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Inactivation of Escherichia coli O157:H7 and Salmonella spp. on alfalfa seeds by caprylic acid and monocaprylin
International Journal of Food Microbiology 144 (2010) 141–146
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Inactivation of Escherichia coli O157:H7 and Salmonella spp. on alfalfa seeds by caprylic acid and monocaprylin Su-sen Chang, Mauricio Redondo-Solano, Harshavardhan Thippareddi ⁎ Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE 68583, United States
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Article history: Received 24 April 2010 Received in revised form 9 September 2010 Accepted 16 September 2010 Keywords: Antimicrobials Caprylic acid Monocaprylin Alfalfa seeds Food safety
a b s t r a c t Alfalfa and other seed sprouts have been implicated in several Escherichia coli O157:H7 and Salmonella spp. human illness outbreaks in the U.S. Continuing food safety issues with alfalfa seeds necessitate the need for discovery and use of novel and effective antimicrobials. The potential use of caprylic acid (CA) and monocaprylin (MC) for reducing E. coli O157:H7 and Salmonella spp. populations on alfalfa seeds was evaluated. The effectiveness of three concentrations of CA and MC (25, 50, and 75 mM) to reduce E. coli O157: H7 and Salmonella spp. populations in 0.1% peptone water and on alfalfa seeds was evaluated. Surviving populations of E. coli O157:H7 and Salmonella spp. were enumerated by direct plating on tryptic soy agar (TSA). Non-inoculated alfalfa seeds were soaked for up to 120 min to evaluate the effect of CA and MC solutions on seed germination rate. For planktonic cells, the efficacy of the treatments was: 75 MC N 50 MC N 25 MC N 75 CA N 50 CA N 25 CA. Both E. coli O157:H7 and Salmonella spp. were reduced to below the detection limit (0.6 log CFU/ml) within 10 min of exposure to 75 MC from initial populations of 7.65 ± 0.10 log CFU/ml and 7.71 ± 0.11 log CFU/ml, respectively. Maximum reductions of 1.56 ± 0.25 and 2.56 ± 0.17 log CFU/g for E. coli O157:H7 and Salmonella spp., respectively, were achieved on inoculated alfalfa seeds (from initial populations of 4.74 ± 0.62 log CFU/g and 5.27 ± 0.20 log CFU/g, respectively) when treated with 75 MC for 90 min. Germination rates of CA or MC treated seeds ranged from 84% to 99%. The germination rates of CA or MC soaked seeds and water soaked seeds (control) were similar (P N 0.05) for soaking times of ≤ 90 min. Monocaprylin (75 mM) can be used to reduce E. coli O157:H7 and Salmonella spp. on alfalfa seeds without compromising seed viability. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Consumption of raw seed sprouts has increased worldwide as a result of changes in consumer preference for healthy, natural foods (Neetoo et al., 2009). While alfalfa sprouts are the primary sprout product associated with foodborne outbreaks, sprouts from other seeds such as clover, mung bean, radish, and mustard cress have also been reported (Scouten and Beuchat, 2002). The United States Food and Drug Administration (FDA) recommends sanitizing seeds with 20,000 ppm of free chlorine from Ca(OCl)2 (or with an equivalent approved antimicrobial treatment) for 15 min and monitoring spent irrigation water for Escherichia coli O157:H7 and Salmonella spp. as a means to reduce the risk of foodborne illness from sprouts (FDA, 1999a,b). Sprouts pose a unique challenge to the fresh produce industry. The temperature and high humidity conditions maintained during the 4–7day sprouting process are favorable for the proliferation of contaminating pathogens (Fett, 2000). Foodborne pathogens can reach very high
⁎ Corresponding author. Tel.: + 1 402 472 3403; fax: + 1 402 472 1693. E-mail address: [email protected] (H. Thippareddi). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.09.011
populations (N5–6 log CFU/g) from low populations (b1 log CFU/g) during the sprouting process (Stewart et al., 2001). In addition, pathogens present on seeds can be internalized within the sprout and become protected from post-harvest sanitation (Itoh et al., 1998; Warriner et al., 2003). Elimination of contaminating foodborne pathogens is necessary to ensure the safety of raw sprouts due to the ability of pathogens to rapidly proliferate during the sprouting process. Non-esterified fatty acids and their monoglycerides are effective against a wide spectrum of pathogens including bacteria, enveloped viruses, and parasites. Although considerable data is available on the antimicrobial properties of fatty acids, most research has focused on either short chain (containing 6 carbons or less) or long chain (containing more than 10 carbons) fatty acids. Caprylic acid (octanoic acid) is an eight carbon fatty acid naturally present in human breast milk, bovine milk, and coconut oil (Jensen et al., 1990; Jensen, 2002; Nair et al., 2005; Sprong et al., 2001), and approved by the FDA under CFR 184.1025 as generally recognized as safe (GRAS) for use in food processing. Caprylic acid and its monoglycerol, monocaprylin, were found to inactivate infant pathogens respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), Haemophilus influenzae, Group B streptococcus (Isaacs et al., 1995), and major bovine mastitis pathogens (Streptococcus agalactiae, S. dysgalactiae, S. uberis, Staphylococcus aureus,
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and E. coli; Nair et al., 2005), and others (Edwardsiella ictaluri, E. tarda, S. iniae, and Yersinia ruckeri; Kollanoor et al., 2007). The effectiveness of CA and MC against foodborne pathogens E. coli O157:H7, L. monocytogenes, Cronobacter sakazakii, and Bacillus cereus in milk has also been reported (Jang and Rhee, 2009; Nair et al., 2004). Despite the extensive research on decontamination of sprout seeds, no chemical sanitizer has been effective in eliminating E. coli O157:H7 or Salmonella spp. from the surfaces of the sprout seeds. The objective of the current study was to evaluate the potential use of caprylic acid and monocaprylin as alternative sanitizers for reducing the E. coli O157:H7 and Salmonella spp. populations on alfalfa seeds for sprout manufacture. 2. Materials and methods 2.1. Bacterial strains and preparation of bacterial cocktails E. coli O157:H7 strains (TR-4, 70/30-4, 70/30-5, 91/9-4, 91/9-5) and Salmonella serotypes (Thompson, Enteritidis PT 4, Hadar, Montevideo, and Heidelberg) were obtained from the Applied Food Safety Laboratory Culture Collection at the University of Nebraska (Lincoln, NE). The cultures were stored in tryptic soy broth (TSB; Difco, Becton Dickinson Co., Sparks, MD) containing 40% glycerol (w/v) at −80 °C prior to use. Frozen stock cultures of each serotype was inoculated individually into TSB (9 ml) and incubated for 24 h at 35 °C. Two subsequent transfers into fresh TSB were performed every 24 h to prepare fresh cultures. Following incubation, strain/serotypes of each bacterium were combined in equal volumes into a sterile centrifuge tube and centrifuged for 10 min at 4 °C, 6000 ×g (Model GS-15R; Beckman Instruments, Palo Alto, CA). The supernatant was discarded and the cells in the pellet were resuspended in 0.1% peptone water (PW) to the original volume. All bacterial cocktails were prepared on the day of the experiment and used immediately. 2.2. Preparation of inoculated alfalfa seeds Alfalfa seeds were obtained from the International Specialty Supply (Cookeville, TN) and stored at room temperature. Bacterial cocktails (10 ml) were transferred to sterile twist tie sample bags (7 in. × 12 in.; Fisher, Pittsburgh, PA) containing 90 ml PW. Alfalfa seeds (100 g) were added to the cell suspension, gently agitated, and soaked at room temperature for 5 min. Seeds were separated from the cell suspension by pouring the mixture over a double layer of cheese cloth and the seeds were dried in a biosafety cabinet (Bellco Glass, Inc., Vineland, NJ) for 24 h with the fan switched on. Dried seeds were transferred into sterile twist tie sample bags (7 in. × 12 in.; Fisher, Pittsburgh, PA) and stored at ambient temperature (25 °C) until use. 2.3. Inactivation of planktonic E. coli O157:H7 and Salmonella spp. by caprylic acid and monocaprylin Caprylic acid (CA) and monocaprylin (MC) used in this study were obtained from Sigma-Aldrich (St. Louis, MO). Caprylic acid and monocaprylin were added to citrate phosphate buffer (pH 7.0, 7.2, 7.8, or 8.0) containing 0.1% Tween 80 to obtain final concentrations of 25, 50, or 75 mM. All solutions were adjusted to pH 7.0 using 1 N NaOH. Citrate phosphate buffer (pH 7.0) and citrate phosphate buffer (pH 7.0) containing 0.1% Tween 80 were used as the control and internal control for CA and MC, respectively. Aliquots (1 ml) of the prepared bacterial cocktails were added to 9 ml of each treatment solution to achieve an initial inoculum concentration of approximately 7.5 log CFU/ml. Surviving populations of each pathogen were enumerated following exposure to treatment solutions for 0, 1 min, 10 min, 1 h, 6 h, or 12 h at ambient temperature (25 °C) by removing 1 ml aliquots and surface plating the appropriate dilution onto tryptic soy agar (TSA; Difco, Becton Dickinson Co., Sparks, MD) and either sorbitol MacConkey agar (Difco,
Becton Dickinson Co., Sparks, MD) or xylose lysine deoxycholate (Difco, Becton Dickinson Co., Sparks, MD) for E. coli O157:H7 and Salmonella spp., respectively. Typical colonies were enumerated following incubation at 35 °C for 24 h. Three independent replications were conducted for each organism. 2.4. Effect of caprylic acid and monocaprylin on alfalfa seed viability Non-inoculated alfalfa seeds (0.25 g; ca. 100 seeds) were combined with 2.5 ml of each treatment solution in nylon pouches (2.5 in. × 5 in.; 3 mil standard barrier, water vapor transmission rate—10 g/L/m2/24 h at 37.8 °C and 100% relative humidity, oxygen transmission rate— 3000 cm3/L/m2/24 h at 23 °C and 1 atm [101.29 kPa]; Prime Source, Kansas City, MO) and soaked at ambient temperature (25 °C) for 0, 30, 60, 90, or 120 m. Water was used as the control treatment. Following each soaking period, the seeds were spread evenly on a pre-wetted germination apparatus. The apparatus was constructed of a pipe (PVC, 7.6 cm dia and 6.0 cm length) and a coupling of the same diameter (PVC, 6.0 cm length). A fiberglass screen was placed around the top of the PVC pipe and the pipe was inserted into the coupling (total length of the apparatus was 9 cm). All materials were obtained from a local home supply store. The fiberglass screen supported the sprout seed, and when the apparatus was placed in the water bath, the screen was ca. 6 cm from the bottom of the water bath and ca. 3 cm from the surface of the water. The apparatus was placed in a water bath and the water level was maintained below the level of the seeds to maintain humidity within the chamber. The water bath was covered with a lid and the seeds were allowed to germinate at room temperature for 5 days. With the exception of day 0, seeds were regularly misted with water 5 times throughout the day and covered immediately to maintain humidity of the germination environment. The number of germinated (characterized by the emergence of the radicle) and non-germinated seeds was enumerated after 5 days, and germination percentage was calculated. 2.5. Inactivation of E. coli O157:H7 and Salmonella spp. on alfalfa seeds by caprylic acid and monocaprylin Inoculated alfalfa seeds (1 g) prepared as previously described were weighed into nylon pouches (2.5 in. × 5 in.; 3 mil standard barrier, water vapor transmission rate—10 g/L/m2/24 h at 37.8 °C and 100% relative humidity, oxygen transmission rate—3000 cm3/L/m2/ 24 h at 23 °C and 1 atm [101.29 kPa]; Prime Source, Kansas City, MO), combined with 10 ml of 25, 50, and 75 mM CA or MC solution and soaked at 4 °C for 0, 30, 60, and 90 min. Peptone water (0.1%) was used as the control. Following each soak period, the treatment solution was discarded, and 10 ml of Dey/Engley (D/E) neutralizing broth (Difco, Becton Dickinson Co., Sparks, MD) was added to each pouch to terminate the antimicrobial effect of CA and MC as suggested by Burnett et al. (2007). The seeds were soaked in the D/E neutralizing broth for an additional 90, 60, 30, or 0 min for initial soak times of 0, 30, 60, and 90 min, respectively, at 4 °C so that the total soak time for each pouch was 90 min. Following treatment, the pouches were stomached in a masticator (Neu-Tech Group, Farmingdale, NY) for 2 min and the resulting solution was used for microbial analysis. Surviving populations of each pathogen were enumerated by surface plating the appropriate dilution onto SMAC or XLD for E. coli O157:H7 and Salmonella spp., respectively. The samples were also plated onto TSA to evaluate potential injury to the cells due to exposure to CA and MC. Typical colonies were enumerated following incubation at 35 °C for 24 h. The experiment was replicated three times. 2.6. Statistical analysis All experiments were replicated three times. Triplicate data were analyzed by the ANOVA procedure (α = 0.05) using Minitab® 15 (Minitab Inc, State College, PA). When significant differences among
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the means were detected, Tukey's pairwise comparison was performed to determine differences in E. coli O157:H7 or Salmonella spp. populations as well as differences in the germination rates of seeds as affected by treatment.
3. Results Inactivation of E. coli O157:H7 and Salmonella spp. by caprylic acid and monocaprylin in suspension (CPB; final pH 7.0) was dependent on the antimicrobial, concentration, and exposure time (Figs. 1 and 2). Addition of 0.1% Tween 80 as an emulsifier to facilitate dispersion of caprylic acid and monocaprylin did not affect (P N 0.05) E. coli O157: H7 and Salmonella spp. populations. E. coli O157:H7 population was reduced to b0.6 log CFU/ml from initial population of 7.60 ± 0.24 log CFU/ml within 1 min of exposure to 50 mM and 75 mM MC, and within 10 min of exposure to 25 mM MC (Fig. 1). The antimicrobial efficacy of caprylic acid was lower (P ≤ 0.05) compared to monocaprylin. E. coli O157:H7 population was reduced (P ≤ 0.05) by 75 mM CA to 1.33 log CFU/ml within 10 min and reached undetectable levels within 6 h. Reductions (P ≤ 0.05) in E. coli O157:H7 were observed with 50 mM CA after 1 h and a maximum reduction level of 2.57 ± 0.45 log CFU/ml was observed. A maximum E. coli O157:H7 reduction of 0.75 ± 0.26 log CFU/ml was observed with 25 mM CA. Compared to E. coli O157:H7, Salmonella spp. was more resistant to monocaprylin and more susceptible to caprylic acid (Fig. 2). Greater reductions of Salmonella spp. were observed with longer exposure times, higher concentrations, and with monocaprylin compared to caprylic acid at similar concentrations. Salmonella spp. populations were reduced to b0.6 log CFU/ml within 10 min, 1 h, and 6 h of exposure to 75 mM MC, 50 mM MC, and 25 mM MC/75 mM CA, respectively. Reduction (P ≤ 0.05) of Salmonella spp. was observed following treatments with 25 and 50 mM CA for 1 h. Maximum Salmonella spp. reductions of 1.28 ± 0.11 log CFU/ml and 2.31 ±0.59 log CFU/ml were achieved with 25 mM CA and 50 mM CA, respectively. The efficacy of treatment solutions on planktonic E. coli O157:H7 and Salmonella spp. in suspension by decreasing order is: 75 MC N 50 MC N 25 MC N 75 CA N 50 CA N 25 CA. Table 1 summarizes the effect of the selected treatment solutions on the viability of alfalfa seeds at room temperature. Differences in germination rates were not observed (P N 0.05) across all treatment solutions and soaking times for up to 90 min. Germination rates of alfalfa seeds were reduced (P ≤ 0.05) with soaking times ≥ 120 min for all treatments except 25 mM MC compared to control (water).
Fig. 2. Mean Salmonella spp. populations in suspension exposed to caprylic acid and monocaprylin at room temperature. Data within each treatment represent Salmonella spp. populations following 0 m (□), 1 m (m), 10 m (l), 1 h (V), 6 h (W), and 12 h (■) of treatment exposure. Treatments include control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA) at 25, 50, 75 mM, and monocaprylin (MC) at 25, 50, and 75 mM. Values are the mean of triplicates. Error bars represent ±standard deviation.
Based on these results, a maximum soak time of 90 min was selected for use in the seed decontamination study. The antimicrobial effect of caprylic acid and monocaprylin was lower on E. coli O157:H7 and Salmonella spp. on alfalfa seeds compared to cells suspended in buffer. Reduction of E. coli O157:H7 on alfalfa seeds by caprylic acid and monocaprylin at 4 °C is illustrated in Fig. 3. Within any given treatment, microbial reductions increased with increasing soak time. E. coli O157:H7 concentrations were lower (P ≤ 0.05) compared to control (time 0) when alfalfa seeds were soaked in 50 mM CA for ≥60 min, 75 mM CA for 90 min, 25 mM MC for ≥30 min, or 75 mM MC for ≥ 30 min. Maximum reduction of 1.56 ± 0.49 log CFU/g was achieved by soaking alfalfa seeds in 75 mM MC for 90 min at 4 °C. Monocaprylin was more effective in reducing E. coli O157:H7 populations on alfalfa seeds compared to caprylic acid. Following a 30 min soaking time, seeds treated with monocaprylin solutions had lower (P≤ 0.05) populations of E. coli O157:H7 than the control and caprylic acid treatments. With longer soaking times (≥60 min), all treatments except 25 mM CA had lower (P ≤ 0.05) E. coli O157:H7 populations compared to the control. For Salmonella spp., at any given treatment soaking time, antimicrobial efficacy of the treatments was 75 mM MC N 50 mM MC ≥ 75 mM CA ≈ 25 mM MC≥ 50 mM CA≥ 25 mM CA. Within a treatment antimicrobial, reductions of Salmonella spp. populations increased with longer soak time though differences (PN 0.05) could not always be detected. Greater reductions (P ≤ 0.05) of Salmonella spp. were observed when seeds were soaked in 75 mM MC for ≥60 min. The maximum Salmonella spp. reduction achieved on alfalfa seeds was 2.43 ± 0.36 log CFU/g. The most effective among all tested treatments for reducing E. coli O157:H7 and Salmonella spp. populations both in suspension and on alfalfa seeds was 75 mM MC. E. coli O157:H7 and Salmonella spp. populations were reduced to b0.6 log CFU/ml within 1 and 10 min of exposure to 75 mM MC, respectively, in CPB. Maximum reductions on alfalfa seeds treated with 75 mM MC for 90 min at 4 °C were 1.56 ± 0.49 log CFU/g for E. coli O157:H7 and 2.43 ± 0.36 log CFU/g for Salmonella spp. 4. Discussion
Fig. 1. Mean Escherichia coli O157:H7 populations in suspension exposed to caprylic acid and monocaprylin at room temperature. Data within each treatment represent E. coli O157:H7 populations following 0 m (□), 1 m (m), 10 m (l), 1 h (V), 6 h (W), and 12 h (■) of treatment exposure. Treatments include control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA) at 25, 50, 75 mM, and monocaprylin (MC) at 25, 50, and 75 mM. Values are the mean of triplicates. Error bars represent ±standard deviation.
The antimicrobial efficacy of fatty acids and their monoglycerides are well established (Bergsson et al., 1998, 1999; Isaacs et al., 1995; Kabara, 1979; Petschow et al., 1996). These antimicrobials occur naturally in foods such as milk, and are assumed to be non-toxic to mucosa (Bergsson et al., 1998). With the trend toward more natural
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Table 1 Alfalfa seed viability on exposure to various concentrations of caprylic acid and monocaprylin at room temperature. Treatment
Germination ratea(%) Soak time (min)
Water (control) 25 mM CA 50 mM CA 75 mM CA 25 mM MC 50 mM MC 75 mM MC a b c
0
30
60
90
120
96.98 ± 4.38Abac 98.14 ± 1.64Aa 96.96 ± 1.38Aa 96.54 ± 0.29Aa 97.98 ± 1.01Aa 96.93 ± 0.92Aa 98.44 ± 0.36Aa
97.44 ± 3.60Aa 97.76 ± 2.23Aa 96.33 ± 1.38Aa 96.19 ± 3.60Aa 94.72 ± 4.51Aa 95.11 ± 1.67Aa 98.67 ± 0.38Aa
97.46 ± 2.32Aa 95.51 ± 2.30Aa 94.68 ± 1.24Aa 96.84 ± 1.06Aa 95.02 ± 3.34Aa 90.67 ± 2.72Aa 96.00 ± 2.10Aa
98.00 ± 2.14Aa 88.76 ± 6.10Aa 96.64 ± 1.17Aa 94.31 ± 4.52Aa 92.99 ± 9.61Aa 88.27 ± 3.32Aa 96.12 ± 1.67Aa
98.39 ± 1.58Aa 84.13 ± 3.59Bb 88.62 ± 7.64Ab 84.25 ± 4.41Bb 96.72 ± 1.39Aa 88.10 ± 2.90Ab 88.07 ± 5.81Ab
Data represent mean ± standard deviation of three independent trials. Means within a row with the same upper case letter are not statistically different (P N 0.05). Means within a column with the same lower case letter are not statistically different (P N 0.05).
foods, there is renewed interest in the use of fatty acids and their derivatives as alternatives to synthetic antimicrobials. Higher biological activity of monoglycerols compared to their corresponding fatty acid is well established (Kabara et al., 1977; Lieberman et al., 2006). The enhanced effectiveness monocaprylin over caprylic acid under identical treatment conditions demonstrated in this study further supports these observations. Nair et al. (2004) reported E. coli O157:H7 surviving populations of 5.44 ± 0.27 log CFU/ ml and b1 log CFU/ml after exposure to 25 mM CA and 25 mM MC for 6 h at 37 °C in milk. Monocaprylin was more effective than caprylic acid in reducing populations of foodborne pathogens E. coli O157:H7 and L. monocytogenes (Nair et al., 2004), the mastitic pathogens S. agalactiae, S. dysgalactiae, S. uberis, S. aureus, as well as the enveloped viruses (Nair et al., 2005; Thormar et al., 1987). The differences in the efficacies of caprylic acid and monocaprylin may be attributed to the differences in their respective modes of action and the effect of substrate pH on their activity. The primary mode of action for microbial inactivation by medium and short chain fatty acids is the diffusion of undissociated acids across the bacterial cells and intracellular acidification as the acids dissociate within the protoplasm (Sun et al., 1998). The acidification of the protoplasm can lead to inactivation of intracellular enzymes (Viegas and Sa-Correia, 1991), inhibition of amino-acid transport (Freese et al., 1973), and dissipation of the electrochemical proton gradient, leading to the depletion of energy reserves in bacterial cells (Galbraith and Miller, 1973). Secondary mechanisms such as disintegration of plasma membrane or fusion of cell membranes have also been reported (Ahkong et al., 1973; Thormar et al., 1987). Monoglycerides of fatty
Fig. 3. Mean E. coli O157:H7 populations on alfalfa seeds exposed to caprylic acid and monocaprylin at 4 °C. Data within each treatment represent E. coli O157:H7 populations following 0 m (□), 30 m (m), 60 m (l), and 90 m (■) of exposure at 4 °C. Treatments include control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA) at 25, 50, 75 mM, and monocaprylin (MC) at 25, 50, and 75 mM. Values are the mean of triplicates. Error bars represent ± standard deviation.
acids, on the other hand, are non-ionic surfactants that function mainly by penetrating and incorporating themselves into the plasma membranes, causing destabilization of the membrane bilayer, changes in membrane permeability (Cullis and Hope, 1978; Nair et al., 2004), and interference with signal transduction during cell replication (Projan et al., 1994). Hence, the antimicrobial mechanism of fatty acid monoglycerides is not pH dependent (Isaacs and Lampe, 2000; Sun et al., 2003). Caprylic acid has a pKa of 4.86 and would exist primarily in the dissociated form at the pH of the medium (pH = 7.0) used in this study. Thus, reduction of bacterial cells by caprylic acid would be limited to secondary mechanisms such as membrane disintegration. Greater release of bacterial cells from seeds with longer soak time is commonly reported in sprout seed-related research (Neetoo et al., 2008; Sharma et al., 2002; Sharma and Demirci, 2003). As seeds imbibe water, swelling exposes and releases microorganisms sequestered in the cracks, crevices, or other microenvironments of seeds, resulting in an “increased” microbial population (Charkowski et al., 2001). To evaluate the true antimicrobial efficacy of caprylic acid and monocaprylin, bacterial reduction should be compared to the total microbial population on the seeds, not only the microbial cells released into the soaking medium for a given exposure time. A preliminary experiment was conducted to evaluate if total soak times of ≥90 min would be necessary to achieve the maximum release of bacterial counts (results not shown). Therefore, a unique soaking process was adopted for this experiment, which included soaking the seeds in the selected treatment for the specified time, decanting the soak solution, and then further soaking the seeds in DE broth until the total solution soak time of 90 min was reached. To minimize the microbial growth during this soak period, the solutions were maintained at 4 °C. This method allowed enumeration of the true reduction based on the total bacterial count. Limited bactericidal effect of caprylic acid and monocaprylin was observed for E. coli O157:H7 and Salmonella spp. in inoculated alfalfa seeds compared to planktonic cells and microbial reductions ranged between 1.56 ± 0.49 and 2.43 ± 0.36 log CFU/g (Figs. 3 and 4). Several factors may limit the antimicrobial effect of caprylic acid and monocaprylin on inoculated alfalfa seeds, including the reduced bioavailability of the compounds at refrigeration temperatures, and the protective effect of the seed structures. As previously discussed, the primary site of action for caprylic acid and monocaprylin at pH 7.0 is the bacterial plasma membrane (Freese et al., 1973; Bergsson et al., 1998, 2001). To achieve microbial reductions, the antimicrobials must remain in solution for attachment to occur (Galbraith et al., 1971). Under refrigeration temperatures, caprylic acid and monocaprylin have lower solubility (Nair et al., 2004), reducing the attachment to microbial cells, thus reducing their antimicrobial efficacy. Changes in the bacterial cell membrane fluidity under refrigeration may also reduce the antimicrobial efficacy of caprylic acid and monocaprylin (Nair et al., 2004). Cell membranes are more rigid at lower temperatures, and are less likely to facilitate the incorporation of
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References
Fig. 4. Mean Salmonella spp. populations on alfalfa seeds exposed to caprylic acid and monocaprylin at 4 °C. Data within each treatment represent Salmonella spp. survival following 0 m (□), 30 m (m), 60 m (l), and 90 m (■) of exposure to control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA; 25, 50, 75 mM), or monocaprylin (MC; 25, 50, and 75 mM). Values are the mean of triplicates. Error bars represent ± standard deviation.
fatty acids or monoglycerides into the membrane structure, making disruption of the plasma membrane less likely to occur. Another plausible explanation for the decreased efficacy could be the reduced bioavailability of the antimicrobials due to the formation of complexes with other compounds (Glassman, 1949; Wang and Johnson, 1992). Proteins, especially the lipophilic proteins such as albumin, and other nutrients, including fat and starch, can interact with the fatty acids and monoglycerides and reduce their bioavailability (Kabara, 1979). In addition, it is well recognized that microorganisms are protected by the seed structure (Sharma et al., 2002; Sharma and Demirchi, 2003; Neetoo et al., 2008). Microbial cells lodged within the crevices or wrinkles of the seeds are inaccessible to the antimicrobials and survive the exposure to chemical disinfectants. In addition, alfalfa seeds (3–85%) may be cracked or may miss part of the testa (Sharma and Demirchi, 2003). Microbial cells could be internalized within the seed structure through these cracks, and shield them from the sanitizing treatments. Reductions in E. coli O157:H7 and Salmonella spp. populations on alfalfa seeds observed in this study are comparable to the reductions reported for other anitmicrobials in the literature. Acidified NaClO2 (500 ppm) was effective in reducing populations of E. coli O157:H7 from 2.7 log CFU/g to b0.5 log CFU/g (Taormina and Beuchat, 1999). When electrolyzed oxidizing water was tested, the most effective treatment without affecting germination was 19 A for 32 min, corresponding to a 91.6% reduction in E. coli O157:H7 on alfalfa seeds (Sharma and Demirci, 2003). Ozone sparging reduced E. coli O157:H7 up to 2.21 log CFU/g after a 64-min contact time (Sharma et al., 2002). A 3-log destruction of Salmonella spp. on alfalfa seed following 10-min dips in 2000 μg/ml of sodium hypochlorite, 6% hydrogen peroxide, or 80% ethanol was reported by Beuchat (1997). Immersion of seeds in 8% H2O2, 1% Ca (OH)2, and 1% calcinated calcium for 10 min also reduced Salmonella spp. by 2.8–3.2 log CFU/g without compromising the germination rate (Weissinger and Beuchat, 2000). Reductions in Salmonella spp. populations on seeds treated with 20,000 ppm of chlorine or Fit (alkaline solution consisting of water, oleic acid, glycerol, ethanol, potassium hydroxide, sodium bicarbonate, citric acid, and distilled grapefruit oil) for 30 min ranged from 2.3 to 2.5 log CFU/g and 1.7 to 2.3 log CFU/g, respectively (Beuchat et al., 2001). To date, chemical sanitizers that can eliminate E. coli O157:H7 and Salmonella spp. from the surface of sprout seeds are not available. However, for practical applicability in the industry, soaking with chemical sanitizers remains the method of choice that is simple and realistic. Monocaprylin (75 mM) can be used as an alternative sanitizer to reduce E. coli O157:H7 and Salmonella spp. population on alfalfa seeds without compromising the seed germination rates.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Inactivation of Escherichia coli O157:H7 and Salmonella spp. on alfalfa seeds by caprylic acid and monocaprylin Su-sen Chang, Mauricio Redondo-Solano, Harshavardhan Thippareddi ⁎ Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE 68583, United States
a r t i c l e
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Article history: Received 24 April 2010 Received in revised form 9 September 2010 Accepted 16 September 2010 Keywords: Antimicrobials Caprylic acid Monocaprylin Alfalfa seeds Food safety
a b s t r a c t Alfalfa and other seed sprouts have been implicated in several Escherichia coli O157:H7 and Salmonella spp. human illness outbreaks in the U.S. Continuing food safety issues with alfalfa seeds necessitate the need for discovery and use of novel and effective antimicrobials. The potential use of caprylic acid (CA) and monocaprylin (MC) for reducing E. coli O157:H7 and Salmonella spp. populations on alfalfa seeds was evaluated. The effectiveness of three concentrations of CA and MC (25, 50, and 75 mM) to reduce E. coli O157: H7 and Salmonella spp. populations in 0.1% peptone water and on alfalfa seeds was evaluated. Surviving populations of E. coli O157:H7 and Salmonella spp. were enumerated by direct plating on tryptic soy agar (TSA). Non-inoculated alfalfa seeds were soaked for up to 120 min to evaluate the effect of CA and MC solutions on seed germination rate. For planktonic cells, the efficacy of the treatments was: 75 MC N 50 MC N 25 MC N 75 CA N 50 CA N 25 CA. Both E. coli O157:H7 and Salmonella spp. were reduced to below the detection limit (0.6 log CFU/ml) within 10 min of exposure to 75 MC from initial populations of 7.65 ± 0.10 log CFU/ml and 7.71 ± 0.11 log CFU/ml, respectively. Maximum reductions of 1.56 ± 0.25 and 2.56 ± 0.17 log CFU/g for E. coli O157:H7 and Salmonella spp., respectively, were achieved on inoculated alfalfa seeds (from initial populations of 4.74 ± 0.62 log CFU/g and 5.27 ± 0.20 log CFU/g, respectively) when treated with 75 MC for 90 min. Germination rates of CA or MC treated seeds ranged from 84% to 99%. The germination rates of CA or MC soaked seeds and water soaked seeds (control) were similar (P N 0.05) for soaking times of ≤ 90 min. Monocaprylin (75 mM) can be used to reduce E. coli O157:H7 and Salmonella spp. on alfalfa seeds without compromising seed viability. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Consumption of raw seed sprouts has increased worldwide as a result of changes in consumer preference for healthy, natural foods (Neetoo et al., 2009). While alfalfa sprouts are the primary sprout product associated with foodborne outbreaks, sprouts from other seeds such as clover, mung bean, radish, and mustard cress have also been reported (Scouten and Beuchat, 2002). The United States Food and Drug Administration (FDA) recommends sanitizing seeds with 20,000 ppm of free chlorine from Ca(OCl)2 (or with an equivalent approved antimicrobial treatment) for 15 min and monitoring spent irrigation water for Escherichia coli O157:H7 and Salmonella spp. as a means to reduce the risk of foodborne illness from sprouts (FDA, 1999a,b). Sprouts pose a unique challenge to the fresh produce industry. The temperature and high humidity conditions maintained during the 4–7day sprouting process are favorable for the proliferation of contaminating pathogens (Fett, 2000). Foodborne pathogens can reach very high
⁎ Corresponding author. Tel.: + 1 402 472 3403; fax: + 1 402 472 1693. E-mail address: [email protected] (H. Thippareddi). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.09.011
populations (N5–6 log CFU/g) from low populations (b1 log CFU/g) during the sprouting process (Stewart et al., 2001). In addition, pathogens present on seeds can be internalized within the sprout and become protected from post-harvest sanitation (Itoh et al., 1998; Warriner et al., 2003). Elimination of contaminating foodborne pathogens is necessary to ensure the safety of raw sprouts due to the ability of pathogens to rapidly proliferate during the sprouting process. Non-esterified fatty acids and their monoglycerides are effective against a wide spectrum of pathogens including bacteria, enveloped viruses, and parasites. Although considerable data is available on the antimicrobial properties of fatty acids, most research has focused on either short chain (containing 6 carbons or less) or long chain (containing more than 10 carbons) fatty acids. Caprylic acid (octanoic acid) is an eight carbon fatty acid naturally present in human breast milk, bovine milk, and coconut oil (Jensen et al., 1990; Jensen, 2002; Nair et al., 2005; Sprong et al., 2001), and approved by the FDA under CFR 184.1025 as generally recognized as safe (GRAS) for use in food processing. Caprylic acid and its monoglycerol, monocaprylin, were found to inactivate infant pathogens respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), Haemophilus influenzae, Group B streptococcus (Isaacs et al., 1995), and major bovine mastitis pathogens (Streptococcus agalactiae, S. dysgalactiae, S. uberis, Staphylococcus aureus,
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and E. coli; Nair et al., 2005), and others (Edwardsiella ictaluri, E. tarda, S. iniae, and Yersinia ruckeri; Kollanoor et al., 2007). The effectiveness of CA and MC against foodborne pathogens E. coli O157:H7, L. monocytogenes, Cronobacter sakazakii, and Bacillus cereus in milk has also been reported (Jang and Rhee, 2009; Nair et al., 2004). Despite the extensive research on decontamination of sprout seeds, no chemical sanitizer has been effective in eliminating E. coli O157:H7 or Salmonella spp. from the surfaces of the sprout seeds. The objective of the current study was to evaluate the potential use of caprylic acid and monocaprylin as alternative sanitizers for reducing the E. coli O157:H7 and Salmonella spp. populations on alfalfa seeds for sprout manufacture. 2. Materials and methods 2.1. Bacterial strains and preparation of bacterial cocktails E. coli O157:H7 strains (TR-4, 70/30-4, 70/30-5, 91/9-4, 91/9-5) and Salmonella serotypes (Thompson, Enteritidis PT 4, Hadar, Montevideo, and Heidelberg) were obtained from the Applied Food Safety Laboratory Culture Collection at the University of Nebraska (Lincoln, NE). The cultures were stored in tryptic soy broth (TSB; Difco, Becton Dickinson Co., Sparks, MD) containing 40% glycerol (w/v) at −80 °C prior to use. Frozen stock cultures of each serotype was inoculated individually into TSB (9 ml) and incubated for 24 h at 35 °C. Two subsequent transfers into fresh TSB were performed every 24 h to prepare fresh cultures. Following incubation, strain/serotypes of each bacterium were combined in equal volumes into a sterile centrifuge tube and centrifuged for 10 min at 4 °C, 6000 ×g (Model GS-15R; Beckman Instruments, Palo Alto, CA). The supernatant was discarded and the cells in the pellet were resuspended in 0.1% peptone water (PW) to the original volume. All bacterial cocktails were prepared on the day of the experiment and used immediately. 2.2. Preparation of inoculated alfalfa seeds Alfalfa seeds were obtained from the International Specialty Supply (Cookeville, TN) and stored at room temperature. Bacterial cocktails (10 ml) were transferred to sterile twist tie sample bags (7 in. × 12 in.; Fisher, Pittsburgh, PA) containing 90 ml PW. Alfalfa seeds (100 g) were added to the cell suspension, gently agitated, and soaked at room temperature for 5 min. Seeds were separated from the cell suspension by pouring the mixture over a double layer of cheese cloth and the seeds were dried in a biosafety cabinet (Bellco Glass, Inc., Vineland, NJ) for 24 h with the fan switched on. Dried seeds were transferred into sterile twist tie sample bags (7 in. × 12 in.; Fisher, Pittsburgh, PA) and stored at ambient temperature (25 °C) until use. 2.3. Inactivation of planktonic E. coli O157:H7 and Salmonella spp. by caprylic acid and monocaprylin Caprylic acid (CA) and monocaprylin (MC) used in this study were obtained from Sigma-Aldrich (St. Louis, MO). Caprylic acid and monocaprylin were added to citrate phosphate buffer (pH 7.0, 7.2, 7.8, or 8.0) containing 0.1% Tween 80 to obtain final concentrations of 25, 50, or 75 mM. All solutions were adjusted to pH 7.0 using 1 N NaOH. Citrate phosphate buffer (pH 7.0) and citrate phosphate buffer (pH 7.0) containing 0.1% Tween 80 were used as the control and internal control for CA and MC, respectively. Aliquots (1 ml) of the prepared bacterial cocktails were added to 9 ml of each treatment solution to achieve an initial inoculum concentration of approximately 7.5 log CFU/ml. Surviving populations of each pathogen were enumerated following exposure to treatment solutions for 0, 1 min, 10 min, 1 h, 6 h, or 12 h at ambient temperature (25 °C) by removing 1 ml aliquots and surface plating the appropriate dilution onto tryptic soy agar (TSA; Difco, Becton Dickinson Co., Sparks, MD) and either sorbitol MacConkey agar (Difco,
Becton Dickinson Co., Sparks, MD) or xylose lysine deoxycholate (Difco, Becton Dickinson Co., Sparks, MD) for E. coli O157:H7 and Salmonella spp., respectively. Typical colonies were enumerated following incubation at 35 °C for 24 h. Three independent replications were conducted for each organism. 2.4. Effect of caprylic acid and monocaprylin on alfalfa seed viability Non-inoculated alfalfa seeds (0.25 g; ca. 100 seeds) were combined with 2.5 ml of each treatment solution in nylon pouches (2.5 in. × 5 in.; 3 mil standard barrier, water vapor transmission rate—10 g/L/m2/24 h at 37.8 °C and 100% relative humidity, oxygen transmission rate— 3000 cm3/L/m2/24 h at 23 °C and 1 atm [101.29 kPa]; Prime Source, Kansas City, MO) and soaked at ambient temperature (25 °C) for 0, 30, 60, 90, or 120 m. Water was used as the control treatment. Following each soaking period, the seeds were spread evenly on a pre-wetted germination apparatus. The apparatus was constructed of a pipe (PVC, 7.6 cm dia and 6.0 cm length) and a coupling of the same diameter (PVC, 6.0 cm length). A fiberglass screen was placed around the top of the PVC pipe and the pipe was inserted into the coupling (total length of the apparatus was 9 cm). All materials were obtained from a local home supply store. The fiberglass screen supported the sprout seed, and when the apparatus was placed in the water bath, the screen was ca. 6 cm from the bottom of the water bath and ca. 3 cm from the surface of the water. The apparatus was placed in a water bath and the water level was maintained below the level of the seeds to maintain humidity within the chamber. The water bath was covered with a lid and the seeds were allowed to germinate at room temperature for 5 days. With the exception of day 0, seeds were regularly misted with water 5 times throughout the day and covered immediately to maintain humidity of the germination environment. The number of germinated (characterized by the emergence of the radicle) and non-germinated seeds was enumerated after 5 days, and germination percentage was calculated. 2.5. Inactivation of E. coli O157:H7 and Salmonella spp. on alfalfa seeds by caprylic acid and monocaprylin Inoculated alfalfa seeds (1 g) prepared as previously described were weighed into nylon pouches (2.5 in. × 5 in.; 3 mil standard barrier, water vapor transmission rate—10 g/L/m2/24 h at 37.8 °C and 100% relative humidity, oxygen transmission rate—3000 cm3/L/m2/ 24 h at 23 °C and 1 atm [101.29 kPa]; Prime Source, Kansas City, MO), combined with 10 ml of 25, 50, and 75 mM CA or MC solution and soaked at 4 °C for 0, 30, 60, and 90 min. Peptone water (0.1%) was used as the control. Following each soak period, the treatment solution was discarded, and 10 ml of Dey/Engley (D/E) neutralizing broth (Difco, Becton Dickinson Co., Sparks, MD) was added to each pouch to terminate the antimicrobial effect of CA and MC as suggested by Burnett et al. (2007). The seeds were soaked in the D/E neutralizing broth for an additional 90, 60, 30, or 0 min for initial soak times of 0, 30, 60, and 90 min, respectively, at 4 °C so that the total soak time for each pouch was 90 min. Following treatment, the pouches were stomached in a masticator (Neu-Tech Group, Farmingdale, NY) for 2 min and the resulting solution was used for microbial analysis. Surviving populations of each pathogen were enumerated by surface plating the appropriate dilution onto SMAC or XLD for E. coli O157:H7 and Salmonella spp., respectively. The samples were also plated onto TSA to evaluate potential injury to the cells due to exposure to CA and MC. Typical colonies were enumerated following incubation at 35 °C for 24 h. The experiment was replicated three times. 2.6. Statistical analysis All experiments were replicated three times. Triplicate data were analyzed by the ANOVA procedure (α = 0.05) using Minitab® 15 (Minitab Inc, State College, PA). When significant differences among
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the means were detected, Tukey's pairwise comparison was performed to determine differences in E. coli O157:H7 or Salmonella spp. populations as well as differences in the germination rates of seeds as affected by treatment.
3. Results Inactivation of E. coli O157:H7 and Salmonella spp. by caprylic acid and monocaprylin in suspension (CPB; final pH 7.0) was dependent on the antimicrobial, concentration, and exposure time (Figs. 1 and 2). Addition of 0.1% Tween 80 as an emulsifier to facilitate dispersion of caprylic acid and monocaprylin did not affect (P N 0.05) E. coli O157: H7 and Salmonella spp. populations. E. coli O157:H7 population was reduced to b0.6 log CFU/ml from initial population of 7.60 ± 0.24 log CFU/ml within 1 min of exposure to 50 mM and 75 mM MC, and within 10 min of exposure to 25 mM MC (Fig. 1). The antimicrobial efficacy of caprylic acid was lower (P ≤ 0.05) compared to monocaprylin. E. coli O157:H7 population was reduced (P ≤ 0.05) by 75 mM CA to 1.33 log CFU/ml within 10 min and reached undetectable levels within 6 h. Reductions (P ≤ 0.05) in E. coli O157:H7 were observed with 50 mM CA after 1 h and a maximum reduction level of 2.57 ± 0.45 log CFU/ml was observed. A maximum E. coli O157:H7 reduction of 0.75 ± 0.26 log CFU/ml was observed with 25 mM CA. Compared to E. coli O157:H7, Salmonella spp. was more resistant to monocaprylin and more susceptible to caprylic acid (Fig. 2). Greater reductions of Salmonella spp. were observed with longer exposure times, higher concentrations, and with monocaprylin compared to caprylic acid at similar concentrations. Salmonella spp. populations were reduced to b0.6 log CFU/ml within 10 min, 1 h, and 6 h of exposure to 75 mM MC, 50 mM MC, and 25 mM MC/75 mM CA, respectively. Reduction (P ≤ 0.05) of Salmonella spp. was observed following treatments with 25 and 50 mM CA for 1 h. Maximum Salmonella spp. reductions of 1.28 ± 0.11 log CFU/ml and 2.31 ±0.59 log CFU/ml were achieved with 25 mM CA and 50 mM CA, respectively. The efficacy of treatment solutions on planktonic E. coli O157:H7 and Salmonella spp. in suspension by decreasing order is: 75 MC N 50 MC N 25 MC N 75 CA N 50 CA N 25 CA. Table 1 summarizes the effect of the selected treatment solutions on the viability of alfalfa seeds at room temperature. Differences in germination rates were not observed (P N 0.05) across all treatment solutions and soaking times for up to 90 min. Germination rates of alfalfa seeds were reduced (P ≤ 0.05) with soaking times ≥ 120 min for all treatments except 25 mM MC compared to control (water).
Fig. 2. Mean Salmonella spp. populations in suspension exposed to caprylic acid and monocaprylin at room temperature. Data within each treatment represent Salmonella spp. populations following 0 m (□), 1 m (m), 10 m (l), 1 h (V), 6 h (W), and 12 h (■) of treatment exposure. Treatments include control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA) at 25, 50, 75 mM, and monocaprylin (MC) at 25, 50, and 75 mM. Values are the mean of triplicates. Error bars represent ±standard deviation.
Based on these results, a maximum soak time of 90 min was selected for use in the seed decontamination study. The antimicrobial effect of caprylic acid and monocaprylin was lower on E. coli O157:H7 and Salmonella spp. on alfalfa seeds compared to cells suspended in buffer. Reduction of E. coli O157:H7 on alfalfa seeds by caprylic acid and monocaprylin at 4 °C is illustrated in Fig. 3. Within any given treatment, microbial reductions increased with increasing soak time. E. coli O157:H7 concentrations were lower (P ≤ 0.05) compared to control (time 0) when alfalfa seeds were soaked in 50 mM CA for ≥60 min, 75 mM CA for 90 min, 25 mM MC for ≥30 min, or 75 mM MC for ≥ 30 min. Maximum reduction of 1.56 ± 0.49 log CFU/g was achieved by soaking alfalfa seeds in 75 mM MC for 90 min at 4 °C. Monocaprylin was more effective in reducing E. coli O157:H7 populations on alfalfa seeds compared to caprylic acid. Following a 30 min soaking time, seeds treated with monocaprylin solutions had lower (P≤ 0.05) populations of E. coli O157:H7 than the control and caprylic acid treatments. With longer soaking times (≥60 min), all treatments except 25 mM CA had lower (P ≤ 0.05) E. coli O157:H7 populations compared to the control. For Salmonella spp., at any given treatment soaking time, antimicrobial efficacy of the treatments was 75 mM MC N 50 mM MC ≥ 75 mM CA ≈ 25 mM MC≥ 50 mM CA≥ 25 mM CA. Within a treatment antimicrobial, reductions of Salmonella spp. populations increased with longer soak time though differences (PN 0.05) could not always be detected. Greater reductions (P ≤ 0.05) of Salmonella spp. were observed when seeds were soaked in 75 mM MC for ≥60 min. The maximum Salmonella spp. reduction achieved on alfalfa seeds was 2.43 ± 0.36 log CFU/g. The most effective among all tested treatments for reducing E. coli O157:H7 and Salmonella spp. populations both in suspension and on alfalfa seeds was 75 mM MC. E. coli O157:H7 and Salmonella spp. populations were reduced to b0.6 log CFU/ml within 1 and 10 min of exposure to 75 mM MC, respectively, in CPB. Maximum reductions on alfalfa seeds treated with 75 mM MC for 90 min at 4 °C were 1.56 ± 0.49 log CFU/g for E. coli O157:H7 and 2.43 ± 0.36 log CFU/g for Salmonella spp. 4. Discussion
Fig. 1. Mean Escherichia coli O157:H7 populations in suspension exposed to caprylic acid and monocaprylin at room temperature. Data within each treatment represent E. coli O157:H7 populations following 0 m (□), 1 m (m), 10 m (l), 1 h (V), 6 h (W), and 12 h (■) of treatment exposure. Treatments include control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA) at 25, 50, 75 mM, and monocaprylin (MC) at 25, 50, and 75 mM. Values are the mean of triplicates. Error bars represent ±standard deviation.
The antimicrobial efficacy of fatty acids and their monoglycerides are well established (Bergsson et al., 1998, 1999; Isaacs et al., 1995; Kabara, 1979; Petschow et al., 1996). These antimicrobials occur naturally in foods such as milk, and are assumed to be non-toxic to mucosa (Bergsson et al., 1998). With the trend toward more natural
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Table 1 Alfalfa seed viability on exposure to various concentrations of caprylic acid and monocaprylin at room temperature. Treatment
Germination ratea(%) Soak time (min)
Water (control) 25 mM CA 50 mM CA 75 mM CA 25 mM MC 50 mM MC 75 mM MC a b c
0
30
60
90
120
96.98 ± 4.38Abac 98.14 ± 1.64Aa 96.96 ± 1.38Aa 96.54 ± 0.29Aa 97.98 ± 1.01Aa 96.93 ± 0.92Aa 98.44 ± 0.36Aa
97.44 ± 3.60Aa 97.76 ± 2.23Aa 96.33 ± 1.38Aa 96.19 ± 3.60Aa 94.72 ± 4.51Aa 95.11 ± 1.67Aa 98.67 ± 0.38Aa
97.46 ± 2.32Aa 95.51 ± 2.30Aa 94.68 ± 1.24Aa 96.84 ± 1.06Aa 95.02 ± 3.34Aa 90.67 ± 2.72Aa 96.00 ± 2.10Aa
98.00 ± 2.14Aa 88.76 ± 6.10Aa 96.64 ± 1.17Aa 94.31 ± 4.52Aa 92.99 ± 9.61Aa 88.27 ± 3.32Aa 96.12 ± 1.67Aa
98.39 ± 1.58Aa 84.13 ± 3.59Bb 88.62 ± 7.64Ab 84.25 ± 4.41Bb 96.72 ± 1.39Aa 88.10 ± 2.90Ab 88.07 ± 5.81Ab
Data represent mean ± standard deviation of three independent trials. Means within a row with the same upper case letter are not statistically different (P N 0.05). Means within a column with the same lower case letter are not statistically different (P N 0.05).
foods, there is renewed interest in the use of fatty acids and their derivatives as alternatives to synthetic antimicrobials. Higher biological activity of monoglycerols compared to their corresponding fatty acid is well established (Kabara et al., 1977; Lieberman et al., 2006). The enhanced effectiveness monocaprylin over caprylic acid under identical treatment conditions demonstrated in this study further supports these observations. Nair et al. (2004) reported E. coli O157:H7 surviving populations of 5.44 ± 0.27 log CFU/ ml and b1 log CFU/ml after exposure to 25 mM CA and 25 mM MC for 6 h at 37 °C in milk. Monocaprylin was more effective than caprylic acid in reducing populations of foodborne pathogens E. coli O157:H7 and L. monocytogenes (Nair et al., 2004), the mastitic pathogens S. agalactiae, S. dysgalactiae, S. uberis, S. aureus, as well as the enveloped viruses (Nair et al., 2005; Thormar et al., 1987). The differences in the efficacies of caprylic acid and monocaprylin may be attributed to the differences in their respective modes of action and the effect of substrate pH on their activity. The primary mode of action for microbial inactivation by medium and short chain fatty acids is the diffusion of undissociated acids across the bacterial cells and intracellular acidification as the acids dissociate within the protoplasm (Sun et al., 1998). The acidification of the protoplasm can lead to inactivation of intracellular enzymes (Viegas and Sa-Correia, 1991), inhibition of amino-acid transport (Freese et al., 1973), and dissipation of the electrochemical proton gradient, leading to the depletion of energy reserves in bacterial cells (Galbraith and Miller, 1973). Secondary mechanisms such as disintegration of plasma membrane or fusion of cell membranes have also been reported (Ahkong et al., 1973; Thormar et al., 1987). Monoglycerides of fatty
Fig. 3. Mean E. coli O157:H7 populations on alfalfa seeds exposed to caprylic acid and monocaprylin at 4 °C. Data within each treatment represent E. coli O157:H7 populations following 0 m (□), 30 m (m), 60 m (l), and 90 m (■) of exposure at 4 °C. Treatments include control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA) at 25, 50, 75 mM, and monocaprylin (MC) at 25, 50, and 75 mM. Values are the mean of triplicates. Error bars represent ± standard deviation.
acids, on the other hand, are non-ionic surfactants that function mainly by penetrating and incorporating themselves into the plasma membranes, causing destabilization of the membrane bilayer, changes in membrane permeability (Cullis and Hope, 1978; Nair et al., 2004), and interference with signal transduction during cell replication (Projan et al., 1994). Hence, the antimicrobial mechanism of fatty acid monoglycerides is not pH dependent (Isaacs and Lampe, 2000; Sun et al., 2003). Caprylic acid has a pKa of 4.86 and would exist primarily in the dissociated form at the pH of the medium (pH = 7.0) used in this study. Thus, reduction of bacterial cells by caprylic acid would be limited to secondary mechanisms such as membrane disintegration. Greater release of bacterial cells from seeds with longer soak time is commonly reported in sprout seed-related research (Neetoo et al., 2008; Sharma et al., 2002; Sharma and Demirci, 2003). As seeds imbibe water, swelling exposes and releases microorganisms sequestered in the cracks, crevices, or other microenvironments of seeds, resulting in an “increased” microbial population (Charkowski et al., 2001). To evaluate the true antimicrobial efficacy of caprylic acid and monocaprylin, bacterial reduction should be compared to the total microbial population on the seeds, not only the microbial cells released into the soaking medium for a given exposure time. A preliminary experiment was conducted to evaluate if total soak times of ≥90 min would be necessary to achieve the maximum release of bacterial counts (results not shown). Therefore, a unique soaking process was adopted for this experiment, which included soaking the seeds in the selected treatment for the specified time, decanting the soak solution, and then further soaking the seeds in DE broth until the total solution soak time of 90 min was reached. To minimize the microbial growth during this soak period, the solutions were maintained at 4 °C. This method allowed enumeration of the true reduction based on the total bacterial count. Limited bactericidal effect of caprylic acid and monocaprylin was observed for E. coli O157:H7 and Salmonella spp. in inoculated alfalfa seeds compared to planktonic cells and microbial reductions ranged between 1.56 ± 0.49 and 2.43 ± 0.36 log CFU/g (Figs. 3 and 4). Several factors may limit the antimicrobial effect of caprylic acid and monocaprylin on inoculated alfalfa seeds, including the reduced bioavailability of the compounds at refrigeration temperatures, and the protective effect of the seed structures. As previously discussed, the primary site of action for caprylic acid and monocaprylin at pH 7.0 is the bacterial plasma membrane (Freese et al., 1973; Bergsson et al., 1998, 2001). To achieve microbial reductions, the antimicrobials must remain in solution for attachment to occur (Galbraith et al., 1971). Under refrigeration temperatures, caprylic acid and monocaprylin have lower solubility (Nair et al., 2004), reducing the attachment to microbial cells, thus reducing their antimicrobial efficacy. Changes in the bacterial cell membrane fluidity under refrigeration may also reduce the antimicrobial efficacy of caprylic acid and monocaprylin (Nair et al., 2004). Cell membranes are more rigid at lower temperatures, and are less likely to facilitate the incorporation of
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References
Fig. 4. Mean Salmonella spp. populations on alfalfa seeds exposed to caprylic acid and monocaprylin at 4 °C. Data within each treatment represent Salmonella spp. survival following 0 m (□), 30 m (m), 60 m (l), and 90 m (■) of exposure to control (citrate phosphate buffer; CPB, pH 7.0), Tween 80 (CPB + 0.1% Tween 80), caprylic acid (CA; 25, 50, 75 mM), or monocaprylin (MC; 25, 50, and 75 mM). Values are the mean of triplicates. Error bars represent ± standard deviation.
fatty acids or monoglycerides into the membrane structure, making disruption of the plasma membrane less likely to occur. Another plausible explanation for the decreased efficacy could be the reduced bioavailability of the antimicrobials due to the formation of complexes with other compounds (Glassman, 1949; Wang and Johnson, 1992). Proteins, especially the lipophilic proteins such as albumin, and other nutrients, including fat and starch, can interact with the fatty acids and monoglycerides and reduce their bioavailability (Kabara, 1979). In addition, it is well recognized that microorganisms are protected by the seed structure (Sharma et al., 2002; Sharma and Demirchi, 2003; Neetoo et al., 2008). Microbial cells lodged within the crevices or wrinkles of the seeds are inaccessible to the antimicrobials and survive the exposure to chemical disinfectants. In addition, alfalfa seeds (3–85%) may be cracked or may miss part of the testa (Sharma and Demirchi, 2003). Microbial cells could be internalized within the seed structure through these cracks, and shield them from the sanitizing treatments. Reductions in E. coli O157:H7 and Salmonella spp. populations on alfalfa seeds observed in this study are comparable to the reductions reported for other anitmicrobials in the literature. Acidified NaClO2 (500 ppm) was effective in reducing populations of E. coli O157:H7 from 2.7 log CFU/g to b0.5 log CFU/g (Taormina and Beuchat, 1999). When electrolyzed oxidizing water was tested, the most effective treatment without affecting germination was 19 A for 32 min, corresponding to a 91.6% reduction in E. coli O157:H7 on alfalfa seeds (Sharma and Demirci, 2003). Ozone sparging reduced E. coli O157:H7 up to 2.21 log CFU/g after a 64-min contact time (Sharma et al., 2002). A 3-log destruction of Salmonella spp. on alfalfa seed following 10-min dips in 2000 μg/ml of sodium hypochlorite, 6% hydrogen peroxide, or 80% ethanol was reported by Beuchat (1997). Immersion of seeds in 8% H2O2, 1% Ca (OH)2, and 1% calcinated calcium for 10 min also reduced Salmonella spp. by 2.8–3.2 log CFU/g without compromising the germination rate (Weissinger and Beuchat, 2000). Reductions in Salmonella spp. populations on seeds treated with 20,000 ppm of chlorine or Fit (alkaline solution consisting of water, oleic acid, glycerol, ethanol, potassium hydroxide, sodium bicarbonate, citric acid, and distilled grapefruit oil) for 30 min ranged from 2.3 to 2.5 log CFU/g and 1.7 to 2.3 log CFU/g, respectively (Beuchat et al., 2001). To date, chemical sanitizers that can eliminate E. coli O157:H7 and Salmonella spp. from the surface of sprout seeds are not available. However, for practical applicability in the industry, soaking with chemical sanitizers remains the method of choice that is simple and realistic. Monocaprylin (75 mM) can be used as an alternative sanitizer to reduce E. coli O157:H7 and Salmonella spp. population on alfalfa seeds without compromising the seed germination rates.
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