Food Protection Trends - September 2010 - 533

INTRODUCTION
The preservation of meat has always been important to the survival of humans. For example, one of the first references to dry-cured pork was reportedly recorded on Sumerian tablets in 2000 BC (28). Historically, cured, fermented and dried meat products are regarded as microbially safe ready-to-eat (RTE) foods because of their low water activity (aw) and low pH as well as the presence of curing salts (15). These products have been consumed throughout history, and often have strong cultural associations. For example, pemmican was a dried meat product that provided Native Americans with protein in the lean months of winter. Similarly, carne seca and machaca in New Mexico, jerky in the United States, charqui in South America, kilshi in Sahel, rou gan in China and biltong in South Africa are popular RTE meats in modern times (16, 29). RTE meats are often produced from meats such as beef, lamb, pork and poultry or mixtures of such meats (15). The Shiga-toxin producing Escherichia coli (E. coli) O157:H7, Listeria monocytogenes (L. monocytogenes), Salmonella and Staphylococcus aureus (S. aureus), have been detected in these types of meat products (22). Indeed, several outbreaks linked with E. coli O157:H7 and Salmonella in RTE meat products have been recorded (15). For example, there have been 9 recorded foodborne illness outbreaks and 5 recalls have been associated with jerky to date (1, 26). Biltong is a traditional South African RTE dried and spiced meat product that is easy to produce (12, 24). All that is required is a selection of beef, game, chicken or ostrich meat, which is then cured with several basic flavoring agents (salt, black pepper, dried and roasted coriander and brown sugar) and vinegars (apple cider, brown spirit, wines), after which it is dried at ambient temperatures for several days (12, 20). As a result, biltong production is often a home industry, and the safety of this commodity is of concern (19). In addition, there are several new international markets for biltong, including Australia, Portugal, the UK and the US (2). However, very little updated survey data has appeared in the literature on the safety of this RTE meat (27, 29). Thus, the aims of this study were to update current knowledge on the

prevalence of bacterial foodborne pathogens associated with this product and to evaluate the in vitro response of selected isolated pathogens to some of the hurdles used during the biltong preparation process.

MATERIALS AND METHODS
Sample collection
One hundred fifty biltong samples were obtained from various geographical locations in the Gauteng province in South Africa during July–September 2008. Suppliers included slaughterhouses (n = 21), biltong bars (small outlets that usually sell biltong as the main source of income, often found in shopping malls) (n = 35), convenience stores (supermarkets) (n = 26), biltong shacks (a biltong bar that sells both raw and dried meat in the same establishment on a small scale) (n = 25), home-based industries (n = 4), shops that sell prepackaged product (n = 19) and sweet (confectionary) shops (n = 10).

counts and expressed as log colony forming unit (log CFU) per gram. Presumptive pathogens were selected for further molecular identification (10). In addition, presumptive isolates of S. aureus were tested for enterotoxin production, using SET-RPLA TD900 kits (Oxoid, London) (20, 21).

Molecular identification of presumptive bacterial pathogens
Presumptive pathogens were further identified using 16S rDNA gene sequencing. Polymerase chain reaction (PCR) amplification was carried out as previously described, using the primer sets U1392R (5’- ACG GGCGGT GTG TRC-3’) and Bac27F (5’-AGA GTT TGA TCM TGG CTC AG\-3’) in combination with Fermentas 2× PCR Master Mix (Fermentas Life Science). The purified PCR product was sequenced and analyzed using BLAST against the 16S rDNA gene sequences from GenBank, and samples were submitted to obtain accession numbers (10).

Sample processing and enumeration
For each biltong sample, 20 g of the product (if not already sliced, then cut from the original sample, using a sterile blade) was transferred into a Whirl-Pak bag (Nasco, USA), combined with 180 ml diluent (0.1% Bacteriological Peptone {BioLab, Midrand, South Africa} + 0.85% sodium chloride {Saarchem-Merck Chemicals, South Africa}) and homogenized for 2 min with a Colworth 400 Stomacher (10). The homogenized biltong samples were serially diluted in diluent and plated in duplicate, using standard plating procedures, as outlined in Table 1. After plating, the pH of the homogenized samples was recorded by placing a pH detection probe of a laboratory pH meter (Metrohm 744) directly into each sample. In addition, biltong samples were concurrently processed with standard methods for the detection of Salmonella, L. monocytogenes and presumptive S. aureus (Table 1). Duplicate plates containing between 30 and 300 colonies, or the highest number if fewer than 30 colonies were obtained, were enumerated for aerobic plate (APC), Enterobacteriaceae (ECB), coliform (CC), E. coli (EC) and coagulase-positive Staphylococcus (SAC)

Isolates selected for growth/ tolerance assays
Eight isolates that were then selected for further work included strains of L. monocytogenes, S. aureus and S. pasteuri [accession numbers FJ160766; FJ160767; FJ392795; FJ392802; FJ392805; FJ392798; FJ392800 and FJ392804]. In order to generate working inocula, each isolate was successively cultured twice (from previously frozen stock cultures) for 24 h at 37°C in Tryptone Soy Broth (TSB) (BioLab, Midrand South Africa), streaked onto Tryptone Soy Agar plates (TSA) (BioLab, Midrand South Africa) and incubated for 48 h at 37°C. Colony morphology as well as Gram-stain reactions were examined to ensure the purity of the cultures of each isolate, and plates were stored at 4°C.

Growth in high salt concentrations
To evaluate salt tolerance, each isolate was plated by the streak plate technique, in triplicate and on four separate occasions, onto TSA plates supplemented with varying concentrations (5, 10, 15, 20, 25%) of sodium chloride (NaCl) (Saarchem, Merck Chemicals-South Africa)

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