nwnsadt euetenmeso ibeluootcbacteria leuconostoc viable of fermentation numbers the malo-lactic reduce bacterial to and delay of Fu- wines to one bacteria. in found some is was of cycle, acid growth acid inhibit maric citric to the reported 2004). compounds on the (Burt intermediate an these com- acid, for de- Fumaric occurring substitute to naturally might efforts of that been pounds activities have antimicrobial there years, the products recent termine in In preservatives undesirable. traditional primary be these the to of are con- use many which However, consider yeasts, products. sumers and these and acid in (LAB) microorganisms of bacteria spoilage preservation growth acid assist inhibit lactic preservatives to These of use vegetables. of and fruits history acidified long a have fite ute erdcinwtotpriso sprohibited is permission without reproduction Further 10.1111/j.1750-3841.2011.02299.x works doi: government US original to claim No suitable. Science be may of that Journal products other of exclusion Research the Agricultural to Carolina approval North imply or it Agriculture does warranty of nor or Dept. Service, guarantee U.S. a the constitute by Bioprocessing, not product does the Food, product of of proprietary 27695-7624. or Department NC trademark a Raleigh, the University, of State of Mention Carolina Series North Sciences, Journal Nutrition the and of FSR09-15 North nr 7624, Paper Box Hall, author to Schaub inquiries Direct 322 U.S.A. 27695-7624, Unit, P NC Raleigh, Research Univ., State Science Carolina USDA- Food with is SAA Author 6/7/2011. ARS, Accepted 8/9/2010, Submitted 20100901 MS Introduction P M. Ilenys Yeasts and Bacteria Acid Lactic Target That Cinnamaldehyde Acid and Fumaric of Combination a with Cucumbers Acidified of Preservation erez-D ´ oimbnot,ptsimsrae n oimmetabisul- sodium and sorbate, , Sodium rcia Application: Practical Keywords: Abstract: eescesul rsre,a niae ylc fyat rLBgot n irba atcai rehnlproduction ethanol or 30 acid at lactic storage microbial during and cinnamaldehyde growth and LAB (3.8 acid or cinnamaldehyde fumaric yeasts agent, of of antimicrobial combination lack The a by inhibitor. by indicated yeast as grade preserved, food successfully a acidified were with in it of a growth combine as prevented to acid mM) fumaric necessary of apply be usefully inhibition To will medium. achieve it juice to cucumber same required the benzoate. was in concentrations sodium concentration saturation of this aciduric presence of extremely the Half an of in 3.8. growth cucumbers inhibit pH to of at medium required juice were preservation strain cucumber acid allow acidified fumaric an could of in millimolars that (LAB) Forty bacteria conditions acid resembling lactic of system growth inhibited model acid Fumaric cucumbers. of storage fccmesi eue alcvrbieslto n ihu hra process. thermal a without and solution brine cover NaCl reduced a in cucumbers of xetdgot ftesolg yeasts spoilage the of growth expected rsdu eaiufiefrpeevto faiie eealswtotatemlprocess. thermal sorbate, a potassium without benzoate, vegetables sodium acidified of of use preservation the to for approach metabisulfite alternative sodium an or as serve may compounds preservative occurring ´ a Emi:Ilenys.P (E-mail: ıaz ´ erez-D h aual curn opud uai cd a vlae saptnilpeevtv o h long-term the for preservative potential a as evaluated was acid, fumaric compound, occurring naturally The inmleye uai cd atcai atra eeal preservation, vegetable bacteria, acid lactic acid, fumaric cinnamaldehyde, ´ ıaz C erez-D ´ 01Isiueo odTechnologists Food of Institute 2011 hssuyeautsteptnilapiaino lentv rsraie o h ogtr storage long-term the for preservatives alternative of application potential the evaluates study This ´ ı[email protected]). .globiformis Z. yoacaoye globiformis Zygosaccharomyces swl steyat htwr rsn nfehccmes cdfidcucumbers Acidified cucumbers. fresh on present were that yeasts the as well as R n tya18) uai cdwsrpre ohv bactericidal a have (Splittstoesser to juice reported against was effect grape acid refrigerated Fumaric inhib- 1989). in also Stoyla LAB It and of 1974). activity others the and ited Pilone 1974; Kunkee and (Ough hc as taoi emnain paeo uai cdand acid 1952), fumaric Etchells of and Uptake fermentation. (Bell ethanolic yeasts very cause spoilage both which of and inhibition LAB require cucum- will acid-tolerant for temperatures method ambient acid preservation at fumaric successful bers by A replaced application. be lower this could or in benzoate 3.6 sodium to if to adjusted undertaken determine was pH investigation This with growth. benzoate microbial mM) prevented sodium (150 acid mM Acetic 12 cucumbers. of and chlo- firmness calcium fer- the containing maintain without solution to storage brine ride bulk a allow in would storage by that mentation way a foods. in in preserved acid be is tartaric and flavor and acid tart/sour a citric substitute has levels to acid minimal used This of often consumables. addition to the acid requests this and regarded of generally status a (GRAS) granted safe Administration as Drug and Food The against active be to shown been botulinum also Clostridium have esters Fumarate 2006). of ( aureus numbers pathogens the inoculated reduced and it microflora and natural 2002) Beelman and (Comes 3.3 pH P acidulent. an as foods in use of history long a has acid Fumaric erez-D ´ ,and and .bailii Z. ´ a n cetr 20)soe htccmescould cucumbers that showed (2008) McFeeters and ıaz o.7,N.7 2011 7, Nr. 76, Vol. shrci coli Escherichia ◦ o o hscmiaino naturally 2 of combination This mo. 2 for C a o niie yfmrcai tnear at acid fumaric by inhibited not was Dmcyadohr 1987). others and (Dymicky p. nfehctltue(od n others and (Kondo lettuce fresh-cut on spp.) .plantarum L. Zygosaccharomyces 17H ncmeca pl ie at cider apple commercial in O157:H7 atbclu plantarum Lactobacillus r ora fFo Science Food of Journal A45a H35 As 3.5. pH at LA0445 .coli E. , Staphylococcus LA0445 M473

M: & Safety Z. Zygosaccharomyces C for 1 wk. Preservative ◦ CFU/mL for bacteria and yeasts, LA0445, and a mixture of 5 strains (Table 1). The mixed yeast C. 4 ◦ in acidified cucumber jars and 10 Z. bailii 5 0.1 by addition of 3 N HCl solution. The ± C and eluted with 0.03 N sulfuric acid at a flow Z. bailii, ◦ and isolates and 2 Lactobacillus plantarum For each treatment, triplicate 15-mL tubes of the EM were in- Size 2B cucumbers (25.4 to 31.8 mm dia) were obtained from Preservation treatments in which there was no detectable mi- respectively. oculated and incubated statically at 30 a local processorThe and cucumber:brine packed ratio in withouttions the contained washing jars commercial was into vinegar 55:45 (20% 46 w/v.chloride acetic Cover acid) such solu- oz that and after calcium jars. equilibration withcentration the of cucumbers acetic the acid con- wasof 0 calcium or chloride 150 was mM eitherzoate and 100 (Fisher the mM Scientific concentration or Co., 30fumarate Pittsburgh, mM. (Alfa Pa., Sodium Aesar, ben- U.S.A.) Ward and Hill,powders sodium to Mass., equilibrated USA) concentrations were ofspectively. 12 added Cinnamaldehyde mM as (Sigma-Aldrich and dry 30mM, Co., re- St.U.S.A.) Louis, was Mo., added toin the the text. equilibrated Thewas concentrations initial adjusted equilibrated indicated to pH 3.5 of the brined cucumbers amount of HClslurry required and was cover determined brinetitrating by solution the mixing in mixture cucumber a withcate 55:45 acid jars (w/v) or of ratio, base 2 andtreatment. to different Jars then lots the were of closed targeta cucumbers with pH. rubber commercial were Tripli- septum prepared lug to for capswith allow each fitted for sterile with inoculation and syringes. samplingsoften The of the the lids plastisol jars werejars. gasket heated Jars and were in incubated immediately at boiling applied 30 water to to the filled Evaluation of the effect of aand combination cinnamaldehyde of on fumaric the acid growth ofmicroorganisms naturally and occurring spoilage yeasts, globiformis bacteria and yeasts, respectively. Bacteriawere incubated and for yeast 16 or broth 72 cultures and h, resuspended centrifuged, with and then saline washed solution twice the prior to cucumber the juice inoculationstrains into experimental of medium. the Mixtures individualinoculate the of microbes EM listed 2 to 10 in to Table 5 1 were used to crobial growth after 3oculating wk of incubationglobiformis were challenged by in- effectiveness was evaluated based upondity the development as of turbi- determinednm by using the measurements Nanodrop ND-1000 ofter UV-Visible (Nanodrop, Spectrophotome- optical Wilmington, density Del.,assessment at of U.S.A.), any and 600 reduction followedulated or by microorganisms increases an by in plating thethe on numbers end either of of MRS inoc- the oranalysis, incubation samples YM period. of agar the In at inoculated additionformance EM were to liquid analyzed microbiological by chromatography high (HPLC) per- and to malic determine acid if werelactic utilized acid during and the ethanol incubationusing were period a formed. and 30-cm Chromatography if HPX-87H wascules, done column Calif., (Bio-Rad U.S.A.) (McFeeters Laboratories,was and Her- heated Barish to 2003). 65 The column rate of 1.0 mL/min.detector A Thermo set to Separations UV6000 collectacids. diode data A array at Waters model 210 410 nmseries refractive was index was used detector used to connected to in detectnal measure standards organic were glucose, used fructose, for calibration andethanol, and ethanol. quantification and of Exter- organic sugars, acids. Amer- = Sevillano variety olives spp. (Etchells and Bell Candida Vol. 76, Nr. 7, 2011 r USDA-ARS Research Unit = NRRL SPY 9NRRL SPY 15NRRL SPY 21 Spoiled sweetNRRL pickles SPY Spoiled 29 sweet pickles NRRL SPY Spoiled 32 sweet pickles NRRL Y7260 Spoiled sweet pickles NRRL Y7258 Spoiled sweet pickles ATCC8287 Spoiled salad dressing Spoiled salad dressing ATCC14869 Green, fermenting ATCC19255ATCC 8293 Human feces ATCC8014 Dairy ATCC14917FSRU Fermenting LA0445 olives ATCC25745 Corn silage Pickled cabbage ATCC33316 Fermented cucumbers Plants Dried beer yeast spp. and C) using an Eppendorf Centrifuge 5810 ◦ Black and Decker, Towson, Md., U.S.A.). , (25 g × Hansenula 0.5%) of 3 N HCl or 5 N NaOH solution and filter < Strains of aciduric microorganisms used in this study. USDA-Agriculture Research Service Culture Collection; ATCC Journal of Food Science = The objective of this investigation was to determine the Microorganisms listed in Table 1 were propagated on deMan The experimental media (EM) consisted of fresh cucumber juice ican Type Culture Collection; FSRU Culture collection. NRRL MicroorganismsZygosaccharomyces globiformis Strains Source Zygosaccharomyces bailii Lactobacillus brevis Leuconostoc mesenteroides Lactobacillus plantarum Pediococcus pentosaceous 1950; Etchells and othersthe ability 1952), of combinations have of beencinnamaldehyde fumaric documented. acid (Tampieri and Thus, the and yeast2008), inhibitor, others to 2005; preserve Jantanhyde cucumbers and constitutes was up others also toactive evaluated. ingredient 75% (Calsamiglia Cinnamalde- of and cinnamon2008). others Cinnamon 2007; oil is Jantan currently and and added ita others to seasoning, selected is primarily pickle the products for as main the sweet varieties. ability of fumariccinnamaldehyde acid to and inhibitcarrying combinations growth out of of lactic LAB fumaricpotential acid that acid spoilage fermentations are and yeastscucumber of capable pickle that cucumbers products. of cause as ethanolic well as fermentations in The extracted juice was frozen untilfor used, 1 thawed h and at centrifuged 18660 sterilized. Rogosa and Sharpe (MRS) or Yeast and Mold (YM) medium for R (WestBury,N.Y.,U.S.A.) to remove solids. Cover brineconsisted solution of 300 mM aceticthat acid the and final 60 acid mM andwere calcium calcium 150 chloride, chloride and so concentrations 30 inLaboratory the mM, Products, EM respectively. Inc., Dry Gardena, fumaricin Calif., acid U.S.A.) the was (Spectrum EM dissolved atpH concentrations was adjusted shown in tovolumes Table ( 3.8 2 or and 3.5 3. as The indicated EM by addition of small M474 Table 1– Materials and Methods Evaluation of the effect of fumaricgrowth acid in on acidified microbial cucumber juice LAB inhibition by fumaric acid. . . its conversion to malic acidSaayman by and yeasts others (Corte-Real 2000), and naturally Leao presenttations in 1990; such cucumber as fermen- and cover brine solution mixedcumber in juice a 50:50 was ratio expressedcumbers (w/w). (25.4 from Fresh to 2 cu- 31.8 lots mm(JM400 dia) of Juiceman using size an Jr. automatic 2A juice pickling extractor cu-

M: Food Microbiology & Safety irognssFmrcai o of Log acid Fumaric Microorganisms yoacaoye bailii Zygosaccharomyces globiformis Zygosaccharomyces mesenteroides Leuconostoc pentosaceous Pediococcus CFU/mL (mM) added brevis Lactobacillus plantarum Lactobacillus tested on ntejr o 5mn usqety aswr oldto cooled were jars Subsequently, min. 15 for jars 40 the in point aktdktl.Pserzto a oet vlaeteability the evaluate to done was Pasteurization kettle. jacketed irogns uai cdLgo CFU of Log acid Fumaric Microorganism ∗ bailii Zygosaccharomyces /mL globiformis Zygosaccharomyces (mM) added brevis Lactobacillus plantarum Lactobacillus tested 3– Table ∗ 2– Table 10 of populations initial give to 10 jars and through sample CFU/mL the inoculated of individually septum were lid the bacterium the and isolates . . acid. fumaric by inhibition LAB aktdktl oatmeaueo 74 of temperature a to kettle jacketed † † kpostinoculation. wk 1 kpostinoculation. wk ‡ ‡ envalues Mean values Mean P P ND ND atra n es utrswr ncltdt 5.0 to inoculated were cultures yeast and Bacterial 5.0 to inoculated were cultures yeast and Bacterial nta lcs,futs,admlcai ocnrtoswr 25.2 were concentrations acid malic and fructose, glucose, Initial 25.2 were concentrations acid malic and fructose, glucose, Initial > > eetdjr eepserzdb etn nwtri steam- a in water in heating by pasteurized were jars Selected ◦ .5.Azr au a sindt hs esrmnsta eeblwtedtcinlmt(ND). limit detection the below were that measurements those to assigned was value zero A 0.05). .5.Azr au a sindt hs esrmnsta eeblwtedtcinlmt(ND). limit detection the below were that measurements those to assigned was value zero A 0.05). = = ncl a ae eoermvn hmfo h steam- the from them removing before water tap cold in C o detected. not detected. not feto uai cdo rwhof growth on acid fumaric of Effect feto uai cdo rwhof growth on acid fumaric of Effect ± ± tnaddvainfrtotil.Ma auswti oun o ahmcoraimwt iia etr r o infiatydfeet(Duncan’s different significantly not are letters similar with microorganism each for columns within values Mean trials. two for deviation standard tnaddvainfr2til.Ma auswti oun o ahmcoraimwt iia etr r o infiatydfeet(ucnsmu (Duncan’s different significantly not are letters similar with microorganism each for columns within values Mean trials. 2 for deviation standard 5 F/Li ahjr respectively. jar, each in CFU/mL 06.09 5.89 6.19 60 6.24 50 5.90 40 6.10 ND 60 ND 50 40 ND ND 20 10 4.89 30 20 10 06.5805.9307.8606.65 06.26 6.07 5.87 60 5.91 50 5.75 40 6.02 ND 60 ND 50 40 ND 20 10 4.77 20 3.08 10 4.62 7.49 30 ND 20 10 3.86 7.57 7.58 40 30 20 10 57.5257.0057.8257.84 06.5207.5607.7906.9707.8007.54 ± ± ◦ . n 4.0 and 0.5 4.0 and 0.5 ttesoetheating slowest the at C Lactobacillus Lactobacillus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.3 0.5 0.6 1.9 0.7 0.4 0.2 0.6 0.2 0.6 1.4 b b b b † 0.1 c c c c 0.5 0.4 0.4 0.1 1.1 0.6 0.8 0.4 0.3 0.1 0.8 0.1 0.3 0.3 0.3 0.3 0.1 0.4 2.9 0.2 0.2 0.1 ± ± a a a a a a a a a a a ± ± † a b b b b b b b b a a a a a a a a a a a a a a .,29.9 0.9, 29.9 0.9, . F/L respectively. CFU/mL, 0.5 respectively. CFU/mL, 0.5 6.7 7.3 12.5 ND 12.1 11.6 10.7 25.5 0.5 14.3 19.3 16.1 23.1 24.2 21.1 23.3 26.5 26.8 18.6 23.1 25.6 19.3 22.3 24.4 22.4 22.2 2.1 9.8 1.2 5.6 3.7 4.6 lcs rcoeFmrcai ai cdLci cdEthanol acid Lactic acid Malic acid Fumaric Fructose Glucose p.and spp. p.and spp. lcs rcoeFmrcai ai cdLci cdEthanol acid Lactic acid Malic acid Fumaric Fructose Glucose ND ND ND ND ± ± ± b ND ND ND ND ND ND ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.6 0.8 0.1 b b b 1.6 1.4 1.6 1.5 1.5 5.5 9.3 c .,ad14.9 and 1.4, 14.9 and 1.4, 0.6 5.3 0.9 0.2 0.6 3.3 b b a a a a 0.9 3.8 1.9 0.9 1.5 2.5 0.8 2.8 7.0 9.9 7.5 5.4 9.7 6.1 8.6 b b b a a a a a ab a b b a a a a b a a a a a a a a a a a a a a Zygosaccharomyces Zygosaccharomyces 4 usrt tlzto (mM) utilization Substrate usrt tlzto (mM) utilization Substrate 28.5 29.6 29.8 34.5 27.2 30.0 30.1 30.1 12.6 19.4 21.5 23.7 11.7 10.1 32.2 32.2 32.2 29.2 34.9 32.3 32.3 32.3 32.3 32.3 32.3 32.3 32.3 32.3 raim aual rsn ntefehcucumbers. fresh the from on interference present without naturally grow organisms to microorganisms inoculated of ilsmlso oe rn ouin eeaetclycollected aseptically were solutions brine cover of samples tial cdfidccme jars from cucumber collected acidified samples for analysis chemical and Microbial 4.2 2.5 5.0 9.5 ND ND ND ND fe qiirto fteaiie uubrjr 2d,ini- d), (2 jars cucumber acidified the of equilibration After ND ND ND ND ND ND ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± .,respectively. 3.3, respectively. 3.3, 0.8 0.6 0.2 6.2 b b b b b b b c a a 6.4 6.4 1.5 8.6 1.5 1.5 1.5 10.4 2.3 6.2 2.9 2.6 5.3 6.7 0.2 0.2 0.2 4.0 6.4 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 b a a a a a a a a a a a a a b c a a a a a a a a a a a a a a a a p.i uubrjiemdu tp . n 30 and 3.8 pH at medium juice cucumber in spp. p.i uubrjiemdu tp . n 30 and 3.5 pH at medium juice cucumber a in spp. 10.5 2.5 5.3 5.7 5.3 3.1 9.8 4.1 o.7,N.7 2011 7, Nr. 76, Vol. ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ± ± ± ± ± ± ± ± –4.1–9.1 10.9 – 12.9 – 11.5 – 15.5 –3.1– 15.2 – 0.3 1.0 0.9 1.0 1.3 2.3 0.5 b b a a a a a a a a a a a a a a a a a a a a a a a a 1.5 a a a a a a a a ‡ ‡ 14.9 7.4 7.5 7.6 7.4 7.3 7.6 10.5 12.4 12.9 12.8 1.8 3.8 3.1 8.3 9.7 ND ND ND ND ND ND ND ND ± ± ± ± ± ± ± ND ND ND ND ND ND ND ND ND ND ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± r 1.2 0.6 1.2 1.1 0.7 1.1 0.6 b b b b 5.8 7.5 7.5 7.5 0.3 2.4 ora fFo Science Food of Journal 0.8 1.7 0.8 1.5 0.4 b b b b a a a a a a a a a a 0.4 1.0 0.7 1.1 1.0 0.7 b b b b b b a a a a a b b a a a a a a a a a a a 102.3 rdc omto (mM) formation Product 86.2 45.2 29.9 39.1 35.4 rdc omto (mM) formation Product 1.64 2.27 51.7 10.5 24.3 34.9 35.4 76.6 88.2 84.4 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± b b b b a a a a a a a a 47.7 10.6 41.7 12.5 14.9 11.2 b b c c a a a a a a a a a a 0.8 0.1 5.1 3.4 6.3 0.4 6.8 9.1 3.1 2.1 b b b a a a a a a a ab a a a a a utpernetest, range multiple tpernetest, range ltiple 49.8 65.1 69.4 72.4 36.5 57.3 58.2 48.8 21.3 54.6 49.9 48.7 48.5 49.2 34.3 48.8 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ± ± ± ± ± ± ± ± ± ± ± ± ± M475 ± ± ± ◦ 1.3 16.7 18.6 5.3 5.2 18.9 18.2 16.1 ∗ C ∗ 10.1 12.6 10.6 14.3 10.5 a a a a a a a a a a a a a a a a a a a a a a a a 9.7 7.5 8.0 a a ◦ ,1 C a a a a a a , a a a a a a a a a a

M: Food Microbiology & Safety ± a a a a a into ,and ND ND ND ND ND 2 (mM) Z. bailii a a a a a ∗ ND ND ND ND and ND Products formed a a a a a 2.1 ± None None None None Z. globiformis into pasteurized cucumber jars 11.9 in pasteurized and acidified cu- a spp. in foods has been traditionally (mM) a a a a 0.7 Z. bailii ± Z. bailli Substrates utilized and and Zygosaccharomyces standard deviation for two trials. Mean values within columns for each Z. globiformis ± Ethanol production and fructose utilization 60 d after inoc- 0.05). A zero value was assigned to those measurements that were below the Z. globiformis not detected. supplemented with the yeast inhibitor, cinnamaldehyde. > 2 = Table 4 shows the results of an initial survey to determine the Table 5 shows the inhibition of microbial growth as indicated ND microorganism with similar letters aretest, not P significantly different (Duncan’s multipledetection range limit (ND). Preservative Mean values 3.8 Cinnamaldehyde0.8 Cinnamaldehyde∗ None 2.15 added (mM)12.0 Sodium benzoate19.0 Cinnamaldehyde7.6 Cinnamaldehyde None Glucose None None Fructose Glycerol Ethanol and a reduced amountfumaric of acid glucose. added, The 60Therefore, highest mM, fumaric concentration is of acid nearwould added its not by solubility be able limit itselfInhibition to in to of prevent water. growth acidified of cucumbers potentiala challenge spoilage due yeasts. tohigh the levels ability of of preservatives, these sugars,and microorganisms , others to 2007). and Additionally, tolerate a acidic variety pH offumaric organic (Martorell acids, acid, including are either(Corte-Real actively and or Leao passively transported 1990).lized by Fumaric to yeasts malic acid acid is viaand fumarase primarily by Leao metabo- a 1990; number Saayman of and yeasts (Corte-Real others 2000). effectiveness of cinnamaldehyde to prevent growthyeasts, of the spoilage cumber jars. Thesodium standard benzoate, preservative prevented forduction acidified by utilization vegetables, the andin inoculated ethanol the yeasts pasteurized pro- at(Table cucumber a 4). jars Among concentration the acidified of cinnamaldehydemM with concentrations 12 was HCl tested, the mM to 3.8 minimaltion pH level and found 3.5 ethanol to production inhibit byof carbohydrate the acetic utiliza- spoilage acid yeasts, and into the sodium absence acidified chloride, cucumbers which are forcinnamaldehyde traditionally preservation at added purposes. 3.8 Jars mM or containing turbidity higher during did the not 60-d develop incubation pressure period. or by sugar utilization and formationcumbers of lactic acidified acid with and 150 ethanol in mM cu- acetic acid, 30 mM CaCl 3.8 mM cinnamaldehyde with an adjustedbers pH were of 3.5. not The pasteurized. cucum- and Under fructose these were conditions, utilized bothduced and glucose in lactic the acid controlHowever, and lactic jars ethanol acid to were was which pro- ing not fumaric detected 30 acid in mM was treatment fumaricat not jars acid. the added. contain- Addition concentrations of foundthe previous 3.8 to experiment mM prevent resulted cinnamaldehyde spoilage incucumbers successful yeast in preservation that growth of there in the waseither no sugar reduction in and the nofrom concentration production of of of ethanol the cucumbers0.3 beyond that in mM). the expected In acidifiedthe brines addition, (4.0 jars no during visiblesuccessful a turbidity upon inoculation 60-d developed of in incubation both any period. of Preservation was also jars containing 30 mM fumaricAlthough, acid and the 3.8 mM ability cinnamaldehyde. of cinnamaldehyde to preserve cucumbers Table 4– ulation of at pH 3.5 withCaCl 0 mM acetic acid, 30 mM fumaric acid, and 100 mM L. L. bre- and Z. bailii gfor10 Pediococcus P. pentosa- × ,werepre- and L. plantarum has been shown L. plantarum L. plantarum Z. globiformis C until analyzed. Samples ◦ L. plantarum 20 Vol. 76, Nr. 7, 2011 r − C for 72 h. MRS plates were in- ◦ were unable to grow and produce died off to nondetectable numbers and and reduced viable cell counts below detectable levels. Leu. mesenteroides Journal of Food Science and produced no detectable lactic acid with 30 mM fumaric acid produced no lactic acid and the inoculated cells died off with When the pH of the EM was reduced to pH 3.5, Table 2 shows viable counts for the inoculated microorganisms Experiments were repeated 2 times with 3 jars per replicate. Log In contrast to LAB, the spoilage yeasts min (Eppendorf Centrifugeand 5810R, the West supernatants Bury, stored N.Y., U.S.A.), at pentosaceous Leuconostoc mesenteroides produced no lactic acidacid in at the pH 3.8. presence of only 10 mMceous fumaric brevis at pH 3.8.lower Viable than the organisms inoculated survived, level.effective This preservative but against showed these fumaric their acid-tolerant acid LAB. numberslimolar Twenty mil- was fumaric an were acid prevented lactic acid production by increased in numbers and produced ethanolfumaric at all acid concentrations at of both pHconditions 3.8 they and utilized 3.5 primarily (Table 2 fructose and from 3). the Under EM these medium cubated under anaerobicgrowth conditions. was also Occurrence evaluatedars, of by organic determination microbial acids, ofof and changes brine ethanol in were using taken sug- HPLC from jars, for centrifuged which at samples 18660 M476 Results and Discussion Statistical analysis LAB inhibition by fumaric acid. . . for microbiological analysis. Jarsdevelopment were of visually turbidity and/or monitored pressure forage. on the Additional the lids brine duringaseptically samples stor- collected, for after microbiologicalwere turbidity analysis plated developed on were MRS inPlates or a YM were jar. for incubated bacteria Samples at and 30 yeasts, respectively. were thawed, spun downappropriately again with water to into remove autosamplerwithin particles, vials so and the that diluted peaks ranges were glucose, of fructose, the ethanol, standards.described and above. HPLC organic analysis acids to was measure performed as to have both fumarasefumaric and acid fumarate as a reductase, substrate whichLAB (Chen can and have utilize McFeeters putative 1986). genes The for other these enzymes. and the utilization ofmation glucose, of fructose, lactic and acidconcentrations malic of and fumaric acid ethanol acid and inacid were added for- the added. to EM With pH 5 3.8 toto EM, mM which produce all fumaric the some different LAB lactic speciesadded acid evaluated fumaric were (Table acid able 2). was Furthermore, metabolized. some of the numbers and measured concentrationswere for analyzed lactic using acid the and analysiswith ethanol of the variance (ANOVA) Duncan’s procedure multipleSystems range version 9.0 test (Statistical ofN.C., Analysis U.S.A.) the using System, a Statistical SAS significance Analysis level Inst., of Cary, 0.05. lactic acid in the absenceproduced of no fumaric detectable acid.below At lactic detectable pH acid 3.5 levels with andvis 20 viable mM cell added fumaric countsonly acid. 10 were mM fumaricLAB, including acid the at highly pHvented acid-tolerant 3.5. from growing These in resultsadded cucumber at showed concentrations juice that that could when reasonably fumaric becucumbers. added acid to acidified was

M: Food Microbiology & Safety opud u otefc htasalfato fpol are people of fraction small a that 1995). (Lester fact sulfites to the sulfite hypersensitive and containing to (Nyman products due processed avoid compounds thermally consumers are Some low 2008). when drinks others juice be of and levels to sodas billion reported per pH been part has produce benzoate to Sodium degraded successful. be to sodium to were or possible benzoate it sodium (P make system without metabisulfite bulk could preservative in treatment useful cucumbers Such preserve a applications. be some could in cinnamaldehyde, as such ihtm sas expected. also is time tank with fermenting volatile open-top the the of loss from significant cinnamaldehyde, (Etchells preservative, more A cucumbers 1952). Etchells in and present Bell 1941; naturally yeasts the film this com- of growth and the abundant for oxidative more in a used promote likely concentrations would jars potentially oxygen tanks mercial cucumber that higher than of experimental Availability higher sealed work. be the to in expected fermenta- present is commercial which in present tanks, content tion oxygen dissolve would scale the such could be at preservation that preventing variable in role A significant scale. a commercial play the at here proposed ment cover brine and the washed-acidified the of packing pickles. for preserved formulation used be the would that on solutions would dependent products final primarily the in be consumers by flavor perceived the Ultimately, be products. would final that into out acid carried fumaric be and would cinnamaldehyde that both of products amount final the reduce making tially before water with P and pickles (McFeeters the out wash be iet ananfimes oee,CaCl However, chlo- calcium firmness. mM maintain 100 of chalky to addition overwhelming the An ride to acid. due acetic detected mM was 350 flavor to up traditionally with pickles acidified brined in observed signifi- that not was from cinnamaldehyde different mM cantly 3.8 and acid, fumaric mM 30 Conclusion None None 29.8 10 None None None None which in 3.5 pH to mM None chloride 150 calcium with 3.8 mM brined 100 inoculated treat- and jars 3.8 preservation acid cucumber effective acetic an 0.0 nonpasteurized not for with ex- combination is ment independent in cinnamaldehyde tested, acid mM fumaric not mM was 2.0 0.0 10 acid that fumaric suggest periments of absence the in 0.0 † (mM) added 30 30 30 benzoate) mM (12 0.0 0.0 (mM) 3.5. added pH at chloride calcium mM 30 and acid acetic mM 150 containing solutions 5– Table . . acid. fumaric by inhibition LAB uai cdCnaadhd Inocula Cinnamaldehyde acid Fumaric envalues Mean h rsre,bie ih10m ctcai,10m CaCl mM 100 acid, acetic mM 150 with brined preserves, the shown). P ND > obnto ffmrcai ihcmonsta agtyeasts, target that compounds with acid fumaric of Combination ute tde ol encsaybfr pligtetreat- the applying before necessary be would studies Further ae npeiiayosrain,teclradtxueof texture and color the observations, preliminary on Based .5.Azr au a sindt hs esrmnsta eeblwtedtcinlmt(ND). limit detection the below were that measurements those to assigned was value zero A 0.05). = o detected. not uubrpeevto ihu atuiainb obnto f3 Mfmrcai n . Mcnaadhd nbrine in cinnamaldehyde mM 3.8 and acid fumaric mM 30 of combination a by pasteurization without preservation Cucumber ± .plantarum L. tnaddvainfr2til.Ma auswti oun o ahmcoraimwt iia etr r o infiatydfeet(ucnsmu (Duncan’s different significantly not are letters similar with microorganism each for columns within values Mean trials. 2 for deviation standard erez-D ´ erez-D ´ ´ a n cetr 08,i agrsaetrials scale larger if 2008), McFeeters and ıaz ´ a 00.Sc ahn tpcudpoten- could step washing Such 2010). ıaz and .globiformis Z. Zygosaccharomyces rlfrtd(aanot (data proliferated 2 ocnrtoscan concentrations atbclu plantarum Lactobacillus 5 /10 p.(F/L lcs rcoeLci cdEthanol acid Lactic Fructose Glucose (CFU/mL) spp. 4 2 , cec eerhUi oae nRlih Cfradn with aiding presented. for data the NC of Raleigh, analysis in statistical Food the located SAA USDA-ARS, Unit the Research also with Science author Thompson The Roger Olive cucumbers. Mr. fresh Mount thanks supplying and for assistance Company technical in Pickle located excellent Unit for Research NC Science Raleigh, Food SAA USDA-ARS, the at oe E ela B 02 diino uai cdadsdu ezaea nalternative an as benzoate sodium and acid fumaric of mannitol Addition anaerobic 2002. RB. for Beelman JE, acceptors Comes electron of Utilization 1986. essential RF. review: Invited McFeeters 2007. K-H, A. Ferre Chen L, a Castillejos – PW, foods Cardozo in M, applications Busquet potential S, and Calsamiglia properties antibacterial their malolactic-deficient oils: marked Essential genetically 2004. S. of sweet-cucumber Burt growth from Competitive yeasts 1992. spoilage HP. of Fleming tolerance F, acid Breidt and Sugar 1952. JL. Etchells TA, Bell References Acknowledgments yik ,Bnieg ,Bcaa L mt L 97 niiinof Inhibition yeast 1987. JL. the Smith in RL, acids Buchanan dicarboxylic M, other Bencivengo and M, Dymicky acid malic of Transport 1990. C. Leao M, Corte-Real aya ,vnVue J,vnZlW,VlonBomM 00 ifrnilutk of uptake Differential 2000. M. Bloom Viljoen WH, Zyl van HJJ, Vuuren van dry M, Australian in Saayman fermentation malo-lactic Inhibiting 1974. DA. Pilone BC, Rankine GJ, Pilone wines in fermentation malo-lactic on acid fumaric P of effect The 1974. RE. Kunkee CS, Ough Survey 2008. KM Morehouse MH, Hiatt TP, McNeal GA, Perfetti GW, Diachenko PJ, Nyman P high-performance RF, by McFeeters vegetables and fruits of analysis Sulfite 2003. AO. Barish RF, McFeeters 14(3):229–32. Nutr Fern Coll H, Am Steels J M, health. Stratford human P, in Martorell significance sensitivity: Sulfite 1995. MR. Lester mild and acid, fumaric hypochlorite, sodium of Efficiency 2006. K. Isshiki chemical M, Murata between N, Correlation Kondo 2008. JA. Jamal J, Santhanam cucumber BA, commercial Moharam from Karim yeasts IB, of Jantan Identification 1952. TA. Bell brine RN, commercially Costilow of JL, fermentation the Etchells from 6(1):95–104. yeasts Res of Food Classification 1950. fermentations. TA. cucumber Bell in JL, yeasts Etchells of Incidence 1941. JL. Etchells apeiM,GlpiR acin ,CrleM,Flin ,CoiP,MrliI 2005. I. Morelli PL, Cioni L, acid Falcioni lactic MS, of Carelle growth F, the Macchioni on R, inhibitors Galuppi various MP, of Tampieri Effect 1989. BO. Stoyla DF, Splittstoesser erez-D ´ ehdt civ -o euto of reduction 5-log a achieve to method 3(1):83–92. Microbiol Food 90:2580–95. Sci by metabolism Dairy J fermentation. microbial rumen of modifiers as oils 94:223–53. Microbiol Food J Int review. plantarum Lactobacillus 6(12):468–72. Technol Food pickles. asnl anomala Hansenula 65(3):476–83. Prot Food J e ie yadn uai cd mJEo ii 25(2):99–107. by Vitic fumarate Enol J Am acid. fumaric 73(6):M287–91. adding Sci by Food wines J red preservatives. food and acid acetic of use the by 25(4):188–90. Vitic Enol J Am areas. warm chromatography/massfrom gas headspace by 56(2):571–6. beverages Chem other Food and Agric drinks J soft spectrometry. in benzene of results 75(3):C291–6. Sci Food J chloride. sodium of instead Food Agric J detection. 51(6):1513–7. spectrophotometric Chem ultraviolet with (HPLC) chromatography liquid 114:234–42. Microbiol Food J Int environments. sugar high of from strains spoilage of acterization and and microflora native killing in heat eight of oils essential 46(6):406–12. the Biol of activity antifungal and composition 4(2):249–64. Farlowia areas. brining northern in fermentations 4(1):87–112. Farlowia cucumbers. constants. physicochemical some to 53(1):110–3. activity Microbiol of Environ relationship Appl and maleates and fumarates by 62A yoahlga159(3):339–45. Mycopathologia of 52(4):240–3. inhibition Prot The Food J system. juice grape model a in bacteria aytak oM.D ehFre n s ae .Hayes S. Janet Ms. and Fornea Seth D. Mr. to thanks Many tpyoocsaureus Staphylococcus / ´ a M cetr F 08 irbooia rsraino uubr o ukstorage bulk for cucumbers of preservation Microbiological 2008. RF. McFeeters IM, ıaz add utilis Candida erez-D ´ usrtsuiie m)Pout omd(mM) formed Products (mM) utilized Substrates atbclu plantarum Lactobacillus None plEvrnMcoil56(4):1109–13. Microbiol Environ Appl . o.7,N.7 2011 7, Nr. 76, Vol. add albicans Candida ± ´ a M 00 emnaino uubr rndwt acu chloride calcium with brined cucumbers of Fermentation 2010. IM. ıaz nccme emnain.Ap nio irbo 58(12):3845–49. Microbiol Environ Appl fermentations. cucumber in odPo 69(2):323–9. Prot Food J . 1.2 b b b b and a cioacaoye pombe Schizosaccharomyces yoacaoye bailii Zygosaccharomyces 25.2 37.6 ne-sia T maoQ 07 hsooia char- Physiological 2007. Q. Amparo MT, andez-Espinar ´ shrci coli Escherichia yslce seta isadtermjrcomponents. major their and oils essential selected by nye n nemdae nteuiiaino citrate. of utilization the in intermediates and Enzymes . None None None shrci coli Escherichia ± ± 1.8 3.6 r c c c ora fFo Science Food of Journal b a O157:H7, plMcoilBoeho 54:792–8. Biotechnol Microbiol Appl . 17H ouain napecider. apple in populations O157:H7 80.8 and ND ND ND ND yoacaoye rouxii Zygosaccharomyces amnlatyphimurium Salmonella ± Cinnamomum † b b b b 2.6 a lsrdu botulinum Clostridium tpernetest, range ltiple 23 49 pce.Pharm species. 4 4 3 . . . . . 3 0 6 9 8 ± ± ± ± ± M477 DT104, isolated 1 0 0 6 11 . . . . 9 3 5 1 . b b b b 3 a

M: Food Microbiology & Safety