The effect of undissociated lactic acid on Staphylococcus aureus growth and enterotoxin A production

International Journal of Food Microbiology 162 (2013) 159–166

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

The effect of undissociated lactic acid on Staphylococcus aureus growth and enterotoxin A production Åsa Rosengren a, b,⁎, Mats Lindblad a, Roland Lindqvist a, b a b

National Food Agency, P.O. Box 622, SE-751 26 Uppsala, Sweden Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Sweden

a r t i c l e

i n f o

Article history: Received 20 February 2012 Received in revised form 20 December 2012 Accepted 7 January 2013 Available online 16 January 2013 Keywords: Staphylococcus aureus Undissociated lactic acid Time to growth Growth rate Lag time Enterotoxin production rate

a b s t r a c t The potential of Staphylococcus aureus cheese isolates to grow and produce staphylococcal enterotoxin A under conditions typical for cheese making was investigated in three broth experiments. The effect of the concentration of undissociated lactic acid (HLac) in conjunction with specific pH values was studied by adjusting pH at a single concentration of lactic acid. First, the time-to-growth of S. aureus was modelled by using survival analysis and absorbance data obtained from an automated turbidity reader. The fitted model describes the time to growth and indicates the growth ⁄ no growth boundary of S. aureus as a function of HLac concentration, temperature and water activity. Second, growth rates and lag times of S. aureus were estimated after two different pre-treatments in skim milk at three HLac concentrations and two temperatures based on optical detection times of serial dilutions of bacterial solutions. Growth rates differed between strains, and increased with increasing temperature and decreasing HLac concentration. Preliminary results indicate that lag times were dependent on pre-treatment suggesting that the growth potential of S. aureus in cheese curd may be greater if milk is used immediately after milking compared to holding at 4 °C after milking. Third, growth, inactivation, and enterotoxin A production of S. aureus strains were investigated at twelve combinations of HLac concentration and temperature. Concentrations of enterotoxin A increased linearly during the first four days, with a production rate increasing with increasing temperature and decreasing HLac concentration. Significant amounts of enterotoxin A were produced during extended incubation, up to 14 days, but then initial pH had changed. This highlights a potential limitation of modelling based on the initial environmental conditions in batch experiments. In summary, ranges of time-to-growth, growth rates, lag times and enterotoxin A production rates of S. aureus in the presence of HLac were estimated. The results can be used together with process data to indicate the range and magnitude of growth and enterotoxin A production during initial stages of cheese production. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Staphylococcus aureus may cause staphylococcal food poisoning (SFP) due to staphylococcal enterotoxins (SET) preformed in foods, typically resulting in sudden onset of nausea, violent vomiting, abdominal cramps and sometimes diarrhoea. It is a ubiquitous pathogen that has the ability to grow and produce SET in many types of food (ICMSF, 1996). Foods commonly implicated in SFP include mixed meals, buffet meals and cheese (Kerouanton et al., 2007; EFSA, 2010). SETs are primarily produced and secreted during late exponential to post-exponential growth phase (Czop and Bergdoll, 1974; Balaban and Rasooly, 2000; Derzelle et al., 2009). Presently, 21 different SETs have been identified. They are named alphabetically enterotoxin A

⁎ Corresponding author at: National Food Agency, P.O. Box 622, SE-751 26 Uppsala, Sweden. E-mail address: [email protected] (Å. Rosengren). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.006

(SEA), B (SEB), C (SEC) etc., and the encoding genes subsequently sea, seb, sec, etc. (Schelin et al., 2011). S. aureus is an udder pathogen and is often isolated from raw milk (Bergonier et al., 2003; Jorgensen et al., 2005; Jakobsen et al., 2011). Other contamination sources are hands and noses of the food handlers as well as the farm or food processing environment (Borch et al., 1996; Le Loir et al., 2003; Jorgensen et al., 2005). Milk makes an excellent substrate for growth of S. aureus and other bacteria due to high nutrient content and pH around 6.7 (Chambers, 2002; Singh and Bennett, 2002). During the initial phase of cheese production, temperature and pH are close to optimal growth conditions for S. aureus, and if present in the milk, it may grow and produce SET. Generally, S. aureus levels peak within the first 24 h followed by a slow decline during ripening (Bachmann and Spahr, 1995; Delbes et al., 2006). A number of SFP outbreaks involving raw and pasteurised milk products have been reported (De Buyser et al., 1984; Evenson et al., 1988; De Buyser et al., 2001; Schønberg and Wåltorp, 2001; Asao et al., 2003; Schmid et al., 2009; Ostyn et al., 2010). In most cases, levels >5 log10 colony

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forming units (CFU) gram −1 have been associated with SFP outbreaks (Kerouanton et al., 2007). Important for limiting S. aureus growth and enterotoxin production in e.g., cheese is the presence of lactic acid bacteria, which can act by bacteriocins, lactic acid, and a decrease in pH during fermentation (Hernandez et al., 2005; Le Marc et al., 2009). Lactic acid is a weak organic acid produced during fermentation of e.g., milk products, and can disturb pH homeostasis of bacteria resulting in stressed cells (Cogan and Beresford, 2002). In the undissociated form, lactic acid and other weak organic acids are lipophilic, which enable acid molecules to freely diffuse across the bacterial cell membrane. Once inside, the acid may dissociate and release protons that acidify the cytoplasm. Energy is diverted to maintain internal pH, and hence, growth is reduced or inhibited (Shelef, 1994). However, the mechanisms of bacterial response to acidic environments are very complex and the tolerance varies widely between strains and species (Foster, 2000; Merrell and Camilli, 2002). Prevention of growth and SET production during cheese making is essential for controlling the safety of cheese, especially when using unpasteurised milk (De Buyser et al., 2001). Knowledge about the conditions in the production process combined with predictive microbiology models can be useful to evaluate the safety of a process (McMeekin et al., 2002). Existing S. aureus models include growth–no growth (GNG) models (Stewart et al., 2002; Valero et al., 2009) and kinetic growth models (Sutherland et al., 1994; Pathogen Modeling Program 7.0, 2007) describing effects of temperature, NaCl/water activity (aw), pH with HCl as acidulant, NaNO2, humectants, but not of lactic acid. Growth models have also been developed in milk-based systems in the presence of lactic acid bacteria (Alomar et al., 2008a; Le Marc et al., 2009), and a model for SEA production in sterilised liquid milk has been developed by Fujikawa and Morozumi (2006). To our knowledge, models describing the effect of undissociated lactic acid (HLac) on growth and enterotoxin production at temperatures and pH relevant for cheese production are lacking for S. aureus. A previous study showed the common occurrence of enterotoxin producing S. aureus in cheese produced on farm-dairies (Rosengren et al., 2010). Strains carrying sec were most common and strains carrying sea were also detected, thus indicating a potential risk with these products. Despite high S. aureus levels, SET was not detected in any of the cheeses (detection limit, ≤0.1 ng g−1; Hennekinne et al., 2003). To support efforts to develop advice to small-scale producers, the purpose of the present study was to improve our understanding of the effect of undissociated lactic acid on the growth and SEA producing potential of S. aureus. Strains isolated from farm-dairy products were studied under conditions relevant for the initial phases of cheese production, i.e., temperatures between 15 and 37 °C, low pH (4.3 to 6.5) and in the presence of lactic acid. The specific objectives were to i) illustrate time-to-growth (TTG) and growth boundaries of S. aureus by developing a TTG model, ii) investigate the growth potential of individual strains by determining growth rate and lag times at two milk pre-treatments, and iii) evaluate the effects of undissociated lactic acid (HLac) and temperature on growth and SEA production. Three broth experiments were done and the effects of HLac were evaluated for one concentration of lactic acid, 0.5% (55 mM), by setting the HLac concentration through adjustment of the pH. The effect of pH was not evaluated separately and the present results apply only to this lactic acid concentration/pH value, which is in the range produced during the first day of fermentation. 2. Methods and materials 2.1. Bacterial strains and growth conditions The S. aureus strains used were isolated and characterised in a survey of Swedish farm-cheese by the National Food Agency (Rosengren et al., 2010). Strains selected for growth experiments represented different

biotypes, different enterotoxin gene profiles and were from cheeses with high S. aureus levels, indicating that they were well adapted to the cheese environment. Enterotoxin A producing strains were chosen for the toxin experiment. To increase the chance of capturing the fastest growth and toxin production potential, strain cocktails were used to estimate time-to growth (TTG) and toxin production (Legan et al., 2002). Strains from −70 °C storage were inoculated on Columbia blood agar (BA) (Oxoid, Basingstoke, UK) and incubated 24 h at 37 °C. Pure colonies were transferred to brain heart infusion (BHI) agar slopes (Becton and Dickinson, Franklin Lakes, NJ, USA), incubated for 24 h at 37 °C, and then stored at 4 °C prior to experiments. In all experiments, BHI broth with a total concentration of 55 mM sterile filtered (0.2 μm, Whatman, Dassel, Germany) lactic acid (Lactot) (88% W/W; BDH, Leuven, Belgium) was used as the basal growth medium. The Lactot concentration was chosen to represent an average in cheese milk during the initial phases of cheese making (Walstra et al., 1993). Depending on experiment, appropriate concentrations of HLac were achieved by adjusting the pH (Radiometer, Copenhagen, Denmark) with 1 M HCl or 1 M NaOH (both Merck, Darmstadt, Germany). BHI broth without lactic acid added was used in combination with 0 mM HLac. NaCl was added as a humectant to obtain the desired aw. HLac concentration in broths was calculated by using the Henderson– Hasselbalch equation and to account for the effect of the ionic environment on pKa, an adjustment of pKa to pK′a was made (Sortwell, 2001). Cells were harvested by centrifugation at 2000 ×g, washed once in 5 mL TSG + G (TTG experiment) or BHI broth (growth rate, lag time experiment) and dissolved in 2 mL BHI broth (Becton and Dickinson). In the TTG and growth rate-lag time experiments, dissolved pellets were diluted and optical density (OD) was checked in a Bioscreen C Analyzer (Oy Growth Curves AB Ltd., Helsinki, Finland) at 420–580 nm. Initial S. aureus concentrations were determined by surface spreading 50 μL or 100 μL of suitable dilutions onto TSA with extra agar (Oxoid) by using a spiral plater (Eddy Jet, IUL Instruments, Barcelona, Spain) followed by incubation at 37 °C for 24 h. 2.2. Time-to-growth To estimate TTG and S. aureus growth boundaries in typical cheese-making conditions, a cocktail of five S. aureus strains was incubated in a total of 105 combinations of incubation temperature (T), aw and HLac concentration. The cocktail included strains nos. SA 31, SA 56, SA 139, SA 158 and SA161, representing different se-genes profiles (sec, sea and seh, and none) and biotypes (Rosengren et al., 2010). Prior to the experiment, strains were pre-cultured separately in 50 mL TSB + G, i.e., tryptone soya broth with 1% sterile filtered glucose (Oxoid; Sigma-Aldrich St. Louis, MO, USA). Since glucose fermentation during growth gradually decreases the pH in the media, strains are allowed to adapt and to induce acid tolerance (Buchanan and Edelson, 1996; Weinrick et al., 2004). The OD of the pre-cultured strains were measured, strains were diluted and pooled in a 1:1 ratio to make a stock solution. Then, 25 μL of the stock solution was added to each of the 5 mL of 21 variants of BHI broths (Becton and Dickinson) prepared as follows: Basal growth medium was dispensed in three separate flasks and aw levels were adjusted to 0.95, 0.97 and 0.99 respectively. Each broth was divided in seven equal volumes, and pH was adjusted to: 7.5, 6.5, 5.5, 5.0, 4.7, 4.5 and 4.3 to obtain HLac concentrations ranging from 0 to 10.2 mM (Table 1). All 21 broths with different HLac x aw combinations were filtered to be sterilised (0.2 μm, Whatman) and checked for sterility 96 h at 37 °C. A volume of 300 μL of each inoculated broth combination was added in triplicate to five identical honeycomb plates, one plate per temperature. Non-inoculated BHI broth (blank) was added to nine wells per plate as sterile controls. At time 0, all plates were measured for OD at 420–580 nm. The initial OD in the inoculated broths was set to approx. 0.1 units above the blank OD and

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corresponding to an initial concentration of around 8.0 log10 CFU mL−1. Contamination was not detected in the controls during the experiments. Honeycomb plates were incubated at 15, 20, 25, 30 and 37 °C respectively. The turbidity of individual wells in the honeycomb plates were measured at intervals 18, 24, 42, 48 h and 6 days in a Bioscreen C Analyzer. Bacterial growth was indicated by an 0.05 OD increase compared to the blank from time 0 (Dalgaard and Koutsoumanis, 2001). In cases of no OD increase, TTG was censored at final measurement of 6 days. After experiments were finished, cultures in all honeycomb wells were checked for purity on Rabbit plasma fibrinogen agar (RPFA) and BA (both Oxoid).

2.3. Growth rate and lag time The purpose of the experiment was to determine the growth potential of five strains isolated from cheese with high S. aureus levels and to investigate the effect of two different milk pre-treatments at temperatures relevant to cheese making, 20 and 37 °C. Growth rates and lag times were determined by using detection times of replicate samples of different dilutions of bacterial solutions recorded by optical turbidity as described in (Baranyi and Pin, 1999). In short, the ratio of the estimated within- and between-dilution group variances (ANOVA) of transformed detection times were minimised by fitting the growth rate (Lindqvist 2006). Strains were grown in basal growth medium with aw of 0.99 and adjusted to three pH-values to obtain three different HLac concentrations. Different HLac concentrations were used at different incubation temperatures. At 20 °C, strains were grown in 6.2 mM HLac (pH 4.5) and 0.7 mM HLac (pH 5.5), and at 37 °C in 0.7 mM HLac and 0.07 mM HLac (pH 6.5). Pre-treatments were chosen to simulate two types of cheese making procedures practised on Swedish small-scale dairies, i.e., using milk directly after milking or after one day of refrigeration. Pre-treatment 1 (Pre-1); one loopful of each strain from BHI agar slopes was inoculated separately into 50 mL of sterile reconstituted skim milk powder (Oxoid) and incubated 20 h at 37 °C. Pre-treatment 2 (Pre-2); first as Pre-1, and then strains were incubated for an additional 24 h at 4 °C. When preparing strains on the day of

Table 1 Concentration of undissociated lactic acid (HLac) (mM) in brain heart infusion broths in relation to pH, total concentration of lactic acid (Lactot) and levels of water activity (aw). Growth (+) or no growth (−) of S. aureus cocktail (SA 31, SA 56, SA 139, SA 158 and SA 161) after 6 days of incubation for each combination of pH/undissociated lactic acid, aw and incubation temperature (T). Samples were analysed in triplicate. pH

4.3 4.3 4.3 4.5 4.5 4.5 4.7 4.7 4.7 5.0 5.0 5.0 5.5 5.5 5.5 6.5 6.5 6.5 7.5 7.5 7.5

aw

0.95 0.97 0.99 0.95 0.97 0.99 0.95 0.97 0.99 0.95 0.97 0.99 0.95 0.97 0.99 0.95 0.97 0.99 0.95 0.97 0.99

Lactot (mM)

HLac (mM)

55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 0 0 0

10.4 9.4 9.3 7.1 6.4 6.3 4.7 4.2 4.1 2.4 2.2 2.2 0.8 0.7 0.7 0.08 0.07 0.07 0 0 0

T (°C) 15 − − − − + + + + + + + + + + + + + + + + +

20 −− −− −− −− +− ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

− − − + + + + + + + + + + + + + + + + + +

25 − − − + + + + + + + + + + + + + + + + + +

− − − + + + + + + + + + + + + + + + + + +

−− −− −− ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

30 − − − + + + + + + + + + + + + + + + + + +

− + + + + + + + + + + + + + + + + + + + +

37 −− −− −− ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

− − − + + + + + + + + + + + + + + + + + +

− − − + + + + + + + + + + + + + + + + + +

− − − + + + + + + + + + + + + + + + + + +

161

experiment start, cultures from Pre-1 were kept at room temperature whereas cultures from Pre-2 were kept on ice. After pre-treatments, cultures were harvested, washed twice in BHI broth and pellets dissolved in 2 mL BHI broth. To make stock solutions, first the dissolved pellets of the ten cultures (5 strains and 2 pre-treatments) were decimally diluted four times. Then, OD was checked and finally, 10 or 20 μL of the dilution closest to 0.05 units above the turbidity of the blank was added to 2 mL basal BHI growth medium with pH values set to 4.5, 5.5 or 6.5. The stock solutions were decimally diluted five times, and 300 μL of each decimal dilution was added in duplicate to wells of honeycomb plates. Plates were put in the Bioscreen C Analyzer and incubated at 20 and 37 °C respectively. Initial concentrations of the S. aureus strains in the undiluted stock solutions were calculated to approx. 7 log10 CFU mL−1. For each strain and pre-treatment, OD value at 420–580 nm of the undiluted stock solution at time 0 was regarded as the detection limit. OD was measured every 10 min with 5 s of shaking prior to measurement. The experiment was run until all dilutions of tested strains had exceeded the detection limit or stopped after 15 days. To indicate the relative energetic burden imposed by the lactic acid at the investigated temperatures and pH values, ΔODs (the difference in initial and final, stationary phases, OD of the appropriate dilutions) were estimated from the growth curves recorded in the Bioscreen instrument. The ΔOD was attributed to the energetic efficiency (Krist et al., 1998). 2.4. Enterotoxin production rate and concentration Growth, inactivation, toxin production rate and concentration were investigated. A cocktail of SEA producing S. aureus strains was grown in 12 combinations of pH (i.e., HLac concentration) and temperature for typical cheese production conditions. The water activity was 0.995. The cocktail consisted of S. aureus strains nos. SA 79, SA 161 and SA168 (Rosengren et al., 2010). Basal growth medium was divided in four equal volumes and the pH in each volume was adjusted to 4.5, 4.7, 5.2 and 6.0 corresponding to 7.6, 4.7, 1.6 and 0.2 mM HLac. Sixty mL pH adjusted broths were dispensed into 100 mL Erlenmeyer flasks. Broths were checked for sterility and stored at 4–5 °C prior to experiments. Strains were pre-cultured in BHI broth and incubated without shaking for 18 h at 37 °C. For the preparation of the S. aureus cocktail, pre-cultures of each strain were diluted to ODs between 0.750 and 0.780 at 550 nm (Hitachi U 2000 spectrophotometer, Tokyo, Japan) with 0.1% peptone water (PW) (Oxoid; Merck) to obtain approx. 8 log10 CFU mL −1 (Stewart et al., 2002). Then, cultures were diluted 1:3 in 0.1% PW and added in equal volumes to a cocktail. The cocktail was decimally diluted three times and finally, for each incubation temperature, 660 μL in triplicate was added to 60 mL of each adjusted basal growth media. The initial concentration of the cocktail was approx. 3 log10 CFU mL − 1 and the inoculated adjusted basal growth media were incubated at 25, 30 and 37 °C respectively. Samples for the determination of S. aureus' growth and SEA production were taken at times: 0, 0.25, 1, 1.25 and 2 days, and for SEA production only also on days 3, 4, 7 and 14. Prior to SEA analyses, samples were centrifuged for 10 min at 3100 ×g and the supernatants were sterile filtered (0.2 μm, Pall Corp. Ann Arbor, MI, USA) and stored at −18 °C. The pH of the medium was measured regularly during the experiment. 2.5. ELISA assays for detection and quantification of SEA The detection of SEA was done by using Ridascreen SET total (R-Biopharm, Darmstadt, Germany) following the manufacturer's instructions. An ELISA protocol for the quantification of SEA based on WallinCarlquist et al.'s (2010) was used with two minor modifications: First, the coating of the microtiter plates was done at 4 °C and, secondly, modified blocking buffer was used (5% milk powder, Semper,

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Sundbyberg, Sweden, added to washing buffer: 0.05% Tween 20 in 100 mM PBS, Merck). The detection limit varied between 0.31 and 0.63 ng SEA mL−1.

(P > 0.05). The resulting model equation for TTG of the S. aureus cocktail was as follows: ln TTG ¼ 2:9−0:43T þ 0:07aw þ 0:76HLac−0:45T⋅HLac

2.6. Data analyses

2

þ 0:10aw ⋅HLac þ 0:14T þ 0:62HLac

2

ð1Þ

TM

All statistical tests were done in MINITAB statistical software version 15 (Minitab Inc. State College, PA, USA). The significance level was set to 0.05. Survival analysis is a branch of statistics which is used for studying the occurrence and timing of events, in this case the growth of S. aureus. The TTG model was developed by using regression with life data in the reliability/survival module of Minitab. TTG of the S. aureus cocktail was modelled as a function of the experiment conditions together with a censoring indicator. Dependent variable in the model was the natural logarithm (ln) of median TTG of three replicates of each combination of aw, HLac concentration and temperature. Quadratic and interaction terms of aw, HLac concentration and temperature were also tested. Modelling was done in a stepwise procedure starting with a model including all variables and then successively removing insignificant variables. Values of aw, HLac concentration and temperature were normalised to make regression coefficients comparable by using the following function: normalised value= (value− mean) / standard deviation (SD). Mean and SD were obtained from the experiment set up; mean (SD) of aw = 0.97 (0.0164), mean (SD) of HLac concentration = 3.39 mM (3.44 mM) and mean (SD) of temperature (T) = 25.4 °C (7.7 °C). A lognormal distribution was selected based on the criterion of normally distributed residuals. Maximum specific growth rates, μmax (h −1), estimated in the natural logarithm (ln) and lag times, λ (h), were determined from the data on the time of detection of serial dilutions of bacterial stock solutions by using the ANOVA approach developed by Baranyi and Pin (1999). The effects of temperature, HLac concentration and strain on μmax, λ, and ΔOD were analysed by using the General Linear Model (GLM) in Minitab. The data were ln-transformed before analysing the growth rate in GLM. Tukey's pair-wise comparisons were used to indicate significant differences between the treatments. The toxin production rate during the first four days at each combination of temperature and pH was determined by using simple linear regression analysis. In the analysis, the data from the last observation with no toxin detected (t0) through the last observation at four days were included. GLM was used for the evaluation of the effect of time and pH on toxin concentrations by using the data from day 7 and day 14. Data were log10-transformed prior to analyses, because toxin concentrations at these times were substantially non-normally distributed. Growth rates were estimated with the Baranyi–Roberts model (Baranyi and Roberts, 1994) in DMfit in the ComBase toolbox (DMfit, 2011). Linear inactivation rates were estimated by using the GInaFit software (Geeraerd et al., 2005).

where ln TTG represents natural logarithm of median TTG (h), and aw, HLac and T are normalised values of water activity, HLac concentration (mM) and incubation temperature (°C). Based on Eq. (1), TTG was plotted from 18 h to 6 days in relation to temperature and HLac at a fixed water activity of 0.99 to illustrate the combined effect of temperature and HLac (Fig. 1). The model correctly predicted growth conditions in 87 of 89 combinations when using the line for 144 h (6 days) as a boundary for growth/no growth conditions. Growth was predicted, but not observed at 37 °C in combinations aw 0.97, 9.4 mM HLac and aw 0.99, 9.3 mM HLac. The model correctly predicted no-growth conditions in all 14 combinations for which growth was not observed in any well, or in only one of the three wells. 3.2. Growth rate and lag time Growth was not detected at 20 °C for any of the five strains at an HLac concentration of 6.2 mM (pH 4.5), although viable S. aureus bacteria were recovered after the experiment (15 days). At the other experimental conditions, growth occurred with significant effects of strain, temperature and HLac concentration on estimated growth rates (P b 0.05, GLM). There was no significant effect of pre-treatment on estimated growth rates (P > 0.05, GLM). The effect of strain was manifested in significant interaction terms with HLac concentration and temperature, which indicates that the responses to these factors differed between strains at the investigated conditions. At HLac concentration 0.7 mM (pH 5.5), maximum specific growth rates ranged from 0.1 to 0.3 h −1 (20 °C) and 0.9 to 1.7 h −1 (37 °C), respectively (Table 2). At HLac concentration, 0.07 mM (pH 6.5), growth rates ranged from 1.9 to 2.2 h −1 (37 °C). As expected, estimated lag times decreased with growth temperature (Table 2, P b 0.05, GLM). In addition, refrigerated storage of cultures in milk prior to experiments (Pre-2, Table 2) increased lag times significantly compared to direct use (Pre-1) at 20 °C (P b 0.05, GLM). This suggests that refrigerated storage of milk before cheese production may increase lag time. However, at 37 °C the lag time difference between pre-treatments was smaller and not significant (P = 0.10, GLM) (Table 2), possibly due to generally shorter lag times at this temperature, making the difference harder to detect. Significant effects of HLac on lag time was not detected at 37 °C either (Table 2) (P > 0.05, GLM).

3. Results

10

3.1. Time to growth

9

Growth was observed in 71% (75 of 105) of all combinations after 24 h. At HLac concentrations less than 5 mM (≥pH 4.7), growth was observed in all but one combination after 24 h, and in all combinations after 48 h. The maximum concentration of HLac at which S. aureus grew within 6 days, was approx. 7.1 mM at all temperatures except 15 °C. At HLac concentrations above 7.1 mM, growth was only observed at 30 °C (Table 1). HLac concentration and incubation temperature mainly determined the growth whereas aw affected growth to a lesser extent. The final TTG model included three main effects, two interaction terms, HLac × T and HLac × aw, and the quadratic effect of T and HLac (Eq. (1)). Other interaction and quadratic terms were not significant

7

6 days

No growth (> 6 days)

48 hours 42 hours

HLac (mM)

8 6

24 hours

5

18 hours

4 3 Growth <18 h

2 1 0 15

20

25

30

35

40

Temperature (°C) Fig. 1. Predicted time to growth of Staphylococcus aureus in relation to temperature and concentration of undissociated lactic acid (HLac) at water activity 0.99.

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To indicate the relative energetic burden imposed under the present, modest, HLac concentrations, ΔOD was analysed as a proxy for energetic efficiency (Table 2). There were significant effects of strain, temperature, and HLac concentration on ΔOD (P b 0.05, GLM), but not pre-treatment (P > 0.05, GLM). At 37 °C, the mean ΔOD for all strains was higher at 0.07 mM HLac compared to 0.7 mM; 1.0 and 0.6, respectively. At 0.7 mM HLac, the mean ΔOD for all strains were higher at 20 °C compared to 37 °C, 1.1 and 0.6, respectively. This indicates that energetic efficiencies decreased with an increase in HLac and temperature. 3.3. Enterotoxin production rate and concentration The inactivation of SEA producing strains of S. aureus was observed at all incubation temperatures at HLac concentrations ≥ 4.7 mM (pH ≤ 4.7). At all growth inhibiting HLac concentrations, inactivation rates were faster at 37 °C compared to 25 and 30 °C, and S. aureus was not detected at 7.6 mM HLac (pH 4.5) after two days (Table 3). At 37 °C, observed growth rates at HLac concentrations b4.7 mM (pH >4.7) were lower than those estimated for

Table 2 Estimated growth rates, lag times and biomass change (ΔOD, turbidity units) of five S. aureus strains isolated from cheese during growth in brain heart infusion broth with 55 mM lactic acid at different conditions of temperature, concentration of undissociated lactic acid (HLac) and pre-treatment.a Strain Temp. HLac (pH)

31

20

0.7 mM (5.5)

56 139 158 161

Pre-treatmentb Growth Lag time rate (h) (h−1)

Mean lag time ΔOD of pre-treatment (h)

1

0.3

4.1

0.8

2 1 2 1 2 1 2 1 2

0.2 0.3 0.2 0.2 0.1 0.2 0.3 0.2 0.2

5.8 0.3 0.6 0.9 1.6 1.2 8.1 0.5 13.4

0.9 1.3 1.3 1.2 1.1 0.8 0.9 1.3 1.2 1.4 (Pre-1) 5.9 (Pre-2)

31

37

0.7 mM (5.5)

56 139 158 161

1

0.9

0.1

0.7

2 1 2 1 2 1 2 1 2

0.9 1.6 1.6 1.7 1.5 1.6 1.6 1.7 1.6

1.0 0.5 1.5 1.1 1.6 1.0 1.9 1.3 1.7

0.7 0.6 0.6 0.8 0.8 0.5 0.6 0.6 0.6 0.8 (Pre-1) 1.5 (Pre-2)

31

56 139 158 161

37

0.07 mM (6.5)

1

1.9

0.7

1.0

2 1 2 1 2 1 2 1 2

2.0 2.0 2.1 2.1 2.2 2.0 1.9 2.2 2.0

1.5 0.3 1.4 0.8 1.6 0.5 1.2 1.0 1.4

1.0 1.1 1.1 1.0 0.9 1.1 1.1 1.1 1.1 0.7 (Pre-1) 1.4 (Pre-2)

a No growth was detected for any of the five strains at 20 °C in 6.2 mM HLac (pH 4.5). b Pre treatment 1 (Pre-1): incubation in skim milk powder 20 h at 37 °C. Pre treatment 2 (Pre-2): first as pre-treatment 1, and then further incubation 24 h at 4 °C.

163

individual strains in the pre-treatment experiment (Tables 2 and 3). The pH in the growth media was not constant during the experiment (Table 4). In general, the pH dropped during the first four days and then increased gradually up to 14 days at all temperatures. The drop in pH was smaller at 1.6 mM HLac compared to 0.2 mM HLac. The final pH increased with the incubation temperature (Table 4). Of the HLac × Temperature combinations showing growth, SEA was detected after 24 h of incubation except at 1.6 mM HLac at 25 °C, where SEA was detected after 48 h (Table 3). At the tested conditions, SEA was first detected at S. aureus levels >6.8 log10 CFU mL−1. There was good agreement between the qualitative and quantitative ELISA methods when indicating the time to the first toxin detection (Fig. 2 and Table 3). SEA production appeared to increase exponentially or to follow two phases over the investigated 14 days period. The latter was indicated when data for days 0–4 and 7–14 days were analysed separately. SEA concentrations increased linearly during the first four days. Under the present conditions, the SEA production rate, i.e., the coefficient in the equations for the lines in Fig. 2, ranged from 4 to 49 ng mL−1 day−1. Further, the rate increased with an increase in temperature, and was at least two times faster at 0.2 mM HLac (pH 6.0) than 1.6 mM HLac (pH 5.2) (Fig. 2). There was a significant effect of time, temperature and HLac on SEA concentrations when comparing concentration days 7 and 14 (P b 0.05, GLM). Since there was a significant interaction between time and temperature (P b 0.05, GLM), data from each temperature were analysed separately. At 37 °C, toxin concentrations increased significantly between day 7 and day 14 and were significantly higher at 1.6 mM HLac (pH 5.2) than at 0.2 mM HLac (pH 6.0) (P b 0.05, GLM) (Fig. 2e–f). At 25 and 30 °C, mean SEA concentrations at day 14 appeared to be greater at 1.6 mM HLac (pH 5.2), but differences were not significant (Fig. 2a–d). SEA concentrations increased significantly from day 7 to day 14 at 30 °C (P b 0.05, GLM), but not at 25 °C (P = 0.08, GLM).

4. Discussion This study investigated the potential of S. aureus cheese isolates from a Swedish survey (Rosengren et al., 2010) to grow and produce SEA under conditions typical for the initial phases of cheese making, addressing the knowledge gap of the effects of HLac at specific pH values on growth boundaries, growth rates and SEA production. Other factors will also have an impact, but these were not evaluated in this study. These results can be used together with process data to indicate the range and magnitude of these processes during the initial stages of cheese production. Significant amounts of SEA were produced during extended incubation (14 days), but then initial pH and most likely several other environmental parameters had changed. This highlights a potential limitation of using initial conditions in uncontrolled batch experiments for modelling since the parameter values may have changed considerably during the experiment. The present TTG model was developed under conditions favouring the potential growth, i.e., using high inocula levels and a cocktail of acid adapted strains. The TTG model (Eq. (1)) indicates growth within the first 18 h at 20–25 °C and between 1.2 and 2.7 mM HLac (pHs 5.3–4.9) (Fig. 1). Growth limiting HLac concentrations at 15–37 °C are indicated at 6–9 mM (pHs 4.5–4.3) (Fig. 1). A successful lactic fermentation decreases the pH in cheese curd from ca 6.7 to ca 4.6–5.3 (Cogan and Beresford, 2002). Charlier et al. (2008) showed the total growth inhibition of lactic acid adapted S. aureus in low heat skim milk medium in the range of 0.5–0.7% lactic acid (pHs 4.5–4.8), corresponding to 6–12 mM HLac. This growth inhibiting concentration is in the same range as indicated in our TTG model, which is developed with 0.5% lactic acid. Thus, results suggests that S. aureus is able to grow or survive within the pH and HLac ranges typical for a large part of the fermentation process at typical cheese making temperatures and aw (Ramsaran et al., 1998; Rosengren et al., 2010).

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Table 3 Levels (log10 CFU mL−1), presence of staphylococcal enterotoxin A (SEA), growth or inactivation rates of S. aureus cocktail (strains SA 79. SA 161 and SA168) grown in brain heart infusion broths simulating conditions typical for cheese production. 7.6 mM HLac (pH 4.5)

4.7 mM HLac (pH 4.7)

1.6 mM HLac (pH 5.2)

Meana (sd) level (log10 CFU mL−1)

Meana (sd) level (log10 CFU mL−1)

Temp (°C)

Days Meana (sd) level (log10 CFU mL−1)

Inactivation rate (SE) (h−1)

25 25 25 25 25 30 30 30 30 30 37 37 37 37 37

0 0.25 1 1.25 2 0 0.25 1 1.25 2 0 0.25 1 1.25 2

3.05 (0.04) 3.01 (0.01) 2.96 (0.05) 2.93 (0.05) −0.034 (0.002) 2.71 (0.06) 3.14 (0.03) 3.09 (0.01) 2.85 (0.06) 2.66 (0.06) −0.045 (0.003) 2.45 (0.16) 3.10 (0.01) 2.99 (0.08) 2.18 (0.09) 1.75 (0.30) −0.140 (0.012) 1.30 (0.00)

3.08 3.01 2.74 2.66 2.38 3.13 3.02 2.73 2.67 2.15 3.01 2.94 1.64 1.30 b1

(0.02) (0.01) (0.08) (0.05) (0.05) (0.08) (0.02) (0.10) (0.08) (0.10) (0.08) (0.05) (0.19) (0.43) (−)

Inactivation rate (h−1)

3.09 (0.07) 3.06 (0.02) 5.26 (0.08) 6.26 (0.56) −0.015 (0.002) >7.32b(−) 3.14 (0.02) 3.22 (0.03) 6.96 (0.04) 7.40 (0.15) −0.034 (0.003) 8.74 (0.70) 3.06 (0.04) 3.60 (0.10) 7.46 (0.10) 7.60 (0.08) −0.094 (0.006) 8.08 (0.15)

Growth rate (SE) (h−1)

0.39 (0.08)

0.46 (0.07)

0.55 (0.04)

0.2 mM HLac (pH 6.0) SEA Meana (sd) level (log10 CFU mL−1)

Growth rate (h−1)

− − − − + − − + + + − − + + +

− − + + 0.46 (0.03) + − − + + 0.76 (0.09) + − − + + 0.78 (0.05) +

3.04 (0.02) 3.79 (0.07) 6.89 (0.08) 8.46 (0.04) 8.96 (0.03) 3.14 (0.01) 3.23 (0.10) 7.64 (1.55) 8.85 (0.14) 8.98(0.24) 3.09 (0.01) 5.11 (0.05) 8.60 (0.03) 8.48 (0.31) 8.71 (0.05)

SEA

+ SEA detected (−, not detected) by qualitative ELISA method. sd standard deviation of mean value. SE, standard error of the estimated rate. a Mean value of triplicate determinations. b Overgrowth on plates.

Several studies report growth during the first phase of cheese manufacture. During the production of raw milk semi hard Saint-Nectaire and Salers cheese, S. aureus grew during the first 6 h (Delbes et al., 2006) and in soft Camembert cheese during the first 22 h (Meyrand et al., 1998). Whether unsafe levels of S. aureus are reached within this time frame, depends among other factors on the initial levels of the bacterium and the growth rate. When using our growth rate data at 30 °C (Table 3), the time taken for a 10-fold increase in S. aureus numbers is 3.0 h at 0.2 mM HLac (pH 6.0) and 5.0 h at 1.6 mM (pH 5.2). HLac is a significant stress factor and the inhibitory effect increases with concentration and temperature. At low HLac concentrations, 0.7 mM or less (Table 2), the effect on growth rate is within the range of experimental and strain variations. For example, the PMP model (Pathogen Modeling Program 7.0, 2007), not including the effect of lactic acid, predicted rates of 0.23 h−1 (20 °C) and 1.2 h−1 (37 °C) at pH 5.5, and 1.7 h−1 (37 °C) at pH 6.5. These rates are similar to those estimated in the presence of HLac (Table 2). However, growth rates at 1.6 mM HLac were 60–85% of the rates at 0.2 mM HLac (Table 3). Similarly, Charlier et al. (2008) showed that S. aureus growth rates in reconstituted low heat skim milk decreased 30–40% in the range from 0.4 to 4.5 mM HLac. The evidence of the stress induced by HLac is the observation that final levels of bacteria (Table 3) and the ΔOD (Table 2) were two to four times higher in cultures with the lower initial HLac concentration.

Table 4 Development of pH during incubation of a cocktail of enterotoxigenic S. aureus strains in growth media with different initial HLac concentrations at three temperatures. The corresponding estimated S. aureus growth rates are shown in Table 3 and SEA concentrations in Fig. 2. Days

0.25 1 4 7 14

1.6 mM, (pH 5.2)

0.2 mM, (pH 6.0)

25 °C

30 °C

37 °C

25 °C

30 °C

37 °C

5.2 5.2 5.2 5.2 5.9

5.2 5.2 4.9 5.3 6.7

5.2 5.1 5.1 5.9 7.8

6.0 5.9 5.3 5.3 5.8

5.9 5.6 5.2 5.4 6.4

6.0 5.2 5.4 5.6 7.4

Further, the inhibitory effect of HLac tended to increase with an increase in temperature. At 1.6 mM HLac (pH 5.2) (Table 3), observed growth rates at 25, 30 and 37 °C were 100, 70 and 55% of PMP predicted rates. In addition, strain inactivation rates increased both with an increase in HLac concentration and with an increase in temperature (Table 3). Increased bacterial inactivation rates with an increase in temperatures are also reported in other studies (Lindqvist and Lindblad, 2009; McQuestin et al., 2009). Several studies have reported that SEA is detected first after growth to high levels of bacteria. In two French studies, SEA was first detected in cheese after 6 days and 12 days, respectively, and not when CFU levels were highest (Meyrand et al., 1998; Vernozy-Rozand et al., 1998). In our study, SEA was first detected when S. aureus levels were above 6.8 log10 CFU mL −1. Our findings agree with the observations from a study of SEA production during 35 h in sterile skim milk (Fujikawa and Morozumi, 2006). These authors reported that SEA concentration increased linearly with time between 14 and 32 °C when the bacterial population exceeded 6.5 log10 CFU mL − 1. The model of Fujikawa and Morozumi (2006) predicts SEA production rate constants in close agreement with our observed rates at the low HLac concentration at 25 °C and 30 °C, (Fig. 2b and d). However, the model of Fujikawa and Morozumi (2006) does not include the effect of HLac or pH. Although SEA production rates were initially faster at lower stress levels (lower initial HLac concentration), final SEA concentrations tended to be higher in samples with a higher initial HLac concentration (Fig. 2e–f). This may support the observations of increased toxin production with increased stress levels (Lovenklev et al., 2004; Wallin-Carlquist et al., 2010). Similarly, it has been suggested that more SEA is produced in acidic environments since they found that sea expression is slightly increased in the mild acidic environment of a cheese matrix and in the presence of Lactococcus lactis (Cretenet et al., 2011). However, in the present study final pH values in the growth media were surprisingly similar regardless of the initial pH (Table 4). The observed pH change over time suggests that the HLac concentration, as well as other chemical compounds in the medium, may have changed during the incubation period. In response to lactic acid exposure, it has been reported that the pH gradually increased during S. aureus' growth from pH 4.5 to 7.5 within 24 h due to ammonium accumulation and removal of acidic groups (Rode et al., 2010). The pH increase in our study was much

Å. Rosengren et al. / International Journal of Food Microbiology 162 (2013) 159–166

(a)

165

(b) 160

25 °C,1.6 mM HLac

160

25°C, 0.2 mM HLac

140

120

SEA ng mL-1

SEA ng mL-1

140

100 80 60 40 y=3.6(t-t0)

20

120 100 80 60 y=9.4(t-t0)

40 20 0

0 0

1

2

3

7

4

14

0

1

2

Days

(c) 30°C, 1.6 mM HLac

7

14

7

14

30°C, 0.2 mM HLac

900

SEA ng mL-1

800

SEA ng mL-1

4

(d) 1000

700 600 500 400 300 200 y=4.8(t-t0)

100 0 0

1

2

3

4

7

1000 900 800 700 600 500 400 300 200 100 0

14

y=15.1(t-t0)

0

1

2

Days

3

4

Days

(e)

(f) 2000 1800 1600 1400 1200 1000 800 600 400 200 0

37°C, 1.6 mM HLac SEA ng mL-1

SEA ng mL-1

3

Days

y=24.7(t-t0)

0

1

2

3

4

7

2000 1800 1600 1400 1200 1000 800 600 400 200 0

14

37°C, 0.2 mM HLac

y=48.9(t-t0)

0

1

Days

2

3

4

7

14

Days

Fig. 2. Concentration of enterotoxin A [SEA] (ng mL−1) (◆) produced by a cocktail of S. aureus strains (SA 79, SA 161 and SA 168) when grown in brain heart infusion broths with two different levels of undissociated lactic acid (HLac), at incubation temperatures 25, 30 and 37 °C. The solid line is the linear relation between [SEA] and time and the fitted equation for the relation is shown as y = p(t − t0), where y = [SEA] (ng mL−1), p = rate constant (ng mL−1 days−1), t= time (days) and t0 = time of the last observation before SEA was detected (days). Error bars illustrate standard error of mean SEA concentrations based on triplicate samples.

slower (Table 4), possibly due to a lower initial concentration in our study, 3 log10 compared to ca 8 log10 CFU mL−1. Thus, it may not be appropriate to relate SEA production to stress levels based on the initial experimental conditions in uncontrolled batch experiments. Other mechanisms than lactic acid can be important for limiting S. aureus' growth during fermentation. Several studies have emphasised the importance of weak lactic acid producing starter strains invoking other mechanisms than acidification during the initial stages of fermentation (Alomar et al., 2008a, 2008b; Charlier et al., 2008; Le Marc et al., 2009). However, those studies focused on lactic acid and not on undissociated lactic acid. The present study investigated the role of HLac concentration, temperature, and aw on S. aureus' growth and SEA production in broth cultures. Findings indicate that if S. aureus is present in the cheese milk, it is capable of both growing and producing SEA within the ranges of temperature and HLac concentration (pH values) combinations typical for the initial phases of cheese making.

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