HEAT SENSITIZATION OF BACILLUS SUBTILIS SPORES BY SELECTED SPICES

GREG BLANK, GEORGE AKOMAS, MICHAEL HENDERSON and JERZY ZAWISTOWSKI

Department o j Food Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 Canada

Accepted for Publication December 2 2 , 1987

ABSTRACT

Commercially sterile garlic, sage, black pepper, cinnamon and clove were investigated as heat sensitizing agents towards Bacillus subtilis spores. Heat treat- ment at 80°C for 10 min of all spice-spore mixtures (0.9 g spice/O.l g spores) in distilled water (9 mL) reduced initial counts (10VmL) by approximately 0.5-1.0 logarithms, with the exception of garlic which showed no effect. The clove-spore mixture exhibited the greatest reduction in survivor counts. Dry spice-spore mix- tures conditioned for 24-96 h in relative humidities (RH) from 64 to 98% and at temperaturesfrom 25 to 35°C. prior to heat treatment, exhibited additional reduc- tion in survivors. The clove-spore mixture exhibited the greatest decrease in heat- treated survivors (approximately 4 logarithms) a f e r conditioning at 96% RH and 35°C for 96 h. Relative humidity conditioning of cinnamon-spore mixtures, regardless of treatmeizt regime, did not reduce the number of heat-treated survivors. Increasing the temperature and/or RH of conditioning for the remaining spice-spore mixtures generally increased heat sensitization. Aw-equilibrated spice-spore mix- tures which were not heat-treated (controls) showed no reductions in viability. Moisture sorption by the spices during conditioning only partially influenced their heat sensitizing effects.

INTRODUCTION

Although organisms of public health significance such as Salmonella and Clostridiu are encountered infrequently in spices (Wilson and Andrews 1976; Bax- ter and Holzapfel 1982). populations of spoilage bacteria ranging from lo2 to 106/g are often reported (Julseth and Deibel 1974; Powers et ul. 1975; DeBoer er al. 1985). Members of the Bucillaceue usually predominate, usually as spores (Powers et al.

Journal of Food Safety 9(1988) 83-96. All Ri@.s Reserved. @Copright I988 hy Food & Nurririon Prc.ss. IJK. , Trutnhull, Comerticur. 83

u4 G. BLANK, G. AKOMAS. M . HENDERSON and J . ZAWISTOWSKI

197s). Since spores are particularly resistant to heat they are of great concern in processed foods. Microbiological evaluation of spices, even after conventional decon- tamination procedures, therefore includes spore counts. Enumeration of spores is normally performed after sublethal heat treatment (10 min at 70-80°C) in order to activate spores and kill vegetative cells (Julseth and Deibel 1974). Heat treatment of vegetative cells in spices has been shown to reduce heat resistance (Kosker et (il. 1951; Anderson et al. 1953; Johnson and Vaughn 1969). Spores also may become sensitired; thus, survivor counts from spices may be misleadingly low.

This investigation was initiated to study the possible heat sensitizing effects by spices on spores. In addition, temperature and relative humidity are known to influence the heat resistance of spores during and/or after sporulation (Roberts and Hitchins 1969). These environmental factors would be especially important in tropical countries, where the majority of spices are grown. Accordingly, the effects of conditioning or equilibrating spice-spore mixtures to different relative humidity ( K H ) and temperature regimes, prior to heat treatment, were also investigated.

MATERIALS AND METHODS

Culture

and maintained on standard plate count agar (Difco, SPC) slants at 4°C. Rtrcillu.\ subtilis (ATCC 6633) was cultured in nutrient broth (Difco; 24 h, 37°C)

Spore Preparation

The preparation, harvesting and cleaning of spores were performed as described by Al-Khayat and Blank (1985). The final spore suspension was frozen (24 h. -40°C) and freeze dried. Spores (stock) were maintained in screw-cap tubes at 0°C.

Heat Treatment of Nonconditioned Spice-Spore Mixtures

The \tack spore preparation was mixed with sterile, silicone treated glass beads (80/100 mesh; Chromatographic Specialties, Brockville, Canada) by gentle mix- ing using a wrist action shaker for 30 min. The spore mixture was standardized to contain lo8 heat treated (10 min at 8OOC) colony forming units (CFU)/g. Commer- cially ground and sterile spices (Stange Canada Inc.) consisting of black pepper, clove, cinnamon, sage and garlic were individually mixed with portions of the spore mixture (9:l,w/w). The spice-spore mixtures (1.0 g) were diluted 1:lO in distilled water and heated (10 min at 80°C). CFU were determined after incubation (48 h at 37°C) using SPC agar and Tryptone Soy Agar (TSA; Difco). Standardized spore mixtures (0.lg) suspended in distilled water were adjusted to the following pH: 4.0. 5.0. 6.0. 7.0 and 8.0 using citrate-Na,HPO, buffer (O.1M). The pH of the unadjusted control was 9.4. These values represent the entire pH range of the spice-spore mix- turc\. All controls were heat treated (10 min at 80°C) in a final volume of 10 mL

HEAT TREATMENT OF SPORES IN SPICES 85

sterile distilled water and enumerated on SPC agar and TSA after incubation (48 h at 37°C). The heat treatment was performed in duplicate. As a control, the preceding experiment was repeated, omitting the heat treatment.

Heat Treatment of Conditioned Spice-Spore Mixtures

Portions of each spice-spore mixture (0.9 g spice plus 0.1 g spore mixture) were maintained on sterile petri plates (60 x 15 mm), above saturated salt solutions in a series of sealed glass desiccators (150 mm, ID; approx. 1.3 L). The following saturated salt solutions (Winston and Bates 1960) were used to obtain the indicated RH environments (25°C): NaBr (57.5 %), NaCl (75.5%), KCl (85.0%), K,SO, (97.5 %). Incubation temperatures of 25, 30 and 35°C were used in combination with relative humidity. CFUs in conditioned spice-spore mixtures were enumerated at 24 h intervals with and without heat treatment using SPC agar (48 h, 37°C). Aw- equilibrated spore mixtures served as controls. The pH was not adjusted. All spices were adjusted with portions of spore mixture such that lo7 CFU/g were obtained following heating (10 min, 80°C). This procedure ensured that the number of CFUs in the spice-spore mixtures following heat treatment at zero time of conditioning would be similar. All analyses were performed in duplicate.

Minimum Inhibitory Spice Concentration

Two-fold increasing amounts of ground spices (0.05 to 5 %, w/v) were initially mixed with sterile, tempered (45°C) SPC agar (15 mL) prior to surface-plating of an aqueous spore suspension (0.1 mL; approximately lo7 CFU/mL). The spore suspension was prepared from the lyophilized stock, which was rehydrated in sterile distilled water (48 h at 7°C). CFU were determined after serial dilution and incuba- tion at 37°C for 48 h. The minimum concentration (%, wlv) of spice inhibiting growth for 48 h at 37°C was defined as the minimum inhibitory spice concentra- tion (MIC). Analyses were performed in duplicate.

Moisture Content

The initial moisture contents (%) of the ground spices were determined as outlined by Karmas (1980) and AOAC (1984) using an azeotropic distillation method.

The initial moisture content (%) of the spore mixture (10 g) was determined as the difference in weight before and after drying (120°C) to constant weight. The moisture content of the conditioned spore mixtures (10 g) was similarly determined at 24 h intervals.

The moisture content of the relative humidity-equilibrated spice-spore mixtures was calculated as:

% moisture content = (g H20) (g dry material)

All analyses were performed in duplicate.

86 G. RI.ANK, G. AKOMAS. M HENDERSON and J. ZAWISTOWSKI

TABLE I . SURVIVOR COUNTS FROM NON-CONDITIONED SPICE-SPORE MIXTURES

ENUMERATED ON SPC A N D TSA AFTER HEAT TREATMENT (10 MIN AT 80°C)

CFU/mLc

Spice-spore mixture PHa SPC TSA

9 . 4 6 . 5 6 . 9 5 1 f > . 0 4 5

RESULTS AND DISCUSSION

Heat treatment of all nonconditioned spice-sporc mixtures, with the exception of garlic, reduced CFUs by approximately 0.5-1.0 logarithms (Table 1). Compared to the control (nonconditioned spore mixture), the clove-spore mixture exhibited the greatest reduction in CFU. All nonconditioned spice-spore mixtures which were not heat-treated exhibited less than 1% reduction in counts (data not shown). The pH ofthe mixtures (Table 1) ranged from 4.5 (cloves) to 6.9 (black pepper); the con- trol had a pH of 9.4. Based on the differences in survivor counts between heat and nonheat treated spice-spore mixtures and their controls, it would appear that all spices. with the exception of garlic, exhibited a sensitizing effect. Bose and Roy (1959) also reported that mustard oils were effective in reducing heat resistance of B. sub- t i l is spores.

Heat treatment of controls (nonconditioned spore mixtures), adjusted from pH 4.0 to 9.5, showed no appreciable difference in survivor counts (data not shown). This observation, in addition to the differences in survivor counts between the garlic and black pepper-spore mixture, despite the neutral pH of the latter, may indicate that the pH of the spices had a minimal effect on nonconditioned spore survival.

The relationship of the pH of the heating menstruum to spore heat resistance has been widely investigated. In general, decreasing the pH of the heating menstruum concomitantly decreases the heat resistance of spores. This decrease in heat resistance appears to be dependent upon several factors including strain (Odlaug and Pflug 19761, menstruum (Cameron er al. 1980) and aw (Harnulv cr d. 1977) and the severity of the heat treatment (temperature and/or time; Roberts and Hit- chins 1969). The latter factor may account for the apparent lack of a pH effect since

HEAT TREATMENT OF SPORES IN SPICES 87

heating was performed for a relatively short time and at a low temperature. It should be pointed out, however, that the antimicrobial effectiveness of some spices and/or their components is pH dependent, usually increasing with decreasing pH (Ander- son et al. 1953; Jay and Rivers 1974; Al-Khayat and Blank 1985). It is possible therefore that some spices may have a greater sensitizing effect with decreasing pH. However, routine microbiological analyses of spices normally involve menstrua of neutral pH.

Studies pertaining to the heat resistance of microorganisms in spices are limited. Kosker et al. (1951) and Anderson et al. (1953) reported, however, that several spice essential oils including garlic, clove and cinnamon reduced the thermal resistance of several yeast and mold species by as much as 97-99 %. Anderson (1953) also reported that garlic oil (62-125 ppm) in tomato juice lowered the thermal resistance of Bacillus thermoacidurans by approximately 42 % . The specific mechanism(s) responsible for the apparent sensitization of spores by spices is unclear, although all spices used in this study have been reported to inhibit microbial growth (Pruthi 1980; Shelef 1983). In addition, garlic, cinnamon and cloves have been reported to affect various stages of spore development (Mantis et al. 1979; Shelef et al. 1984; Al-Khayat and Blank 1985). Based on the MIC values of the spices: 0.13,0.25, 1.50, 4.0 and > 5.0%, w/v for sage, clove, cinnamon, garlic and black pepper respec- tively, it would appear that carry-over concentrations during serial dilution and plating would be insufficient to prevent spore outgrowth and replication.

The survivor counts from conditioned spice-spore mixtures, enumerated after heat treatment, are given in Fig. 1-3; note differences in scale of the y axes. CFUs in cinnamon (Fig. l), regardless of the incubation conditions, remained relatively con- stant. In the remaining spices, CFUs gradually decreased with an increase in temperature and/or relative humidity. Conditioning spice-spore mixtures for 96 h at 35°C in a relative humidity of 96% decreased the CFUs in clove, sage, black pep- per, garlic and cinnamon by 47, 32, 27, 13 and 1%, respectively. All conditioned spice-spore mixtures enumerated without heat treatment exhibited a negligible decrease in CFUs (approximately 1%, data not shown). The conditioned spore mix- tures (controls) enumerated with and without heat treatment also exhibited a negligi- ble decreae in CFU (less than 3%, data not shown).

Based on these results it would appear that conditioning, at least for 96 h, under the described RH and temperature conditions had no effect on the reduction of CFUs unless followed by heat treatment. In this respect, sensitization may result from a chemical inactivation enhanced by heat.

The varying degrees of effectiveness for the different spices may be due to their diverse chemical and physical properties. The concentration of active agent(s) in the spice would also be an important factor. Clove, which exhibited the greatest sen- sitizing effect, is known to contain a relatively high essential oil content (16-18%) consisting of approximately 95 % eugenol (Pruthi 1980). In contrast, cinnamon,

xx G BLANK, G. AKOMAS. M . HENDERSON and J. ZAWISTOWSKI

I 0'

- E lo6

$ I B i I I l , 0 lo6

*--K NO& Z H 2 0

m - - + N a C I o-----a KCL D - - -A K,SO,

J lo5 i 10'

10'

F n

I 0 24 48 72 96 120

l o 6 u ,041 I I 1 I I 0 24 48 72 96 120

INCUBATION TIME (h)

FIG. 1. SURVIVOR CURVES OF B. SL'BTILIS FROM CONDITIONED CINNAMON- AND SAGE-SPORE MIXTURES FOLLOWING HEAT TREATMENT (10 MIN, 80°C) Cinnamon-spore mixture conditioned at 25°C (A) 30°C (B) and 35°C (C). Sage-spore mlxture conditioned at 25°C ( I ) ) 10°C (E) and 35°C (F). Relative humidities during conditioning (25°C): 57.5% (NaBr.2H20). 75.5% (NaCl). 85.0% (KCI) and 97.5% (K,SO,); (30°C): 56.0% (NaBr.2H20), 75.5%. (NaCl), 84.5% (KCI). and 965% (K,SOo); (35°C): 54.5% (NaBr.2H20), 75.5% (NaCI). 83.0% (KCI) and 96.0% (K,SO,).

HEAT TREATMENT OF SPORES IN SPICES 89

d

E

Io4 lo3 L-LL 0 24 48 72 96 120

1 0 4

INCUBATION TIME (h)

FIG. 2. SURVIVOR CURVES OF B. SUBTILIS FROM CONDITIONED CLOVE- AND BLACK PEPPER-SPORE MIXTURES FOLLOWING HEAT TREATMENT (10 min, 80°C) Clove-spore mixture conditioned at 25°C (A) 30°C (B) and 35°C (C). Black pepper-spore mixture conditioned at 25°C (D) 30°C (E) and 35°C (F). Relative humidities during conditioning (25°C): 57.5% (NaBr.2H20), 75.5 % (NaCl), 85.0% (KCl) and 9 . 5 % (K,SO,); (30°C): 56.0% (NaBr.2H20), 75.5% (NaCl), 84.5% (KC1) and 96.5% (K,SO,); (35°C): 54.5% (NaBr.2H20), 75.5% (NaCl), 83.0% (KC1) and 96.0% (K2S04).

90 G BLANK. G AKOMAS. M HENDERSON and J. ZAWISMWSKI

A

I o6

I o5

lo5 0 24 48 7 2 96 120

INCUBATION TIME ( h ) FIG. 3. SURVIVOR CURVE OF B. SUgTILfS FROM CONDITIONED GARLIC-SPORE MIXTURES FOLLOWING HEAT TREATMENT (10 MIN. 80°C) CONDITIONED AT 25°C (A) 30"(B) AND 35°C (C) Relative humidities during conditioning (25°C): 57.5% (NaBr 2H,O). 75.5% (NaCI), 85.0%(KCI) and Y7.5% (K,SO,); (30°C): 56.0% (NaBr.2H20). 75.5% (NaCI). 84.5% (KCI). and 96.5% (K,SO,);

(35°C): 54.5% (NaBr.ZH,O). 75.5% (NaCI), 83.0% (KCI) and 96.0% ( K 2 S 0 , ) .

HEAT TREATMENT OF SPORES IN SPICES 91

which showed no sensitizing effect, contains relatively low levels of essential oil (0.5 to 2.0%) of which 65-75 % consists of cinnamaldehyde (Shelef 1983). The prox- imate essential oil content of these spices should not, however, be used as the sole criterion to assess their activity. For example, sage which showed similar sensitiz- ing effects to clove, has an approximate essential oil content of 0.7 to 2.0% which is similar to that of cinnamon (Shelef 1983). Active spice agents, like many other antimicrobials, may also be expected to vary with respect to host specificity and target site(s). These factors not only contribute to their potency, but may also explain the lack of correlation between the MIC level and the sensitizing activity of some spices. Black pepper, for example, exhibited heat sensitization activity yet had an MIC greater than 5 %; cinnamon, with an MIC approximately 3.5 times lower than black pepper, however, was not an effective sensitizer.

Of the recognized antimicrobial compounds found in spices, the majority are phenolic (Jurd et al. 1971) and are sparingly soluble in water. Adsorption of the active agent(s) onto the spore, which appears essential for heat-sensitization would therefore not only depend on their concentration and solubility but also on the substrate moisture level. The moisture levels of the conditioned spice-spore mixtures are given in Fig. 4-6. The garlic-spore mixture conditioned at all temperatures and at RHs from 75.5 to 96% contained the highest level of moisture (Fig. 4). When conditioned for 96 h at 25, 30 and 35"C, the spice-spore mixture contained 52, 61 and 70% moisture, respectively. In contrast, the cinnamon-spore mixture contained the lowest level of moisture (less than 20%) at all temperatures under RHs of 96-97.5 % (Fig. 4). Equilibrium moisture content was reached by all spice-spore mixtures within 2448 h when conditioned at RHs from 57 to 85 %. The moisture content of the spore mixtures conditioned at 35°C in all RHs was less than 0.1 % at 96 h (data not shown). The garlic-spore mixture exhibited the highest level of moisture at all RHs, but its sensitizing effects were less than those of sage or cloves, both of which showed substantially lower levels of moisture. The high moisture sorption of garlic, like onion powder may result from its high concentration of water-soluble material (Peleg and Mannheim 1977). It is possible that a large proportion of the moisture was in a bound form and therefore was unavailable for microbial interaction. The difference in heat sensitization activity of garlic in the conditioned and nonconditioned spore mix- tures may have resulted from an increased contact time, prior to heat treatment. In contrast, the low moisture sorption by cinnamon, particularly at the highest RH, may account for its poor sensitizing activity. The higher heat-sensitization activity of clove and black pepper, despite moisture sorptions similar to cinnamon may, however, indicate the involvement of additional factors. Excluding differences in the concentration and/or potency of spice active agents, it is possible that they may have deteriorated during conditioning and/or heating. For example, Shelef et al. (1980) reported that the antibacterial effectiveness of sage was more stable over time than rosemary. Instability of spices through the loss of volatiles would then render them less effective. This may have occurred with cinnamon.

92 G BLANK, G. AKOMAS, M. HENDERSON and J. ZAWISTOWSKI

30 r

1 120

. A *--* NOCl

0-----a KCL P -- 6o 5 0 I D - - - A K ~ S O , NaEr 2H10 ,,-

30 ...... 0 ----U-----Q

I :, 20

0 24 48 72 96 120

TIME (h)

FIG. 4. MOISTURE SORPTION OF CONDITIONED CINNAMON- AND GARLIC- SPORE MIXTURES Cinnamon-spore mixture conditioned at 25°C (A) 30°C (B) and 35°C (C). Garlic- spore mixture conditioned at 25°C (D) 30°C (E) and 35°C (F). Relative humidities during conditioning (25°C): 57.5% (NaBr.ZH,O), 75.5% (NaCI), 85% (KCI) and 97.5% (K,SO,); (30°C): 56.0% (NaBr,2H20), 75.5% (NaCI), 84.5% (KCI), and 96.5% (K2S04); (35°C): 54.5% (NaBr.2H20), 75.5%

(NaC1). 83.0% (KCI) and 96.0% (K,SO,).

HEAT TREATMENT OF SPORES IN SPICES 93

40

25

20

-* a-- P'

4 0

25 20

TIME (h)

FIG. 5. MOISTURE SORFI'ION OF CONDITIONED BLACK PEPPER- AND CLOVE-

AND 35°C (C). Clove-spore mixture conditioned at 25°C (D) 30°C (E) and 35°C (F). Relative humidities during conditioning (25°C): 57.5% (NaBr.2H20), 75.5% (NaCl), 85.0% (KCI) and 97.5% (K2S0,); (30°C): 56.0% (NaBr.2HzO), 75.5% (NaCl), 84.5% (KCl), and 96.5% (K2S04); (35°C): 54.5%

(NaBr.2HzO). 75.5% (NaCI), 83.0% (KCI) and 96.0% (K,SO,).

SPORE MIXTURES. BLACK PEPPER-SPORE MIXTURE CONDITIONED AT 25°C (A) 30°C (B)

94 G. BLANK. G. AKOMAS. M. HENDERSON and J. ZAWISTOWSKI

50 45 40 35 30 25 20 15 10 5 - 0

- A

,A- -- -

,+ -

9' - - I

- I I

I - I I

;

&-- I I I I 1 I

0 ..-..- .c.--.-.a _ _ _ _ _ -4 ; ,./.p--4-.-&--a

- - c /,p ., I K

FIG. 6. MOISTURE SORRION OF CONDITIONED SAGE-SPORE MIXTURE AT 2S"C ( A ) 30°C (B) AND 35°C (C) Relative humidities during conditioning (25°C): 57.5 % (NaBr,2H20). 75.5% (NaCI), 85% (KCI) andY7.5% (K,SO,); (30°C): 56% (NaBr.2H20), 75.5% (NaC1). 84.5% (KCI) and 96.5% (K,SO,); (35°C): 54.5% (NaBr.2H2O), 75.5% (NaCI). 83.0% (KCI) and 96.0% (K,SO,)

HEAT TREATMENT OF SPORES IN SPICES 95

In summary, evaluation of spore microflora in spices, following heat activation, may result in misleadingly-low counts. Although the presence of spice components which sensitize spores to heat appears to be involved, caution is needed in inter- preting these results. A clear distinction should be made between substances which are capable of reducing heat resistance and substances which do not alter heat resistance but prevent outgrowth and cell division following germination. In this respect, microscopic studies are needed to evaluate in further detail the potential of spice sensitizers.

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