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Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea. Introduction. The microbiology ...
Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork Y.M. Choi, O.Y. Kim, K.H. Kim, B.C. Kim and M.S. Rhee Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea

Abstract

Keywords acetic acid, Escherichia coli, foodborne pathogenic bacteria, lactic acid, pork, supercritical carbon dioxide. Correspondence Min Suk Rhee, Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, 5-1 Anamdong, Sungbuk-gu, Seoul 136-713, South Korea. E-mail: [email protected]

2009 ⁄ 0471: received 13 March 2009, revised and accepted 6 July 2009 doi:10.1111/j.1472-765X.2009.02702.x

Aims: To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO2) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork. Methods and Results: The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO2 at 12 MPa and 35C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO2. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO2, micro-organism levels were maximally reduced, ranging from 2Æ10 to 2Æ60 log CFU cm)2 (E. coli, 2Æ58 log CFU cm)2; Listeria monocytogenes, 2Æ60 log CFU cm)2; Salmonella typhimurium, 2Æ33 log CFU cm)2; E. coli O157:H7, 2Æ10 log CFU cm)2). Conclusions: The results of this study indicate that the combined treatments of SC-CO2 and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO2 or organic acids alone. Significance and Impact of the Study: The combination treatment of SC-CO2 and organic acids may be useful in the meat industry to help increase microbial safety.

Introduction The microbiology of meat and meat products is most often investigated to determine their potential safety and keeping quality. All fresh meat can have some level of microbial contamination present, and sometimes the contamination acts as a vector for pathogens of both animal and human origin (Coll Cardenas et al. 2008). The most important pathogens associated with meat and meat products include Listeria monocytogenes, Salmonella typhimurium and Escherichia coli O157:H7 (Mead et al. 1999). However, despite improvements in meat-processing hygiene in recent years, concerns about meat products serving as vehicles of foodborne pathogenic micro-organisms are increasing rather than diminishing. 510

Supercritical carbon dioxide (SC-CO2) is an alternative nonthermal technique for sterilization (Wei et al. 1991; Lin et al. 1992, 1994); SC-CO2 not only is a powerful solvent for a wide range of compounds of interest in food processing, but is relatively inert, inexpensive, nontoxic, nonflammable, recyclable and readily available in high purity and leaves no residue (Clifford and Williams 2000). Moreover, SC-CO2 is an effective treatment for destroying foodborne pathogens when it is applied to meat and meat products (Sirisee et al. 1998; Spilimbergo and Bertucco 2003). For example, Sirisee et al. (1998) achieved a microbial reduction of Staphylococcus aureus in ground beef by applying SC-CO2 at 31Æ5 MPa and 42Æ5C for 180 min. In addition, when subcritical CO2 was applied at 6Æ05 MPa and 45C for 150 min, the reduction

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level of Brocothrix thermosphacta was about 5Æ9 log in meat (Erkmen 2000). However, many earlier studies seem to have no practical use (Sirisee et al. 1998; Erkmen 2000), because the treatments consisted of pressures or temperatures that were too high or exposure times that were too long and the muscle sample size was too small. The most common and classic preservative agents are food-grade organic acids. Especially acetic and lactic acids, which are environmentally friendly, naturally occurring and have generally recognized as safe (Brul and Coote 1999). However, although some studies have reported the decontamination effects of weak organic acid treatments on micro-organisms in meat, opinions among scientists on this remain divided. For example, according to Castillo et al. (2001), the treatment of 2% lactic acid reduced levels of Salm. typhimurium and E. coli O157:H7 on carcass surfaces that had been inoculated. Moreover, they reported that spraying chilled carcasses with 4% lactic acid in a commercial setting caused about 3 log reductions in aerobic plate counts. In contrast, Acuff et al. (1987) applied organic acid treatments to beef strips and found no difference between aerobic plate counts of the control and treated strips after various time periods of storage and retail display. Therefore, for the purpose of practical use, this study was conducted to evaluate the effects of organic acids and SC-CO2 treatments as well as their combined treatments on fresh pork for the reduction of nonpathogenic E. coli and three pathogenic bacteria, including L. monocytogenes, Salm. typhimurium and E. coli O157. Materials and methods Bacterial strains Four bacterial strains each, including nonpathogenic E. coli (ATCC 25922, BE4a and K-12 2B), L. monocytogenes (ATCC 191413, ATCC 19114 and ATCC 19115), Salm. typhimurium (ATCC 19585, ATCC 43174 and DT104 Killercow) and E. coli O157:H7 (ATCC 35150, ATCC 43889 and ATCC 43890), were obtained from the Food Microbiology Culture Collection at Korea University (Seoul, Korea). All cultures were suspended in fresh tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) containing 10% glycerol and were stored at )80C until subsequent analysis. The cultures used in each experiment were freshly prepared using the same procedure. Culture and cell suspension Each strain of nonpathogenic E. coli, L. monocytogenes, Salm. typhimurium and E. coli O157:H7 was activated by

Combination effect of organic acids and SC-CO2 in fresh pork

two successive transfers in TSB, and incubated overnight at 37C. The cultures from each group were combined in plastic centrifuge tubes (Corning Inc., Corning, NY, USA), and the cells were harvested by centrifugation (Centri-CL2; IEC, Needham Heights, MA, USA) at 2600 g for 30 min. After the supernatant was discarded, the pellet was washed twice with 0Æ85% saline solution. The final pellet was resuspended in 0Æ85% saline solution to a concentration calculated to yield c. 107 CFU ml)1 of sample. Each culture cocktail of E. coli, L. monocytogenes, Salm. typhimurium and E. coli O157:H7 was used in further experiments. Treatment of pork Twenty-four boneless pork loins, the longissimus dorsi muscle at the eighth to thirteenth thoracic vertebra, were obtained from a porcine carcass at 24 h postslaughter at a local abattoir and were transported to the laboratory under refrigerated conditions (4C) within 1 h. Samples of muscle were cut into 20 mm of intact shaped thickness (each weighing 70 ± 2 g) and were randomly selected to minimize bias. In each experiment, the meat samples were inoculated with each micro-organism (nonpathogenic E. coli or the three pathogens) cocktail (0Æ7 ml) to yield c. 107 CFU cm)2. The inoculated meat samples were randomly assigned to treatments. After spot inoculation, each micro-organism cocktail was spread using a sterilized spatula, and each sample was put into culture flask and incubated at 4C overnight. The samples were then subjected to organic acid and ⁄ or SC-CO2 treatments. For the organic acid treatments, each pork chunk soaked with acetic or lactic acid solutions in a 1 : 1 ratio in polyethylene bags at 4C for 1 min. The pH levels of the organic acid solutions ranged from 2Æ80 to 2Æ55 (acetic acid) and 2Æ29 to 2Æ02 (lactic acid) (Table 1). The SC-CO2 treatments were performed with a SC-CO2 system (Supercritical system; Tharex Co., Seoul, Korea). The system consists of two vessels Table 1 pH levels of acetic and lactic acid solutions Concentration (%) Acetic acid 1Æ0 2Æ0 3Æ0 Lactic acid 1Æ0 2Æ0 3Æ0

pH

2Æ80 ± 0Æ05 2Æ63 ± 0Æ05 2Æ55 ± 0Æ04 2Æ29 ± 0Æ04 2Æ13 ± 0Æ02 2Æ02 ± 0Æ13

Results are expressed in mean values ± SD.

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(2 · 12 l), and was performed at 12 MPa and 35C for 30 min. The various treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO2 only and (iii) treatment with acetic (3%) or lactic acid (3%) followed by treatment with SC-CO2. The whole experiment was replicated three times. After each run, the SC-CO2 system was cleaned with SC-CO2, and then with ethanol by sterile distilled water to prevent possible contamination. Microbiological analysis The treated pork samples were placed in stomacher bags containing 180 ml of buffered peptone water and immediately homogenized for 2 min (Stomacher 400; Seware, London, UK). After homogenization, the samples underwent serial tenfold dilutions with 9 ml of sterile buffered peptone water. Eosin-Methylene Blue agar (Difco Laboratories), Oxford agar base (Difco Laboratories), Xylose lysine desoxycholate agar and Sorbitol MacConkey agar (Difco Laboratories) were used as selective media for the enumeration of nonpathogenic E. coli, L. monocytogenes, Salm. typhimurium and E. coli O157:H7, respectively, and were incubated at 37C for 24 h. These enumerations were performed before and after the treatment for each treatment condition.

Y.M. Choi et al.

statistical package (SAS Institute, Cary, NC, USA, 2001). When the effect was significant (P < 0Æ05), mean separation was accomplished with the probability option (pdiff, p-value differentiation a pairwise t-test). Results Effect of food-grade organic acid treatments Table 2 shows the effects of the food-grade organic acid treatments at various conditions on the reduction of nonpathogenic E. coli and the three pathogenic bacteria in fresh pork. For any given organic acid concentration, the levels of E. coli, L. monocytogenes, Salm. typhimurium and E. coli O157:H7 were significantly reduced by the acetic and lactic acid treatments. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions, with the exception of the 2% organic acid treatments for the level of L. monocytogenes. In the samples that were treated with acetic acid, the reduction levels of nonpathogenic E. coli and three foodborne pathogens were higher by the 3% solution than by the 1% solution, with the exception of Salm. typhimurium. Also, in the case of the lactic acid treatments, the reduction levels of nonpathogenic E. coli and three pathogens were higher by the 3% solution than by the 1% lactic acid solution.

Statistical analysis The entire experiment was conducted three times, and the average of the duplicate plate counts for the three replications was converted to units of log CFU cm)2. The least squares analysis was conducted with the General Linear Model procedure program available in the sas

Combination effects of organic acids and SC-CO2 treatments The effects of the organic acid and ⁄ or SC-CO2 treatments under various conditions on the reduction of nonpathogenic E. coli and the three pathogenic bacteria in fresh

Organic acid concentration Control E. coli Acetic acid Lactic acid L. monocytogenes Acetic acid Lactic acid Salm. typhimurium Acetic acid Lactic acid E. coli O157:H7 Acetic acid Lactic acid

1%

2%

3%

SE

Level of significance

6Æ84a

6Æ34b 6Æ32b

6Æ24b,c 6Æ19c,d

6Æ09d,e 6Æ05e

0Æ29

***

6Æ40a

5Æ54c 5Æ49c,d

5Æ65b 5Æ40d,e

5Æ33d,e 5Æ22f

0Æ30

***

7Æ02a

6Æ56b 6Æ44b,c

6Æ38b,c,d 6Æ20c,d

6Æ27c,d 6Æ16d

0Æ11

***

6Æ80a

6Æ26b 6Æ19b,c

6Æ12b,c 6Æ09c,d

5Æ94d,e 5Æ86e

0Æ25

***

Table 2 Effects of acetic or lactic acid treatments on the reduction of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 (log CFU cm)2) in pork

Least-square means with different superscripts in each micro-organism significantly differ (P < 0Æ05). Level of significance: ***P < 0Æ001.

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Combination effect of organic acids and SC-CO2 in fresh pork

E. coli 9·0

L. monocytogenes 9·0

a

Log CFU cm–2

8·0

8·0 a

b

7·0

7·0 c d

6·0

5·0

6·0

b

5·0

c c

4·0

4·0

Salm. typhimurium 9·0

Log CFU cm–2

8·0

E. coli 0157:H7 9·0

8·0

a

7·0

a

7·0 b

6·0

6·0 c

b c

c

c

5·0

5·0

4·0

4·0

Figure 1 Effects of supercritical carbon dioxide (SC-CO2) at 12 MPa and 35C for 30 min and ⁄ or organic acid treatments on the reduction of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in pork. Bars indicate standard errors. Different letters denote significant differences (P < 0Æ05). ( ) Control; ( ) SC-CO2; ( ) AA (acetic acid) + SC-CO2 and ( ) LA (lactic acid) + SC-CO2.

pork are shown in Fig. 1. When SC-CO2 was applied to the samples at 12 MPa and 35C for 30 min, significantly lower numbers of micro-organisms were observed compared to the control. The levels of E. coli, L. monocytogenes, Salm. typhimurium and E. coli O157:H7 were more rapidly reduced by the combined treatment of 3% acetic acid and SC-CO2 than by the SC-CO2 treatment alone. However, no significant differences were observed between the samples combined treated with acetic acid and SC-CO2, and the samples combined treated with lactic acid and SC-CO2, with the exception of the level of nonpathogenic E. coli. Finally, a significantly lower level of E. coli was observed in the samples combined treated with lactic acid and SC-CO2 than the samples combined treated with acetic acid and SC-CO2. Discussion In the meat industry, organic acid solutions are being increasingly applied as sequential interventions for meat

decontamination, because their application to fresh meat can reduce foodborne pathogenic bacteria (Bacon et al. 2000). However, a number of researchers have investigated the effects of using organic acids for sterilization purposes in terms of the technical aspects and sensory quality of meat and poultry, and some have found unacceptable colour scores (Del Rio et al. 2007). Van Netten et al. (1995) reported that the application of 5% lactic acid resulted in an unacceptable sensory quality, which included pork color. In contrast, when ground beef was treated with 2% lactic acid, the treatment group maintained relatively similar sensory colour and beef odour characteristics as the control (Jimenez-Villarreal et al. 2003). Moreover, the treatment of buffalo steaks with 3% solutions of acetic and lactic acid had no adverse effect on surface colour (Surve et al. 1991). For this reason, in the current study, the treatment concentrations of the organic acid solutions were minimized, and the organic acid applications ranged from 1% to 3%.

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Organic acid molecules inhibit the outgrowth of both bacterial and fungal cells (Stopforth et al. 2003), and these inhibitory effects are caused by the induction of the following phenomena (Brul and Coote 1999): cell membrane disruption (Bracey et al. 1998), inhibition of essential metabolic reactions (Krebs et al. 1983) and stress on intracellular pH homeostasis (Bracey et al. 1998; Stopforth et al. 2003). Thus, pH decreases as the antimicrobial activity of the organic acids increases. However, in this study, although the pH of the lactic acid solution was lower than that of the acetic acid solution, no significant differences were observed between the acetic acid treatments and the lactic acid treatments within the same concentration, with the exception of the 2% organic acid treatments for the level of L. monocytogenes. When acetic or lactic acids were applied to the fresh meat, the reductions in the levels of nonpathogenic E. coli and the foodborne pathogens ranged from 0Æ75 to 1Æ07 log CFU cm)2 or 0Æ79 to 1Æ18 log CFU cm)2, respectively. In general, the application of SC-CO2 on solid foods compared with that on liquid foods, including meat and meat products, is more difficult with regard to microbial safety because of a more limited diffusion of CO2 into the solid matrices (Garcia-Gonzalez et al. 2007). Moreover, when SC-CO2 is applied to solid foods, both the treatment pressure and temperature can affect molecular interactions and protein conformation, and certain food samples show colour changes after treatment. For this reason, SC-CO2 should not be applied at too high temperatures or for too long, because it can deteriorate meat quality. However, according to the report of Choi et al. (2009), the level of nonpathogenic E. coli was reduced in SC-CO2 treatment at 12 MPa and did not differ among marinated pork samples following treatment at 12 and 14 MPa at same temperatures for 30 min. Moreover, when SC-CO2 was applied at 15Æ2 MPa and 31Æ1C, quality traits of fresh pork, such as muscle pH, weight loss and tenderness, were generally unaffected by the treatment (Choi et al. 2008). Therefore, from a meat quality point of view, the SC-CO2 treatment conditions in this study were also minimized (at 12 MPa and 35C for 30 min). Moreover, the meat sample size and SC-CO2 system were scaled up for the purpose of practical use compared with previous studies (Choi et al. 2008, 2009), the micro-organism reduction levels in the samples ranged from 1Æ29 to 1Æ59 log CFU cm)2. Moreover, although combined treatments had no synergistic effect, samples from the combined treatments had clearly different levels of nonpathogenic E. coli and the three pathogenic bacteria. For the combined treatment of 3% lactic acid and SC-CO2 at 12 MPa and 35C for 30 min, the levels of nonpathogenic E. coli and foodborne pathogens showed maximum reductions and were reduced in the range of 514

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2Æ10 to 2Æ60 log CFU cm)2 (E. coli, 2Æ58 log CFU cm)2; L. monocytogenes, 2Æ60 log CFU cm)2; Salm. typhimurium, 2Æ33 log CFU cm)2, E. coli O157:H7, 2Æ10 log CFU cm)2). Organic acids are inexpensive and environmentally friendly compounds; for these reasons, weak organic acids are widely used in food preservation. On the other hand, until now, the use of SC-CO2 as a preservation technique is operative and near to be employed at commercial scale, but has not been implemented on a large scale by the food industry (Garcia-Gonzalez et al. 2007). Moreover, the expenses associated with the contraction have constituted a significant barrier to the application of this technology, although the operation cost is inexpensive. Thus, the economics of the process must be assessed before the construction of SC-CO2 system. In conclusion, based on these results, the combined treatments of SC-CO2 and the organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO2 or the organic acids alone. Therefore, the results of this study should be useful to help increase microbial safety within the food industry. Acknowledgements This study was supported by the Agricultural R&D Promotion Center (Korea). The authors also thank the Korea University Food Safety Center for allowing the use of their equipments and facilities. References Acuff, G.R., Vanderzant, C., Savell, J.W., Jones, D.W., Graffin, D.B. and Ehlers, J.G. (1987) Effect of acid decontamination of beef subprimal cuts on microbiology and sensory characteristics of steaks. Meat Sci 19, 217–226. Bacon, R.T., Belk, K.E., Sofos, J.N., Clayton, R.P., Reagan, J.O. and Smith, G.C. (2000) Microbial population on animal hides and beef carcasses at different stages of slaughter in plants employing multi-sequential interventions for decontamination. J Food Prot 63, 1080–1086. Bracey, D., Holyoak, C.D. and Coote, P.J. (1998) Comparison of the inhibitory effect of sorbic acid and amphotericin B on Saccharomyces cerevisiae: is growth inhibition dependent on reduced intracellular pH? J Appl Microbiol 85, 1056–1066. Brul, S. and Coote, P. (1999) Preservative agents in foods. Mode of action and microbial resistance mechanisms. Int J Food Microbiol 50, 1–17. Castillo, A., Lucia, L.M., Roberson, D.B., Stevenson, T.H., Mercado, I. and Acuff, G.R. (2001) Lactic acid sprays reduce bacterial pathogens on cold beef carcass surfaces and in subsequently produced ground beef. J Food Prot 64, 58–62.

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