The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores

The causes of Bacillus spore resistance remain unclear. Many structures including a highly compact envelope, low hydration of the protoplast, high concentrations of Ca-chelated dipicolinic acid, and the presence of small acid-soluble spore proteins seem to contribute to resistance. To evaluate the role of internal protoplast composition and hydration, spores of Bacillus subtilis were produced at different osmotic pressures corresponding to water activities of 0.993 (standard), 0.970, and 0.950, using the two depressors (glycerol or NaCl). Sporulation of Bacillus subtilis was slower and reduced in quantity when the water activity was low, taking 4, 10, and 17 days for 0.993, 0.970, and 0.950 water activity, respectively. The spores produced at lower water activity were smaller and could germinate on agar medium at lower water activity than on standard spores. They were also more sensitive to heat (97 degrees C for 5-60 min) than the standard spores but their resistance to high hydrostatic pressure (350 MPa at 40 degrees C for 20 min to 4 h) was not altered. Our results showed that the water activity of the sporulation medium significantly affects spore properties including size, germination capacity, and resistance to heat but has no role in bacterial spore resistance to high hydrostatic pressure.     

THE INCIDENCE AND THERMAL RESISTANCE OF MESOPHILIC SPORES FOUND IN MILK AND RELATED ENVIRONMENTS

SUMMARY: A spore 'spectrum' is described of aerobic mesophiles capable of resisting different heat treatments. It is shown that B. licheniformis is the most common spore former found in bulk milk but since its spores are rapidly destroyed at 100°, the more heat resistant B. subtilis is the dominant surviving spore former in commercial sterilized milk. The thermal resistance of strains of B. subtilis and B. licheniformis isolated from different sources has been investigated and the strains of B. subtilis typed according to the behaviour of their spores when heated at 100°. All strains of B. licheniformis were destroyed more rapidly by boiling for 2 min than strains of B. subtilis but only those strains of the latter which showed some degree of heat activation were more resistant than B. licheniformis . The 'resistant' and heat activated strains of B. subtilis appear to be sparsely distributed in nature and were only isolated from sterilized milk where the heat treatment applied would tend to eliminate other strains. The spore content of bovine faeces was similar to that in bulk milk and the total spore content varied seasonally, the spore content of faeces being on the average a hundred times greater during indoor feeding than during the period when the cattle were fed outside. A faecal infection of the milk in the ratio of 1:10⁴ would infect the milk with spores at about the same concentration as they are found in bulk raw milk, and it is suggested that bovine faeces could be a primary source of spore formers in milk supplies.     

An evaluation of the sporicidal activity of ozone

This study was undertaken to determine the feasibility of sterilizing surfaces with ozone-saturated water by the methods of the Association of Official Analytical Chemists (AOAC). Initially, it was determined that there was no apparent difference in ozone resistance between spores of Bacillus subtilis and Clostridium sporogenes when they are suspended in water. Both species were inactivated by a 10-min exposure at ambient temperature. Resistance was increased when the spores were dried on AOAC carriers. Viable organisms were recovered after an exposure of 40 min at ambient temperature. An increase in the reactor water temperature to 60 degrees C did not improve the effectiveness of the ozone in sterilizing AOAC carriers. Dried spores of C. sporogenes were more resistant than B. subtilis spores because of a greater accumulation of organic matter on the carriers. No significant sporicidal activity was demonstrated after 40 min for spores of either species when they were inoculated on silk suture loops. The data suggest that organic loading and poor ozone penetrability are key factors in effecting the ability of ozone to sterilize surfaces rapidly.     

An evaluation of the sporicidal activity of ozone.

This study was undertaken to determine the feasibility of sterilizing surfaces with ozone-saturated water by the methods of the Association of Official Analytical Chemists (AOAC). Initially, it was determined that there was no apparent difference in ozone resistance between spores of Bacillus subtilis and Clostridium sporogenes when they are suspended in water. Both species were inactivated by a 10-min exposure at ambient temperature. Resistance was increased when the spores were dried on AOAC carriers. Viable organisms were recovered after an exposure of 40 min at ambient temperature. An increase in the reactor water temperature to 60 degrees C did not improve the effectiveness of the ozone in sterilizing AOAC carriers. Dried spores of C. sporogenes were more resistant than B. subtilis spores because of a greater accumulation of organic matter on the carriers. No significant sporicidal activity was demonstrated after 40 min for spores of either species when they were inoculated on silk suture loops. The data suggest that organic loading and poor ozone penetrability are key factors in effecting the ability of ozone to sterilize surfaces rapidly.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. III. LABORATORY DETERMINATIONS OF THE HEAT RESISTANCE OF SPORES OF BACILLUS SUBTILIS IN WATER AND IN MILK

SUMMARY: Thermal death curves for spores of Bacillus subtilis 786 have been determined in water and in milk. Generally a non-logarithmic order of death was observed. Numbers of survivors were lower in milk than in water, suggesting that there may be inhibitory factors in UHT sterilized milk which affect the germination and/or subsequent growth of heated spores.
The thermal death curves for spores suspended in milk yielded Q₁₀ values of about 30 in the range 110–120°. This is higher than the figures previously reported in the literature for R. subtilis spores. Spores of a number of strains of B. subtilis were compared with strain 786 and all gave high Q₁₀ values.
The results obtained in this work have been used to predict the destruction of spores at higher temperatures in a UHT plant (Burton et al. 1958). The calculated values agree well with the results obtained in the plant by Franklin et al. (1958).     

Heat Resistance of Bacillus subtilis Spores at Various Water Activities

S ummary : The heat resistance of spores of Bacillus subtilis was determined in water vapour and in aqueous solutions of NaCl, LiCl, glucose and glycerol at various water activities. In water vapour and in glycerol solutions, maximal resistance appeared at low, but not zero water activity. In NaCl and glucose solutions only small variations in heat resistance occurred with decreasing water activity although a small increase was observed at the highest concentrations tested. With increasing concentrations of LiCl, heat resistance at first decreased but increased at water activities below 0·5. The possible interaction of compounds controlling water activity and water activity per se on heat resistance is discussed.     

Pressure inactivation of Bacillus endospores

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80 degrees C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70 degrees C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60 degrees C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. II. EXPERIMENTS WITH SUSPENSIONS OF SPORES IN MILK

SUMMARY: The sporicidal efficiency of an ultra-high-temperature (UHT) milk processing plant has been tested using spores of a strain of Bacillus subtilis in milk. With the inoculum and volume of milk adjusted to obtain a countable number of survivors by a conventional dilution counting method, a temperature of 130·5° with the minimum time setting of the plant was found necessary to give a destruction of 99·99999%. This temperature was lower than that found previously (135°) for spores suspended in water and evidence is produced to support the suggestion that UHT milk may be inhibitory to the germination and/or subsequent growth of heated spores.     

Repair and calcium dipicolinate release of bioindicators in propylene glycol and propylene glycol—water mixtures

B. PHILIPP AND H. SUCKER. 1992. The heat sterilization of spores of Bacillus stearothermophilus ATCC 7953 in propylene glycol (PG) and PG—water mixtures was investigated. Unusual non-logarithmic survival curves with a marked long shoulder, designated as the lag-time, were observed at the maximum of resistance. When the number of colony-forming units were determined after intervals of incubation, the growth curves of spores previously treated at 121°C in PG or PG-4% water varied considerably. Spores receiving a heat treatment that was longer than the lag-time showed no growth. When the heat treatment was shorter than the lag-time, spores showed a delayed growth phase. When spores received heat treatment that was long but which fell within the lag-time, there was a greater decrease in spore counts before the exponential growth phase began. In PG and PG with low water concentrations, a suppressed release of the specific spore substance calcium dipicolinate was observed during sterilization at 121°C of B. stearothermophilus ATCC 7953 and B. subtilis var. niger ATCC 9372. Calcium dipicolinate release, however, was observed in water.     

Radiation Resistance of Spores of Bacillus subtilis and B. stearothermophilus at Various Water Activities

S ummary . The radiation resistance of spores of Bacillus subtilis and B. stearothermophilus was determined under anoxic conditions in water vapour and in aqueous solutions of glucose and glycerol at various water activities. In water vapour, resistance of both kinds of spores increased slightly with decreasing water activity ( a _w). In glycerol solutions, a phase of rapid increase in resistance with decreasing a _w was followed by a slower increase on further reduction of a _w. This second phase was almost parallel to the water vapour curve, which, however, showed considerably lower resistance. The initial phase of rapid increase in resistance was also observed in glucose solutions.     

Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures

Dried preparations with Streptococcus faecium, strain A(2)1, and spores of Bacillus sphaericus, strain C(I)A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R(1), and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain C(I)A, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment.     

Resistance of Bacillus Spores to Combined Sporicidal Treatments

S ummary . Moist heat at 82° (100° for Bacillus stearothermophilus ) and solutions of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride at 24° separately showed no sporicidal activity against B. pumilis, B. stearothermophilus, B. subtilis and B. subtilis var. niger . Spores of the last organism were the most sensitive to γ radiation, the D value being 0.16 Mrad. Prior irradiation with a dose of 0.16 Mrad brought about only a slight increase in the sensitivity of the spores to moist heat. The presence of bactericide during irradiation did not affect radiation resistance. Inactivation rates were greater when the spores were heated in the presence of a bactericide than in aqueous suspension and benzalkonium chloride was more active than chlorocresol. Chlorocresol enhanced the heat activation of B. stearothermophilus at 100°. Irradiation in the presence of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride had no effect on the subsequent resistance of the spores when heated in the presence of these bactericides. It is concluded that it is unlikely that combinations of moist heat, radiation and bactericides, each less severe than when used in an accepted sterilization process, will lead to an alternative process which, while less damaging to the materials being sterilized, would still maintain the accepted standards of freedom from contamination.     

Effect of water activities of heating and recovery media on apparent heat resistance of Bacillus cereus spores

Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.     

Potential occurrence of adhering living Bacillus spores in milk product processing lines

AIMS: The hygienic risk associated with microbial soil on surfaces of milk processing lines was evaluated, based on experimental results. METHODS AND RESULTS: From a panel of Bacillus spores isolated from milk products, B. cereus CUETM 98/4, was found to be highly resistant to heat (D100=3.32 min in whole milk) and oxidant disinfectant (70% lethality of adherent spores with Ikalin 2%). From adhesion trials, up to 1.1 x 10(7) spores cm(-2) were found to be adherent to solid surfaces when suspended in saline or in custard (10(5) and 10(7) cfu ml(-1)), and over 10% of these adherent spores would resist the cleaning procedure. CONCLUSION: A highly contaminated milk (10(5) cfu ml(-1)) subjected to a current sterilization process (8 log reduction) led to a residual contamination of less than 1 cfu in the representative processing line after a complete production run. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that under appropriate processing conditions (efficient sterilization and cleaning procedures), even disinfection would be sufficient to eliminate any contamination risk. Conversely, the disinfection procedure becomes an essential step under inappropriate processing conditions.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Comparison of Pressure Resistances of Spores of Six Bacillus Strains with Their Heat Resistances

The pressure resistances of the spores of six Bacillus strains were examined at 5 to 10(deg)C and were compared with their heat resistances. The pressure treatments (at 981 MPa for 40 min and at 588 MPa for 120 min) did not inactivate the spores of B. stearothermophilus IAM12043, B. subtilis IAM12118, and B. licheniformis IAM13417. However, these spores had large differences in heat resistance. The spores of B. megaterium IAM1166 were 9.3 times more pressure resistant but 246 times less heat resistant than those of B. stearothermophilus IAM11001. The spores of B. coagulans IAM1194 were activated by the pressure treatments. There was no correlation between these pressure and heat resistances.     

Effect of lipid materials on heat resistance of bacterial spores

The apparent heat resistance of spores of Bacillus megaterium, B. subtilis, B. cereus, B. stearothermophilus, and Clostridium botulinum type E in lipids was investigated and compared with the resistance of the spores in phosphate buffer solution. The most pronounced increase in heat resistance was noted for B. subtilis and C. botulinum type E, the increase varying with the type of lipid used. A high water content of the lipids used as heating menstruum lowered the heat resistance of the spores. Possible explanations for the high heat resistance of spores in lipids are discussed.