au 803,Snig,Cie ietiqiist uhrP author to inquiries Direct Seville [email protected]). Chile. 46, and Santiago, Enngineering Building 7820436, Chemical Biotechnology, Olavide, of Macul Dept. Cat Food de the Universidad Pablo of at Pontificia currently Campus Dept. Bioprocesses, NC is Univ. the Franco Hill, CSIC, Author Spain. Chapell at Grasa, 41013, Carolina, currently la North is de of Inst. Medina Univ. Biology Author Cell Medicine, U.S.A. of of Dept. Authors 27599, Facility, U.S.A. Science School Core 27695, Microbiome Physiology, Food the NC SAA, and with Raleigh, are Service, Azcarate-Peril Hall-NCSU, Research and Schaub Butz USDA-Agriculture 322 the Unit, with Research are Hayes and C a cainlypoeddrn uksoaea h eutof result the as storage bulk that during suggested proceed has fiberglass occasionally research may Previous top FCS 8%. NaCl open equilibrated to an 5.8% L with of solution concentration 40000 brine fer- cover in Commercially a containing packed dollars. tanks million are a cucumbers of of quarter mented one type than this estimated more activities losses to prevented at research be (FCS) due the spoilage to alone, cucumber season season fermented had pickling on pickling tanks 2010 the top the of which In open middle in spoilage. 800 instance the of historical in composed 1 closed yard is tank of There entire U.S. appearance an the pick- the in the for industry and problem tangible ling pH a is aromas rising cheese-like and manure- by characterized cucumbers Introduction P M. Ilenys Medina, Eduardo Nonculture-Based Methods by Determined as Spoilage Rising pH Cucumber Fermented of Ecology Bacterial ulse 05 hsatcei ..Gvrmn okadi ntepbi oani h USA. the in domain prohibited public is permission the without in reproduction is Further and work Government 10.1111/1750-3841.13158 U.S. doi: a is article This 2015. Published aoiaSaeUi. aeg,N 79,USA uhr P Authors U.S.A. North 27695, Sciences, Nutrition NC and Raleigh, Bioprocessing, Univ., Food, of State Dept. Carolina the with were Franco and S2116 umte /421,Acpe 02/05 uhr Medina Authors 10/22/2015. Accepted 6/24/2015, Submitted 20151060 MS cainlsolg fsdu hoie(al fermented (NaCl) chloride sodium of spoilage Occasional C olce rmtnsntsbetdt i ugn o oeta o hs ti pcltdta xgnavailability microbiota. that leading speculated the is and it spoilage Thus, mo. of 4 initiation than the more in for factor purging determining air a to be subjected may contrast, not In tanks agar. from Peptone collected Yeast FCS Mannitol on plating by purging pH. higher the at Dialister pasteurianus atbclu namurensis C ie hi blt omtblz atcai.I a h betv fti td oagettecretkoldeof knowledge current the augment to data study Torrent of this Ion samples. of spoilage objective commercial the 16S studies characterize was and microbiologically It culture-based to acid. methods Previous lactic culture-independent industry. using metabolize FCS to pickling ability the their given for FCS impact economical significant parrafaraginis of challenge yeasts the a identified is aromas like rcia Application: Practical Keywords: Abstract: nacn h oimclrd NC)fe uubrfretto ehooyadi rvniglse asdb it by caused losses preventing NaCl. in with fermented and cucumbers technology of fermentation tanks in cucumber development (NaCl)-free its from chloride sodium the enhancing .rapi L. nsolg ape ihp bv . n ofimdtepeec of presence the confirmed and 4.0 above pH with samples spoilage in rRNA and ctcai atra uubr,fretdvegetables fermented cucumbers, , acid acetic emne uubrsolg FS hrceie yrsn Hadteapaac fmnr-adcheese- and manure- of appearance the and pH rising by characterized (FCS) spoilage cucumber Fermented , tp eo ..Teclueidpnetaaye lorvae h rsneo pce of species of presence the revealed also analyses culture-independent The 3.4. below pH at Clostridium .buchneri L. lnn irr nlsso ape olce rmcmeca emnaintnscnre h presence the confirmed tanks fermentation commercial from collected samples of analyses library cloning ihamanshurica Pichia , atbclu acetotolerans Lactobacillus nesadn fteudryn irbooyadbohmsr rvn C sesnilto essential is FCS driving biochemistry and microbiology underlying the of Understanding p,and sp., ´ erez-D lc eCie v,Vcn akn 4860, Mackena Vicuna Ave, Chile, de olica ´ n eeldtepeec fadtoa pce novdi h eeomn fFSsc as such FCS of development the in involved species additional of presence the revealed and p.wr ucsflyioae rmcmeca ape olce rmtnssbetdt air to subjected tanks from collected samples commercial from isolated successfully were spp. ´ a,Fe rit ae ae,WnyFac,NtsaBt,adMar and Butz, Natasha Franco, Wendy Hayes, Janet Breidt, Fred ıaz, Propionibacterium and sacekaoccidentalis Issatchenkia erez-D ´ erez-D ´ , atbclu panis Lactobacillus ´ a.(E-mail: ıaz. n rmngtv , Gram-negative 1 and ´ a,Breidt, ıaz, estv op eo ..Teeoe dnicto forganisms of identification Therefore, 3.5. are that below microbes pH of proliferation to the sensitive for allows which pH, in ment are acids butyric and propionic and 5, to produced. 3.2 around the from after rises species pH Gram-positive of supersede anaer- analysis Gram-negative bacteria that the obic confirmed Additionally, libraries 2013). cloning pH rRNA others at 16S acid and propionic (Breidt to acid 4.2 lactic above converting in role a that play revealed species step and enrichment us- techniques, an bacteria culture-independent spoilage and culture-dependent fermentation ing cucumber charac- NaCl The of 2013). in terization McFeeters and 3.2 Johanningsmeier and of 2012; aerobic P pH others both and acidic under (Franco initial medium acid the the lactic of of regardless expense conditions anaerobic the only but at sugars, acid from lactic produce to annsee n cetr 03.Prle tde determined Jo- studies spoilage-associated 2012; Parallel that others 2013). McFeeters and and Johanningsmeier hanningsmeier 2012; others and (Franco ethanolidurans coccus parrafaraginis tobacillus ain n aoaoyrpoue piaewt alidentified NaCl with manshurica spoilage Pichia reproduced laboratory and tations 1989). others acetic, and of (Fleming acids formation propionic and and acid butyric, lactic of utilization microbiological rmpstv bacteria, Gram-positive 4 , ntaino atcai tlzto etil nue h incre- the induces certainly utilization acid lactic of Initiation nlsso omrilsolg ape rmNC fermen- NaCl from samples spoilage commercial of Analysis Lactobacillus , ctbce peroxydans Acetobacter o.8,N.1 2016 1, Nr. 81, Vol. , p.wr rmrl dnie nsmlsof samples in identified primarily were spp. Pectinatus sacekaoccidentalis Issatchenkia sptnilyrlvn odfeetsae fFCS of stages different to relevant potentially as Pectinatus , atbclu rapi Lactobacillus .buchneri L. p.drn atcai degradation acid lactic during spp. erez-D ´ , atbclu buchneri Lactobacillus srlvn nvrossae of stages various in relevant as , ctbce aceti Acetobacter r ora fFo Science Food of Journal ´ a 02 oannsee and Johanningsmeier 2012; ıaz Propionibacterium and ,a , ´ aAde Azcarate-Peril Andrea ıa Clostridium atbclu buchneri Lactobacillus .ethanolidurans P. .buchneri L. ,and Veillonella , Lactobacillus p,and sp., Acetobacter and Pectinatus produces r able are and M121 Pedio- , Lac-

M: Food Microbiology & Safety cover 2 LA0045 in- at the commer- were inoculated ıaz and McFeeters ´ 2 C for 48 h. Yeasts were ´ erez-D L. plantarum ° cover brine samples were aseptically 0.2. In addition to pH, the detection of manure-like CFU/mL for a 30400 L total volume. Preparation of 9 ± CFU/mL by using 1850 mL of 6 A set of 10 commercial cover brine samples were provided Cover brine samples for microbiological, molecular, and chemical analyses Detection of fermented cucumber rising pH spoilage For the microbial analysis Cucumber fermentations brined with CaCl the cover brines hadpurging moved had through been the applied for sampling 15 toconsisted tubing 20 of and min. a The buffer air siphon sampling PVC apparatus pumpInc., (BSP-1000; San CBS Scientific Diego, Calif.,tygon U.S.A.) tubing connected with to a a thinglass 1.3 wall. jars The cm and samples diameter immediately wereday placed transported processing. in to 1.3 our kg laboratory for same by processors duringsons. These the samples 2010, were collectedspoilage 2012, that from had tanks and been with packed symptoms 2013 with of the pickling experimental CaCl sea- starter cultures was done as described by(2011). P Tanks packed with thenot traditional inoculated. NaCl formulation were were collected on days 1,120. 2, On 3, each 7, sampling 14, day, approximatelysamples 21, 500 were 30, mL taken 60, of from 75, an cover brinesurface 90, average of 105, via 1 a and m perforated below pipein the placed the cover next brine tank. to the Cover air brine purging samples system were collected after 100 mL of to 10 collected as described above,lution, serially and diluted spiral in platedNorwood, 0.85% using Mass., saline an U.S.A.). so- Autoplateing Enumeration 400 of deMan, (Spiral Biotech, LAB Rogosa,son was and and done Sharpe Co., us- 1% agar Franklin cycloheximide (0.1% Lakes, (MRS, solution, N.J., Becton Oxoid,vent U.S.A.) growth New Dickin- of supplemented England) yeasts. to with MRS pre- cally agar using plates a were incubated CoyInc., anaerobi- anaerobic Grass chamber Lakes, (Coy Mich., Laboratory U.S.A.) Products, at 30 brine formulation or the traditionalas NaCl indicated cover on brine the formulation text. The NaClair commercial purged fermentations were continuously forfermentation the 14 occurred; to and 21for sporadically d the during in which incorporation storage the ofaseptically to primary rain collected allow from 1 intoin m the sterile under tank. 50 the Samples mLour cover were conical facilities. boards, tubes collected and refrigerated until delivered to cial scale wasabove based 3.3 mainly onand cheesy the aromas measurement andindicated of the tanks surfacing pH may of be values bubblesondary in in fermentation the the was process tanks of confirmedchromatography also spoiling. by (HPLC) Suspected analysis high-performance sec- of liquid organicdescribed acids below. and alcohols as oculum (USDA-ARS Food Science Research Unit culturetion) collec- at 10 brine solution components withoutthe exposing top the of fresh thefor fruits 4 tank h on to in air. thelactic morning The acid and experimental 4 fermentation tanks hof to in were the the prevent purged season afternoon bloater (summer during damage. months),within the the In active 2 primary the wk fermentation peak occursair-purged and as during described this above. The7 time tanks d the were and purged fermentation for on8, tanks the days 10, 1st were 9, 12, 11,after and and after primary 13. day fermentation Tanks 14.and were to prior not Air allow to purged purging the for onin collection was mixing days by of minimally air samples. of applied purging Rain preservatives circulation. water was not mixed 2 L. were Pecti- 2 Clostridium as a tool to ıaz 2012). ´ 2 spp., and ´ erez-D Commercial cucum- , and NaCl to achieve 2 Vol. 81, Nr. 1, 2016 r Propionibacterium ıaz 2012; Breidt and others 2013). ´ , ´ erez-D studied during the 2013 pickling season. 2 , to utilize has been observed under and 6 mM (0.1%) potassium sorbate (equilibrated 2 , ıaz and others 2015). Thus, the objective of this study ´ and closely related species seem to be unique among sev- Journal of Food Science ) systems. The approach taken in this study was the utiliza- 2 Commercial Cucumber Fermentation Tanks: Even though significant progress has been made in understand- ´ erez-D ferent sizes. Two tanks were packed with the experimental CaCl equilibrated concentrations of(0.9%) mM 15 and (0.09%), 1.03was 2 M added into (6%), (0.03%), the respectively. tanks, Cushion and prior cover to brine 40 the addition of the fruits of dif- fruits; and 40% cover(0.9%) brine CaCl solutions containing either 100 mM ber fermentations were carried outtanks in containing 40000 L 60% open 3A top38 fiberglass (39 mm to diameter) 51 mm whole diameter) cucumbers or 2B or (27 pieces to of variable size spoiled cucumber fermentations brined with NaCl or CaCl stable NaCl cucumber fermentationby chemical system and as microbiological control;such analyses followed tanks to monitor during the experimentation state (Figure of 1). Particularities of consistently reproduce FCS at the commercial scale, and the more trated , potassium sorbate, CaCl concentrations) or a combination of acetic acid, added as concen- M122 from multiple farmers primarily locatedIn-tank in North fruits Carolina, U.S.A. were immediatelyprevent them covered from with floating, wooden after adding boards thevolume, to to remaining allow cover brine for the equilibration between the fruits and cover (tanks 3 and 4) coverditional brine NaCl and cover 2 brine tanks (tanks were 1 packed and with 2). the Fresh tra- cucumbers came Material and Methods The need totation understand of FCS NaCl-free isscale, cucumber which augmented fermentations is by more at vulnerable the(P the to implemen- commercial FCS during long-term storage ing the underlying microbiologya and comprehensive biochemistry study driving ofcial FCS; the NaCl dominant FCS microbiota using in culture-independent commer- techniques is lacking. above 4.5 (Franco and P Therefore, these microbes are considered asin secondary FCS. microbiota natus sottacetonis conditions similar tocucumber those tanks present containing in NaCl, such commercially activity fermented proceeds at pH design of strategies forability the prevention of of spoilage-associated the undesiredconditions yeasts FCS. similar to The to degrade thosementations lactic present has in acid been confirmed commercial under (Franco cucumber and fer- P capable of utilizing lactic acid at pHpresent 3.0 in to 3.5 commercial under cucumber the conditions fermentations is essential in the Fermented cucumbers spoilage . . . brined with CaCl documented by comparing samples oftations spoiled brined cucumber with fermen- NaCl provided(2009 by to processors 2012), in with the previous characteristics years of cucumber fermentations tion of cucumber fermentations brined with CaCl nity present in commercial FCS in(CaCl the NaCl and calcium chloride was to apply nonculture-basedrent techniques, sequencing such technology aslibraries, and the to analysis obtain Ion a of Tor- comprehensive view 16S of the rRNA bacterial cloning commu- acetic acid and 1,2-propanediol at pH6% 3.8 NaCl in the (Johanningsmeier presence and ofability 2% McFeeters to of 2013). other Althoughbifermentans the spoilage-associated bacteria, such as buchneri eral lactic acid bacteriaacid (LAB) in in fermented their ability cucumber to slurry, metabolize with lactic concurrent increases in

M: Food Microbiology & Safety iue1Wr o ceai o h xeietlapproach. experimental the for schematic flow 1–Work Figure genomic total resulting 0.6 and the isolates, (5-AGAGTTTGATCCTGGCTCAG-3 of bacterial RBUP uL from (Bio- 10 extracted kit 2X DNA (Bio-Rad), contained extraction mixture mix DNA PCR master The U.S.A.). Matrix Calif., InstaGene of Hercules, Rad, using amplication partial done the was microscope by 16S ran- a identification were the for in colonies selected Such motility Gram-negative. domly showed were positive, and preparation, catalase were that ing Pa., (Pittsburgh, MP MYP 825 water. model distilled meter, U.S.A.). pH of Accumet liter Fisher per a agar with 30 of at extract, g incubated yeast 15 were of plates and g peptone, 5 agar of mannitol, Peptone g of and 3 g 25.0 Extract Yeast contained which Mannitol, (MYP), in were plating plates by chlortetracycline YMA merated 0.01% 30 growth. at aerobically and bacterial incubated U.S.A.) inhibit Mo., to (Sigma-Aldrich) Louis, Dickin- St. (Sigma- Becton chloramphenicol (YMA; 0.01% Aldrich, with agar supplemented Co.) mold and and son yeast using enumerated . . . spoilage cucumbers Fermented ylso i t94 94 at at min min 4 1 of of consisted cycles cycle PCR The GGTTACCTTGTTACGACTT-3 unedtbs odtrieteiett ftesris(accession strains KT074257). the to of KT074234 se- identity # RNA the ribosomal determine 16S to the database using quence others 1997) and others Altschul and (BLAST; Benson tool 1990; search to alignment subjected local were were 20 obtained basic to sequences the equal The or alignment. than the greater for scores used quality had (www.mbio.ncsu.edu/bioedit). that bases software Only and BioEdit formatted using were data analyzed Sequence U.S.A.). N.C., (Durham, Inc. 4 72 ° dnicto faei cdbcei AB y1SrN sequenc- rRNA 16S by (AAB) bacteria acid acetic of Identification ni sd C rdcswr eune yEo Bioscience Eton by sequenced were products PCR used. until C ° ,wt nletninse f7mna 72 at min 7 of step extension final a with C, a opee rmioae ooisgoigo MYP on growing colonies isolated from completed was rRNA eunig N slto rmsnl colonies single from isolation DNA sequencing. ° ,2mna 57 at min 2 C, ° ° o 8h. 48 for C o 4h h Hwsdetermined was pH The h. 24 for C ʹ isnadohr 1990). others and Wilson ; ° ° olwdb 30 by followed C ,ad2mnat min 2 and C, ʹ n 42 (5 1492r and ) ° µ n trdat stored and C Mofprimers eeenu- were ʹ - codn otemnfcue ntutos uie N samples K DNA Purified proteinase instructions. with manufacturer U.S.A.) the to Wis., according Madison, (Epicentre, Kit cation eesoe at stored were eesn oEo isine(tnBocec n. Research clones Inc., rRNA Bioscience the (Eton with Bioscience plates Eton to petri sent analysis, were clone instructions. rRNA manufacturer For the to Grand Technologies, according U.S.A.), Life N.Y., (Invitrogen, Island, sequencing for kit cloning e,Vlni,Clf,USA)adterslig1SrN PCR in rRNA (Qia- 16S cloned kit resulting were purification the products and PCR U.S.A.) QIAquick Calif., Valencia, a gen, using products up PCR above. cleaned described 1492r were and RBUP primers the ing n iea ecie.PAtetdsmlswr hnsoe as stored then were at samples pellets PMA-treated a cell PMA DNA. described. with temperature, as the treated room time pellet with at 2nd the min dye and 5 removed the were for of supernatants rpm the cross-linking 10000 at for spun tubes, allow were microcentrifuge Samples to 650-watt the min a of to top 5 exposed the for above and cm open tops 20 lamp, the halogen and with were ice speed Samples temperature. on high room placed at at min then 5 s for 10 dark the for in incubated vortexed 57 were of concentration samples final Treated a PMA. achieve to resuspension cells 490 to the of added of were microliters U.S.A.) Ten Calif., bacterial Inc., 2007). (Biotium, PMA Breidt dead mM and 2.5 eliminate (Pan DNA to extracellular (PMA) and monoazide propidium 490 Fisher with in Thermo resuspended min (Sorvall, were 10 spun for rpm were 4 10000 mL at at U.S.A.) 400 Mass., Waltham, Inc., to Scientific 10 of those aliquots only positive. catalase and as colonies classified were the bubbles of on production motility. showing added cell detect was to order peroxide in Hydrogen microscope a in observed and slide, h 6 RAlbaywsconstructed was library rRNA 16S The N slto a oeuigaMasterPure a using done was isolation DNA extraction: DNA genomic Total a on mounted picked, were plates MYP from AAB of Isolates ° ,adwse n1m f08%sln ouin Pellets solution. saline 0.85% of mL 1 in washed and C, − o.8,N.1 2016 1, Nr. 81, Vol. − 20 20 ° ni N xrcinwsperformed. was extraction DNA until C ° ni use. until C shrci coli Escherichia µ trl aieslto n treated and solution saline sterile L omrilsolg oe brine cover spoilage Commercial r ora fFo Science Food of Journal yPRapicto us- amplification PCR by sn h TOPO the using TM N Purifi- DNA M123 R µ TA µ M L

M: Food Microbiology & Safety ± 0.3 fer- 2 ± 0.3, 13.2 16.5 mM was ,however,an ± 2 ± 0.1 Log CFU/mL ± 2.5 to 30.3 ± 0.1 for the NaCl and CaCl cucumber fermentation in which cover brines to 6.3 salt were delivered by independent 2 2 ± 2 3.4 mM in the sodium and calcium sys- presenting an increase in pH to 4.3 2 ± 0.4 Log CFU/mL (Table 1). An increase in ± 0.1 and 3.3 2 mM, respectively. ± ± 7.9 and 13.1 ± 7.7 mM were reached after 60 d in all the tanks studied. The tra- Commercial FCS samples collected from tanks brined with ei- The characteristics of the commercial fermentations studied 1, and 15.4 and a decrease inafter lactic 120 acid d concentration of toof storage undetectable LAB at levels ambient and temperaturesby yeasts (Table day remained 1). 60 Counts stable in at the 4.9 fermentations brined with CaCl ther the sodium or the CaCl were in agreementfermentation with and the for expectations a CaCl for a NaCl primary metabolites such(4.58 as mM; Table propionic 2).taining In acid NaCl contrast, (sample (12 acounts F) second of mM) showed sample LAB, a of and andthough, pH FCS relatively the low propanol con- differences of lactic in 4.26,containing acid the NaCl relatively concentration. characteristics are high Al- of primarilypH the attributed values FCS to of samples the the variabilitysuggest samples of that at the the the course moment ofthese of the tanks. analysis; initial spoilage it stage could was also different in processors to our facilities2013. during Air purging the was seasons not of applieding the to 2010, course the 2012, of NaCl spoilage. and spoiling Acetic tanks25 acid dur- mM) was detected in (approximately the samplesthe collected concentrations from added tanks from brined vinegar.values The with (Table detection NaCl, 2), of at manure-like and high cheesy pH of aromas, and bubbles the on presence thestarted surface in of the theseNaCl corresponding tanks cover tanks indicated brine (Franco that sampledetectable and FCS with counts others of had a LAB, 2012). unusually pH highrelatively A of numbers high of concentration 3.22 yeast of counts, (sample lactic acid, E), and had spoilage-associated no of the fermentation. In contrast,tions FCS brined proceeded with in CaCl the fermenta- mentations, respectively. Acetic acid wasof 5.6 produced to antems, average respectively. Maximum lactic± acid concentrations of 101.9 ditional NaCl fermentations were stableas during evidenced long-term by storage undetectable levels of LAB and yeasts after day 28 Samples of commercial FCS provided by processors Results Attributes of fermentations spoilage took place. Mostin the lactic 1st acid 28 productionvalues d was of of completed 3.2 fermentation, generating postfermentation pH increase in theter counts 120d of to LABacetic 5.6 from acid MRS concentration from plates 5.0 was noted af- arations UV6000 diode arrayFisher detector Scientific (Spectra Inc.) System, setdetect Thermo to malic, collect lactic, data acetic,ters at model propionic, 410 210 and refractive nm index butyricCorp., detector Billerica, was acids. ( Mass., used Corp., U.S.A.) A connected Millipore to in Wa- array series detector with was the used diode toExternal measure , standardization fructose, of and the . centrations detectors of the was standard done compounds. using 4 con- also evident after 76of d propionic of acid, fermentation butyric (Table100 1). acid, d The of and formation storage propanol in was the detected CaCl after - - ʹ ʹ tar- Cfor60s, 20, and any ° ); where the ʹ < C followed by ° -3 C and eluted with ° prior to analysis with dis- ). PCR reactions contained 5 × ʹ Vol. 81, Nr. 1, 2016 Cfor60s,and72 r ° C for 10 min. Negative controls, not ° HotStar Hi-Fidelity PCR buffer con- M of each primer. Reaction conditions µ × Samples of commercial cover brines were col- C for 60 s, 57 ° AGAGTTTGATCCTGGCTCAG Journal of Food Science dimensional principal coordinate analysis plots. QIIME was - raw Ion Torrent fastq files, which were demultiplexed, C (ambient temperature) to remove residual particulate matter ° 16S rRNA amplicon sequencing analysis using Ion Torrent HPLC analysis: Amplicon high-throughput sequencing data analysis was conducted TGCTGCCTCCCGTAGGAGT-3 to 10 ng of DNApolymerase template, 2.5 (Qiagen), units 1 of HotStar Hi-Fidelity DNA M124 taining dNTPs, and 0.6 the universal primer 8F. The reverse primer was 338R (5 xxxxxxxxxx underlined sequence containedcleotides barcodes (Table consisting S1) of 10 and nu- the sequence in italics contained geted theplification V1–V2 was fragment performedisolated of from from the purified each total 16S bacterial sample rRNA. DNA PCR using am- the forward primer (5 ribosomal RNA (Bacteriawere and submitted Archaea) to database. the Nationalmation All Genbank Center sequences database for (accession Biotechnology # Infor- KF998365 - KF998547). ware (www.mbio.ncsu.edu/bioedit). Only basescaller that quality have value base- the greater alignments. than Sequence similarities orNCBI BLASTN were equal algorithm determined (www.ncbi.nlm.nih.gov) with using to the 16S the 20 were used for Triangle Park, N.C., U.S.A.).Eton The Bioscience sequence were formatted data and obtained analyzed from using BioEdit soft- Fermented cucumbers spoilage . . . sured using a 30-cm HPX-87H columnBarish (Bio-Rad; McFeeters 2003). and The column0.03 was N sulfuric heated acid to at 37 a flow rate of 0.6 mL/min. A Thermo Sep- 22 (Eppendorf Benchtop Refrigerated Centrifuge 5810R, Hamburg, Germany). Samples were dilutedtilled 10 water. Organic acids and sugars concentrations were mea- sampling depth coveragephylogenetic using diversity metrics observed (Table 2). species, Shannon and lected as described above and spun at 12000 rpm for 10 min at coordinates were computed toand 3 compress dimensionality intoalso 2- used to calculate alpha diversity on rarefied OTUs to assess for assignment. Beta diversitywithin estimates QIIME were calculated using weighted and(Lozupone unweighted and Unifrac Knight distances 2005)sequences between per samples sample. at From a these depth estimates, of jackknifed 989 principal units (OTUs) wereof assigned UCLUST using the (Edgaridentity, QIIME 2010), and implementation representative with sequences a from threshold each of OTU 97% selected pairwise 2010). The 400-bpthan 3 reads sequential were basesread receiving truncated a containing quality at ambiguous scorewere base any of discarded, calls as site or were if truncated barcode/primer reads. more errors Operational taxonomic from quality-filtered, and analyzed using QIIME (Caporaso and others All kits were usedstructions. according Purified to libraries the were respectivebiome submitted manufacturer’s Core to in- Facility the UNC (Chapelon Micro- Hill, the Ion N.C., Torrent U.S.A.) platform. for sequencing vitrogen, Life Technologies). DNA concentrations were quantified using PicoGreen dsDNA reagenton (Invitrogen, a Life 96-well Technologies) plate reader and mixed at equimolar concentrations. and a final extension ofcontaining 72 template, were amplifiedThe PCR for products with all approximately 400 barcode-primer nucleotidesfirmed were sets. and con- purified by gel electrophoresis in an E-gel system (In- consisted of an initial denaturation35 for cycles 5 min of at 94 94

M: Food Microbiology & Safety n nrae np tre fe a 0 hstesmlsfo day community from bacterial samples the the regarding thus information 60, provide day should after 76 started disappearance pH acid in lactic increases concen- example, and acid For organic counts. in bacterial days changes or these trations that significant given of selected initiation were the 120 samples and mark FCS 105, technology. 90, Torrent pop- 76, of days Ion from analysis the the for using used diversity were E, values, ulation B, pH (A, different processors with F) by and collected samples brine cover commercial CaCl ihrp Tbe2.A nraei h ctcai concentration CaCl the acid the with acetic of all the samples in in the detected increase was in An de- detected 2). (Table was and pH tanks acid higher these reduction organic A in the 2). observed of (Table was 4.50 pletion to concentrations 3.05 acid from lactic range in pH a with FCS of alE NaCl tn )o CaCl or 1) (tank hc a atal nune ydfeecsi h i purging air the in differences routine. tanks, by and the rate in influenced oxygen dissolved partially of was levels the which in presumably variations varied, the YMA to fermen- from due the counts of yeast However, Counts regardless pH. values. high, relatively tation pH were higher MRS and from LAB acidity lower of the in with detected were samples propanol and the acids butyric and propionic of levels hne nbceildvriyo uubrfermentation spoilage cucumber of diversity bacterial in Changes available. not samples. N/A, these limits; ∗ in detection detected below not BDL, were fructose and Glucose samples brine cover type in CFU/mL) (log brine processors. Cover population by microbial provided and spoilages (mM), pH concentrations rising acid cucumber fermented organic commercial value, of pH in 2–Changes Table CaCl with brined 2 and NaCl with brined (alpha fermentations means commercial squares 2 of least deviations treatment standard for and different. Grouping Average Tukey significantly the not of are results column the represent alphabets Superscript NaCl samples brine cover CaCl in CFU/mL) (log populations type microbial brine and Cover (mM), spoilages. concentrations pH acid rising cucumber organic fermented pH, commercial of in 1–Changes Table . . . spoilage cucumbers Fermented D,blwdtcinlmt;N,ntavailable. not samples. NA, these limits; in detection detected below not BDL, were fructose and Glucose aafo ritadohr (2013). others and Breidt from Data eetdsolg ape olce rmtnsbie ihNaCl with brined tanks from collected samples spoilage Selected CaCl 2 2 2 omrilsolg ape ersne ifrn stages different represented samples spoilage commercial Sample 2 tns3ad4 ndy 6 0 0,ad10and 120 and 105, 90, 76, days on 4) and 3 (tanks ID identification F 940 . . D 05 76 D D 15.21 BDL BDL 47.61 30.53 BDL 2.4 6.2 4.04 49 H 234 . . . 93 18 .4BL8.42 BDL 3.74 31.83 29.36 4.1 4.9 6.4 3.40 72 D 740 . . . 86 80 .9BL11.92 BDL 3.49 48.08 28.61 4.0 2.8 6.1 4.01 57 G 830 . . . 00 11 D D BDL BDL BDL 11.14 90.07 4.3 5.5 5.2 3.05 78 C 540 . . / 86 19 98 D BDL BDL 19.81 41.98 28.64 N/A 2.6 4.6 4.03 75 A 2 .05249NABL5.82.674 7.54 7.46 24.86 58.48 BDL N/A 4.9 5.2 4.50 120 B 838 . . . 60 30 .3BL11.05 BDL 3.63 43.07 46.09 1.6 1.3 6.1 3.88 48 I 439 . . D 59 39 .3BL15.61 BDL 3.93 53.91 25.96 BDL 4.8 6.7 3.90 64 J ∗ ∗ a d 03.4 60 3and4 a d 03.1 60 1and2 Tanks Fermentation g d HMSYAMPLci cdAei cdPoincai uyi cdPropanol acid Butyric acid Propionic acid Acetic acid Lactic MYP YMA MRS pH (d) age 2 2 .66454NA3.02.460 D BDL 4.58 BDL BDL 6.00 12.00 22.34 31.00 39.00 91.00 N/A 5.4 N/A 5.6 6.4 BDL 4.26 3.22 420 150 pie emnain.Significant fermentations. spoiled Fermentation g d HYAMSLci Acetic Lactic MRS YMA pH (d) age 2 3.1 3.1 120 4.3 105 4.0 120 105 03.1 3.1 90 3.9 76 3.7 90 76 o F/LOgncaiscnetain(mM) concentration acids Organic CFU/mL Log ± ± ± ± ± ± ± ± ± ± 0.1 0.1 0.7 0.5 0.1 0.1 0.3 0.6 0.3 0.1 A A A A A C B AB AB AB h eeac farprigi h oiatpritn microbiota persisting storage. dominant long-term the in during purging predominant air suggest of may the relevance growth the that for environment aerobic fact an The demand respectively. microbes 7.59%, and 73.65% genus the the of composed and was family and 120 day from samples of order the However, detected. eepriual bnat(62% nsmlsfo a 6with era, 76 day from age. samples Acetobacteraceae in fermentation (66.23%) the abundant with particularly were varied groups these of of orders the to Rhodospirialles belonging pre- tanks of control groups NaCl main 3). 2 in (Table sented increased participating pH species the of as number spoilage the algo- the in Shannon decline and a Chao1 showed the rithms, on based indices, storage. bulk diversity during General fermentations were of diversity types obtained 2 bacterial were the the between reads observed in Differences 21592 amplicon. of or average of sample An length per average obtained. an were A with bp sequences samples. 400 high-quality independent 302295 from of derived total amplicons PCR storage. bulk bacterial of d 120 after evolved propanol 105, of day by production detected and was acids 90, butyric day by and evident propionic more of were production counts MRS the in produc- increases acid and tion acetic Similarly, changes. such in involved potentially eunigraswr bandfo igernwt 14 with run single a from obtained were reads Sequencing 3.6 3.1 3.6 3.3 3.3 Acetobacter D D 84.8 85.6 85.8 BDL 101.9 BDL BDL BDL BDL BDL BDL BDL AN BDL NA NA o fCUm rai cd ocnrto (mM) concentration acids Organic CFU/mL of Log ± ± ± ± ± . D 84.5 BDL 0.1 5.6 5.1 4.6 0.3 4.9 0.2 0.1 0.6 2 = epciey r hw dt dpe rmP from adapted (data shown are respectively, , .5.Vle dnie ihtesm uesrp lhbtwti a within alphabet superscript same the with identified Values 0.05). and o.8,N.1 2016 1, Nr. 81, Vol. and ersnigteol aiyietfid w gen- Two identified. family only the representing Rhizobiales ± ± ± ± Methylobacterium . 18.0 35.0 56.6 0.4 91.6 0.2 0.2 0.1 Fgr ) h eaiedistribution relative The 2). (Figure rdmntdi h ed were reads the in predominated , r Rhizobiales ora fFo Science Food of Journal ± ± ± ± ± ± ± ± ± ihrltv bnacsof abundances relative with 9.1 5.5 7.0 7.7 25.4 49.5 39.5 24.6 8.1 D BA A BA BA BA C B BA A erez-D ´ rdmntdi the in predominated Aurantimonodaceae ´ a n tes 2015). others, and ıaz 53.2 54.8 44.5 30.3 21.6 Rhodospirialles 3.3 5.8 4.4 5.5 6.8 ± ± ± ± ± ± ± ± ± ± 4.7 8.2 6.2 7.8 9.6 7.5 6.1 20.7 16.5 7.3 M125 D AD AD D AD C C AB B AB

M: Food Microbiology & Safety . L. A. 0.0 0.0 0.0 0.1 0.1 0.1 0.0 0.1 0.0 0.1 and fol- ± ± ± ± ± ± ± ± ± ± and ,and Firmicutes and A. peroxydans A. aceti . The rest of the , Pectinatus sp. (Table 4). A clone L. acetotolerans L. buchneri 29.129.1 3.8 4.0 26.9 2.8 25.924.634.324.0 2.2 56.8 1.7 1.6 2.2 2.4 20.6 3.7 24.5 1.7 ± ± ± ± ± ± ± ± ± ± genus. Additionally, 6 and L. panis L. parabrevis genusfollowedby13cloned A. peroxydans and ,and Dialister fermentation sample (tank 4) collected Prevotella 2 Figure 2–Bacterial taxonomic compositions in cucumber fermentation spoilage samples. L. buchneri genus, with , genus, mostly composed of sp. was also identified in sample D with 10 clones. Acetobacter peroxydans Acetobacter genus with 12 clones, including being the most abundant. The second group was the commercial sample B collected on day 120 with a pH spp., respectively, were detected (Table 4). 2 L. namurensis during the 2013 pickling season (C, D, G, H, I, and J). Lactobacillus Genus Methylobacterium Family Auranmonadaceae Unclassified Rhizobiales Unclassified Bacteria Lactobacillus brevis Genus Lactobacillus Family Lactobacillales Unclassified Genus Pecnatus Family Veillonellaceae Genus Prevotella Unclassified Bacteroidales Acetobacteraceae Family Pectinatus 2 . Plating on MYP was completed to confirm the presence of of 4.5 (Table 2), had 26those (55.3%) clone sequences sequences from with the similaritysequences to with similarity to the species being clones had sequence similarity with the phyla. The spoiled CaCl AAB were detected inCFU/mL; while 3 below detection out levels, ofwere or observed numbers 6 close in to tanks the that, MYP additional at tanks. plates approximately The 4 were colonies all log and observed motile. Gram-negative on Twenty-four bacilli, isolates positive subjected for to catalase partial sequencing after 76 dto presented the 47 (94%)lowed clones by with sequence similarities 2 clone sequences withAcetobacter similarities to those of AAB in FCS samplesCaCl collected from fermentations brined with panis The CaCl sequence from this sameSample D sample showed a was pH identified ofAAB 3.4 as on and day high 76 counts (Table 4). ofin Three LAB, main this yeasts, groups and commercial have brine. been identified members Twenty-seven clones of were identified as pasteurianus Lactobacillus NaCl Tank F Tank Ace- NaCl Tank E Tank , which in- CaCl2 Tank B Tank dominated. cloning library. Tank B 12920 172 157.9 Tank A 40080 365 207.4 NaCl Tank 1 Tank Tanks 3 and 4Tanks 3 and 4Tanks 3 and 4Tanks 3 and 4 26192 38372 36080 23343 301 357 360 235 203.1 199.5 220.6 220.6 , regardless of the fer- 2 rRNA Bacteroidales & 4 CaCl2 Tank 3 Tank Acetobacteraceae & 4 CaCl2 Tank 3 Tank At the lower pH values and in Vol. 81, Nr. 1, 2016 r 2 2 2 2 2 2 & 4 was dominantly detected in the sam- CaCl2 Tank 3 Tank with 53.06% relative abundance. Also, was identified in the samples from 120 Fermentaon Age Fermentaon Age (days) genus, respectively; with the majority of spp. were uniquely identified in 1 sample Prevotella. CaCl2 Tank A Tank Prevotella & 4 spp. Samples collected at a later time point, had CaCl2 Tank 3 Tank Acetobacteraceae Lactobacillus Lactobacillus 75 75 75 90 105 120 120 120 150 420 150 120 120 120 105 90 75 75 75 Veillonellaceae NaCl Tank 1 Tank spoiled cover brine collected on day 76 of the fermen- 2 Journal of Food Science and Pectinatus 0

50 40 30 20 10 90 80 70 60

100 Relave Abundance (%) Abundance Relave Four samples were selected to represent the different stages of The family tobacter fermentations. The control sample collectedcally from dormant a microbiologi- NaCl commercial fermentationpresented (tank 10 1) (26%) on and day 9 76, (23%) clones with sequences of the Three samples represented thepH values initiation between 3.0 of andvance spoilage 3.4, stage (day and of 1 76) spoilage samplepresents (day at represented the 120) the clones with ad- obtained a for the pH commercial value spoiled of cucumber 4.5. Table 4 spoilage in pickles for the creation of a 16S ples collected from 2 tanks brined with CaCl 150420 NaCl NaCl Tank E Tank F 6407 7465 317 239 369.4 257.0 mentation age, reaching relative abundance values between 71.55% and 99.9%. of CaCl M126 Analysis of the 16S rRNA cloning library age (d)7676 salt NaCl CaCl Tank Tank 1 Reads 19999 OTUs 301 Chao1 224.8 Shannon Table 3–Summary sheet with Chao1 and ShannonFermentation alpha diversity metrics for the Ion Torrent sequencing data analysis. Cover brine Fermented cucumbers spoilage . . . 76105120120 CaCl CaCl CaCl NaCl Tank 1 25572 254 173.1 for the proliferation of the family d of the fermentationcluded with a 7.3% relativepH abundance, which values in- above 4.5, which presumably predominantly allowed bacterial diversity was detectedwhen on the day predominant 120 ordercluded of the found the genus was fermentation, tation with a 25% relative abundance. A significant change in the 90 CaCl 120 CaCl the presence of air, members of the

M: Food Microbiology & Safety rbst h pce ee.TeInTretaayi eeldthat revealed analysis Torrent Ion The level. species the mi- to relevant crobes identifying information this deepened taxonomically h ata eunigo 16S from of obtained sequencing data partial the level; the family the to community higher bacterial the values pH at organisms utilization these acid that lactic 3.5. suggest than in pH, implicated higher be a may with spoilage of of identification stage the Additionally, spoilage. Prevotella of development the Discussion samples. the of collection observed, the were to symptoms spoilage prior once tanks and ben- sodium brines selected preservative, cover the in the of to AAB addition zoate, the of to absence attributed The be may databases 1997). archaea others and and bacteria (Sasaki for sequences RNA query ribosomal NCBI the 16S the with of alignment 98% the to and respectively, according 100% sequences, over respectively, similarities, 98% to sp. Lysinibacillus 16S the of b a 4.5 3.4 B 3.33 120 D 3.08 72 4 75 komagatae Methylobacterium 1 75 Delftia Granulicella ID Tentative pH (d) samples. age brine Fermentation spoilage from ID results Sample/tank BLASTN sequence clone rRNA 16S 4–The Table . . . spoilage cucumbers Fermented h oiatbceilgop dnie ntesmlsstudied samples in the variability in The identified either that brines. groups of suggests cover bacterial age the dominant the of the on pH the depending and abundance relative fermentation variable in present Microorgan- oxygen. the of role absence of the the isms in confirms and primarily oxygen, LAB, of selected presence of the in FCS of initiation Sphingomonas ilse succinatiphilus Dialister propionicifaciens Dialister fluviatilis Prosthecobacter baltica Rhodopirellula yabuuchiae resiniphila testosteroni Comamonas lusitana Aquicella Achromobacter ilse microaerophilus Dialister naganoensis Pullulanibacillus Prevotella Pectinatus pasteurianus Acetobacter namurensis Lactobacillus brevis/hammesii Lactobacillus parabrevis Lactobacillus Lactobacillus ctbce peroxydans Acetobacter rapi Lactobacillus acetotolerans Lactobacillus atbclu panis Lactobacillus buchneri Lactobacillus h ettv dnicto fteseisbsdo h LSNainetuigNB atra n rheldatabase. Archaeal and Bacterial NCBI using alignment BLASTN the on based species the of identification tentative The h ecnaesqec iiaiisfrec lndsqec ihtecoetmthi h CI1SBceiladAcaa database. Archaeal and Bacterial 16S NCBI the in match closest the with sequence cloned each for similarities sequence percentage The hl h o orn aagnrlydsrbdtedvriyof diversity the described generally data Torrent Ion the While identified study This p 9t 100 to 99 2 sp. p 5t 99 to 95 2 2 sp. p 68 o94 to 86 26 1 sp. p 068 o99 to 89 6 10 sp. and p 97 2 sp. p 99 8 1 sp. rRNA p 99 1 sp. p 98 2 sp. Lactobacillus a Dialister rGram-negative or eeietfida cditlrn Gram-positive intolerant acid as identified were , Acetobacter pce nsolg ape rmtelater the from samples spoilage in species and .pasteurianus A. Acetobacter rRNA p.o eetdlcoail a lead may lactobacilli selected or spp. 196 186197199298to99 8t 99 to 98 2 100 1 1971 698to99 329 o100 to 95 2 23 1 9 196 5 96to99151 198 ubro lnssqecd%similarity % sequenced clones of Number .pasterianus A. lnn irr ofimdand confirmed library cloning eeawr lotalways almost were genera samjrpae nthe in player major a as 419 o99 to 92 1 34 4t 99 to 94 1 1 698to99 ih9%ad97% and 99% with 294to99199 194490to93 893to95 piae uhas of development such 16S the spoilage, in involved the potentially in of species lactobacilli FCS analysis cific drive The 2), microbes. could relevant (Figure family rRNA tested other this samples of of 8 absence members the the of that out suggest 5 which in analysis Torrent Ion l oueue oetatttlgnmcDAfo h NaCl CaCl the the of from sam- that DNA 50-fold the genomic was Additionally, total samples collected. tank extract control were to used samples volume the to ple unlikely time fermentation and the the of minimal biochemistry at was the tanks in micro- control changes the significant NaCl reflect that the suggesting in YMA, load experimental and bial the MRS to on compared samples as spoilage samples control it were the above, counts in microbial stated lower observed significantly as However, that highlighted 3.4. be below must values pH with analyzed of as species that revealed analysis such Additionally, Moreover, family genus the hncmae ota fteCaCl the of that to insignificant been compared have when could samples fermentation NaCl the to from corresponding abundance relative the Thus, samples. hc atbcliwr on odmnt,wr olce by collected were in dominate, F, to and found the E were and samples corre- spoilage lactobacilli of fermentation the which initiation Spoilage in the in microbiota. purging factor leading air determining availability a oxygen of that be speculated absence may is it the tanks, and originating sponding FCS of samples ie h aalli h oiac of dominance the in parallel the Given peroxydans Acetobacteriaceae lnn irr umne hspcueb eeln spe- revealing by picture this augmented library cloning Lactobacillus Acetobacteriaceae , aceti o.8,N.1 2016 1, Nr. 81, Vol. b namurensis ,and oiae n2solg ape,wiethe while samples, spoilage 2 in dominated oiae n8ohrsmls(iue1). (Figure samples other 8 in dominated pasteurianus hlm ls,order class, Phylum; rtoatra eartoatra Burkolderiales ; Proteobacteria; Acidobacteriaceae Acidobacteriales; Acidobacteria; rtoatra ampoebcei;; ; Proteobacteria; Burkolderiales Betaproteobacteria; Proteobacteria; ; Proteobacteria; lntmcts lntmcta Planctomycetales Planctomycetia; Planctomycetes; Legionellales Gammaproteobacteria; Proteobacteria; Betaproteobacteria; Proteobacteria; imcts eaiiue;Selenomonadales Negativicutes; Firmicutes; imcts ail;Lactobacillales Bacilli; Firmicutes; Bacillales Bacilli; Firmicutes; Verrucomicrobiales Verrucomicrobiae; Verrucomicrobia; Burkolderiales Betaproteobacteria; Proteobacteria; Rhizobiales Alphaproteobacteria; Proteobacteria; atriee;Bceoii;Bacteroidales Bacteroridia; Bacteroidetes; rtoatra lhpoebcei; Alphaproteobacteria; Proteobacteria; Lactobacillales Bacilli; Firmicutes; imcts eaiiue;Selenomonadales Negativicutes; Firmicutes; Selenomonadales Negativicutes; Firmicutes; Clostridiales Clostridia; Firmicutes; rtoatra lhpoebcei;Rhodospirillales Alphaproteobacteria; Proteobacteria; Lactobacillales Bacilli; Firmicutes; Bacillales Bacilli; Firmicutes; imcts ail;Lactobacillales Bacilli; Firmicutes; Lactobacillales Bacilli; Firmicutes; Lactobacillales Bacilli; Firmicutes; rtoatra lhpoebcei;Rhodospirillales Alphaproteobacteria; Proteobacteria; Lactobacillales Bacilli; Firmicutes; imcts ail;Lactobacillales Bacilli; Firmicutes; , a h nyfml eetdb the by detected family only the was acetotolerans r ora fFo Science Food of Journal eepeeti h samples 3 the in present were , 2 emnainsamples. fermentation , buchneri Lactobacillus , Acetobacter panis 2 fermentation ,and Acetobacter p.in spp. M127 ,such rapi .

M: Food Microbiology & Safety A. Di- Dial- Pecti- . The Pectina- Dialister spp. nor isolates in cloning li- spp. to ful- in vitro is related to species, spp. and rRNA spp. seems to play Pectinatus Pectinatus Acetobacter Acetobacter Prevotella Dialister propionicifaciens Pectinatus Veillonella spp. in the initiation of sec- Lactobacillus , sharing the same metabolic pro- Acetobacter spp. After understanding that this bac- Veillonella Acetobacter spp. are able of converting lactic acid to propionic , was isolated from a commercial pickle spoilage genome is extremely unstable, has experience intensive spp. Thus, previous studies of FCS neither enriched Prevotella species have been collected from various human clinical or spp. are commonly isolated from unpasteurized beer spoilage Bacterial diversity identified from commercial spoilage samples The culture-independent approach taken in this study not only humans and has beenand isolated urogenital from tracts. oral The cavity, majorand upper fermentation succinic respiratory, acids products and, are occasionally, acetic isovaleric lower levels acids of isobutyric are and tus produced (Shah andcausing the Collins formation of 1990). off-flavors derivedsulphuric from compounds organic and acids are and considered strictand anaerobes Suihko (Juvonen 2006). The recentlynatus proposed sottacetonis tank in a previous study (Breidtby and others biochemical, 2013) physiological, and characterized and(Caldwell molecular and biological others methods 2013).propionic The acid isolate from produces fructose aceticpH and acid values close glucose and to as neutrality. However, neither majorister metabolites at using high-throughput technology as Ioncloning Torrent and 16S is rRNA present comprehensively in FCS. representative Spoilage involvesmicroorganisms, cooperative of whose among the contributionsbrine are pH. microbiota The defined presence of by AABa and the relevant role cover inpresumably the initiation depending of fermentedpresent on cucumbers in spoilage the the tankslactic when amounts spoilage acid is of to initiated. acetic They dissolved can acid convert oxygen during the 1st stage of the secondary acid at pH below 4.8. Conclusion spp. to the listthe of spoilage and bacteria confirmed potentially thealister participation active of in thesamples later and stage never of frompossesses fermented some foods. physiological characteristicsmembers in of the common genus withfile, the with the(Jumas-Bilak production and others of 2005). acetic, The propionic, genus and lactic acids ondary fermentation, but also added they may lose their resistance2000). to Within the the organic vinegar acid industry (Holt iting is and fermentation customary others to to inoculate use a anpreserving new ongo- viability batch, in given frozen the stocks difficulties (personal in communicationvinegar with processors). Azuma and others (2009)pasteurianus reported that the reduction, and that cellstially may explain the have difficulties up in growing tostandard this microbiological bacterium 6 testing plasmids, of whichbrine cucumber par- fermentation samples cover inmedia our or laboratory incubation do conditionsAcetobacter not to include successfully detect thenor and appropriate selected isolate for terium was present intheir commercial DNA cucumber signature in fermentations the from brary Ion analyses, Torrent and we 16S were able to recoverculture-based viable colonies characterization. on MYP However, for attemptsthe spoilage to in the reproduce laboratoryfill by Koch inoculating postulates and to studyvatives were their hindered sensitivity by to lack selected of preser- growtha of cucumber the juice medium containing 0not or shown). 10 Certainly, mM this acetic acid aspect (data needs to be furtherrevealed evaluated. a relevant role for , spp. . Acetobacter spp. were de- spp. has been Acetobacter cloning library A. pasteurianus at pH 3.2 if less , spp. are naturally A. pasterianus rRNA A. aceti Acetobacter Acetobacter , L. buchneri Acetobacter , were suppressed and, thus, Vol. 81, Nr. 1, 2016 r accumulation in the fermentation spp. in the high salt cucumber fer- 2 A. liquefaciens fermentations (Figure 2). Most 2 Aurantimonadaceae Acetobacter spp. as a relevant causative agent. It has been re- are resistant to 0.5% and 2% NaCl, respectively. and spp. are known for colonizing lactic acid fermented Journal of Food Science Acetobacter cucumber fermentation contains 0% NaCl and reaches pH 2 A. hansenii Although AAB were detected in cucumber fermentations with In contrast, the use of open-top tanks and air-purging to control It is relevant to mention that the lactobacilli species identified in Application of culture-independent methods for the study of both cover brinenotable types, in the the production CaCl of acetic acid was only fermentation when the pHof is still lactic acidic. Thus, acid thepartially and disappearance or consequent fully attributed formation to AAB, of primarily acetic acid may be tanks prevents anaerobiosis in thefavor commercial the tanks, proliferation which of could AAB in the earliest stages of secondary Acetobacter foods given their(Gossele ability and to others 1984). catabolize The lactic fact that acid to acetic acid as low as 3.0. While thetranslate absence into of a NaCl in highera this susceptibility new final system to could pH the of development of 3.0 FCS, could be a hurdle for selected M128 bloater damage due to CO than 5.8% NaCl concentrationsLactic acid was disappearance present was in3.8 observed and in the above, the cover regardless same of brine. system the at salt concentration. pH mentation pH (3.5 to 3.8),others and (2012) reduced salt. reported Johanningsmeier that and mented lactic cucumber acid slurry degradation containing occurs in fer- sarily universally presentpromoting in their proliferation, all shouldcucumber fermentation they microbiota, be tanks. may part Conditions described of include the above, anaerobiosis nutrient natural in limitation, the unusually context high end of fer- tank. This observation suggestsan that important these role species in mayconditions be the that playing development promotes of their spoilage proliferation, under specific but are not neces- not detected in these tanks. spoilage samples by thewere analysis not of the detected 16S in the sample representing the NaCl control that the facultative anaerobic lactobacilliated could in have the been absence potenti- of airAcetobacteraceae purging, and that aerobic bacteria, such as (personal communication withtime processor). between the During months such ofnot May storage and applied September, to air purging theseamounts was were 2 present tanks, in which the corresponding implies tanks. that It reduce is oxygen presumed processors from commercial tanksmore that than 4 had months been due inaccessible to for construction in their nearby location Fermented cucumbers spoilage . . . fermentation pH of 3.3of and 6% proliferation NaCl of may bementations, as a suggested factor by in the dataCaCl the presented lack here. The proposed While the optimum pHidentified between for 6.3 and growth 5.4, of as they 3.6 seem to (De tolerate Ley pH and as others acidic 1984). The combination of an end of pH values below 4.5,fermentations conditions brined met withported NaCl. in that De commercial 37% Ley and cucumber 2% andand of others (1984) re- spp. are sensitive to the presence of NaCl in the medium and the bacterial community present in FCStifying was instrumental in iden- ported that unless AAB are cultivated in a medium with acetic acid, controlled NaCl tanks, suggestspresent that in cucumber fermentations, butLAB are outcompeted spearheading by primary those fermentation. tected in most of the samples tested, including those obtained from

M: Food Microbiology & Safety ihsaitclanalysis. assistance statistical for Sciences with Nutrition and Bioprocessing Food, of Dept. aoaoJ,KcysiJ tmag ,BtigrK uha D otloE.21.QIIME 2010. EK. Costello DeLeyJ,SwingsJ,Gossel FD, Bushman K, Bittinger J, Stombaugh J, Kuczynski JG, Caporaso 2013. F. Breidt J, Brown R, Juvonen JM, Caldwell 25:1–6. Res Acids P Nucleic D, GenBank. Wafa 1997. E, J. Medina Ostell F, DJ, Breidt Lipman MS, Boguski DA, Benson epwt PCaayi.W lotakD.Jsei D Joscelin Dr. thank technical also We for analysis. HPLC Unit, with Fornea Research help Seth Science Mr. Food thank USDA-ARS We the Medina. with E. Dr. funded fully (MECD) performed Azcarate-Peril data. the sequencing A. Torrent of bioin- Ion M. 10% processed managed and executed and and Franco Butz W. data N. and the experimentation; Hayes J. of con- analysis; interpretation Breidt formatics F. the experimentation; to the of tributed 90% manuscript; executed the Medina wrote and E. data, interpreted experimentation, the zm ,Hsym ,MtuaiM uuaN oiaaH aaaT iaaaH Kuhara H, Hirakawa T, search Harada alignment H, Horikawa local N, Basic Furuya M, 1990. Matsutani DJ. A, Hosoyama Lipman Y, EW, Azuma Meyers W, Miller W, Gish SF, Altschul References Acknowledgments Contributions Author FCS. Further in proliferation involved 4.5. the microorganisms above control of is to activity pH strategies metabolic the and define when to FCS needed of are genus stage studies later the the of in Members acid 3.4. below pH Pectinatus at initiated fermentation . . . spoilage cucumbers Fermented da C 00 erhadcutrn reso antd atrta LS.Bioinformatics BLAST. than faster magnitude of orders clustering and Search 2010. RC. Edgar losaayi fhg-hogptcmuiysqec aa a ehd 7(5):335–6. Methods Nat data. sequence community high-throughput of analysis 63(10):3609–16. allows Microbiol Evol Syst J Int tank. spoilage pickle commercial 78(3):470–6. Sci Food J culture enrichment cloning. by rRNA bacteria 16S spoilage and fermentation cucumber of Characterization 2013. JH. ,Mtuht ,Fjt ,Sia .20.Woegnm nlssrva eei instability genetic reveal analyses Whole-genome 2009. M. Shirai N, of Fujita K, Matsushita S, 215:403–10. Biol Mol J tool. R otJ,eios egysmna fsseai atrooy o.1 atmr,Md.: Baltimore, 26:2460–1. 1. Vol. bacteriology. systematic 267–74. of p Wilkins. manual Bergey’s and Williams editors. JG, Holt NR, ototrlFlosi rmteSaihGovernment Spanish the from Fellowship Postdoctoral A P M. I. and Medina E. ctbce pasteurianus Acetobacter , Veillonella uli cd e 37(17):5768–83. Res Acids Nucleic . .18.GnsI ctbce ejrnk19,215 1898, Beijerinck Acetobacter I. Genus 1984. F. e ´ ,and erez-D ´ ´ a M rnoW un ,JhnigmirS,Kim, SD, Johanningsmeier H, Huang W, Franco IM, ıaz erez-D ilse propionifacien Dialister ´ ´ a ocpulzdadplanned and conceptualized ıaz etntssottacetonis Pectinatus a tlz lactic utilize can p o. sltdfo a from isolated nov., sp. ´ a ihthe with ıaz A .In:Krieg isnK,Bicigo B reeR.19.Apicto fbceil1Sribosomal 16S bacterial of Amplification 1990. RC. Greene RB, Blitchington KH, Wilson Gossel P W, Franco fer- of spoilage acid Butyric P 1989. W, MD. Franco Pierson RF, McFeeters MA, Daeschel HP, Fleming al S1. Table Information Supporting cetr F P high-performance RF, by McFeeters vegetables and fruits of analysis Sulfite 2003. AO. microbial Barish comparing RF, for method McFeeters phylogenetic new a UniFrac: 2005. R. Knight CA, Lozupone F, Morio J, Campos B, Gay K, Bernard C, Teyssier JP, Carlier H, Jean-Pierre E, Jumas-Bilak P W, cucum- Franco fermented SD, in Johanningsmeier acid lactic manual of Berge’s Metabolism 2000. 2013. editors. RF. ST, McFeeters Williams SD, Johanningsmeier JT, Staley PHA, Sneath NR, Krieg JG, Holt a ,Bed .20.Eueaino viable of Enumeration 2007. F. Breidt Y, Pan hhH,ClisD.1990. DM. Collins HN, Shah identification for method new a of Evaluation 1997. T. Kenri M, Shintani T, Nishiyama T, Sasaki P P uoe ,Sik L 2006. ML. Suihko R, Juvonen erez-D erez-D ´ ´ N ihplmrs hi ecin lnclMcoil28:1942–6. Microbiol Clinical J reaction. chain polymerase with DNA piaeascae emne uubrrsn Hsolg.Ap nio Microbiol Environ Appl spoilage. pH rising cucumber Microbiol 78(4):1273–4. Food fermented fruit. spoilage-associated fermented the of spoilage with 32:338–44. associated fermentation secondary 54(3):636–9. Sci Food J cucumbers. mented eae pce omrycasde ntegenus the in classidies formerly species related iudcrmtgah HL)wt lrvoe pcrpooercdtcin gi Food Agric J detection. 51(6):1513–7. spectrophotometric Chem ultraviolet with (HPLC) chromatography liquid 71:8228–35. 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L, Sci berg Food J guidelines. kosher meet can that fermentations cucumber genus 55:2471–8. haikarae Microbiol Pectinatus Evol Syst J Int samples. clinical human from isolated ,Sig ,Mse A,D e .18.Ietfiainof Identification 1984. J. Ley De DAA, Mossel J, Swings F, e ´ ´ ´ a M cetr F ole ,JhnigmirS,HysJ onaD,Rosen- DS, Fornea J, Hayes SD, Johanningsmeier L, Moeller RF, McFeeters IM, ıaz a of Preparation 2011. RF. McFeeters IM, ıaz Pectinatus atbclu buchneri Lactobacillus erez-D ´ erez-D ´ upeetr Material. Supplementary erez-D ´ n ytEo irbo 56:695–702. Microbiol Evol Syst J Int . ´ a M 02 oeo eetdoiaieyat n atrai cucumber in bacteria and yeasts oxidative selected of Role 2012. IM. ıaz ´ o.8,N.1 2016 1, Nr. 81, Vol. a M oannsee D cetr F 02 hrceitc of Characteristics 2012. RF. McFeeters SD, Johanningsmeier IM, ıaz p o. sltdfo rwr ape,adeedddsrpino the of description emended and samples, brewery from isolated nov., sp. ´ a M 00 emnaino uubrbie ihCaCl with brined cucumber of Fermentation 2010. IM. ıaz ilse microaerophilus Dialister eapar paucivorans Megasphaera Prevotella n eae pce,ptnilsolg raim nrdcdsalt reduced in organisms spoilage potential species, related and erez-D ´ e eu oinclude to genus new a , ´ a M cetr F 02 nuneo NaCl, of Influence 2012. RF. McFeeters IM, ıaz itramonocytogenes Listeria r Bacteroides p o.and nov. sp. ora fFo Science Food of Journal p nov., sp. atbclu plantarum Lactobacillus n ytBceil40(2):205–8. Bacteriol Syst J Int . eapar sueciensis Megasphaera ilse propionicifaciens Dialister atrie melaninogenicus Bacteroides el yra-iePRwith PCR real-time by cells Acetobacter tre utr for culture starter p o.and nov. sp. 2 tan iso- strains nta of instead M129 p nov., sp. and

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