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Hindawi Journal of Food Quality Volume 2018, Article ID 8051435, 13 pages https://doi.org/10.1155/2018/8051435

Research Article Quality of Cucumbers Commercially Fermented in without

Erin K. McMurtrie1 and Suzanne D. Johanningsmeier 2

1 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, 400 Dan Allen Drive, Raleigh, NC 27695-7642, USA 2USDA-ARS,SEAFoodScienceResearchUnit,322SchaubHall,Box7624,NorthCarolinaStateUniversity, Raleigh, NC 27695-7624, USA

Correspondence should be addressed to Suzanne D. Johanningsmeier; [email protected]

Received 1 October 2017; Revised 5 December 2017; Accepted 20 December 2017; Published 26 March 2018

Academic Editor: Susana Fiszman

Copyright © 2018 Erin K. McMurtrie. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dr. Suzanne D. Johanningsmeier’s contribution to this article is a work of the United States Government, for which copyright protection is not available in the United States. 17 U.S.C. §105.

Commercial cucumber fermentation produces large volumes of salty wastewater. Tis study evaluated the quality of fermented cucumbers produced commercially using an alternative calcium chloride (CaCl2) process. Fermentation conducted in calcium (0.1 M CaCl2, 6 mM sorbate, equilibrated) with a starter culture was compared to standard industrial fermentation. Production variables included commercial processor (� = 6), seasonal variation (June–September, 2 years), vessel size (10,000–40,000 L), cucumber size (2.7–5.1 cm diameter), and bulk storage time (55–280 days). Cucumber mesocarp frmness, color, bloater defects, pH, and organic acids were measured. Complete lactic acid fermentation was achieved, resulting in terminal fermentation pH values of 3.23 ± 0.09 and 3.30 ± 0.12 for CaCl2 and NaCl processes, respectively. On average, CaCl2 brined, fermented cucumbers were 1.8 N less frm, which remained signifcant in the fnished product (� < 0.0001). Color diferences evidenced by higher hue and lower chroma values (� < 0.0269) were consistent with increased photooxidation in CaCl2 brined cucumbers. Commercial implementation of CaCl2 brines for cucumber fermentation in open tanks variably resulted in texture and color defects that can impact product quality. Additional research is needed to understand the atypical sofening observed at the commercial scale and identify process controls for quality improvements.

1. Introduction the EPA’s Secondary Drinking Water Standards of less than 250 ppm or 7 mM chloride [1]. To lessen the amount of Commercial cucumber fermentation is a preservation meth- in wastewater, spent fermentation brines are now commonly od that depends on high (NaCl) concentra- stored and reused in subsequent fermentations [2]. Despite tions. Each fermentation is conducted in a 10,000 to 40,000 L extensive reuse of spent brines, processors still routinely open-top tank containing at least 0.86 M (5%) NaCl afer produce high volumes of salty wastewater from the desalting equilibration with the fresh cucumbers. Tis salt concentra- process. Perez-D´ ´ıaz et al. [3] estimated that the production tion enables a natural fermentation to occur by selecting for of fermented cucumber pickles in the United States resulted the lactic acid bacteria present on the cucumbers and inhibit- in 45 million liters of wastewater containing 0.58 M NaCl ing salt-sensitive spoilage bacteria. Te fermented cucumbers from desalting processes, 1.3 billion liters of wastewater with are then desalted to palatable salt levels by removing the 79 mM , and 12,000 tons of salt sludge solid waste. fermentation brine and soaking the fermented cucumbers in An alternative fermentation process utilizing 0.1 M cal- fresh water. Tis process reliably produces high quality, fer- cium chloride (CaCl2), 0.025 M acetic acid, and a Lactobacil- mented pickle products but generates signifcant volumes of lus plantarum starter culture was developed by McFeeters high salt wastewater with chloride well above and Perez-D´ ´ıaz [4]. Complete fermentation with fermented 2 Journal of Food Quality cucumbers that maintained their frmness was achieved with- and 2014 seasons were fermented in 10,000 to 40,000 L open- out using any NaCl and an 80% lower chloride content. Since top tanks at six processing facilities. Fermentation was initi- the development of this process, attempts to commercialize ated between the months of June and September each year. thetechnologyhaveresultedinslightadjustmentsofthe Fermentation tanks contained size 2A (2.7–3.5 cm diameter), brineformulationandprocess.Teseadjustmentsincluded 2B (3.5–3.8 cm diameter), 3A (3.8–4.4 cm diameter), or 3B the elimination of acetic acid from the brine formulation, (4.4–5.1 cm diameter) cucumbers covered with a fermenta- addition of 6 mM potassium sorbate, and introduction of a tion brine. Fermentation brines for the control fermentation minimal air-purging routine [3]. A typical purging routine were prepared using the current commercial process and utilizes the fow of air or nitrogen gas through the brine were comprised of recycled brines with additional NaCl, during the active fermentation period to help encourage the CaCl2, and potassium sorbate such that the cucumbers removal of carbon dioxide [5–7]. Carbon dioxide (CO2)is equilibrated to approximately 1.0 M NaCl, 40 mM CaCl2, produced as a product of heterofermentative fermentation, and 2 mM, respectively. Fresh brines for the CaCl2 brining malolactic fermentation, and cucumber respiration [5, 6, 8– process were prepared to equilibrate with the cucumbers to 11]. An excess of CO2 can cause hollow cavities concentrations of 100 mM CaCl2 and 6 mM potassium sor- in the fesh of the cucumber that constitute physical defects bate. In total, 41 tanks of cucumbers were fermented using called bloaters. Both air and nitrogen purging have been the CaCl2 brining process along with 27 tanks of cucumbers suggested for use and implemented in commercial fermen- fermented in NaCl brines using standard industry practices. tation to remove CO2 from the brine to minimize bloater Each fermentation process began with the addition of damage. Nitrogen purging is ideal since air purging can cushion brines (18–20 inches deep) to the tanks to minimize introduce oxygen into the fermentation tanks allowing aero- damage as the cucumbers were added. Cucumbers were bicmicroorganismstofourishandreducingthetexturequal- added to the tanks and wooden boards were placed over the ity of the fermented cucumbers [12, 13]. However, air purging cucumbers to ensure that they stayed submerged under the is less costly and used by most processors in combination brine during fermentation. Brines were then added to the tanks and the CaCl2 brined cucumbers were inoculated with potassium sorbate to inhibit yeasts and molds. During 5 6 the early stages of commercialization, fermentations using with 10 –10 CFU/mL L. plantarum starter culture (LA0445 the alternative CaCl2 process were found to spoil much more or other presumptive L. plantarum isolates from cucum- easily than in the current commercial process. Terefore, the ber fermentation (unpublished), USDA-ARS, Food Science air-purging routine was altered to minimize the introduction Research Unit, Raleigh, NC, USA, culture collection) pre- of oxygen and potassium sorbate was added as a preservative pared according to the kosher process described by Perez-´ against yeast spoilage [3]. Acetic acid can serve as a substrate D´ıaz and McFeeters [16]. Sodium chloride brined cucumbers for oxidative spoilage yeasts such as Issatchenkia occidentalis underwent a natural fermentation and were air-purged for and Pichia manshurica [14], so it was removed from the brine thedurationofactivefermentationincyclesof8hfollowed by a 4 h reprieve (processor 1) or 20 h per day (all other pro- formulation as well [3]. cessors). During the 2013 season, CaCl2 brined cucumbers at Pickles produced commercially using this modifed, alter- processor 1 received a minimal purge cycle of only 2 h, twice native fermentation process have shown variable results with aday,butfollowedthesamepurgeroutineastheNaCltanks regard to cucumber frmness. In the laboratory development during the 2014 season. All other processors used their typical of the process, mesocarp frmness of cucumbers fermented in air-purging routine of 20 h per day for all fermentations. this CaCl2 brinewasfoundtobeconsistentlycomparableto the cucumbers fermented in a commercial brine formulation Fermented cucumbers were bulk stored in the fermenta- [4]. In contrast, cucumbers fermented in 12,500 L open-top tionbrinesfor55to280daysandthenprocessedintofnished tanks with the postcommercialization formulation changes products according to routine processing schedules. Prior to showed variability in mesocarp frmness values as well as processing,a5gallonsampleofcucumbersandbrinewas collected for analysis, hereafer referred to as “fermented” lower sensory hardness, fracturability, crispness, and crunch- samples. Processing of the CaCl2 brined cucumbers utilized iness scores in some pickle products fermented and stored in a two-step desalting procedure that entailed the pumped CaCl2 brines [3]. Consumer testing also showed a decreased removal of the fermentation brine and replacement with fresh texture liking for cucumber pickles commercially produced water. Tis was allowed to equilibrate for a day, during which with the CaCl2 brining process in some, but not all, experi- the cucumbers were air-purged for 2 h before the liquid was ments [15]. Te objective of this study was to perform a large pumped out again. Freshly made 0.2% alum water was added scale evaluation of commercial cucumber fermentation to to the tanks and allowed to equilibrate with the fermented determine the variability in physical, textural, and color qual- cucumbers for a second day, again with 2 h of purging. Te ity of cucumbers fermented using the CaCl2 brining process NaCl brined tanks followed a one-step desalting procedure and typical NaCl brining process during routine commercial where the fermentation brine was removed and replaced with production of hamburger dill chips. 0.2% alum water and allowed to equilibrate with the fer- mented cucumbers for 1 day. A 5 gallon sample of cucumbers 2. Materials and Methods and brine was collected for analysis just before packaging into fnished product, referred to as “Desalted” samples. 2.1. Commercial Scale Cucumber Fermentation. Pickling Tealumwaterwasthenpumpedoutofthetankandthe cucumbers grown throughout the United States over the 2013 cucumbers were conveyed to the packing line for cutting, Journal of Food Quality 3 packing, addition of freshly prepared cover liquor, capping, where hue indicates the pigment of the color and chroma ∘ ∘ ∘ and pasteurization using standard practices. Cover liquors indicates the intensity. A hue of 0 /360 represents red, 90 , ∘ ∘ were formulated for each fermentation process to create yellow, 180 ,green,and270,blue. fnished products that equilibrated to the processor’s desired product specifcations such that cucumbers fermented in 2.5. Quantifcation of Fermentation Metabolites. Concentra- CaCl2 and cucumbers fermented in NaCl would be stored tions of organic acids, residual sugars, and some spoilage as fnished product with equivalent concentrations of NaCl, metabolites were quantifed by High Performance Liquid CaCl2,vinegar,andfavors.Finishedproducts(atleast3jars Chromatography analysis using an Agilent 1260 Infnity per fermentation) were collected near the beginning, middle, HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) and end of processing of each tank of fermented cucumbers equipped with an HPX-87H exchange column for organic and stored for 6 months at ambient temperature prior to acid analysis (Bio-Rad Laboratories, Hercules, CA, USA). analysis as “fnished product” samples. Chromatography was conducted with 0.03 N sulfuric acid mobile phase at a fow rate of 0.6 mL/minute and column ∘ 2.2. Bloater Defects. Bloater type and severity were recorded temperature of 37 C. Organic acids were quantifed using an for 25 fermented and 25 desalted cucumbers per fermenta- Agilent 1260 Infnity DAD detector (Agilent Technologies tion. Each cucumber was sliced longitudinally and evaluated Inc.) at 210 nm. Monomeric sugars and were quanti- according to the bloater chart developed by Etchells and fed using an Agilent 1260 Infnity RI detector (Agilent Tech- others [17]. Bloater indices were calculated as described by nologies Inc.) connected in series with the DAD detector. Fleming and others [18]. 2.6. Chemical Analyses. A Fisher Accumet (model AR25) pH meter equipped with a pencil-tip gel-flled electrode 2.3. Texture Analysis. Instrumental frmness measurements calibrated with standardized pH 2, 4, and 7 bufers (Fisher were performed using a mesocarp puncture test. A 6.7 ± Scientifc, Waltham, MA, USA) was used for pH measure- 0.1 mm slice was taken from the center of 30 cucumbers for ment. Calcium concentrations were determined by titration the raw, fermented, and desalted samples. Finished products with 0.025 N disodium ethylenediaminetetraacetate dihy- were crinkle cut using a commercial slicer, packed into jars, drate (EDTA) with hydroxy naphthol blue as the indicator and pasteurized. Afer ambient storage for 6 months, 30 according to AOAC Method 968.31 [21]. Sodium chloride crinkle cut slices were collected from the top, middle, and concentrations were estimated by Fajan’s chloride titration bottom of 3 jars as the test samples for fnished products. using 0.171 N silver nitrate (AgNO3) and a dichlorofuores- Te mesocarp puncture test was conducted on a TA.XT cein indicator [22]. Reported values for NaCl were calculated Plus Texture Analyzer (Texture Technologies Corp, Scarsdale, by subtracting the chloride contributed by CaCl2 from the NY/Stable Micro Systems, Godalming, Surrey, UK) using a − total chloride content using the formula [NaCl]=[Cl ]− 3 mm probe to puncture the mesocarp tissue at a test speed of 2∗[CaCl2], where the molar concentration of CaCl2 was 2.5 mm/sec [19, 20]. Te mesocarp of one lobe of each slice presumed to be equal to the molar concentration of calcium. wascenteredabovea3.1mmholeonthebaseplateand the probe was lowered through the sample. Te test was conducted and data was analyzed using Texture Expert sof- 2.7. Polygalacturonase Activity. Polygalacturonase (PG) acti- ware (version 6.1.3.0, Texture Technologies Corp.). Te peak vity was assayed using the difusion plate assay developed force measured during the puncture test and the thickness by Buescher and Burgin [23]. Polygalacturonase activity was of each slice were determined and recorded to create an assayed for both brines and cucumbers. Cucumber homoge- average peak force and average slice thickness for each tank nates were produced by blending the cucumbers from texture of fermented cucumbers at fermented, desalted, and fnished and color analysis in a Waring Blender. Briefy, an aliquot product processing stages. of 30 �L of each brine or cucumber supernatant was pipetted intoawellcreatedbyasize4corkborerinanagarplate containing polygalacturonic acid substrate. Te agar plates ∘ 2.4. Color Analysis. Cucumber mesocarp color was measured were incubated at 38 C for 48 h. Afer incubation, agar was using a Minolta Chroma Meter model CR-300 (Minolta Co., stained for 30 minutes using ruthenium red which binds to �∗ �∗ �∗ Ltd., Osaka, Japan) to record the , ,and values of a dif- polygalacturonic acid remaining in the medium. Afer stain- ferent lobe of each of the 30 slices used for frmness measure- ing, the plates were rinsed and destained in deionized water ments. Each slice was placed on a representative fermented for20h.Tediameteroftheclearzonearoundeachsample cucumber background at least 1.5 inches high to account for well was measured using calipers. Clear zones indicated the translucency of the slices. Values of hue and chroma were activity of the enzyme as the dye will not bind to galacturonic calculated to convert the CIELAB color variables to the polar acid residues. coordinate system CIELCH as follows: 2.8. Statistical Analysis. All results were analyzed using SAS �∗ = −1 ( ) statistical sofware (version 9.4, SAS Institute, Cary, NC, Hue tan �∗ (1) USA). A mixed model analysis of variance was used with least square means. Fermentation process (NaCl or CaCl2), cu- ∗2 ∗2 1/2 Chroma =(� +� ) , cumber size, and stage in processing were designated as fxed 4 Journal of Food Quality

35 fermentation in NaCl brines had an average brine pH of 3.6 ± 30 0.4 afer 1 day of fermentation, while the experimental 25 fermentation in CaCl2 brines had an average brine pH of 4.9 ± 20 0.2 afer 1 day based on the quality control data collected by 15 the commercial processors. 10 Bloater index Bloater 5 A desalting step was used in both fermentation processes 0 to lower the NaCl to palatable concentrations or to lower the CaCl2 concentration to below the legal limit of 36 mM

Desalt Desalt Desalt Desalt Desalt Desalt Desalt Desalt in the fnished product (21 CFR 184.1193) [24]. Te pH

Ferment Ferment Ferment Ferment Ferment Ferment Ferment Ferment values, CaCl2, NaCl, lactic acid, and acetic acid concentra- tions for CaCl2 and NaCl brined fermented cucumbers were n=3 n=3 n=9 n=7 n=3 n=1 n=2 n=2 n=3 n=2

n=11 n=10 n=10 n=14 n=18 afected by the desalting process as expected (Table 1). During n= 25 2A 2B 3A 3B 2A 2B 3A 3B desalting, the pH rose to 3.47 ± 0.28 for cucumbers fermented ± NaCl CaCl2 in CaCl2 brines and 3.40 0.19 for those in NaCl brines as the lactic acid and acetic acid concentrations were diluted along Honeycomb with the salts (Table 1). Although the desalted, NaCl brined Lens cucumbers contained signifcantly more lactic and acetic Balloon acids than the desalted CaCl2 brined cucumbers, the pH Figure 1: Total, balloon, lens, and honeycomb bloater indices for values did not difer signifcantly, likely due to the bufering fermented and desalted cucumbers by cucumber size and brining capacity of the system. Te lactic and acetic acid concentra- process. Error bars represent standard error of the mean. tions were much lower in the desalted cucumbers fermented in CaCl2 brines, because they were desalted twice to reduce the CaCl2 concentrations to that of the typical commercial products. efects and the individual tanks of fermented cucumbers were In the production of fnished pickle products, desalted designated as random efects. Statistical signifcance was indi- cucumbers were separated from the desalting water before � < 0.05 ± cated at . Values are presented as means standard being cut and packed with freshly made cover liquor contain- deviation in Table 1. Data in Table 2 and Figure 1 are pre- ing sufcient NaCl, CaCl2,vinegar,andfavors,suchasdill, ± sented as means standard error. to equilibrate to each processor’s desired formulation. Proces- sors measured salt and acid concentrations in the desalting 3. Results and Discussion solutions to calculate the concentrations of ingredients for the cover liquor to equilibrate with the desalted cucumbers to Raw cucumber samples, representative of those fermented, the specifcations for the product they were making. Because had initial mesocarp frmness of 8.7 ± 0.9 N, sugar content of these specifcations vary based on the type of pickle product 54.2 ± 12.0 mM glucose and 65.2 ± 14.1 mM fructose, and cal- made and between brands, there was greater variability in cium content of 3.0 ± 0.8 mM. Te chemical compositions of the composition of the fnished product samples (Table 1). fermented, desalted, and fnished cucumber pickles produced However, since the cover liquors were made for each batch, with the typical, NaCl-dependent commercial process and the concentrations of acetic acid, NaCl, and/or CaCl2 were the alternative CaCl2 brining process are shown in Table 1. able to equilibrate to the processors’ target concentrations. Completefermentationwasachievedwithbothprocesses, Despite the diferences in the composition of the desalted fer- resulting in safe, insignifcantly diferent terminal pH values mented cucumbers from the two processes, processors were of 3.23 ± 0.09 in the CaCl2 brined, fermented cucumbers able to produce hamburger dill chips within the normal com- and 3.30 ± 0.12 in cucumbers fermented with the current positional range of current products. commercial process using NaCl. Lactic and acetic acids were signifcantly higher in the NaCl fermentation process despite 3.1. Bloater Defects. Bloater defects are formed by an excess the insignifcant diference in pH values, likely because recy- of CO2 in the fermenting cucumbers that builds pressure, cled brines were used that contained residual acid generated breaking cell walls [8]. Bloater indices indicating the type in previous fermentation. It is possible that the terminal and severity of bloaters were calculated for balloon, lens, and pH values were not signifcantly diferent from each other honeycombtypebloatersaswellastotalbloaters(Figure1) because of the diferent ratio of lactic to acetic acid. Lactic according to Fleming et al. [18]. Overall, the fermentation acid has a pKa of 3.86 which is much lower than the pKa in CaCl2 brines produced fermented cucumbers with higher of acetic acid, 4.76. Since the NaCl brined cucumbers had bloater indices (CaCl2:10.5± 1.7; NaCl: 9.8 ± 1.9); however, greater acetic acid to lactic acid ratio, the pH could be insigni- this diference was not statistically signifcant due to large fcant because of greater bufering. Due to the presence of tank to tank variability. Although the brining process alone residual acid in recycled brines, the NaCl brines started with did not signifcantly afect lens, balloon, or total bloaters alowerinitialpHthantheCaCl2 brines which were not (� > 0.3878),itwassignifcantforhoneycombbloaters(� = acidifed prior to fermentation. Te additional acid in the 0.0226). Size 3B, NaCl brined cucumbers had signifcantly NaCl brines may contribute to higher bufering capacity and higher honeycomb bloater indices than any other treatment mayinfuencethepHduringearlyfermentation.Indeed,the (� < 0.0006). However, there were only a few fermentation Journal of Food Quality 5 ± 1 b b a c ( mM ) 36.7 36.7 ± ± 4.0 ± 2.0 114 26.9 ± 14 15.0 ± 6.3 11.7 ± 3.7 105.4 Acetic acid 1 a c b d brine. 2 ( mM ) 8.4 13.5 ± ± 66.4 ± 21.5 92.2 ± 17.8 26.7 ± 11.2 111.7 ± 12.8 Lactic acid 3 64 44 33.7 29 176 45.4 ± ± ± - ± ( mM ) NaCl titrations and include natural cucumber calcium concentrations of 3.0 2+ 2 1.9 240 3.3 739 6.7 240 6.9 - 4.9 4.6 1088 ± ( mM ) ± ± ± ± ± 2 37.9 31.4 26.7 85.2 CaCl subtracted from the total chloride concentration. 2 1 c a bc ab 0.08 24.8 0.05 28.4 ± ± 3.35 3.40 3.23 ± 0.09 3.47 ± 0.28 3.30 ± 0.12 3.40 ± 0.19 concentrations were estimated from Ca 2 CaCl 2 � < 0.05 ; 14 41 27 36 26 20 2 2 2 NaCl NaCl CaCl CaCl Table 1: Fermentation chemistry for cucumber pickles commercially produced in either 1.03 M NaCl recycled brine or 100 mM CaCl NaCl concentrations were estimated from chloride Fajan’s titrations with the chloride contributed by CaCl 3 Means for diferent treatments followed by diferent letters are signifcant at Processing stageFermented Fermentation brineFermented Desalted Number ofDesalted fermentation processesFinished product Terminal pH NaCl Finished product CaCl 1 0.8 mM; 6 Journal of Food Quality 2, 4 ( N ) a a c b b b 7.3 ± 0.1 9.5 ± 0.2 8.9 ± 0.2 9.0 ± 0.2 10.8 ± 0.2 10.8 ± 0.3 brine. 2 Mesocarp frmness 3 average mesocarp tissue frmness presented as the 4 None to slight None to slight None to slight None to slight PG activity level Slight to moderate Slight to moderate means followed by diferent lowercase letters represent treatments 2 1, 2 ( mm ) a a b b ab ab 2.8 ± 0.6 3.4 ± 0.6 0.2 ± 0.5 0.7 ± 0.3 1.2 ± 0.8 2.4 ± 0.6 14 41 27 36 26 20 Polygalacturonase (PG) activity presented as a relative level that is commonly used by processors that recycle brines; 3 plus/minus the standard error of the mean. 2 2 2 ( N ) NaCl NaCl NaCl CaCl CaCl CaCl � < 0.05 . Table 2: Polygalacturonase activity and texture quality of cucumber pickles commercially produced in either 1.03 M NaCl recycled brine or 100 mM CaCl Results of the difusion plate assay for polygalacturonase activity presented as the mean net clear zone plus/minus the standard error of the mean; Processing stageFermented Fermented Fermentation brineDesalted Desalted Number ofFinished fermentation product processesFinished product Difusion plate assay units mean peak puncture force in Newton 1 that are signifcantly diferent at Journal of Food Quality 7 tanks of this type and a single fermentation with high occur- 45 rence of honeycomb bloating may have skewed the results. 40 35 Other bloater indices were highly variable and not accounted 30 for solely by brine type (Figure 1). In the 2013 season, the 25 occurrence of moderately to severely bloated cucumbers was 20 15 signifcantly higher in fermentation in CaCl2 brines (data not Index Bloater 10 shown). Te alternative purging routine used in the experi- 5 mental tanks was suspected to afect the incidence of bloaters 0 2 hrs, 8 hrs, 20 hrs, 8 hrs, 20 hrs, becauseoflessenedremovalofCO2 from the fermentation 2 × day 2 × day 1 × day 2 × day 1 × day brines, so processors increased the air-purging routine in n = 25 n = 2 n = 12 n = 15 n = 12 CaCl2 brined cucumbers to match that of their NaCl brining CaCl2 NaCl process for subsequent fermentations. Brining season was foundtobesignifcantlycorrelatedwiththebloaterindex Figure 2: Bloater indices for fermented cucumbers by brining (� = 0.0019).Tiscouldbeimpactedbyanumberofuncon- process and purging procedure. Te minimal purging routine of 2 h, trolled production variables, such as cultivar, farm location, 2 times a day was only used in CaCl2 brines. Te 8 h, 2 times per day (2 × day) treatment was used by processor 1 and the 20 h, 1 time per and rainfall, but was more likely due to the modifcation in day (1 × day) treatment was used by the remaining processors. the purging routine between seasons. Te use of the minimal purging routine with the CaCl2 process was found to signi- fcantly correlate with the total bloater indices (� = 0.0258, Figure 2). Using CaCl2 brines with standard air-purging rou- had no correlation with mesocarp frmness in this study (� = tines reduced bloating to levels much closer to the current 0.9917). Tus, there is likely another factor afecting the tex- commercial process. Purging procedures were developed to ture quality of cucumbers fermented with the CaCl2 brining minimize the accumulation of CO2 gas that can cause bloat- process on the commercial scale. ers, but the introduction of oxygen into the nearly anaerobic Othercommerciallyrelevantvariableswereinvestigated fermentation environment can be an impediment to success- for explaining the variability in fermented cucumber frmness ful fermentation by LAB in the CaCl2 fermentation process. independently of the brining process. Time spent in bulk However, the use of a minimal purge routine appears to be a storage was found to negatively correlate with fermented cu- detriment to cucumber pickle quality. Te variability in qual- cumber mesocarp frmness relating to a loss of ∼0.7 ± 0.2 N ity associated with the use of the minimal purging routine over 100 days regardless of fermentation process (Figure 4). indicates a need for further optimization of purging routines Te efect of the time spent in bulk storage on texture became to consistently obtain a high quality product with this fer- more discernible as the cucumbers were processed into mentation process. desalted and fnished product samples with 0.8 ± 0.2 N and 1.1 ± 0.2 N lower peak forces for every 100 days, respectively. 3.2. Texture Quality. Raw cucumber mesocarp frmness was Firmness was higher in smaller diameter cucumbers (� = 8.7 ± 0.9 N and the frmness afer fermentation and bulk 0.0187), consistent with previous fndings by Sistrunk and storage was 9.0 ± 1.7 when brined with CaCl2 or 10.8 ± 1.1 Kozup [25]. Rosenberg [26] also found that size 2B cucum- afer natural fermentation in recycled NaCl brines. Tese dif- bershadsignifcantlyfrmermesocarptissuethansize3Acu- ferences between brining processes were frst detected at the cumbers in both the NaCl and CaCl2 fermentation processes, fermented stage and were detected thereafer at the desalted but the diference in texture was not detected with a whole and fnished product stages (Figure 3). Cucumbers brined in cucumber fruit pressure test that is commonly used by the CaCl2 for fermentation and storage had an average peak force industry for monitoring texture quality during processing. 1.7 N lower than those brined in NaCl across all processing Polygalacturonase, an enzyme known for sofening of stages. Average calcium concentrations of 85.2 ± 6.9 and cucumber pickles [27–29], was assayed, but no signifcant dif- 37.9 ± 4.6 mM were slightly lower than the intended 100 mM ferences between brining processes (� = 0.4470) nor correla- and40mMfortherespectivetreatments.Variabilityinthe tions between PG and mesocarp frmness were found for ratio of cucumbers to brine used in the fermentation and the fermented, desalted, or fnished product samples in this study purity of the CaCl2 ingredient could contribute to the lower (Table2).Onaverage,PGactivitylevelswerelowinboth than targeted calcium content measured in the commercially brining processes and further decreased in samples afer fermented cucumbers. In laboratory experiments, cucumbers desalting and fnished product processing. Furthermore, the fermented and stored in 100 mM, 200 mM, and 300 mM levels of calcium in the two processes should be sufcient to CaCl2 brines without NaCl have been shown to produce prevent enzymatic tissue sofening at these low PG activities fermented cucumbers with mesocarp frmness comparable [29, 30], which is congruent with the lack of correlation be- to cucumbers fermented and stored in a 1.03 M NaCl and tweenPGactivityandfrmnessvaluesobserved. 40 mM CaCl2 brine for 8 months [4]. Te range observed in Organic acids have been shown to cause nonenzymatic our commercial study (70.5–98.3 mM CaCl2)wasbelowthe sofening of fresh pack cucumber pickles [31], so it was of targeted100mM,knowntomaintainfrmnessinlaboratory interest whether the diferent concentrations of lactic and studies[4].However,theminimumCaCl2 concentration to acetic acids in industrial fermentation would be related to tex- retain frmness in the absence of NaCl is unknown, and the ture quality. Lactic acid (as little as 26 mM) has been shown CaCl2 concentration in CaCl2-brined, fermented cucumbers to cause sofening over time in low salt (2% NaCl) conditions 8 Journal of Food Quality

14 13 12 11 10 9 8 7

Average peak force (N) peak force Average 6 5 4 2 2 2 Raw NaCl NaCl CaCl CaCl NaCl product product CaCl Finished Finished Desalted Desalted Fermented Fermented b b a b a c b

CaCl2 NaCl

Figure 3: Mesocarp frmness (N) for cucumbers brined in CaCl2 compared with NaCl at the various stages in processing. Peak force values were signifcantly diferent between brining processes at each stage but were not signifcantly diferent between fermented and desalted stages. Peak forces were signifcantly diferent at � < 0.05 for treatments as signifed by diferent lowercase letters below the �-axis treatment labels.

13 and/or the inclusion of CaCl2 in the fermentation brine mini- 12 mized acid-induced sofening. Only brining process, cucum- 11 ber size, and time in bulk storage were signifcantly infu- encing the texture quality of the commercially fermented cu- 10 cumbers in this study. 9 Two major diferences between the laboratory fermenta- 8 tion in CaCl2 brines that maintained equivalent texture qual- 7 ity and the commercial fermentation in this study are the level Average peak force (N) peak force Average 6 of oxygen exposure and initial brine acidifcation. Both of these factors could infuence the microbiota and biochemical 5 0 50 100 150 200 250 300 changes during the initiation of fermentation. Commercial Bulk Storage Time (days) fermentation is conducted in open-air tanks and air-purging is applied during the active fermentation period. Te addi- CaCl2 fermentation tional oxygen from the air could alter the microbiota, allow- NaCl fermentation ing salt-sensitive, aerobic organisms with pectinolytic activi- Figure 4: Diferences in mesocarp frmness associated with in- ties to infuence product quality of CaCl2 brined cucumbers. creased time spent in bulk storage. Each data point represents the Te fermentation microbiota as well as enzymatic and nonen- average mesocarp frmness for a 30-cucumber sample from each zymatic sofening activities in cucumber tissue could also independent commercial cucumber fermentation tank. be infuenced by the lack of initial brine acidifcation in the commercial CaCl2 brines. Te greater availability of oxygen in combination with higher levels of calcium may also induce oxidative sofening. Tere is some evidence that calcium can act indirectly as a prooxidant by preferentially binding to nat- with higher concentrations of lactic acid causing greater ural chelators, making them unavailable to bind prooxidant sofening [31]. In this commercial study, lactic acid concen- metals such as [34]. Further research is needed in two key trations varied between CaCl2 brined cucumbers (92.22 ± (1) ± areas: to determine the infuence of oxygen and initial 17.83 mM) and NaCl brined cucumbers (111.7 12.8) during brine acidifcation on the microbiota of low or no salt fermen- bulk storage, but the frmness of fermented cucumbers was tation during the frst few days afer brining; and (2) to inves- �= not correlated to lactic acid concentration (CaCl2: tigatethecombinedefectsofoxygen,sodium,andcalcium 0.2071 � = 0.4636 ,NaCl: ) or acetic acid concentration on chemical and biochemical reactions during cucumber (CaCl2: � = 0.3647;NaCl:� = 0.1577). Calcium has previ- fermentation that impact product quality. ously been shown to minimize the sofening induced by acid No degradation or enhancement of texture quality was [32, 33]. Although sofening has been shown in fresh pack observed during desalting. Desalting did not signifcantly cucumbers at the concentrations of lactic acid in these fer- afect the mesocarp frmness for either brining process (� = mentation brines, it is possible that the fermentation process 0.2891). Te inclusion of alum in desalting water has been Journal of Food Quality 9 shown to increase the frmness of fermented cucumbers, but showedamarkedincreaseintheintensityofthemesocarp this has been found to be insignifcant if 0.3% CaCl2 is in- color (� < 0.0001,Figure7)thatmaskedsomeofthevariabil- cluded in the fermentation brine [35]. At least 0.3% CaCl2 was ity in appearance of the fermented cucumbers. Te cucum- included in the fermentation brine of both treatments, so it is bers fermented in NaCl brine also had signifcantly higher not surprising that the mesocarp frmness of desalted cucum- chroma values than their counterparts in the CaCl2 brine bers was not signifcantly diferent from the fermented cu- (� = 0.0269); however, this diference was no longer statis- cumbers. It is also reassuring that no further degradation in tically signifcant afer the desalting procedure (� = 0.1807). texture quality occurred at this stage of processing. Color changes occurring during cucumber fermentation Finishedhamburgerdillchipproductsfrombothbrining are primarily a result of the degradation of chlorophyll. Te treatments were considerably lower in mesocarp peak force degradation of chlorophyll to pheophorbide has been pro- (�) than the fermented and desalted cucumbers. All raw, posed to occur in plant products by two pathways, through fermented, and desalted samples were obtained as whole pheophytin as an intermediate or through chlorophyllide as cucumbers and cut manually by a hand operated food slicer an intermediate [39]. Color changes in cucumbers exposed to to slices measured to be 6.7 ± 0.1 mm. However, the fnished acidic brines have been shown to occur by the production of products were cut into crinkle cut chips by commercial slicers a combination of pheophorbide and pheophytin pigments as is sold on store shelves. Te commercial slicers were less [40]. Tis degradation of brightly colored green chlorophyll precise and produced thinner chips that were 3.8 ± 0.7 mm to pheophorbide and pheophytin produces a dull olive- thick. Tompson et al. [19] found that slices less than 3.2 mm green or olive-yellow color in food products [41]. Fermenting had signifcantly reduced peak force measurements, but slice cucumbers have very small concentrations of chlorophyllide, thicknesses between 4.8–9.5 mm did not signifcantly afect but pheophytin accumulates in large concentrations, so it has peak puncture force measurement. Since most of the fnished been modeled that the accumulation of pheophorbide occurs product slices were between 3.2 mm, where the efect of predominantly using chlorophyllide as an intermediate, while thickness on frmness is known to be signifcant, and 4.8 mm, the pathway from pheophytin to pheophorbide is rarely used the range where the efect of thickness on frmness is known [39, 42]. At the beginning of fermentation in fresh, nonacidi- to be insignifcant, it is unclear whether slice thickness may be fed brines, the pH is neutral, allowing the enzymatic con- infuencing the lower instrumental frmness of fnished prod- version of chlorophyll to chlorophyllide by chlorophyllase to ucts. An alternate explanation for the overall lower frmness dominate the degradation reactions. Once the pH is lowered 2+ of fnished products could include the sofening of fnished by fermentation, the acid causes the loss ofmagnesium (Mg ) products during the pasteurization process. Higher pasteur- from the center of the chlorin ring of the remaining chloro- ization temperatures and longer processes have been previ- phyll or newly formed chlorophyllide to produce pheophytin ously correlated with sofer fnished product [36]. Despite and pheophorbide, respectively. Tis results in chlorophyll the overall reduction in mesocarp frmness in the fnished being converted to pheophytin directly or being converted to products, the cucumber pickles fermented in NaCl brine were pheophorbide from the intermediate chlorophyllide, but the still signifcantly frmer than those fermented with the CaCl2 high acid environment makes the fnal concentration of chlo- process by 1.6 N. Instrumental mesocarp frmness values are rophyllide minimal. well correlated with sensorial measurements of frmness, Te proposed mechanism was for nonacidifed brines; crispness,hardness,andcrunchiness[3,19,20,37,38].Wilson however, commercial production of fermented cucumber et al. [15] found signifcant diferences in hedonic texture lik- pickles currently utilizes recycled acidic brines. Tis acid still ingforhamburgerdillchipsproducedfromcucumberspre- must equilibrate into the cucumbers to degrade their chloro- served by NaCl and CaCl2 fermentation processes that were phyllmolecules,buttheacidifedbrineswouldlikelyresult 1.1 N diferent in instrumental frmness values. Terefore, it in less product proceeding through the enzymatic process is likely that a consumer would notice the 1.6 N diference in to form chlorophyllide before acid reactions became more frmnessvaluesforpickleproductsfromtheNaClandCaCl2 favorable. Te brines for the CaCl2 process were not acidifed fermentation processes. and would more closely follow the previously detailed mech- anism. Tis could, therefore, result in a higher ratio of pheo- 3.3. Mesocarp Color. Cucumber mesocarp color was evalu- phorbide: pheophytin in the cucumbers undergoing CaCl2 ∗ ∗ ∗ ated by colorimetric analysis using � � � color space and fermentation process. It is worth noting that pheophytin can converted to hue and chroma values. Cucumbers fermented also be converted to pheophorbide by chlorophyllase, but this with the CaCl2 process had signifcantly higher hue values reaction has not been measurably observed in fermenting compared to those fermented in NaCl brines at each stage in cucumbers [39, 42]. processing. Fermented cucumbers with average hue, chroma, In this study, the mesocarp chroma (intensity of the color) ∗ and � values very close to the means for each treatment was lower with longer time spent in bulk storage and was are shown in Figure 5 to illustrate the signifcant diference generally lower in cucumbers fermented in CaCl2 brines in appearance. Te diferences in hue and chroma caused by (Figure 8). Tis decrease in chroma corresponds with the idea brining treatment at diferent processing stages can be seen in that the pigments may be degrading with greater exposure Figures 6 and 7, respectively. Chroma was signifcantly higher time to sunlight and oxygen. While the opaque tanks and the in the fnished product for each treatment than in the fer- use of wooden boards to submerge the cucumbers minimize mented and desalted stages. Chroma measures the intensity the exposure of the cucumbers to sunlight, the cucumbers are of a color and the addition of yellow 5 dye to the cover liquor exposed during the frst day, while tanking and the ultraviolet 10 Journal of Food Quality

CaCl2 process L∗ 42.71 NaCl process : ∗ Hue: 107.75 L : 41.18 Chroma: 6.45 Hue: 101.19 Chroma: 10.89

Figure 5: Fermented cucumbers representative of the average color of each fermentation process. Te average hue value for the cucumbers in the CaCl2 fermentation process was signifcantly higher than the average hue of the cucumbers in the NaCl fermentation process (� < ∗ 0.05). Te chroma values were also signifcantly diferent (� = 0.0269),butthe� values were not signifcantly diferent from each other (� = 0.9432).

135 34 130 30 125 26 120

) 22 ∘ 115 110 18

105 Chroma 14 100 Hue angle ( angle Hue 10 95 6 90 85 2 2 2 2 2 2 2 NaCl NaCl CaCl CaCl NaCl NaCl CaCl CaCl NaCl product product CaCl NaCl Finished Finished Desalted Desalted product product CaCl Finished Finished Desalted Desalted Fermented Fermented Fermented Fermented b d a cd b c cbc bc a a

CaCl2 CaCl2 NaCl NaCl

Figure 6: Mesocarp hue for cucumbers brined in CaCl2 and Figure 7: Mesocarp chroma for cucumbers brined in CaCl2 and cucumbers brined in NaCl at the various stages in processing. Hue cucumbers brined in NaCl at the various stages in processing. is measured as the degrees from red on a polar coordinate system Chroma is measured radially from the pole and indicates the inten- ∘ ∘ with 90 being in the yellow direction and 180 being in the green sity of the color. Chroma values were signifcantly diferent at �< direction. Hue values were signifcantly diferent at � < 0.05 for 0.05 for treatments signifed by diferent lowercase letters below the treatments as signifed by diferent lowercase letters below the treat- �-axis. ment labels on the �-axis.

overall lower chroma value of the cucumbers fermented in (UV) light from the sun would penetrate the surface of the CaCl2 brines (Figure 8). Tey also found that lightening of brine. Eisenstat and Fabian [43, 44] used salt stock of 16% cucumber color increased from 1 to 6 weeks in the 3.8% salt and desalted stock of 3.8% salt to show that direct sunlight desalted cucumbers. More recently, Buescher and Hamilton ∗ ∘ ∘ caused severe bleaching (loss of characteristic color) in a [45]foundanincreasein� value by 10 and a 5 (100 to 95 ) matter of hours unless the cucumber pickles were completely change toward yellow in hue value for cucumbers fermented submerged in the 16% salt stock brine. Te process of flling under incandescent lights compared to those fermented in a fermentation tank with cucumbers would result in a short the dark in brines composed of 5% NaCl, 0.35% CaCl2, period of sun exposure time, and it may be enough to initiate 0.1% acetic acid, and 0.05% potassium sorbate. Additionally, photooxidation.EisenstatandFabian[44]alsofoundthat Guillou et al. [46] found the hue of cucumbers fermented exposuretoCaCl2 could cause slight bleaching of cucumbers in 5% NaCl, 0.2% CaCl2, and 0.2% potassium sorbate to be ∘ ∘ in jars exposed to difused light which is consistent with the between 105 and 125 during 18 weeks (126 days) of storage Journal of Food Quality 11

16 fermentation and to identify process changes or controls that 14 will improve the quality of cucumbers fermented and stored 12 with reduced salt. 10 Disclosure 8

Chroma 6 Mentionofatrademarkorproprietaryproductdoesnot 4 constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or North Carolina Agricultural 2 Research Service nor does it imply approval to the exclusion 0 of other products that may be suitable. USDA is an equal 0 50 100 150 200 250 300 opportunity provider and employer. Bulk storage time (days)

CaCl2 fermentation Conflicts of Interest NaCl fermentation Figure 8: Diferences in mesocarp chroma associated with greater Te authors declare no conficts of interest. length of time spent in bulk storage for fermented cucumbers brined in CaCl2 or NaCl. Acknowledgments Tis study was funded by Mount Olive Pickle Company under 254 nm UV lights, while chroma decreased from 20 to (Mt. Olive, NC) and the USDA-ARS Food Science Research 7.Tishueisgreenerthanthehueofthesecommerciallyfer- Unit (Raleigh, NC) with partial support from Pickle Pack- mented cucumbers, but the decrease in chroma matches our ers International (Washington, DC). Te authors gratefully observed values fairly well (Figure 8). Eisenstat and Fabian acknowledge Bay View Foods (Pinconning, MI), Gedney [43, 44] found diferences in observed bleaching defects Foods (Chaska, MN), Mount Olive Pickle Company (Mt. depending on the light source: direct sunlight, an IR lamp, Olive, NC), Hartung Brothers (Imlay City, MI and Bowling or a 210–328.7 nm UV lamp. Terefore, it is possible that Green, OH), and Swanson Pickle Company (Ravenna, MI) there are multiple mechanisms occurring that afect the color for conducting commercial scale fermentation in CaCl2 of fermented cucumber. Further research to identify the brines and providing samples for quality evaluation. Tey also mechanisms of color changes during fermentation and other thank Mrs. Sandra Parker for excellent administrative assis- products of photooxidation reactions could lead to the pos- tance and editorial review of the manuscript. Tis manuscript sibility of manipulating chlorophyll degradations to enable isbasedonaportionofthemaster’sthesisresearchconducted reduction or elimination of yellow 5 in cucumber pickle byErinK.McMurtrieunderthedirectionofDr.SuzanneD. products. Johanningsmeier at North Carolina State University, Raleigh, NC [47]. 4. Conclusions References Commercialization of the CaCl2 fermentation process ena- bles elimination of sodium and an 80% reduction of chlorides [1] United States Environmental Protection Agency, “National in wastewaters. Tis study found that the color and texture primary drinking water regulations,” EPA 816-F-09-004, 2009. quality were compromised when this process was imple- [2] R. F. McFeeters, W. Coon, M. P. Palnitkar, M. Velting, and N. mented in a typical commercial environment using open Fehringer, “Reuse of fermentation brines in the cucumber pickl- ing industry,” Cincinnati, Ohio; Springfeld, Va.: EPA-600/2-78- tanks and air-purging. Te quality changes occurred during 207 Environmental Protection Agency, 1978. fermentation and bulk storage and remained evident in the desalted cucumbers and fnished pickle products. It appears [3] I. M. Perez-D´ ´ıaz, R. F.McFeeters, L. Moeller et al., “Commercial Scale Cucumber Fermentations Brined with Calcium Chloride that no further degradation of quality takes place in the Instead of Sodium Chloride,” Journal of Food Science,vol.80, later processing steps with the possible exception of the no.12,pp.M2827–M2836,2015. efects of pasteurization on texture. Signifcant evidence of [4]R.F.McFeetersandI.Perez-D´ ´ıaz, “Fermentation of cucumbers photooxidation was apparent in the CaCl2 brined, fermented brined with calcium chloride instead of sodium chloride,” cucumbers, and there was a reduction in texture quality that Journal of Food Science,vol.75,no.3,pp.C291–C296,2010. could not be explained by compositional variation. Bloater [5] H.P.Fleming,R.L.Tompson,J.L.Etchells,R.E.Kelling,andT. defects were related to the minimal air-purging routine with A. Bell, “Bloater formation in brined cucumbers fermented by the CaCl2 process, but implementation of typical purging lactobacillus plantarum,” Journal of Food Science,vol.38,no.3, routines in CaCl2 brines resulted in reduced bloater defects, pp. 499–503, 1973. complete fermentation, and stable bulk storage, so the min- [6] R.N.Costilow,C.L.Bedford,D.Mlngus,andD.Black,“Purging imal purging routine may not be necessary for future use of of natural salt-stock pickle fermentations to reduce bloater this process. Additional research is warranted to understand damage,” Journal of Food Science, vol. 42, no. 1, pp. 234–240, the tissue sofening observed in the CaCl2 brined cucumber 1977. 12 Journal of Food Quality

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