Food Protection Trends - September 2010 - 535

TABLE 2. Amendments made to the composition of mock biltong agar (MBA) to create variations that highlight growth susceptibilities to each component Variation of MBA MBA 1 MBA 2 MBA 3 MBA 4 MBA 5 MBA 6 MBA 7 Modification of components No modifications made to original media Exclusion of brown sugar Exclusion of beef extract Exclusion of sodium chloride Exclusion of both beef extract and biltong spice Exclusion of both biltong spice and brown sugar Exclusion of biltong spice

South Africa), brown sugar (3 g/l) (Selati, South Africa), sodium chloride (5 g/l) and traditional biltong spice (40 g/l) and was referred to as mock biltong agar (MBA 1). 	 •	 Six	variations	of	MBA	were	also	 created to highlight growth or inhibition of the various components, as shown in Table 2. Bacterial isolates were streak plated, in triplicate and on four separate occasions, onto TSAB and all variations of MBA (Table 2), incubated at 37ºC and observed every 24 h for 7 days for bacterial growth.

RESULTS AND DISCUSSION
Overall counts from biltong samples linked to points-of-sale

(9). Inoculated plates were incubated at 37ºC and qualitatively inspected every 24 h for 7 days for signs of bacterial growth.

Growth at various temperatures
A loopful of each isolate was inoculated into 20 ml of TSB, as well as plated by the streak plate technique onto TSA plates, in triplicate and on four separate occasions, and samples were incubated at 4, 25, 30, 37 and 45ºC for 7 days. At 24 h intervals, TSA plates were observed for bacterial growth. In addition, a loopful from each inoculated TSB broth was streak plated onto TSA plates and incubated for 24 h at the appropriate temperature. For example, TSB-grown cultures incubated at 4°C were plated and incubated again at 4°C. These plates were also observed to confirm any bacterial growth.

prepared by pour and spread plating of 1 ml of this overnight bacterial culture mixed with TSA to a final colony count of ca. 105–106 CFU/ml (3). Plates were allowed to stand for 5 h at ambient temperature to allow drying of the surface. Indicator plates were divided into sections and 50 µl of sterile distilled water (negative control) or undiluted apple cider vinegar (Safari SAD, South Africa) or brown spirit vinegar (Safari SAD, South Africa) or 99.7% glacial acetic acid (Associated Chemical Enterprises, South Africa) (positive control) were spotted into each section. Plates were incubated at 37ºC and observed every 24 h for 7 days for zones of clearing that would indicate inhibition of bacterial growth (3).

Growth in the presence of biltong spice
To determine the growth of bacterial isolates in the presence of traditional biltong spice (commercially available product, www.biltongmakers.com), eight different agar combinations were prepared as follows: 	 •	 TSA	was	supplemented	(prior	to	 auto-claving) with traditional biltong spice (40 g/l) and was referred to as TSAB. 	 •	 Bacteriological	 agar	 (13	 g/l)	 (Merck, South Africa) was supplemented with a combination of beef extract (10 g/l) (BioLab,

Growth in the presence of organic acids
To determine if bacterial strains were tolerant to the organic acids used in the biltong manufacturing process, spoton-lawn assays (3) were conducted in triplicate and on four separate occasions. A colony for each isolate was selected and inoculated into 50 ml of TSB, and samples were then incubated at 37ºC for 18–20 h. Bacterial lawns and indicator plates were

Overall, biltong samples obtained from biltong bars in this study had the highest associated APC counts (ca. 7.01 log CFU/g) (Fig. 1). This product was often sold uncovered, and handling of the product at point-of-sale may have contributed to an overall higher APC (19). In contrast, pre-packaged samples had the lowest APC counts (ca. 6.14 log CFU/g) (Fig. 1), probably because of the protective barrier provided by the packaging (14). Biltong is produced under several microbial growth-limiting conditions such as curing (salts, spices and vinegars), refrigeration and drying (19). Traditional biltong reportedly has a water activity (aw) of 0.74–0.77 and pH of 5.5–5.8 associated with the final product (12, 29). Although these factors reduce the presence of several microbial populations, biltong thus favors the prevalence of heat-tolerant and salttolerant microorganisms. EBC, CC and EC were used in this study to assess the overall hygiene of the production process, as high EBC and ECs are indicative of enteric pathogens (5). From results obtained in this study, it was evident that biltong samples from convenience stores showed the highest associated EBC, CC and EC counts (3.94, 3.03 and 1.57 log CFU/g, respectively) (Fig 1). In contrast, pre-packaged samples had the lowest associated EBC and CC values (2.21 and 1.73 CFU/g, respectively) (Fig. 1), and were the only samples with EC counts

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Food Protection Trends - September 2010

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