thermal resistance of bacillus cereus spores as
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THERMAL RESISTANCE OF BACILLUS CEREUS SPORES AS AFFECTED BY ADDITIVES IN THE RECOVERY MEDIUMISAAC GONZALEZI,MERCEDES LOPEZ~,MARGARITAW A S , G O N Z k E Z I and ANA BERNARD01.3'Dpto. de ffigiene y Tecnoiogia de Ios Alimentos
Universidad de Ledn, Spain 2Area Tecnologia de 10s Alimentos Universidad de Vigo, SpainReceived 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 agentsfor heat injured B. cereus spores especially for strain 9818, although only monopotassium and disodium phosphate caused a signifcant reduction ($0.05) in D-values obtainedfor 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 ratesfor strains 9818 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 (p0.05). 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 Strain4342
Recovery (%)* on NA NA+Starch'107.8* 13.6" 129.8i18.6" 134.7*21.4" 132.7*17.9"
Time (min)1.66 2.66 3.66
NA+Glucose'l04.1* 9.65' 116.2*15.6" I09.7*13.6" 72.4* 13 .7b 91.3* 7.3" 84.1*12.9" 125.9*18.4"b 116.6*17.0b l10.7*12.4"
1oo.oi 9.2"lOO.oi12.4" 100.oil2.9" 100.M13,6ab 1oo.oi 9.2" 100.oi16.8" 100.M 7.1" lOO.O* 7.9"IOO.Ok 6.8"
0.33 0.66I .oo
1.66 2.66 3.66
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 = CFU per plate (NA)
HEAT RESISTANCE OF B. CEREUS
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 Strain 4342 7004 9818 D-value* 1.26iO.15" 0.29*0.03 Ib 4.52*0.36' z-value 7.77 7.75 7.39 NA+Starch' D-value* 1.45k0.05" 0.31i0.024b 4.99i0.50" z-value 8.14 7.80 7.61 NA+Glucose' D-value* 1.43*0.15" 0.28*0.034b 4.76k0.49" z-value 8.11 7.88 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
I . GONZALEZ ETAL.
characteristicsof 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 (p0.05). 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. CEREUSJ 51
-_s 7 j
FIG. 1 . SURVIVAL CURVES FOR BACILLUS CEREUS SPORES RECOVERED IN NUTRlENT 2% AGAR WITH DIFFERENT SODIUM CHLORIDE LEVELS: 0%(x), 0.5% (A), 1% (0). (o),3% (B) AND 4% (+)
I. WNZALEZ ETAL
TABLE 3. EFFECT OF SODIUM CHLORIDE IN THE RECOVERY MEDIUM ON THE APPARENT HEAT RESISTANCE OF BACILLffSC E E U S SPORES NaCl concentration (%)0 0.5
strain 4342 1.26i0.15' 1.12i0.10'
strain 7004 0.29i0.03 1" 0.16*0.03k 0.13i0.008' N.T. 0.056*0.002d N.T.
strain 981 8 4.52i0.36" 4.09i0.40" 3.95*0.34"
1.04i0.09"0.68*0.06b 0.24i0.02"0. 15*0.005d
2.0 3.0 4.0' M a of three experiments en
1.15io.l o b0.48*0.05" 0.19=t0.01d
N.T. tested. No'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
Time (rnin)0 0.66 1.33 2.0 0 0.33 0.66 1 .o 01
NA 100.h13.6' 100.O.t 9.8"100.O.t10.2"1 O O . h 7.9'1OO.o-k 9.4' lOO.o-kl4.2" 1OO.o-kl1.4" 1OO.o-k 9.1" 1O0.o-k16.l0
NA+C 91.3* 7.6" 82.7*12.6"b 84.9.t 8.Qb 68.5i~12.7~ 108.o-kl3.3" 8 4 3 7Snb 80.7*13.6b 76.3* 6.9b 81.o-k 7.9'8 0 . 8 i 7.6" 62.1i 5.4b 54.2* 4.8b 53.3i 7.1b
NA+PK 89.6i11.6n 73.3*1 1.2h 73.4* 7Sb 62.4* 5.6b 101.3i 9.6' 77.7i 8.4b 75.7*10.2b 79.9i 8.9b 92.7i12.6" 45.7* 5.6b 21.4i 2.0" 14.7* 1.7" 9 . 2 1.0"
NA+PNa 94.1*15.7" 75.65*10.9b 72.95* 8.8b 57.7* 6.Sb 105.5i13.6' 74.8i 8.6b 76.6* 9.4b 77.0i10.0b 97.5* 9.8" 48.5* 4.1b 28.1* 3.1d 20.7*2.1d 16.7k 1.9d
1.66 2.33 3.0
100.0.tlS.2 1OO.O.t 8.9" 1O0.O.t13.Oa1OO.o-k 4.9"
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