Abstract: The objective of this research was to evaluate the effect of ozone gas and potassium lactate on lipid oxidation and survival of Salmonella typhimurium on fresh pork. A total of 144 samples of fresh pork samples were cut into pieces approximately (8x5x0.6) cm in size, then (0, 2 and 4%) potassium lactate (KL), inoculated with S. typhimurium was applied by spreading a 0.5 mL cell suspension over each sample. The pork samples were then packed in (6"x8") airtight polyethylene bags with a thickness of 87.5 μm and a volume of 3.5 L with and without ozone. Ozone gas was injected into the plastic bags at a rate of 0, 200, 500 and 1,000 mg h-1 and the samples later stored at 8°C. Thio-barbituric Acid Reactive Substance (TBARS) and microbial loads were determined on days 0, 5, 10 and 15. Data was statistically analyzed using SPSS software and differences among means detected at the 0.5% confident level using the Scheffe’s test. Samples treated with 2 and 4% KL had significantly (p<0.01) lower TBARS value than non-treated samples. Combination of ozone and KL showed inhibitory effects on S. typhimurium in samples. S. typhimurium was sensitive to 4% KL with and without ozone. At 1,000 mg h-1, ozone improved KL inhibitory effect on S. typhimurium. Ozone and KL are potential substances for inhibition of S. typhimurium.
INTRODUCTION
Fresh pork is a source of protein often contaminated with pathogen microorganisms such as Salmonella sp. and Listeria monocytogenes. Meat and meat products contaminated with these pathogens can create a considerable hazard for public health. Salmonella is a common contaminant of raw pork (Escartin et al., 2000). S. typhimurium was the most frequently isolated serotype (Korsak et al., 2003). Survival of Salmonella depended on environmental conditions including pH, temperature and relative humidity (Soliman et al., 2009). In an attempt to eliminate the pathogens from carcasses, various applications such as washing and sanitizing with chilled water, hot water, chlorinated water, steam, steam vacuuming, organic acids and salt alone and in combination have been developed and used. Another more recently proposed treatment involves the utilization of ozone, either as a washing tool or as a gaseous treatment (Rice, 2002). Ozone is one of the most powerful oxidants that leaves no detectable residues in or on treated produce and is a powerful antimicrobial agent (Mudway and Kelly, 2000). The ozone has since received full U.S. FDA approval as a direct contact food-sanitizing agent (U.S. FDA, 2001). Stivarius et al. (2002) reported that 1% ozonated water decreased S. typhimurium by 0.78 log CFU g-1. Williams et al. (2004) reported that ozone and treatment temperature reduced S. typhimurium in apple cider and orange juice. This has raised commercial interests in the deployment of ozone for various applications in the food industry such as food surface hygiene, sanitation of food plant equipment, treatment and lowering BOD and COD of food plant waste. The bactericidal effects of ozone on a wide variety of organisms, including Gram positive and negative bacteria as well as spores and vegetative cells have been documented (Guzel-Seydim et al., 2004).
Potassium lactates are already widely used to extend the shelf life and increase the safety of meat and poultry products (Aran, 2001; Knock et al., 2006; Quilo et al., 2010). Quilo et al. (2010) reported that 200 ppm peroxyacetic acid followed with 3% potassium lactate reduced Salmonella on ground beef and Kim et al. (2010) reported that lactate with phosphate enhancement had beneficial effects on color and lipid oxidation stability of beef cuts under high oxygen conditions. The US Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) approved use of potassium salts (up to 4.8% w/w) in meat products as an antimicrobial ingredient to control pathogens (USDA FSIS, 2000). The aim of this research was to study the effect of ozone gas and potassium lactate on lipid oxidation and survival of Salmonella typhimurium on fresh pork.
MATERIALS AND METHODS
Raw pork samples were purchased from a local vendor in Maha Sarakham, Thailand during January 2009 to March 2010. Ozone gas was generated from a laboratory corona discharge ozone generator (HGOZ-1000, Enaly M and E, China) using oxygen gas as a substrate, with a working voltage of 220 volt, 50 Hz. Salmonella typhimurium was obtained from a stock culture of the Foodborne Pathogens and Biofilm Research Laboratory (FBRL), Mahasarakham University, Thailand.
Bacterial strain: S. typhimurium was activated in a tryptic soy broth (Criterion, Santa Maria, USA) and grown in a tryptic soy agar at 37°C for 24 h. Cell suspension was prepared by adding 0.1% sterile peptone water onto culture plates, which was further diluted with 0.1% sterile peptone water to obtain a cell density of 9 Log CFU mL-1.
Inoculation and treatments: Samples of fresh pork were cut into pieces approximately (8x5x0.6) cm in size, then treated with 0, 2 and 4% potassium lactate (KL), (Fluka, Netherlands) by dropping 0.5 mL on each side and inoculated with 0.5 mL S. typhimurium suspension on both side by spreading with sterile brush. The samples were then packed in customized airtight polyethylene plastic bags (6"x8") in size with a thickness of 87.5 μm. Ozone gas was injected into the plastic bags at a rate of 0, 200, 500 and 1,000 mg h-1. Controls were uninoculated samples. Total of 144 samples (48 pieces/replicates) were stored at 8°C. The analyses for survival of S. typhimurium and Thiobarbituric Acid Reactive Substance (TBARS) were determined on days 0, 5, 10 and 15.
Survival study: Twenty five grams of fresh pork samples were aseptically placed in a sterile stomacher bag containing 225 mL of sterile 0.1% peptone water. Samples were stomached for 2 min. Serial dilution was prepared using 0.1% peptone water and plated in duplicate on Xylose Lysine Desoxycholate agar (XLD) (Criterion, U.S.A). The numbers of colonies were counted after incubation at 37°C for 24 h.
Evaluation of lipid oxidation Secondary lipid oxidation products were evaluated using 2-thiobarbituric Acid (TBA) as described by Vyncke (1970, 1975) and with modifications by Sorensen and Jorgensen (1995). Briefly, 5 g of meat was stomached in 15 mL of 7.5% Trichloroacetic Acid (TCA) with 0.1% propylgallate and 0.1% ethylenediaminetetraacetic acid (EDTA) for 5 min at room temperature and filtered. Filtrate 5 mL was mixed with 5 mL of 0.02 M Thiobarbituric Acid (TBA) and incubated at 100°C in a water bath for 40 min. Absorbance was measured at 532 nm (PU8625 UV/VIS Spectrophotometer, Phillips, Cambridge and England). Results are expressed as 2-thiobarbituric Reactive Substances (TBARS), namely malonaldehyde (mg kg-1 dry matter) using a standard curve prepared from 1,1,3,3-tetraethoxypropane (TEP). Mean values of two independent determinations were obtained.
Statistical analysis: A 4x3 factorial design in a randomized completed block design with three replications was used. The same culture, chemical stock and equipment were used in all replicates. The main effects were ozone injection rate (0, 200, 500 and 1,000 mg h-1) and KL concentration (0, 2 and 4%). Data was analyzed with SPSS for windows software (Version 12.0, SPSS Inc, U.S.A.). Significant differences between means were determined using the Scheffe’s method (Norusis, 1990). Significance was determined by least square means at p = 0.05.
RESULTS AND DISCUSSION
Survival of S. typhimurium: With the initial microbial load of 8.0 Log CFUg-1 on day zero, S. typhimurium decreased by 1 Log CFU g-1 (p<0.01) after treated with 2% KL or more with and without ozone (Table 1). Stivarius et al. (2002) observed similar results, reporting beef trimmings inoculated with S. typhimurium then treated with either 1% ozonated water for 7 min (7O) or 15 min (15O). The 15O treatment reduced S. typhimurium 0.78 Log CFU g-1 (p<0.05), respectively in ground beef. However, Castillo et al. (2003) reported that the aqueous ozone treatment had no significant improvement over a water wash in reducing S. typhimurium and Escherichia coli O157:H7 on beef carcass surfaces. In this study, ozone and KL showed inhibitory effects on S. typhimurium in samples. S. typhimurium was sensitive to 4% KL with and without ozone. At 1,000 mg h-1 , ozone improved KL inhibitory effect on S. typhimurium. It is postulated that the primary attack of ozone was on the cell wall or membrane of the bacteria, probably by reaction with the double bonds of lipids and that leakage orlysis of the cells depended on the extent of that reaction (Scott and Lesher, 1963).
| Table 1: | Survival of S. typhimurium at 8°C during 15 days of storage after treatment of ozone and potassium lactate |
| KL: Potassium lactate a-cMeans with common superscript in the same column are not significantly different (p>0.01); A-CMeans with common superscript in the same row are not significantly different (p>0.01) | |
| Table 2: | Lipid oxidation as determined by malonaldehyde at 8°C during 15 days of storage after treatment of ozone and potassium lactate |
| TBARS: 2-thiobarbituric reactive substances; a-cMeans with no common superscript in the same column differ at (p<0.01); A-CMeans with no common superscript in the same row differ at (p<0.01); ns Means in this column values are not significantly different at (p>0.01) | |
It is possible that the antimicrobial effect of KL may be due to the lactate anions attack on cell wall or membrane leading to malfunction of enzyme activity (Reddy et al., 2007). Reddy et al. (2007) observed that in addition of 1% calcium lactate or 2% of potassium and sodium lactate to injected pork was able to control Clostridium perfringens germination and outgrowth to <1 log CFU g-1. Shelef and Potluri (1995) studied the effect of different lactates on the listeriostatic activity in cooked liver sausage and found that Calcium Lactate (CaL) was consistently more inhibitory than sodium lactate (NaL). They also observed that growth of S. typhimurium was abated in sausage by 3% sodium or calcium lactate and indicated that the antibacterial activity could be due to lactate rather than Ca or Na ions. Maas et al. (1989) studied the effect of lactate and sodium ion on botulinum toxin formation and noted that lactate concentration, rather than sodium ion concentration was the antibotulinal factor.
Lipid oxidation: Resultant thiobarbituric acid reactive substance (TBARS) value (Table 2) was not significant on day 0 but decreased (p<0.01) significantly on day 5 in samples using 2% KL or more with and without ozone and the same on days 10-15. Sample treated with 200 mg h-1 ozone without KL had the highest TBARS value on every day of storage. Samples treated with 2 and 4% KL had significantly (p<0.01) lower TBARS value than untreated samples. Similar results were observed by Quilo et al. (2009) who reported the effect of antimicrobial treatment on pH and lipid oxidation on ground beef. In that study, the control had the highest (p<0.05) lipid oxidation compared to samples treated with 3% potassium lactate, sodium metasilicate, 200 ppm peroxyacetic acid, 1000 ppm acidified sodium chlorite on all days of display. Mancini et al. (2010) observed the effects of lactate and modified atmospheric packaging on premature browning in cooked ground beef patties. The data showed lactate decreased lipid oxidation in ground beef patties in all packaging types. Nnanna et al. (1994) reported similar results where sodium lactate was effective in controlling lipid oxidation in ground pork and in a model system containing methyl linoleate, Tween-20, ascorbate and ferric chloride. In addition, the antioxidant activity of lactate also has been reported in pork sausage, chicken sausage and chicken patties (Bloukas et al., 1997; Choi and Chin, 2003; Naveena et al., 2006; Cegielska-Radziejewska and Pikul, 2004). Lactate’s role in minimizing lipid oxidation may be due to its ability to scavenge superoxide (O2¯) and Hydroxyl radicals (OH¯) (Groussard et al., 2000). Ozonated samples were higher in TBARS than control and lactate treated samples because ozonation increased oxygen on samples. Similarly, further high-oxygen packaging systems provide favorable environments for oxidation to occur (Jackson et al., 1992; Taylor et al., 1990). Seyfert et al. (2005) reported that high-oxygen atmospheres increased TBARS values throughout storage and display.
CONCLUSIONS
Ozone and KL showed inhibitory effects on S. typhimurium in samples. S. typhimurium was sensitive to 4% KL with and without ozone. At 1,000 mg h-1, ozone can improve KL inhibitory effect on S. typhimurium. Potassium lactate retarded lipid oxidation. Ozone and KL are potential substances for inhibition of S. typhimurium.
ACKNOWLEDGMENTS
This research was financially supported by Thailand Research Fund Master Research Grants (Grant No. MRG-WI515S093).