Antagonistic effect of acetic acid and salt for inactivating Escherichia coli O157:H7 in cucumber puree

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Antagonistic effect of acetic acid and salt for inactivating Escherichia coli O157:H7 in cucumber puree S.Y. Lee 1, M.S. Rhee, R.H. Dougherty 3 and D.H. Kang 3, 1 Department of Food and Nutrition, Chung-Ang University, Anseong-si, Gyeonggi-do, South Korea Division of Food Science, College of Life and Environmental Sciences, Korea University, Anam-dong, Sungbuk-ku, Seoul, South Korea 3 School of Food Science, Washington State University, Pullman, WA, USA Department of Food and Animal Biotechnology, Seoul National University, Sillim-dong, Gwanak-gu, Seoul, South Korea Keywords acetic acid, combination treatment, cucumber, Escherichia coli O157:H7, salt. Correspondence Dong-Hyun Kang, School of Food Science, Washington State University, Pullman, WA , USA. dhkang@wsu.edu 9 93: received June 9, revised 1 August 9 and accepted 13 August 9 doi:1.1111/j x Abstract Aim: This study was conducted to investigate the effects of acetic acid alone or combined with salt for inhibiting Escherichia coli O157:H7 in cucumber puree during storage time at 5 and C. Methods and Results: Samples at various concentrations were prepared by the addition of acetic acid (%, Æ5%, Æ5%, Æ75%, 1%, %, Æ5%, or 3%) along with NaCl (% or 3%) and pureed cucumber (5% or 9%). The effect of storage temperature (5 and C) on the survival of E. coli O157:H7 was also tested. The numbers of E. coli O157:H7 were reduced as the amount of acetic acid increased (P <Æ1) and rate of reduction was more rapid at C than at 5 C. Combinations of acetic acid and salt were less effective at killing E. coli O157:H7 in simulated pickled cucumber samples stored at 5 and C than did treatment of acetic acid alone. The concentration of pureed cucumber affected the reduction rate of E. coli O157:H7. When stored at 5 C, E. coli O157:H7 survived for up to 3 days in a mixture of 9% cucumber puree, 1% acetic acid, and 3% NaCl. Conclusions: There was the antagonistic effect of acetic acid and 3% NaCl with regard to the reduction of E. coli O157:H7. Significance and Impact of the Study: Our results suggest that pickled cucumbers prepared with at least % acetic acid and stored at room temperature ( C) after 3 days storage can free from E. coli O157:H7 without heating. Introduction Fruits and vegetables can become contaminated with pathogenic micro-organisms from different sources such as soil, irrigation water, washing and postharvest contamination (Beuchat and Ryu 1997). Pickling or fermentation is one traditional method used to preserve fruits and vegetables. Acidified pickled food products are generally considered safe due to low ph and relatively high salt content. Recently, however, outbreaks of foodborne pathogens such as Escherihcia coli O157:H7 and Salmonella spp. in acidified foods, such as apple cider and orange juice (that have ph values similar to many acidified pickled products) suggest that pickled vegetables may not be free from these pathogens (CDC 199, 1999). Escherichia coli O157:H7 could be a pathogen of concern in acidified pickled vegetables because of its low infectious dose (Gorden and Samll 1993; Keene et al. 199), high acid tolerance (Glass et al. 199; Miller and Kaspar 199; Benjamin and Datta 1995; Leyer et al. 1995; Tsai and Ingham 1997) and severe consequences of infection such as hemorrhagic colitis and hemolytic uremic syndrome (Griffin and Tauxe 1991; Padhye and Doyle 199). The growth or survival of E. coli O157:H7 in acid and acidified foods (ph Æ5) has been reported in a variety of products, including mayonnaise (Weagant et al. 199; Raghubeer et al. 1995), apple cider (Besser et al. 1993; Zhao et al. 1993) and fermented dairy products (Arocha et al. 199). Also, consumers have been moving away from salty foods over the past 15 years, and the pickling industry has been changing to accommodate them. Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 1 (1)

2 Antagonistic effect of acetic acid and salt S.Y. Lee et al. The involvement of some pathogens including E. coli O157:H7 in outbreaks associated with the consumption of acidic foods has drawn attention to the acid adaptation response of these pathogens and its impact on resistance to environmental stresses, especially acid. Acid-adapted cells have increased resistance to inactivation by organic acids and therefore acid-adapted cells can survive better than nonadapted cells in various low-ph foods (Glass et al. 199; Leyer et al. 1995; Tsai and Ingham 1997; Berry and Cutter ). Moreover, several works have revealed that acid-adapted E. coli O157:H7 strains survive longer under refrigeration than at room temperature in mayonnaise (Zhao et al. 1993; Zhao and Doyle 199; Erickson et al. 1995; Raghubeer et al. 1995), ketchup (Tsai and Ingham 1997), mustard (Mayerhauser 1) and unpasteurized apple cider (Zhao et al. 1995). These pathogens are often exposed to such environments during both food processing and food preservation. Understanding the survival of these pathogens in acidic foods may have important implications in food production. Pickled vegetables are made by immersing raw vegetables in brine containing acetic acid and salt then heated. Heat processing or pasteurization is performed to increase microbial food safety. However, some vegetables used in pickling are very heat sensitive and indicators quality such as colour and texture are usually degraded to a large extent during thermal treatment (Lau et al. ). Therefore, to maintain product quality, thermal treatment may be undesirable. In fact, some pickled vegetables such as fresh-pack pickles were produced without heat processing (Miller and Wehner 199). Moreover, consumers insist upon the freshness of food products. The demand for high-grade, mild (low salt and low acid), refrigerated pickles has increased over the past 1 years, and the pickling industry has responded with a range of products. Without heat treatment, organic acid (i.e. acetic acid) and salt are major factors that contribute to the microbial safety of acidified pickled vegetables. Sometimes other preservatives (such as sodium benzoate and potassium sorbate) are added to increase microbial safety. However, the US Food and Drug Administration (FDA) regulations do not allow the use of preservatives as the primary barriers to the growth of microbial pathogens in acidified foods (Breidt et al. ) and consumers also regard the use of synthetic preservatives in their food products undesirable. For acidified pickled food products, the Code of Federal Regulations (CFR) (1 CFR Part 11) states only that acid or acid ingredients must be added such that the ph is maintained at or below Æ; a heat treatment must be included in the process, if necessary, to prevent the growth of vegetative pathogens (such as E. coli) and spoilage organisms, including yeasts and lactic acid bacteria. Since organic acid (acetic acid) and salt is used in combination, the safety of pickled vegetables against food-borne pathogens could be achieved by the hurdle effect that exploits synergistic interactions between acetic acid and salt. Generally speaking, the combination of preservative factors has greater lethality for cells than one treatment alone. However, in our previous study, the combination of acetic acid and salt showed an antagonistic effect, that is, the combination had less effectiveness at killing E. coli O157:H7 in laboratory media compared to acetic acid treatment alone (data not shown). A similar antagonistic effect observed for the combination of lactic acid and salt for E. coli and E. coli O157:H5 has been reported by Casey and Condon (). However, these studies were conducted in laboratory media and results may differ in food samples. The effect of organic acids and low ph values on the growth, survival, and death of E. coli or E. coli O157:H7 has been examined in other studies. However, to date, no study has investigated the effect of combined acid and salt in pickled vegetables. Also, the behaviour of E. coli O157:H7 when subjected to combined preservation factors, particularly acidic ph and reduced water activity (a w ), needs to be more clearly determined. Therefore, this study was conducted to investigate the combined effect of acetic acid and salt on killing E. coli O157:H7 in simulated pickled cucumbers. Also, the effect of concentration of pureed cucumber (5% or 9%) and the effect of storage temperature (5 and C) on the survival of E. coli O157:H7 were tested in this study. Materials and methods Bacterial strains and culture conditions Three strains of E. coli O157:H7 (ATCC 3515, ATCC 39 and ATCC 39) were used to inoculate pureed cucumber. Each strain of E. coli O157:H7 was cultured in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) at 37 C for h, harvested by centrifugation at g for min at C, and washed twice with buffered peptone water. The final pellet was resuspended in buffered peptone water, corresponding to c. 1 9 CFU ml )1. Next, three strains were mixed to produce culture cocktails. The mixed culture cocktail of E. coli O157:H7 was used as an inoculum. Peoxidase assay To measure peroxidase activities, blanched cucumber tissue (5 g) from six slices was homogenized using a Waring blender. Peroxidase activity was measured by the procedures of MacAdam et al. (199). The assay mixture consisted of Æ5 mol l )1 sodium phosphate buffer (ph 7Æ), Æ1 mol l )1 H O, Æ7 mol l )1 guaiacol was added into blanched and homogenized cucumber sample. Colour changes were observed visually. 13 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 1 (1)

3 S.Y. Lee et al. Antagonistic effect of acetic acid and salt Sample preparation and treatment Figure 1 shows the process of cucumber sample preparation and treatment. Commercial cucumbers (Cucumis sativus, size B, 35 3 mm diameter) were purchased from local stores (Pullman, WA, USA). To inactivate peroxidase, cucumbers were placed in a steam cooker and blanched for 1 min at 1 C (the minimal time determined necessary to inactivate peroxidase enzyme activity). Following blanching, samples were homogenized in a Waring blender. A sample of cucumber puree was refrigerated for h (5 ± 1 C). In order to produce a puree containing 5% or 9% cucumber, a portion of puree (5 or 9 g) was placed in a presterilized 5 ml glass bottle with a screw cap (Corning Inc., NY). After that, samples were amended with various concentrations of acetic acid [, Æ5, Æ5, Æ75, 1,, Æ5 and 3 ml (v v)] by adding the proper volume of glacial acetic acid along with or 3 g of salt (w v) and bringing the volume up to 1 ml with sterile distilled water. Fifty and ninety-four per cent pureed cucumber was chosen because the concentration of commercial pureed cucumber ranged from 5% to 95%. The samples were prepared h prior to inoculation and held at 5 ± 1 or ± 1 C for equilibration, and a prepared culture cocktail of E. coli O157:H7 was inoculated into samples. Samples stored at C were examined at, 1,, 7, 1, and 1 h, respectively. Samples stored at 5 C were examined at, 1, 3, 5 and for up to 3 days at day intervals, respectively. Bacterial enumeration Fresh cucumber Washing with tap water Blanching using steam for 1 min Blending and homogenizing Distribution into sterile bottles Mixing with acetic acid and/or salt Inoculation of E. coli O157:H7 Storage at 5 C or RT ( C) Bacterial enumeration Figure 1 Preparation of cucumber samples and storage at room temperature. Aliquots (1 ml) of sample were tenfold serially diluted with 9 ml of sterile buffered peptone water and Æ1 mlof sample or diluent was spread plated onto Sorbitol MacConkey agar (Difco). All samples were incubated at 37 C for h, and then colonies were enumerated. Isolates from plates were randomly selected and subjected to serological conformation as E. coli O157:H7 (E. coli O157:H7 Latex Agglutination Test; Remel, Lenexa, KA, USA). ph measurement The ph values of samples were determined with a flatsurface combination probe (Model 3; Corning Inc.) before and after inoculation and during the time course. Statistical analysis All experiments were repeated three times with duplicate samples. Before analysis, the average of duplicate plate counts from three replications was converted to log 1 CFU ml )1 for analysis of variance (anova). Data were analysed by anova by the anova procedure of sas (SAS Institute, Cary, NC, USA) for a completely randomized design with three way analysis (acid concentration, temperature, storage time). When the effect was significant (P Æ5), least-squares means were separated by the probability option (a pairwise t-test). Results ph values The initial ph of the samples ranged from Æ to 5Æ (Table 1). There was little change (<Æ1 ph units) in ph after bacterial inoculation and during storage except for the control (Table ). Samples containing 3% salt had lower ph values at the same concentrations of acetic acid for both 5% and 9% cucumber puree than did those without salt, and samples containing 5% cucumber had lower ph values at the same levels of acetic acid and salt than did those containing 9% cucumber, except for the control and no acetic acid treatments. The addition of 3% salt had a significant effect (P <Æ1) on ph and the same results of lowering ph by adding salt were observed in laboratory media. From the Debye Hückel limiting law, lower ph values were calculated in acetic acid solution when the ionic strength of solution was increased by adding potassium chloride compared to solution without potassium chloride (Blackburn 199). Survivals of Escherichia coli O157:H7 at 5 C The survival of E. coli O157:H7 in samples containing pureed cucumber, acetic acid, and salt at 5 C is shown in Figs and 3. The numbers of E. coli O157:H7 were Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 1 (1)

4 Antagonistic effect of acetic acid and salt S.Y. Lee et al. Table 1 Initial ph values of cucumber samples before storage at room temperature ( C). (A) 5% cucumber (w v); (B) 9% cucumber samples (w v) Acetic acid (%, v v) Æ Æ5 Æ5 Æ75 1Æ Æ Æ5 3Æ A No salt 5Æb* Æc 3Æ9d 3Æ7e 3Æf 3Æ3g 3Æh 3Æ19i 3% Salt 5Æ3j 3Æ1e 3Æ5k 3Æ3g 3Æ7h Æ99l Æ9m Æn B No salt 5Æ77b Æ3c Æ15d 3Æ99e 3Æf 3Æ59g 3Æ9h 3Æ39i 3% Salt 5Æ3j Æk 3Æ7l 3Æ59g 3Æ5h 3Æ19m 3Æ9n Æ99o *Means within both row and column with different letters are statistically different (n = 3,P < Æ5). Table ph values of cucumber samples during storage at room temperature ( C). (A) 5% cucumber (w v); (B) 9% cucumber samples (w v) Acetic acid alone Acetic acid with 3% salt 1 Æ5 3 1 Æ5 3 A 5Æ 3Æ5 3Æ37 3Æ7 3Æ17 5Æ3 3Æ7 3Æ1 3Æ5 Æ Æ5 5Æ 3Æ 3Æ1 Æ9 Æ 5Æ13 3Æ Æ7 Æ5 Æ3 1 Æ9 3Æ51 3Æ1 3Æ1 3Æ Æ 3Æ5 Æ7 Æ3 Æ 5Æ 3Æ77 3Æ 3Æ33 3Æ5 Æ 3Æ37 3Æ1 3Æ1 3Æ 3 5Æ 3Æ71 3Æ1 3Æ33 3Æ Æ3 3Æ9 3Æ Æ9 Æ91 5 5Æ1 3Æ3 3Æ35 3Æ 3Æ1 3Æ 3Æ1 Æ3 Æ Æ1 7 5Æ 3Æ7 3Æ1 3Æ33 3Æ5 3Æ9 3Æ3 3Æ Æ97 Æ91 B 5Æ 3Æ 3Æ 3Æ51 3Æ 5Æ 3Æ51 3Æ 3Æ1 3Æ Æ5 5Æ 3Æ7 3Æ33 3Æ3 Æ9 5Æ1 3Æ Æ95 Æ7 Æ59 1 5Æ 3Æ75 3Æ5 3Æ3 3Æ17 Æ99 3Æ9 Æ9 Æ9 Æ7 5Æ31 3Æ97 3Æ9 3Æ5 3Æ9 Æ9 3Æ1 3Æ35 3Æ7 3Æ5 3 5Æ3 3Æ91 3Æ9 3Æ59 3Æ7 Æ5 3Æ5 3Æ3 3Æ1 3Æ3 5 Æ 3Æ9 3Æ3 3Æ53 3Æ Æ11 3Æ5 3Æ11 3Æ 3Æ 7 Æ73 3Æ95 3Æ73 3Æ 3Æ53 Æ3 3Æ9 3Æ 3Æ1 3Æ reduced as the amount of acetic acid increased (P <Æ1). Additionally, 9% pureed cucumber (Fig. 3) was a more hospitable environment (P <Æ1) for the survival of E. coli O157:H7 than was 5% pureed cucumber (Fig. ). The growth of E. coli O157:H7 was inhibited when Æ5% acetic acid was used irrespective of addition of salt and concentration of pureed cucumber. For 5% pureed cucumber, E. coli O157:H7 was not detected by direct plating after 1 day in samples containing Æ5% acetic acid and 3% salt, whereas it was detected after 1 day in samples with Æ5% acetic acid alone. In contrast, with the addition of % acetic acid, although there were no clear differences between the salt and no salt added treatments at the same acetic acid concentration after 1 day, the numbers of E. coli O157:H7 decreased more rapidly in the no salt added treatment than in the 3% salt added treatment at the same acetic acid concentration, except for Æ5% acetic acid. This phenomenon was observed in 9% pureed cucumbers with either Æ75% or 1Æ% acetic acid. For 9% pureed cucumber, E. coli O157:H7 was detected for up to 1 days even in samples containing 1% acetic acid and 3% salt. However, the growth of E. coli O157:H7 was inhibited in samples containing 3% salt with no acetic acid added (Fig. 3). Survivals of Escherichia coli O157:H7 at C Room temperature storage resulted in reduction of foodborne pathogens. The survival of E. coli O157:H7 in various samples containing pureed cucumber, acetic acid and salt storage at C is shown in Figs and 5. Escherichia coli O157:H7 was not detected after 7 days storage in samples containing Æ5% acetic acid regardless of the concentration of pureed cucumber. Differences in the combined antimicrobial effect of acetic acid and salt for the survival of E. coli O157:H7 were more clearly 13 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 1 (1)

5 S.Y. Lee et al. Antagonistic effect of acetic acid and salt (a) (a) (b) (b) Figure Survival of Escherichia coli O157:H7 in 5% cucumber containing various concentrations of acetic acid alone (a) and various concentrations of acetic acid with 3% salt (b) during storage at 5 C. ( )Æ%; ( )Æ5%; ( )Æ5%; ( )Æ75%; ( )1Æ%; ( )Æ%; ( )Æ5% and ( )3Æ% Figure 3 Survival of Escherichia coli O157:H7 in 9% cucumber containing various concentrations of acetic acid alone (a) and various concentrations of acetic acid with 3% salt (b) during storage at 5 C. ( )Æ%; ( )Æ5%; ( )Æ5%; ( )Æ75%; ( )1Æ%; ( )Æ%; ( )Æ5% and ( )3Æ%. Discussion observed for samples stored at C than those stored at 5 C. Although these differences were not clear for samples amended with Æ5% and 3% acetic acid, E. coli O157:H7 survived longer in samples containing 3% salt than in those with no salt at the same concentrations of acetic acid. This phenomenon was observed in both 5% and 9% pureed cucumbers. For both 5% and 9% pureed cucumber controls, E. coli O157:H7 grew rapidly, and their numbers reached > log 1 CFU ml )1 and decreased during the time course. In the presence of 3% salt, the growth of E. coli O157:H7 was inhibited at an early stage but the trends were similar to those of the controls. From our results, combinations of acetic acid and salt were less effective at killing E. coli O157:H7 in simulated pickled cucumber samples stored at 5 and C than did treatment of acetic acid alone. However, this antagonistic effect was not apparent in samples containing Æ5% or Æ5% acetic acid. In samples contained higher than Æ5% acetic acid, the rate of reduction of E. coli O157:H7 was rapid, moreover, results obtained using our time intervals for sampling could not determine enough differences between samples with or without 3% salt. Our results show enhanced susceptibility of E. coli O157:H7 in pickled cucumbers held at C, rather than Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 1 (1)

6 Antagonistic effect of acetic acid and salt S.Y. Lee et al. (a) 1 (a) Storage time (h) Storage time (h) (b) 1 (b) Storage time (h) Figure Survival of Escherichia coli O157:H7 in 5% cucumber containing various concentrations of acetic acid alone (a) and various concentrations of acetic acid with 3% salt (b) during storage at C. ( )Æ%; ( )Æ5%; ( )Æ5%; ( )Æ75%; ( )1Æ%; ( )Æ%; ( )Æ5% and ( )3Æ% Storage time (h) Figure 5 Survival of Escherichia coli O157:H7 in 9% cucumber containing various concentrations of acetic acid alone (a) and various concentrations of acetic acid with 3% salt (b) during storage at C. ( )Æ%; ( )Æ5%; ( )Æ5%; ( )Æ75%; ( )1Æ%; ( )Æ%; ( )Æ5% and ( )3Æ%. at 5 C. Reduced survival of E. coli O157:H7 in acidic foods at higher temperatures has also been seen in studies investigating other food products, including apple juice and cider (Miller and Wehner 199; Zhao et al. 1995; Uljas and Ingham 199), mayonnaise (Raghubeer et al. 1995) and ketchup (Tsai and Ingham 1997). Survival of E. coli O157:H7 and Salmonella typhimurium in model brines (3% salt and ph 7) and cheese brine ( 9% salt and ph 5Æ3 or 5Æ7) was significantly increased when stored at C compared to 15 C (Ingham et al. ). Clavero and Beuchat (199) observed, that regardless of the ph (Æ, 5Æ, and Æ) and a w (Æ99, Æ95, and Æ9) of TSB, survival of E. coli O157:H7 was better at 5 C than at or 3 C. Breidt et al. () investigated the effect of acetic acid on survival of E. coli in simulated acidified pickled products. They found that acetic acid has a higher inhibitory effect for killing E. coli compared with the effect of ph alone (glutamic acid was used) in the ph range of 3Æ1 Æ1, and the inhibitory effects of acetic acid were increased with increasing treatment temperature (1,, and 3 C). This may occur because pathogenic bacteria may be protected at low temperatures due to the production of cold shock proteins. However, further study of these possibilities is warranted (Han and Linton ). Since E. coli survived much longer in samples stored at 5 C compared to room temperature ( C) in this study, a higher temperature, such as room temperature, for storage of acidified pickled vegetables is recom- 13 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 1 (1)

7 S.Y. Lee et al. Antagonistic effect of acetic acid and salt mended rather than refrigeration for increasing product safety against E. coli O157:H7. The US FDA recommends 5 log unit reductions of food-borne pathogens by preservation technology for packaged fruits and vegetables products to ensure microbial safety although the contaminated levels of pathogens are usually low. Higher than 5 log reductions were observed when concentrations of acetic acid were higher than Æ5% and Æ75% in samples containing 5% and 9% pureed cucumber, respectively and % salt, and stored for h at C (Figs and 5). However, in the same samples containing 3% salt, 5 log reductions resulted only when samples contained higher than % acetic acid (Figs and 5). These results indicate that acetic acid in combination with salt is less bactericidal against E. coli O157:H7 in simulated pickled cucumber than is acetic acid alone. Therefore, although pickled cucumbers are commonly stored at room temperature, if they are produced without proper pasteurization processing and contain some amount of salt (such as 3%), a high concentration ( %) of acetic acid is required to ensure adequate destruction of E. coli O157:H7. Several studies have investigated the combined effect of acid or low ph and salt in laboratory media or food samples. However, they have conflicting results. It was the synergistic effect in some studies but it also shown the antagonistic effect in others. These differences may be due to differences of acidulant, media composition, different strains or species and growth phases of target microorganisms. However, the antagonistic effect of combining acetic acid and NaCl observed in this study using simulated cucumber samples was the same as in a previous study using laboratory media. Survival mechanisms of E. coli O157:H7 in acetic acid could be enhanced in the presence of NaCl. However, to date there is no direct evidence of how E. coli O157:H7 could become more resistant in the presence of salt. When acetic acid and salt are combined, acetic acid is the major factor that can cause the greatest reduction of E. coli O157:H7 in samples. The inhibitory effect of organic acids against bacterial cells is based upon the food products ph, the pk a of the acid, the antimicrobial activity of the undissociated form of the acid and specific effects of particular acids (Silliker et al. 19). An undissociated organic acid can readily permeate the cell membrane, whereupon dissociation occurs and the internal ph of the cell drops (Wart 199). Thus, the effectiveness of organic acids in retarding growth or killing foodborne pathogenic bacteria may vary among treatments, depending upon the percentage of undissociated acid at a given ph. However, this study demonstrated that survival of E. coli O157:H7 did not depend on the environmental ph if NaCl was added, since cucumber was a good medium for the growth of E. coli O157:H7. The complexity of acetic acid and NaCl environments may increase microbial cell tolerance to these harsh environments and E. coli O157:H7 may survive longer in lower ph environments than in higher ph environments with no added NaCl. The extent of survival of these foodborne pathogens also depends on the amount of nutrients available, acetic acid concentrations, amount of NaCl, and storage temperature. This study indicates that survival of E. coli O157:H7 in various acetic acid and NaCl environments could be changed if the level of cucumber solids was altered. The ph and or acidity of a food are generally used to determine processing requirements for regulatory purposes. However, these results indicate the importance of a combination of additives and nutrients, not only the ph of the products necessary to reduce the risk of foodborne pathogens survived in pickled cucumbers regardless of subsequent heat treatment. Since pickled cucumber is normally consumed without further heat treatment by the consumer, the results of this study are potentially important to the food industry. Our results suggest that pickled cucumbers prepared with higher than % acetic acid and held room temperature can be free of foodborne pathogens without heating, regardless of concentration of cucumber solids. Mild heating processing may further increase the safety of these products. Acknowledgement This work was supported by grant no. R from the World Class University (WCU) project of the Ministry of Education, Science & Technology (MEST) and the KOSEF through Seoul National University. References Arocha, M.M., McVey, M., Loder, S.D., Rupnow, J.H. and Bullerman, L.B. 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