Brazilian Journal of Medical and Biological Research (2005) 38: 1259-1265 High pressure effects on pathogens in caviar 1259 ISSN 0100-879X Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing F. Fioretto1-3, C. Cruz1, 1ERAP, IUT Périgueux, Bordeaux IV, Périgueux, France A. Largeteau2, T.A. Sarli3, 2Groupe Hautes Pressions, Institut de Chimie de la Matière Condensée de Bordeaux G. Demazeau2 and et Ecole Nationale Supérieure de Chimie et de Physique de Bordeaux, Pessac, France A. El Moueffak1 3Department of Inspection of Foods from Animal Origin, University “Federico II” of Naples, Naples, Italy Abstract Correspondence We studied the action of high pressure processing on the inactivation Key words F. Fioretto of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and • High pressure processing ERAP, IUT Périgueux Salmonella enteritidis ATCC 13076, suspended in a culture medium • Staphylococcus aureus Bordeaux IV, and inoculated into caviar samples. The baroresistance of the two • Salmonella enteritidis Rue Paul Mazy, 39 pathogens in a tryptic soy broth suspension at a concentration of 108- • Tryptic soy broth 24019 Périgueux Cedex • 109 colony-forming units/ml was tested for continuous and cycled Caviar France • Pressure cycles Fax: +33-5-5306-3143 pressurization in the 150- to 550-MPa range and for 15-min treatments E-mail: [email protected] at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microor- Presented at the 3rd International ganisms in the tryptic soy broth suspension, whereas the effect of Conference on High Pressure different procedure times on complete inactivation of the microorgan- Bioscience and Biotechnology, isms inoculated into caviar was similar. Rio de Janeiro, RJ, Brazil, September 27-30, 2004. Received January 17, 2005 Introduction son, fish is frequently contaminated with Accepted May 18, 2005 Salmonella. Generally fish products such as The handling of fish products during the fish eggs are involved in a secondary con- manufacturing process involves a risk of tamination process. Recent studies have contamination with Staphylococcus aureus, pointed out the increase in the detection of a Gram-positive microorganism causing Salmonella in fish products (2) and the inci- foodborne human intoxication (1). These dence of foodborne human diseases caused bacteria are salt-tolerant and therefore can by Salmonella (3,4). contaminate all cured preparations such as “Caviar” is the product obtained from caviar and fish-based preserves. fish eggs, separated from the connective tis- Salmonella enteritidis is a Gram-nega- sue of ovaries and subjected to salting, to the tive microorganism inhabiting the intestinal addition of additives and sometimes to pas- tract of a wide range of animals that can be teurization for its preservation. Caviar is a isolated close to the coast from animal and foodstuff of high nutrient value because fish human waste-polluted waters. For this rea- eggs are a rich source of vitamins and min- Braz J Med Biol Res 38(8) 2005 1260 F. Fioretto et al. eral nutrients and, on the basis of their amino Samples of “caviar d’Aquitaine” (eggs acid profile, their protein quality is consider- from Siberian sturgeon: Acipenser baerii), able (5). Generally fish eggs do not remain free from the additive borax, packaged into microbiologically sterile after the different 2-part metal tins of 30 g, were obtained from procedures involved in caviar fabrication. S.C.E.A. Sturgeon, St. Fort sur Gironde, Egg screening is a critical step during pro- France. Samples of aquacultured trout caviar cessing which contributes to bacterial con- (eggs from rainbow trouts: Onchorinchus tamination due to the lack of proper hygiene mykiss) in glass jars (about 30 g) were pro- often occurring in this case. Caviar can be vided by Viviers de France, Sarrance (France). contaminated with various species of spoil- The samples were stored at 0-4ºC until use. age bacteria, but also with pathogens (S. aureus, Salmonella sp, Vibrio sp, Aeromonas Preparation of bacterial suspensions in sp, Clostridium botulinum) (6). Thus, caviar tryptic soy broth solution can pose food safety risks if not properly handled. The cultures were rehydrated by the ad- Caviar can be pasteurized, but the changes dition of 0.3 ml tryptic soy broth (TSB, in sensory properties of the thermically Merck, Darmstadt, Germany) and stock cul- treated product are not appreciated by con- tures of both strains were then prepared on sumers (7). The salt concentration and stor- tryptic soy agar (Difco, Detroit, MI, USA), age temperature do not always represent incubated at 37ºC for 24 h and stored at 4ºC. preventive measures to preserve caviar. To revive the microorganisms, a single pel- Therefore, it is necessary to add chemical let of the cultures was resuspended in 5 ml preservatives such as borax because of their TSB, followed by incubation at 37ºC for 48 antimicrobial effect. New mild technologies h. Further dilutions in TSB were necessary are being evaluated for their effectiveness in to achieve a concentration of approximately making caviar safe for consumers and in 108-109 colony-forming units (CFU)/ml extending its shelf life. High pressure is an S. aureus ATCC 6538 and S. enteritidis interesting technique for the preservation of ATCC 13076 at the beginning of high pres- caviar, not only because it inactivates patho- sure processing. To determine that the mi- gens and spoilage bacteria, but also because crobial load was in the same range for each it can avoid the use of chemical additives assay, absorbance at 600 nm was checked such as borax, although its potential risks with a spectrophotometer. The cell suspen- have not been fully determined. sions (25 ml) were vacuum sealed in double sterile polyethylene pouches (PA/PE 20/70 Material and Methods µm) and subjected to the high-pressure treat- ments. Material Caviar inoculated with Staphylococcus aureus S. aureus ATCC 6538 and S. enteritidis and Salmonella enteritidis ATCC 13067 were obtained as freeze-dried pellet cultures in thermosealed vials from The assays were carried out separately Institut Pasteur, Paris, France. These cul- for the broth culture of each strain. A 1-ml tures were selected because they are well- cell suspension was added to 25 g of caviar defined typed strains which have been used and the mixture was gently shaken by hand in many previous investigations including for 5 min. The inoculated samples were high pressure processing (8-10). The vials vacuum sealed in double sterile polyethyl- were stored at freezing temperature until use. ene pouches (PA/PE 20/70 µm) and sub- Braz J Med Biol Res 38(8) 2005 High pressure effects on pathogens in caviar 1261 jected to high pressure processing. Each treat- of the pressure values was based on the ment was carried out in duplicate. analysis of preliminary results obtained for TSB suspensions subjected to only two pres- High pressure processing surization times (15 min, 5 min x 3 cycles). Five hundred and 400 MPa were the pres- The equipment used for high pressure sures able to destroy S. aureus and S. enter- processing was a discontinuous isostatic press itidis, respectively, after a treatment of 15 specifically used for applications in the food min, while 450 and 350 MPa were the lethal science field at IHP (“Interface Hautes pressures for the treatment of 5 min for 3 Pressions”) - ENSCPB (“Ecole Nationale cycles. All high-pressure treatments were Supérieure de Chimie et de Physique”), Uni- tested twice. Control samples were held at versity Bordeaux I: Sciences and Technolo- atmospheric pressure. After processing, all gies, France. The hyperbaric apparatus (NFM- samples were stored at refrigeration temper- Technologies, Le Creusot, France) and the ature until analysis. Framatome device (Paris, France) commer- cialized by Clextral (Firminy, France) are Enumeration of viable cells by microbiological designed to attain a maximum pressure of analysis of tryptic soy broth suspension 800 MPa. Pressure and temperature were samples constantly monitored and recorded during the process. Water was used as the pressur- Appropriate 10-fold serial dilutions (10-8) ization fluid. were made in sterile-buffered peptone-water (AES Laboratoire, Route de Dol, Combourg, Objective of our study and parameters of France). One milliliter of each dilution was high pressure processing plated onto plate count agar (AES Labora- toire) in duplicate. The plates were incu- The aim of the present investigation was bated at 37ºC for 72 h and the average num- to study the application of high-pressure ber of colonies from the duplicate plates was treatments able to destroy a large load of recorded for each sample. The number of pathogens potentially present in fish eggs, survivors was reported as log CFU/ml. and to find the optimal pressure levels nec- essary to preserve the organoleptic charac- Inoculated caviar samples teristics of fresh caviar. Duplicate samples of S. aureus ATCC 6538 and S. enteritidis Twenty-five grams of each sample and ATCC 13076 in TSB suspension were sub- 225 ml of buffered peptone water were ho- jected to pressure treatments in the 150-550 mogenized using a Stomacher®. Further 10- MPa and 150-450 MPa range, respectively, fold dilutions (10-8) were made in sterile- at room temperature for different times (15 buffered peptone water (AES Laboratoire) min, 5 min x 3 cycles, 3 min x 5 cycles, 2 min and 0.1 ml of each dilution was plated in x 7 cycles). The pressure used for the treat- duplicate onto selective media: Baird-Parker ment of caviar samples inoculated with S. (AES Laboratoire) for the enumeration of S. aureus ATCC 6538 was 500 MPa at room aureus ATCC 6538 and Rambach (from temperature for 15 min and 450 MPa for 3 Merck) for the enumeration of S.