Pathogens Associated with Biltong Product and Their in Vitro Survival of Hurdles Used During Production

Pathogens Associated with Biltong Product and Their in vitro Survival of Hurdles Used during Production

KESHIA NaidoO and Denise Lindsay* School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, South Africa

ABSTRACT Biltong is a traditional South African spiced and dried, ready-to-eat meat product, and is an increasingly popular commodity worldwide. As few studies have evaluated its safety, this study evaluated 150 samples of South African biltong for aerobic bacteria, Enterobacteriaceae, coliforms, Escherichia coli, coagulase-positive Staphylococcus, Salmonella and Listeria monocytogenes. Selected strains of potential pathogens were further identified by use of 16S rDNA gene sequencing methods. In addition, the in vitro antimicrobial properties of each primary ingredient component of the biltong-making process was tested against selected bacterial isolates. Plate counts were the highest for aerobic bacteria (ca. 7 log CFU/g), followed by Enterobacteriaceae (ca. 4 log CFU/g), coliforms (ca. 3 log CFU/g), presumptive Staphylococcus (ca. 3 log CFU/g) and E. coli (ca. 1 log CFU/g) counts. All samples tested negative for Salmonella, while 2 samples tested positive for L. monocytogenes and 3 samples for enterotoxin-producing Staphylococcus strains. Results also showed that 25% of the isolates grew in the presence of up to 20% NaCl. Apple cider vinegar and brown spirit vinegar inhibited the growth of 63 and 50% of the isolates, respectively, while all 8 isolates (2 L. monocytogenes, 3 S. aureus and 3 S. pasteuri) showed the same growth patterns in the presence and absence of spices traditionally used in manufacturing biltong. Overall, strains of L. monocytogenes, Staphylococcus aureus and Staphylococcus pasteuri showed the most growth in all assays conducted. Results from this study highlighted biltong as a potential reservoir for foodborne pathogens, which have implications for foodborne illness.

A peer-reviewed article

*Author for correspondence: +27.11.717.1000; Fax: +27.11.717.6351 E-mail: [email protected]

532 FOOD PROTECTION TRENDS | SEPTEMBER 2010 INTRODUCTION prevalence of bacterial foodborne patho- counts and expressed as log colony form- gens associated with this product and to ing unit (log CFU) per gram. The preservation of meat has always evaluate the in vitro response of selected Presumptive pathogens were select- been important to the survival of hu- isolated pathogens to some of the hurdles ed for further molecular identification mans. For example, one of the first refer- used during the biltong preparation pro- (10). In addition, presumptive isolates of ences to dry-cured pork was reportedly cess. S. aureus were tested for enterotoxin pro- recorded on Sumerian tablets in 2000 duction, using SET-RPLA TD900 kits BC (28). Historically, cured, fermented MATERIALS AND METHODS (Oxoid, London) (20, 21). and dried meat products are regarded as microbially safe ready-to-eat (RTE) Sample collection Molecular identification foods because of their low water activity One hundred fifty biltong samples of presumptive bacterial (a ) and low pH as well as the presence were obtained from various geographi- w pathogens of curing salts (15). These products have cal locations in the Gauteng province been consumed throughout history, and in South Africa during July–September Presumptive pathogens were fur- often have strong cultural associations. 2008. Suppliers included slaughterhouses ther identified using 16S rDNA gene For example, pemmican was a dried meat (n = 21), biltong bars (small outlets that sequencing. Polymerase chain reaction product that provided Native Americans usually sell biltong as the main source (PCR) amplification was carried out as with protein in the lean months of win- of income, often found in shopping previously described, using the primer ter. Similarly, carne seca and machaca in malls) (n = 35), convenience stores sets U1392R (5’- ACG GGCGGT GTG New Mexico, jerky in the United States, (supermarkets) (n = 26), biltong shacks TRC-3’) and Bac27F (5’-AGA GTT charqui in South America, kilshi in (a biltong bar that sells both raw and TGA TCM TGG CTC AG\-3’) in com- Sahel, rou gan in China and biltong in dried meat in the same establishment bination with Fermentas 2× PCR Master on a small scale) (n = 25), home-based South Africa are popular RTE meats in Mix (Fermentas Life Science). The pu- industries (n = 4), shops that sell pre- rified PCR product was sequenced and modern times (16, 29). packaged product (n = 19) and sweet analyzed using BLAST against the 16S RTE meats are often produced from (confectionary) shops (n = 10). rDNA gene sequences from GenBank, meats such as beef, lamb, pork and poul- and samples were submitted to obtain try or mixtures of such meats (15). The accession numbers (10). Shiga-toxin producing Escherichia coli Sample processing (E. coli) O157:H7, Listeria monocyto- and enumeration Isolates selected for growth/ genes (L. monocytogenes), Salmonella and For each biltong sample, 20 g tolerance assays Staphylococcus aureus (S. aureus), have of the product (if not already sliced, been detected in these types of meat then cut from the original sample, us- Eight isolates that were then products (22). Indeed, several outbreaks ing a sterile blade) was transferred into selected for further work included strains of linked with E. coli O157:H7 and Salmon- a Whirl-Pak bag (Nasco, USA), com- L. monocytogenes, S. aureus and S. pasteuri ella in RTE meat products have been bined with 180 ml diluent (0.1% Bac- [accession numbers FJ160766; FJ160767; recorded (15). For example, there have teriological Peptone {BioLab, Midrand, FJ392795; FJ392802; FJ392805; FJ392798; been 9 recorded foodborne illness out- South Africa} + 0.85% sodium chloride FJ392800 and FJ392804]. In order to breaks and 5 recalls have been associated {Saarchem-Merck Chemicals, South generate working inocula, each isolate was with jerky to date (1, 26). Africa}) and homogenized for 2 min successively cultured twice (from previous- Biltong is a traditional South Afri- with a Colworth 400 Stomacher (10). ly frozen stock cultures) for 24 h at 37°C in can RTE dried and spiced meat product The homogenized biltong samples were Tryptone Soy Broth (TSB) (BioLab, Mid- that is easy to produce (12, 24). All that serially diluted in diluent and plated rand South Africa), streaked onto Tryptone is required is a selection of beef, game, in duplicate, using standard plating Soy Agar plates (TSA) (BioLab, Midrand chicken or ostrich meat, which is then procedures, as outlined in Table 1. South Africa) and incubated for 48 h cured with several basic flavoring agents After plating, the pH of the homo- at 37°C. Colony morphology as well as (salt, black pepper, dried and roasted co- genized samples was recorded by placing Gram-stain reactions were examined to riander and brown sugar) and vinegars a pH detection probe of a laboratory pH ensure the purity of the cultures of each (apple cider, brown spirit, wines), after meter (Metrohm 744) directly into each isolate, and plates were stored at 4°C. which it is dried at ambient temperatures sample. In addition, biltong samples were for several days (12, 20). As a result, bil- concurrently processed with standard tong production is often a home indus- methods for the detection of Salmonella, Growth in high salt try, and the safety of this commodity is L. monocytogenes and presumptive S. aureus concentrations of concern (19). In addition, there are (Table 1). several new international markets for bil- Duplicate plates containing be- To evaluate salt tolerance, each iso- tong, including Australia, Portugal, the tween 30 and 300 colonies, or the high- late was plated by the streak plate tech- UK and the US (2). However, very little est number if fewer than 30 colonies nique, in triplicate and on four separate updated survey data has appeared in the were obtained, were enumerated for occasions, onto TSA plates supplement- literature on the safety of this RTE meat aerobic plate (APC), Enterobacteriaceae ed with varying concentrations (5, 10, (27, 29). Thus, the aims of this study (ECB), coliform (CC), E. coli (EC) and 15, 20, 25%) of sodium chloride (NaCl) were to update current knowledge on the coagulase-positive Staphylococcus (SAC) (Saarchem, Merck Chemicals-South Africa)

SEPTEMBER 2010 | FOOD PROTECTION TRENDS 533 Table 1. Culture methods, media and the aerobic incubation time and temperatures utilized in analysis of the 150 biltong samples obtained

Incubation and growth media Time Temp Plating (h) (°C) method

Aerobic Plate Count (APC) 48 30 Pour (101) Tryptone Soy Agar aND (TSA) (BioLab, Midrand South Africa). Spread Enterobacteriaceae 48 37 Pour RAPID’E. coli 2™ Agar (Bio-Rad, (EBC), France). All colonies. Blue-green Coliform Count (CC), colonies. Purple colonies. E. coli Count (EC) Coagulase-positive 48 37 Spread Baird-Parker Agar plus Egg Yolk Tellurite Staphylococcus (SAC) (0.5% w/v) (Scharlau, Spain). Count Black colonies with halo selected and gram stained. 24 37 Spread Selected isolates – DNase agar (Scharlau, Spain). Flooded with 1 ml hydrochloric acid (1M) aND to show clearing. 24 37 Streak Isolates showing clearing on DNAse plates selected for Rapid Staph’ Agar (RSA)(Bio-Rad, France) plus Egg Yolk Tellurite (0.5% w/v) (Scharlau, Spain). Listeria monocytogenes 24 30 Pre- Fraser ½ (Bio-Rad, France). detection enrichment 48 37 Enrichment Fraser 1 (Bio-Rad, France). 24 37 Streak RAPID’ L. mono™ Agar (Bio-Rad, France), Blue colonies selected and Gram stained. Salmonella detection 18 37 Pre- Buffered Peptone Water (Scharlau, Spain). enrichment 24 37 Enrichment Müller Kauffmann Medium plus Brilliant green Cycloserine supplement (1 vial/ aND 500 ml) plus 200 μl Gram's Iodine/ 10 ml (Scharlau, Spain). 24 41.5 Enrichment Rappaport-Vassiliadis Broth (Scharlau, Spain). 24 37 Streak Brilliant Green Agar Modified (Scharlau, Spain). Red colonies. aND 24 37 Streak Xylose Lysine Deoxycholate Agar Scharlau, Spain). Black colonies.

534 FOOD PROTECTION TRENDS | SEPTEMBER 2010 South Africa), brown sugar (3 Table 2. Amendments made to the composition of mock g/l) (Selati, South Africa), so- biltong agar (MBA) to create variations that highlight growth dium chloride (5 g/l) and tra- susceptibilities to each component ditional biltong spice (40 g/l) and was referred to as mock Variation of MBA Modification of components biltong agar (MBA 1). • Six variations of MBA were also created to highlight growth or MBA 1 No modifications made to original media inhibition of the various components, as shown in Table 2. MBA 2 Exclusion of brown sugar Bacterial isolates were streak plated, MBA 3 Exclusion of beef extract in triplicate and on four separate occasions, onto TSAB and all variations of MBA 4 Exclusion of sodium chloride MBA (Table 2), incubated at 37ºC and MBA 5 Exclusion of both beef extract and biltong spice observed every 24 h for 7 days for bacterial growth. MBA 6 Exclusion of both biltong spice and brown sugar MBA 7 Exclusion of biltong spice RESULTS AND DISCUSSION Overall counts from biltong samples linked to points-of-sale (9). Inoculated plates were incubated prepared by pour and spread plating of Overall, biltong samples obtained at 37ºC and qualitatively inspected ev- 1 ml of this overnight bacterial culture from biltong bars in this study had the ery 24 h for 7 days for signs of bacterial mixed with TSA to a final colony count highest associated APC counts (ca. 7.01 5 6 growth. of ca. 10 –10 CFU/ml (3). Plates were log CFU/g) (Fig. 1). This product was allowed to stand for 5 h at ambient tem- often sold uncovered, and handling of perature to allow drying of the surface. Growth at various the product at point-of-sale may have Indicator plates were divided into sec- temperatures contributed to an overall higher APC tions and 50 µl of sterile distilled water (19). In contrast, pre-packaged samples A loopful of each isolate was inocu- (negative control) or undiluted apple ci- had the lowest APC counts (ca. 6.14 lated into 20 ml of TSB, as well as plated der vinegar (Safari SAD, South Africa) or log CFU/g) (Fig. 1), probably because by the streak plate technique onto TSA brown spirit vinegar (Safari SAD, South of the protective barrier provided by plates, in triplicate and on four separate Africa) or 99.7% glacial acetic acid the packaging (14). Biltong is produced occasions, and samples were incubated at (Associated Chemical Enterprises, South under several microbial growth-limiting 4, 25, 30, 37 and 45ºC for 7 days. At Africa) (positive control) were spotted conditions such as curing (salts, spices 24 h intervals, TSA plates were observed into each section. Plates were incubated and vinegars), refrigeration and drying for bacterial growth. In addition, a loop- at 37ºC and observed every 24 h for (19). Traditional biltong reportedly has a ful from each inoculated TSB broth 7 days for zones of clearing that would water activity (aw) of 0.74–0.77 and was streak plated onto TSA plates and indicate inhibition of bacterial growth pH of 5.5–5.8 associated with the final incubated for 24 h at the appropriate (3). product (12, 29). Although these fac- temperature. For example, TSB-grown tors reduce the presence of several mi- cultures incubated at 4°C were plated Growth in the presence of crobial populations, biltong thus favors and incubated again at 4°C. These plates the prevalence of heat-tolerant and salt- biltong spice were also observed to confirm any bacte- tolerant microorganisms. rial growth. To determine the growth of bacte- EBC, CC and EC were used rial isolates in the presence of traditional in this study to assess the overall Growth in the presence biltong spice (commercially available hygiene of the production process, as product, www.biltongmakers.com), eight high EBC and ECs are indicative of of organic acids different agar combinations were pre- enteric pathogens (5). From results To determine if bacterial strains pared as follows: obtained in this study, it was evident that were tolerant to the organic acids used in • TSA was supplemented (prior to biltong samples from convenience stores the biltong manufacturing process, spot- auto-claving) with traditional showed the highest associated EBC, CC on-lawn assays (3) were conducted in biltong spice (40 g/l) and was and EC counts (3.94, 3.03 and 1.57 log triplicate and on four separate occasions. referred to as TSAB. CFU/g, respectively) (Fig 1). In contrast, A colony for each isolate was selected and • Bacteriological agar (13 g/l) pre-packaged samples had the lowest as- inoculated into 50 ml of TSB, and samples (Merck, South Africa) was sup- sociated EBC and CC values (2.21 and were then incubated at 37ºC for 18–20 h. plemented with a combination 1.73 CFU/g, respectively) (Fig. 1), and Bacterial lawns and indicator plates were of beef extract (10 g/l) (BioLab, were the only samples with EC counts

SEPTEMBER 2010 | FOOD PROTECTION TRENDS 535 Figure 1. Bacterial populations of aerobic bacteria (black bar), total Enterobacteriaceae (dotted bar), coliforms (checked bar), E. coli (striped bar) and presumptive Staphylococcus aureus (white bar) counts obtained from 150 biltong samples (lower detection limit = 1 log CFU/g). {pH ranges associated with samples.}

below the lower detection limit (Fig. 1). FJ392795, FJ392802 and FJ392805). of a moister biltong product, especially The presence of E. coli, an index organ- The other 12 were identified as S. past- since strains of S. aureus are capable of ism, traditionally highlights the suspected euri (FJ392791 – FJ392793, FJ392796 growth and enterotoxin production at presence of other pathogens, such as Sal- – FJ392799, FJ392801, FJ392803 and aws of 0.85 (24, 25). It is reported that monella, as they are capable of surviving FJ392804), S. saprophyticus (FJ392800) the modern consumer markets favor in the same environmental niches (17). and Macrococcus caseolyticus (FJ392794). more moist biltong, which is considered However, Salmonella was absent from all All 15 isolates were also tested for entero- more appealing to the palate (12). Tradi- biltong samples tested in this study. toxin production. Results showed that 3 tionally, dried biltong has a water activity isolates produced enterotoxin B, includ- SAC counts showed that conve- (aw) of 0.77 (12); however, the favored nience stores, followed closely by slaugh- ing 2 strains (FJ392795 and FJ392802) biltong has 40% more moisture than identified as S. aureus (99% seq-uence traditional biltong and an a of between terhouses and biltong bars, were associat- w similarity to S. aureus ATCC 14458). ed with samples with higher SAC counts 0.85 and 0.93 (12, 24) which supports Enterotoxin B-producing strains are the growth of several pathogens. than the other points-of-sale; indeed, all reportedly the serotypes that are the SAC counts observed were below 3 log third most common in terms of being, CFU/g (Fig. 1). As Staphylococcus popu- associated with food poisoning events, L. monocytogenes lations are often native to the human after enterotoxins A, and D (4). Interest- The findings of this study showed nose, throat and skin, high Staphylococ- ingly, a strain of S. pasteuri (FJ392798) that 2 of the 150 (1.33%) biltong cus counts are often indicative of poor (99% sequence similarity to S. pasteuri samples tested positive for L. monocyto- human handling practices (15). AF041361) also produced a positive re- genes (accession numbers FJ160766 and action to enterotoxin B. Although en- Presence of bacterial pathogens FJ160767). In comparison, the preva- terotoxin production is often character- lence of L. monocytogenes observed in in biltong istic of coagulase-positive Staphylococcus this study was 1% higher than that ob- S. aureus strains such as S. aureus, it is not limited served in a study on jerky (1). Although to these organisms (20, 25). Even though the minimum dose of L. monocytogenes After the screening of 159 presum- S. pasteuri strains are often associated cells required to cause foodborne illness ptive Staphylococcus isolates obtained from with food commodities (18), there is no is variable, foodborne illness has often biltong samples, 15 isolates were singled record of this species having been impli- been coupled with elevated levels of this out as presumptive S. aureus strains and cated in foodborne illness outbreaks. pathogen in a consumed food product further identified with 16SrDNA sequenc- The presence of enterotoxin produc- (22). ing. Results from molecular analysis showed ing S. aureus in biltong could potentially Biltong would generally be consid- that of the 15 isolates, only 3 were con- be attributed to the high concentration ered a potentially unfavorable environ- firmed as (accession numbers S. aureus of salts, acidic pH (25) and increased aw ment for L. monocytogenes because of its

536 FOOD PROTECTION TRENDS | SEPTEMBER 2010 low aw and high salt concentrations (7, the lowest concentrations, vinegar had 4. Balaban, N., and A. Rasooly. 2000. 8), and it would therefore be less likely to bacteriocidal properties against Escheri- Staphylococcal enterotoxins. Int. harbor and support its growth. However, chia coli O157:H7. Although apple cider J. Food Microbiol. 61:1–10. strains of L. monocytogenes are often as- vinegar exhibited enhanced antimicro- 5. Barros, M. A. F., L. A. Nero, A. A. sociated with raw poultry meat (7, 15). bial properties compared to brown spirit Monteiro, and V. Beloti. 2007. Iden- In this study, this pathogen was isolated vinegar, both vinegars were inadequate tification of main contamination from chicken biltong. The presence of to cause complete growth inhibition of points by hygiene indicator micro- these strains could be attributed to the foodborne pathogenic and enterotoxin organisms in beef processing plants. contamination of biltong prior to and at producing strains. Vinegar marination is Ciênc. Tecnol. Aliment. 27:856–862. production, and distribution and within an important component of the biltong 6. Billing, J., and P. W. Sherman. 1998. the retail environments of this commod- manufacturing process, and survival of Antimicrobial functions of spices: ity, due to the ubiquitous prevalence of potential foodborne pathogens during Why some like it hot. Q. Rev. Biol. this foodborne pathogen (15). this process is cause for concern. 73: 3–49. Furthermore, results also showed 7. Buncic, S., and S. M. Avery. 2004. that all 8 isolates exhibited the same Listeria monocytogenes. (pp. 804- Effect of in-process hurdles growth patterns in the presence and ab- 814) In W. K. Jensen, C. Devine and applied to biltong on selected sence of traditionally used biltong spice. M. Dikeman (eds.). Encyclopedia Spices such as black pepper and corian- foodborne pathogens of meat sciences, vol. 2. Elseveir der, which are the predominant spices Academic Press, London. Overall, strains of L. monocytogenes in the traditional biltong spice mix, are 8. Burnham, G. M., D. J. Hanson, (n = 2), S. aureus (n = 3) and S. pasteuri known to possess weak to mild antimi- C. M. Koshik, and S. C. Ingham. (n = 3) showed growth in most of the as- crobial properties in general (6, 11). 2008. Death of Salmonella serovars, says conducted. Seven of the 8 isolates Escherichia coli O157:H7, Staphylo- grew in the presence of 15% NaCl. In CONCLUSION coccus aureus and Listeria monocy- addition, it was evident that isolates be- This study highlighted biltong at togenes during the drying of meat: longing to the genus were Staphylococcus point-of-sale as a potential vehicle for A case study using biltong and droë- the only isolates that showed growth at ≥ foodborne pathogens and showed that wors. J. Food Safety 28:198–209. 15% NaCl. This was not unexpected, as S. aureus, S. pasteuri and L. monocytogenes 9. Chesneau, O., A. Morvan, F. Grimont, several strains of Staphylococcus have been may also survive the hurdles used during H. Labischinski, and H. El Solh. 1993. shown to survive in environments con- biltong production. However, the find- Staphylococcus pasteuri sp. nov., taining high salt concentrations (9). isolated from human, animal, and Both the plate and the broth ings in this study were based on in vitro results. Thus the question remains as to food specimens. Int. J. Syst. Bacteriol. method used in this study showed 43: 237–244. the same qualitative growth patterns. whether the same foodborne pathogens are able to survive the biltong manufac- 10. Christison, C. A., D. Lindsay, and Only strains of L. monocytogenes grew A. von Holy. 2007. Cleaning and at 4ºC, while all other isolates grew turing process in situ, a question which is currently being investigated. handling implements as potential optimally in the temperature range reservoirs for bacterial contamina- of 25–37ºC. In addition, none of the ACKNOWLEDGMENTS tion of some ready-to-eat foods in 8 isolates tested showed growth at 45ºC. retail delicatessen environments. It is important to note that during the The authors thank the University J. Food Prot. 70:2878–2883. biltong manufacturing process, meat of the Witwatersrand and the Friedel 11. De, M., A. K. De, and A. B. Banerjee. slices are often marinated at refrigera- Sellschop Award, and Carnegie Corp- 1999. Antimicrobial screening of tion temperatures of ca. 4ºC. Although oration Transformation Programme for some Indian spices. Phytother. Res. this temperature does not favor the project funding, as well as the National 13:616–618. growth of 80% of the isolates evaluated Research Foundation, for student fund- 12. Dzimba, F. E. J. M., J. A. F. Faria, in this study, it did support the growth ing. and E. H. M. Walter. 2007. Testing of strains of L. monocytogenes. Such find- the sensory acceptability of biltong ings are not uncommon, as strains of REFERENCES formulated with different spices. Afri. J. Agric. Res. 2:574–577. L. monocytogenes are known to proliferate 1. allen, K., D. Cornforth, D. Whittier, 13. Entani, E., M. Asai, S. Tsujihata, at refrigeration temperatures (15). M. Vasavada, and B. Nummer. 2007. Y. Tsukamoto, and M. Ohta. 1998. 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