF STARCH AND GLUCOSE ON THE RECOVERY* OF BACILLUS CEREUS

SPORES

Experimental Condition Recovery (%)* on

Strain H.T. Time (min) NA NA+Starch' NA+Glucose'

4342 IOOC 1.66 1oo.oi 9.2" 1 07.8* 13.6" l04.1* 9.65'

2.66 lOO.oi12.4" 129.8i18.6" 116.2*15.6"

3.66 100.oil2.9" 134.7*21.4" I09.7*13.6"

7004 96C 0.33 100.M13,6ab 132.7*17.9" 72.4* 13 .7b

0.66 1oo.oi 9.2" 15 1.5i16.9b 91.3* 7.3"

I .oo 100.oi16.8" 1 58.8*27.9b 84.1*12.9"

9818 104C 1.66 100.M 7.1" 139.2*17.6b 125.9*18.4"b

2.66 lOO.O* 7.9" 142.6*24.6b 116.6*17.0°b

3.66 IOO.Ok 6.8" 1 72.9*36.gb l10.7*12.4"

NA:nutrient agar. I Starch and glucose were added at 0.1% (w/v). H.T. Heating time Mean of three experiments f C.V.

Values in the same row which have the same superscript were not significantly different at 5% level.

CFU per plate N A o r N A + S o r N A + G )

'Expressed as: plating efficiency = x 100 CFU per plate (NA)

HEAT RESISTANCE OF B. CEREUS 5

Mallidis and Scholefield 1986), although some authors (Cook and Gilbert 1968; Mallidis and Scholefield 1986) found that this compound exerted considerably greater stimulatory effect and that such protection caused an increase in D-values obtained. There seems to be no information published on the effect of glucose into the plating medium on heat resistance of B. cereus spores. For other Bacillus species, authors disagree on the influence of this compound. Although Richardson (1965) reported that the addition of glucose had a protective effect on B. subtilis, others (Zechman and Pflug 1991 for B. stearothermophilus) described that the recovery was less effective using a medium which contained glucose. These authors proposed as a possible explanation the formation of inhibitory brown compounds or intermediary reducing compounds during medium preparation and sterilization due to the presence of glucose and phosphate. In our experimental conditions and although the medium was darker after sterilization, it was free of potassium phosphate, which exerts an inhibitory effect on recovery of B. cereus (see below). Recently, L6pez (personal communication) observed a similar effect on B. stearothermophilus.

TABLE 2. EFFECT OF STARCH AND GLUCOSE IN THE RECOVERY MEDIUM ON THE APPARENT

HEAT RESISTANCE OF BACILLUS CEREUS SPORES D,, (min) AND z-VALUES (C)

Thermal resistance parameters obtained on

NA NA+Starch' NA+Glucose'

Strain D-value* z-value D-value* z-value D-value* z-value

4342 1.26iO. 15" 7.77 1.45k0.05" 8.14 1.43*0.15" 8.11

7004 0.29*0.03 I b 7.75 0.31i0.024b 7.80 0.28*0.034b 7.88

9818 4.52*0.36' 7.39 4.99i0.50" 7.61 4.76k0.49" 7.43

NA: nutrient agar. I Starch and glucose were added at 0.1% (w/v). * Mean of three experiments f S.D. ""Values obtained for each strain with the same superscript were not significantly different at 5% level.

Effect of Sodium Chloride on Heat Resistance

Figure 1 shows one of the survival curves obtained for B. cereus strains using as plating medium nutrient agar with different NaCl concentrations. These curves are representative examples of those obtained in all the range of temperatures studied. The presence of NaCl in the recovery medium had a marked effect on the thermal

6 I . GONZALEZ ETAL.

characteristics of B. cereus spores. While for unheated spores, salt concentrations of 3% for strain 4342, 4% for 7004 and 6% for 9818 were required to cause significant differences in counts (data not shown), for heated spores, salt concentrations as low as 0.5% caused a significant reduction in the recovery efficiency (see Fig. 1). As the salt concentration in the medium rose, the recoverability efficiency progressively dropped for all strains tested. The tail population of 7004 strain was also affected by the level of salt in the recovery medium, but this second most resistant fraction did not disappear even at a concentration as high as 3% (w/v).

Table 3 shows the D,, values average obtained for the different strains at the previously cited conditions. The salt content in the recovery medium also influenced D-values obtained which progressively decreased as the NaCl concentration increased. The extent of this reduction and the salt concentrations in which statistically differences (p<0.05) were obtained varied among the strains. Thus, D-values for 7004 strain were reduced at concentrations as low as 0.5% while for the other two strains, at least 2% salt was necessary to have significant effect on decimal reduction times. However, at higher levels (3%), the drop observed was much more marked for strain 9818.

Figure 2 shows the thermal death time curves obtained when sodium chloride was added to the recovery medium. The corresponding statistical analysis showed that in no case were the z-values affected (p>0.05).

These fmdings show that in order to improve the effectiveness of media used for detection and enumeration of B. cereus in heat processed food, it would be wise to bear in mind the inhibitory effect caused by sodium chloride on the recovery of surviving heat damaged spores. This is of particular importance given that the most of media used for the isolation and identification of B. cereus contain salt in fairly high concentrations. This could have a considerable inhibitory effect. On the other hand, in the practical canning operation it is common to use salt and taking into account its inhibitory effect, it could be expected that this compound plays a critical role in the germination and/or outgrowth of B. cereus, regardless of the temperature of treatment.

Effect of Different Stabilizing Additives on Heat Resistance Table 4 shows the recovery percentages obtained for each strain at different

treatment times after the addition of sodium citrate, monopotassium phosphate and disodium phosphate. None of these compounds had any significant effect (p>0.05) in the counts of unheated spores. After heating, the addition of these compounds resulted in a drop in the recovery efficiency of the three strains, being more pronounced for the more heat-tolerant strain (98 18 ATCC) when disodium phosphate and monopotassium phosphate were used giving way to significant lower D-values (Table 5). z-Values obtained in medium containing these additives were 7.82 f 0.26C for strain 4342, 8.10 f 0.18C for strain 7004 and 6.95 f 0.05C for strain 98 18.

HEAT RESISTANCE OF B. CEREUS

J 51

7

ATCC 7oW 4

- _ s 7 j z P y 3. 3 co

2.5

2

FIG. 1 . SURVIVAL CURVES FOR BACILLUS CEREUS SPORES RECOVERED IN NUTRlENT AGAR WITH DIFFERENT SODIUM CHLORIDE LEVELS: 0% (x), 0.5% (A), 1% (0). 2% (o),

3% (B) AND 4% (+)

8 I. WNZALEZ ETAL

TABLE 3. EFFECT OF SODIUM CHLORIDE IN THE RECOVERY MEDIUM ON THE APPARENT

HEAT RESISTANCE OF BACILLffS C E E U S SPORES

NaCl D,,* (min) ~

concentration (%) strain 4342 strain 7004 strain 981 8

0 1.26i0.15' 0.29i0.03 1" 4.52i0.36" 0.5 1.12i0.10' 0.16*0.03k 4.09i0.40"

1 .o 1.04i0.09" 0.13i0.008' 3.95*0.34"

2.0 0.68*0.06b N.T. 1.15io. l o b

3.0 0.24i0.02" 0.056*0.002d 0.48*0.05"

4.0 0. 15*0.005d N.T. 0.19=t0.01d ' Mean of three experiments * S.D. N.T. No tested. 'dValues obtained for each strain with the same superscript were not significantly different at 5% level.

TABLE 4. EFFECT OF DIFFERENT STABILIZING ADDITIVES ON THE RECOVERY' OF BACILLUS CEREUS SPORES

Strain H.T. Time (rnin) NA NA+C NA+PK NA+PNa

4342 lOOC 0 100.h13.6' 91.3* 7.6" 89.6i11.6n 94.1*15.7" 0.66 100.O.t 9.8" 82.7*12.6"b 73.3*1 1.2h 75.65*10.9b 1.33 100.O.t10.2" 84.9.t 8.Qb 73.4* 7Sb 72.95* 8.8b 2.0 1 O O . h 7.9' 68.5i~12.7~ 62.4* 5.6b 57.7* 6.Sb

7004 96C 0 1OO.o-k 9.4' 108.o-kl3.3" 101.3i 9.6' 105.5i13.6' 0.33 lOO.o-kl4.2" 8 4 3 7Snb 77.7i 8.4b 74.8i 8.6b 0.66 1OO.o-kl1.4" 80.7*13.6°b 75.7*10.2b 76.6* 9.4b 1 .o 1OO.o-k 9.1" 76.3* 6.9b 79.9i 8.9b 77.0i10.0b

9818 104C 0 1O0.o-k16.l0 81.o-k 7.9' 92.7i12.6" 97.5* 9.8" 1 .o 100.0.tlS.2° 80.8 i 7.6" 45.7* 5.6b 48.5* 4.1b 1.66 1OO.O.t 8.9" 62.1i 5.4b 21.4i 2.0" 28.1* 3.1d 2.33 1O0.O.t13.Oa 54.2* 4.8b 14.7* 1.7" 20.7*2.1d 3.0 1OO.o-k 4.9" 53.3i 7.1b 9 . 2 1.0" 16.7k 1.9d

NA:nutrient agar; NA+C: nutrient agar supplemented with 0.1% of sodium citrate; NA+PK: nutrient agar supplemented with 0.1% of monopotassium phosphate; NA+PNa: nutrient agar supplemented with 0.1% of disodium phosphate. H.T.: Heating time Mean of three experiments i C.V. "Values with the same superscript within rows and strain were not significantly different at 5% level. * Expressed as:

CFU per PIate ( N A ~ ~ N A ~ c ~ ~ N A ~ P K ~ ~ N A + P N . )

plating efficiency = X I 0 0 CFU per plate (NA)

HEAT RESISTANCE OF B. CEREUS

'1 ATCC 4342

7.74 -1.5 7.88

. 5

.2s 0. ....

5.25. 0 - -.s -.75,

-1.

~I .25

-1.5

92 94 96 98 100 102 104 106 108 Temperature (T)

-1.75'

1.251

ATCC 9818

- ".,, 98 100 102 104 106 10R 110 l l Z 114 116

Temperature (T)

9

FIG. 2. THERMAL DEATH TIME CURVES FOR BACILLUS CEREUS SPORES RECOVERED

1% (O) , 2% (m), 3% (m) AND 4% (+) IN NUTRIENT AGAR WITH DIFFERENT SODIUM CHLORIDE LEVELS: 0% (x), 0.5% (A),

10 I . GONZALEZ ETAL

TABLE 5. EFFECT OF DIFFERENT STABILIZING ADDITIVES IN THE RECOVERY MEDIUM ON THE

APPARENT HEAT RESISTANCE OF 5AClLLUS CEREUS SPORES

Dim* (min)

Strain NA NA+C NA+PK NA+PNa

4342 1.26*0.15" 1.19*0.11" I .07*0.06' I. 18*0.07'

7004 0.2%0.03 1" 0.32*0.043" 0.34*0.058' 0.3W0.040'

9818 4.52*0.36" 5.34*0.56" 3.79i0.29b 3.9W0.23b

NAmutrient agar: NA+C: nutrient agar suaalemented with 0.1% of sodium citrate: NA+PK: nutrient I , - ..

agar supplemented with 0. I % of monopotassium phosphate; NA+PNa: nutrient agar supplemented with 0.1% of disodium phosphate. * Mean of three experiments * S.D. a-bValues obtained for each strain with the same superscript were not significantly different at 5% level.

The heat resistance of different B. cereus spores showed a considerable range. In this study we found that the D,,, values obtained in standard conditions ranged from 0.29 min for ATCC 7004 to 4.52 min for ATCC 9818. From our data and others (Burgos et al. 1972; Johnson et al. 1982; Wong et al. 1988; Dufrenne et al. 1994) it could be concluded that thermal sterilization treatments would be sufficient to guarantee a good degree of safety with regard to the B. cereus in sterilized food. Furthermore, results obtained here and the data previously published by our group (Gonzblez et al. 1995; Gonzhlez et al. 1996) showed that heat resistance of B. cereus is influenced by recovery conditions, especially medium pH and presence of salt. We have found that D-values for the strain that exhibits the higher heat resistance (ATCC 98 18) decreased 4-fold when the medium was acidified up to pH 5.4 (Gonzhlez et al. 1996) and about 25-fold in presence of 4% of sodium chloride. This effect could give an additional security margin in certain products. However, one of the strains tested (ATCC 7004) exhibits subpopulations with greater heat resistance. The existence of tails has been reported for others strains of B. cereus (Johnson et al. 1982; Rajkowski and Mikolajcik 1987). Although these tails represented a minority lkaction of the population, they were present in all recovery conditions studied.

ACKNOWLEDGMENTS

This work was supported by CICYT, project ALI 90-0555 and by a grant to I.G. from the Gobiemo Vasco.

HEAT RESISTANCE OF B. CEREUS 11

REFERENCES

BRADSHAW, J.G., PEELER, J.T. and TWEDT, R.M. 1975. Heat resistance of ileal loop reactive Bacillus cereus strains isolated from commercially canned food. Appl. Microbiol. 12,943-945.

BRIGGS, A. and YAZDANY, S. 1970. Effect of sodium chloride on the heat and radiation resistance and on the recovery of heated or irradiated spores of the genus Bacillus. J. Appl. Bacteriol. 33,62 1-632.

BURGOS, J., ORDOaZ, J.A. and SALA, F. 1972. Effect of ultrasonic waves on the heat resistance of Bacillus cereus and Bacillus licheniformis spores. Appl. Microbiol. 24,497-498.

CHUMEY, R.K. and ADAMS, D.M. 1980. Relationship between the increased sensitivity of heat injured Clostridium perpingens spores to surface active anti- biotics and to sodium chloride and sodium nitrite. J. Appl. Bacteriol. 49, 55-63.

CONDON, S., LOPEZ, P., ORIA, R. and SALA, F.J. 1989. Thermal death determination: design and evaluation of a thermoresistometer. J. Food Sci. 54,

CONDON, S., O M , R. and SALA, F.J. 1987. Heat resistance of microorganisms: an improved method for survival counting. J. Microbiol. Meth. 7, 37-44.

COOK, A.M. and GILBERT, R.J. 1968. Factors affecting the heat resistance of Bacillus stearothermophilus spores. I. The effect of recovery conditions on colony count of unheated and heated spores. J. Food Technol. 3,285-293.

DUFRENNE, J., SOENTORO, P., TATINI, S., DAY, T. and NOTERMANS, S. 1994. Characteristics of Bacillus cereus related to safe food production. Int. J. Food Microbiol. 23,99-109.

FEEHERRY, F.E., MUNSEY, D.T. and ROWLEY, D.B. 1987. Thermal inactivation and injury of Bacillus stearothermophilus spores. Appl. Environ. Microbiol. 53, 365-370.

FRANKLIN, J.G. 1970. Spores in milk: Problems associated with U.H.T. processing. J. Appl. Bacteriol. 33, 180- 191.

GONZALEZ, I., LOPEZ, M., MAZAS, M., BERNARDO, A. and MARTIN, R. 1996. Effect of pH of the recovery medium on the apparent heat resistance of three strains of Bacillus cereus. Int. J. Food Microbiol. 31,341-348.

GONZALEZ, I., LOPEZ, M., MAZAS, M., GONZALEZ, J. and BERNARDO, A. 1995. The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores. J. Appl. Bacteriol. 78,548-554.

HUTTON, M.T., KOSKINEN, M.A. and HANLIN, J.H. 1991. Interacting effects of pH and NaCl on heat resistance of bacterial spores. J. Food Sci. 56,821-822.

JARVIS, B., RHODES, A.C., KING, S.E. and PATEL, M. 1976. Sensitization of heat-damaged spores of Clostridium botulinum, type B to sodium chloride and sodium nitrite. J. Food. Technol. 11,41-50.

45 1-457.

12 I. GONZALEZ E T A L .

JOHNSON, K.M., NELSON, C.L. and BUSTA, F.F. 1982. Germination and heat resistance of Bacillus cereus spores from strains associated with diarrheal and emetic food borne illnesses. J. Food Sci. 47, 1258-1271.

LABBE, R.G. 1979. Recovery of spores of Bacillus stearothermophilus from thermal injury. J. Appl. Bacteriol. 47,457-462.

LOPEZ, M., MAZAS, M., GONZALEZ, I., BERNARDO, A. and GONZALEZ, J. 1994. Effect of pH and sodium chloride on the thermal resistance of Bacillus stearothermophilus spores. Microbiol. Alim. Nut. 12, 3 17-322.

MALLIDIS, C.G. and SCHOLEFIELD, J. 1986. Evaluation of recovery media for heated spores of Bacillus stearothermophilus. J. Appl. Bacteriol. 61, 5 17- 523.

MCKENZIE, H.A. and DAWSON, R.M.C. 1969. pH, buffers and physiological media. In Data for Biochemical Research. (R.M.C. Dawson, D.C. Elliot and K.M. Jones, eds.) pp. 484-485, Oxford at the Carendon Press, Oxford.

MOSTERT, J.F., LUCK, H. and HUSMANN, R.A. 1979. Isolation, identification and practical properties of Bacillus species from UHT and sterilized milk. S . Afr. J. Dairy Technol. 11, 125-131.

OLSEN, A.M. and SCOTT, W.J.1950. The enumeration of heated bacterial spores. Australian J. Sci. Res. B, 3,219-233.

RAJKOWSKI, K.T. and MIKOLAJCIK, E.M. 1987. Characteristics of selected strains of BaciIlus cereus. J. Food Prot. 50, 199-205.

RICHARDSON, G . 1965. The viability of spores of some Bacillus species. J. Pharm. Pharmac. 17, 12s.

STEEL, R.G.D. and TOFUUE, J.H. 1986. Bioestadistica: principios y procedimientos. pp. 83-1 16; 23 1-261, McGraw-Hill, MCxico.

SUGIYAMA, H. 1951. Studies on factors affecting the heat resistance of spores of Clostridium botulinum. J. Bacteriol. 62, 8 1-96.

WONG, H.C., CHANG, M.H. and FAN, J.Y. 1988. Incidence and characterization of Bacillus cereus isolates contaminating dairy products. Appl. Environ. Microbiol. 3, 699-702.

ZECHMAN, L.G. and PFLUG, I.J. 1991. Bacillus stearothermophilus spore recovery altered by media concentration and formulation. J. Food Sci. 56, 1408- 14 14.