3. ผลลัพธ์ และสนทนา3.1 การกิจกรรมเพาะเลี้ยงจุลินทรีย์ของสารจากธรรมชาติกับโรค foodborneUntilafewyearsago,fruitjuiceswerenotrecognizedasvehicles of foodborne illness because of their low pH and high organic acid levels (Mazzotta, 2000). However, several outbreaks associated with unpasteurized fruit juices have been reported in the last decade,andasaconsequence,issuessurroundingthesafetyoffruit products started to be an issue of concern (Anderson & Bailey, 2001). Therefore, four different pathogens were carefully chosen to evaluate the antimicrobial capacity in vitro of the natural com- pounds. Two Gram positive bacteria: L. monocytogenes and S. aureus; and two Gram negative: E. coli O157H:7 and P. aeruginosa. MIC values found in the in vitro study are shown in Table 1. Neither of the solvents used in this study (DMSO and tween 80) showed antimicrobial activity by themselves. Vanillin showed the same level of effectiveness against all the tested bacteria. However, geraniol showed less effectiveness against P. aeruginosa (MIC > 2.4 mL/mL), than E. coli, L. monocytogenes and S. aureus. Vanillin MIC values found by Vasantha Rupasinghe et al. (2006) are in accordance with our findings. Vasantha Rupasinghe et al. (2006) studied antimicrobial activity of vanillin in vitro against E. coliandP.aeruginosa,amongothersfoodpathogens,andfoundthat most microorganisms tested showed MIC values between 6 and 18 mM vanillin (between 0.9 and 2.7 mg/mL). In accordance with our results Scortichini and Rossi (2008) studied the in vitro antimicrobial activity of geraniol towards seven strains of Erwinia amylovora, and all of the strains tested at 105 CFU/mL were inhibited for 24 h by geraniol in the range 0.6e1.5 mL/mL.3.2. In vivo antimicrobial activity of natural compounds against E. coli O157:H7 inoculated in strawberry juiceIn order to evaluate the effectiveness of the proposed treat- ments in the inactivation of E. coli O157:H7 in strawberry juice, samples were inoculated with the pathogen. Fig. 2 shows the effect of vanillin treatment on E. coli survival throughoutrefrigeratedstorageat5C.TheinitialcountofE.colion untreated sample was 5.4 log CFU/mL. Immediately after the application of the treatments (day 0), Van1MIC had no effect on E. coli counts, while the rest of the treatments (Van2MIC, Ger1MIC, Ger2MIC) showed significant reductions of 2.2e2.4 log. At day 3, Van1MIC reached E. coli counts of 2.4 log. The rest of the treatments showed counts below the detection limit (<2 log), while the untreated sample showed counts of 5 log. Until the end of the refrigerated storage, untreated sample showedasignificantdecreaseinE.colicounts,reaching3.7logCFU/ mL by day 14. Those reductions in E. coli observed in untreated strawberry juice along storage time could be due to effects such as storage temperature (5 C) and competitive microflora (native flora). On the other hand, every treated sample (Van1MIC, Van2- MIC, Ger1MIC, and Ger2MIC) maintained the counts belowdetection limit showing an important bactericidal effect of both natural antimicrobial agents tested on the control of the pathogen inoculated. Inaccordancewithourresults,Moon,Delaquisb,Toivonenb,and Stanich (2006) found significant reductions (5 log cycles) on L. monocytogenes and E. coli O157:H7 counts adding 6 mg/mL of vanillin to apple juice, after 24 h of storage at 4 or 15 C. According to Fitzgeraldetal.(2004),basedoninvitrostudiesconductedusing E. coli, Lactobacillus plantarum, and Listeria innocua, the inhibitory activity of vanillin resides primarily in its ability to detrimentally affect the integrity of the cytoplasmic membrane, with the resul- tant loss of ion gradient, pH homeostasis and inhibition of respi- ratory activity. On the other hand, with respect to geraniol, few studies were found were this natural compound was tested in vivo. However, it was tested in vitro in several studies, and its antimicrobial activity has been associated with its ability to partition into membranes where it alters bilayer properties, making the membrane more fluid, increasing general permeability (Bard, Albrecht, Gupta, Guynn, & Stillwell, 1988) leading to potassium loss from within the yeast cells. The antimicrobial activity can be ascribed to the combinedmembraneeffects suchasincreased bilayerdisorderand ion leakage. These effects disturbed the osmotic balance of the cell through loss of ions, making its membrane associated proteins inefficient due to increased membrane disorder eventually leading to inhibition of cell growth or cell death (Dalleau, Cateau, Berges, Berjeaud, & Imbert, 2008).4. ConclusionsThe results found are promising. Vanillin and geraniol antimi- crobialactivitywasproveninvitro,againstfourdifferentpathogens of great importance in the food industry. Both natural compounds studied showed great efficacy in reducing E. coli counts even when applied in vivo on inoculated strawberry juice (more than 2 log cycle reductions, compared to untreated sample). Furthermore, throughout storage both biopreservatives were able to reduce the pathogen counts below detected limit, showing an important bactericidal effect. Vanillin and geraniol are both GRAS and can easily replace the traditional thermal treatment in the beverage industry. Further studies are necessary, in order to evaluate theTable 1 In vitro antimicrobial activity of vanillin and geraniol against Gram negative and Gram positive pathogenic bacteria: MIC values.E. coli L. monocytogenes S. aureus P. aeruginosa Vanillin 2.5 mg/mL 2.5 mg/mL 3 mg/mL 3 mg/mL Geraniol 0.6 mL/mL 0.5 mL/mL 0.6 mL/mL >2.4 mL/mLFig. 2. Effect of vanillin and geraniol on Escherichia coli O157:H7 in strawberry juice through refrigerated storage at 5 C. Bars indicate standard deviations. (Van1MIC) vanillin application at 1 MIC; (Van2MIC) vanillin application at 2 MIC; (Ger1MIC) ge- raniol application at 1 MIC; (Ger2MIC) geraniol applicarion at 2 MIC.B. Tomadoni et al. / LWT - Food Science and Technology 64 (2015) 554e557
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