combined-effects-of-heat-acetic-acid-and-salt-for-inactivating-escherichia-coli-o157-h7-in-laboratory-media_2009_food-control

1、Food Control 20 (2009) 10061012Combined effects of heat, acetic acid, and salt for inactivating Escherichia coliO157:H7 in laboratory mediaSun-Young Lee c, Dong-Hyun Kang a,b,*a Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376, USAb Department of Fo

2、od and Animal Biotechnology, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742, Koreac Department of Food and Nutrition, Chung-Ang University, 72-1 Nae-ri, Daedeok-myeon, Anseong-si, Gyeonggi-do 456-756, South Koreaa r t i c l e i n f oa b s t r a c tArticle history:Received

3、 25 August 2008Received in revised form 2 December 2008 Accepted 11 December 2008Heat, acid, and salt have been commonly used to ensure the microbial safety of foods and are often used in combinations in many food products. When combined, they can produce different results, such as additive, synergi

4、stic, and antagonistic effects. However, there has been little investigation into the effect of these combination treatments. Therefore, in this study, the effect of combined treatment of heat, acid, and salt was investigated in laboratory media. All possible paired combinations among three factors,

5、 heat (55 C), acid (0.25% acetic acid, v/v) and salt (3%, w/v) were tested and compared with individual treat- ments for killing E. coli O157:H7 in laboratory media. When salt was combined with heat, there was no signicant difference in reduction of E. coli O157:H7 (additive effect). However, when a

6、cid was combined with heat, there was a higher reduction of E. coli O157:H7 (synergistic effect). When salt was combined with acid treatment, salt gave protection against acid treatment (antagonistic effect), thus, there was lower reduction of E. coli O157:H7 in the combined treatment than in the si

7、ngle acid treatment. Depend- ing on the combination of preserving factors, results were different.2009 Published by Elsevier Ltd.Keywords: Combined effect HeatAcetic acid SaltE. coli O157:H71. Introduction(McMeekin et al., 2000). The essence of this approach is that foods can remain stable and safe

8、even without refrigeration, and are acceptable organoleptically and nutritionally due to the mild pro- cesses applied (Leistner, 1978). Consumers demand fresher and more natural products. This prompts food manufacturers to use milder preservation techniques and could be stimulating the cur- rent tre

9、nd to hurdle technology. The mode of action of combined hurdles may be additive or even synergistic with the latter deserv- ing particular attention as a means to select constraints that best achieve microbial stability and safety (Leistner, 1992). That syner- gism is anticipated derives from the ef

10、fect of hurdles on separate targets within the cell which disturb homeostasis by different mechanisms (Leistner, 2000).Hurdle technology is employed because we expect that a com- bination of two or more factors is more inhibitory effect than any one agent alone. However, recently, some studies showe

11、d that combination treatments were less effective at reducing levels of microorganism than were single treatments alone (Casey & Con- don, 2002) therefore application of the hurdle concept for preser- vation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in foods (Uyt

12、tendaele, Taverniers, & Debevere, 2001). The various responses of microorganisms under stress might hamper food preservation and could turn out to be problematic for the application of hurdle technology. Heating, acidication, and adding salt is widely used in the food industry, and are commonly used

13、 in combination for food preservation. For an example, acidied pickled vegetables are made byEscherichia coli O157:H7 is a member of the enterohemorrhagic group of pathogenic E. coli that has emerged as a foodborne and waterborne pathogen of major public health concern. A wide vari- ety of foods hav

14、e been implicated as vehicles of E. coli O157:H7 infection, including meat, milk, fruit juices, and vegetables (Bucha- nan & Doyle, 1997). Unlike most foodborne pathogens, E. coli O157:H7 is tolerant of acidic environments. Survival in apple cider (pH 3.64.0) and mayonnaise (pH 3.63.9) has been repo

15、rted andE. coli O157:H7 survived fermentation of buttermilk (pH 4.4) and drying and storage of fermented sausage (pH 4.5) (Buchanan & Doyle, 1997). These organisms cause a spectrum of disease increas- ing in severity from a mild diarrheal illness to hemorrhagic colitis, hemolytic uremic syndrome, an

16、d, in some cases, death (Bolton & Aird, 1998).Several years ago, hurdle technology was developed as a new concept for the realization of safe, stable, nutritious, tasty, and eco- nomical foods. This approach uses a combination of suboptimal growth factors, e.g. heating, chilling, drying, salting, co

17、nserving, acidication, oxygen-removal, fermenting, adding various preser- vatives, to establish growth inhibition of microorganisms in foods* Corresponding author. Address: Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376, USA. Tel.: +1 509 335 3937

18、; fax: +1 509 335 4815.E-mail address: (D.-H. Kang).0956-7135/$ - see front matter 2009 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2008.12.002Contents lists available at ScienceDirectFood Controljournal homepage: /locate/foodcontS.-Y. Lee, D.-H. Kang / Food Contr

19、ol 20 (2009) 100610121007immersing raw vegetables in brine containing vinegar (acetic acid) and salt, and then heat-treated. However, the relationships of these hurdles on the survival of pathogens are less clear. Therefore, in this study, the effect of combined treatment of heat, acid, and salt, wh

20、ich are major inhibitory factors used in many types of foods including pickled vegetables, was investigated in laboratory media.counts from three replications was converted to log10 CFU/ml for analysis of variance. To determine the slope of the regression line and corresponding standard error, the L

21、INEST function from Excel (Microsoft Ofce XP, Microsoft, Redmond, WA, USA) was used. In simple linear regression, the least squares and maxi- mum likelihood estimate of the slope are the same. To deter- mine the upper 95% condence limit for D-values (decimal reduction time) at 55 C or at pH 4.2 (0.2

22、5% acetic acid), the neg- ative inverse of condence limits for the slope was used. Statis- tical inferences concerning the similarity of D-values were determined with the general linear models (GLM) procedure of SAS (Version 8.1, SAS Institute, Cary, NC) for a completely ran- domized design. When th

23、e effect is signicant (P 0.05), mean separation was accomplished with the probability option (PDIFF, a pairwise t test).2. Materials and methods2.1. Bacterial culturesFive strains of E. coli O157:H7 (ATCC 35150, ATCC 43889, ATCC 43890, ATCC 43894, and ATCC 43895) were obtained from the Food Science

24、and Human Nutrition Culture Collection at Washington State University (Pullman, WA, USA). All cultures were maintained on tryptic soy agar (TSA; Difco laboratories, Detroit, MI, USA) slants and subcultured monthly.3. Results and discussion2.2. Cell suspensionIn this study, the combined effect of eac

25、h of two factors of heat, acid, and salt for reducing E. coli O157:H7 were investigated in lab- oratory media. LB broth containing 1% tryptone and 0.5% yeast ex- tract was used as a control (none of treatment), and for acid and salt treatment, 0.25% acetic acid and 3% salt were added into LB broth.

26、When 0.25% acetic acid was added into LB, pH was 4.2. Add- ing 3% salt decreased pH from 7.0 and 4.2 to 6.8 and 3.9 for LB and LB containing 0.25% acetic acid, respectively (data not shown). Fur- ther decrease of pH when 3% salt was added could be due to a change of ionic strength of solution. From

27、the DebyeHckel limit- ing law, lower pH values were calculated in acetic acid solution when the ionic strength of solution was increased by adding potas- sium chloride compared to solution without potassium chloride (Blackburn, 1969).Each paired combination among three factors (heat, acid, and salt)

28、 was compared with heat treatment alone or acid treatment alone. For combination of heat and salt or heat and acid, it was ob- served that 0.25% acetic acid and 3% salt did not affect the levels ofE. coli O157:H7 during heat treatment (up to 30 min) at 55 C; heating was the major factor that reduced

29、 levels of E. coli O157:H7 inoculated into broth. Therefore, the result of these two combinations (combination treatment) was compared with that of heat treatment alone (single treatment). For the same reason, since 3% salt did not affect levels of E. coli O157 during acid treat- ment time, the comb

30、ination of acid and salt was compared with acid treatment alone.Fig. 1 shows the effect of combined heat and salt combination and heat alone on killing ve different strains of E. coli O157:H7 in laboratory media. Initial levels of all ve strains of E. coli O157:H7 in laboratory broth before treatmen

31、ts were approxi- mately 108 CFU/ml. There were signicant decreases in the levels of E. coli O157:H7 with each increase in treatment time when they were treated with both heat alone and heat combined with salt and enumerated on both TSA and SMAC (P 0.05). The method of enumerating survivors of heat i

32、nactivation experi- ments can affect the percent recovery of heat-injured cells and hence has a bearing on the calculated heat resistance of the organ- ism. Enumeration on TSA led to D-values up to twice as great asEach strain of E. coli O157:H7 was cultured separately in Tryptic soy broth (TSB; Dif

33、co) at 37 C for 22 h, harvested by centrifugation at 9000g for 5 min at 4 C, and washed twice with buffered peptone water (Difco). The nal pellet was resuspended in buffered peptone water to a concentration calculated to yield approximately1010 CFU/ml. Then, each of the ve strains was used asinoculu

34、m.an2.3. Heat treatment with or without combination of acid or saltIn this study, individual heat treatment (55 C) was compared with heat treatment combined with 3% (w/v) sodium chloride or 0.25% (v/v) acetic acid. LuriaBertani (LB) broth containing 1% (w/v) tryptone (Difco) and 0.5% (w/v) yeast ext

35、ract (Difco) without salt and with 3% salt or with 0.25% acetic acid was prepared. Each cell suspension was 100-fold diluted into 2 ml of the three differ- ent media and subjected to heat treatment. For heat treatment, inoculated tubes were completely immersed in a water (55 C) using water bath. At

36、selected time intervals (0, 5, 10, 20, and 30 min), tubes were removed, cooled in ice water and used for the enumeration of survival bacteria as describe below.2.4. Acid treatment with or without combination of saltTwo milliliters of LB broth without salt and with 3% salt was prepared. Acetic acid (

37、0.25%) was incorporated into both LB and LB containing 3% salt using 25% acetic acid solution. Each cell sus- pension was added to the media as described previously and stored at room temperature (22 C). At selected time intervals (0, 1, 5, and 7 days), samples were enumerated as described below.2.5

38、. Bacterial enumerationTreated samples were serially 10-fold diluted with 9 ml sterile buffered peptone water. Diluted samples were spread-plated onto TSA as a non-selective agar and Sorbitol MacConkey agar (SMAC; Difco) as a selective agar for E. coli O157:H7 and incubated at 37 C for 24 h before c

39、ounting. The difference of numbers between TSA and SMAC were used to calculate the level of sublethally in- jured E. coli O157:H7.2.6. Statistical analysisAll experiments were performed in duplicate and repeated three times. Before analysis, the average of duplicate plate1008S.-Y. Lee, D.-H. Kang /

40、Food Control 20 (2009) 10061012those from enumerated on SMAC and there was a signicant differ- ence of D-values calculated from recovery levels of E. coli O157:H7 from TSA and SMAC (P 0.01) for all ve strains of E. coli O157:H7.Therefore, there was no signicant reduction of E. coli O157:H7 when 3% s

41、alt was combined with 55 C treatment, thus the combi- nation of 55 C and 3% salt showed the additive effect.BA 101088664422000510152025303505101520253035Incubation time (min)Incubation time (min)D 10C 1088664422000510152025303505101520253035Incubation time (min)Incubation time (min)E1086420051015202

42、53035Incubation time (min)Fig. 1. Survival curves for E. coli O157:H7 ATCC strains (A, 35150; B, 43889; C, 43890; D, 43894 and E, 43895) in laboratory media with or without 3% NaCl treated with heat at 55 C. E. coli O157:H7 was enumerated on both sorbitol MacConkey agar (SMAC) and tryptic soy agar (

43、TSA) for enumeration of healthy cells and recovery of injured cells, respectively. d, TSA (heat alone); s, TSA (heat combined with salt); ., MAC (heat alone) and 5, SMAC (heat combined with salt).Log10CFU/mlLog10 CFU/mlLog10CFU/mlLog10CFU/mlLog10CFU/mlS.-Y. Lee, D.-H. Kang / Food Control 20 (2009) 1

44、00610121009There are several studies that have studied the combination ef- fect of salt on heat treatment. In many studies, adding salt or other ingredients increases the heat resistance of pathogenic bacteria including E. coli O157:H7 because they decrease water activity (aw) of samples. Blackburn,

45、 Curtis, Humpheson, Billon, and McC- lure (1997) reported that heat resistance of E. coli O157:H7 in- creased with increasing NaCl up to 8.5% (w/w) and the resultant model predicted maximum D-values at about 57% (w/w) NaCl. A similar effect has been seen for Listeria monocytogenes at NaCl concentrat

46、ions up to 4.5% (Cole, Davies, Munro, Holyoak, & Kilsby, 1993). For Salmonella spp., heat tolerance increases as the aw de- creases in other studies as well (Corry, 1974; Goepfert, Iskander, & Amundson, 1970; Kirby & Davies, 1990; Sumner, Sandros, Har- mon, Scott, & Bernard, 1991; Mattick et al., 20

47、01). In this study, adding 3% salt did not signicantly increase the heat resistance of ve strains of E. coli O157:H7 upon treatment at 55 C. This re- sult may have occurred because 3% salt was not enough to show the synergistic effect, as only small decreases of aw in media were observed. In fact, 3

48、% salt added into LB decreased aw from 0.99 to0.97 (data not shown). In one study which investigated the effect of 3% and 8.5% salt on heat treatment, the effect of 3% salt was small and 3% salt did not increase the heat resistance of E. coli O157:H7 as much as did 8.5% salt (Blackburn et al., 1997)

49、.Fig. 2 shows the survival of ve strains of E. coli O157:H7 after subjection to heat treatment alone and combination of heat (55 C) and acetic acid. There was a signicant decrease in the levels ofE. coli O157:H7 with each increase in treatment time when E. coli strains were treated with both heat al

50、one and heat combined with acetic acid and enumerated on both TSA and SMAC (P 0.05). Treatment with heat combined with acetic acid signicantly in- creased the reduction of E. coli O157:H7 compared to heat alone for all ve strains of E. coli O157:H7 both on TSA and SMAC (P 0.01). Also, there were sig

51、nicantly levels of E. coli O157:H7 recovered on TSA and SMAC (P 0.01). D-values at 55 C for the combination of heat and acetic acid were in the range of 2.53.0 min and 2.32.6 min when they recovered on TSA and SMAC, respectively, and there was no signicant difference among all ve strains of E. coli

52、O157:H7. Compared to D-values at 55 C in LB (control; 7.49.6 min and 4.86.5 min enumerated on TSA and SMAC, respectively), D-values were signicantly decreased (P 0.01) when heat treatment was combined with acetic acid and this indicates that the combination of heat and acetic acid is very effective

53、at killing all ve strains of E. coli O157:H7 compared to heat alone. Therefore, the combined effect of heat and acetic acid showed the synergistic effect.The pH of the heating menstruum affected thermal inactivation, with low and high pH values generally decreasing heat resistance (Blackburn et al.,

54、 1997). A similar synergistic effect of low pH on heat treatment has been observed with the inactivation of E. coli O157:H7 and S. enteritidis in buffer (Teo, Raynor, Ellajosyula, & Knabel, 1996), Salmonella in liquid whole egg (Anellis, Lubas, & Rayman, 1954) and L. monocytogenes in beef gravy (Jun

55、eja & Eblen, 1999). In fact, temperature is a primary factor inuencing organic acids activity and there are several studies reporting that increas- ing temperature increased the effectiveness of organic acids (Brud- zinski & Harrison, 1998; Presser, Ross, & Ratkowsky, 1998; Uljas & Ingham, 1998). Ho

56、wever, the effect of combination of heat and acid could be affected by several factors such as type of acidulent and microorganism. Citric acid was shown to increase the heat resis- tance of spoilage yeasts in concentrated orange juices (Juven, Kan- ner, & Weisslowiez, 1978) and AbdulRaouf, Beuchat, and Ammar (1993) demonstrated that the rate of thermal inactivation of E. coli O157:H7 in acidied beef slurry was dependent on the acidulent an