Death Kinetics for Escherichia coli Cells Heated in the Presence of Short- and Medium-Chain Fatty Acids

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1 Biocontrol Science, 2000, Vol.5, No.1, Original Death Kinetics for Escherichia coli Cells Heated in the Presence of Short- and Medium-Chain Fatty Acids HIROYUKI YAMANAKA1 AND TETSUAKI TSUCHIDO2* 1Basic Research Laboratory, Prima Meat Packers, Ltd., Nakamukaihara, Tsuchiura and 2Department of Biotechnology, Faculty of Engineering and High- Technology Research Center, Kansai University, Yamate-cho, Suita , Japan Received 27 May 1999/Accepted 21 July 1999 We investigated the death kinetics for Escherichia coli K12 cells heated at different temperatures in the presence of saturated fatty acids having short and medium alkyl-chain lengths at various concentrations. Apparent first-order death kinetics were observed with cells heated in the presence of caproic acid, caprylic acid, capric acid, or lauric acid. Values of activation enthalpy and activation entropy of the death reaction decreased with an increase in the concentration of caprylic acid, capric acid, and lauric acid, but did not decrease much for caproic acid. Also, the value of dilution coefficient decreased with increasing heating temperature for the former three fatty acids but was not noticeably temperature dependent for caproic acid. These results suggest that there are two distinct modes for the death reaction of E. coli cells heated in the presence of fatty acids and that the hydrophobicity based on the alkyl-chain length of fatty acid molecule is responsible for such a difference in the death kinetics. Key words : Fatty acid/escherichia coli/thermal death/alkyl-chain length/combined treatment/death kinetics. INTRODUCTION *Corresponding author. Tel : , Fax : +81- The pasteurization process has been widely used for killing vegetative cells in food but often damages to food components. One of the possible methods for reducing the deleterious effect of heat on the food quality is the addition of an antimicrobial compound. Many investigators have so far reported the enhancing effect of a variety of such compounds on the thermal death of microorganisms (Shibasaki and Tsuchido, 1973; Tsuchido, 1977). Tsuchido and Shibasaki (1980) compared the death kinetics of Escherichia coli K12 between cases in which sorbic acid or an amphoteric surfactant preparation was added in combination with heat. They found that the temperature dependency of the killing action of the amphoteric surfactant was dependent upon the concentration of the compound, whereas no such dependency on the concentration was observed with sorbic acid. On the basis of these results, they have proposed two modes of action of antimicrobial compounds in their enhancing action on the thermal death (Tsuchido and Shibasaki, 1980). Fatty acids are widely distributed in organisms and known to possess antimicrobial activity. They have also been reported to possess an enhancing action on the thermal death of some bacteria including Escherichia coli and Pseudomonas aeruginosa (Kato and Shibasaki, 1975; Tsuchido and Takano, 1988). We are interested in investigating how the kinetics of thermal death are affected by fatty acids present in the heating menstruum. In this study, we then compared the kinetic characteristics of the enhancing effect among fatty acids having different alkyl-chain lengths. MATERIALS Organism and cultivation AND METHODS

2 18 H. YAMANAKA AND T. TSUCHIDO Cells of E. coli K12 from the laboratory stock culture Thermodynamic relationships were grown at 37 C for 18 h in nutrient broth consisting of 1 % (w/v) beef extract (Riken Vitamin Co., Ltd., Tokyo), 1 % (w/v) Polypepton (Nihon Seiyaku Co., Ltd., Tokyo), and 0.5 % (w/v) NaCI (ph 7.0). After being harvested, cells were washed with 50mM phosphate buffer at ph 7.0 and then resuspended in an appropriate volume of fresh buffer at 37 Ž to adjust the cell density to 2 ~ 108 per ml. Treatments with heat and fatty acids For heat treatment, a portion (1 ml) of the cell suspension was poured into a flask containing 19 ml of 50mM phosphate buffer at ph 7.0 preheated to 48, 50, 52, or 54 Ž. An appropriate volume of an aqueous solution of the sodium salt of a fatty acid was added to the buffer to obtain a required final concentration before the addition of cell suspension. Sodium salts of caproic (C6), caprylic (C8), capric (C10), and lauric acid (C12) were all purchased from Wako Pure Chemical Industries (Osaka). During the heating period, a sample portion (1 ml aliquot) was periodically taken out and added to a test tube containing 9 ml of a solution of 0.1 % (w / v) Polypepton (Nihon Seiyaku) at room temperature. The tube was then cooled in an ice-water bath. Viability measurement Samples were diluted with sterile deionized water and plated on nutrient agar. The plates were incubated at 37 Ž for 20 h and the resultant colonies were then counted. Kinetic analysis The viability data obtained by the above experiments were analyzed by using the theory described below. The lines in each plot were drawn by using the least-square analysis. THEORY First order kinetics of thermal death The thermal death of a microorganism in general follows the first-order kinetics as described by equation (1), -dn / dt = kn (1) where N and t are the viable cell number and time Concerning the dependency of k on the heating temperature, T (K), two equations, the Arrhenius equation (3) and the Eyring's absolute reaction rate theory (5), are well known. The former is k = A e-ea/rt (3) where Ea is the activation energy of death reaction (J /mole), R is the gas constant, A is the frequency factor, and T is the absolute temperature (K). Taking the logarithm of both sides of the equation (3) log k =-(Ea / 2.303R) (1 / T) + log A (4) When log k is plotted against the reciprocal absolute temperature, Ea is calculated from the slope of the line obtained. Eyring's equation is expressed as k = (kb T / h) e- H*/ R T e s* / R (5) where H* and S* are the activation enthalpy (J /mole) and activation entropy (J/mole E deg) of death reaction, respectively, and kb and h are the Bolzman's constant and Planck's constant. T is the absolute temperature (K). From the equation (5), the following is derived: log (k/ T) = -( H*/ R) (1 / T)+ S* / R + log (kb / h) (6) When log k/t is plotted against 1/T, H * and S* can be calculated from the slope of the line obtained and the intercept of the line with y-axis, respectively. Furthermore, the activation free energy (J/mole) of death reaction, G*, has the following relationship with H* and S*, G* = H* T- S* (7) Between Ea and H*, the relationship of equation (8) is introduced. Ea = H* +R T(8) Dependency of the death rate on the concentration of antimicrobial compound In killing of microbial cells with an antimicrobial compound, the death rate is affected by the concentration of the compound present, C, as expressed by the equation (9), k = a Cn (9) where n is the dilution coefficient (or concentration exponent) and a is a constant. Therefore, log k = n log C + log a (10) When log k is plotted against log C, n can be calculated from the slope of the line obtained. (min), respectively, and k is the death rate constant (min-1). When taking the integral of both sides of equation (1), equation (2) is obtained. log N = -(k / 2.303)t + log N0 (2) where N0 is the initial viable cell number, k is calculated from the slope of the line obtained from the plot of log N against t. RESULTS Enhancement of the thermal death of E. coli cells by fatty acids First-order death kinetics were observed with E coli

3 DEATH KINETICS FOR E. COLI HEATED WITH FATTY ACIDS 19 K12 cells heated in the presence of caproic acid, caprylic acid, capric acid, or lauric acid. All of these fatty acids were found to enhance the thermal death at heating temperatures tested. An example of a set of data in the case of heating at 50 Ž with caprylic acid at concentrations of 5, 10, 20, and 30 mm is shown in Fig. 1. Treatment of cells with fatty acids at room temperature caused no reduction in viability. From the survival curves for cells heat-treated at temperatures of 48, 50, 52, and 54 Ž in the presence of each fatty acid at different concentrations, the death rate constant, k, was calculated. In part of the data, however, either the shoulder or tailing-off was Heating time (min) FIG.1. Thermal death of E coli cells at 50 Ž in 50mM phosphate buffer at ph 7.0 in the presence of caprylic acid at 5 ( ž), 10 ( ), 20 ( ) and 30 mm ( ). Data for control cells heated without the fatty acid are also shown ( œ). FIG. 2. Eyring's plots of the death rate constant for the death of E. coli cells heated in the presence of fatty acids at different concentrations. Symbols: (a) caproic acid at 20 ( ž), 50 ( ), 100 ( ) or 200 mm ( ) ; (b) caprylic acid at 5 ( ž), 10 ( ), 20 ( ) or 30 mm ( ) ; (c) capric acid at 1 ( ž), 2 ( ), 3 ( ) or 5 mm ( ) ; (d) lauric acid at 0.1 ( ž), 0.2 ( ), 0.5 ( ) or 1.0 mm ( ). TABLE 1. Thermodynamic parameters for the death reaction of E. coli cells heated in the presence of fatty acids.

4 20 H. YAMANAKA AND T. TSUCHIDO seen in the survival curves obtained. In these cases, k was calculated from the linear portion of the survival curve. Temperature dependency of death kinetics Figure 2 indicates Eyring's plots for the thermal death in the presence of caproic acid, caprylic acid, capric acid, and lauric acid. For the latter three fatty acids, the slope of the line decreased with increasing concentration, while, for caproic acid, it seemed constant irrespective of the concentration used. From these data, the values of Ea,. H*, S* and G* were calculated by using equations (6), (7), and (8), and listed in Table 1. These results indicated that Ea, H *, and S* were decreased with an increase in the concentration of caprylic acid, capric acid, or lauric acid, whereas they did not vary with the concentration in the case of caproic acid, which had the shortest alkyl-chain among the fatty acids tested. The dependency of H* on the concentration was shown in Fig. 3 and its linearity was seen for the former three fatty acids but not for caporic acid. The value of AG* decreased only slightly with increasing concentration for all fatty acids. As described above, the thermodynamic behavior patterns of thermal death in the presence of various saturated fatty acids were found to be different between the three fatty acids having medium alkyl-chain lengths, caprylic acid, capric acid, and lauric acid, and the shortest alkyl-chain acid tested, caproic acid. FIG. 3. Dependency of the activation enthalpy of the death reaction for E. coli cells heated in the presence of fatty acids. Symbols: ž, caproic acid;, caprylic acid;, capric acid;, lauric acid. Dependency of the cell death on the concentration of fatty acid Using equation (10), the effect of the concentration of fatty acid on the death rate constant was analyzed by using the same data as those for Eyring's plot, and the results are shown in Fig. 4. From the slope of lines obtained, we calculated the dilution coefficient, n, and the minimum effective concentration for enhancing cell death, which was obtained from the average of the intercept of each line with log k value in the absence of fatty acid (Table 2). Also, here, a difference in the dependency of the dilution coefficient on the heating temperature was observed between the three TABLE 2. The dilution coefficient and the minimum effective concentration for the death of E. coli cells heated in the presence of fatty acids. a Average value } S. D.

5 DEATH KINETICS FOR E. COLI HEATED WITH FATTY ACIDS 21 concretely the above differences in kinetic behavior. One possible reason for such a difference in the mode of enhancing action may be the degree of hydrophobicity of the fatty acid molecule dependent upon its alkyl-chain length. Heating E. coli cells causes damage to the outer membrane which is known to play a role as a permeability barrier against a variety of hydrophobic compounds (Katsui et al., 1982; Tsuchido et al., 1985 and 1989). As a result, these cells have been reported to become sensitive to these inhibitory compounds (Tsuchido and Takano, 1988), since the compounds can enter the cells because of the disruption of the outer membrane structure (Tsuchido et al., 1985 and 1989). Considering this physiological event, it is likely that the difference in death kinetics among the fatty acids observed in this study is derived from the difference in levels of penetration and partition of fatty acid molecules into cells or cell membranes. In agreement with the results obtained here, it has been reported that the inhibition on the regrowth of heat-injured E. coli cells, as compared with that on the FIG.4. The dependency of the death rate constant on the concentration of fatty acid present when E. coli cells are heated at different heating temperatures. (a) caproic acid; (b) caprylic acid; (c) capric acid; (d) lauric acid. Symbols: ž, 48 Ž;, 50 Ž;, 52 Ž;, 54 Ž. medium-chain acids and caproic acid. The value of n was lower at higher temperatures for the former three acids, whereas it was not much changed in the case of caproic acid. The minimum effective concentration for enhancing cell death was decreased with an increase in the number of carbon atoms of fatty acids from 11 mm for caporic acid to mm for lauric acid. DISCUSSION As has already been reported in regards to various antimicrobial compounds (Tsuchido, 1977), it was confirmed in this study that short- and medium-chain fatty acids also have an enhancing effect on the thermal death of E. coli cells. A similar action has also been obtained with a derivative of a fatty acid, monolaurin (Tsuchido et al. 1981). From the kinetic analysis of the death reaction, the mode of the enhancing action of fatty acids was found to be different between a short-chain acid, caproic acid, and three medium-chain acids, caprylic acid, capric acid, and lauric acid. The values of thermodynamic parameters, Ea, H* and S*, of the death reaction, as influenced by the concentration of fatty acids, indicate growth of intact cells, is drastically enhanced by medium-chain acids, caprylic acid, capric acid, and lauric acid, whereas it is only slightly enhanced by short-chain acids, butylic acid and caproic acid (Tsuchido and Takano, 1988). Leao and van Uden (1982) have investigated the enhancement of the thermal death of Saccharomyces cerevisiae by alkanols having relatively short alkyl-chain lengths, such as ehanol, isopropanol, propanol, and butanol, and they have found that the value of H* did not significantly change, indicating the parallelism of Eyring's plot. Although they did not examine the effects of alkanols having longer alkyl-chain lengths, their results coincide with our data obtained with caproic acid having a relatively short alkyl-chain length. Tsuchido and Shibasaki (1980) have indicated that the difference in kinetic behavior in the enhancement of thermal death varies with the antimicrobial compound and microorganism, and they have proposed a classification of those antimicrobial compounds into two groups, S type (surfactant as a representative) and A type (low-molecular weight organic acid as a representative). The former type of compounds, such as sodium hydroxide, a preparation of amphoteric surfactant (Tego 15DL), sodium dodecylsulfate and cetyltrimethylammonium bromide, demonstrated a convergence of lines at a temperature higher than the observation temperatures on Erying's plot, whereas the latter, including benzoic acid, sorbic acid, and epoxides, tended to show a parallelism of lines on the plot (Tsuchido and Shibasaki, 1980). Based on this

22 H. YAMANAKA AND T. TSUCHIDO classification, therefore, caprylic, capric, and lauric acids seem to be of the S type, while caproic acid seems to be of the A type.

6 22 H. YAMANAKA AND T. TSUCHIDO classification, therefore, caprylic, capric, and lauric acids seem to be of the S type, while caproic acid seems to be of the A type. Monolaurin has also been reported to be a S-type compound (Tsuchido et al., 1981). These two distinct modes of the enhancing action of antimicrobial compounds can also be characterized numerically by use of the compensation temperature (Tc). Tc is calculated based upon the Ho*- S* compensation phenomenon (Krug et al. 1976a and 1976b; Labuza, 1980), which was a correlation between these two thermodynamic parameters as expressed by the H* = Tc S* + b, where b is a constant. From the values of H* and S* described in Table 1, the values of Tc were calculated to be } 7.1 K, } 4.8 K, and } 3.7 K for caprylic acid, capric acid, and lauric acid, respectively. For caproic acid, on the other hand, no significant value was obtained because there were almost identical values at all concentrations of the acid tested. This Tc value can also be obtained as the temperature at which the lines intersect when they are extrapolated to the side of higher temperature on Eyring's plot (Barnes et al., 1969; Exner, 1970). This temperature can also be defined as the minimum temperature for enhancing the thermal death. For fatty acids, we obtained the values of Tc by taking averages of the temperature at the intersection of each set of two lines extrapolated in Fig. 2, being (59.8 Ž) 4.6 (S.D.) K for caprylic acid, (63.2 Ž) } } 5.7 K for capric acid, and (58.1 Ž) } 2.0K for lauric acid. These values were relatively close to those obtained from the H* - S* plot. For caproic acid, however, the lines seemed to be parallel and Tc could thus not be obtained as a significant value. The above compensation effect has been attributed to the role of water in a number of chemical, biological, and biochemical reactions (Elizondo and Labuza, 1974; Fisher, 1979; Labuza, 1980). If Tc is in the temperature range of 320 to 350K as a significant value, some common mechanism of water structure interaction has been suggested to be involved in the reaction (Labuza, 1980). Also in the enhancement of thermal death by three medium-chain acids employed here, water may play a significant role in the death reaction relating to the involvement of the hydrophobicity of the fatty acid molecule described earlier. In practical use of fatty acids in combination with heat treatment, the results obtained in this study indicate the following predictive principles, as also described in the previous papers (Tsuchido and Shibasaki, 1980; Tsuchido et al. 1985). First, as deduced from Eyring's plot, theoretically, for mediumchain fatty acids, the efficiency of enhancement of, thermal death (the increment of death rate) by increasing the concentration may be marked at lower temperatures, whereas, for short-chain fatty acids, an identical enhancement efficiency may be obtained at any temperature. Second, as deduced from the plot of the death rate constant against the fatty acid concentration, the efficiency of enhancement of death by elevating the heating temperature is striking at lower concentrations for medium-chain fatty acids, whereas it may be identical at any concentration for shortchain fatty acids. Third, for the enhancing activity of medium-chain acids, there should be a critical temperature, which corresponds to the compensation temperature. At temperatures above this defined temperature, no enhancing effect may be expected. Fourth, there should be a minimum effective concentration for all fatty acids as depicted in the plot of the death rate constant against the fatty acid concentration. These principles may be useful for the application of a combined treatment with heat and fatty acids to the pasteurization of food. REFERENCES Barnes, R., Vogel, H., and Gordon, I. (1969) Temperature of compensation. Significance for virus inactivation. Proc. Natl. Acad. Sci. U.S.A., 62, Elizondo, H., and Labuza, T.P. (1974) Death kinetics of yeast in spray drying. Biotechnol. Bioeng., 16, Exner, O. (1970) Determination of the isokinetic temperature. Nature, 227, Fisher, J.L. (1979) An entropy-enthalpy compensation law model analysis of the thermal shock bioassay procedure for certain species of fishes. In Cell Associated Water. (Drost-Hansen, W., and Clegg, J.S., ed.), pp , Academic Press, London. Kato, N., and Shibasaki, I. (1975) Enhancing effect of fatty acids and their esters on the thermal destruction of Escherichia coli and Pseudomonas aeruginosa. J. Ferment. Technol., 53, Katsui, N., Tsuchido, T., Hiramatsu, R., Fujikawa, S., Takano, M., and Shibasaki, I. (1982) Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli. J. Bacteriol., 151, Krug, R.R., Hunter, W.G., and Grieger, R.A. (1976a) Enthalpy-entropy compensation. 1. Some fundamental statistical problems associated with the analysis of van't Hoff and Arrhenius data. J. Phys. Chem., 80, Krug, R.R., Hunter, W.G., and Grieger, R.A. (1976b) Enthalpy-entropy compensation. 2. Separation of the chemical from the statistical effect. J. Phys. Chem., 80, Labuza, T.P. (1980) Enthalpy-entropy compensation in food reactions. Food Technol., 34, (2), Leao, C., and van Uden, N. (1982) Effects of ethanol and other alkanols on the kinetics and the activation parameters of thermal death in Saccharomyces cerevisiae. Biotechnol. 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7 DEATH KINETICS FOR E. COLI HEATED WITH FATTY ACIDS 23 Lumry, R., and Rajender, S. (1970) Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules. A ubiquitous property of water. Biopolymers, 9, Shibasaki, I., and Tsuchido, T. (1973) Enhancing effect of chemicals on the thermal injury of microorganisms. Acta Alimentaria, 2, Tsuchido, T. (1977) Combined effects of antimicrobial compounds and heating on microorganisms (in Japanese). Hakkokogaku, 55, Tsuchido, T., and Shibasaki, I. (1980) Kinetics of microbial death by combined treatment with heat and antimicrobial compounds. Biotechnol. Bioeng., 22, Tsuchido, T., and Shibasaki, l. (1983) Thermochemical treatment for sterilization and inhibition of microorganisms. In Heat Sterilization of Food (Motohiro, T., and Hayakawa, K., eds.), pp , Koseisha-koseikaku, Tokyo. Tsuchido, T., and Takano, M. (1988) Sensitization by heat treatment of Escherichia coli K-12 cells to hydrophobic antibacterial compounds. Antimicrob. Agents Chemother., 32, Tsuchido, T., Aoki, I., and Takano, M. (1989) Interaction of fluoresent dye 1-N-phenylnaphthylamine with Escherichia coli cells during heat stress and recovery from heat stress. J. Gen. Microbiol., 135, Tsuchido, T., Saeki, T., and Shibasaki, I. (1981) Death kinetics of Escherichia coli in a combined treatment with heat and monolaurin. J. Food Safety, 3, Tsuchido, T., Katsui, N., Takeuchi, A., Takano, M., and Shibasaki, I. (1985) Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment. Appl. Environ. Microbiol., 50,

Comparison of Anti- Vibrio Activities of Potassium Sorbate, Sodium Benzoate, and Glycerol and Sucrose Esters of Fatty

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1980, p. 1178-1182 0099-2240/80/06-1178/05$02.00/0 Vol. 39, No. 6 Comparison of Anti- Vibrio Activities of Potassium Sorbate, Sodium Benzoate, and Glycerol