Inactivation of Bacillus spores by the combination of moderate heat and low hydrostatic pressure in ketchup and potage

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International Journal of Food Microb

Inactivation of Bacillus spores by the combination of moderate heat and

low hydrostatic pressure in ketchup and potage

Md. Shahidul Islam, Ayaka Inoue, Noriyuki Igura *, Mitsuya Shimoda, Isao Hayakawa

Laboratory of Food Process Engineering, Division of Food Biotechnology, Department of Bioscience and Biotechnology,

Faculty of Agriculture, Kyushu University 6-10-1, Hakozaki, Higashi-ku, Fukuoka-shi, 812-8581, Japan

Received 23 May 2003; received in revised form 13 July 2005; accepted 17 August 2005

Abstract

The combination effect of moderate heat and low hydrostatic pressure (MHP) on the reduction of Bacillus subtilis, Bacillus coagulans and

Geobacillus stearothermophilus spores in food materials (potage and ketchup) was investigated. These bacterial spores were suspended in potage

(pH 7), acidified potage (pH 4), neutralized ketchup (pH 7) and ketchup (pH 4). The suspensions were treated with and without pressure (100

MPa) and temperatures of 65–85 -C for 3 to 12 h. The bacterial spores were inactivated by 4–8 log cycles during MHP treatment in potage,

acidified potage and ketchup, whereas the spores were highly resistant to long time heat treatment in potage and neutralized ketchup. The degrees

of spore destruction were mostly dependent on pH and medium composition during MHP treatment. The inactivation effect in MHP treatment was

higher at the pH 7 than at pH 4 both in ketchup and potage. The bacterial spores showed higher inactivation in potage than ketchup during MHP

treatment.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Bacterial spores; Low hydrostatic pressure; Moderate heat; Potage and ketchup

1. Introduction

High pressure processing is an alternative technology for

preservation of foods. The destruction of microorganisms by

high pressure was reported 100 years ago (Hite, 1899). Many

studies indicated that the hydrostatic pressure can inactivate

microorganisms without altering the flavor and nutrient

components of foods (Cheftel, 1992). At present, retort

processing, using high temperature, such as 121–135 -C for

20 min holding time followed by 20 min cooling, is frequently

employed to kill bacterial spores in food. Such high tempera-

tures cause losses in nutrients, produce burnt flavor and allergic

components (Jankiewicz et al., 1997; Codina et al., 1998;

Chung and Champagne, 1999).

In high pressure sterilization, bacterial spores are more

resistant than vegetative bacteria (Timson and Short, 1965;

Cheftel, 1992) and spores can survive up to 1200 MPa (Larson

et al., 1918; Johnson and Zobell, 1949; Timson and Short,

0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2005.08.021

* Corresponding author. Tel.: +81 92 642 3016; fax: +81 92 642 3016.

E-mail address: [email protected] (N. Igura).

1965; Sale et al., 1970). Hence, it has been suggested that

bacterial spores are poorly inactivated by the hydrostatic

pressure treatment at room temperature (Sonoike, 1997). On

the other hand, combination of high pressure with heat is

effective in increasing the inactivation of bacterial spores

(Gould, 1973; Mallidis and Drizou, 1991; Roberts and Hoover,

1996). Several studies have been carried out for inactivation of

Bacillus spores by heat with pressure (Okazaki et al., 1996;

Moerman et al., 2001), mild heat and chemical preservatives

including nisin (Roberts and Hoover, 1996; Capellas et al.,

2000; Shearer et al., 2000), heat with pressure at low pH

(Wuytack and Michiels, 2001), reciprocal pressurization

(Furukawa et al., 2003), CO2 and high pressure (Spilimbergo

et al., 2002; Park et al., 2003) and rapid decompression

(Hayakawa et al., 1998).

Pressure above 600 MPa combined with mild or moderate

heat is required to inactivate bacterial spores (Hayakawa et al.,

1994a,b; Mills et al., 1998). Depending on the material

strength, it is possible to make pressure equipment with a

vessel size over 10 tons under reasonable cost if operating

pressure is smaller than 100 MPa. On the contrary, small size

pressure vessels (<100 kg) can be made if the operating

iology 107 (2006) 124 – 130

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Md.S. Islam et al. / International Journal of Food Microbiology 107 (2006) 124–130 125

pressure is larger than 600 MPa. Considering these facts,

several attempts have been made in our laboratory to reduce the

pressure by combining heat treatment (Furukawa and Haya-

kawa, 2000, 2001; Furukawa et al., 2001). Furukawa and

Hayakawa (2001) suggested that low hydrostatic pressure from

60 to 100 MPa is highly effective in sterilizing Geobacillus

stearothermophilus spores when used in combination with long

time heating at 75–95 -C.We examined Bacillus subtilis spores as they are extensively

studied, and Bacillus coagulans spores as they are pressure

resistant and relatively heat resistant at acidic pH (Palop et al.,

1999). Mallidis et al. (1990) demonstrated that B. coagulans

spores are able to germinate and grow at pH values as low as 4,

and are the microorganisms most frequently isolated from

spoiled canned vegetables acidified to pH values between 4

and 4.5. We also examined G. stearothermophilus spores, as

they belong to the most heat-tolerant species among aerobic

spore-forming bacteria.

Combination of moderate heat and long time pressurization

(MHP) with dormant bacterial spores in buffer solution, and a

model liquid food system has been studied by some

researchers (Furukawa and Hayakawa, 2000, 2001; Furukawa

et al., 2001). Ananta et al. (2001) studied the inactivation of

G. stearothermophilus spores suspended in mashed broccoli

and in cocoa mass exposed to high pressure for short time.

However, there are no available data on the inactivation of

bacterial spores in food materials under low pressure (100

MPa) for long time treatment. Therefore, in the present study,

we investigated the inactivation effect of combined treatment

of low hydrostatic pressure, moderate heat and long time on

the inactivation of B. subtilis , B. coagulans and G.

stearothermophilus spores in potage, acidified potage, ketch-

up and neutralized ketchup.

2. Materials and methods

2.1. Bacterial spores

The bacteria used were Bacillus subtilis NBRC 13722,

Bacillus coagulans NBRC 12583, and Geobacillus stearother-

mophilus NBRC 12550, obtained from the Institute of

Fermentation Osaka (Osaka, Japan).

The stationary-phase cultures of B. subtilis, B. coagulans

and G. stearothermophilus grown in nutrient broth (Eiken

Chemical Co., Ltd., Tokyo, Japan) were transferred to nutrient

agar plates (Eiken Chemical Co. Ltd.) with 0.1 mM MnSO4

(Nacalai Tesque, Inc., Kyoto, Japan). The plates for B. subtilis

and B. coagulans were incubated at 37 -C for 10 days and G.

stearothermophilus was incubated at 55 -C for 10 days. Spores

were collected by flooding the surface of the agar culture with

sterile distilled water, and then scraping the surface with a

sterile microscope glass slide. The spores were washed three

times in sterile distilled water by centrifugation at 7000�g for

10 min, followed by heat treatment at 70 -C for B. subtilis and

B. coagulans and 90 -C for G. stearothermophilus for 30 min

in order to kill the vegetative cells. The concentration of

prepared spore suspension of B. subtilis was 108–109 CFU/ml

and those of B. coagulans and G. stearothermophilus were

107–108 CFU/ml determined as described below.

2.2. Sample preparation

Potage (pH 7, Nagoya Seiraku Co., Ltd., Japan) and tomato

ketchup (pH 4, Kagome Co., Inc., Tokyo, Japan) were obtained

from the local market and kept at 4 -C until use. To investigate

the effect of pH on heat and pressure inactivation of the

bacterial spores, potage was acidified to pH 4 and ketchup was

neutralized to pH 7. Acidification was carried out with 0.6 M

hydrochloric acid (Nacalai Tesque, Inc.) and neutralization

with 5 M sodium hydroxide (Nacalai Tesque, Inc.).

Each spore suspension was mixed with the potage and

ketchup to a concentration of 107–108 CFU/ml for B. subtilis

and 106–107 CFU/ml for B. coagulans and G. stearothermo-

philus. The spore suspension mixed with foods was sealed into

a germ-free plastic tube (volume=1.5 ml, Greiner Labortech-

nik Co., Ltd., Germany) and kept at 4 -C until use.

2.3. Heat treatment

Bacterial spores in the sealed tubes were heated in a water

bath (model-DTS 100D, Kyoto Electronics, Kyoto, Japan) at

65, 75, and 85 -C for 3, 6, 9 and 12 h. After heat treatment, the

tubes were cooled immediately in crushed ice with water and

the spores were subjected to viable count immediately, as

described below.

2.4. MHP treatment

Spores in the sealed tubes were exposed to hydrostatic

pressure treatment at 100 MPa at the same temperature and

time as the heat treatment using a prototype pressurization

apparatus (Yamamoto Suiatsu Kogyosho Co., Ltd., Osaka,

Japan) with a cylindrical pressure chamber (inside volume=8

L). The pressurization rate was 20 MPa/min, and decompres-

sion time from 100 MPa to 0.1 MPa was less than 20 s.

Adiabatic heat generated during pressurization was about 3 -C.The temperature variation was regulated to T2 -C by a voltage

controller (type: S-130, Yamabishi Co., Ltd., Tokyo, Japan).

Treatment temperature was monitored by a digital temperature

controller (type: SR-62, Shimaden Co., Ltd., Tokyo, Japan)

with a thermocouple placed inside the top of pressure chamber.

Deionized water was used as the pressure medium. After MHP

treatment, surviving spores were enumerated by the same

procedure as in heat treatment experiment.

2.5. Counting of surviving spores

Survivors after pressurization and heat treatment were

estimated by the viable count method using nutrient agar

media (Eiken Chemical Co., Ltd.). The plates for B. subtilis

and B. coagulans were incubated at 37 -C for 24 h and 72 h,

respectively, and those for G. stearothermophilus were

incubated at 55 -C for 48 h and then the colonies were

enumerated.

Md.S. Islam et al. / International Journal of Food Microbiology 107 (2006) 124–130126

2.6. Statistical analysis

All experiments were carried out at least in three different

experiments and the standard deviations were calculated from

the triplicate experiments.

3. Results

In MHP treatment, B. subtilis spores were inactivated by

6.5–8 log cycles at pH 7 in potage and in neutralized ketchup

at 65 -C for 3 h, whereas the spores were inactivated by only 1-

log cycle at pH 7 after heat treatment at 65 -C for 12 h (Fig. 1).

On the other hand, 6-log and 5-log reduction of spores were

achieved in the acidified potage (pH 4) during MHP and heat

treatment at 65 -C for 6 h, respectively. In ketchup (pH 4),

there were 4.2-log reduction of spores in both MHP and heat

treatments at 65 -C for 12 h. The inactivation effect of heat

treatment for B. subtilis spores was higher at pH 4 compared to

pH 7 in both ketchup and potage, whereas that of MHP

treatment was higher at pH 7 than pH 4. The effect of

inactivation was higher in acidified potage (pH 4) compared to

ketchup (pH 4) during MHP treatment (Fig. 1).

Fig. 2 shows the inactivation behavior of B. coagulans

spores subjected to the MHP and heat treatment in ketchup and

potage. Heat treatment alone did not decrease the initial

number of spores in potage and ketchup at pH 7 even after

heat treatments at 85 -C up to 12 h. On the other hand, a 4-log

reduction was achieved in potage (pH 7) during MHP treatment

0 3 6 9 12

65°C (Heat)65°C (MHP)




-8-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Potage, pH 7

0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Potage, pH 4

Fig. 1. Comparison between MHP (65, 75 -C, 100 MPa, 0–12 h) and heat (65, 75 -Cspores in potage (pH 4, 7) and ketchup (pH 4, 7).

at 85 -C for 12 h, although there was no reduction of spores in

neutralized ketchup (pH 7) at 85 -C during MHP treatment. In

ketchup (pH 4), MHP and heat treatments showed only about

1-log reduction at 75 -C for 12 h, whereas there were 6.4-log

reductions in ketchup after heat and MHP treatment at 85 -Cfor 12 h. However, spore reduction after heat treatment for 3–9

h was more pronounced than that after MHP treatment at 85

-C. In acidified potage (pH 4), MHP treatment was effective to

reduce the spores compared to heat treatment, more than 6-log

reduction was achieved by MHP treatment at 85 -C for 9 and

12 h. Our results showed that B. coagulans spores tended to be

more resistant in neutralized foods than acid foods during heat

and MHP treatment, and to be more resistant in ketchup than

potage during MHP treatment.

G. stearothermophilus spores were not inactivated in potage

at 85 -C for 12 h during heat treatment and reduced only 2.2-

log cycles in neutralized ketchup at 85 -C for 12 h and 5 to 6-

log cycles at 85 -C for 12 h in both acidified potage and

ketchup (Fig. 3). On the contrary, about 6-log reduction of G.

stearothermophilus spores was achieved during MHP treat-

ment at 85 -C for 3 h in all food systems we used. The results

show that the spores were more sensitive in ketchup than in

potage at pH 7, and inactivated more effectively at pH 4 than

pH 7 in potage and ketchup during heat treatment. While, in

MHP treatment, the spores were more sensitive in potage than

ketchup at pH 7, and inactivated more effectively at pH 7 than

pH 4. The spores were highly resistant to heat treatment in both

potage and ketchup at pH 7.




75°C (Heat)75°C (MHP)0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N/N

o)

Treatment time (h)

Ketchup, pH 7

0 3 6 9 12

-8-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Ketchup, pH 4

0 3 6 9 12

-8-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Ketchup, pH 4

, 0.1 MPa, 0–12 h) treatments on the inactivation behavior of Bacillus subtilis

0 3 6 9 12









-7-6-5-4-3-2-101

Log

(N/N

o)

Treatment time (h)

Potage, pH 7

0 3 6 9 12

-7-6-5-4-3-2-1

0

Log

(N

/No)

Treatment time (h)

Ketchup, pH 7

0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N/N

o)

Treatment time (h)

Potage, pH 4

0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N

/No)

Ketchup, pH 4

0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Ketchup, pH 4

Fig. 3. Comparison between MHP (75, 85 -C, 100 MPa, 0–12 h) and heat (75, 85 -C, 0.1 MPa, 0–12 h) treatments on the inactivation behavior of Geobacillus

stearothermophilus spores in potage (pH 4, 7) and ketchup (pH 4, 7).

03 6

912

85°C (MHP)

85°C (Heat)-7-6-5-4-3-2-101

Log

(N/N

o)

Treatment time (h)

Ketchup, pH 7

0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Potage, pH 4

0 3 6 9 12

-7-6-5-4-3-2-101

Log

(N

/No)

Treatment time (h)

Ketchup, pH 4

0 3 6 9 12







-7-6-5-4-3-2-10

1L

og (

N/N

o)

Treatment time (h)

Potage, pH 7

Fig. 2. Comparison between MHP (75, 85 -C, 100 MPa, 0–12 h) and heat (75, 85 -C, 0.1 MPa, 0–12 h) treatments on the inactivation behavior of Bacillus

coagulans spores in potage (pH 4, 7) and ketchup (pH 4, 7).



4. Discussion

Furukawa and Hayakawa (2001) have studied the combined

effect of low hydrostatic pressure and heat treatments on the

inactivation of G. stearothermophilus spores in standard buffer

solutions and suggested that treatment at 100 MPa and 80 -Cfor 12 h could reduce the spores by 5.5-log. These results are

similar to the data obtained in the present study for potage at

pH 7 (Fig. 3).

The pH of the heating medium is one of the most important

factors influencing the heat resistance of microorganisms

(Xezones and Hutchings, 1965; Lowick and Anema, 1972;

Alderton et al., 1976). Microorganisms usually have their

maximum heat resistance at pH values close to neutrality.

Condon and Sala (1992) demonstrated that the heat resistance

of B. subtilis spores in foods was mostly determined by the pH

of food. Present data also show that these spores in potage and

neutralized ketchup (pH 7) are more heat-resistant than in the

acidified food systems (pH 4). This may be due to the fact that

in high acid environments, the spores are demineralized and

changed to H-spores, which are replaced by their minerals with

protons (Igura et al., 2003). Some researchers showed that

demineralization of spores markedly reduce their heat resis-

tance of bacterial spores (Marquis et al., 1981; Bender and

Marquis, 1985).

Furukawa and Hayakawa (2000) showed that addition of

12% (w/v) glucose and 3% (w/v) or more NaCl decreased the

G. stearothermophilus spore inactivation, indicating that these

food additives had a protective effect on the spore resistance

to hydrostatic pressure treatment. Several reports have

suggested that carbohydrates and salts increase the heat

resistance of microorganisms (Harnulv et al., 1977; Mazas et

al., 1999). Addition of glucose and NaCl decreased inactiva-

tion of bakers yeast by hydrostatic pressure (Hayakawa et al.,

1992). Raso et al. (1998) also reported that high concentra-

tions of sucrose protected Bacillus cereus spores from the

germinating and inactivating effect of high hydrostatic

pressure. Jordan et al. (2001) demonstrated that NaCl and

ascorbic acid might have a deleterious effect on microorgan-

isms pressure treated under acidic conditions. The results

from the MHP and heat treatments of the bacterial spores are

in agreement with previous studies on the effect of sugar and

salt on spore inactivation. As we can see, B. subtilus spores

during MHP treatment at 65 -C for 6 h (Fig. 1) were

inactivated 6-log cycles in acidic potage, whereas the spores

were inactivated 2-log cycles in acidic ketchup. Bacillus

coagulans and G. stearothermophilus spores also have shown

similar behavior. The spores of G. stearothermophilus were

reduced by 6.5-log cycles in acidic potage at pH 4, whereas

they were reduced by 2-log cycles in ketchup (pH 4) during

MHP treatment at 75 -C for 12 h (Fig. 3). This protection

against the inactivation of spores by pressure treatment may

be due to the high amount of sugar and salt in ketchup as

compared to acidic potage.

Besides the effect of carbohydrates and NaCl, germinants

(amino acid content such as l-alanine, etc.) may also influence

the germination and inactivation of spores during MHP

treatments. Hydrostatic pressure initiates germination of

dormant bacterial spores in germinant-free solution, and

germinated spores are inactivated by hydrostatic pressure and

heat (Clouston and Wills, 1969; Gould and Sale, 1970). Under

low pressurization, it is considered that the germinated spores

are not only inactivated by pressurization but also by heat

because hydrostatic pressure below 100 MPa is not enough to

inactivate vegetative cells (Sonoike, 1997). In this study, higher

inactivation of B. subtilis and B. coagulans spores was

achieved in potage compared to neutralized ketchup during

MHP treatment. Raso et al. (1998) reported that the combina-

tion of pressurization at 250 MPa with l-alanine were found to

give an additive inactivation response. Thus, there is a

possibility that germinants containing in the potage promoted

the pressure-induced germination during the MHP treatment.

At the present time, we did not found what ingredients affects

the additive inactivation response.

The germination of spores might be related to the lower

resistance of these spores to MHP treatment in neutralized food

systems than acidified food systems, because the germination

of spores, including the pressure-induced germination, is

inhibited in acidic conditions (Raso et al., 1998; Wuytack

and Michiels, 2001). Considering these facts, the higher

inactivation of spores in neutralized food than acidified food

system, and in potage than in ketchup during MHP treatment,

was attained by the pressure-induced germination of the spores,

and the lower sugar and NaCl contents which protect microbial

inactivation from heat and pressure, respectively.

The present study showed that B. coagulans spores are more

resistant in potage and ketchup compare to other two species of

spore-forming bacteria used in the experiments. Heat resistance

of B. coagulans has been extensively studied in tomato

products. So far we know that there are no data available in

the literature regarding the behaviour of B. coagulans in the

MHP treatment temperature in the range of 75–85 -C for

longer time, to which the present results can be compared.

Although Palop et al. (1999) demonstrated that the acidification

of the heating medium leads to a decrease in the heat resistance

of B. coagulans, its heat resistance is also influenced by the

composition of the medium and the treatment temperature.

Condon and Sala (1992) showed that apart from pH, the

composition of heating medium (buffer, tomato paste) can also

influence the heat resistance of microorganisms. However,

possible differences in behavior among different species of

microorganism in MHP and heat treatment should also be

considered.

5. Conclusions

In order to introduce pressure processing into the food

industry, treatment conditions, pressure and temperature,

should be optimized with eliminating sufficient bacterial spores

in processed foods. Low pressure (100 MPa) treatment will

reduce the equipment and manufacturing cost and be possible

help to make large-scale pressurization equipment. From the

results of the present study, it is concluded that moderate heat

and low pressure treatment for long time (MHP treatment)


could sufficiently inactivate bacterial spores in low acid foods.

In high acid foods, reductions of spores can be achieved by

moderate heating for a longer time instead of high temperature

heating. This study shows that MHP treatment can be used as

an effective alternative method to ultra high pressure or high

temperature (retort) treatment.

Acknowledgements

One of the authors (Islam, M.S.) expresses his gratitude to

the Ministry of Education, Culture, Spores, Science and

Technology for its scholarship to conduct this research. The

authors would like to thank Dr. T. Kato for his technical help

for this research.

References

Alderton, G., Ito, K.A., Chen, J.K., 1976. Chemical manipulation of the heat

resistance of Clostridium botulinum spores. Appl. Environ. Microbiol. 31,

492–498.

Ananta, E., Heinz, V., Schluter, O., Knorr, D., 2001. Kinetic studies on high-

pressure inactivation of Bacillus stearothermophilus spores suspended in

food matrices. Innov. Food Sci. Emerg. Technol. 2, 261–272.

Bender, G.R., Marquis, R.E., 1985. Spore heat resistance and specific

mineralization. Appl. Environ. Microbiol. 50, 1414–1421.

Capellas, M., Mor-Mur, M., Gervilla, R., Yuste, J., Guamis, B., 2000. Effect of

high pressure combined with mild heat or nisin on inoculated bacteria and

mesophiles of goat’s milk fresh cheese. Food Microbiol. 17, 633–641.

Cheftel, J.C., 1992. Effects of high hydrostatic pressure on food constituents: an

overview. In: Balny, C., Hayashi, R., Heremans, K., Masson, P. (Eds.), High

Pressure Biotechnology Colloque INSERM. John Libbey and Co. Ltd.,

London, pp. 195–205.

Chung, S.Y., Champagne, E.T., 1999. Allergenicity of Maillard reaction

products from peanut proteins. J. Agric. Food Chem. 47, 5227–5231.

Clouston, J.G., Wills, P.A., 1969. Initiation of germination and inactivation of

Bacillus subtilis spores by hydrostatic pressure. J. Bacteriol. 97, 648–690.

Codina, R., Oehling, A.G., Lockey, R.F., 1998. Neoallergens in heated soybean

hull. Int. Arch. Allergy Immunol. 117, 120–125.

Condon, S., Sala, F.J., 1992. Heat resistance of Bacillus subtilis in buffer and

foods of different pH. J. Food Prot. 55, 605–608.

Furukawa, S., Hayakawa, I., 2000. Investigation of desirable hydrostatic

pressure required to sterilize Bacillus stearothermophilus IFO 12550 spores

and its sterilization properties in glucose, sodium chloride and ethanol

solutions. Food Res. Int. 33, 901–905.

Furukawa, S., Hayakawa, I., 2001. Effect of temperature on the inactivation of

Bacillus stearothermophilus IFO 12550 spores by low hydrostatic pressure

treatment. Biocontrol Sci. 6, 33–36.

Furukawa, S., Matsuoka, A., Nakamichi, Y., Kato, T., Shimoda, M., Hayakawa,

I., 2001. Inactivation of spores of three Bacillus strains by low hydrostatic

pressure (LHP) treatment. J. Fac. Agric., Kyushu Univ. 45, 525–530.

Furukawa, S., Shimoda, M., Hayakawa, I., 2003. Mechanism of the inactivation

of bacterial spores by reciprocal pressurization treatment. J. Appl.

Microbiol. 94, 836–841.

Gould, G.W., 1973. Inactivation of spores in food by combined heat and

hydrostatic pressure. Acta Aliment. 2, 377–383.

Gould, G.W., Sale, A.J.H., 1970. Initiation of germination of bacterial spores by

hydrostatic pressure. J. Gen. Microbiol. 60, 335–346.

Harnulv, B.G., Johanson, M., Snygg, B.G., 1977. Heat resistance of Bacillus

stearothermophilus spores at different water activities. J. Food Sci. 42,

91–93.

Hayakawa, I., Oda, M., Kajihara, J., 1992. Study of pressure bearability of

baker’s yeast under buffer conditions. In: Hayakawa, I. (Ed.), Food

Processing by Ultra High Pressure, Twin-Screw Extrusion. Technomic

Publishing Co. Inc., Lancaster, pp. 47–56.

Hayakawa, I., Kanno, T., Tomita, M., Fujio, Y., 1994a. Application of high

pressure for spore inactivation and protein denaturation. J. Food Sci. 59,

159–163.

Hayakawa, I., Kanno, T., Yoshiyama, K., Fujio, Y., 1994b. Oscillatory compared

with continuous high-pressure sterilization on Bacillus stearothermophilus

spores. J. Food Sci. 59, 164–167.

Hayakawa, I., Furukawa, S., Midzunaga, A., Horiuchi, H., Nakashima, T.,

Fujio, Y., Ishikura, T., Sasaki, K., 1998. Mechanism of inactivation of heat-

tolerant spores of Bacillus stearothermophilus IFO 12550 by rapid

decompression. J. Food Sci. 63, 371–374.

Hite, B.H., 1899. The effect of pressure in the preservation of milk. Bull. West

Virginia Univ. Agric. Exp. Station 58, 15–35.

Igura, N., Kamimura, Y., Islam, M.S., Shimoda, M., Hayakawa, I., 2003.

Effects of minerals on resistance of Bacillus subtilis spores to heat and

hydrostatic pressure. Appl. Environ. Microbiol. 69 (10), 6307–6310.

Jankiewicz, A., Baltes, W., Bogl, K.W., Dehne, L.I., Jamim, A., Hoffmann, A.,

Haustein, D., Vieths, S., 1997. Influence of food processing on the

immunochemical stability of celery allergens. J. Sci. Food Agric. 75,

359–370.

Johnson, F.H., Zobell, C.E., 1949. The retardation of thermal disinfection of

Bacillus subtilis spores by hydrostatic pressure. J. Bacteriol. 57, 353–358.

Jordan, S.L., Pascual, C., Bracey, E., Mackey, B.M., 2001. Inactivation and

injury of pressure-resistant strains of E. coli 0157 and Listeria monocyto-

gens in fruit juices. J. Appl. Microbiol. 91, 463–469.

Larson, W.P., Hartzell, T.B., Diehl, H.S., 1918. The effect of high pressure on

bacteria. J. Infect. Dis. 22, 271–279.

Lowick, J.A., Anema, P.J., 1972. Effect of pH on the heat resistance of

Clostridium sporogenes spores in minced meat. J. Appl. Bacteriol. 35,

119–121.

Mallidis, C.G., Drizou, D., 1991. Effect of simultaneous application of heat and

pressure on the survival of bacterial spores. J. Appl. Bacteriol. 71, 285–288.

Mallidis, C.G., Frantzeskakis, P., Balatsouras, G., Katsabotxakis, C., 1990.

Thermal treatment of aseptically processed tomato paste. Int. J. Food Sci.

Technol. 25, 442–448.

Marquis, R.E., Carstersen, E.L., Child, S.Z., Bender, G.R., 1981. Prepara-

tion and characterization of various salt forms of Bacillus megaterium

spores. In: Levinson, H.S., Sonenshein, A.L., Tipper, D.J. (Eds.),

Sporulation and Germination. American Society for Microbiol, Washing-

ton, DC, pp. 266–268.

Mazas, M., Martınez, S., Lopez, M., Alvarez, A.B., Martin, R., 1999. Thermal

inactivation of Bacillus cereus spores affected by the solutes used to control

water activity of the heating medium. Int. J. Food Microbiol. 53, 61–67.

Mills, G., Earnshaw, R., Patterson, M.F., 1998. Effects of high hydrostatic

pressure on Clostridium sporogenes spores. Lett. Appl. Microbiol. 26,

227–230.

Moerman, F., Mertens, B., Demey, L., Huyghebaert, A., 2001. Reduction of

Bacillus subtilus, Bacillus strarothermophilus and Streptpcoccus faecalis in

meat batters by temperature–high hydrostatic pressure pasteurization. Meat

Sci. 59, 115–125.

Okazaki, T., Kakugawa, K., Yamauchi, S., Yoneda, T., Suzuki, K., 1996.

Combined effects of temperature and pressure on inactivation of heat-

resistant bacteria. In: Hayashi, R., Balny, C. (Eds.), High Pressure

Bioscience and Biotechnology. Elsevier, Amsterdam, pp. 415–418.

Palop, A., Raso, J., Pagan, R., Condon, S., Sala, F.J., 1999. Influence of pH on

heat resistance of spores of Bacillus coagulans in buffer and homogenized

foods. Int. J. Food Microbiol. 46, 243–249.

Park, S.J., Park, H.W., Park, J., 2003. Inactivation kinetics of food poisoning

microorganisms by carbon dioxide and high pressure. J. Food Sci. 68,

976–981.

Raso, J., Gongora-Nieto, M.M., Barbosa-Canovas, G.V., Swanson, B.G., 1998.

Influence of several environmental factors on the inhibition of germination

and inactivation of Bacillus cereus by high hydrostatic pressure. Int. J. Food

Microbiol. 44, 125–132.

Roberts, C.M., Hoover, D.G., 1996. Sensitivity of Bacillus coagulans spores to

combinations of high hydrostatic pressure, heat, acidity and nisin. J. Appl.

Bacteriol. 81, 363–368.

Sale, A.J.H., Gould, G.W., Hamilton, W.A., 1970. Inactivation of bacterial

spores by hydrostatic pressure. J. Gen. Microbiol. 60, 323–334.


Shearer, A.E.H., Dunne, C.P., Sikes, A., Hoover, D.G., 2000. Bacterial spore

inhibition and inactivation in foods by pressure, chemical preservatives, and

mild heat. J. Food Prot. 63, 1503–1510.

Spilimbergo, S., Elvassore, N., Bertucco, A., 2002. Microbial inactivation by

high-pressure. J. Supercrit. Fluids 22, 55–63.

Sonoike, K., 1997. High pressure sterilization technology: subject for the

application to food (in Japanese). J. Jpn. Soc. Food Sci. Technol. 44,

522–530.

Timson, W.J., Short, A.J., 1965. Resistance of microorganisms to hydrostatic

pressure. Biotechnol. Bioeng. 7, 139–159.

Wuytack, E.Y., Michiels, C.W., 2001. A study on the effects of high pressure

and heat on Bacillus subtilis spores at low pH. Int. J. Food Microbiol. 64,

333–341.

Xezones, H., Hutchings, I.J., 1965. Thermal resistance of Clostridium

botulinum (62A) spores as affected by fundamental food constituents: 1.

Effect of pH. Food Technol. 19 (6), 113–115.

Documents

Inactivation of Bacillus spores by the combination of moderate heat and low hydrostatic pressure in ketchup and potage