JP2016195575A - Method for preparing microorganism liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To unify bacterial liquid in sample liquid for inoculation.SOLUTION: There is provided a method for preparing microorganism liquid that includes the steps of: (1) preparing microorganism liquid containing microorganism, nonionic surfactant acceptable as a food additive selected from the group consisting of glycerol fatty acid ester, sorbitan fatty acid ester, and sucrose fatty acid ester, and polymer acceptable as food or food additive; and (2) irradiating the microorganism liquid with ultrasonic wave.SELECTED DRAWING: None

Description

  The present invention relates to a method for preparing a microbial solution that can be suitably used for verifying the sterilization effect of a plastic container before the beverage is filled into the plastic container when a beverage in a plastic container is produced. The present invention also relates to a method of applying a microbial fluid produced by the method of the present invention to a plastic container. The present invention also relates to a method for sterilizing a plastic container to which a microbial solution is applied according to the method of the present invention. The present invention also relates to a method for producing a plastic container-containing beverage in which a plastic container sterilized by the method of the present invention is filled with a beverage.

When microorganisms are mixed in food and drink, it increases exponentially when conditions such as nutrient source, temperature, water activity, and presence / absence of oxygen are met. As a result, food and drink are quickly degraded due to contamination with microorganisms. When people ingest foods and drinks that have been degraded by microorganisms, they feel uncomfortable due to off-flavors. In addition, it may cause physical harm such as food poisoning.
There are a plurality of routes through which microorganisms enter foods and drinks. One of the routes is a container for storing food and drink. Since this container is in direct contact with food and drink, it causes deterioration due to contamination with microorganisms and subsequent growth. Therefore, it is required that the inner surface of the container for storing food and drink that comes into contact with the food and drink is sufficiently sanitary.
There are several methods for ensuring the hygiene of the inner surface of the container. One of them is a method in which the inner surface of the container is sterilized in advance before storing food or drink. For example, a technique called aseptic for a beverage in a plastic bottle container corresponds to this.
By this technique, a plastic container for storing food, called a preform (a plastic container before blow molding, which may be referred to as PF hereinafter) can be sterilized. This PF is designed to have a small volume. Therefore, a sufficient amount of beverage cannot be stored as it is. This PF is used after expanding the volume of the container in the blow molding process to form a plastic container. When food and drink are stored in a plastic container molded from PF, microorganisms remaining on the inner surface of the plastic container must be reliably removed in advance by sterilization.
Here, the operation of sterilizing the microorganisms remaining on the inner surface of the plastic container is very important in preventing the deterioration of food and drink due to the growth of the microorganisms. Therefore, establishing a technique for strictly setting sterilization conditions for microorganisms remaining on the inner surface of the plastic container is the key to properly operating the entire aseptic technique.
In order to experimentally verify the bactericidal effect on the inner surface of the plastic container, first, a bacterial solution serving as an indicator is prepared, and this is uniformly inoculated on the inner surface of the PF or the inner surface of the plastic container after molding (hereinafter referred to as “injection”). , Referred to as “inoculation”). In consideration of efficiency such as ensuring the uniformity of microorganisms, work speed, and work place, it is preferable to use PF.
In Patent Document 1, when evaluating the sterilization effect by various sterilization devices, it is possible to create a subject assuming a state in which a sterilization process is actually performed, and an experimental model in accordance with the actual situation Discloses a sterilization rate measuring method, a fungus planting / recovery rate measuring method, a fungus planting system, and a sterilization rate measuring system that can evaluate the sterilization effect.
Patent Document 2 discloses a liquid inoculum inoculation method and an inoculation apparatus capable of performing stable inoculum filling while keeping the supply amount of the inoculum substantially constant.

JP 2011-130672 A JP 2003-134934 JP

1) Problems related to the homogeneity of microorganisms in the microorganism liquid Some microorganisms have high hydrophobicity. For this reason, when a solution in which microorganisms are dispersed (hereinafter referred to as “microorganism liquid”) is prepared, an event occurs in which the cells aggregate and do not uniformly disperse. For this reason, there was doubt about the validity of the verification result of the bactericidal effect. This is because, as a technique for experimentally verifying the bactericidal effect on the inner surface of a conventional plastic container, a method for ensuring the uniformity of microorganisms in the microorganism liquid is not provided. In the present invention, when a beverage in a plastic container is produced, before the beverage is filled in the plastic container, the microorganisms in the microorganism liquid that can be suitably used for verifying the sterilizing effect of the plastic container are made uniform. An object of the present invention is to provide a method for preparing a microbial solution in which microorganisms are uniformly dispersed.
2) Problems related to uniformity of spraying of microbial liquid Conventionally, as a method of spraying the microbial liquid uniformly, it has been usually performed manually using a method similar to a sprayer used for home use. For this reason, adjustments such as pressure and flow rate when spraying are inaccurate. Because of this, the amount of microbial liquid sprayed varies depending on the examiner, and there has been doubt about the validity of the verification result of the bactericidal effect. This is because there is no provision for a method of spraying the microbial liquid uniformly on the entire inner wall surface of the PF. Moreover, it is because it is not provided about the method of fixing a microbe uniformly on the whole PF inner wall surface. The present invention relates to these problems, and sprays the microbial liquid produced by the method of the present invention uniformly over the entire inner wall surface of the PF, and further uniformly distributes the microbial fluid produced by the method of the present invention over the entire inner wall surface of the PF. It is an object to provide a highly versatile inoculation method for experimentally verifying the bactericidal effect on the inner surface of a plastic container.
Another object of the present invention is to provide a method for producing a sterilized plastic container.
Another object of the present invention is to provide a method for producing a beverage in a plastic container.

As a result of intensive studies by the present inventors, it has been found that a uniform microbial solution can be obtained by irradiating ultrasonic waves while containing a specific nonionic surfactant and a specific polymer. The present invention has been made based on such knowledge. That is, the present invention provides the following method:
1. (1) a microorganism; a nonionic surfactant acceptable as a food additive selected from the group consisting of glycerin fatty acid ester, sorbitan fatty acid ester and sucrose fatty acid ester; and a polymer acceptable as food or food additive Preparing a microbial fluid containing
(2) a step of irradiating the microorganism liquid with ultrasonic waves,
A method for preparing a microbial fluid, comprising:
2. 2. The method according to 1 above, wherein the nonionic surfactant is a sorbitan fatty acid ester.
3. The method of claim 1, wherein the nonionic surfactant is polysorbate 80.
4). 4. The method according to any one of 1 to 3, wherein the content of the nonionic surfactant is 0.05 to 0.1% by mass based on the total mass of the microbial solution.
5. The method according to any one of 1 to 4, wherein the polymer is an adhesive polysaccharide.
6). 6. The method according to any one of 1 to 5, wherein the polymer is agar.
7). The method according to any one of 1 to 6, wherein the content of the polymer is 0.05 to 0.1% by mass based on the total mass of the microorganism solution.
8). 8. The method according to any one of 1 to 7, wherein the number of microorganisms contained in the microorganism solution is 1 × 10 ⁵ to 1 × 10 ⁸ / mL.
9. 9. The method according to any one of 1 to 8, wherein the microorganism is selected from the group consisting of the genus Aspergillus and the genus Bacillus.
10. 10. The method according to any one of 1 to 9, wherein the microorganism is selected from Aspergillus niger and Bacillus subtilis.
11. A step of storing the microbial liquid prepared by the method according to any one of claims 1 to 10, further comprising a microbial liquid stirring device, and preparing a spraying device capable of spraying while stirring the microbial liquid.
Spraying the microbial liquid stored in the spraying device so that the amount sprayed on the inner wall is 20 to 50 μL with respect to the inner wall of the blow-moldable plastic container having a volume of 10 to 100 cm ³ ;
Applying a microbial fluid to a plastic container.
12 12. The method according to 11 above, wherein the spraying device comprises a nozzle and a pressure generating means, and the pressure by the pressure generating means is 0.05 to 0.1 MPa.
13. 13. The method according to 11 or 12 above, wherein the spraying device is installed so that a distance from the tip of the opening of the plastic container to the nozzle is -10 to 0 mm.
14 14. The method according to any one of 11 to 13, wherein a discharge time per 100 μL of the microorganism liquid is 0.1 to 1 second.
15. The step of drying the inner surface of a blow-moldable plastic container, wherein the microorganism liquid is applied to the inner wall by the method according to any one of 11 to 14 above,
Sterilizing the dried plastic container,
A method for producing a sterilized plastic container.
16. A step of obtaining a blown plastic container by heating the plastic container sterilized by the method of 15.
Filling the blown plastic container with a beverage,
A method for producing a beverage in a plastic container, comprising:

  According to the present invention, it is possible to prepare a uniform microbial solution capable of uniformly inoculating the entire inner wall of a PF container for experimentally verifying the sterilizing effect on the inner surface of the plastic container. In addition, the microbial solution can be inoculated uniformly over the entire inner wall of the PF. From these things, it becomes easy to determine whether or not the microorganisms present on the inner surface of the container have been sterilized.

FIG. 1a is an overall view of the bacterial solution spray apparatus used in the examples. FIG. 1b is an enlarged view around the preform of the device. FIG. 2 is a photograph showing that no bacteria were generated after washing the bacterial solution spray device used in the examples.

[Method of preparing microbial fluid]
The present invention includes (1) a microorganism; and a nonionic surfactant acceptable as a food additive selected from the group consisting of glycerin fatty acid ester, sorbitan fatty acid ester and sucrose fatty acid ester; and acceptable as food or food additive A step of preparing a microbial solution containing a macromolecule,
(2) a step of irradiating the microorganism liquid with ultrasonic waves,
The present invention relates to a method for preparing a microbial fluid.

  Examples of microorganisms that can be used in the step (1) include bacteria, molds, and yeasts. It can be selected from the genera Aspergillus, Bacillus, Clostridium, Saccharomyces, and Chaetomium. Examples of the Aspergillus genus include Aspergillus niger, A. flavus, A. nidulans, and A. oryzae. Examples of the genus Bacillus include Bacillus subtilis, B. anthracis, and Bacillus coagulans. Examples of the genus Clostridium include C. botulinum, C. perfringens, and tetanus (C. tetani). Examples of the genus Saccharomyces include budding yeast (Saccharomyces cerevisiae). Examples of the genus Chaetomium include C. globosum. From the viewpoint of ease of handling and safety of the microorganism, the microorganism is preferably selected from Aspergillus niger and Bacillus subtilis.

The nonionic surfactant that can be used in the present invention is selected from the group consisting of sorbitan fatty acid esters and sucrose fatty acid esters, and is a nonionic surfactant that is acceptable as a food additive.
Examples of sorbitan fatty acid esters include mono-, di- or tri-esters of sorbitan and saturated or unsaturated fatty acids having 12 to 18 carbon atoms. Specific examples include sorbitan lauric acid monoester, sorbitan palmitic acid monoester, sorbitan stearic acid monoester, sorbitan stearic acid triester, sorbitan oleic acid monoester, sorbitan oleic acid triester, and the like. An ethylene oxide adduct of sorbitan fatty acid ester can also be used. Those having an average added mole number of ethylene oxide of 20 to 90 mol can be suitably used. As the sorbitan fatty acid ester used in the present invention, polysorbate 80 is preferable.

Examples of the sucrose fatty acid ester include mono-, di-, and tri-esters of sucrose and a saturated or unsaturated fatty acid having 12 to 18 carbon atoms. Specific examples include sucrose lauric acid monoester, sucrose palmitic acid monoester, sucrose stearic acid monoester, sucrose stearic acid triester, sucrose oleic acid monoester, sucrose oleic acid triester, etc. .
It is preferable that the nonionic surfactant is a sorbitan fatty acid ester. More preferred is polysorbate 80.
The content of the nonionic surfactant is preferably 0.05 to 0.1% by mass, more preferably 0.07 to 0.08% by mass, based on the total mass of the microbial fluid. . When the content of the nonionic surfactant is in such a range, it is preferable because cell aggregation in the microorganism liquid can be prevented without damaging the microorganism.

The polymer that can be used in the present invention is not particularly limited as long as it is acceptable as a food or food additive. The weight average molecular weight of the polymer is preferably 30,000 to 1,000,000, and more preferably 50,000 to 400,000. Within such a range, the viscosity of the microbial solution can be increased without impeding the dispersion of the bacterial cells in the microbial solution, and the colonization of the microorganisms on the inner wall surface of the PF can be promoted.
Examples of the polymer include adhesive polysaccharides, agar, and carboxymethyl cellulose. The polymer is preferably agar.
The content of the polymer is preferably 0.05 to 0.1% by mass based on the total mass of the microbial solution.

The microorganism, the nonionic surfactant, and the polymer are prepared, and these are put into physiological saline. Ultrasound is irradiated to disperse the microorganisms in physiological saline. Those skilled in the art will be able to set the conditions of ultrasonic waves to be irradiated as appropriate. For example, a microbial solution can be prepared by irradiating 100 mL of physiological saline solution with ultrasonic waves at a frequency of 15 to 50 kHz for 60 to 600 seconds. Whether the microorganisms are uniformly dispersed can be confirmed by microscopic observation.
Depending on the microorganism used, such as mold, yeast, bacteria, etc., hyphae and viscous substances (polysaccharides, etc.) possessed by these microorganisms may be removed before irradiation with ultrasonic waves. The removal can be performed by a known means such as filtration. In addition, by performing homogenization before removal, the microorganisms can be more uniformly dispersed.
The number of microorganisms contained in the microorganism solution is preferably 1 × 10 ⁵ to 1 × 10 ⁸ cells / mL, and more preferably 1 × 10 ⁶ to 1 × 10 ⁷ cells / mL. When the number of microorganisms is in such a range, the cell number in the optimum range for calculating the sterilizing effect of the PF inner wall surface sterilizing device without excessive aggregation of the bacterial cells in the microbial fluid. Since it can be made to adhere to, it is preferable.

[Method of applying microbial fluid to plastic container]
The present invention also contains a microbial liquid prepared by the method of the present invention, further comprising a microbial liquid stirring device, and a step of preparing a spraying device capable of spraying while stirring the microbial liquid.
Spraying the microbial liquid stored in the spraying device so that the amount sprayed on the inner wall is 20 to 50 μL with respect to the inner wall of the blow-moldable plastic container having a volume of 10 to 100 cm ³ ;
The present invention relates to a method for applying a microbial fluid to a plastic container.

In order to suppress the aggregation of microorganisms in the microorganism liquid and keep it uniform, it is preferable to have a stirring device for the microorganism liquid, and the stirring means for the microorganism liquid is not particularly limited, but stirring is performed using a stirring blade. It can also be performed by blowing air (bubble) or applying vibration. Stirring may be performed continuously or intermittently during the spraying operation. Stirring conditions such as the rotational speed and size of the stirring blade, the number of blades, the distance from the tip of the blade to the inner wall, the blowing speed and amount of air, the tool that gives vibration, the magnitude of vibration, etc. are not particularly limited. Those skilled in the art can appropriately set.
Moreover, since there is a possibility that the amount of sprayed bacteria varies between workers when inoculating the preform, it is preferable that the spraying device used in the present invention can be controlled by a minute amount of fine pressure. The spray device used in the present invention is preferably small. Specifically, it is preferable that the height from the nozzle tip of the spraying device to the uppermost part of the microorganism liquid storage container is about 30 cm because workability is good.

The preform used in the present invention has an average inner diameter of 20 to 30 mm and a length of 50 to 150 mm. The preform is not a cylinder, but has one or more constrictions on the side surface, and the portion that becomes the bottom surface after molding is hemispherical. The height of the preform refers to the length from the apex of the hemisphere to the point that intersects the preform mouth end in the vertical direction.
The material of the container used in the present invention can be molded from a preform and is not particularly limited as long as the effects of the present invention are obtained, but is preferably polypropylene, polyethylene, or polyethylene terephthalate, more preferably polyethylene terephthalate. is there.
The amount sprayed on the inner wall of the preform is an amount of 10 to 100 μL with respect to a volume of 10 to 100 cm ³ . It is preferably 20 to 50 μL. It is preferable that the spray amount be in such a range because the microbial liquid sprayed on the PF inner wall can be fixed to the bottom surface of the PF inner wall without agglomeration and dripping.

The spraying device further includes a nozzle and a pressure generating means, and the pressure by the pressure generating means is preferably 0.05 to 0.1 MPa.
As a pressure generating means, aseptic gas can be used. Aseptic gas can be obtained by passing high-purity air, nitrogen gas, or the like through a filter.
The spraying device may be installed so that the distance from the tip of the opening of the plastic container to the nozzle is preferably −10 to 0 mm, more preferably −10 to −5 mm. It is preferable that the distance from the tip of the container opening to the nozzle is in such a range because the microbial solution can be fixed to the bottom surface of the PF inner wall without excessively scattering around the PF.
The discharge time per 100 μL of the microorganism solution is preferably 0.1 to 1 second, and more preferably 0.5 to 1 second. It is preferable that the discharge time be in such a range because the microbial liquid can be uniformly fixed on the inner wall of the PF while performing the inoculation work quickly.

[Sterilization method]
The present invention also includes a step of drying the inner surface of a blow-moldable plastic container, wherein the microbial liquid is applied to the inner wall by a method of applying the microbial liquid to a plastic container.
And a step of sterilizing the dried plastic container.

Drying is preferably performed by leaving the preform at room temperature without heating. Thereby, it is possible to prevent the microorganisms applied to the inner wall of the preform from being killed by heat at the time of blow molding.
Sterilization can be performed by applying hydrogen peroxide to the dried plastic container. The density | concentration of hydrogen peroxide is a density | concentration normally used as a disinfectant, and is 2.5-3.5 w / v% specifically ,. A higher concentration may be used.

[Beverage filling method]
The present invention also includes a step of obtaining a blown plastic container by heating the plastic container sterilized by the sterilization method,
Filling the blown plastic container with a beverage,
The present invention relates to a beverage filling method.
Those skilled in the art will be able to appropriately set the conditions and means for blow molding, and the conditions and means for filling the blow-molded plastic container with the beverage.

1. Preparation of Microbial Solution (1) A. niger was inoculated into potato dextrose agar medium (PDA medium) and cultured at 25 ° C. for 7 days.
(2) A dispersion was prepared by adding 0.07 w / w% of Tween 80 and 0.07 w / w% of agar (weight average molecular weight 116,000) to physiological saline, and after culturing for 7 days using this dispersion A.niger spores were scraped off with a large stick.
(3) Mycelia were removed by glass homogenization and Kimwipe filtration.
(4) The above microorganism solution was treated with an ultrasonic cleaning device (30 kHz) for 10 minutes to disperse A. niger spores.
(5) The dispersibility of A. niger spores was confirmed by microscopic observation.

2. Application of microbial solution to inner wall of preform (1) 100 preforms made of polyethylene terephthalate were prepared.
(2) The spraying device used was an improved version of the Saneitec Co., Ltd. two-fluid nozzle SV91 type.
Specifically, the preform 1 was attached to the deformed joint clip in the form of a neck grip, and the spray nozzle 2 and the fluid pressure adjusting valve were installed using a clamp. They were installed using a square muff and a stand while keeping balance so that each member was vertical and parallel. Safety measures and risk of process contamination were reduced by covering the entire device with a simple clean booth (with negative pressure / sterilization device) 3 so that the bacterial solution would not splash outside. Further, pure air alpha gas 2 (Air Liquide) was used as the supply air, and a pore size 0.003 μm in-line filter (Entegris) was attached immediately after the regulator 4. A general view is shown in FIG.
(3) The microbial solution prepared above was sprayed into the preform. The spray solution was stirred with a stirring blade throughout the test. The pressure was applied using sterile air.
(4) Various items shown in the following table were changed to determine whether or not the entire inner wall of the preform was sprayed uniformly.
(5) The determination was made based on the following criteria by visual observation. In addition, since PF is transparent, it is possible to easily identify the state of adhesion of the microbial fluid to the inner surface by visual observation. From this, the uniformity of spraying on the inner wall of the PF was evaluated by visual observation based on the following method.

  The results are shown in the table below. In the step of preparing the microbial solution, if Tween 80 or agar is not used, the microorganisms in the microbial solution aggregate, and if only Tween 80 is used, the microbial solution is drained in the preform. In addition, when the microbial solution was not irradiated with ultrasonic waves, the dispersibility of the microorganisms was worse than when the ultrasonic waves were irradiated.

  The influence of spraying conditions on the spray amount was evaluated by washing out the bacteria in the preform, measuring the number of recovered bacteria, and comparing it with the calculated number of attached bacteria.

  The recovery rate of the bacteria was higher when the spray amount was 25 μL than 35 μL.

In addition, the following washing | cleaning operation | work was performed at the time of switching of bacteria amount levels, and the spray apparatus was sterilized.
(1) The part (SUS member) which was autoclaved after ultrasonic cleaning was replaced.
(2) Packings were sterilized with alcohol and replaced.
(3) The liquid stirring blade was flame sterilized with a burner.
(4) After applying the special grease, various parts were mounted.
(5) The liquid agent pressure was set to 0.2 MPa, and the valve stroke was set to 15 scales open.
(6) The spray pressure was set to 0 Mpa (in some cases, only the spray pipe was removed).
(7) A cleaning cylinder was attached, and 50 ml of sterilized water was allowed to flow through the liquid agent passage in the apparatus.
(8) 50 ml of 70% ethanol was allowed to flow through the liquid agent passage in the apparatus.
(9) Bactericidal air was continuously passed through the liquid agent flow path in the apparatus for 3 minutes or more.

The A.niger spore solution (1 × 10 ⁷ cells / ml) was poured into the spray device, and then the above washing operation was performed. After washing, about 50 ml of sterilized water was poured into the spray device, and sterilized water that passed through the device was collected. The whole amount was filtered through a 0.45 μm filter, and cultured in PDA medium at 25 ° C. for 72 hours. As a result, generation of bacteria was not observed (FIG. 2).

1 Preform 2 Spray nozzle 3 Simple clean booth (with negative pressure and sterilizer)
4 Regulator 5 Liquid cylinder with stirring blades

Claims (16)

(1) a microorganism; a nonionic surfactant acceptable as a food additive selected from the group consisting of glycerin fatty acid ester, sorbitan fatty acid ester and sucrose fatty acid ester; and a polymer acceptable as food or food additive Preparing a microbial fluid containing
(2) a step of irradiating the microorganism liquid with ultrasonic waves,
A method for preparing a microbial fluid, comprising:   The method of claim 1, wherein the nonionic surfactant is a sorbitan fatty acid ester.   The method of claim 1, wherein the nonionic surfactant is polysorbate 80.   The method according to any one of claims 1 to 3, wherein the content of the nonionic surfactant is 0.05 to 0.1% by mass based on the total mass of the microorganism liquid.   The method according to claim 1, wherein the polymer is an adhesive polysaccharide.   The method according to claim 1, wherein the polymer is agar.   The method according to any one of claims 1 to 6, wherein the content of the polymer is 0.05 to 0.1% by mass based on the total mass of the microorganism solution. The method according to claim 1, wherein the number of microorganisms contained in the microorganism solution is 1 × 10 ⁵ to 1 × 10 ⁸ cells / mL.   The method according to any one of claims 1 to 8, wherein the microorganism is selected from the group consisting of the genus Aspergillus and the genus Bacillus.   The method according to any one of claims 1 to 9, wherein the microorganism is selected from Aspergillus niger and Bacillus subtilis. A step of storing the microbial liquid prepared by the method according to any one of claims 1 to 10, further comprising a microbial liquid stirring device, and preparing a spraying device capable of spraying while stirring the microbial liquid.
Spraying the microbial liquid stored in the spraying device so that the amount sprayed on the inner wall is 20 to 50 μL with respect to the inner wall of the blow-moldable plastic container having a volume of 10 to 100 cm ³ ;
Applying a microbial fluid to a plastic container.   The method according to claim 11, wherein the spraying device includes a nozzle and pressure generation means, and the pressure generated by the pressure generation means is 0.05 to 0.1 MPa.   The method of Claim 11 or 12 which installs the said spraying apparatus so that the distance from the opening part front-end | tip of the said plastic container to the said nozzle may be -10-0 mm.   The method according to any one of claims 11 to 13, wherein a discharge time per 100 µL of the microorganism liquid is 0.1 to 1 second. A step of drying the inner surface of a blow-moldable plastic container, wherein the microorganism liquid is applied to the inner wall by the method according to any one of claims 11 to 14.
Sterilizing the dried plastic container,
A method for producing a sterilized plastic container. A step of obtaining a blown plastic container by heating the plastic container sterilized by the method according to claim 15;
Filling the blown plastic container with a beverage,
A method for producing a beverage in a plastic container, comprising: