JP2773493B2 - High pressure sterilization indicator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an indicator used for high-pressure sterilization in which a hydrostatic pressure of 1000 atm or more is applied to an object to be treated, and particularly used for high-pressure sterilization of food having a pH of 4.5 or less. The present invention relates to an indicator for high-pressure sterilization.

[0002]

2. Description of the Related Art Conventionally, when sterilizing foods, medicines, medical devices, and the like, an indicator is used to confirm whether sterilization has been performed reliably.

[0003] As such a sterilization indicator,
There is a chemical indicator that informs that sterilization has been achieved by discoloration, etc. when the specified sterilization conditions have been reached, and a biological indicator that confirms whether or not sterilization has been completed using microorganisms.High-temperature heating such as retort sterilization Sterilization,
Alternatively, they are used in gas sterilization using a gas having a sterilizing property such as ethylene oxide gas, radiation sterilization using radiation such as γ-rays, and the like.

For example, JP-A-62-228147, JP-A-1-107765, and JP-A-2-195.
No. 898 discloses a biological indicator using a microorganism.

On the other hand, chemical indicators have been known for a long time, but Japanese Patent Application Laid-Open Nos. Hei 1-166759, Hei 2-180973 and Hei 2-221162 are known.

In the case of biological indicators, it is necessary to use microorganisms that are most resistant to the target sterilization method. At present, practical indicators for heat sterilization include wet heat sterilization. Bacillus stearothermophilus
us), but Bacillus s subtilis (Bacillus s.
ubtilis).

By the way, recently, 1,000-10,000
Technology for processing and sterilizing food products by applying a very high hydrostatic pressure of 0 atm to the material to be processed has been developed and has already been partially put into practical use, but an indicator that can be used for high-pressure sterilization using this technology Has not been developed yet.

[0008] This is because autoclaving is a relatively new technology, and the processing conditions are not fixed like heat sterilization, etc., and are processed under various conditions. Therefore, an indicator that can be used in a wide range of conditions is required. In addition, it is considered that microorganisms that can be used as an indicator and that are resistant to high pressure and chemical substances that change visually and irreversibly at high pressure have not been found.

Japanese Patent Application Laid-Open No. 1-240831 discloses a pressure indicator using a substance which changes color reversibly with pressure. However, the pressure indicator is reversible and is applicable to high-pressure sterilization as in the present invention. Is impossible.

[0010] Here, the biological indicator used for sterilization can be proliferated in a food or the like to be sterilized, and
If a bacterium having the highest resistance to the set sterilization conditions is found and used, the object can be achieved. Therefore, the indicator for high-pressure sterilization is preferably a biological indicator. And if it is a biological indicator, it is possible to adapt to various conditions by a combination of a bacterial species and a bacterial amount.

In the case of heat sterilization, bacterial spores are the most resistant bacteria and can be used as indicator bacteria of biological indicators for heat sterilization, but bacterial spores cannot be completely sterilized by high-pressure sterilization. At present, foods subjected to high-pressure sterilization are foods having a pH of 4.5 or less, at which bacteria cannot grow. Microorganisms that can grow at such a low pH are fungi such as filamentous fungi and yeast, and it is necessary to use the microorganism that is most resistant to high pressure among these fungi as an indicator.

[0012]

SUMMARY OF THE INVENTION The present invention has a pH of 4.
It is an object of the present invention to provide a microbial indicator for confirming sterilization certainty when sterilizing microorganisms capable of growing in foods of 5 or less by high-pressure sterilization.

[0013]

Means for Solving the Problems The present inventors have found that conidia of yeasts of the genus Candida and filamentous fungi of the genus Penisillium have remarkably high high pressure resistance compared to other fungi. And found that it could be used as an indicator for high-pressure sterilization. Above all, in Candida, Candida cruzei (C
and Candida tropicalis, and in the genus Penicillium, Penicillium citrinum (Peni)
(Cillium citrinum), Penicillium.
Sticky (Penicillium Sticki)
It has been recognized that the spores i), i.e., conidia, have particularly high resistance (hereinafter sometimes simply referred to as fungi or fungi). Therefore, the present invention has been conceived to solve the above-mentioned problems, and the invention of claim 1 is an indicator for high-pressure sterilization using yeast of the genus Candida as an indicator bacterium. The invention of claim 2 is that the yeast of the genus Candida is Candida krusei or Candida tropicalis.
The indicator for high-pressure sterilization according to claim 1, wherein the indicator is any one of the following. The invention of claim 3 is an indicator for high-pressure sterilization, wherein a conidia of a filamentous fungus of the genus Penicillium is used as the indicator bacterium. The invention according to claim 4 is characterized in that the penicillium filamentous fungus is Penicillium citrinum.
The indicator for high-pressure sterilization according to claim 3, wherein the indicator is any one of llium citrinum) and Penicillium sticky. The invention according to claim 5 is characterized in that the indicator bacterium is supported on a substrate made of a water-absorbing material, a water-impermeable material, or a complex thereof. An indicator for high-pressure sterilization according to any one of the above. The invention of claim 6, indicator bacteria, an indicator for a high pressure sterilization according to claim 5, characterized in that it is 10 ⁵ or more carrier per indicator.

The indicator may be in the form of a suspension of bacteria, or a powder or a granule obtained by vacuum or freeze-drying, but is composed of a water-absorbing material, a water-impermeable material, or a complex thereof. It is preferable that the fungus is supported on a base material because handling becomes easy.

That is, in the case of a powdery, granular, or liquid indicator, the indicator may be scattered during use,
The product and the operator may be contaminated.

Such an indicator in which the fungus is carried on a substrate can be prepared, for example, by the following procedure.

When a yeast of the genus Candida is used as an indicator bacterium, first, the yeast of the genus Candida is cultured to a stationary state and collected, and then various sugars, skim milk, sodium glutamate, serum, peptone, dextrin, etc. It is suspended at a high bacterial concentration in a dispersion medium containing various dry protective agents. This bacterial suspension is impregnated or dripped into various substrates so as to have a certain number of bacteria, and then is naturally dried (air-dried), freeze-dried, and dried.
-It is dried by a method such as vacuum drying, and the bacteria are supported (fixed) on the substrate to form an indicator.

When a conidia of a Penicillium sp. Is used as an indicator bacterium, Penicillium sp. Is cultured until conidia are sufficiently formed, the conidia are collected, and various sugars, skim milk, glutamic acid, It is suspended at a high conidia concentration in a dispersion medium containing various dry protective agents such as sodium, serum, peptone, dextrin and the like. This conidia suspension is impregnated or dripped into various substrates so as to have a certain number of conidia, and then dried by a method such as natural drying (air drying), freeze drying, or L-vacuum drying. Is carried (fixed) on a substrate to form an indicator.

The number of bacteria per indicator depends on the processing conditions of high-pressure sterilization, but it is necessary to make the number of bacteria higher than the actual maximum contamination level of the product to be sterilized.
For example lowering of survival bacterial count due to drying, in order to increase reliability of the results also, it is desirable that the 10 ⁵ or more.

The substrate used for the indicator is not particularly limited as long as it can support bacteria or conidia. However, the substrate is preferably used because it has little detachment of the supported bacteria or conidia and is easy to handle. Paper, especially filter paper, is preferred.

Further, one side of the paper is made of a water-impermeable material such as a plastic film such as polyethylene or polypropylene or an aluminum foil so as to easily impregnate and carry a certain amount of the bacterial suspension or conidia suspension on the paper. Materials may be attached.

FIGS. 1A and 1B are cross-sectional views of the indicator.

FIG. 1A shows a case where filter paper is used as a substrate (1), and a bacterium or a conidia (10) is carried in and on the surface of the substrate (1). FIG. 1B shows filter paper (1).
Substrate (1) obtained by laminating water-impermeable material (12) to 1)
Is used.

Further, as shown in FIG. 1 (c), a water-impermeable material (12) is used as a substrate (1), and a bacterial suspension or a conidia suspension is thinly spread thereon. It is also possible to prepare an indicator that is dried and carries the fungi or conidia (10). In this case, as the substrate (1), a plastic film or sheet such as polyethylene, polypropylene, polyethylene terephthalate, or nylon is suitable.

In the above embodiment, the fungus or conidia (10)
As shown in FIG. 2, bacteria or conidia (10) are supported on a part of the substrate (1), and the other part is used as a knob (2) as shown in FIG. The item may be printed or a part to be filled in by the user.

The dispersion medium used for suspending and dispersing the bacteria or conidia (10) and the substrate (1) are preferably sterilized in advance.

The indicator also includes a barrier layer made of aluminum foil, polyvinylidene chloride, saponified ethylene-vinyl acetate copolymer or the like to prevent the indicator from wetting and to prevent contamination by other microorganisms.
It is preferable to store in a sealed package with a moisture-proof packaging material.

In using the indicator obtained as described above, the indicator is enclosed in a flexible container such as a plastic film bag together with the food to be sterilized or an appropriate medium. Then, predetermined high-pressure processing is performed. After the high-pressure treatment, the container enclosing the indicator is taken out as it is or aseptically, and cultured under suitable growth conditions for the indicator bacterium, for example, at 25 to 30 ° C., and the presence or absence of growth of microorganisms is observed, and the bactericidal effect is evaluated. By making the determination, it is possible to evaluate whether the sterilization target has been completely sterilized or whether the necessary sterilization conditions have been given.

[0029]

By using the fungus having high pressure resistance as an indicator, in a food having a pH of 4.5 or less, if the indicator microorganism is completely sterilized, other fungi which are problematic in food hygiene are also sterilized. To be
High-pressure sterilization can be reliably performed.

[0030]

【Example】

<Experiment 1> As a microorganism, Candida tropicalis (Can
dida tropicalis: IFO 1400 (fungus 1), Candida krusei: IFO 0584 (fungus 2), and Candida maltosa:
IFO 1977 (bacterium 3) and Saccharomyces cerevisiae (Saccharomyces cerevisiae): A bacterium (yeast) obtained by culturing IFO 0251 (bacterium 4) for 3 days in a glucose / peptone medium, respectively.

This bacterium was suspended as a freeze-drying protective agent in a dispersion medium containing 10% by weight of skim milk powder and 1% by weight of sodium glutamate (other components were water) at a concentration of 10 ⁸ cells / ml (suspension). Suspension 1), and further using the same dispersion medium,
It was diluted to 10 ⁶ cells / ml (suspension 2).
After 0.1 ml of each of the suspensions 1 and 2 of each bacterium was dropped on the substrate, lyophilization was performed to obtain samples in which the number of bacteria per sample was 10 ⁷ and 10 ⁵ cells, respectively. .

As a base material, filter paper (Toyo Roshishi, No.
6), 50μ thickness on one side of filter paper (Toyo Filter Paper, No. 6)
m low density polyethylene film (PE) and a 70 μm polypropylene film (P
Each of the three types P) was cut into 5 mm × 20 mm. That is, four kinds of bacteria, two stages of the number of bacteria, substrate 3
A total of 24 samples of the species were prepared.

Each of these samples was put together with a glucose / peptone medium (10 ml) in a bag-like container made of stretched nylon / unstretched polypropylene, sealed so that no air remained, and then sealed at 3000, 4000 and 5000 atm. High pressure treatment was performed at 20 ° C. for 10 and 20 minutes. After the treatment, the sample was cultured in a bag-like container at 30 ° C., and the presence or absence of bacterial growth (turbidity and gas generation) was confirmed up to 5 days later. In addition, the same sample was processed for each condition by 5 sheets.

Further, in addition to the above, similar experiments were carried out for the following yeasts.

Bacteria 5: Candida utilis: IFO 0988 Bacteria 6: Candida salmon: IFO 1981 Bacteria 7: Zygosaccharomyces rouxii: IFO 0505 Bacteria 8: Hansenula anomola: IFO 0122 ・ Bacteria 9: Rhodotolula glutinis: IFO 1125

Table 1 shows the number of bag-shaped containers in which the growth of bacteria was observed by day 5 in the sample using the filter paper. In addition,
Filter paper / PE, or even samples based on PP,
The results were almost the same.

[0037]

[Table 1]

From the above results, it can be seen that Saccharomyces cerevisiae, Hansenula anomala and Rhodotorula glutinis are sterilized under any conditions and have low pressure resistance. In addition, T. saccharomyces luxii, Candida utilis and Candida salmon showed intermediate pressure resistance between Candida maltosa and Saccharomyces cerevisiae, and all had low pressure resistance.

On the other hand, Candida kulzei and Candida tropicalis may not be able to sterilize even if the number of bacteria is low, the treatment is long, or the pressure is high. You can see that it is doing.

From the above results, Candida cruzei and Candida tropicalis have extremely high resistance, and require at least 4000 atm or more for sterilization, and are optimal as indicators. In addition, Candida maltosa may not be sterilized when the treatment condition is 3000 atm, so as an indicator for confirming that at least 3000 atm has been applied by confirming that Candida maltosa has been killed. Can be used.

<Experiment 2> The following filamentous fungi were used as microorganisms, and each was cultured on a potato / dextrose agar medium for 10 times.
After culturing for days, well-formed spores were collected. -Bacteria 11: Penicillium citrinum: IFO 6352-Bacteria 12: Penicillium steckii: IFO 6028-Bacteria 13: Rhizopus delemar: OUT 1150-Bacteria 14: Mucor jabanics ( Mucor javanicus): OUT 1056 ・ Bacteria 15: Tricoderema viride: IFO 30498 ・ Bacteria 16: Aspergillus niger: IAM 2020 ・ Bacteria 17: Penicillium funiclosum: IFO 6345

Using this spore as a lyophilization protective agent, 10% by weight of skim milk powder, 1% by weight of sodium glutamate, polyoxyethylene (20) -sorbitan monooleate (surfactant, trade name Tween 80, I.C. the eye Co.) dispersion medium containing 0.01 wt% (other components water), were suspended so as to be 10 ⁸ spores / ml (suspension 11), further using the same dispersion medium, 10 ⁶ Spores / ml
(Suspension 12). After 0.1 ml of each of the suspensions 11 and 12 of each spore was dropped on the substrate, lyophilization was performed to obtain samples in which the number of spores per sample was 10 ⁷ and 10 ⁵ respectively. .

As a base material, filter paper (Toyo Roshishi, No.
6), 50μ thickness on one side of filter paper (Toyo Filter Paper, No. 6)
m low density polyethylene film (PE) and a 70 μm polypropylene film (P
Each of the three types P) was cut into 5 mm × 20 mm. That is, a total of 42 kinds of samples including seven kinds of filamentous fungi, two stages of spore count, and three kinds of base materials were prepared.

These samples were used in orange juice 1
0 ml together with one piece in a bag-like container made of stretched nylon / unstretched polypropylene, sealed so that air does not remain, and then sealed at 3,000, 4,000 and 5,000 atm.
The high pressure treatment was performed at 20 ° C. for 0 and 15 minutes. After the treatment, the sample and the contents were transferred aseptically to a sterilized flask and cultured at 25 ° C. for 7 days to confirm the presence or absence of mold growth. In addition, the same sample was processed for each condition by 5 sheets.

Table 2 shows the number of samples using filter paper in which the growth of filamentous fungi was observed by day 7. It should be noted that almost the same results were obtained with the samples using the filter paper / PE or PP as the base material.

[0046]

[Table 2]

As is clear from the above results, Rhizopus
Delemar, Mucor Javanics and Tricho delema viride were relatively resistant to pressure, but penicillium
Slightly less resistant than citrinum or penicillium sticky. Penicillium funiculosum had lower pressure resistance than these. Aspergillus niger was all sterilized under the conditions described above and had low pressure resistance.

On the other hand, Penicillium citrinum,
Penicillium sticky, as compared to the above filamentous fungi, may not be able to sterilize even if the number of bacteria is low, the treatment is long, or the pressure is high, indicating that the penicillium sticky sufficiently functions as an indicator.

From the results of Experiments 1 and 2, Candida cruzei, Candida tropicalis, Penicillium citrinum, and Penicillium sticky have extremely high pressure resistance, and at least 400% for sterilization.
It was necessary that the pressure be 0 atm or more, which proved to be optimal as an indicator.

In addition, it was confirmed that Candida maltosa, Rhizopus delemar, Mucor Javanix, and Trichodelema viride could not be sterilized when the treatment conditions were 3000 atm. Therefore, it was confirmed that at least 3000 to 4000 atm was applied. It can be used as an indicator for

It has been reported that increasing the temperature of the pressurizing treatment enhances the sterilizing effect. However, it is considered that there is no influence on the relative pressure resistance of the bacteria. It is considered that the temperature condition of the pressure treatment does not affect the use of the yeast of the genus Candida and the filamentous fungus of the genus Penicillium for the indicator of the spores.

[0052]

Industrial Applicability The present invention provides fungi having extremely high resistance to high pressure, especially yeasts of the genus Candida,
It is an indicator for high-pressure sterilization using Kuruzei or Candida tropicalis, or a conidia of a filamentous fungus of the genus Penicillium, especially Penicillium citrinum, Penicillium sticky, and is used for high-pressure sterilization of foods having a pH of 4.5 or less. Thereby, the effect of sterilization can be reliably grasped, and high reliability can be obtained in the sterilization treatment.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a cross-sectional view showing an embodiment of an indicator for high-pressure sterilization of the present invention. FIG. 1 (a) shows a case where filter paper is used as a substrate carrying bacteria, and FIG. (C) shows a case where a water-impermeable material is used when the combined base material is used.

FIG. 2 is a sectional view showing another embodiment of the indicator for high-pressure sterilization of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Base material 10 ... Bacteria 11 ... Filter paper 12 ... Water-impermeable material

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12Q 1/22 C12R 1:74) (C12Q 1/22 C12R 1:80) (58) Investigated field (Int.Cl. ⁶ , DB name) C12Q 1/22 A23L 3/015 A61L 2/26 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. Candid (Candid) is used as an indicator bacterium.
a) An indicator for high-pressure sterilization using yeast of the genus. 2. The yeast of the genus Candida is Candida krusei or Candida tropicalis.
The indicator for high-pressure sterilization according to claim 1, wherein the indicator is any one of the following. 3. A penicillium (Penicium) as an indicator bacterium.
An indicator for high-pressure sterilization using a conidia of a filamentous fungus of the genus (illium). 4. The method of claim 1, wherein the penicillium filamentous fungus is Penicillium citrinum.
m), Penicillium Sticky
4. The indicator for high-pressure sterilization according to claim 3, wherein the indicator is one of um stickii). 5. The method according to claim 1, wherein the indicator bacterium is carried on a substrate made of a water-absorbing material, a water-impermeable material, or a complex thereof. The indicator for high-pressure sterilization according to the above. 6. The indicator for high-pressure sterilization according to claim ⁵ , wherein at least 105 indicator bacteria are supported per indicator.