Inactivation of Staphylococcus aureus and Salmonella enteriditis in tryptic soy broth and caviar samples by high pressure processing

Abstract We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10 8 -10 9 colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.


Introduction
The handling of fish products during the manufacturing process involves a risk of contamination with Staphylococcus aureus, a Gram-positive microorganism causing foodborne human intoxication (1). These bacteria are salt-tolerant and therefore can contaminate all cured preparations such as caviar and fish-based preserves.
Salmonella enteritidis is a Gram-negative microorganism inhabiting the intestinal tract of a wide range of animals that can be isolated close to the coast from animal and human waste-polluted waters. For this reason, fish is frequently contaminated with Salmonella. Generally fish products such as fish eggs are involved in a secondary contamination process. Recent studies have pointed out the increase in the detection of Salmonella in fish products (2) and the incidence of foodborne human diseases caused by Salmonella (3,4).
"Caviar" is the product obtained from fish eggs, separated from the connective tissue of ovaries and subjected to salting, to the addition of additives and sometimes to pasteurization for its preservation. Caviar is a foodstuff of high nutrient value because fish eggs are a rich source of vitamins and mineral nutrients and, on the basis of their amino acid profile, their protein quality is considerable (5). Generally fish eggs do not remain microbiologically sterile after the different procedures involved in caviar fabrication. Egg screening is a critical step during processing which contributes to bacterial contamination due to the lack of proper hygiene often occurring in this case. Caviar can be contaminated with various species of spoilage bacteria, but also with pathogens (S. aureus, Salmonella sp, Vibrio sp, Aeromonas sp, Clostridium botulinum) (6). Thus, caviar can pose food safety risks if not properly handled.
Caviar can be pasteurized, but the changes in sensory properties of the thermically treated product are not appreciated by consumers (7). The salt concentration and storage temperature do not always represent preventive measures to preserve caviar. Therefore, it is necessary to add chemical preservatives such as borax because of their antimicrobial effect. New mild technologies are being evaluated for their effectiveness in making caviar safe for consumers and in extending its shelf life. High pressure is an interesting technique for the preservation of caviar, not only because it inactivates pathogens and spoilage bacteria, but also because it can avoid the use of chemical additives such as borax, although its potential risks have not been fully determined.

Material
S. aureus ATCC 6538 and S. enteritidis ATCC 13067 were obtained as freeze-dried pellet cultures in thermosealed vials from Institut Pasteur, Paris, France. These cultures were selected because they are welldefined typed strains which have been used in many previous investigations including high pressure processing (8)(9)(10). The vials were stored at freezing temperature until use.
Samples of "caviar d'Aquitaine" (eggs from Siberian sturgeon: Acipenser baerii), free from the additive borax, packaged into 2-part metal tins of 30 g, were obtained from S.C.E.A. Sturgeon, St. Fort sur Gironde, France. Samples of aquacultured trout caviar (eggs from rainbow trouts: Onchorinchus mykiss) in glass jars (about 30 g) were provided by Viviers de France, Sarrance (France). The samples were stored at 0-4ºC until use.

Preparation of bacterial suspensions in tryptic soy broth solution
The cultures were rehydrated by the addition of 0.3 ml tryptic soy broth (TSB, Merck, Darmstadt, Germany) and stock cultures of both strains were then prepared on tryptic soy agar (Difco, Detroit, MI, USA), incubated at 37ºC for 24 h and stored at 4ºC. To revive the microorganisms, a single pellet of the cultures was resuspended in 5 ml TSB, followed by incubation at 37ºC for 48 h. Further dilutions in TSB were necessary to achieve a concentration of approximately 10 8 -10 9 colony-forming units (CFU)/ml S. aureus ATCC 6538 and S. enteritidis ATCC 13076 at the beginning of high pressure processing. To determine that the microbial load was in the same range for each assay, absorbance at 600 nm was checked with a spectrophotometer. The cell suspensions (25 ml) were vacuum sealed in double sterile polyethylene pouches (PA/PE 20/70 µm) and subjected to the high-pressure treatments.

Caviar inoculated with Staphylococcus aureus and Salmonella enteritidis
The assays were carried out separately for the broth culture of each strain. A 1-ml cell suspension was added to 25 g of caviar and the mixture was gently shaken by hand for 5 min. The inoculated samples were vacuum sealed in double sterile polyethylene pouches (PA/PE 20/70 µm) and subjected to high pressure processing. Each treatment was carried out in duplicate.

High pressure processing
The equipment used for high pressure processing was a discontinuous isostatic press specifically used for applications in the food science field at IHP ("Interface Hautes Pressions") -ENSCPB ("Ecole Nationale Supérieure de Chimie et de Physique"), University Bordeaux I: Sciences and Technologies, France. The hyperbaric apparatus (NFM-Technologies, Le Creusot, France) and the Framatome device (Paris, France) commercialized by Clextral (Firminy, France) are designed to attain a maximum pressure of 800 MPa. Pressure and temperature were constantly monitored and recorded during the process. Water was used as the pressurization fluid.

Objective of our study and parameters of high pressure processing
The aim of the present investigation was to study the application of high-pressure treatments able to destroy a large load of pathogens potentially present in fish eggs, and to find the optimal pressure levels necessary to preserve the organoleptic characteristics of fresh caviar.

Enumeration of viable cells by microbiological analysis of tryptic soy broth suspension samples
Appropriate 10-fold serial dilutions (10 -8 ) were made in sterile-buffered peptone-water (AES Laboratoire, Route de Dol, Combourg, France). One milliliter of each dilution was plated onto plate count agar (AES Laboratoire) in duplicate. The plates were incubated at 37ºC for 72 h and the average number of colonies from the duplicate plates was recorded for each sample. The number of survivors was reported as log CFU/ml.

Inoculated caviar samples
Twenty-five grams of each sample and 225 ml of buffered peptone water were homogenized using a Stomacher ® . Further 10-fold dilutions (10 -8 ) were made in sterile-buffered peptone water (AES Laboratoire) and 0.1 ml of each dilution was plated in duplicate onto selective media: Baird-Parker (AES Laboratoire) for the enumeration of S. aureus ATCC 6538 and Rambach (from Merck) for the enumeration of S. enteritidis ATCC 13076. The plates were incubated at 37ºC for 24-48 h. The average number of colonies on the duplicate plates was then recorded for each sample. The number of survivors is reported as log CFU/g.

Results and Discussion
Effect of high pressure on Staphylococcus aureus ATCC 6538 in tryptic soy broth suspension The rate of microorganisms able to survive the treatment is reported by the N/N 0 ratio which in our study ranged from 1 to 10 9 . The DE of high-pressure treatment is shown in the graphs in Figures 1 and 2 and is correlated with pressure and with treatment times. For S. aureus ATCC 6538 the inactivation rates after treatment for 15 min at 150, 350, 450, and 500 MPa were 0.7, 1.9, 1.7, and 8.9 log 10 , respectively. We observed a destruction threshold value of 500 MPa, with no significant destruction occurring up to this pressure.  (9), who showed that 3 cycles of 10 min at 500 MPa and at room temperature were sufficient to obtain a 6.6-log 10 reduction, while 6 cycles of 5 min completely destroyed S. aureus (8.6 log 10 ). Under the same conditions of pressure and temperature, for a continuous treatment of 30 min the loss of microorganism viability was only 1.42 log 10 . After electron microscopy analysis of the changes occurring in cells after pressurization, the author observed irreversible modifications of the intracellular material and also a perturbation in cell division concerning the septum and the cell membrane (9). These phenomena can explain the lethal effect of pressure cycles on the microorganisms. Processing conditions (pressure, temperature, time), microorganism strain, and the kind of substrate (composition, pH, Aw) obviously influence the inactivation caused by pressure treatment. In general, the sensitivity of microorganisms to pressure is lower in foods than in buffer suspensions (11) and can depend on the food type (12,13).

Effect of high pressure on Salmonella enteritidis ATCC 13076 in tryptic soy broth suspension
The effect of high pressure in a continuous 15-min treatment on S. enteritidis ATCC 13076 in TSB suspension was obtained in the 150-450 MPa range. The choice of this range was based on literature data (14,15) showing that S. enteritidis is less baroresistant than S. aureus. Based on these preliminary results, the study was carried out in the same way as in the experiments on S. aureus, with the application of various pressure cycles. 2 and 2 present the inactivation rates of S. enteritidis after continuous and cycling pressure treatments. The total destruction of S. enteritidis was obtained for 3, 5, and 7 cycles, respectively, at 350, 300, and 250 MPa. The increase of cycle numbers permitted us to reduce the pressure level needed to destroy S. enteritidis. The effectiveness of the pressure cycles is evident when compared to continuous pressure under the same treatment conditions. Few studies have been done about the effect of high pressure processing on S. enteritidis, which is the main food poisoning Salmonella serotype. The results of the present study underline the higher baroresistance of S. aureus ATCC 6538 compared to S. enteritidis ATCC 13076 and the effectiveness of pressure cycles in the destruction of both pathogens at weak pressure levels. Using several cycles it was possible to reduce the pressure able to inactivate high loads of S. aureus and S. enteritidis (10 8 -10 9 CFU/ml). In fact, after 7 pressure cycles of 2 min at room temperature the total destruction of the pathogens in TSB suspension was obtained at 350 and 250 MPa, respectively. The destruction threshold value was high especially in the case of S. aureus ATCC 6538 for the continuous treatment, while for S. enteritidis ATCC 13076 this phenomenon did not occur. The different features of the two microorganisms could explain our results.

Destruction by high pressure of Staphylococcus aureus and Salmonella enteritidis inoculated into caviar
Based on the analysis of preliminary results of the TSB models, the pressurization of caviar samples inoculated with S. aureus ATCC 6538 and S. enteritidis ATCC 13076 was carried out at the threshold values obtained for the suspensions for the 15-min treatment and for 3 pressure cycles of 5 min at room temperature. The effect of high-pressure treatments on the strains inoculated into caviar is shown in Tables  3 and 4. For the samples inoculated with S. aureus ATCC 6538, both pressure treatments (450 MPa/5 min x 3 cycles and 500 MPa/15 min) significantly reduced the counts.
Also the number of S. enteritidis ATCC 13076 inoculated into caviar samples, obtained after pressurization, were <2 log CFU/g for the continuous treatments (400 MPa/15 min) and for the treatments in cycles (350 MPa/5 min x 3 cycles). Thus, at the pressure levels used for the experiments no difference was found between the continuous and cycling treatments. In addition, the substrate did not affect the effect of high pressure processing on the microorganisms.
The present results confirm that treatments in cycles improve the effect of pressure on S. aureus and Salmonella enteritidis. Also, the application of cycles permitted us to reduce the pressure level necessary to preserve the sensory properties of caviar. Therefore, high pressure processing represents an interesting procedure for microbial stabilization in fish products because it can eliminate pathogens or spoilage bacteria, potentially present in the product, and can also reduce the use of chemical preservatives which have potential and still undetermined risks.

Perspectives
Our study of the inactivation of S. aureus ATCC 6538 and S. enteritidis ATCC 13076 in caviar samples has raised different problems involving both basic science and food processing. In order to obtain an explanation about the pressure behavior in caviar an investigation will be conducted on the proteins. In addition, the role of cycling pressure compared to continuous pressure must be investigated. In parallel, regarding food processing conditions, it would be necessary to reduce the pressure level and consequently improve the efficiency of cycling processes.