Practical and Novel Sterilization Approach for the Pathogenic Staphylococcus aureus Bacteria

Problem statement: Decontaminating meat surfaces has been the big concern of meat industry. Thus, various intervention strategies have been studied to reduce the level of bacteria on animals’ carcass surfaces. Approach: Mixture of different concentrations 1, 1.5 and 2% of acetic, lactic, propionic and formic acids at 1:1 ratio were spray washed on inoculated meat to evaluate their efficacy in reducing numbers of Staphylococcus aureus on meat tissue at 4±1°C. The beef pieces were decontaminated with hot water and then inoculated with S. aureus which then were spray washed with treatments for 15 sec separately. Results: Spray wash combinations of acetic and formic, lactic and formic and propionic and formic acids reduced the number of S. aureus at a range of 1.18-1.43 log cfu mL more than combinations of acetic and lactic, acetic and propionic and lactic and propionic acids on meat tissue. Increasing the concentration of used acids increased the lethality of treatments as lethal effect of 2% concentration >1.5% concentration >1% concentration. Conclusion: Lactic and formic acids Combination showed the strongest lethal effect on S. aureus among other treatments. Moreover, this study showed that the combination of lactic and formic acids treatment is a feasible, safe, and economical decontamination method which is highly recommended for use rather than other combinations or single organic acids.


INTRODUCTION
Organic acids are weak acids that are commonly found in fruit juices and fermented foods [1] . Organic acids have a long history of being applied as food additives and preservatives for preventing food deterioration and extending the shelf-life of perishable food ingredients [2] . Organic acids are Generally Recognized As Safe (GRAS) antimicrobial agent and the dilute solutions of organic acids (1-3%) are generally without effect on the desirable sensory properties of meat when used as a carcass decontaminant [3,4] .
Various researchers indicated the antibacterial effect of different types of organic acids [5][6][7][8] . Usage of organic acids could reduce the population of bacteria on meat surface, even though the reductions were statistically significant, but they did not yet found sufficient, therefore, researchers attempted to find the new treatments, which can increase the lethality effect of organic acids. To this end, organic acids were mixed with each other or with other antibacterial agents.
The effect of combination of organic acids with other antibacterial agents such as silver ions [9] copper [10] and hydrogen peroxide [11] has been studied. The results of these studies indicated stronger antibacterial effect compared with organic acids alone. However, these treatments might have undesirable effects caused by the residual trace of silver, copper and hydrogen peroxide on meat surface. Therefore, there is a grave need to test multiple combinations of different organic acids spray washed on inoculated meat.
To the best of our knowledge, there was no previous study investigated a large number of two organic acids combinations at different concentrations for controlling different bacteria on meat all at once. Previous studies focused on limited treatments for controlling bacteria in which results were inconsistent because of the extensive variations in conditions of experiments. Accordingly, this study compared the antibacterial effect of different two acid combinations of acetic, lactic, propionic and formic acids at 1:1 ratio on inoculated S. aureus on meat. The objective of this study was to investigate, compare and adjust the antibacterial effect of the studied treatments on the inoculated S. aureus on meat at 4±1°C.
Meat preparation: Fresh meat was obtained from a local butchery in Serdang, Selangor, Malaysia. Having been packed in sterile bags, the meat was transported to laboratory in a cool box. The samples were prepared immediately after transferring meat to the laboratory. Several 10-gram pieces of meats were procured from freshly slaughtered cow.
Bacterial strains: Staphylococcus aureus ATCC 29247 obtained from the American Type Culture Collection (ATCC).
Sample preparation: S. aureus was cultured on standard plate count agar (Merck, Germany) and was then incubated for 24 h at 37°C. After 24 h of incubation, a number of colonies were inoculated in sterile DW and the cell concentration was adjusted to10 3 bacteria mL −1 .
The prepared 10 g pieces of meat were decontaminated by washing with hot sterile DW (80°C) for 30 sec [12] then they were kept for few minutes to reach room temperature. At this stage, about 10 3 bacteria mL −1 [13] of S. aureus was inoculated on decontaminated meat by pouring and swabbing over the meats surfaces [14] . Subsequently, the inoculated meats with selected bacterium were kept for 20 min to allow attachment and absorption of bacteria however, some of the inoculated meats were kept as an inoculation control [6] .
After 20 min, the inoculated meat was spray washed with organic acids for 15 sec individually [11] . Once the inoculated meat was spray washed and drained, they were packed in sterile bags that were stored at 4±1°C. Another set was also prepared at the same time as a replicate.
Microbiological analyses were carried out immediately after spray washing until the 12th day of refrigeration. The surface pH of samples was measured by using flat probe pH meter (Prescisa, Switzerland) on 0, 2nd, 6th and 12th days of storage. At this step, each piece of meat (10 g) was aseptically blended with 90 mL of sterile peptone water (Merck, Germany) in a laboratory blender [15] . After that, 1 mL of the blended sample of each inoculated meat with S. aureus was transferred onto Petri dishes for pour plate culturing with standard plate count agar (Merck, Germany) individually. Again, another one ml of the same suspension was cultured as a duplicate. The Petri dishes were then incubated for 24 h at 37°C. After 24 h of incubation, the number of colonies was enumerated in each Petri dish.

Statistical analysis:
The bacterial population (CFU mL −1 ) was obtained from four replications performed on separate days and their means were converted to log 10 CFU mL −1 . Differences between log 10 CFU mL −1 of untreated beef carcass tissue and log 10 CFU mL −1 of treated beef carcass tissue were calculated as log reduction [16,11] . Log reductions of treatments were compared by Analysis Of Variance (ANOVA) test using the general linear models of SPSS 12.0 for windows, P value<0.05 was considered as significant.

RESULTS
The initial surface pH of meat decreased directly after spray washing with treatments. With progress of storage, it increased while the pH of untreated decreased. A significant (p<0.05) reductions were found in the population of S. aureus after being spray washed with all treatments.
As 3.16±0.5, 3.16±0.5 and 3.16±0.5 log 10 cfu mL −1 reductions after spray washed with PAFA at 1, 1.5 and 2% concentrations (Fig. 1f) and at pH rang 4.18-5.40, 4.06-5.29 and 3.95-5.20 respectively. The untreated meat showed no significant changing in the population at pH range 6.12-4.30. The mean log reductions of S. aureus spray washed with AAFA, LAFA and PAFA at 1, 1.5 and 2% concentrations were similar, but they could be distinguished by three way interaction analysis (acid × concentration × day). Three-way interaction analysis showed that these treatments had different log reductions levels on different days. AAFA at 1, 1.5 and 2% concentrations on 5th, 4th and 3rd, LAFA at 1% on 4th, 1.5 and 2% concentrations on 3rd, PAFA at 1, 1.5 and 2% concentrations on 6th, 5th and 4th day of storage respectively. For the treatments which caused 3.16 log 10 cfu mL −1 reduction of S. aureus on 4th day of storage, differences were found in comparison the level of log reduction on 3rd day of storage, which were 1.88, 1.91 and 2.16 log 10 cfu mL −1 reduction for AAFA 1.5%, LAFA 1% and PAFA 2% respectively, also the same analysis showed differences of log reductions on 5th day of storage.

DISCUSSION
The main goal of this study was to investigate the antibacterial effect of combinations of various organic acids and find a spray wash treatments that decrease the microbial loads of bacteria most efficiently on beef tissue.
The mean log reductions analysis of bacterial population showed that various organic acids exerted different lethal effects on bacterial population; also, various bacterial species showed different sensitivity to organic acids. Divers factors can be effective on antibacterial activity of organic acids. A study [17] indicated that the degree of bactericidal activity of the different organic acids on the bacterial cell would most probably depend on the presence of the organic compounds, acid concentration, structure of the acid and capacity of a cell to alkalinize the cytoplasm.
Results of various studies showed that the combination of antibacterial agents have stronger antibacterial effect in contrast with each one alone. A study [6] found that the spray wash of contaminated meat with combination of 1.5% acetic and 1.5% propionic acids had better lethal effect on S. aureus, L. monocytogenes, E. coli and S. typhimurium in contrast with lactic acid 2%. It was indicated that combination of acetic and hydrogen peroxide had greater reduction effect on population of E. coli, Listeria innocua and Salmonella wentworth than each one alone [11] . In another research [18] , it was found that combination of 2% lactic acid and 2% acetic acid reduced population of bacteria on beef more than each one alone.
The log reductions of bacterial population spray washed with combination of two acids in this study support the findings of previous research. The mean log reductions of S. aureus showed that the combination of two organic acids had stronger lethal effect on selected bacteria than each acid individually, which was studied in this laboratory (unpublished data).
Combination of organic acids can cause synergistic antibacterial effect on bacteria. Some researchers explained the mechanism of this synergistic effect is yet unknown [19,17] . Some hypotheses can be made for the reason of stronger lethality effect of combination of organic acids in comparison with each one alone. The stronger lethality effect of combination of two organic acids maybe due to the release of more proton ions by acids in aqueous environment when compared with each one alone, or maybe because of increase in the amount of undissociated form of organic acids in the aqueous environment when they are combined together. Another possibility can be hypothesized, when acids are combined together, the resulting suspension possess mixture of different structures of acid molecules. This helps each pair of acids to compensate for the inherent deficit present in the other, thereby augmenting the inoculating power of the combination.
The antibacterial effect of the organic acids was found to be caused mainly by the undissociated form of organic acids [20][21][22] . Two studies [23,24] individually reported that short chain organic acids such as acetic, lactic and citric acids possesses higher bactericidal activity than the non-organic acids such as hydrochloric acid and that bactericidal activity of the organic acids depends mainly on their undissociated form.
Non-dissociated organic acids can passively diffuse through a bacterium's cell wall and once internalized into the neutral pH of the cell cytoplasm, they dissociate into anions and protons, both of which exert an inhibitory effect on bacteria. Releasing the proton ions cause the internal pH to decrease, which is incompatible with certain categories of bacteria that do not tolerate an important gradient of transmembranous pH [21,[25][26][27][28] ; also, leading to disruption of proton motive force and inhibiting substrate transport mechanisms [26,28] . All these actions of organic acids can negatively affect cell viability.
The reduction rate of S. aureus was proportional to the type and the concentration of each treatment. Analysis of variance (ANOVA) for log reduction of S. aureus showed that there was a significant difference (p<0.05) between 1, 1.5 and 2% concentrations of each organic acid. Log reductions analysis showed that the increase in the concentration of organic acids resulted in increasing the antibacterial effect of organic acids.
These findings were similar to another study [29] which scrutinized the reduction in the microbial population of E. coli and S. typhimurium exposed to 1, 2 and 3% concentrations of lactic acid. They found that population reduction of E. coli rose by increasing concentration of lactic acid. It was also observed that both 2 and 4% concentrations of mixture of acetic and lactic acids had inhibitory effect on growth of E. coli O157:H7 in ground beef [30] . They indicated that 4% acetic and lactic acids caused stronger reduction effect on population of bacteria [30] .
The mean log reductions analysis of S. aureus showed that the treatments, which involved formic acid, had stronger reduction effect on the population of studied bacteria. The main reason of the stronger antibacterial effect of these treatments was the existence of formic acid in the mixture. Formic acid is the shortest chain organic acid, which could be beneficial for its diffusion into the cell and cause acidification of the cytoplasm [17,22,31] .
There were some differences between antibacterial effect of combinations of acetic and formic, lactic and formic and propionic and formic acids on S. aureus. Two-way interaction (bacteria × acid) analysis of log reduction of S. aureus treated with combinations of two acids individually showed that there is no significant difference (p<0.05) in the lowering effect of LAFA, AAFA and PAFA treatments. However, they both showed higher (p<0.05) lowering effect as compared to AAPA, LAPA, AALA treatments.
Analysis of mean log reductions of S. aureus population spray washed with all treatments showed that LAFA had the best reduction effect on population of S. aureus with more than 3 log reduction in this study. To the best of our knowledge, it was the first time such finding is published in decontaminating meat surface. Interestingly, this treatment showed remarkable antibacterial effect on S. aureus inoculated on meat, which is one of the most problematic bacteria in meat industry.
The initial surface pH of meat, for LAFA treatment showed the lowest pH value compared with other treatments, so the low pH, which can be because of releasing high number of proton ions in aqueous environment showed the considerable synergistic effect of these two acids on each other which resulted in the strongest lethal effect.

CONCLUSION
Taken together, the population of S. aureus decreased remarkably after spray washing with AALA, AAPA, AAFA, LAPA, LAFA and PAFA treatments.
Among the treatments, these involved formic acid, showed the stronger lethal effect on S. aureus than others. LAFA showed the best antibacterial effect on selected bacterium. Collectively, it was concluded that the combination of lactic and formic acids treatment is a feasible and economical method of decontaminating meat.
intervention technologies for reducing Escherichia coli O157:H7 on beef cuts and trimmings. Food Protect. Trends