etto omnmz reigo h ak nwne.Aot3 y 30 About winter. in (CaCl tanks chloride calcium the ago, fer- of after freezing added minimize is 12%, to to mentation up salt, flavor additional (Fleming salty climates fruit cold provides cucumber In it fermented 1982). the and from tanks, made fermentation products the in in year stored a are to they up while cucumbers for fermented of texture firm the tain tarum as such tolerant bacteria, salt acid for lactic selects homofermentative It process. fermentation multi- the serves in Salt functions 2007). ple others and (Breidt volume in L 40000 about oee,tesl ocnrto nfretdccmesi above is cucumbers fermented in concentration Pal- industry. salt the 1973; by the practiced However, Henne widely Recy- now and is process. 1975) (Geisman McFeeters this and brines by nitkar fermentation generated the waste of salt high cling the to due limits and Tang 1983). 1980; McFeeters alone Buescher and NaCl Hudson than 1979; others better and cucumbers (Thompson fermented of firmness the main- helped tain it because brines fermentation cucumber to added be hoieIsedo oimChloride Sodium R of Instead Calcium Chloride with Brined Cucumbers of Fermentation JFS ute erdcinwtotpriso sprohibited is permission without reproduction Further exclusion suitable. be the may to that approval or Carolina products imply North guarantee other it or of does a Agriculture nor of constitute Science, Service, Dept. Research U.S. not Food the Agricultural by does of of product product the Mention Dept. of proprietary U.S.A. warranty the 27695-7624, or of N.C. trademark Series Raleigh, a Univ., Journal State the Carolina of North FSR09-24 Nr Paper 7624, [email protected]). Box (E-mail: McFeeters inquiries Hall, Direct author U.S.A. Schaub to 27695-7624, 322 NC with Raleigh, Unit, Univ., State are Research Carolina North Authors Science 1/19/2010. Food Accepted SAA USDA-ARS, 9/15/2009, Submitted 20090912 MS C o:10.1111/j.1750-3841.2010.01558.x doi: C OGER otnigisefrtepcl nutyi ome discharge meet to is industry pickle the for issue continuing A 00Isiueo odTechnologists Food of Institute 2010 eprtr (45 temperature fsolg atra imesrtnini uubr emne ih10t 0 MCaCl growth mM indicate 300 stable would to were that 100 acid with pH fermented butyric and cucumbers or concentration in acid retention acid propionic Firmness of Lactic occurs bacteria. production salt. that spoilage detectable of pattern brining no same major was the there the is and This as storage 3.5. NaCl during than with less to fermented decrease are to fresh cucumbers pH the caused when in which sugars acid of lactic conversion were to NaCl primarily without cucumbers fermentation during changes metabolite major The mentations. ubr emne nbie otiigbt aladCaCl and NaCl both containing brines in fermented cumbers n 0m CaCl mM 40 and iia ohi h hmclcagscue ytefrettv irognssadi h eeto ffirmness of retention the in and microorganisms fermentative the cucumbers. by fermented caused the changes in chemical the in both similar rbe o h ike eeal nuty ao euto nwsesl ol eahee fNC eeelimi- brine were in NaCl cucumbers ferment if to achieved were continuing be project a could this is salt of yards objectives waste The tank CaCl in process. containing fermentation reduction fermentation cucumber major cucumber A from the industry. from NaCl nated vegetable of pickled levels the high for containing problem water Waste ABSTRACT: ocmaefrns eeto fccmesfretdi CaCl in fermented cucumbers of retention firmness compare to ih10 6)NC noe o ak htaetypically are that tanks top open solution in NaCl brine (6%) a M in 1.03 fermented with commercially are ucumbers htaerqie o omlfrettos thlsmain- helps it fermentations, normal for required are that , Keywords: .M F. :Fo Chemistry Food C: C F EESAND EETERS uui sativus Cucumis 2 2 ◦ steol at odtrietecus ffretto eaoimi h bec fNC,and NaCl, of absence the in metabolism fermentation of course the determine to salt, only the as ncoe as uubrfrettoswt n ihu ali h emnainbiewere brine fermentation the in NaCl without and with fermentations cucumber jars, closed In . )wsntsgiiatydfeetfo htotie nfretdccmeswt .3MNaCl M 1.03 with cucumbers fermented in obtained that from different significantly not was C) Introduction 2 qiirtda 0t 0m ea to began mM 40 to 20 at equilibrated ) I LENYS P R atcai fermentation, acid lactic , EREZ ´ -D ´ IAZ atbclu plan- Lactobacillus atbclu plantarum Lactobacillus uubrfrettosi rn otiigCaCl containing brine in fermentations cucumber processors. for issue an of less be would salt major ann ohNC n CaCl and NaCl both con- brines taining in fermented were cucumbers when retention firmness ec fNC,adt opr imesrtnino cucumbers of retention ab- firmness the CaCl compare in in to fermented metabolism and fermentation NaCl, of of sence course the determine to adfrettost edn ihu NaCl. without done be to fermentations yard eswt CaCl cucum- with of storage bers and fermentation If fermentations. commercial acu ocnrtos u oNC ntefretto brine fermentation the CaCl in 2009). McFeeters higher NaCl and with (Maruvada no fermentations has but cucumber salt concentrations, doing waste of of calcium disposal concept of the (McFeeters problem to fruits the led cucumber and of 1990) Fleming firmness and the preserving on effect firmness adequate obtain to cucumbers. the fermentation of retention to blanch to prior necessary it cucumbers found fermen- but 2002), scale others pilot and in (Fleming (4%) tations NaCl reduced with fermen- cucumbers demonstrate of did tation they However brining NaCl. in for without successful used cucumbers not be were (1995) to others low and too Fleming oxy- concentrations recycling. biological salt high and with (BOD) brine demand of gen volume This large products. a consumer in washed into results made partially process are be cucumbers must the it before so out foods in consumed normally levels snttxct lns ipslo rae at ihCaCl with waste treated of disposal plants, calcium to Since used. toxic is not that is NaCl of lower concentration much current be the to than expected be would firmness cucumber maintain emnain ihNC stemjrbiigsl ncombination in salt brining CaCl major with the as NaCl with fermentations h betvso hspoetwr ocryotlbrtr scale laboratory out carry to were project this of objectives The h atta acu osa o ocnrto aeamajor a have concentration low at ions calcium that fact The 2 tcnetain yial sdi omrilfer- commercial in used typically concentrations at 2 rn uigsbeun trg oprdt cu- to compared storage subsequent during brine o.7,N.3 2010 3, Nr. 75, Vol. 2 hnefrscudb aet dp omriltank commercial adapt to made be could efforts then , 2 steol ati h oe rn eesmlrto similar were brine cover the in salt only the as 2 rn uigsbeun trg oprdto compared storage subsequent during brine pickles , — 2 ORA FFO SCIENCE FOOD OF JOURNAL tcnetain yial sdin used typically concentrations at 2 uigsoaea high a at storage during 2 ocnrtosrqie to required concentrations 2 steol salt, only the as 2 sthe as C 291

C: Food Chemistry , 2 along . After .Three 2 Cincu- 2 2 ◦ that equilibrated to final 2 . 2 C, the fermented jars were trans- ◦ CFU/mL, the jars were incubated at 6 . Due to the ratio of water to cucumbers 2 concentration on the concentration of 2 Size 2A cucumbers were packed into jars, inoc- Triplicate 1360 mL jars were filled with size 2B cu- along with control fermentations in which cucum- . C incubator to accelerate softening (McFeeters and culture at 10 2 2 ◦ C and stored for 7 mo. C for 48 h and mixed 3 to 4 times each day. Liquid sam- o ◦ was added at concentrations from 50 to 500 mM. Analy- 2 C for 110 d. For comparison with fermentations with NaCl, ◦ Fermented size 2A cucumbers were removed from duplicate fer- Effect of CaCl Changes in firmness of cucumbers fermented without The same experimental design and analysis was used for fer- fermentation metabolites andfermentation. pH after the completion of 3 jars were filledwith with brine cucumbers from solution the to same equilibrate lot at and 1.03 covered M NaCl, 40 mM CaCl cumbers from each treatment were evaluatedsampling for time. A firmness 2nd at lot each cucumbers of were size also 2A fermented and cucumbers evaluated and forness a changes during in lot firm- of storage. size Fermentations 1A were200 mM only CaCl done with 100 and Fleming 1990; McFeeters and others 1995). Duplicate jars of cu- bers were brined to equilibrate at 1.03 M NaCl, 40 mM CaCl sis of the fermented cucumber brines wasjars done and 95 adding d starter after culture. filling the NaCl compared to40 cucumbers mM fermented CaCl with 1.03 M NaCl, cumbers and cover solutions with CaCl 30 Calcium washout that were not desalted waspare with determined cucumbers at after the the washout same process.of The time calcium concentration and to lactic com- acid into the the liquid amount at expected each at stepbration equilibrium had was been to compared reached. determine when equili- in the jars, at equilibration the concentrationponents of that water are soluble not com- bound to insoluble componentsbers of should the cucum- have been reducedin to the 40% cucumbers of with each the change initial of water.cess concentration After was the completed, washout slices pro- were cut from eachfor cucumber firmness in measurement. each jar The remainder offrom the a cucumber jar was pieces blended into a slurryysis. for The calcium firmness and of lactic cucumbers acid in anal- duplicate jars of each treatment ulated, and fermented with 100, 200, and 300 mM CaCl months after packing, the jars were transferred from a 30 mentation jars after 6 wktained and 40% transferred to by a volumesample 2nd of fermented fermented jar cucumbers from which cucumbers each jar con- was andof taken transfer at the 60% for time calcium and water. lactic A acidstored analysis. at Jars 30 were closed and with control jars fermented with 1.03 M NaCl, 40 mM CaCl and 8 mM acetic acid.from each At jar the was end analyzed of forsubstrates pH the and and incubation products for period, the by brine majorevaluate HPLC. fermentation changes Linear in regression pH waschanges and used in metabolite the to concentration concentrations of related CaCl to concentrationsof5to100mM.Vinegarwasaddedtoallthecover solutions to equilibrate at 25 mM aceticL. acid. After plantarum inoculation with bator to a 45 a 1 mo fermentation period at 30 ferred to 45 ples were taken after 24ysis. and After 48 48 h h for calcium thevolume and liquid of lactic fresh was water. acid discarded This anal- process and wascontaining replaced repeated 300 by a 3rd mM an time CaCl equal for jars mentation of size 1A cucumbers exceptand that CaCl 710 mL jars were used , , . 2 2 2 L. 32 mm, ≤ -test to de- CFU/mL t 6 C in MRS (Difco, ◦ 25 mm and ≥ Lactobacillus plantarum Vol. 75, Nr. 3, 2010 — C. Duplicate jars fermented with ◦ 19 mm, 2A ≤ , vinegar (20% acetic acid), glass jars and lids 2 38 mM) from different growing areas in the , which was used as a control for comparison CFU/mL. The bacteria were removed from the 2 ≤ Materials and Methods 9 compared to fermentations with 1.03 M NaCl, Twenty 1360 mL jars were packed with size 2A cu- 2 . 2 and incubated at 30 JOURNAL OF FOOD SCIENCE 32 mm and CFU/mL. > Time course of metabolite changes in fermentations with Fermentations were done by filling 55% of the volume of glass A broad range of cucumber sizes are fermented commercially 6 292 grade anhydrous CaCl 2B United States and Mexico weresmall supplied by size a 1A local cucumbers processor.sis. have The The size the 2A highest and 2B valueume cucumbers on are commercially. sizes Cucumber a fermented cultivars weight in were large ba- vol- not identified. Food 18 mM CaCl 100 mM CaCl cumbers and covered with brine to equilibrate at 100 mM CaCl C Experiments Fermentations Cucumber fermentation without NaCl . . . Cucumbers, ingredients, and chemicals plantarum Detroit, Mich., U.S.A.) broth withapproximately 0.342 10 M NaCl to a population of broth by centrifugation, re-suspended in aniological equal volume saline of and phys- then10 inoculated into the fermentation jars at and covered with brine to equilibrate at8 1.03 M mM NaCl, 18 acetic mM CaCl acid. All jars were inoculated with 10 25 mM acetic acid. Another 20 jars were packed with cucumbers LA0445 (USDA-ARS, Food Science Research Unit,ture Raleigh, collection), which NC was cul- grown overnight at 30 After filling jarsclosed with with a metal cucumbers lid that and was heatedsealing in brine liner boiling and water applied solution, to to soften the the they jarswas while inserted were still in hot. each lid A to rubber allow septum pling inoculation of the the brine jars with and a for sam- syringebrined without cucumbers opening the were containers. inoculated The with jars (710 mL forcucumbers) size with 1A cucumbers cucumbers; and 1360 45%brine containing of mL all components the for at volume concentrations size 2.22-fold with higher than 2A a the and cover intended 2B concentrations afterbers equilibration with in cucum- the containers.NaCl, 40 For mM example, CaCl fermentations with 1.03 M from 10 mm upbased to upon 51 fruit diameter mm (1A diameter. For these experiments sizes termine when components equilibratedprocess. during the fermentation ysis. Brine fromcumber each and container brine was samplesand for frozen fermentation from substrates separately. and each products The by jar high-performance liquid cu- were chromatography analyzed (HPLC) to foroutside determine pH the changes cucumbers inside during and pH the and first metabolite 17 concentrations d insidebers of and at fermentation. outside each the The cucum- sampling point were compared using a and without NaCl in thepling cover times solution from were analyzed 1cumbers at from to a 10 jar 17 sam- were d blendedsample together of after without the added inoculation. slurry liquid. from Sections A each jar of was all frozen for cu- subsequent anal- ing cover brine that contained 2.28 M NaCl and 88.9 mM CaCl with fermentations carried out without NaCl, were done by prepar- were obtained from the same processor.purchased Morton’s pickling at salt local was grocery stores. Otherfrom chemicals Sigma-Aldrich were (St. purchased Louis, Mo., U.S.A.). C: Food Chemistry ifrn rmzr tthe was at regression zero the from of different slope the if doing determining by and evaluated regression linear was products and substrates fermentation and eedfeecsi eaoiecnetain n imesa the at firmness and P concentrations metabolite in differences there if were determine to treatments among comparisons included This rmsae aswt yig n rznutlaayi.FrHPLC For analysis. until frozen and syringe a with jars sealed from Duplicate slice. point. a sampling on each test at measured punch were a treatment each be- do of cucumber to jars the small of too center were the they at cause outside jar the a cucumbers from from 2B cucumbers punched and 1A were size 2A Fifteen size 1982). for from others jar and slice a (Thompson in thick cucumber 6-mm each single Firm- of center a (N). the on Newtons done in were expressed measurements and ness cu- recorded a of was tissue slice mesocarp cumber the puncture to required The force Systems). maximum diame- Expert Micro Texture Corp./Stable mm using Technologies analyzed 3 (Texture and software a collected were using Data U.K.) punch. ter Surrey, Godalming, U.S.A./Stable Systems, N.Y., Micro Scarsdale, Corp., Technologies (Texture alyzer eso .. fSAS of 9.1.3 version to heated was column The 2003). 65 Barish and Calif., (McFeeters Hercules, Laboratories, U.S.A.) (Bio-Rad column major HPX-87H 30-cm in on a Changes chromatography standards. by so determined the were vials of metabolites fermentation ranges autosampler the into within water were and peaks with particles appropriately remove diluted to U.S.A.) then thawed, Calif., were Oakland, LLC, samples Instrument, slurry cucumber 13000 at centrifuged and mixed, liquid the analysis hmclanalyses Chemical firmness Cucumber . . . NaCl without fermentation Cucumber D analysis Statistical etaino C a ntesadr ouin o h y binding dye the for solutions assay. standard the in con- analysis. was same TCA the calcium of that centration so to solutions calcium prior standard to added particles were was TCA samples slurry The remove tissue. to cucumber be centrifuged the might of 4% that components ions a to calcium give bound of to release assure (TCA) slurry to acid concentration Cucumber trichloroacetic TCA (1972). with mixed King were and samples Gindler binding dye by the described using procedure method washout calcium the during ples with calibrated buffers. 7 was and 4 which pH U.S.A.) Pa., (Pittsburgh, 825MP, model standard the of concentrations to 4 used using compounds. done was was standardization detector detectors External the array ethanol. of diode and fructose, the glucose, with measure series in acid, U.S.A.) connected Mass., acetic Billerica, in- Corp., acid, refractive Millipore 410 Corp., lactic (Waters model acid, detector Waters dex A malic acid. detect butyric and to acid, used propionic data was collect nm to set 210 U.S.A.) at Sys- Mass., (Spectra Waltham, detector Scientific, array Thermo tem diode UV6000 Separations Thermo A ≤ ttsia nlsso xeiet a oeuigPo L in GLM Proc using done was experiments of analysis Statistical removed aseptically were fermentations from samples Liquid An- Texture TA.TX2 a with made were measurements Firmness esrmnso Hwr oewt ihrAcmtp meter, pH Accumet Fisher a with done were pH of Measurements acu eemntoswr efre ietyo iudsam- liquid on directly performed were determinations Calcium ◦ n ltdwt .3Nslui cda lwrt f1mL/min. 1 of rate flow a at acid sulfuric N 0.03 with eluted and C .5lvl h feto CaCl of effect The level. 0.05 et nieadotietefut hnteccmesare cucumbers the When fruit. the environ- outside distinct and 2 inside are ments there fermentations cucumber uring eut n Discussion and Results TM otae(A nt n. ay .. U.S.A.). N.C., Cary, Inc., Inst. (SAS software P ≤ × .5level. 0.05 g naRii ircnrfg (Rainin microcentrifuge Rainin a in 2 ocnrto ncagsi pH in changes on concentration esta 0m nte10m CaCl but mM NaCl, M 100 1.03 the of in presence mM the 10 in mM than 50 less over were d 17 after ing Hws29ad3 epciey iue2as hw h utilization the shows also 2 Figure respectively. 3, and 2.9 was pH CaCl mM 100 and NaCl M 1.03 the in pH in ue ihtebiecnann alcmae o4 Mlactic mM 40 CaCl to pro- mM 100 compared was the NaCl acid in lactic containing acid mM brine 10 the than with less duced In fermentation NaCl. of without day first and CaCl the with mM fermentations 18 between and differences NaCl tial M 1.03 with librate uubr oeulbaewt atcai ntebiei h CaCl the in brine the in acid lactic with equilibrate to cucumbers etain flci cd( acid lactic of centrations is a,p elndt . nieteccmesfretdwith fermented cucumbers the inside 4.2 CaCl to declined pH day, first alad5dit h emnain ihCaCl with fermentations the with into fermentations d the 5 into and NaCl d 4 significant not of were outside cucumbers and the inside the Dif- between expected, declined. concentrations be cucumbers sugar in the would ferences inside As fructose) fermentation. and of (glucose d sugars cucumbers 17 the first of the outside and throughout inside the between signif- different not was icantly acid lactic fermentations NaCl the In fermentations. n usd ie2 uubr emne n10mM 100 in fermented cucumbers inside 2A metabolites size CaCl fermentation outside in and --- Changes 1 Figure CaCl oeulz o ohteCaCl cucumbers the fermenting both the outside for and equalize inside to pH the for d 3 took It niefretn uubr.Oe ybl hwcon- ( show acid lactic symbols of Open centrations cucumbers. fermenting inside hl ie2 uubr ihabiemd pt qiirt at equilibrate to up made brine CaCl a mM with 100 cucumbers of 2A course outside cucum- and time size inside the the sugars whole and show acid, from 2 malic acid, and lactic and pH, 1 in cucumbers Figure changes occurs. the brine the into into brine bers the components from soluble water both of diffusion brine, with covered initially h Dfo h nlsso ulct asa ahsam- each at jars duplicate of analysis show time. bars the pling Error from cucumbers. SD the the surrounding brine the in 2 2 2

Fermentation metabolites, mM Fermentation metabolites, mM NaCl. with fermented cucumbers the in 4.8 pH to compared rn ouin(ae )adi .3MNC,1 mM 18 NaCl, M 1.03 in and A) (Panel solution brine rn ouin(ae ) ildsmosso con- show symbols Filled B). (Panel solution brine 100 120 140 160 100 120 140 160 180 20 40 60 80 20 40 60 80 o.7,N.3 2010 3, Nr. 75, Vol. 0 024681012141618 2 oprdt uubr akdi rn oequi- to brine in packed cucumbers to compared A. B. 100mMCaCl 1.03MNaCl, 18mMCaCl 2 Fermentation time, days rn.I ok5dfrlci cdisd the inside acid lactic for d 5 took It brine. — Glucose +Fructose 2 ORA FFO SCIENCE FOOD OF JOURNAL n alfrettos fe 7d 17 After fermentations. NaCl and ◦ r Glucose +Fructose n glucose and ) 2 n glucose and ) Lactic acid 2 Lactic acid .Figure2showsthechange 2 2 hr eesubstan- were There . emnain.I the In fermentations. 2 2 . h uasremain- sugars The + + rcoe( fructose rcoe( fructose C 293 2 ) )

C: Food Chemistry 2 2 in the 2 in the fer- 0.09, a lactic 2 ± and no NaCl re- 2 =0.1360 2 0.21. The mean lactic acid =0.0311 =0.0023 2 2 ± =0.0117 2 =0.9453 2 brine concentration on the pH concentrations in the cover solutions 2 17 mM and no detectable residual sug- 2 had a mean pH of 3.50 pH R Lactic acid R Glucose + fructose R Ethanol R Acetic acid R 2 ± Calcium chloride, mM Calcium chloride, 15 mM over the range of 50 to 500 mM CaCl ± 0 20406080100 0

80 60 40 20

3.6 3.4 3.2 3.0 2.8 160 140 120 100

pH Metabolite concentration, mM concentration, Metabolite Fermentations carried out in brines with CaCl mentation cover solutions.the Error bars analysis show of the SD triplicate from fermentations at each CaCl and fermentation metabolitethe concentrations beginning of 110 the dregression fermentations. after Lines of show metabolite theto linear concentrations the and pH equilibrated related concentrations of CaCl acid concentration of 105 ars. The absence of anygardless detectable of residual the glucose NaCl or or fructose CaCl re- Figure 3 Effect --- of CaCl concentration. concentrations from 50 totected 500 in mM. these No fermentations. As residualcumbers, the was sugars slopes the of were the case linear de- with regressionethanol of the lactic concentrations larger acid acetic produced cu- and werefrom not zero (Figure significantly 4). The different final pHchange significantly of in the relation fermentations to also the did concentration not of CaCl in the cover solutions1.03 for M the NaCl, 40 fermentations. mM Fermentations CaCl with sulted in nearly completefresh cucumbers fermentation of different of sizes even thebuffer without sugars to addition enhance of present sugar acetate utilization in (Etchellsis and others beneficial 1974). for This cucumberas fermentations was because present residual in sugar, of the cucumbers, 1.03 can serve Mmentations as NaCl after a fermentations the substrate of for lactic larger undesirable acid sizes yeast fermentation fer- has been finished. was due to the lowthe initial 1A sugar size concentrations cucumbers. that Forcontained are example, only typical the 83 in fresh mM size glucosesugars and 1A fructose in cucumbers compared the to size 154 2Aported mM in cucumbers Figure fermented 1. in the experiment re- cover solution. The mean pH wasproduction 3.74 was 96

2

±

pH pH ) and brine brine ◦ 2 2 declined 5.0 4.5 4.0 3.5 3.0 2.5 2.0 5.0 4.5 4.0 3.5 3.0 2.5 2.0 2 2 2.7 mM lactic acid and ± 2 Vol. 75, Nr. 3, 2010 — fermentations. The concentration 2 ) inside fermenting cucumbers. 0.6mM.Nodetectablepropionicacid 1.1 mM) and ethanol formation (5.6 produced 99.4 1.03 M NaCl, 18 mM CaCl 1.03 M NaCl, 100 mM CaCl ± concentration increased even though the 2 pH ± used (Fleming and others 1989). In com- 2 B. A. 2 Fermentation time, days time, Fermentation Malic acid Malic acid pH concentration was not significantly different from 2 fermentations. ) and pH ( r 2 2.8 mM residual sugars after fermentation and storage 19 mm diameter) were fermented over a range of CaCl 6 mM in these CaCl concentrations from 5 to 100 mM in the fermentation ± < ± 024681012141618 JOURNAL OF FOOD SCIENCE 2 8 6 4 2 0 2 0 8 6 4 alone. The final pH of fermentations with CaCl ) in the brine surrounding the cucumbers. Error bars

14 12 10 12 10 14

2 Fermentation metabolites, mM metabolites, Fermentation Fermentation metabolites, mM metabolites, Fermentation ♦ The smallest size cucumbers used for commercial fermentations CaCl 294 C brines had no significantlactic effect acid, ethanol, upon and acetic the acid 110 finaltations d using concentrations after size initiation 2B of of (32 fermen- to 38The mm slope diameter) of cucumbers the (Figure linear 3). regressionfunction of of CaCl metabolite concentration as a solution (Panel A) andsolution (Panel in B). 1.03 Filled Mmalic symbols NaCl, show acid 18 concentrations ( of mM CaCl of residual sugars was 1.6 or butyric acid wasindicated detected that in microbial any spoilageconcentration of did of the not CaCl fermentations, occur regardless which of the Figure 2 Changes --- inside size malic 2A acid cucumbers and fermented in pH 100 inside mM CaCl and out- Cucumber fermentation without NaCl . . . of malic acid, an important source ofcumber carbon fermentations dioxide gas (McFeeters during and cu- others 1984).the It malic acid took to 5 equalize d in for concentrationNaCl at and 1 CaCl to 2 mM in both the was 132 zero for all of these components. The mean lactic acid production strength increases (Butler and Cogley 1998). (size 1A, production of acids did not change.that This dissociation may constants for be carboxylic related acids to increase as the the fact ionic parison, the fermentations with1.03 cucumbers M NaCl, from 40 the mM CaCl same lot in had 19.0 for 110 d. Acetic acid1.1 (9 mM) were also lowerCaCl than when cucumbers were brined with Open symbols show concentrations of malic acid ( from 3.4 to 3 as the CaCl pH ( show the SDsampling from time. the analysis of duplicate jars at each C: Food Chemistry o bet rwi uubr emne ihCaCl with fermented cucumbers in or grow absent to either able were not microorganisms in- spoilage acid, butyric viable or that acid dicated propionic of production detectable no was hwteS rmteaayi ftilct fermentations triplicate of CaCl analysis each the at from SD the show ih10 al 8m CaCl mM fermented 18 cucumbers NaCl, M that 1.03 found the with (1987) in others storage and prod- of Fleming consumer more into ucts. converted or are year they before a tanks for fermentation of maintained be firmness to the cucumbers requires fermentation commercial fermentation, yards. tank may eval- commercial NaCl in toward of possible step use be first without fermentations the cucumber is whether This uating containers. closed in stored and rto fCaCl of tration imesdrn trg qa oo etrta uubr fer- cucumbers than better or their to maintain equal that storage temperatures during elevated (1987), firmness others at and stored Fleming of cucumbers results previous the Given erations. eentdtcal nayo h treatments. the of any in detectable not were emne ih10 al 0m CaCl mM 40 NaCl, cucumbers M to 1.03 compared with was fermented salt of retention without con- texture fermented for the suitable cucumbers Therefore, be products. would consumer they to so temperature version room at year full a m nteUie tts h atta h atcai concentra- 30 acid at lactic months several the for that stable remained fact pH The and tions States. vol- United greatest the in in fermented slightly cucumbers ume the was are cu- larger pH which the sizes, in final NaCl cumber M the 1.03 with and fermented cucumbers higher than lower was production acid Lactic . . . NaCl without fermentation Cucumber iue4--Efc fCaCl of --- Effect 4 Figure in n Hrltdt h qiirtdconcentrations equilibrated the to CaCl related fermentation. of pH of concentra- and beginning metabolite of tions the regression linear after the show d Lines 95 size cucumbers small of 1A concentrations metabolite fermentation and nadto oteasneo irba piaeatrteprimary the after spoilage microbial of absence the to addition In Fermentation metabolites, mM pH 100 120 140 3.0 3.5 4.0 4.5 20 40 60 80 0 2 0 0 0 0 500 400 300 200 100 0 ntefretto oe ouin.Errbars Error solutions. cover fermentation the in 2 2 nln ihcretcmeca emnainop- fermentation commercial current with line in ocnrto.Nt:Guoeadfructose and Glucose Note: concentration. LacticacidR Ethanol R Acetic acidR Calcium chloride, mM pH R 2 rn ocnrto ntepH the on concentration brine 2 anandsfiin imesfor firmness sufficient maintained 2 =0.2205 2 =0.1237 2 2 =0.0460 =0.1130 2 hc saconcen- a is which , 2 n oNaCl no and ◦ n there and C n usn18;MFeesadFeig19) eeal the products Generally vegetable pickled 1990). in Fleming calcium of and concentration McFeeters maximum 1986; (Buescher polysaccharides Hudson wall cell to and bind to ability its to related is r3seso acu ahu sn 0 ae 0 cucumber 40% : water 60% a using washout calcium of steps 3 2 after or cucumbers and the predicted inside the shows concentration 1 calcium Table final less. measured or would h cucumbers 24 cu- in the M 40% in 0.41 salt and to equilibrate the water NaCl, 60% M with 1.03 filled containing is cumbers tank desalting If, a pro- NaCl. remove example, washout to for commercially 24-h used currently a that to using to similar reduced products cess be consumer cucumbers could for fermented concentration suitable the the levels in so calcium washout readily if was would determine it walls, to cell plant interest to bind of can cal- calcium of Since concentration increased. the is as cium flavor peo- chalky of undesirable proportion increasing an an detect because ple mM 25 about than less is sccmesfretdi .3MNC,4 MCaCl mM 40 NaCl, M 1.03 in well fermented as cucumbers cucumbers as fermented of firmness the maintain to sufficient aptsu fccmesfretdwt CaCl with meso- fermented the cucumbers of of tissue firmness carp the in ambient changes normal shows at 5 y Figure 1 temperatures. least at for storage during firmness sufficient infcnl ifrn rmta fteccmesfretdand CaCl mM fermented 40 NaCl, cucumbers M the 1.03 with stored of that from different significantly .3MNC,4 MCaCl mM 40 NaCl, M 1.03 t30 at ih10ad20m CaCl mM 200 and 100 with 1989). others and period (Fleming extended time an for of microbiologically NaCl spoil not without did fermented jars closed cucumbers in the indicating storage mo, the of 8 end at the period until fermentation of beginning from the acid after butyric wk or 6 acid propionic of formation detectable no with 3.11 a otoesoni iue3 atcai rdcinwshigher was production acid Lactic 3. CaCl Figure the in in shown those to lar ifrn rmccmesfretdi .3MNC,4 MCaCl mM 40 NaCl, M 1.03 in fermented cucumbers from different 0,20 n 0 MCaCl mM 300 and 200, 100, 5moat45 h eprtr a nrae o45 to increased was temperature the andthenstoredfor7moat45 CaCl and NaCl of combination the with mented etdadsoe nbie otiigol CaCl only fer- containing cucumbers brines 2A in size stored in and mented retention --- Firmness 5 Figure eefretdadsoe o 0da 30 at d 90 for stored and fermented were 5 fsoae ro asso h Do h analysis the of time. SD sampling the each show at bars fermentations Error duplicate storage. of of d 150 ae obiigi .3MNC,4 MCaCl mM 40 NaCl, M 1.03 in brining to pared h blt fclimt ananccme imesa o pH low at firmness cucumber maintain to calcium of ability The n o fsz Accmesadsz Accmesfermented cucumbers 1A size and cucumbers 2A size of lot 2nd A ±

Firmness, N ◦ .Teerslssoe ht10m CaCl mM 100 that showed results These C. .1 ohtelci cdcnetainadp eestable were pH and concentration acid lactic the Both 0.01. 10 12 14 16 18 0 2 4 6 8 o.7,N.3 2010 3, Nr. 75, Vol. 0101020250 200 150 100 50 0 2 ◦ .Teotoe ftefrettoswr eysimi- very were fermentations the of outcomes The C. emnain ihp f2.94 of pH with fermentations 300 mMCaCl 200 mMCaCl 100 mMCaCl 1.03 MNaCl, 40mMCaCl Storage time, days 2 2 — 2 lortie imesntsignificantly not firmness retained also emnain hr h ia Hwas pH final the where fermentations ORA FFO SCIENCE FOOD OF JOURNAL eutdi eeto ffrns not firmness of retention in resulted ◦ fe -ofretto period fermentation 1-mo a after C 2 2 2 2 o oo trg,including storage, of mo 8 for 2 ± ◦ o h remaining the for C .5cmae othe to compared 0.05 2 emnainwith Fermentation . 2 2 2 Cucumbers . ◦ rhge was higher or hudretain should n then and C 2 . 2 com- C 295 2

C: Food Chemistry a a a b 0.90.2 12.8 4.5 0.91.0 20.9 16.8 ± ± ± ± ˜ niz I. 2007. Fermented vegetables. In: Doyle MP, 0.44.4 12.1 4.6 2.12.0 20.8 16.0 . J Food Sci 51:135–7. 2 References ± ± ± ± ıaz-Mu ´ Acknowledgments 80.0 70.7 130.4 104.9 for cucumber fermentations would be a reduction of 2 b a a a and texture during cucumber fermentation and storage. J Food Sci 48(1):66–70. ness of brined cucumbers. J Food Sci 44(3):843–6. of firmness in cucumber slices. J Text Studies 13(3):311–24. York: John Wiley and Sons. 559 p. bacteria during theWhiting fermentation GC, of editors. Lactic brined acidYork: cucumbers. bacteria Academic in Press In: beverages Inc. Carr and food JG, (Chapter Cutting V.2). New CV, 7, Chapter 7). New York: Academic Press Inc. p 227–58. tration on firmness retentionchloride. J of Food Sci cucumbers 52(3):653–7. fermented and stored withfermented cucumbers. calcium J Food Sci 54(3):636–9. mentation of cucumbers without sodium chloride. J Food Sci 60(2):312–5, 319. 2002. Bag-in-box technology: pilotbers. Pickle system Pak Sci for VIII(1):1–8. process-ready, fermented cucum- 45(11):119–21. fluids with methylthymol blue. Am J Clin Pathol 58:376–82. cucumber pickles by calcium. J Food Sci 45(5):1450–1. ening in low salt cucumber fermentations. Int J Food Sci Technol 44:1108–17. cucumber tissue softening. J Food Sci 55(2):446–9. performance liquid chromatography (HPLC)detection. with J Agric ultraviolet Food spectrophotometric Chem 51(6):1513–7. cucumber bloating. J Food Sci 49(4):999–1002. cucumber tissue: effects of pH,793. calcium, and temperature. J Food Sci 60(4):786–8, J Food Sci 40(6):1311–5. ington, D.C.: ASM Press. p 783–93. as affected by brining and CaCl Beuchat LR, editors. Food microbiology: fundamentals and frontiers. 3rd ed. Wash- Tang HCL, McFeeters RF. 1983. Relationships among cell wallThompson constituents, RL, calcium Fleming HP, Monroe RJ. 1979. Effects ofThompson RL, storage Fleming HP, conditions Hamann DD, on Monroe RJ. firm- 1982. Method for determination Butler JN, Cogley DR. 1998. Ionic equilibrium:Etchells solubility JL, and Fleming pH HP, calculations. Bell New TA. 1974. Factors influencing the growth of lactic acid Fleming HP. 1982. Fermented foods. In: Rose AH, editor. Economic microbiology (Vol. Fleming HP, McFeeters RF, Thompson RL. 1987. Effects of sodium chloride concen- Fleming HP, Daeschel MA, McFeeters RF, Pierson MD. 1989. ButyricFleming acid HP, spoilage McDonald of LC, McFeeters RF, Thompson RL, Humphries EG.Fleming 1995. HP, Fer- Humphries EG, Fasina OO, McFeeters RF, Thompson RL,Geisman Breidt JR, F Henne Jr. RE. 1973. Recycling foodGindler brine EM, eliminates King pollution. JD. 1972. Food Rapid Eng colorimetric determinationHudson of JM, calcium Buescher RW. in 1980. Prevention biologic of soft center development in large whole Maruvada R, McFeeters RF. 2009. Evaluation of enzymatic andMcFeeters RF, non-enzymatic soft- Fleming HP. 1990. Effect of calciumMcFeeters ions RF, on Barish the thermodynamics AO. of 2003. Sulfite analysis of fruitsMcFeeters RF, and Fleming vegetables HP, Daeschel by MA. high- 1984. MalicMcFeeters acid RF, degradation Brenes Balbuena and M, brined Fleming HP. 1995. Softening rates of fermented Palnitkar MP, McFeeters RF. 1975. Recycling spent brines in cucumber fermentations. Buescher RW, Hudson JM. 1986. Bound cations in cucumber pickle mesocarp tissue The excellent technical assistanceSeth of Fornea Dr. during Sabine the Morrison courseedged. and of D. this study is gratefully acknowl- Breidt Jr F, McFeeters RF, D 200 mM CaCl chlorides from tank yard wastes byfermentations 60% in to 1.03 80% M compared to NaCl. doing fermentations While show these cucumbers results can from be fermented laboratory andout stored the with- use of NaCl in thethese cover results brine, to it brining will in be commercial a tanktion challenge yards. will to Major apply be considera- the ability toare prevent air spoilage in purged open during top the tanks,carbon period which dioxide of gas active from the fermentation fermentations tonot tanks suffer remove so bloating cucumbers damage. do 0.00.0 6.6 14 0.7 29 0.7 16.6 , which ± ± ± ± 2 2 300 mM CaCl 2 brine containing no 2 Calcium in cucumbers, mM Lactic acid in cucumbers, mM 0.01.4 7 15 2.1 30 9.2 17 200 mM CaCl Vol. 75, Nr. 3, 2010 ± ± ± ± — 41 88 181 2 after washing after washing before washing washing before 100 mM CaCl 2 2 from the fermented cucumbers. Error bars 2 2 2 40 mM CaCl 1.03 N NaCl,

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20 15 10 Firmness, N Firmness, Figure 6 shows the firmness of the cucumbers before and after Fermentation of cucumbers in CaCl 296 Expect an 84% reductionExpect after a 2 93.4% successive reduction equilibrations after with 3 40% successive cucumbers equilibrations : with 60% 40% water. cucumbers : 60% water. C water after 24 and 48 hbrated showed after that 24 both h. components were equili- calcium removal. There was no significantresult change of in the washout firmness procedure. as a was very close to predicted.ble Lactic and acid, which which is is notalso very known was water to solu- bind removed substantially fromtions. to the This plant washout cucumbers tissues, procedure in was2A the repeated cucumbers with predicted and a propor- a 2nd lot lotFor of of the size size 1A washout cucumbers experiments with48 similar cucumbers h results. at were each held step. in Analysis water of calcium for and lactic acid in the wash ratio at each step.upon The the assumption predicted that finalbrate the concentration with calcium the was would water based addedof completely to fresh equili- the water. cucumbers If aftercumber some cell each calcium walls addition that was iting so in did tightly the not cucumber bound equilibrate, would theIt to be calcium greater can the than remain- be cu- the seen predicted amount. that the calcium remaining inside the cucumbers Figure 6 Firmness --- different of size brine 2Asalt solutions and cucumbers CaCl before fermented and in after washing out 1.03 M NaCl, 40 mM100 CaCl mM CaCl 200 mM CaCl 300 mM CaCl2a b 259 Cucumber fermentation without NaCl . . . Table 1 --- Washout of lactic acid and calcium ions from sizeFermentation brine 2A fermented cucumbers. After fermentation After washout Predicted After fermentation After washout Predicted show the SDof of cucumbers the before analysis and of after firmness washout of of duplicate calcium jars ions. centrations of lactic acid,Firmness low of pH, the stored, and fermented low cucumbersas was or well retained no at as least residual cucumbers sugars. stored in 1.03 M NaCl, 40 mM CaCl sodium chloride resulted in fermented cucumbers with high con- dustry to remove sodium chloride. The advantage to using 100 to suggests that they would retainfor commercially acceptable at firmness least 1bound y at in ambient fermented temperatures. cucumbersmoved Calcium by was so a washout not excess procedure tightly such calcium as is could currently be used in re- the in- C: Food Chemistry