JPH05227925A - Method for sterilization - Google Patents

Abstract

PURPOSE:To effectively sterilize at relatively low pressures such bacterial spores of esp. high pressure and heat resistance as to have been hard to make high- pressure sterilization, without impairing the ingredients and flavor of a food to be sterilized. CONSTITUTION:According to the pressure resistance of spores, the spores in a food to be sterilized is put to germination heat activation at 60-100 deg.C followed by high pressure treatment at >=100MPa.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for sterilizing bacterial spores which may cause problems in hygiene or quality preservation in foods, medicines and the like. The present technology is particularly applicable to sterilization of bacterial spores having high heat resistance and pressure resistance.

[0002]

2. Description of the Related Art Since the spoilage of foods and pharmaceuticals and the contamination of pathogenic microorganisms pose a public health problem, control of microorganisms by heat sterilization has been widely practiced in the fields of food industry, medical industry and the like. The types of bacteria that attach to foods or medicines differ slightly depending on the types, but various bacteria may attach via air or water. From the viewpoint of sterilization, pathogenic bacteria, Gram-negative bacteria, yeasts, etc. are generally weak to heat and can be easily treated by ordinary heat sterilization and can be easily controlled, but Gram-positive bacteria and molds, especially those that form spores. Is a problem because it has high heat resistance and does not easily die by heat sterilization. For example, a gram-positive bacterium, Bacillus subtilis, exhibits a heat resistance of several minutes to 1,000 minutes or more at 100 ° C., although it varies depending on the strain.

However, heat sterilization causes a change in components such as proteins and vitamins and a change in flavor at the same time as sterilization.
Depending on the type of food, there are many cases where implementation is not appropriate. Taking milk as an example, UHT sterilization method, UHT
Sterilization method, pasteurization method, HTST sterilization method and the like are adopted. The UHT sterilization method (120 to 130 ° C., 2 to 4 seconds) kills a part of vegetative cells, thermostable bacteria, and spores of thermostable bacteria in milk, and the UHT sterilization method (135 to 150 ° C., 2
In ~ 4 seconds), all microorganisms including spores of thermostable bacteria can be killed, but changes in the components and changes in flavor cannot be avoided. In addition, low temperature sterilization method (62 ~ 65 ℃, 30 minutes), HT
In the ST sterilization method (72 to 85 ° C., 16 seconds), there is little change in components and flavor, and the original flavor of milk is maintained, but heat-resistant bacteria and spores of heat-resistant bacteria cannot be sterilized sufficiently and thus cannot be stored for a long time.

Therefore, the high pressure sterilization method has been attracting attention in recent years. For example, in high-pressure sterilization of soft drinks, vegetative cells such as Escherichia coli, mold and yeast are 300 to 600 M at room temperature.
Pa-A report that it can be sterilized with a holding time of 10 minutes (Takahashi Yasuo et al., Fruit Juice Association Bulletin Vol 370 pp6-13, 1988) and a report that heat treatment combined with high pressure sterilization improves the sterilization effect (Takahashi Yasuo et al. Juice Association Bulletin Vol 381 pp 41-46, 1989). In addition, regarding sterilization of B. subtilis, all of the bacteria except B. subtilis were killed by the hydrostatic pressure of 4,000 kg / cm ² (about 400 MPa) for 10 minutes in these reports, and 47 ℃ and 57 ℃. Heat treatment of 6,000kg
It was reported that B. subtilis was killed by the combined use of hydrostatic pressure treatment for 10 minutes / cm ² (about 600 MPa).

However, according to the tests conducted by the present inventors, spores of B. subtilis having high heat resistance and pressure resistance are 600 MPa-
In some cases, the spores were not completely killed even when kept at 60 ° C for 60 minutes. This is B.subt which generally has high heat resistance and pressure resistance.
There are various strains of ilis, some of which are particularly resistant, and the fact that the resistance of the bacterium changes depending on the type of treated material, and foods with low pH such as juices ( Generally, foods having a pH of 4.6 or less are called highly acidic foods), which is because these spores do not germinate and grow. That is, the conventional high-pressure sterilization generally targets low-pressure resistant microorganisms such as Escherichia coli, mold, and yeast, but in many low-acid foods (pH ≧ 4.6) such as milk and soups. In order to realize long-term storage, bacterial spores with high heat resistance should be used as an indicator bacterium, but sterilization of such highly resistant spores by high pressure was difficult. Further, since these spores are resistant not only to temperature but also to pressure, high-pressure treatment conditions are likely to become severe. Therefore, in high-pressure sterilization, food ingredients,
The challenge is to develop technology to kill bacterial spores with lower pressure so that the scent does not change.

On the other hand, a device for high-pressure sterilization was developed by Mitsubishi Heavy Industries, Ltd., Kobe Steel Ltd., etc.
As a testing machine, up to about 1000 MPa is possible,
It is said that operation at 400 to 500 MPa is suitable as a production machine dedicated to food processing in consideration of safety and durability of the apparatus. However, 400-500M
E. coli, mold, yeast, and staphylococcus can be sterilized at a pressure of Pa, but it may not be possible to sterilize bacterial spores having high heat resistance and high pressure resistance even by heating at 60 ° C.

[0007]

DISCLOSURE OF THE INVENTION The present invention, in view of the actual conditions of the conventional high-pressure sterilization technique described above, is a component of an object to be sterilized, in particular, a bacterial spore that has high pressure resistance and heat resistance and is difficult to sterilize under high pressure. It is an object to provide a method for effectively sterilizing without deteriorating the flavor.

[0008]

This object is achieved by the following means. That is, the present invention is a method for sterilizing bacterial spores, which comprises subjecting bacterial spores in an object to be sterilized to germination activation by heat (hereinafter referred to as germination thermoactivity), and then rapidly performing high-pressure treatment. The present inventors pay attention to the fact that bacterial spores are activated by germination when they are heated, and their heat resistance and pressure resistance are weakened. By applying a constant heat treatment before high-pressure treatment, a pressure of about 400 to 500 MPa is obtained. It was found that the bacterial spores and the like can be sterilized by the method of the present invention, and the present invention has been completed. According to the present invention, it is possible to effectively sterilize bacterial spores, which were conventionally difficult to sterilize with high pressure, without causing harmful effects such as a change in the components of the sterilized product, and to those which were conventionally capable of sterilizing with high pressure. As a result, the high-pressure processing conditions can be eased,
As a result, it is possible to improve the quality of food and medical products and at the same time extend the shelf life.

The present invention will be described in detail below. First, as the sterilization object targeted in the present invention, heat resistance, there is a possibility of contamination by spores having high pressure resistance, food that can be processed under high pressure, medical products, etc., regardless of its form, shape, etc. Is also applicable. Of course, if the above-mentioned high-pressure treatment conditions are alleviated, the present method is also effective for spores that are not so resistant to pressure. From the viewpoint of ease of high-pressure treatment, various juices, soft drinks, liquids such as milk, various soups, sauces, fruit jams, seafood / meat pastes, eggs and other fluids and semi-fluids are preferable. Furthermore, even a solid substance can be treated if it is immersed or dispersed in a liquid. Further, from the viewpoint of the possibility of contamination by bacterial spores and easy heat treatment, low-acid foods such as milk and various soups have a practical benefit.

Next, the target bacterial spores are B. su.
Spores of all spore-forming bacteria such as btilis, B. cereus, B. coagulans. From the object of the present invention, conventional high-pressure sterilization requires sterilization and extremely high pressure for sterilization,
In addition, it is useful to target spores of strains for which sufficient sterilization has not been practically possible. For example, B. subtilis is relatively heat resistant, there are strains with low pressure resistance, heat resistance, there are strains with high pressure resistance, the former can be effectively sterilized by high pressure sterilization combined with conventional heat treatment, The latter does not die even with such high pressure sterilization.

The first step of the sterilization method of the present invention is germination heat activation treatment of bacterial spores. Germination heat activation means that germination is affected by heat to induce germination and becomes a germination state, and the outer membrane of the spores gradually becomes clear (decrease in optical density as described below). By entering the preparatory stage for cellularization, heat resistance generally decreases. The spores activated by germination gradually start activities such as absorption of nutrients and enzymes, but in the present invention, the treatment just before the spores start germination is sufficient. The purpose of heat activation for germination in the present invention is to reduce the heat resistance and pressure resistance of the outer membrane of spores, and to enable subsequent sterilization by high-pressure treatment.
Further, it is not necessary that all spores are activated by germination heat, and it is appropriately set depending on the conditions of high-pressure treatment, the intended degree of sterilization, the type of target spores, the type of sterilized object, and the like. However, the degree of germination heat activation has a critical effect on the bactericidal effect in high-pressure treatment, so it is necessary to set it to an appropriate degree in consideration of other conditions.

The germination heat activation treatment is carried out at a critical temperature (this is called activation critical condition) at which the spore activation critical temperature or higher exerts a bactericidal promotion effect in high pressure treatment. The activation critical condition is individually determined by the type of spores, environmental conditions, etc., but below the critical temperature, no effect appears even if treated for a long time, and similarly, below the critical time, even if the temperature is high. No effect. Generally 40 ° C or higher, preferably 6
At a temperature of 0-100 ° C for 1 minute or more, preferably 5-60
Minutes are good. Within this range, the higher the processing temperature, the shorter the holding time. Also, if the temperature is high, the activation rate will be large for about 5 to 10 minutes after the start of the treatment and then decrease. Therefore, if the temperature is too high, the activation treatment will be more efficient, but if the temperature is too high, the spores will be activated. Not done. Spores with relatively low heat resistance may partially die during the activation process, but the purpose of the activation process is not sterilization, so even if the temperature is raised higher than necessary at this stage, it is not effective, Due to the heat resistance of the spores, the bactericidal effect is not so noticeable. On the other hand, if the holding time is too long, the spores will germinate and start activities such as oxygen absorption, possibly promoting the decomposition of the sterilized material. Further, the spores are activated even when kept at a relatively low temperature for a long time, but in this case as well, spoilage due to other microorganisms may occur.

The degree of activation treatment has a great influence on the bactericidal effect in relation to the high-pressure treatment conditions. When the pressure of the high-pressure treatment is the same as that of the non-activation treatment, there are roughly three modes of the bactericidal effect depending on the degree of the activation treatment. First, the gradient of viable cell count decrease is large at the early stage of high-pressure treatment and thereafter becomes gentle, and the final viable cell count is almost the same as that of the unactivated treatment. (FIG. 1 (A)).
This is because only a relatively weakly resistant spore among the spores was activated by the activation treatment, and a strong one was not activated, so that a strong spore remained until the end. in this case,
There is an advantage that a similar bactericidal effect can be obtained even if the high-pressure treatment time is shortened. Secondly, the gradient of viable cell count decrease at the early stage of the high-pressure treatment is relatively large, and then decreases with the same gradient as the inactivation treatment (FIG. 1 (B)). this is,
This is because the less resistant one of the spores is more activated and the more resistant one is also activated to some extent. In this case, the same bactericidal effect can be obtained even if the high-pressure treatment time is shortened.
Further, there is an advantage that a larger bactericidal effect can be obtained when the high pressure treatment is performed for the same time. Third, there is a large gradient of decrease in viable cell count from the beginning to the end of high-pressure treatment (Fig. 1).
(C)). This is due to activation of many spores, including highly resistant spores. In this case, not only the high-pressure treatment time can be shortened, but also spores, which have been difficult to sterilize by high-pressure treatment, can be sterilized. In any of the above aspects (A)-(C), the sterilizing effect equivalent to or higher than that of the non-activation treatment can be obtained even if the pressure of the high pressure treatment is lowered. As described above, since the degree of activation treatment is related to the bactericidal effect, it may be relatively set in relation to the desired sterilization degree, high-pressure treatment conditions, target spores, sterilized objects, and the like.
For example, when sterilizing relatively weakly resistant spores, a sterilizing effect can be obtained only by high-pressure treatment, so a relatively weak activation treatment is carried out to achieve the mode (A) to shorten the high-pressure treatment time and reduce the pressure. Can be promoted. On the other hand, when sterilizing relatively highly resistant spores, the bactericidal effect cannot be obtained only by high-pressure treatment, so a relatively strong activation treatment should be carried out to achieve the mode (C) to achieve the desired bactericidal effect. You can

Optical density of spores (hereinafter referred to as “O”) is one of the indicators of the strength of the activation treatment.
(D)). Here, OD is defined as the absorbance of the spore suspension with respect to visible light (here, light having a wavelength of 440 nm is used). In the following germination heat activation treatment, It will be explained using the relative OD defined in. The OD measured here is the observed value of OD at a certain time during the activation of germination heat, and the initial OD is O of the spore suspension that has not been activated.
The D value and the final OD are the OD values when all the spores in the suspension were germinated and activated. In the case of B. subtilis, for example, those with relatively weak tolerance require less activation, so
Even if the relative OD is reduced by about 4-6%, the high-pressure treatment can improve the sterilization effect. 15-20 for relatively strong ones
The effect will be great if you activate it until it decreases by about%. 2 and 3 show an example of measurement of relative OD, B.I. subtilis ATCC
6633 and B.I. The case of subtilis NCDO 2130 is shown. As shown in the figure, the magnitude of the decrease in relative OD is not always proportional to the treatment time, but as a guideline for the degree of activation treatment, spores with weak resistance show a decrease of about 4-56% in resistance. With strong spores, the reduction is about 15-50%.

The activation can be promoted not only by heat but also by the presence of reducing substances, ethyl alcohol, amino acids, sugars, etc., pH, etc., so that the pH can be adjusted if necessary,
You may heat-process by adding ethyl alcohol, an amino acid, sugar, etc.

Next, the high pressure processing will be described. The activated bacterial spores germinate and proliferate or are deactivated again depending on storage conditions. Therefore, it is desirable that the high-pressure treatment be carried out immediately after the activation treatment. As far as the condition of the high-pressure treatment can be taken, it is not much different from the range in the conventional high-pressure range. That is, 100-1000
It can be said that the range is MP, room temperature-100 ° C, and within several hours. However, this is a range and does not mean that spore sterilization can be carried out in the prior art, for example, at 100 MP, room temperature and minutes. That is, if the pressure is low, it is indispensable to increase the temperature or the processing time, and it is not possible to set the conditions arbitrarily within the above range.

The feature of the present invention in high-pressure treatment is that spores which could not be sterilized by high-treatment in the above range can be sterilized sufficiently in the same range in the present invention. In the present invention, it is possible to sterilize under milder pressure conditions. The latter feature is, for example,
It can be specified as follows. In order to obtain the same bactericidal effect, it is possible to reduce the high-pressure treatment time to about 1 / 2-1 / 5 and the pressure to about 1 / 2-1 / 3 under the same conditions. .. Although the range of the high-pressure treatment is possible, in consideration of practicality in the present invention, 40 ° C. or higher, preferably 50-60 ° C., 100 MPa or higher, preferably 400-700 MPa, 0-60 minutes or so. ..
The treatment time can be 0 minutes because the spores are weakened by the activation treatment, and the spores may be killed in the step of increasing the pressure until a predetermined pressure is reached.

On the other hand, the operating pressure is 500 to 6 from the viewpoints of durability of the high-pressure device and changes in the content components of the sterilized object.
It is considered that 00 MPa is a preferable upper limit in actual operation, but it is possible to sterilize spores that could not be sterilized by heating at such a pressure and prolonging the holding time.

When the temperature of the high pressure treatment is high, the effect of the high pressure can be promoted. When the temperature is low, the effect of high pressure is reduced, but in the case of treating spores having relatively low pressure resistance, the pressure resistance is further lowered by the activation treatment. On the other hand, when the temperature is high, the bactericidal effect is improved, so the upper limit of the temperature is not particularly limited. However,
Due to the structural restrictions of commercially available high-pressure processing equipment, the above range is reached. Further, it is effective to carry out the high-pressure treatment at a rising temperature, and conversely, it may be carried out at a high temperature at the start of the high-pressure treatment and thereafter at a lowering temperature.

It is preferable that the rate of pressure increase up to the predetermined pressure is high, but it is not particularly limited. 20-150M as a guide
It can be adjusted, for example, by changing the processing amount at about Pa / min.
The spores with weak pressure resistance may die during pressurization, but the spores with high pressure resistance may not change at all. However, by strengthening the degree of activation treatment, it is possible to obtain a sufficient bactericidal effect only at the time of pressurization, which is a unique effect of the present invention that has never been seen in the prior art. In other words, depending on the type of bacterial spores, the degree of activation treatment can be adjusted so that sterilization can be carried out to the desired degree only at the time of pressurization.

[0021]

EXAMPLES The present invention will be specifically described below with reference to examples. 1. Method of implementation (1) Test bacterium As a test bacterium, relatively heat-resistant among spores of the genus Bicillus,
Two strains of Bacillus subtilis ATCC 6633, which has low pressure resistance, and Bacillus subtilis NCDO 2130, which has high heat resistance and pressure resistance, were used.

(2) Preparation of test bacterial solution In the experiment, a phosphate buffer solution and Snow Brand Milk Products Co., Ltd.
Milk (non-fat milk solid content 8.3% or more, milk fat content 3.5% or more) was used. Each concentrated bacterial suspension was diluted with phosphate buffer and milk so that the number of test bacteria would be 10 ⁶ cells / ml. The phosphate buffer is phosphate buffer-saline (pH 7.0). The pH was adjusted using sodium hydroxide and hydrochloric acid. The pH of milk was not adjusted, but it was about 6.8. B.subtilis A
The preparation methods for TCC 6633 and B. subtilis NCDO 2130 spores are shown in Table 1.

[0023]

[Table 1] (3) Activation treatment and high-pressure treatment 10 times the bacterial solution prepared using phosphate buffer and milk
Each ml was dispensed into a polypropylene bag, filled so that air did not enter, and the mouth was heat-sealed to obtain a sample. The samples were always stored in ice water except during activation and high-pressure treatment. In the case of B. subtilis ATCC 6633, the activation treatment was carried out at 60 ° C, 80 ° C, 90 ° C for 15 minutes in B. subtilis ATCC 6633.
In the case of NCDO 2130, it was performed at 80 ° C., 90 ° C. and 100 ° C. for 15 minutes.

A high-pressure tester MCT150 manufactured by Mitsubishi Heavy Industries Ltd. was used for the high-pressure treatment. The capacity of the sample chamber is 5301.4 cm ^{3 for} B. subtilis ATCC 6633,
In the case of B. subtilis NCDO 2130, it was 196.4 cm ³ . Although the heating at 60 ° C. was used together during the high-pressure treatment, the temperature of the sample chamber was adjusted by circulating hot water in the constant temperature bath.

(4) Measurement of viable cell count The test bacterial solution was activated, and then the sample was subjected to high pressure treatment. The number of viable bacteria was measured after activation, after pressurization, and after high-pressure treatment. In the case of B. subtilis, the number of grown colonies was counted after diluting with sterile phosphate-buffered saline and culturing for 48 hours at 35 ° C. using a standard agar medium. 2. Implementation Results The results are shown together in FIGS.

(1) Killing of spores during activation treatment B. subtilis ATCC 6633 was heated at a temperature of 60 to 90 ° C for 15 minutes, and the viable cell count after the treatment was measured. 4 and 6 show spore killing in phosphate buffer, and FIGS. 5 and 7 show spore killing in milk. Initial viable cell count (N0) and viable cell count after activation (N
When comparing 1), there was a case where a difference close to one order was observed in some cases, but when repeated experiments were performed, no large difference was observed between the phosphate buffer solution and the milk due to heating. Usually, spores are not easily killed by heating at 60 to 90 ° C for 15 minutes, but the viable cell count (N1) after activation may change somewhat depending on the state of the bacteria during storage or preparation of bacterial solution. it is conceivable that. Then B. subtilis NCDO 2130 at a temperature of 80-100
After heat treatment at 15 ° C. for 15 minutes, the number of viable bacteria (N1) after the treatment was measured. Figures 8 and 10 in phosphate buffer, Figures 9 and 11
It showed that spores were killed by heating in milk. B.su
Similar to btilis ATCC 6633, there was no difference from the initial viable cell count (N0), and no bactericidal effect by heating was observed. As a result, B. subtilis at 110 ℃ calculated by heat sterilization experiment
NCDO 2130 has a D value of 63.4 minutes and a temperature of 80-1
The spores were not killed by heat treatment at 00 ° C. for 15 minutes (the D value means the treatment time until the viable cell count becomes 1/10).

The treatment time of the above-mentioned activation treatment was 15 minutes, but the result of measuring the decrease in the relative OD of the spores due to the activation treatment time was shown in FIG. 2 (B. subtilis ATCC 663).
3) and FIG. 3 (B. subtilis NCDO 2130), in any strain, the higher the activation temperature, the faster the transparency changes and the higher the transparency. However, the transparency was increased, and it was recognized that it was activated.

(2) Effect of activation treatment on killing of spores at pressurization 200MP when high-pressure treatment of B. subtilis ATCC 6633
a, 3 minutes each for reaching a pressure of 400 MPa 3
The pressurization time of 0 seconds and 17 minutes and 30 seconds was required. B.subtilis A
Since TCC 6633 has lower heat resistance and pressure resistance than B. subtilis NCDO 2130, it was pretreated by heating at 60, 80 and 90 ° C. for 15 minutes, and then treated at a pressure of 200 MPa and 400 MPa. FIGS. 4 and 6 show spore killing in phosphate buffer, and FIGS. 5 and 7 show spore killing activation in milk. The number of viable bacteria after activation treatment is N1, and the number of viable bacteria after pressurization is N2 (for convenience, symbols such as N1 and N2 are shown on the horizontal axis in the figure, but this is on the vertical axis. Shows the correspondence with the number.). Without activation treatment, the spores of 1 order at 200 MPa and 2 orders at 400 MPa died in both the phosphate buffer and the milk during pressurization.
The activation temperature for both phosphate buffer and milk is 60 ° C → 8
As the temperature increased from 0 ° C to 90 ° C, the decrease in N2 increased. In particular, at 400 MPa, the effect of the activation treatment was remarkable, and the activation treatment at 80 ° C. reduced it by 4 orders and at 90 ° C. by 5 orders, and sufficient sterilization could be carried out only at the time of pressurization. From this result, it is understood that the spores having relatively low pressure resistance can be sterilized only at the time of pressurization. In addition, 60 ℃ -15
There was no difference in the case of activating treatment for 10 minutes compared to the case of not activating, but it is considered that the difference appeared when spores with lower heat resistance and pressure resistance were targeted. Be done.

Next, in the case of B. subtilis NCDO 2130, 4
It took 5 minutes and 30 seconds and 6 minutes and 30 seconds to increase the pressure to 00 MPa and 600 MPa, respectively. 8
Heat treatment at 0, 90, 100 ℃ for 15 minutes, then 4
High-pressure treatment of 00 MPa and 600 MPa was performed. 8 and 10 show the death of spores in the phosphate buffer, and FIGS. 9 and 11 show the death of spores in milk. 40 in phosphate buffer
Both 0MPa and 600MPa did not differ from those without activation treatment even after treatment for 15 minutes in the temperature range of 80 to 100 ° C, but in milk, 90 ° C at the time of pressurization up to 600MPa.
By the above heating pretreatment, a bactericidal effect close to one order was observed.

Generally, heat resistance and pressure resistance have a high correlation, and B.su
Since btilis ATCC 6633 has lower heat resistance and lower pressure resistance than B. subtilis NCDO 2130, B. subtilis ATCC 6633 is easily germinated and the germinated activated spores are more resistant to pressure than the original spores. It is considered that the bactericidal effect was high even at low pressure because it was low. On the other hand, B. subtilis NCDO 2130
Although the germination was partially activated, it could not be killed only by increasing the pressure because of its high pressure resistance. However, it is possible to obtain a sufficient bactericidal effect at the time of pressurization by further strengthening the activation treatment.

(3) Effect of heat pretreatment on killing of spores during pressure holding (heating at 60 ° C.) The holding time is 0 minutes when the predetermined pressure is reached (N2),
The viable cell count after the pressure was maintained was measured (N3). As shown in FIGS. 4 and 6, B. subtilis ATCC 6633 was
There was a tendency for the bactericidal effect to increase as the holding time became longer, but this tendency was not so different between with and without the activation treatment. This is because B. subtilis ATCC 6633 spores were germinated and activated by the activation treatment, and those whose resistance became weaker had already died at the time of pressurization, and only spores with high pressure resistance close to the original state remained without germination activation. This is because the decrease in the number of viable bacteria when the pressure was maintained was reduced. In other words, the bactericidal effect of the activation treatment has already become apparent when the pressure is increased,
It becomes less noticeable when held.

5 and 7 show the bactericidal effect of B. subtilis ATCC 6633 in milk, the same tendency was observed in milk, and the bactericidal effect was high at the time of pressurization at 80 ° C. for 15 minutes, 9 minutes.
The heating pretreatment at 0 ° C. for 15 minutes did not show a great effect when the pressure was maintained.

Incidentally, the D of N3 / N2 standard under each condition
Value (indicates the time (minutes) until the viable cell count becomes 1/10,
The effect of the activation treatment was not significant on the D value when calculated as (indicator of bactericidal effect). The reason for this is that, in the case of B. subtilis ATCC 6633, the bacteria die when the pressure is increased, as described above.

For B. subtilis NCDO 2130, FIG.
00 MPa) and FIG. 10 (600 MPa) show the bactericidal effect in the phosphate buffer. At 400 MPa, no bactericidal effect was observed even if the holding time was extended when only the pressure treatment was performed, but the bactericidal effect increased as the holding time was prolonged by performing the activation treatment. Moreover, the sterilization effect at the time of maintaining the pressure increased as the activation treatment temperature increased. At 600 MPa, a slight bactericidal effect was recognized by holding for 40 minutes only by pressure treatment, but the decrease was only one order or less. On the other hand, in the case of the activation treatment, the sterilizing effect was surely obtained with the holding time, and the reduction of 2 to 3 orders was observed after the holding for 40 minutes. As described above, spores that do not die at the time of pressurization can also be made to die at the time of holding the pressure by the activation treatment. The bactericidal effect in milk is almost the same.
0 MPa) and FIG. 11 (600 MPa). However, in the case of B. subtilis NCDO 2130, more bacteria were killed in milk than in phosphate buffer, despite the same treatment conditions, which was also observed during pressurization. It is considered that the milk containing more components than the buffer solution is more likely to be activated in germination, but the sterilizing effect was high even without pretreatment, so the components in milk have some influence on sterilization. It is thought that

Table 2 shows D in B. subtilis NCDO 2130.
Indicates a value. As shown in Table 2, B. subtilis A with weak pressure resistance
Compared with TCC 6633, the effect of activation treatment during holding is remarkable, and generally the D value decreases as the degree of activation treatment increases, and the D value decreases as the pressure increases. However, the effect of the activation treatment becomes insignificant in the case of 600 MPa, which has a sterilizing effect only by pressure treatment such as milk. This is because the bactericidal effect is already manifested to some extent when the pressure is increased. In addition, the phosphate buffer solution is 400M, which was activated at 80 ℃.
At Pa and 600 MPa, the bactericidal effect is greatly different, and similarly at 400 MPa, 80 ° C. and 90 ° C. are greatly different. This is 600MP in activation treatment at 80 ℃
The pressure around a is a critical condition value, and 400 MPa
Shows that the temperature around 90 ° C. is a critical condition value. On the other hand, even at 400 MPa and 80 ° C, the bactericidal effect was significantly improved compared to the control, and B. subtilis A
At 60 ° C with TCC6633, the effect was not significant regardless of pressure, so there is a first critical condition that can improve the bactericidal effect between the degree of activation treatment and pressure. It can be seen that there is a second critical condition that can be greatly increased. These conditions can be set depending on the type of spores to be treated, the substance to be sterilized, and the like.

[0036]

[Table 2] As described above, when the changes in the viable cell count from N0 to N3 are generally viewed from the viewpoint of the mode of the bactericidal effect described above, B. subtilis ATCC 66
The sterilization effect of 33 at 200 MPa is 40 (40%).
At 0 MPa it is embodiment (B), B. subtilis NCDO
It can be said that the aspect of 2130 at 400 MPa and 600 MPa is the aspect (C).

[0037]

As described above, by performing the activation treatment before the high-pressure treatment, the spores having relatively low heat resistance and pressure resistance can be activated (or even the spores having high heat resistance and pressure resistance can be activated. It can be sterilized at the time of pressurization (by sufficient implementation), and spores that do not die at the time of pressurization can be sterilized at the time of holding the pressure. That is, there is a technical significance that spores that have not been killed by a single operation of high-pressure treatment can be sterilized, and that high-pressure treatment conditions can be relaxed for those that can be sterilized only by high-pressure treatment. Therefore,
According to the sterilization method of the present invention, it is possible to efficiently perform sterilization of spores including highly resistant spores without inducing a change in components of the sterilization target, and the sterilization method is widely used in food and pharmaceutical fields. is there.

[Brief description of drawings]

FIG. 1 is a conceptual diagram showing modes (A), (B) and (C) of a bactericidal effect by the sterilizing method of the present invention.

Fig. 2 optica during activation of B. subtilis ATCC 6633
It is a graph which shows l density change.

Fig. 3 optica during activation of B. subtilis NCDO 2130
It is a graph which shows l density change.

FIG. 4: 20 of B. subtilis ATCC 6633 in phosphate buffer
It is a graph which shows a change in the number of viable bacteria under 0 MPa. In the figure, (a) shows the activation temperature of 60 ° C., (b) shows the same 80
° C, (c) is the same 90 ° C, (d) is a control.

FIG. 5: 200 MPa of B. subtilis ATCC 6633 in milk
It is a graph which shows the viable cell count change below. (A) in the figure
Has an activation temperature of 60 ° C, (b) has the same temperature of 80 ° C, (c)
Is 90 ° C. and (d) is a control.

FIG. 6: 40 of B. subtilis ATCC 6633 in phosphate buffer
It is a graph which shows a change in the number of viable bacteria under 0 MPa. In the figure, (a) shows the activation temperature of 60 ° C., (b) shows the same 80
° C, (c) is the same 90 ° C, (d) is a control.

Figure 7: 400 MPa of B. subtilis ATCC 6633 in milk
It is a graph which shows the viable cell count change below. (A) in the figure
Has an activation temperature of 60 ° C, (b) has the same temperature of 80 ° C, (c)
Is 90 ° C. and (d) is a control.

FIG. 8: 40 of B. subtilis NCDO 2130 in phosphate buffer
It is a graph which shows a change in the number of viable bacteria under 0 MPa. In the figure, (a) shows an activation treatment temperature of 80 ° C., (b) shows the same 90 °
° C, (c) is the same 100 ° C, (d) is a control.

Figure 9: 400 MPa of B. subtilis NCDO 2130 in milk
It is a graph which shows the viable cell count change below. (A) in the figure
Has an activation temperature of 80 ° C., (b) has the same temperature of 90 ° C., (c)
The same is 100 ° C., and (d) is a control.

FIG. 10: 6 of B. subtilis NCDO 2130 in phosphate buffer
It is a graph which shows the viable cell count change under 00 MPa.
In the figure, (a) shows an activation treatment temperature of 80 ° C., (b) shows the same 90 °
° C, (c) is the same 100 ° C, (d) is a control.

FIG. 11: 600MP of B. subtilis NCDO 2130 in milk
It is a graph which shows the viable cell count change under a. In the figure, (a) shows the activation treatment temperature of 80 ° C, (b) shows the same 90 ° C,
(C) is the same 100 degreeC, (d) is a control.

Claims (2)

[Claims] 1. A method for sterilizing bacterial spores, which comprises subjecting bacterial spores in a substance to be sterilized to germination activation treatment by heating, and then subjecting to high pressure treatment. 2. The germination activation treatment by heating is performed at 40 ° C. or higher for 1 minute or longer, and the pressurization pressure in the high pressure treatment is 10
The sterilization method according to claim 1, wherein the sterilization method is 0 MPa or more.