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Combined Water Activity and Solute Effects on Growth and Survival of Listeria Monocytogenes Scott A

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Combined Water Activity and Solute Effects on Growth and Survival of Listeria Monocytogenes Scott A 414 Journal of Food Protection, Vol. 55, No. 6, Pages 414-418 (June 1992) Copyright©, International Association of Milk. Food and Environmental Sanitarians Combined Water Activity and Solute Effects on Growth and Survival of Listeria monocytogenes Scott A A. J. MILLER* Eastern Regional Research Center, Agricultural Research Service U. S. Department of Agriculture, 600 E. Mermaid Lane, Philadelphia, P ennsylvaina 19118 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/6/414/1664071/0362-028x-55_6_414.pdf by guest on 02 October 2021 (Received for publication October 8, 1991) ABSTRACT al. (14) found that L. monocytogenes strain No. 18 (sero­ type l/2a) remained viable at 37°C for 15 d in 10.5% NaCl, Water activity and solute effects were determined on the growth and survival of Listeria monocytogenes Scott A in bacte­ 10 d in 13% NaCl, and 5 d in 20-30% NaCl. Survival times riological media. Media were adjusted to water activity (a ) levels more than doubled at 22°C. The organism survived longer of 0.99-0.80 with NaCl, glycerol (GLY), or propylene glycol than 100 d in 30.5% NaCl (aw = 0.80) at 4°C. (PG). Minimum aw levels for growth occurred at 0.90, 0.92, and There is a need to obtain a better understanding of the 0.97 for GLY, NaCl, and PG, respectively. Survival was related effect of a on the growth kinetics and survival of L. to the a level, as well as the solute employed. Cells survived the monocytogenes. The aim of this study, therefore, is to longest in GLY and died earliest in PG; NaCl was intermediate. establish how a - as modulated by different solutes affects These results provide quantitative isothermal criteria for estimat­ growth and survival of L. monocytogenes strain Scott A. ing the aw level necessary to prevent growth of L. monocytogenes in food. MATERIALS AND METHODS Strain and maintenance conditions Studies on the effect of water activity (a ) on the Listeria monocytogenes Scott A, from the culture collection growth and survival of the foodborne intracellular pathogen at the U.S. Department of Agriculture, Agricultural Research Listeria monocytogenes have been limited principally to the Service, Eastern Regional Research Center, was maintained at use of salt as a solute. Simon (15) was among the first to 4°C on brain heart infusion (BHI) agar streak plates and subcul- tured monthly. Culture purity and identity were confirmed at the report L. monocytogenes halotolerance. Seeliger (13) deter­ time of each subculturing by Gram stain, catalase test, hemolytic mined that the organism is capable of growing to 10% activity on sheep red blood cell agar plates, and mobility test NaCl (aw = 0.93). Others have demonstrated that tempera­ using phase contrast microscopy. For the experiments described ture and salt combinations affected its growth. For ex­ here, a single colony was picked and grown overnight in 100 ml ample, Conner et al. (5) showed that L. monocytogenes pH 7.4, BHI broth at 30°C at 300 rpm. One-ml portions of the Scott A and LCDC 81-861 grew in cabbage juice contain­ culture were distributed into sterile glass vials and mixed with 20 ing 2% NaCl (aw = 0.99) but did not grow at 5% NaCl (aw u.1 dimethyl sulfoxide as a cryoprotectant. Vials were frozen = 0.97) at 30°C. Yet, Benganovic et al. (2) reported that a immediately in dry ice and stored at -70°C. For each experiment strain of the organism which was isolated from sheep grew a vial was removed, thawed at 4°C, and 10 u.1 transferred to 100 ml fresh BHI broth. The inoculum was grown overnight as at 6°C in 5% NaCl. Petran and Zottola (11,12) studied the described above. For growth/survival studies, the overnight cul­ effect of other humectants on growth or inhibition of L. ture was diluted to log10 3-4 CFU/ml in 0.1% peptone (Difco monocytogenes, including propylene glycol, lactose, fruc­ Laboratories, Detroit, MI). tose, galactose, and sucrose. Knowledge of the effect of water activity on survival Experimental procedure of the pathogen is similarly limited to NaCl studies. Seeliger BHI broth (Difco Laboratories, Detroit, MI) containing 0.5% (13) demonstrated that some strains tolerated 20% NaCl (a NaCl was adjusted to aw levels of 0.99, 0.97, 0.93, 0.90, 0.87, 0.83, and 0.80 with NaCl"(Mallinckrodt, Inc., Paris, KY), glycerol = 0.86) for short periods and remained viable after 1 year (GLY) from Sigma Chemical Co., St. Louis, MO or propylene in 16% NaCl (a = 0.90) at pH 6.0. Beganovic et al. (2) w glycol (PG) from Fisher Scientific Co., Fairlawn, NJ. Salt contain­ reported that the organism died in 10% NaCl in 15-30 d, at ing samples were prepared to a^ levels of 0.92 and 0.91, as well. a rate which was related to the inoculum size. Shahamat et All chemicals were used as received from the manufacturer or distributor. Solute levels for aw modulations were obtained from Niven (10), Grover and Nicol (7), and Sloan and Labuza (16) for Reference to a brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not NaCl, GLY, and PG, respectively. Water activities were con­ mentioned. firmed with a Rotronic Hygroskop DT water activity meter JOURNAL OF FOOD PROTECTION, VOL. 55, JUNE 1992 WATER ACTIVITY AND SOLUTE EFFECTS ON LISTERIA 415 (Rotronic Instrument Corp., Huntington, NY), equipped with a humidity sensor. Triplicate measurements were performed on each treatment combination. One hundred ml of each medium in 250-ml flasks was adjusted to pH 7.4 with dilute HC1 or NaOH, prior to sterilization. Flasks were prepared in triplicate and were frozen and stored at -18°C. Prior to each experiment, one of the three replicate flasks was thawed overnight at 4°C and a culture was prepared as described above. Experimental flasks were equilibrated to 28°C, inoculated with log10 3-4 CFU/ml, and incubated at 28°C at 150 rpm. Samples were taken periodically, diluted serially with 0.1 % peptone, and plated onto tryptose phosphate (TP) agar (Difco) using a Spiral Plater (Spiral Systems Inc., Cincinnati, OH). Petri 200 400 600 800 dishes were incubated inverted for 48 h at 35°C and then enumer­ TIME (hrs) ated using a Model 500 A Bacteria Colony Counter (Spiral Figure 1. Combined NaCl and a effects on growth and inhibition Systems Instruments, Inc.). The threshold level of detection was of L. monocytogenes Scott A in BHI medium at 28°C. Dotted 21 CFU/ml. Survival was defined as the ability of the organism to lines are extrapolations of enumeration data. The minimum level Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/6/414/1664071/0362-028x-55_6_414.pdf by guest on 02 October 2021 be enumerated under the conditions detailed above. of detection was 21 CFU!ml. Curve fitting Glycerol Growth curves were fitted for solute and a treatments using the Gompertz equation (3,6). Gompertz coefficients were used to The effect of GLY on growth or inhibition of L. derive values of more practical use, including: monocytogenes is presented in Fig. 2 and Table 1. In Exponential growth rate (EGR), log]0 CFU/ml/h contrast to NaCl, growth occurred in GLY to an a = 0.90. Generation time (GT), h ' ° w Lag-phase duration (LPD), h, and GT increased (0.8-17 h) over the permissive growth aw Maximum population density (MPD), log CFU/ml. range of 0.99 to 0.90, as did LPD (2-53 h); EGR decreased" Where there was evidence of cell death, decimal reduction times from 0.4 to 0.02 log10 CFU/h. MPD decreased from log10 10 (D, and D2 values) were calculated using the biphasic logistic CFU/ml for 0.99 to 8.5 at the minimum permissive aw equation proposed by Kamau et al. (8) and modified by Dr. (0.90). The most significant difference between data from Richard C. Whiting (ERRC, Microbial Food Safety Research NaCl and GLY grown L. monocytogenes was a pattern of Unit) to include a lag factor (TL). The equation is: shorter lag time durations in GLY grown cells. At aw levels of < 0.87 there was no growth in a GLY containing F,(l+e-(i'TL ) (l-F,)l+e log N = log medium. The death curves were generally parallel for aw = .(l+eW-u)) (l+ep=(t-TL)) 0.87-0.80. Differences observed in the shoulder (TL) or D H m value (D ) did not show an obvious trend, in contrast to the where: N = surviving population (CFU) at time t; N0 = initial NaCl data. Where death occurred at common aw levels, the population (CFU); F] = population fraction in major group; 1- F shoulders (TL) for the GLY treatments were uniformly = F2 = population fraction in minor group; $} = constant for major greater than any of the NaCl treatments and L. monocyto­ group; (32 = constant for minor group; TL = lag time, and e is the genes survived longer in GLY than in NaCl. Survival in base of the natural logarithm. The D values were calculated by the GLY was 400-500 h at 28°C (Fig. 2). equation D. = 2.3/(3.. RESULTS Sodium chloride Growth/inhibition curves are presented in Fig. 1 and j-A- 0.99 -*- 0.87 results from the Gompertz curve fitting are presented in -o- 0.97 -*- 0.83 Table 1. Growth occurred at water activities of 0.99, 0.97, -•- 0.93 -D- 0.80 :-•- 0.90 0.93, and 0.92.
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