KR20070004216A - Method for sterilizing microorganisms using supercritical carbon dioxide - Google Patents

Abstract

A method for sterilizing microorganisms with super-critical carbon dioxide is provided to obtain satisfactory result of efficient extinction for microorganisms even when pressure and temperature as processing factors are relatively low in level by employing supercritical carbon dioxide with principle of extracting constitutional elements at low temperature without alteration of pH effect and shape of cells. The method comprises the steps of: entering a sample reactor at a setup temperature above supercritical temperature of 31.1deg.C; introducing liquid carbon dioxide into the reactor through a duct and pressurizing the reactor above supercritical pressure of 73.8 bar through a pump; closing a valve to maintain the desired temperature and pressure for a constant time in order to extinct microorganisms; opening the valve after the constant time, to remove supercritical carbon dioxide and to keep internal condition of the reactor under ordinary pressure; and opening the reactor and give the sample without the microorganisms.

Description

Method for sterilizing microorganisms using supercritical carbon dioxide}

1 shows a microbial treatment process facility using supercritical carbon dioxide.

Figure 2 shows the killing curves and coefficients of microorganisms in the modified Gompertz equation.

Figure 3 shows the effect of the pressure according to the treatment time when supercritical carbon dioxide treatment in Listeria monocytogenes at 40 ℃. The mean value at each treatment time was expressed as Exp (Experimental), and the death curve obtained by calculating it by the Gompertz equation was expressed as Cal (Calculated).

Figure 4 shows the effect of the pressure on the rate of microbial death according to the treatment time when supercritical carbon dioxide treatment in Salmonella typhymurium at 40 ℃.

Figure 5 shows the effect of the pressure on the microbial killing rate with treatment time when treated with supercritical carbon dioxide in Escherichia coli O157: H7 at 40 ℃.

Figure 6 shows the effect of the pressure on the microbial killing rate with treatment time when treated with supercritical carbon dioxide in Escherichia coli (Generic) at 40 ℃.

Figure 7 shows the effect of temperature on the rate of microbial killing with treatment time when supercritical carbon dioxide was treated in Listeria monocytogenes at 100 bar.

FIG. 8 shows the effect of temperature on microbial killing rate with treatment time when Salmonella typhymurium was treated with supercritical carbon dioxide at 100 bar.

Figure 9 shows the effect of temperature on the microbial killing rate with treatment time when treated with supercritical carbon dioxide in Escherichia coli O157: H7 at 100bar .

Figure 10 shows the effect of temperature on the rate of microbial killing with treatment time when treated with supercritical carbon dioxide in Escherichia coli (Generic) at 100bar .

Figure 11 shows the pH value of the suspension according to the treatment time when treated with supercritical carbon dioxide in E. coli (Generic) under various pressure and temperature conditions.

Figure 12 is a comparison of the changes in cell morphology by scanning electron microscopy (Scanning Electron Microscopy) when the supercritical carbon dioxide treatment for Listeria monocytogenes at 35 ℃, 100bar.

Figure 13 is a comparison of the changes in cell morphology when scanning supercritical carbon dioxide in Salmonella typhymurium at 35 ℃, 100bar by scanning electron microscopy.

Figure 14 is a comparison of the changes in cell morphology when the supercritical carbon dioxide treatment of Escherichia coli O157: H7 at 35 ℃, 100bar by scanning electron microscopy.

Figure 15 shows the change in absorbance of the suspension with treatment time when the Listeria monocytogenes was treated with Listeria monocytogenes at 35 ° C. and 100 bar as a ratio after and before treatment.

Figure 16 shows the change in absorbance of the suspension with treatment time when treated with supercritical carbon dioxide in Salmonella typhymurium at 35 ℃, 100bar as a ratio after and before treatment.

Figure 17 shows the change in absorbance of the suspension with treatment time when treated with Escherichia coli O157: H7 supercritical carbon dioxide at 35 ℃, 100bar as a ratio after and before treatment.

The present invention relates to an effective sterilization method of microorganisms using supercritical carbon dioxide, and more particularly, to provide a mathematical model capable of effectively killing major food poisoning microorganisms through supercritical carbon dioxide treatment in an actual sterilization system.

The most commonly used heating process for food sterilization has the problem that it not only destroys the nutrients of the food but also denatures the flavor or texture. Radiation, cold storage, fermentation, pickling, drying, and gas replacement, including heat treatment, have also been used in the past either alone or in combination. However, as new food poisoning microorganisms are discovered and resistance to existing sterilization methods increases, research on new sterilization methods is urgently needed. In addition, as consumers prefer foods that have been processed with a minimum of processing to ensure that they are of higher quality and no nutrients are destroyed, the development of non-heating treatments to supply them is in many ways. In particular, fresh foods, which are in increasing demand in recent years, are virtually impossible to use by heat treatment.

Non-heating methods currently being studied include high hydrostatic pressure, ultra high pressure, pulsed electric field, radiation, and natural antibacterial agents, but they have limitations in terms of installation cost, safety, and treatment target effects. In particular, high hydrostatic pressure or ultra high pressure treatment has been studied as a method of maintaining or improving the quality while obtaining sufficient sterilization effect, but it is difficult to commercialize in the technical aspect of manufacturing a container that can withstand a very high pressure.

On the other hand, carbon dioxide exists in a supercritical state, not liquid or gas, above the critical point of 31.1 ° C near the room temperature and 73.8 bar rather than relatively high pressure. Such supercritical carbon dioxide has been used in various extraction processes because of its high solvent-like properties because of its high diffusivity, low viscosity, and high density, such as liquid. If supercritical carbon dioxide is applied to food sterilization instead of the conventional sterilization method using only high pressure, it is possible to kill microorganisms effectively with less energy because it can simultaneously obtain not only physical effects but also chemical effects.

Therefore, the present inventors used the physicochemical properties of carbon dioxide having supercritical points at relatively low temperature and pressure in order to effectively kill food poisoning microorganisms present in food. In other words, by treating supercritical carbon dioxide in the suspension of food poisoning microorganisms and killing them, the killing trend was analyzed kinetically using a formula that can adequately reflect the killing effect of each microorganism according to the treatment conditions. The present invention has been accomplished by systemizing mechanisms to establish effective minimum processing conditions for the complete killing of foodborne organisms.

Accordingly, an object of the present invention is to effectively kill food poisoning microorganisms by using supercritical carbon dioxide in all foods including liquid foods, and to effectively destroy microorganisms and minimize sensory changes and to produce safe and fresh foods. To provide.

In order to achieve the object of the present invention, the present invention provides a method for sterilizing microorganisms, comprising the following steps in a method for killing microorganisms using supercritical carbon dioxide:

a) placing the sample in a reactor having a temperature above the supercritical point (31.1 ° C.);

b) closing the reactor and introducing liquid carbon dioxide, which has undergone a cooler, into the reactor through a conduit and pressurizing the reactor to a pressure above the supercritical point (73.8 bar) by means of a pump;

c) maintaining the pressure and temperature by closing the valve for the time necessary to completely kill the target microorganism;

d) opening the valve to remove supercritical carbon dioxide when the required time has elapsed and returning the reactor to atmospheric pressure; And

e) opening the reactor to obtain a sample free of live target microorganisms.

In the sterilization method of the present invention, the sample may be a liquid or a solid, and may be directly placed in the reactor or may be placed in a reactor while the lid is in a loosely closed container.

In the sterilization method of the present invention, the target microorganism may be any microorganism, but preferably, it is a food poisoning microorganism such as Listera monocytogenes, Salmonella typhymurium, Escherichia coil O157: H7.

In the sterilization method of the present invention, when the target microorganism is Listeria monocytogenes , which is a major food poisoning microorganism, it is treated with 23-27 minutes at 33-37 ° C. and 95-105 bar supercritical carbon dioxide, or 38-42 ° C., 75-85 bar seconds. Characterized in 13-17 minutes in critical carbon dioxide. The temperature and pressure conditions are effective minimum treatment conditions for the complete killing of the microorganism.

In the sterilization method of the present invention, when the target microorganism is Salmonella typhymurium which is the main food poisoning microorganism, the treatment is performed at 33-37 ° C. and 95-105 bar supercritical carbon dioxide for 18-22 minutes, or at 38-42 ° C. and 75-85 bar seconds. Characterized in 13-17 minutes in critical carbon dioxide. The temperature and pressure conditions are effective minimum treatment conditions for the complete killing of the microorganism.

In the sterilization method of the present invention, when the target microorganism is Escherichia coli O157: H7, which is a major food poisoning microorganism, or Escherichia coli (Generic), which is an indicator microorganism, treatment is performed at 33-37 ° C. and 95-105 bar supercritical carbon dioxide for 28-32 minutes. Or, 38-42 ℃, characterized in that the treatment for 18-22 minutes in supercritical carbon dioxide of 75-85bar. The temperature and pressure conditions are effective minimum treatment conditions for complete killing of the microorganism.

In order to achieve another object of the present invention, the present invention is applied to the modified Gompertz equation of the following formula ⑥ by applying the reduced log level (y-axis) of the microorganism according to the treatment time (x-axis) of the supercritical carbon dioxide to kill the microorganisms The curves were obtained, and coefficients A (maximum killing level) , k _dm (maximum killing rate) , and λ (killing induction time) were obtained through the nonlinear least square method of Levenberg-Marquardt. It provides a method for analyzing the resistance of microorganisms to supercritical carbon dioxide, including determining the tendency and resistance of the microorganisms to die.

- 식 ⑥

-Equation ⑥

In equation ⑥, y is the rate of reduction of the commercial log of the microorganism (N / N ₀ ), t is the treatment time of supercritical carbon dioxide, λ is the low killing rate after supercritical carbon dioxide treatment, and until the killing rate increases rapidly. K _dm is the negative value (maximum killing rate) of the slope of the section where the number of microorganisms rapidly decreases through the initial stage, and A is the level at which almost all microorganisms are killed except for some resistant microorganisms. The commercial logarithm of the ratio to the initial bacterial count (maximum killing level).

In the resistance analysis method of the present invention, Levenberg-Marquardt nonlinear least square method is a statistical method to obtain the most suitable regression relationship to minimize the RSS (Residual Sum of Squares) through the Levenberg-Marquardt algorithm, SigmaPlot known in the art Through a statistical program such as 2.0, steps 1 and 2 of the analysis method may be simultaneously performed. In general, with the exception of some resistant microorganisms, the time taken for nearly all microorganisms to kill ( t _t ) and λ increases, and as the k _dm values decrease, the resistance to supercritical carbon dioxide is greater. When the time taken to kill t _t is similar, k _dm may tend to increase even if λ increases due to resistance.

In the analysis method of the present invention, the activation energy ( E _d ) for the killing of each microorganism is obtained by applying the k _dm value of the temperature-specific microorganisms calculated in supercritical carbon dioxide at the same pressure to the Arrhenius equation of Equation (8) below. It is characterized in that it further comprises determining the dependence on temperature and resistance to death during the supercritical carbon dioxide sterilization of microorganisms from the activation energy ( E _d ) value. The greater the activation energy ( E _d ), the more microorganisms affected by temperature during supercritical carbon dioxide treatment.

k _dm = αexp (-E _d / RT) -equation ⑧

In equation (8), k _dm is the temperature inactivation rate constant of the microorganism by temperature, α is the frequency factor, E _d is the activation energy for microbial inactivation for supercritical carbon dioxide, R (gas constant) is 8.31447 JK ^-1 mol ^-1 and T is the absolute temperature.

In order to achieve another object of the present invention, the present invention obtains the killing tendency and resistance of the microorganism according to the temperature and pressure of the supercritical carbon dioxide treatment according to the analysis method of the present invention, and from the results obtained above It provides a method for establishing the minimum treatment conditions for supercritical carbon dioxide for microbial sterilization, including determining the minimum treatment conditions for obtaining killing levels.

In the establishment method of the present invention, further experiments such as measuring the pH of the suspension after supercritical carbon dioxide treatment, observing physical modification of the microbial cells by scanning electron microscopy, or confirming the extraction of proteins and nucleic acids by measuring the absorbance of the suspension, etc. Through this analysis, the main mechanisms of microbial killing by supercritical carbon dioxide can be analyzed microbiologically, physiologically and physicochemically, and these results can be used to establish the minimum treatment conditions for supercritical carbon dioxide. Therefore, the supercritical carbon dioxide can be applied to sterilization of various foods and medical devices with various conditions such as moisture content, pH, nutrients, etc. as well as microbial killing of other species not presented in the present invention. Basic data can be presented.

In the method of establishing the present invention, the minimum treatment condition means a minimum temperature, pressure and time condition for obtaining a desired level of killing of microorganisms, and the minimum temperature and pressure can be directly obtained from the above test results, and the minimum time is According to the method of the present invention, the tendency of killing microorganisms according to the temperature and pressure of supercritical carbon dioxide treatment is determined, and the coefficients A , k _dm and λ are applied to the following equation ⑦ to determine the minimum treatment time for killing all microorganisms. Can be.

- 식 ⑦

-Equation ⑦

At this time, substituting the initial bacteria count (N ₀₎ logarithm value (LogN _s / N ₀₎ of the ratio for a particular microbial class (N _s) of death in the supercritical state at a specific pressure and temperature and characters on A of formula ⑦ This can calculate the processing time required for this.

Hereinafter, the present invention will be described in more detail.

In the method for killing microorganisms using supercritical carbon dioxide, the microbial suspension is placed in a reactor having a temperature set, and the supercritical carbon dioxide is added and allowed to die for a period of time necessary for killing. Using the process of obtaining the suspension and the killing level of microorganisms according to each process variable in the suspension, the modified Gompertz equation shown in Example 4 was used to prepare a death curve, and based on this, the resistance of each bacteria to supercritical carbon dioxide was compared. Minimal treatment of supercritical carbon dioxide aimed at effectively and completely killing food poisoning microorganisms established on the basis of the analytical methods, the main mechanisms of microcritical killing by supercritical carbon dioxide revealed through several additional experiments, and the experimental results presented above. The condition is characterized by that.

Centrifuge the selected target harmful food microorganisms three times at 2800 rpm for 20 minutes to remove the supernatant and suspend in 0.85% saline (pH 6.5) to adjust the total bacterial count to 10 ⁷ -10 ⁸ . After mixing well, add 2 mL of the microbial suspension to the 5 mL cryogenic freezing tube to avoid overflow during supercritical carbon dioxide treatment. At this time, take 1mL separately and use it for initial bacterial count identification.

The microbial treatment process facility using the supercritical carbon dioxide of FIG. 1 is designed from a supercritical carbon dioxide extracting apparatus having the following configuration. 99.9% purity carbon dioxide cylinder (1), cooler (2), injection pump (3), cosolvent cylinder (4), cosolvent pump (5), thermostat (6), circulation pump (7), control device ( 8), extractor (9), first separator (10), second separator (11). In the present invention, only the extractor of 9 while blocking the 10 and 11 is used as a high pressure reactor for treating the microbial suspension, and 4 and 5 are excluded because no solvent is used. The high pressure reactor (9) withstands up to 6300 psi, 80 ^o C in 304 stainless steel with an internal capacity of 150 mL and is opened and closed with bolts. The carbon dioxide supplied from the cylinder 1 is supplied from the cyphon cylinder with liquid carbon dioxide having a purity of 99.5%.

When the reactor is set to the temperature set by the thermostat, the sample prepared above is placed in the reactor with the lid loosely closed to prevent breakage of the tube due to the pressure difference, and the bolt is completely locked by a wrench. When liquid carbon dioxide is injected by a pump through a cooler through the bottom conduit of the reactor and reaches a certain pressure level, the valves V-1 and V-2 shown in FIG. 1 are closed to maintain the pressure in the reactor for a certain time. . After the treatment time has elapsed, V-3 is immediately opened to remove supercritical carbon dioxide and the reactor is brought to atmospheric pressure. At this time, it takes about 1 minute, take the sample immediately and measure the number of viable cells. After each treatment the extractor was washed with 70% (v / v) ethanol and the next experiment was carried out and the experiment for each treatment was repeated three times.

By applying the log reduction ratio (Log (N / N ₀ ), y ) of microorganisms over time ( t ) to the modified Gompertz equation, as shown in Reference Example 4, the values of the kill curves and coefficients for each microorganism's treatment conditions are determined. Can be obtained (N ₀ = initial number of bacteria, N = number of bacteria after treatment). The λ value represents the time taken for the reduction level to reach very low intervals of less than 1 Log even after the supercritical carbon dioxide has been treated with the microorganism. The period of rapid decrease of microbial count after the initial stage is almost straight and the slope is represented by- k _dm . In addition, the A value does not have much significance since it is similar to the level at which almost all microorganisms are killed, i.e., the value of -Log at the initial bacterial count, except for some highly resistant microorganisms, but the λ and k _dm values do not depend on the supercritical It is an important basis for comparing resistance. _Limiting the maximum value of A to -Log (N _0max ) and matching the killing trend to the modified Gompertz equation, the higher the resistance to supercritical carbon dioxide, the higher the λ value and the lower the k _dm value. When the time taken to kill them is similar, k _dm tends to increase even if λ increases due to resistance (where N _0max is the maximum initial bacterial count).

CO ₂ dissolves under pressure to form carbonic acid (H ₂ CO ₃ , weak acid). Further acid is below and as bicarbonate (HCO ₃ ^-) and carbonate (CO ₃ ^2-) to the second because after processing the supercritical carbon dioxide microorganism suspension ionization will have a pH value lower than the suspension prior to processing.

Microorganisms usually have optimum pH values for growth, depending on the strain. For most bacteria, the optimal pH is usually between 6.8 and 7.2, and is known to survive at least pH 4.0 to 4.5, but lower pH modifies the protein structure on the microbial cell membrane and inhibits enzyme activity. In comparison, the pH inside the cell should always be kept close to pH 7.0 through a hydrogen ion pump. As the acid in the undissociated state can pass through the phospholipid cell membrane, when it enters the neutral cell, it dissociates to give hydrogen ions. When the hydrogen ion pump exceeds the concentration that the cell can accept, the pH inside the cell decreases. It affects nucleic acids such as enzymes and DNA inside the cell, leading to cell death. In particular, supercritical carbon dioxide can obtain the effect of lowering the pH inside the cell as well as the suspension during supercritical treatment due to the high solubility and diffusion, which can be confirmed indirectly by measuring the pH of the microbial suspension before and after the treatment. As soon as the supercritical microbial suspension is decompressed to atmospheric pressure, dissolved carbon dioxide begins to be converted to gas, so the pH meter is measured using a pH meter immediately after taking out of the reactor.

The microbial suspensions treated with or without supercritical carbon dioxide are centrifuged to recover only the bacteria, and then subjected to pretreatment for scanning electron microscopy to observe the physical deformation of the cells. In this case, only the deformation by supercritical carbon dioxide is considered except for the modification by pretreatment by comparing the untreated cells with the cell types after treatment.

Physicochemical modifications of cells also cause the outflow of cellular internals. Before and after the treatment of supercritical carbon dioxide, the microbial suspension is centrifuged to take only the supernatant and measure the absorbance. At this time, adjust the zero point with physiological saline and take the ratio of the absorbance value of the supernatant after treatment to the absorbance value of the supernatant before treatment. By measuring the absorbance at 260 nm, the outflow of biological material such as DNA and RNA from the cell can be observed. By comparing the measured absorbance at 280 nm, the degree of protein or amino acid leakage can be compared from the cell or the cell membrane.

As can be seen from the above additional experiments, supercritical carbon dioxide is physically and chemically inactivated by membranes and metabolism-related enzymes due to acidification inside and outside the cells, physical destruction of cells by internal pressure, extraction of cell wall lipids and components within cells. These mechanisms allow the microorganisms to die through complex mechanisms, so they can better express the killing trend when applied to the Gompertz model, rather than simply applied to the exponential growth model. Therefore, based on experimental results from the Gompertz model, supercritical carbon dioxide can be used to establish effective minimum processing conditions for complete killing of foodborne organisms.

Therefore, the present invention will be described in more detail based on the embodiments of the main food poisoning microorganisms and indicator microorganisms, but the present invention is not limited to the embodiments, and is applicable to microorganisms or spore-producing microorganisms with resistance.

Reference Example 1: Selection of Major Food Poisoning Microorganisms for Supercritical Carbon Dioxide Sterilization

Among the major food poisoning microorganisms, Gram-positive bacteria Listeria monocytogenes and Gram-negative bacteria Salmonella typhymurium, Escherichia coli O157: H7, Escherichia coli (Generic) were selected for treatment, and each of the three strains shown in Table 1 was mixed. By preparing the suspension, it was considered that there may be a difference in resistance to supercritical carbon dioxide even among bacteria in the same species.

[Table 1] Target food harmful microorganisms applied to inactivation process using supercritical carbon dioxide

¹ ATCC: American Type Culture Collection (Manassas, VA, USA)

² WSU: Food Science and Human Nutrition Bacteria Collection at Washington State University (Pullman, WA, USA)

Reference Example 2: Setting Treatment Conditions for Supercritical Carbon Dioxide

Using the physicochemical properties of supercritical carbon dioxide, we tried to set process variables to effectively kill microorganisms. At 31.1 ° C and 73.8 bar or more, the critical point of supercritical carbon dioxide, the process variables (pressure, temperature, time) determined in consideration of deterioration of the actual food quality were as follows and were repeated three times under the same conditions.

Table 2: Treatment Conditions of Supercritical Carbon Dioxide for Pressure Variables

[Table 3] Treatment of Supercritical Carbon Dioxide for Temperature Variables

Reference Example 3: Determination of viable cell count before and after treatment of microbial suspension

1 mL of each sample was treated before and after the treatment and 0.1 mL was taken with or without dilution with physiological saline and plated on a suitable medium for each bacterium. Incubate at 37 ℃ for 24 hours to determine the number of colonies and to calculate the average of the two repeated values as the experimental results. The selective medium used for each bacterium is shown in the following [Table 4].

[Table 4] Selection medium and screening method for measuring the number of colonies of each bacteria

Reference Example 4: Derivation of Modified Gompertzt Equation and Determination of Analytical Method for Microbial Death by Supercritical Carbon Dioxide

The process of converting mathematical coefficients such as a, b, c into biologically meaningful values ( A , k _dm , λ ) by modifying the Gompertz equation of Equation ① below is as follows.

- 식 ①

-Equation ①

Differentiate both sides with t ,

Differentiate both sides again with respect to t

일 때의 시간을 t _i 라 하면, t=t _i 에서 변곡점(inflection point)을 지나므로,

If the time at is t _i , we pass the inflection point at t = t _i ,

The slope of the tangent at t = t _i is a negative value for the maximum kill rate ( k _dm ),

- 식 ②

-Equation ②

when t = t _i ,

Therefore, the tangent equation at the inflection point is

- 식 ③

-Equation ③

The t- axis intercept (when y = 0 ) of the equation of the tangent can be defined as the death induction time ( λ ),

- 식 ④

-Equation ④

If you combine equation ② and equation ④,

- 식 ⑤

-Equation ⑤

Substituting equation ② and equation ⑤ into equation ①,

일 때,

이므로 a=A(asymtote)

when,

A = A (asymtote)

- 식 ⑥

-Equation ⑥

In conclusion, in the above formula ⑥, y is the log reduction level of the microorganism ( y = Log (N / N ₀ ), N ₀ = initial number of bacteria, N = number of bacteria after treatment), t is the treatment time, λ is supercritical carbon dioxide Characterized by a low killing rate after treatment, the time required before the killing rate increases rapidly (killing induction time), k _dm is a negative value (maximum killing rate) of the slope of the section where the number of microorganisms rapidly decreases through the initial stage, A Is the commercial log (maximum killing level) of the level at which almost all microorganisms are killed and the percentage of initial bacterial counts, except for some highly resistant microorganisms.

In each treatment according to temperature and pressure variables, the y-value over time is applied statistically by nonlinear least square method by applying to the above equation (6). The nonlinear least square method is a method of obtaining the most suitable regression relationship that minimizes the RSS (Residual Sum of Squares) through the Levenberg-Marquardt algorithm. Through this, the death curve and coefficients A , k _{dm ,} λ and R ² (Regression coefficient), RSS and the like are determined. All of these processes can be performed via SigmaPlot 2.0 (Jandel Scientific, San Francisco, CA, USA).

In the tangent equation of (3), the value of t when y = a is defined as the time ( t _t ) that almost all of the microorganisms die except for some resistant microorganisms.

- 식 ⑦

-Equation ⑦

Substituting the logarithmic log value (LogN _s / N ₀ ) of the ratio of the initial microbial count (N ₀ ) of the specific microbial level (N _s ) to be killed in a supercritical state at a certain pressure and temperature into A in equation ⑦ The required processing time can be calculated.

Example 1

Supercritical carbon dioxide was treated according to the treatment conditions of the pressure variables set in Table 2 of Reference Example 2, and the viable cell count was measured by the method of Reference Example 3, and the measured value in the formula shown in Reference Example 4 was used. Substituting can obtain a graph as shown in Figs. 3-6. At this time, the three viable cell counts for each treatment were directly applied to the formula, and the dots indicate the mean values and the standard error values for the three repetitions. In addition, the coefficient values obtained through the graphs of FIGS. 3-6 shown above are shown in Table 5 below.

Listeria monocytogenes took 15 minutes to kill all bacteria at each pressure treatment. Since the λ value was more than 7 minutes at 80 bar treatment, the λ value was about 2.4 ~ 3.4 minutes and the k _dm value was about 0.8 ~ 0.9 at the remaining 100, 120, 150 bar except when the k _dm was relatively high. There was a similar trend. In particular, the λ value was reduced by 1 minute at 120 bar compared to 100 bar, and the k _dm value was increased by 0.1 at 150 bar, and the viable cell count was decreased at a faster rate as the pressure increased.

Salmonella typhymurium also took 15 minutes to kill all bacteria at each pressure treatment. At 80, 100, and 120 bar pressure treatments, the λ values continued to decrease to 4.34, 3.99 and 2.45 minutes, and the k _dm values were very similar at about 1. At 150 bar treatment, the k _dm value increased by about 0.5 than that at the lower treatment, but the λ value was increased. After 10 minutes of treatment, some resistant microorganisms survived even though almost all bacteria died. Because it is reflected in the sudden inflection of

In the case of Escherichia coli O157: H7, the time taken to kill all bacteria was reduced from 20 minutes to 15 minutes when treated with increasing pressures of 80 and 100 bar to 120 and 150 bar. As the pressure increased to 80, 100, 120 bar, the k _dm value increased to 0.85, 0.83, 1.69, and the λ value decreased to 7.44, 8.43, 8.22 minutes, and the death was faster. However, there was no significant difference in killing effect between treatments of 120 bar and 150 bar.

In the case of Escherichia coli (Generic), all bacteria were killed, and the time to release was the same as that of E. coli O157: H7. As the pressure increased to 80, 100, and 120bar, the k _dm values increased to 1.07, 1.24, and 1.62, and the λ values decreased to 9.36, 9.01, and 8.32 minutes. However, as in E. coli O157: H7, there was no significant difference in killing effect between 120 and 150 bar treatments.

Comparing the results according to the treatment pressure, the λ values were similar in all 100, 120 and 150 bar, but the gram positive bacteria L. monocytogenes had lower k _dm values than the gram negative bacteria S. typhimurium , which is the thick cell wall of gram positive bacteria. One reason is that the structure inhibits the uptake of supercritical carbon dioxide into cells. In addition, Gram-negative bacteria are generally 10-20% higher in lipid content of cell walls than positive bacteria, and lipoproteins and lipopolysaccharides, which are constituents of these cell walls, are extracted by non-critical supercritical carbon dioxide. It is feed. At 120 and 150 bar, E. coli (Generic) has a higher killing rate and lower λ value than E. coli O157: H7, so Generic is more sensitive to supercritical carbon dioxide. However, it is difficult to compare k _dm values with the previous two microorganisms due to their relatively high λ values. Overall, the time required for total killing of L. monocytogenes and S. typhimurium is sufficient for 15 minutes at all pressures, whereas E. coli (Generic) and E. coli O157: H7 require a maximum of 20 minutes and the λ value is 2 ~ It takes four minutes and at least seven minutes in the latter case, so that the first two microbes are more sensitive to supercritical carbon dioxide than the next two. This is not simply explained by the above-mentioned distinction between Gram-positive and Gram-negative bacteria, which may be because supercritical carbon dioxide causes microbial death by such a complicated mechanism.

[Table 5] Comparison of A , k _dm and λ values of each microorganism according to pressure variables during supercritical treatment at 40 ℃

Example 2

When the strains presented in Reference Example 1 were carried out as in Example 1 according to the processing conditions of the temperature variables set in Table 3 of Reference Example 2, a graph as shown in FIGS. 7-10 can be obtained. In addition, the coefficient values obtained through the graphs of FIGS. 7-10 shown above are shown in Table 6 below.

In case of Listeria monocytogenes , as the treatment temperature increased to 35 ℃, 40 ℃ and 45 ℃, the time to kill all bacteria was significantly reduced to 25, 15 and 10 minutes. The k _dm value also increased to 0.46, 0.80, and 1.80 as the treatment temperature increased, and the λ value was decreased from 7 minutes to 4 minutes.

In Salmonella typhymurium , as the treatment temperature increased to 35 ℃, 40 ℃, and 45 ℃, the time taken for all bacteria was reduced to 20, 15 and 10 minutes. The k _dm value also increased to 0.63, 1.18, and 1.66 as the temperature increased, and the λ value decreased to 5.84, 3.99, and 2.91 minutes.

In case of Escherichia coli O157: H7, as the treatment temperature increased to 35 ℃, 40 ℃ and 45 ℃, the time taken to kill all bacteria was reduced to 30, 20 and 15 minutes. The k _dm value was also increased to 0.97, 1.24, 1.62 with increasing temperature, and the λ value was decreased to 15.74, 9.01, 5.06 minutes.

In the case of Escherichia coli (Generic), as the treatment temperature increased to 35 ℃, 40 ℃ and 45 ℃, the time taken to kill all bacteria was reduced to 20, 15 and 10 minutes. The k _dm value increased to 0.46, 0.83 and 1.10 with increasing temperature, and the λ value decreased to 12.57, 8.42 and 5.53. E. coli (Generic) was also rapidly killed as the temperature increased.

[Table 6] Comparison of A , k _dm , λ values of each microorganism according to temperature variables during supercritical treatment at 100 bar

The activation energy ( E _d ) for microbial killing can be obtained by applying the inactivation rate constant ( k _dm ) of microorganisms by temperature at the same pressure to the following Arrhenius equation.

| k _dm | = αexp (-E _d / RT) -equation ⑧

ln | k _dm | = lnα- (E _d / R) 1 / T

In equation (8), k _dm is the temperature inactivation rate constant of the microorganism by temperature, α is the frequency factor, E _d is the activation energy for microbial inactivation for supercritical carbon dioxide, R (gas constant) is 8.31447 JK ^-1 mol ^-1 and T is the absolute temperature.

The Arrhenius equation is used to indicate the temperature sensitivity of the microbial killing reaction using the logarithmic relationship between the rate constant and the inverse of the absolute temperature. The activation energy value can explain how sensitively the killing rate of microorganisms increases with increasing temperature at a constant supercritical pressure. Therefore, a large activation energy value can be said to be sensitive to temperature during supercritical carbon dioxide treatment.

Table 7 shows the activation energy, frequency factor, and R ² (regression coefficient) for the microbial killing reaction from the inactivation rate constant for each microorganism when treated with supercritical carbon dioxide at different temperatures at a constant pressure of 100 bar. The larger the activation energy ( E _d ), the more microorganisms affected by temperature during supercritical carbon dioxide treatment. In the order of L. monocytogenes > S. typhimurium > E. coli (Generic)> E. coli O157: H7, the activation energy was increased in response to the increase in temperature. This is because the increase in temperature increases the diffusivity of the supercritical carbon dioxide, which makes it possible to effectively penetrate the cell membrane of the Gram-positive bacteria L. monocytogenes , and this effect is relatively low in Gram-negative bacteria. As such, the value of E _d at constant pressure may be used as an indicator of temperature dependence and resistance to death when microorganisms are killed by supercritical carbon dioxide.

[Table 7] Inactivation energy ( E _d ), frequency factor and ( α ) and regression coefficient (R ² ) of each microorganism in 100 bar supercritical carbon dioxide treatment

Example 3

11 shows the results of measuring the pH of the suspension before and after treatment with E. coli (Generic). The average pH of the microbial suspension before treatment was 5.8 and the pH after treatment decreased differently with treatment pressure or temperature regardless of treatment time. At the same temperature, the higher the pressure, the greater the change in pH after treatment. At 80 bar, the pH was reduced to 4.5, but after 150 bar, it was reduced to pH 4.0. In particular, the lower the treatment temperature, under a pressure of 100 bar, the lower the final pH. This is because the solubility of supercritical carbon dioxide increases at higher pressures and lower temperatures.

Example 4

In FIG. 12-14, the morphology of the cells observed through the scanning electron microscope was presented before and after treatment (left) and after treatment (right) with supercritical carbon dioxide at 35 ° C. and 100 bar until all bacteria were killed. Gram-positive bacteria, Listeria monocytogenes , remained intact, whereas gram-negative bacteria Salmonella typhymurium and Escherichia coli O157: H7 were contracted, and cell membranes were impregnated. In particular, S. typhymurium had a much higher deformation of the cell membrane than E. coli O157: H7, and numerous droplets were formed on the cell membrane which are thought to be due to the extraction of the intracellular material. Therefore, the morphological mechanism of cell death by supercritical carbon dioxide is limited to Gram-negative bacteria, and the cell wall of S. typhymurium is more sensitive to supercritical carbon dioxide than E. coli O157: H7. The degree of deformation was worse. As described above, it is thought that cell wall components such as lipoprotein and lipopolysaccharide of gram-negative bacteria having higher lipid content of cell wall than gram-positive bacteria were dissolved and extracted by non-critical supercritical carbon dioxide.

Example 5

Absorbance values of suspensions of three kinds of food poisoning microorganisms treated at 35 ° C. and 100 bar are shown in FIGS. 15-17. In both Gram-negative bacteria Salmonella typhymurium and Escherichia coli O157: H7, the absorbance tended to increase at 260 nm (maximum absorption wavelength of DNA) and 280 nm (maximum absorption wavelength of protein) depending on the treatment time, but the Gram-positive bacteria Listeria monocytogenes The case was only at 260 nm. In particular, the absorbances of L. monocytogenes and S. typhymurium increased rapidly between 15 and 20 minutes, and E. coli O157: H7 between 20 and 25 minutes. It includes. Therefore, after the cell membrane is damaged by the supercritical carbon dioxide and begins to die rapidly, it is thought that protein or DNA, which is an internal component of the cell, is extracted from the cell.

Example 6

The damage of the cell membrane composed of phospholipids by supercritical carbon dioxide at the early death is thought to be the biggest cause of deterioration of the function of the cell membrane and inactivation of enzymes and proteins by decreasing the pH inside and outside the cells presented in Example 3 above. This is the basis for the rapid killing rate of L. monocytogenes , which have a thick cell wall and do not cause physical deformation. In addition, the cell membranes of L. monocytogenes may be damaged by extraction of phospholipids by the lipophilic properties of supercritical carbon dioxide, and the increase of temperature improves the diffusion instead of reducing the solubility of supercritical carbon dioxide. It becomes possible. This is consistent with the experimental results in which L. monocytogenes reacted most sensitively to temperature.

S. typhymurium and E. coli (O157: H7, Generic) have several factors that work together such as the physical deformation caused by the pressure of supercritical carbon dioxide and the pH lowering effect, as well as the extraction of phospholipids of cell membranes and lipid components of cell walls. Is killed. Therefore, S. typhymurium showed the fastest killing for all treatment conditions among the tested microorganisms, but E. coli (O157: H7, Generic) showed strong resistance to supercritical carbon dioxide despite being Gram-negative. Since the λ value is usually required for more than 7 minutes and is less sensitive to temperature than other microorganisms, it is thought to have a stronger resistance to pH lowering effect than the extraction effect of phospholipid among the factors causing microbial death early on. An unknown mechanism of death may have been included.

Table 8 shows the effective minimum treatment time for killing more than 8 Log of food poisoning microorganisms based on the main mechanism of microcritical killing by supercritical carbon dioxide revealed in Example 3-5 and the results obtained in Example 1-2.

TABLE 8 Effective minimum processing time (min) for killing food poisoning microorganisms above 8 Log

() ^* : Treatment time with more than 8 Log killing time in supercritical carbon dioxide sterilization experiment conducted at actual 5-minute intervals

As described above, according to the present invention, the process using supercritical carbon dioxide utilizes the physical and chemical properties of the supercritical carbon dioxide, so that sufficient results can be obtained even at relatively low levels of pressure and temperature, which are important process variables. This can save energy and increase the economics of the process. In particular, by using the principle of extracting the components at low temperature without changing the pH effect and cell type, it can be applied not only to liquid food but also to surface sterilization of fresh foods and processed foods. It can also be applied to sterilization of tools. In addition, since supercritical carbon dioxide is inexpensive, has a high recovery rate, and is not toxic, it will be an environmentally friendly future facility for the process. In particular, the present invention has proposed a mechanism of microbial killing to consider the physicochemical and biological conditions of the supercritical carbon dioxide treatment conditions, the food and the target microorganism sterilization efficiency in combination, the supercritical carbon dioxide treatment in actual sterilization system The mathematical model suggests the effective killing of major food poisoning microorganisms. In addition, the present invention has been systematically analyzed by analyzing the causes of various factors affecting the killing effect of microorganisms, by comparing the resistance of the supercritical carbon dioxide between the microorganisms by mathematically modeling the killing trends according to various treatment conditions of supercritical carbon dioxide. The evidence is presented.

Claims (10)

In the method of killing microorganisms using supercritical carbon dioxide, a) placing the sample in a reactor having a temperature above the supercritical point (31.1 ° C.); b) closing the reactor and introducing liquid carbon dioxide, which has undergone a cooler, into the reactor through a conduit and pressurizing the reactor to a pressure above the supercritical point (73.8 bar) by means of a pump; c) maintaining the pressure and temperature by closing the valve for the time necessary to completely kill the target microorganism; d) opening the valve to remove supercritical carbon dioxide when the required time has elapsed and returning the reactor to atmospheric pressure; And e) microbial sterilization method comprising the step of opening a reactor to obtain a sample free of live target microorganisms. The sterilization method of claim 1, wherein the sample is placed in a reactor while the lid is in a loosely closed container. The method according to claim 1, wherein the target microorganism is a food poisoning microorganism. The method of claim 1, wherein the target microorganism is Listeria monocytogenes , the main food poisoning microorganism, 23-27 minutes at 33-37 ℃, 95-105 bar supercritical carbon dioxide, or 38-42 ℃, 75-85 bar supercritical carbon dioxide Sterilization method characterized in that the treatment for 13-17 minutes. The method according to claim 1, wherein the target microorganism is Salmonella typhymurium , which is a major food poisoning microorganism, treated at 33-37 ° C. and 95-105 bar supercritical carbon dioxide for 18-22 minutes, or 38-42 ° C. and 75-85 bar supercritical carbon dioxide. Sterilization method characterized in that the treatment for 13-17 minutes. The method of claim 1, wherein the target microorganism is Escherichia coli O157: H7 as the main food poisoning microorganism or Escherichia coli (Generic) as the indicator microorganism is treated for 28-32 minutes in supercritical carbon dioxide at 33-37 ℃, 95-105 bar, Sterilization method characterized in that the treatment for 18-22 minutes at 38-42 ℃, 75-85 bar supercritical carbon dioxide. The microbial death curve is calculated by applying the log reduction level (y-axis) of microorganisms according to the treatment time (x-axis) of supercritical carbon dioxide to the modified Gompertz equation of Equation (6) below, nonlinear least square method to obtain coefficients A (maximum killing level) , k _dm (maximum killing rate) , and λ (killing induction time), and to determine the killing tendency and resistance of microorganisms from the coefficients. Analysis of microorganisms' resistance to supercritical carbon dioxide:

-Equation ⑥ In equation ⑥, y is the rate of reduction of the commercial log of the microorganism (N / N ₀ ), t is the treatment time of supercritical carbon dioxide, λ is the low killing rate after supercritical carbon dioxide treatment, and until the killing rate increases rapidly. K _dm is the negative value (maximum killing rate) of the slope of the section where the number of microorganisms rapidly decreases through the initial stage, and A is the level at which almost all microorganisms are killed except for some resistant microorganisms. The commercial logarithm of the ratio to the initial bacterial count (maximum killing level). The method according to claim 7, wherein the activation energy for the killing of each microorganism by applying the k _dm value of the temperature-specific microorganisms calculated in the supercritical carbon dioxide of the same pressure obtained by the modified Gompertz equation ( E _d) ) And the activation energy ( E _d ) Analyzing the method further comprises determining the dependence of the temperature on the microbial supercritical carbon dioxide sterilization from the value: k _dm = αexp (-E _d / RT) -equation ⑧ In equation ⑥, k _dm is the temperature inactivation rate constant of the microorganism by temperature, α is the frequency factor, E _d is the activation energy for the killing of microorganisms against supercritical carbon dioxide ( E _d ), and R (gas Constant) is 8.31447JK ^-1 mol ^-1 and T is the absolute temperature. According to the method of claim 7, the tendency of killing microorganisms according to the temperature and pressure of supercritical carbon dioxide treatment is calculated, and the minimum treatment time for killing all microorganisms by applying the coefficients A , k _dm and λ values to the following equation ⑦ Method of establishing the minimum treatment conditions for supercritical carbon dioxide for microbial sterilization comprising determining t _t ):

-Equation ⑦ 10. The method according to claim 9, wherein after substituting the coefficients k _dm and λ into Equation ⑦, the initial microbial count (N ₀ ) of the specific microbial level (N _s ) to be killed in the supercritical state at a specific pressure and temperature. And applying the common log value (LogN _s / N ₀ ) of the ratio to A of Equation (7) to determine the minimum processing time required for this.

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