JP6796830B2 - Method for quantifying the degree of microbial damage - Google Patents

Description

The present invention relates to a method for quantifying the degree of microbial damage using a genetic technique.

Various microbial controls (including sterilization) such as heating and pH adjustment are performed on microorganisms, especially harmful microorganisms, in order to reduce the risk. At that time, it has been pointed out that if the microbial control treatment is insufficient, the microorganisms may survive as stressed damaged bacteria. When such damaged bacteria contaminate products such as foods, it is presumed that they behave differently from healthy bacteria, and in order to evaluate the safety and storage stability of the product, the behavior of the damaged bacteria is considered. It is necessary to do.

The prior art of Patent Document 1 below describes a method of predicting the growth of the number of microorganisms in foods and drinks in consideration of the extension time of the induction period of damaged bacteria with respect to healthy bacteria, and is a digital microscope type bacterial inspection apparatus. It has been shown that the time required for damage recovery of damaged bacteria is determined by the colony counting method using.

Japanese Patent No. 5456219

When a product such as a food product contaminated with microorganisms is subjected to a microbial control treatment such as heating or pH adjustment, it is necessary to consider the deterioration of the quality of the product itself due to the microbial control treatment. For example, in the case of heat treatment of food contaminated with microorganisms, in order to completely kill harmful microorganisms, it is sufficient to take a sufficient heating time at a high heating temperature, but this will impair the flavor and texture of the food itself. Become. That is, there is a trade-off relationship between the degree of microbial control treatment and product quality maintenance, and it is necessary to appropriately determine the degree of microbial control treatment in consideration of both quality maintenance and safety. At this time, if the degree of microbial control treatment and the behavior of the surviving damaged bacteria can be quantified and grasped, the microbial control treatment will be performed when the microbial control treatment is performed to the minimum necessary to ensure quality maintenance. It will be an effective index to determine the safety guaranteed by.

On the other hand, according to the above-mentioned conventional technique, although it is possible to predict the growth of the number of damaged bacteria, the difference in the growth rate of the damaged bacteria depending on the degree of microbial control treatment (for example, heat treatment) can be determined. It cannot be obtained directly and cannot be used as an effective index for determining the safety guaranteed by microbial control treatment.

Further, in the above-mentioned conventional technique, the number of damaged bacteria is measured by a colony counting method using a digital microscope type bacterial inspection device, so that the target bacteria are selected from foods and environmental samples in which many germs and impurities are present. It is difficult to extract only and measure the number of bacteria. Therefore, there is a problem that it is not possible to identify harmful microorganisms such as Salmonella and enterohemorrhagic Escherichia coli O157 and accurately and quantitatively grasp the relationship between the degree of microbial control treatment and the behavior of damaged bacteria. Although it is conceivable to use a selective medium, when the selective medium is used, the damaged bacteria do not form colonies and cannot be detected. Therefore, the damaged bacteria of the specific microorganism cannot be measured by the above-mentioned conventional technique.

An object of the present invention is to deal with such a problem. That is, the present invention quantifies the degree of microbial damage by identifying the target bacterium from a sample such as a food containing various bacteria and impurities, and quantitatively determines the relationship between the degree of microbial control treatment and the behavior of the damaged bacterium. By doing so, when the minimum necessary microbial control treatment is performed to ensure the quality maintenance of the product, it is possible to obtain an index to determine the safety guaranteed by the degree of the microbial treatment. It is a subject of the invention.

In order to solve such a problem, the method for quantifying the degree of damage to microorganisms according to the present invention has the following constitution.

A sample in which the target bacterium is present is subjected to a set degree of microbial control treatment, and then cultured, and the number of the target bacterium in the sample having a different culture time is monitored by the target bacterium gene quantification method.
The time required for the number of target bacteria to grow due to the change in culture time to shift to the logarithmic growth phase is determined as the growth delay time, and the maximum specific growth rate is obtained, and the degree of degree set by the growth delay time is determined. The degree of damage to the target bacterium by the microbial control treatment is determined as the first degree of damage, and the degree of damage to the target bacterium by the microbial control treatment set by the maximum specific growth rate is set as the second degree of damage. The degree of damage to the target bacterium by the microbial control treatment obtained and set is set as one dimensional variable with the first degree of damage as one dimensional variable and the second degree of damage as another dimensional variable.
It is safe to consider the deterioration of the quality of the product itself when the degree of the microbial control treatment is set as a multidimensional variable of two or more dimensions and the degree of the microbial control treatment is set to the product in which the target bacterium is present. A method for quantifying the degree of damage of a microorganism, which comprises obtaining the degree of damage that is an index for determining the sex .

According to the present invention having such a configuration, it is possible to identify the target bacteria from a sample such as a food containing various bacteria and impurities and quantify the degree of damage of the microorganisms. By obtaining the degree of microbial damage for each degree of microbial control treatment, the relationship between the degree of microbial control treatment and the behavior of damaged bacteria can be quantitatively grasped. In addition, the degree of damage for each type of various microbial control treatments can be compared (for example, the relationship between the degree of damage at the heating temperature T and the degree of damage at the salt concentration N%). As a result, when the microbial control treatment is performed to the minimum necessary to ensure the quality maintenance of the product, it is possible to obtain an index for determining the safety guaranteed by the microbial control treatment.

It is explanatory drawing which showed the flow of the damage degree quantification method of the microorganism which concerns on embodiment of this invention. It is explanatory drawing which showed the growth curve example of a damaged bacterium. It is explanatory drawing which showed the flow of the damage degree quantification method of the microorganism which concerns on embodiment of this invention. It is explanatory drawing which showed the calculation example of the growth delay time. It is explanatory drawing which showed the damage degree profile (one-dimensional) by the growth delay time. It is explanatory drawing which showed the damage degree profile (two-dimensional) by the growth delay time. It is explanatory drawing which showed the quantification example of the degree of damage by the maximum specific growth rate.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows a flow of a method for quantifying the degree of damage of microorganisms according to an embodiment of the present invention. In this method, first, a sample containing a microorganism is sampled (FIG. 1: S1). The sample here has a predetermined number of target bacteria to be evaluated for the degree of damage, whereas the sample has a set degree of microbial control treatment (for example, heat treatment, pH adjustment, NaCl). Processing, etc.) is performed (Fig. 1: S2). The degree of microbial control treatment is a treatment parameter that affects the severity of damage. For example, in the case of heat treatment, the length of the heating time and the height of the heating temperature correspond to this.

Specimens that have undergone microbial control treatment such as heat treatment are cultured as they are or in an appropriate preculture medium (FIG. 1: S3). When culturing is started, the number of target bacterium is monitored by the target bacterium gene quantification method for samples having different culture times. Then, from the monitoring result, the number of target bacteria grown after the microbial control treatment is determined for each culture time (FIG. 1: S4). The culture temperature at this time may be any temperature suitable for recovery from damage of the target microorganism.

In the present invention, the number of target bacteria is monitored by a gene quantification method. That is, the number of bacteria is monitored by extracting nucleic acid from the sample being cultured and quantifying the number of genes of the target bacterium. As the gene quantification method used here, various methods such as a method based on the real-time PCR method and an emulsion PCR method can be adopted. Further, the nucleic acid extraction method may be any ordinary nucleic acid extraction method that can be utilized in the gene method, including the extraction method using an existing column.

Specifically, all nucleic acids are extracted from the sample at regular intervals during the continuation of culture, and the gene amount of the target bacterium is measured by a quantitative PCR method or the like. Then, the number of bacteria is obtained from the correlation between the amount of gene and the number of bacteria. As a result, as shown in FIG. 2, a growth curve of the target bacterium that grows with the lapse of the culture time is obtained (FIG. 1: S5).

In this way, when a growth curve is obtained after the target bacterium is damaged by the microbial control treatment, as shown in FIG. 2, the growth is delayed as the time until the number of the target bacterium shifts to the logarithmic growth phase. The time can be calculated (Fig. 1: S6). The growth delay time can be calculated as a parameter by using conventional microbial growth curve fitting software such as DMfit, an algorithm, or a calculation formula based on ordinary microbiological knowledge. Since the damage caused by the microbial control treatment to the target bacteria can be macroscopically grasped as the degree of influence on the subsequent growth, it can be said that the growth delay time quantitatively represents the degree of damage caused by the microbial control treatment.

According to such a process, by measuring the number of target bacteria for each culture time by a gene quantification method, even a sample contaminated with various bacteria and impurities is a target necessary for evaluating the safety. The growth delay time can be determined by focusing on the fungus (Salmonella, enterohemorrhagic Escherichia coli O157, etc.). The short growth delay time obtained in this way means that the degree of damage due to the degree of the added microbial control treatment is low, and the storage period in a state where the growth of the target bacterium is suppressed is shortened. Quantitatively, a long growth delay time means that the degree of damage due to the degree of microbial control treatment applied is high, and the storage period in a state where the growth of the target bacterium is suppressed can be long. This is shown quantitatively.

In order to quantitatively grasp the relationship between the degree of microbial control treatment and the degree of damage, as shown in FIG. 3, a step of changing the degree of microbial control treatment in steps S1 to S6 described above (FIG. 3: S7). ) Is added, and the above-mentioned steps S1 to S6 are repeated for each degree of the changed microbial control treatment. According to this, when the degree of the microbial control treatment is changed, the degree of damage caused by the change can be quantitatively grasped by the growth delay time.

FIG. 4 shows an example in which heat treatment is performed as the microbial control treatment and the heating time is changed as the degree of the microbial control treatment, and the growth delay time for each heating time is obtained from the flow shown in FIG.

In this example, first, a sample inoculated with a target bacterium having a set number of bacteria (initial number of bacteria) is cultured without heat damage, and the number of target bacterium genes is quantified to measure the number of target bacterium. Then, the culture time until the target bacterium shifts to the logarithmic growth phase is obtained, and this is set as the reference growth time T0. In the embodiment, a normal target bacteria by culturing the inoculated sample so that 10 ^4, and reference growth time T0 culture time to transition to the logarithmic growth phase.

Next, the sample inoculated with the target bacteria of the set number of bacteria (initial number of bacteria) is heat-treated for a heating time of F1 (12 min in the example), and then cultured to quantify the number of target bacteria genes. By doing so, the number of target bacteria is measured, the culture time until the number of target bacteria reaches the logarithmic growth phase is determined, and this culture time is defined as the growth delay time T1.

In the example shown in FIG. 4, the heating time (degree of microbial control treatment) is changed from the first stage to the fourth stage (heating time F1, F2, F3, F4), and the growth delay time T1, T2 at each stage. , T3, T4 are required. The heating time can be shown, for example, as F2 = F1 × 2, F3 = F1 × 3, F4 = F1 × 4.

In addition, in calculating the growth delay time, it is also possible to obtain the maximum specific growth rate by using conventional microbial growth curve fitting software such as DMfit, algorithms, and calculation formulas based on ordinary microbiological knowledge. It is also possible to more accurately correct the growth delay time obtained by measuring the values according to the existing literature (Journal of Fermentation and Bioengineering 67: 132-134. 1989), if necessary.

In this way, by obtaining the reference growth time T0, the degree of damage due to the heating time of each stage is quantified as standard by the difference between the reference growth time T0 and the growth delay times T1 to T4 obtained in each stage. can do. That is, when the degree of damage k1 to k4 due to the heating time of each stage is expressed by the difference between the growth delay time T1 to T4 and the reference growth time T0, k1 = T1-T0, k2 = T2-T0, k3 = T3-T0, k4 = T4-T0.

As shown in FIG. 4, the number of target bacteria in the heat-treated sample decreases depending on the degree of heating time, but remains as damaged bacteria, and the time of the subsequent storage time (culture time). The initial number of bacteria is reached again by proliferation over time. The growth process during that period is considered to correspond to the recovery of the damaged state, and the storage time (culture time) required for the recovery of the damaged state has a different value depending on the length of the heating time (the amount of damage applied). Therefore, the growth delay times T1 to T4 or the degree of damage k1 to k4 described above quantitatively represent the degree of damage of the damaged bacteria according to the length of the heating time.

FIG. 5 shows an example profile of the degree of damage to a specific degree of microbial control treatment. The horizontal axis in the figure shows the heating time (degree of microbial control treatment), and the vertical axis shows the degree of damage (growth delay time). In the figure, when the heating time is excessively increased, the growth delay time cannot be obtained (a dead state in which no growth is observed after heating), but the heating time up to that point is the degree of damage. It becomes the evaluation range. In the damage degree evaluation range, when the heating time (degree of microbial control treatment) is set, it is possible to quantitatively evaluate how much the damage degree is due to it.

Such a damage profile shows a unique curve depending on the type of target bacterium and the method of microbial control treatment to be added. This profile defines the safe storage period after treatment in relation to the degree of damage (growth delay time) when the degree of microbial control treatment is suppressed to some extent in order to maintain the quality of foods and the like. It becomes an index such as whether it should be done.

FIG. 6 shows a profile of the degree of damage when the degree of microbial control treatment is a multidimensional variable. In the illustrated example, the microbial control treatment is a heat treatment, and the degree thereof is a two-dimensional variable of a heating time (Heating time (sec)) and a heating temperature (Temperature (° C.)). According to such a damage degree profile, it is possible to visualize the relationship between the setting of each variable and the damage degree (growth delay time) for the heat treatment that combines the heating time and the heating temperature, and the quality of foods and the like. It is an effective indicator of how to adjust the microbial control treatment considering both safety and safety.

Further, as described above, the maximum specific growth rate can be determined from the growth curve (FIGS. 1, FIG. 2: S5) obtained by monitoring by the target bacterium gene quantification method using existing software such as DMfit. .. This maximum specific growth rate, together with the above-mentioned growth delay time, is an effective parameter for quantifying the degree of damage depending on the degree of microbial control treatment.

FIG. 7 shows an example in which enterohemorrhagic Escherichia coli O157, which is a target bacterium, is exposed to a medium of pH 3.0 as a microbial control treatment, and is calculated from the growth curve after the microbial control treatment. This is an example of determining the maximum specific growth rate to be achieved. In the graph shown, the horizontal axis represents the time of exposure to the medium of pH 3.0 (exposure time: degree of microbial control treatment), and the vertical axis represents the maximum specific growth rate. Here, a large maximum specific growth rate means a low degree of damage, and a small maximum specific growth side road means a high degree of damage. In the illustrated example, the maximum specific growth rate becomes zero when the exposure time (Hr) exceeds 40 hours, and the target bacterium, enterohemorrhagic Escherichia coli O157, becomes dead, but when the exposure time exceeds 36 hours. The maximum specific growth rate drops sharply. As described above, the maximum specific growth rate, together with the above-mentioned growth delay time, is an effective index for quantifying the degree of damage.

As described above, according to the present invention, it is possible to identify a target bacterium from a sample such as a food containing various germs and impurities, and quantitatively grasp the degree of damage to the target bacterium with respect to the degree of microbial control treatment. .. Then, since it is possible to quantitatively grasp the recovery of damaged bacteria when a predetermined microbial control treatment is applied, the minimum degree of microbial control treatment required to ensure safety and the subsequent storage period can be determined. , Can be determined in detail based on objective data.

Claims (5)

A sample in which the target bacterium is present is subjected to a set degree of microbial control treatment, and then cultured, and the number of the target bacterium in the sample having a different culture time is monitored by the target bacterium gene quantification method.
The time required for the number of target bacteria to grow due to the change in culture time to shift to the logarithmic growth phase is determined as the growth delay time, and the maximum specific growth rate is obtained, and the degree of degree set by the growth delay time is determined. The degree of damage to the target bacterium by the microbial control treatment is determined as the first degree of damage, and the degree of damage to the target bacterium by the microbial control treatment set by the maximum specific growth rate is set as the second degree of damage. The degree of damage to the target bacterium by the determined and set degree of the microbial control treatment is defined as the first degree of damage as one dimensional variable and the second degree of damage as another dimensional variable.
It is safe to consider the deterioration of the quality of the product itself when the degree of the microbial control treatment is set as a multidimensional variable of two or more dimensions and the degree of the microbial control treatment is set to the product in which the target bacterium is present. A method for quantifying the degree of damage of a microorganism, which comprises obtaining the degree of damage that is an index for determining the sex . The first aspect of claim 1 is characterized in that the degree of the microbial control treatment is changed, the degree of damage is obtained for each degree of the microbial control treatment, and the profile of the degree of damage with respect to the degree of the microbial control treatment is obtained. A method for quantifying the degree of damage to microorganisms. The method for quantifying the degree of microbial damage according to claim 1 or 2, wherein the microbial control treatment is a heat treatment, and the degree of the microbial control treatment is a heating time. The method for quantifying the degree of microbial damage according to claim 1 or 2, wherein the microbial control treatment is a heat treatment, and the degree of the microbial control treatment is a two-dimensional variable of a heating time and a heating temperature. The culture time until the number of target bacteria reaches the logarithmic growth phase after culturing the sample not subjected to the microbial control treatment is set as the reference growth time.
The method for quantifying the degree of damage of a microorganism according to any one of claims 1 to 4 , wherein the degree of damage is determined by the difference between the reference growth time and the growth delay time.

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