IIIIIIIIIIIIIIIIIIIIIIIIII IIEIIIIIIIIIIIIIIII 00084 3643 THE” - m-r. armrmm- mm; 9 u .. an E- .1“ n? )1; ii 33" L! ...~.. .3 .‘ 7‘“; 7. Mix: W” 31 Sam .U;« a, Y W; M‘M “2M see~umr~ This is to certify that the thesis entitled AlR-PURGING 0F SALT-STOCK CUCUMBER FERMENTATIONS T0 CONTROL BLOATER (HOLLOW PICKLE) SPOILAGE presented by KAREN CHRISTINE GATES has been accepted towards fulfillment of the requirements for M.S. BIOLOGY degree in Jam/Y KZM Major professor Date 2-25-82 0-7639 MSU RETURNING MATERIALS: P ace in boo rop to remove this checkout from LIBRARIES . F . m your record. FINtS W111 be charged if book is returned after the date stamped below. n‘ ‘M '- ‘I‘lril. . 9- 193 AIR-PURGING OF SALT-STOCK CUCUMBER FERMENTATIONS TO CONTROL BLOATER (HOLLOW PICKLE) SPOILAGE by Karen Christine Gates A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Interdepartmental Biology 1981 ABSTRACT AIR-PURGING OF SALT-STOCK CUCUMBER FERMENTATIONS TO CONTROL BLOATER (HOLLOW PICKLE) SPOILAGE by Karen Christine Gates Comparison of eighteen air—purged salt—stock pickle fermentations with those nitrogen purged showed air-purging as effective in preventing bloater formation. No significant effects of the various treatments were observed. In two tanks, some softening had occurred directly under the purger outlet where disolved oxygen concentrations were highest. In laboratory studies, cucumber fermentations were purged at high air flow rates to study factors influencing softening in cucumbers and treatment which might prevent softening. These results indicated that softening of air— purged cucumbers is caused by some oxygen-dependent, sorbic acid—sensitive microorganism(s) growing in or on the cucumbers. Pectinolytic enzyme activities were not well correlated with the extent of softening. Further laboratory studies showed the causative organism(s) to be mold growing in the brined cucumbers. DEDICATION To Charlie, Scott and Amy for their support and understanding and to Ralph Costilow for his incredible patience. ii ACKNOWLEDGMENTS I wish to express my sincere gratitude to my major professor, Dr. Ralph N. Costilow, for his guidance through- out the course of this investigation and the preparation of this thesis. I also wish to thank Dr. Melvyn L. Lacy for use of laboratory facilities and his advice and consul. Thanks to Green Bay Foods Co., Eaton Rapids, MI for providing cucumbers, H. J. Heinz Co., Lakeview, MI for use of facilities and the Department of Food Science and Human Nutrition, Michigan State University, for the use of their facilities. This research was partially financed by the Industry Division of the Ad Hoc Pickle Research Committee for Michigan State University, Pickle Packers International, Inc., St. Charles, IL. iii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . Vi LIST OF FIGURES . . . . . . . . . . . . . . . . . viii INTRODUCTION. . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . 4 MATERIALS AND METHODS . . . . . . . . . . . . . . 7 Commercial Fermentations. . . . . . . . . . . . 7 Fermentations Studied. . . . . . . . . . . . 7 Analysis . . . . . . . . . . . . . . . . . . 8 Laboratory Fermentations. . . . . . . . . . . . 10 Microbiological. . . . . . . . . . . . . . . 10 Cucumber Fermentations . . . . . . . . . . . ll Non-fermented Brined Cucumbers . . . . . . . 12 Pectinase Assays . . . . . . . . . . . . . . 13 Evaluation of Fermented Cucumbers. . . . . . 14 RESULTS . . . . . . . . . . . . . . . . . . . . . 15 Commercial Fermentations. . . . . . . . . . . . 15 Effect of Various Schedules for Air-purging on Quality of Salt-Stock . . . . . . . . . . 15 Dissolved Oxygen in Brines . . . . . . . . . 20 Laboratory Fermentations. . . . . . . . . . . . 27 Effect of Aeration Rate and Initial Acidification. . . . . . . . . . . . . . . . 27 Effect of Salt Concentration . . . . . . . . 35 Effect of Initial Limitation of Oxygen . . . 35 iv TABLE OF CONTENTS (cont'd.) RESULTS (cont'd.) Page Laboratory Fermentations (cont'd.) Effect of Blanching Fresh Cucumbers. . . . . . 43 Microbes Isolated From Brines. . . . . . . . . 43 Development of Pectinolytic. . . . . . . . . . 45 Molds in Brined Cucumber Tissues . . . . . . . 47 Identity and Softening Activity of Molds Isolated From Cucumber Tissues . . . . . . . . 54 DISCUSSION. . . . . . . . . . . . . . . . . . . . . 56 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 64 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . 65 Table LIST OF TABLES Page INFLUENCE OF VARIOUS PURGING TREATMENTS ON THE QUALITY OF SALT-STOCK AND THE MAXIMUM AND MINIMUM C02 LEVELS IN THE BRINES. . . . . . l6 PRESSURE TESTS OF SALT-STOCK REMOVED FROM DIFFERENT AREAS OF TANK NO. 19 WHICH HAD BEEN PURGED WITH AIR. 0 O O O O O O O O O O O 0 l8 COMPARISON ON AVERAGE SALT-STOCK QUALITY FROM TANKS PURGED WITH NITROGEN AND WITH AIR 0 O O O O O O C O O O O O O O 0 O O O O 0 O 2]- EFFECT OF AIR FLOW RATE ON DISSOLVED OXYGEN CONCENTRATION IN BRINE FROM DIFFERENT AREAS IN THE TANK . . . . . . . . . . 22 EFFECT OF ACIFIFICATION AND OF AERATION RATES ON ACID DEVELOPMENT AND SOFTENING (PRESSURE TEST) OF BRINED CUCUMBERS . . . . . . 28 EFFECT OF SALT CONCENTRATION ON THE DEVELOPMENT OF ACID AND PECTINASE ACTIVITY IN THE BRINE, AND ON SOFTENING AND EXTENT OF CURE OF BRINED CUCUMBERS. . . . . 36 EFFECT OF INITIAL NITROGEN PURGING ON THE DEVELOPMENT OF ACID AND PECTINASE ACTIVITY IN THE BRINE, AND ON SOFTENING AND CURING OF BRINED CUCUMBERS . . . . . . . . . . . . . . 38 EFFECT OF BRINE STRENGTH AND INITIAL NITROGEN PURGING ON ACID AND PECTINASE ACTIVITY DEVELOPMENT IN THE BRINE, AND ON SOFTENING AND EXTENT OF CURE OF BRINED SUCUMBERS . . . . . . . . . . . . . . . . . . . 39 vi LIST OF TABLES (cont'd.) Table 9. 10. ll. 12. 13. 14. EFFECT OF PARTIAL AIR LIMITATION, CON- TROLLED FERMENTATION, AND ADDITION OF POTASSIUM SORBATE ON DEVELOPMENT OF ACID AND PECTINASE IN BRINES AND ON SOFTENING OF THE CUCUMBERS. . . . . . . . . . . . . EFFECT OF BLANCHING CUCUMBERS PRIOR TO BRINING ON SOFTENING DURING FERMENTATIONS WITH AIR-PURGING o o o o o o o o o o o o PECTINASE ACTIVITIES IN BRINES FOR AIR- PURGED CUCUMBER FERMENTATIONS AT VARIOUS PERIODS AFTER BRINING AND IN SHAKE-FLASK BRINE CULTURES INOCULATED WITH FERMENTING BRINES. . . . . . . . . . . . . . . . . . EFFECT OF SPENT BRINES FROM AIR-PURGED FERMENTATION OF DIFFERENT BRINE STRENGTHS ON THE PRESSURE TEST OF NON-FERMENTED BRINED CUCUMBERS. . . . . . . . . . . . . AVERAGE PRESSURE TESTS OF CUCUMBERS EQUILIBRATED AND STORED IN BRINES AT VARIOUS SALT CONCENTRATIONS WITH AND WITHOUT HIGH LEVELS OF PECTINASE ENZYME(S) ACTIVITY. . . . . . . . . . . . PRODUCTION OF PECTINASE ACTIVITY AND SOFTENING OF CUCUMBERS IN BRINES BY MOLDS ISOLATED FROM SURFACE-STERILIZED SLICES OF CUCUMBERS FROM AIR-PURGED CUCUMBER FERMENTATIONS . . . . . . . . . . . . . . vii Page 42 44 46 48 49 55 Figure LIST OF FIGURES Page RATES OF DEPLETION OF DISSOLVED OXYGEN FROM AIR-PURGED CUCUMBER BRINES AT VARIOUS FERMENTATION AGES. . . . . . . . . . . . . . 25 AVERAGE DISSOLVED OXYGEN LEVELS AND AVERAGE YEAST COUNTS IN SALT-STOCK PICKLE BRINES . . . 3O EFFECT OF AERATION RATE AND OF INITIAL ACIDIFICATION ON THE DEVELOPMENT OF PECTINASE ACTIVITIES IN CUCUMBER BRINES. . . 33 MOLD HYPHAE IN THE TISSUE OF SOFT CUCUMBERS FROM AIR-PURGED FERMENTATIONS. . . . . . . . 51 CULTURES ON 2% AGAR AND POTATO DEXTROSE AGAR OF SLICES OF SURFACE STERILIZED CUCUMBERS FROM FERMENTATIONS PURGED FOR 5 DAYS . . . . . . . . . . . . . . . . . . . 52 CULTURES OF SLICES FROM SURFACE STERILIZED CUCUMBERS FROM AIR-PURGED FERMENTATIONS. . . 53 viii INTRODUCTION Etchells et_gl. (9) and Fleming gt El° (l2) demon- strated that nitrogen purging of controlled fermentations of brined cucumbers greatly reduced the formation of bloaters (hollow pickles). Subsequently, it was found that equally satisfactory results could be obtained with natural fermentations by continuously purging them with nitrogen at 20 ft3/h using an apparatus designated as a sidearm purger (5). This system is now widely used in commercial fermenta- tions. Use of air instead of nitrogen would greatly reduce the cost of purging. Fleming 33 a1. (12) reported that continuous air-purging of fermentations in 5 gal pails at air flow rates of 25 to 100 ml/min resulted in soft and off-colored pickles. However, intermittent air-purging at even 425 m1 of air/min for 2 h/day did not result in soft pickles. Purging of pails from the bottom is not comparable to purging large tanks with a sidearm. Even at 25 ml/min, the air flow rate per unit volume of brine in a 5 gal pail is approximately 5X that when 20 ft3/h air is used in a 10,000 gal tank. In purging from the bottom, the cucumbers are in direct contact with the cucumbers and gas bubbles may be entrapped in the cucumber mass; this does not occur with the sidearm purger. The softening observed in air-purged laboratory fermentations was believed to have been caused by molds .growing on the brine surface along with oxidative yeasts (9). This Could not be true with most commercial tanks, however, since their surfaces are exposed to sun and no films of microbes develop on them. In addition, if the softening resulted from pectinolytic or cellulolytic enzymes produced in the brine, essentially all of the cucumbers in the tank would be expected to become soft. This does occur when high levels of these enzymes are present (3,7); how- ever, it has not been reported in air-purged commercial tanks. The principle objectives of this investigation were: 1. To determine, under properly controlled condi- tions, if air could be substituted for nitrogen as a purging gas and to measure the amounts of dissolved oxygen in the brine. To study factors which influenced softening in fermentations at high air flow rates, and to investigate possible procedures to prevent such spoilage. Determine what microorganisms are responsible for softening of brined cucumbers during air- purging. LITERATURE REVIEW Bloaters (hollow pickles) are believed to be caused by CO diffusion into the cucumber tissues from the brine 2 (11,12,13) and can be prevented by purging of the brines with nitrogen (12). Costilow et_al. (5) described a purg- ing system using an apparatus designated as a sidearm purger which circulates the brine and purges it with nitrogen. This purging system is widely used in the pickle industry and has dramatically reduced bloater-type spoilage. A significant cost savings to industry could be achieved if air could be used in place of nitrogen as the purging gas. Fleming gt El' (12) reported continuous air-purging of fermentations in 5 gal pails at air flow rates of 25 to 100 ml/min resulted in soft and off—colored pickles. How- ever, intermittent air-purging at even 425 ml of air/min for 2 h/day did not result in soft pickles. Even at 20 ml/min, the air flow rate per unit volume of brine in a 5 gal pail is approximately 5 times that when 20 ft3/h air is used in a 10,000 gal commercial tank. The purging units in the pails, unlike those in commercial tanks, are located in the bottom allowing direct contact of the gas bubbles with the cucumbers and gas bubbles may become entrapped in the cucumber mass. Costilow 35 31. (6) reported air—purging in nine commercial tanks. Because of difficulty with an interrupted electrical supply and breakdown problems with air compres- sors, the air supply was shut off for variable and unknown periods of time during the nights. When the salt-stock was evaluated, there were no significant differences between the air-purged tanks and the nitrogen-purged controls. There was no evidence of softening, bleaching, or off- coloration of the pickles from any of these fermentations. The lactic acid fermentations deve10ped normally in all tanks. Potential adverse effects on quality factors such as texture and appearance are not the only converns in using air as a purging gas (12,13). The rate of acid development (titratible acidity as lactic acid) has also been demonstrated to develop more slowly resulting from a lower growth rate of lactic acid bacteria (16) in air- purged fermentations. This could indicate an increase in a competitive advantage of yeasts and other nondesirable aerobic microorganisms. Oxygen is supplied for these organisms from the air being circulated in the brine and from the fresh tissues in which it is dissolved. Large cucumbers were found to contain 45-50 ul gas/gram in the tissues at brining. The gas contained about 23% oxygan (15), however, the amount of oxygen in the tissues at brining would vary with the way the fruit had been handled. Softening of cucumbers purged with air has been reported to occur directly under the purger outlet (19) where dissolved oxygen would be expected to be highest. Polygalacturonase has been shown to be a major factor in breakdown of pectic substances and high pectinolytic acti- vity in the brine can cause loss of texture during storage (2). Softening due to storage in brines with high pectinolytic activity is retarded by addition of calcium chloride (4). MATERIALS AND METHODS Commercial Fermentations Fermentations Studied The 20 commercial cucumber fermentations studied were brined at the H. J. Heinz Company station at Lakeview, MI. All tanks were 8 ft. high x 14 ft diameter (ml,000 bu capacity), and were filled with size 3A (1.5 x 1.75 in. diameter) cucumbers unless noted. They were all brined according to plant procedures. The cucumbers were covered with 45° salometer brine acidified with about 0.05% acetic acid. Sufficient dry salt was added to each tank to equil- ibrate at 25 to 28° salometer. After the acidity in the brines had attained about 0.6% lactic acid, the brine strength was slowly increased to 45—50° salometer. The standard sidearm purger equipped with a 2 in. x 12 in. ceramic gas sparger (5) was used for sparging all commercial tanks. Gas flow rates were controlled by use of Brooks gas flow meters, Model 1510B, with a capacity of 5-45 standard cubic ft/h (scfh) (Brooks Instrument Division, Emerson Electric Co., Hatfield, PA). With all tanks, the tubes to the spargers were equipped with quick disconnects and needle valves. Gas flow rates were checked and adjusted at least daily by insertion of a flow meter into the line. This procedure results in actual gas flow rates somewhat higher than measured with the flow meters in the line. Therefore, the actual gas flow rates in those tanks were probably 2-3 ft3/h higher than recorded. A high capacity, heavy duty air compressor equipped with an effi— cent air dryer was used for an air supply. Purging was discontinued when the brine samples were found to have less than 0.05% reducing sugar as determined by the quality con- trol personnel of H. J. Heinz Company. On an average, these tanks were purged for 9 days. Analysis Brine samples from the air-purged and from two nitrogen-purged tanks were plated on dextrose agar (Difco) acidified with 2 ml of 5% tartaric per 100 ml in attempts to isolate molds or unusual yeasts. Aliquots (0.1 ml) of serial dilutions of the brines were spread on pre—poured plates within 4 h after removing from the tanks. Dissolved oxygen concentrations were determined with a dissolved oxygen meter YSI Model 57 (Yellowsprings Instru- ment Co., Inc., Yellow Springs, OH) equipped with an oxygen probe, YSI 5739, and submersible stirrer, YSI 5791A. The instrument was calibrated daily in air-saturated brine at the same salometer (:2°) and temperature as the fermenting brines which were to be measured. Consistent meter read- ings of 0.1 to 0.3 ppm oxygen were obtained with brines in nitrogen-purged tanks which should have no dissolved oxygen in them. Therefore, all readings were corrected by -0.2 ppm. Measurements made in brine near the surface of tanks (above the headboards) were made by submerging the probe and stirrer in the brine in the tank. Center of the tank determinations were made by insertion of a stainless steel tube to the approximate geometric center of a tank and siphoning brine into the bottom of a tall 1-1iter beaker containing the probe and stirrer. The beaker was allowed to overflow until at least 1 liter of brine had passed through it before the dissolved oxygen reading was taken. Brines were analyzed daily for titratable acidity, salometer, and CO concentrations. The CO was measured 2 2 as described previously (5). 10 The salt—stock pickles were evaluated 2-3 months after brining. Routinely, samples were taken from near the surface of the brine directly under the purger outlet. Samples of 100 pickles were sliced lengthwise and evaluated for bloater damage, and other defects, and 10 pickles were tested for firmness by puncturing with a USDA fruit pres- sure tester equipped with a 5/16 in. tip (2). Personnel of the H. J. Heinz Co. conducted the evaluation of the pickles and used the evaluation procedure of Shoup et_§l. (18). Laboratory Fermentations Microbiological Dilutions of brine samples of cucumber fermenta- tions were plated on dextrose agar (Difco) acidified with 2 ml of 5% tartaric acid per 100 ml to isolate yeasts and molds; on nutrient agar (Difco) to detect aerobic non- fastidious bacteria such as the endospore producers; and on trypticase soy broth (BBL) plus 1% dextrose and 0.2% yeast extract with the pH adjusted to 5.6 to detect more fastidious bacteria. 11 Thin sections of cucumber tissues (10-30 pm in thick- ness) were sliced by use of a Hooker microtome (Labline Instruments, Inc., Melrose Park, IL). They were mounted in water on a microscope slide under a coverslip, and examined with a light microscope at 100x magnification. Photomicro- graphs were taken with a 35 mm camera. To culture molds from the tissue, cucumbers were surface sterilized by immersion for 10 min in a 10% v/V solution of household bleach (Chlorox, $5.5% hypochlorite). Using Chlorox-treated forceps, tweezers, and scalpels, cross-cut slices about 2-3 mm in thickness were cut from near each end and the center of the cucumber. All three slices were pressed onto the surface of agar medium in a petri dish and incubated at 30°C for 5 days. Both 2% agar in water, and potato dextrose agar (Difco) were used as cultural media. Mold cultures isolated from cucumber slices were isolated and maintained on potato dextrose agar. Cucumber Fermentations The cucumbers (size 3A, 3.8-4.5 cm diameter) used in this study were obtained fresh from the Green Bay Food Co., Eaton Rapids, MI. They were packed in 1 gal glass jars fitted with a gas sparger (3/4 in. diameter, 2.5 in. 12 long) made of ultra high molecular weight polyethylene (10-20 pm pore size). A l in.-wide strip of semi-rigid polyethylene was placed under the neck of the jars to hold the cucumbers under the brine. Salt brines of the desired equilibrated salometer were poured over the cucumbers. The .9 cucumber/ml brine ratios varied from 55/45 to 60/40. Dry salt necessary for equilibration at the desired salometer was added in three equal amounts over a period of 1 day. Other additions were as indicated in the results. The con— trolled fermentation was established according to the pro- cedure of Etchells 32 El- (9). The brined cucumbers were incubated under ultra— violet light at room temperature (ZS-30°C). Unless stated otherwise, they were purged continuously with air at ~20 ml/min. Air flow rates were controlled by use of gas flow meters (Brooks Instrument Division, Emerson Electric Co., Hatfield, PA) having maximum air flow rates of 146 ml/min. Non—fermented Brined Cucumbers Cucumber brines and brined cucumbers were prepared by equilibration of cucumbers in brines in l-qt jars with enough salt added to achieve the desired final salometer. All were acidified with 0.04% acetic acid. After addition 13 of toluene, the jars were sealed, and stored in a refriger— ator for a minimum of 2 weeks before use. Equilibrated brines used for shake—flask cultures were pasteurized in a steamer for 15 min. Brined cucumbers to be inoculated with various isolated microorganisms were all equilibrated at 25° salometer and 0.04% acetic acid. For individual tests, 1 brine-equilibrated cucumber was placed in each l-qt jar and 50-100 ml of the equilibrated brine added. This resulted in partial submersion of the cucumber. The jars were placed in a steamer for 15 min before inoculation. Pectinase Assays Brines were assayed for pectinolytic enzyme activi— ties by the procedure of Bell 3: a1. (1), except that viscosity measurements were made with Ostwald pipettes having an upper bulb capacity of m3.8 ml. The flow time for water through these pipettes was about 1 min. Four ml of sodium polypectate solution and 1 m1 of dialyzed brine sample were used in each pipette. The percentage losses in viscosity were measured after 20-22 h incubation at 30°C. There is considerable variability in these assays, and any value less than 10% is of doubtful significance. 14 Evaluation of Fermented Cucumbers All of the pickles were removed from each jar and pressure tested with a USDA fruit pressure tester (5/16 in. tip) (2) 14 days after brining unless stated otherwise. They were then sliced lengthwise, observed for any ususual color changes, and the percent cure estimated. The latter was based on the percentage of the tissue which had lost its white color. RESULTS Commercial Fermentations Effect of Various Schedules for Air-purging on Quality of Salt-Stock The purging treatments used, the quality of the salt-stock pickles, and the highest and lowest concentra- tions of CO2 observed in each tank are recorded in Table l. Bloater control was excellent in all tanks even through maximal CO2 concentrations greater than 70 mg/100 ml were observed in three of the tanks (Nos. 9, l7, and 18) during the fermentation. Some softening was observed in two tanks. In one of these tanks, No. 13, a number of pickles in the first sample taken were found to have soft blossom ends. However, this was obviously restricted to a very small area of the tank since no evidence of softening was found in three additional samples from the same general area. Softening was more extensive in tank No. 19, but was restricted to pickles in the purger outlet side of the tank (Table 2). 15 l6 4N mm NH am am «a N smc\ua G NH ms ma «a me NH 2 Baas smomammu Baa ON me ma mm mm NH N smw\ua m ma me as am am NH 2 spas omomammu “Ha 4H Nm NH mm mm OH smcxnn m ma as SH vm mm a Loo amass» Mus «m we as mm em m sms\un m mN ms ma Na Hm 5 who wmcusu “Na NH mm ma Nm es s smw\un N mN N% «a pa mm m mmo wmcusu Ham SH ms mN Ha Na 4 NH NS .. mm mm m nuflm msoscaucoo OH mN ma mm Na N ma ow NH om cm a cmmouuNc msoscapcoo sou swam . umoe >Ho>ooo won om .o m cmE mow HE ooa\mou ma muSmmon w m m MIN moo Mama u p B mxmsmav mmszm may zH mum>mu N 00 EDSHZHS QZ< EDZHx¢Z mmB 92¢ MUOBmlfiqfim m0 MBHQ m0 MUZMDAMZH H mqmfifi l7 .mmmo m pmuflm msflnsw Ho>ma pan“ Umnomms Mann Hospflm: p59 iHmquonm com pm wswun ou omscmHm .mHmEmm ocoomm m mo mpHSmwu ucommummu mHmmnusmHmm CH mnwnasz .m magma mmm m .mmum um0m Eoum mamfimmm U .onEmm Usoomm m CH msos usn .mHmEMm umuflm CH canon mama paw 80mmoan pm mcfismumom £DH3 meonm Hmum>mmo Eoum cmxmu oumB mmaxowm ooa mo moamamm .HmuoEmHo CH m.H|v\H H onB mxcmo 03¢ mmwnu CH mumnfiooso 038 b .ooflm umavso Homnsm map so Mama may mo mou mnu .mmOHHm usolmmono muflamso ooom came» oasoz soans pom mc0flumummmm nonuo no muoxoom mmm mo moum mwz soazz Moogmlpawm mgu mo Haw mo mmwucmouom may musmmmumwu muo>oown usmoumm ompommxw mna .xmav .Nm pm .msoam mo omsomooum map an mammfioo NcHom .h .m onu mo HmGsOmnmm an Umuwsam>m was xoovmluammm ma ma ma mm mm ma mm m¢ mm on mo mm ma mm ABHVNH Amavma ha ma mm mm mm Avmvhm mm mm mm uh om UAvmvmm om mm ON ma ma 5H 0H ma HWMU\HS m HMO UOGHDH Hflfi smw\un N Loo Ham .Hmuofioamm omm smc\us m Loo Ham swap .mwo umnflw Nz 18 Icoo m>ms .oommusm map How: Eonm monOHm oEom omcflmp hoe xcmu mo was: HQBOH Eoum mmamamm .muommumaa .ums m Qua? cmxwu mmamfimmm m.ma Hm ma SH ma m.mH m.wa ma m.ma ma m.ma was: szoq m.hH ma ma m.wH ma m.nH m.>H SH m.oH wa wa xsmu mo mos "opfim ummusm m.HH SH m.mH m.mH m.ma m.ma m.ma Ha m.> m.w m was: Hosoq m.m ma vH ma HH Ha Ha m m.v m m gasp mo mos "woam umauso Hmmnsm $2. 3 m m s m m a m N N cmmflmsmwé saw: x m mummy oHSmmmHm HMDUH>HUGH de EBHB Omwmbm mem Dfim EUHEB mH .OZ MZflB m0 mmmmfl BzmmmmmHQ 20mm Qm>02mm MUOBmlfiqdm m0 mBmWB mMDmmmmm N mqmdB 19 When this tank was emptied the number of pickles affected was estimated to amount to less than 50 bu (17). Of parti- cular interest is the fact that the softening was not uni- form from pickle to pickle even in a very restricted area of the tank. Firm pickles were found adjacent to mushy ones, and a part of a single pickle was frequently soft and the rest firm. There was no evidence that the softening would spread through the tank. No pectinolytic enzyne activity was detectable in the brines (assay conducted by H. J. Heinz Co.). All of these facts suggest that the softening enzyme (5) is produced by an oxygen requiring organism growing on or in the surface tissue of individual cucumbers. Analysis of all the data failed to reveal any dif— ference between tank No. 19 and other airapurged tanks. The dissolved oxygen levels were not higher than normal, the acid fermentation developed normally, the salt concen- tration was not unusually low, and no molds or unusual appearing yeasts were detected by plating of five brine samples during the active fermentation period. Unfortun— ately, pH of the brine was not measured and we cannot be sure that the initial pH was not unusually high. 20 As evident in Table 1, no significant differences in salt-stock quality resulting from the different purging treatments was noted. The salt-stock from the two tanks which were continuously purged with air was excellent, and the soft pickles were from a tank in which the air was turned off 3 h per day. The average quality of the pickles from all 18 air-purged tanks was as good as that for 23 tanks at the same location purged continuously with nitrogen (Table 3). Dissolved Oxygen in Brines The air flow rate has a pronounced effect on the dissolved oxygen levels in brines (Table 4). In some instances, the oxygen concentrations were doubled by increasing the flow rate from 10 ft3/h to 30 or 40 ft3/h (see data for fermentations 2, 4, and 5 days old). As expected, the highest levels were found in brines directly under the purger outlet. However, significant amounts of oxygen were consistently found on the opposite side (purger side), and the concentrations found were also influenced by air flow rate. In most instances, no dissolved oxygen was detectable in samples taken from the center of the tank. 21 .mmOAHm psonmmouo o: oamflw UH503 mmeon mwmsam malm m.mH «.mm ma w.m ma HH< palms o.ms o.Nm ms o.m mN ammouuaz mmsmm smmz mum>oomm w HmMOB mouo>wm mxcms mow puma ousmmoum Umuommxm muommmo Sufi: w mo .02 msflmnsm mMZdB MH< EBHB 92¢ ZmoomBHZ EBHB QMUmDm 20mm WEHAfiDO MUOBmlfiqdm mo¢mm>¢ ZO ZOme4mSOU m mqm.o 0H nmoflm ummusm .QOB mm.m mm.m II mm.m om.N mv.m In I: mo.m om.m as hm.m I: mm.m I: I: II mH.N on.~ II I: om mm.a om.m ow.m mm.H mm.N N.m mo.H I: mo.H mm.m om o>.H on.H oo.N om.a mv.m o.m mm.o om.H mm.o mw.H 0H npwavso Homusm .QOB No 5mm saw: 0H m m n m m v N H Ham xGMB as Ammmtv mom soHDMpsoEme u£\mvm cofluwooq MMZ¢B mmB ZH mfimm< BzmmmMMHQ 20mm HZHmm ZH mZOHefimBZMUZOU mewxo Qm>qommHQ ZO mBaommflo was HHucs ohms 30am Hflm comm pm Ummnsm was mmm COHDMuswEumm swbflm m Mo MCMD mamsflw do - mN.o -- o o o - - o o ow - - mo.o -- I- - o o - - om - os.o o o o mo.o o I- o 0 ON II mo.o o o o o o o o 0 0H Mach mo kucwo 24 There was a trend for the oxygen concentration to increase as the fermentations progressed in age. This undoubtedly results from differences in the oxygen demand in the brines. The actual demand varied widely from tank to tank, but brines in older tanks (7-10 days) usually had a lower demand than those in young ones (Fig. l). The cucumbers will take up oxygen rapidly immediately after brining, and the lactic acid bacteria will also use oxygen during their most active fermentation period. Most of the demand in the older fermentations probably results from the development of yeasts. In most instances, the oxygen con- centrations in brines from tanks of all ages studied were reduced to essentially zero (less than 0.2 ppm) within 1 h after turning off the air. When nitrogen was used to replace air for purging, the oxygen levels declined very rapidly. The dissolved oxygen values obtained for bine from one tank (No. 7, Table 1) at 2, 3 and 4 days after brining were very different from those observed in any other tank. The brine did not appear to have any oxygen demand. In fact, readings of samples taken from the center of the tank after 3 days of fermentation appeared to increase in dis- solved oxygen from 2.2 ppm when the air was turned off to 25 FIGURE 1 Rates of depletion of dissolved oxygen from air—purged cucumber brines at various fermentation ages [h or days (d) after brining]. The air was turned off at time zero and measurements made at the points indicated. The dashed line (N2, 1 d) represents the depletion rate when air was replaced with N for purging. 2 PERCENT OF NITIAL .AG. 02 PER IITER 100 80 60 4O 26 \ /7d 0 V V \- 201- \ o 7 /8d ‘ \ 0 10+- \ ‘ 10d _' 8(- \ I _ 6— \ _ \ [Id 4_ _ \\ -3.- Nzfld' .4 2- \4-5hr 1 4 l J 2d 4 I 1 10 2o 30 46 so 60 TIME -MINUTE FIGURE 1 27 2.95 ppm 3 h later. Samples taken 1 day and 5-9 days after brining were normal with respect to oxygen demand. The oxygen meter appeared to be functioning normally, since brines in other tanks were found to respond in a normal manner. No reason for this anomalous response was apparent, and it was of much concern. However, as indicated in Table 1 (Tank No. 7), no effect on the salt—stock was evident. It is probable that there was something in that particular brine which was interfering with the meter readings. Laboratory Fermentations Effect of Aeration rate and Initial Acidification As expected, the rate of aeration had pronounced effects on the development of acid fermentation and the rate and extent of softening of air-purged cucumbers (Table 5). Practically all of the cucumbers purged at 100 ml air/min were very soft after 7 days of fermentation, and there was little or no acid development. Those purged at 20 ml air/ min all underwent some acid fermentation, and 47-89% of the cucumbers were still reasonably firm after 14 days fermenta- tion. .Aummu mHCmmmHm mnH Hva HHOm mHmHm>mm mum3 CUHCS COHpmquEHmm mCC CH mumnECoso HHm Ho mOMUCmonm mCu qummHmmH mHmmCUCmHmm CH mHmQECZQ .UmumsHm>m CmC3 moCmummmmm Umuso mHmumHmEoo m OMC mnmnfisoso HHC .mmmo b Hmumm omHmCHm>m mum: CHE\HE OOHe um omumnmm mmonu “COHummCmEHmm mmmo MH Hmumm omumCHm>m mums CHE\HE owe um omumnmm mumnfisosum 28 Awhmv m.> m~.O m.m m.w OOH Awmmv O.mH mm.O m.m n.v ON O0.0 Awmmv m.m v0.0 ¢.m m.¢ OOH Awmmv ¢.m mm.O m.m m.v ON v0.0 Awmmv H.v O0.0 >.v m.m OOH Awmmv N.h ON.O m.m m.m ON N0.0 Awmmv O.m mH.O m.v >.o OOH QAwHHV N.nH om.O m.m m.m ON mCoz mQH OHCOMH w CHE\HE UHumoC m amma proHod HCCHm mmo H mumm omoom musmmmum ECEmeS . , mm. COHumHmC oHoC mmmmmsoooo amszm mo lemme mmommmmmv ozHZCBCOm ozm azmsmoqm>mo oHoC zo mma._._m00w_> z. mwOJ .k DAYS FIGURE 3 35 Effect of Salt Concentration Cucumbers brined to equilibrate at salt concentra- tions between 20° and 35° salometer underwent extensive softening during air-purging for 2 weeks (Table 6). Brines in three of the fermentations develOped very high pectinolytic enzyme activities. However the 35° salometer brine had only a marginal level of activity even though many of the cucumbers in the brine were very soft. Air-purged cucumbers appear to cure (tissue becomes opaque) rapidly. Those in brines aerated at 100 ml/min (Table 5) were cured within 7 days of fermentation, and those in brines up to 25° salometer and aerated at 20 ml/min in Tables 5 and 6 were cured within 14 days. How— ever, high brine strength did delay cure (Table 6). Effect of Initial Limitation of Oxygen As noted above in the discussion of the data in Table 5, when the dissolved oxygen level dropped to a low level within 1 day after brining, little or no softening occurred. Many oxygen requiring microbes will die rather quickly in the absence of oxygen. Therefore, experiments 36 CoHHMHCmEHmm me CH mHmQECoCo HHm mo mmmquoumm mCu qummHmmu mHmmCquHmm CH mumnfisz Ummusm mum3 CCm .Aummu mHCmmmHm th HHvO “mom mHmHm>mm mHmB CUHCz a .CHE\HE ONe mo mumn 30H“ m um uHm CuH3 mHmCOCCHuCoo .sHom OHnwom Nmo.o :an sHHmHuHaH omeHcHom mums maoHumucmsumm HHam ON Awmmv m pH mm.o m.m m.w mm Om Awmwv m Hm SH.O O.v m.v Om OOH AwOOV HH mm mv.o m.m m.¢ mm OOH bAwmmv HH mm mm.o ¢.m m.v ON .mnH INuHmoomH> < w OHHOMH w muso Home MHHSHuoC, muHoHUC HMCHm mmo H HmumEOHmmo w mHCmmmCm mmMCHpomm HMCHE ECEmez I. ma ImCHHm mmmmmZDooo omszm mo ammo mo Bzmexm azm oszmemom zo ozm .mszm may zH weH>HeoC mmmzHaomm QZC aHoC Co ezm2moam>mo may zo oneamezmozoo eqam mo sommmm w mqmfifi 37 were conducted to determine the effects of limiting oxygen early in the fermentation. Cucumbers from brines purged with nitrogen the first 2, 3, or 4 days followed by air-purging until 14 days of fermentation were all firm in texture (Table 7). The extent of softening was reduced by purging with nitrogen for 1 day, but 25% of the cucumbers were seriously affected. In another series of fermentations at different brine strengths (Table 8), there was essentially no softening found in fermentations which had been nitrogen-purged for even 1 day before purging with air. No soft pickles were found in brines which had been initially purged with nitro— .gen for 2 days. It was not possible to correlate pectinolytic enzyme activities in the brines with the extent of softening observed in these experiments (Table 7 and 8). The 35° salometer brine purged continuously with air (Table 8) had only marginal enzyme activity but 20% of the pickles had pressure tests less than 11 lbs. In contrast, the 30° salometer brines purged with nitrogen for the first 2 days had reasonably high pectinase levels (46-59% but none of the pickles were soft. 38 .mCHHn mCu mo mommusm mCu HmmC Hmnfisoso CHOm mum> mCo mm: mHmCBo .Aummu mHCmmmHm mnH Hva UHOm meCOHHmm mums CUHCB mumnfisoso HHm mo mmmmHCmonm me m>Hm mHmmCUCmHmm CH wumnaszo .quHH umH0H>muuHC an wmuomuoum 90C mHmB mmCu mmsmomn ummmm mo mECom momwusm >>mmC Ummon>mo mCOHumquEHmm 03¢ mmmCBb .omHCOHUCH mHmCB ummoxm CHE\HE owe um mHmCosCHHCoo Umumnmm mHmB mCoHHmquEHmm HHC .oHom OHHmom wwo.o oCm HmvaOHmm 0mm um muwHQHHHCvm op omCHHQ mumz mumnfisosom mm Amos ON mH ms.o H.N msme s - Nz mm ANoO mH NH ms.o m.m msmw m I Nz mm lwov NN Hm HN.o m.m mama N - Nz mm meNO mH SH NN.o G.N sac H - Nz on Awmmv m mm O0.0 w.¢ CHHm mCosCHuCoo mH s.oHva mH m HH.o m.m nnmmusaIaoz .mnH HHHmoomH> H N .mmmmmm-m mHCO umme mmMCHpomm muHoHUC , mm quEummHB w musmmmum HMCHm ECEmez HMCHC mmmmmEDUDU QMZHmm m0 UZHmDU DZd UZHZmBmOm ZO QZ< .mZHmm HEB ZH NEH>HBU< mm¢ZH80mm QZfl DHUfl m0 BzmszHm>ma mma ZO OZHUMDQ meomBHZ H.m H om om mev mH hm mm.o m.¢ O mN om on Hwov NN OH Nm.O v.m N on om Hwov HN O Nm.o m.m N VN om HNoO HN HN mm.o ¢.m H on om Awov HN ON Nv.O m.m H «N om CAwOHV OH me mm.O n.w O mN mN .mnH muHmoomH> < w UHHOMH w mama HCCHm HCHuHCH mHCO Hmma muH>Huo< MHHUHUC. mm Ummhsm Hmmeonmo w musmwmum mmmCHuomm HMCHm ECEmez HMCHm NZ mCHHm mmmmmZDUDU 9m2Hmm 20 9290 mo BZMme 92¢ UZHZQBmom 20 92¢ .mZHmm mmB 2H 929290H9>99 NBH>HBU¢ mm¢2Hfiumm 92¢ 9HU¢ 20 UZHUmbm 2MOOMBH2 H¢HBH2H 92¢ EBOZWmBm HZHmm mo BUMhhm w MHm¢B 40 .HHMm mum mo CoHunom an COHumquEHmm Ho Mmms pCoomm mCu mCHHCU mmCHm> mmmCu ou ommHmu mHHmComnm mm3 CumCmHum mCHHn mCBo .Hpmmu mHCmmmHm mbH HHvV HHOm mHmHm>mm mums CUHCS COHHMHCmEHmm mCm CH mquECoCo mo mmmquonm mCu pCmmmHmmH mHmmCquumm CH mumnadzn .quHH umH0H>ImnuHC mg Umpomu Iona HOC mums mme mmsmomn mmCHHn mmme Co omQOHm>mU meson m>mmm .mCHmHCm NZ How ompmoHoCH mUOHHmm mCu How ummoxm ACHE\HE ONev HHm CuHB mHmCOCCHHCoo ommusm mums HHC .oHom OHHmom va.O CuHB omHHHtHom mums mCOHumpCmEHmm HH¢ .mCumCmHum mCHHn HMHHHCH UmHMOHOCH mCH um UmCHHC mums AmCHCHHn um umm>HmC pmom mmmw m-NO CmmHCOHE on mHCHmHH> Eonm ommmHCm Cmmb UMC CoHCz mumbECoCo HumumEmHo =N\H HI¢\H HO mN mNHmm OH Awov HN OH I mm.O >.m N 00¢ ON Awov NN NH mv.O m.m N mm ow Awmv mH HN ON.O O.v H 0mm om ANOV HN v ON.O m.m H vm om ANONV NH O hN.O O.v O mm mm 41 The effect of air-purging on curing is evident in Table 7 when compared to a non—purged control. Failure of the smaller sized cucumbers used in the experiments reported in Table 8 to cure rapidly is not known. Effect of Controlled Fermenta- tion of Potassium Sorbate Pickles from a controlled fermentation which was brined and treated as described by Etchells §E_al. (9) but purged constantly with air were all firm (Table 9). In addition, no softening enzyme was detectable in this brine after 2 weeks fermentation. The results were exactly the same when 0.035% potassium sorbate was added and a natural fermentation allowed to develop. As the concentration of potassium sorbate was lowered, more extensive softening occurred and higher pectinolytic activities were observed. A fermentation which had not been purged for 1 day (static), and another which had been held static 6 h each day served as controls in this experiment (Table 9). Both of these fermentations contained some soft cucumbers and significant pectinase activities. 42 .uamHH HmHoH> ImuuHC HmUCC omumnCoCH HOC mums mpmnuom ECHmmMHOQ Umopm CHHB mCOHumquEHmm U .>m© Comm C m 990 UmCHCu mm3 HHm UCm ~mCHCHHQ Hmumm C m UmuHMHm mmB COHpmHm¢o .Aummu musmmmum mnH HHvO “mow mHmCOHHmm mums COHC3 mHmQECoCo HHm mo mmmmquoumd mum mmmmCquHmm CH mHmQECz n .omHCOHoCH mHmCB pdmoxm CHE\HE ONe um mHmCOCCHuCoo omumnmm mum3 mCoHHMUCmEHmH HHC .oHom oHumom wwO.O OCm HmumEonm omN um mumunHHHsom on omCHHb mumnasosom Hmov mH O mm.o s.m wmmo.o Awva mH II mm.O >.m wN0.0 Awnnv OH In me.o o.¢ wH0.0 HNva s - II m.m osmoo.o .mumnuom M msHm Hwov mH O mm.o m.m omHHoupCoo ANHV HH mm ms.o N.m osmw\a m .OHumum nxwmmv eH 0N om.o N.m mac umuHm .oHumum .mnH NHHmoomH> MIN mmmmmmlm Home mmMCHuomm muHoHo¢ mm quEpmmHB mnsmmmnm HMCHm ECEmez HMCHm mmmmmzbuDU HEB mo UZHZmBhOm 20 92¢ mmZHmm 2H mm¢2HBUmm 92¢ 9HU¢ 90 829290Hm>99 20 ma¢mmom ZDHmm¢Bom mo 20HBH99¢ 92¢ .20HB¢8292299 99H9028200 .20HB¢BHEHH MH¢ H¢Hfim¢m 90 909999 m MHm¢B 43 Effect of Blanching Fresh Cucumbers Cucumbers which had been blanched at 65°C (149°F) for 5 min before brining did not show significant softening during 2 weeks air-purging (Table 10). This was true even though the cover brine was inoculated by washing unheated cucumbers. Two of three non-blanched cucumbers brined in a jar with blanched cucumbers all became severely soft. Not one of three blanched cucumbers included in a fermentation of non-blanched cucumbers became soft. These data indicate that either the microbe(s) which cause softening of air- purged pickles have infected the cucumber tissue prior to brining and do not readily reinfect after brining, or that blanching the cucumber prevents the development of the microbe(s) in some way. Microbes Isolated From Brines A number of yeasts and bacteria were isolated from air-purged cucumber fermentations in which there was exten- sive softening of the cucumbers. An occasional mold colony was observed on plates, but their frequency of occurrence on platings of various brine dilutions indicated that they were contaminants. None of the yeasts, two mold cultures, i 44 .omCHHumoCC mum muHCHm mmHCH mmme 90 mummu mHCmmmHm .on CH omQHuommo mm mmCo omCoCmHQICOC CHH3 omxomd mnmz mumbfisoso omCOCMHQ mmHCBm .UmCHHHmoCC mum muHCHm mmHCu mmmCu mo mummu musmmmum .HMn CoHHmm ms» 90 do» mCu um mCo oCm .HmHCmo me CH mCo .Eouuon mCu Co omxomm wok mCo .uHCHm omCoCmHQ CUH3 Umxomm oCm mCHHpm m CuHB mCHmp Na omHmHquoH mHmB mHmCECoCo omCOCMHnICOC mmHCBo .mHmQECoCo omCoCmHn mCu Hm>o mCHHCOQ mnommn wumnECoCo UmCoCmHQICOC Cmmnm mCHCmm3 an nmumHCUOCH mm3 mCHHn Hm>oo mCBo .Hmm Comm mo Coupon 0p 90» Eonm Hmouo CH ompmmu musmmmud UCm om>oEmH mums mumnasooo HMCUH>HUCHn .oHom 0Humom weo.o OCm HmumEOHmm omN um mumunHHHCqm o» omCHHb HHm mums mnmnesoso H=N|v\m HO mm .02 mNHmm NH m .NH .0 .HH .m .NH .NH .HH .oN .mH .4H .NH momHUCMHa m NH NH .NH .mH .HH .NH .NH .N .NH .HH .H .ON .mN c.0umaoamHn-aoa m NH NH .NH .ON .NH .mH .NH .mH .NH .NH .0H .NH .oH .GH owmnoamHn HHN m m .o .e .o .o .NH .0 .N .m .o .o .o Houuaoo - maoz mmmum>¢ bmHmbECoCU HmsoH>H©CH quEummHB AmQHV mammp musmmmum 002H0299I2H¢ mBHZ mZOHB¢BZMSmmm OZHMD9 UZHZMBkOm 20 OZHZHmm OB mOHmm mmmmZDUDU 02H202¢Hm 90 809999 OH 9H9¢B 45 and only an occasional isolate of an endospore-forming bacterium were found to produce pectinolytic enzyme activ- ity when inoculated into pasteurized brined cucumbers. The spore-forming bacteria were never present at pOpula- tions over 1,000 per ml in the fermenting brines and the isolates obtained may have arisen from endospores. Development of Pectinolytic Activities There was an increase in pectinase activities in brines in most, but not all air-purged fermentations in which extensive cucumber softening occurred as noted above. Therefore, an attempt was made to demonstrate production of such activity in aerated non-fermented cucumber brines inoculated with brine samples from fermentations in which cucumbers did become very soft. No significant pectinase activities were detected in shake—flask brine cultures, while high activities developed during the same period in three of the four air-purged fermentations from which inocula were taken (Table 11). Extensive softening of cucumbers occurred within 14 days after brining in all four of the air—purged fermen- tations referred to in Table 11. The average pressure 46 TABLE 11 PECTINASE ACTIVITIES IN BRINES FOR AIR-PURGED CUCUMBER FERMENTATIONS AT VARIOUS PERIODS AFTER BRINING AND IN SHAKE-FLASK BRINE CULTURES INOCULATED WITH FERMENTING BRINESa °Salometer Brines 20 25 30 35 Samples % loss in viscosityb Fermenting brines: 5 days 6 5 24 2 7 days 24 ll 37 5 12 days 64 43 67 0 Shake-flask cultures inoculated with:C 5-day brines 0 3 6 4 7-day brines 3 7 0 7 aSee Materials and Methods for procedures used for brining and purging of cucumbers, and for determining pectinase activities. bAny values below 10% are of doubtful significance. CErlenmyer flasks (250 ml) contained 50 ml of cucumber brines at the indicated salometer levels prepared as des— cribed in Materials and Methods. Flasks were inoculated with 5 ml samples of corresponding fermenting brines at the fermentation ages indicated. These cultures were incubated on a rotary shaker at 30°C until the original fermentations were 12 days old. 47 tests of all of the cucumbers were 11, 11, 5, and 8 lbs from fermentations at 20, 25, 30, and 35° salometer respectively. Of particular interest was the failure to detect signi- ficant pectinase activity in the brine from the fermentation at 35° salometer. Furthermore, this brine failed to soften non—fermented cucumbers after 40 days storage at 30°C, while the brines from the other three fermentations with high enzyme levels had very significant effects (Table 12). The failure to find pectinolytic or cucumber soften- ing activities in the 35° salometer brine was not likely the result of either inactivation or inhibition. The enzyme(s) activity in a pooled lot of cucumber brine was stable for four months at 50° salometer, and there was no obvious inhibition of softening until the brine strength exceeded 35° salometer (Table 13). Similar effects of salt on pectinolytic enzyme(s) have been reported (2). Molds in Brined Cucumber Tissues The above results indicated that the initial soften- ing of cucumbers in air-purged fermentations resulted from the growth of microorganisms in very close association with the cucumbers, not free in the brines. Microscopic 48 TABLE 12 EFFECT OF SPENT BRINES FROM AIR-PURGED FERMENTATION OF DIFFERENT BRINE STRENGTHS ON THE PRESSURE TEST OF NON-FERMENTED BRINED CUCUMBERSa Pressure Tests Brine Controls + Spent Brine °Salometer 20 18 (0%)b 12 (74%)b 25 22 (0%) 14 (63%) 30 19 (0%) 8 (61%) 35 20 (0%) 20 ( 8%) aCucumbers in quart jars were brined to equilibrate at the salometers indicated and at 0.04% acetic acid as described in Materials and Methods. At the start of the experiment, the brine in one of two jars at each salometer was replaced with spent brines from an air- purged fermentation conducted at the same brine strength (see Table 11). One jar at which salometer was held as a control. Toluene was added, the jars sealed, and Stored for 40 days at 30°C. bNumbers in parenthesis are the final pectinase activities (% loss in viscosity) in the brines after storage. 49 TABLE 13 AVERAGE PRESSURE TESTS OF CUCUMBERS EQUILIBRATED AND STORED IN BRINES AT VARIOUS SALT CONCENTRATIONS WITH AND WITHOUT HIGH LEVELS OF PECTINASE ENZYME(S) ACTIVITY_ °Salometer Controls + Enzyme (s) PPS..- 9.10.8.1 25 13 (0%) 4 (53%)b 30 16 8 (60%) 35 15 (0%) 7 (56%) 40 ll (0%) 9 (42%) 45 12 10 (57%) 50 ll (2%) 12 (54%) aCucumbers in quart jars were equilibrated in brine at the various salometers and at 0.7% lactic acid as described in Materials and Methods. At the start of the experiment, 450 ml equilibrated brine in one of 2 jars at each salometer were replaced with 450 ml of spent brine from air purged fermentations which had been adjusted to 0.7% lactic acid and the appropriate salo- meter. The spent brine used was from a pooled lot with very high pectinase activity (m80% loss in viscosity). One jar at each salometer was saved as a control. Toluene was added, the jars sealed and stored at room temperature (m22°C) for 4 months. bNumbers in parenthesis are the final pectinase activities (1% loss in viscosity) in the brines after storage. 50 observations of thin tissue sections of soft cucumbers from air-purged fermentations demonstrated extensive invasion by mold hyphae (Fig. 4). Molds were consistently observed in sections from soft tissue, while none were found in sections from fresh fruit or from brined cucumbers which had not undergone some softening. Cultures of cross-cut slices of surface sterilized brined cucumbers which were undergoing softening confirmed the presence of molds in the tissues (Figs. 5 and 6). In both figures, the cucumbers cultured in (A) were from fermentations in which extensive softening had occurred within 14 days after brining; those in (B) from fermen- tations in which some soft cucumbers were found; and those in (C), and (D) (Fig. 6) from lots in which all of the fruit was still firm after 2 weeks fermentation. Platings of slices of surface sterilized fresh cucumbers were usually free of molds (m10% of slices with mold colonies). In contrast, after holding fresh cucumbers in the refrigerator for 6 days, molds develOped on about 90% of cultured slices. Slices of cucumbers from untreated natural fermentations which had been air-purged for even 1 day usually were found to contain viable molds. 51 .E: m H HmCHoo ummH HmBOH CH Hmm .mCoHumquEHmw UmmHCm-HHM Eon“ mumnfisoso HHOm Ho mCmmHu mCu CH Amzonymv mMCQMC UHoS-I.v .meI ..fx. .. v w . fiff V“ ,' . . .Aui N A... ,. .M.a.\.v... I. .. . g \I. . 52 .HHH swap Sam msmu N Com Nz HOV OCH .HHm Cme UCm mmv H How Nz Hmv .HHm mCOCCHHCoo Adv "mBOHHow mm mmmv m How ommusm mCOHumquEHmm Eoum mumnfisoso OmNHHHHmum mommnsm Ho mmoHHm mo ASOH Eouuonv Hmmm mmOprmC OHCHOQ UCm H30H mouv Hmmm wN Co mmHCHHCUII.m .lo 0.1.1 M41“) nil ..Iumu) .. 11.. (IHIII-Il-I-I'1II Ill 53 .OmOOm mHmQHOm ECHmmmuom wmmo.o AQV OCH .COHHOHCmEHmm OmHHOHuCoo HOV .MMO Hmm C m Mom OCm C O umHHw mCH How ummoxm OmmHCQIHHm >HmCOCCHHCoo Amv .HHO ma OmonHom NCO H How mHHmum A¢v "mSOHHom mm Omummnu mCOHumquEHmm Eoum mHma mumnfisoso .ASOH EOHHOQV Hmmm mmOHume OHOHOQ OCm ASOH QOHV Hmmm wN Co AmCHCHHQ Hmpmm mmmO my COHumquEHmH OmmHCQIHHw Eoum mHmQECoso OmNHHHCmum mommHCm Eoum mmOHHm mo mmuCHHCoII.O .OHM 54 Identity and Softening Activ- ity of Molds Isolated From Cucumber Tissues Fifteen cultures of molds were isolated from plated slices of cucumbers from air—purged fermentations. These were all identified as members of one of three genera; viz., Alternaria, Fusarium, or Mucor. These all grew when inoculated into pasteurized nonfermented cucumbers equi- librated in brines at 25° salometer and 0.04% acetic acid, and they also softened the cucumbers (Table 14). Most of the cultures produced relatively high activities of pectinolytic enzyme(s) which were found in the brines. However, in a few cases, no significant enzyme activity was detected, but extensive softening of the brined cucumbers occurred. 55 TABLE 14 PRODUCTION OF PECTINASE ACTIVITY_AND SOFTENING OF CUCUMBERS IN BRINES BY MOLDS ISOLATED FROM SURFACE-STERILIZED SLICES OF CUCUMBERS FROM AIR-PURGED CUCUMBER FERMENTATIONSa Culture Pectinase Observation of No. Genus Activity Brined Cucumber % A Viscosity CM-l Fusarium 12 very soft -2 M3295 91 very soft -3 Fusarium 37 very soft -4 Alternaria 92 very soft -5 Alternaria 8 soft area -6 Fusarium 10 very soft -7 Alternaria 88 very soft -8 Fusarium 47 very soft -9 Alternaria 78 very soft -10 Fusarium 23 very soft —11 M3393 92 very soft -12 Fusarium 0 Very soft -13 Muggr 0 very soft -14 Fusarium 0 very soft -15 M3395 93 very soft aIndividual molds were inoculated by use of a needle into a cucumber equilibrated and partially submerged in brine at 25° salometer and 0.04% acetic acid. See Materials and Methods for procedure for preparation of pasteurized brined cucumbers. The inoculated cucumbers were incubated at room temperature for 9 days. bThere was visible mold growth and softening of the tissue in the area where the cucumber had been inoculated. DISCUSSION Air-purging, as demonstrated by these results, can and is being used successfully to prevent bloater formation in commercial salt-stock pickle fermentations. During the last 3 years, industry representatives attending meetings at Michigan State University have reported experiences with air-purging about seven million bushels of salt-stock pickles. A few companies are now using air for purging all fermentations of large Size cucumbers; one company has air- purged all tanks of cucumbers regardless of cucumber size during the last 3 years (19). Despite the success of prevention of bloater forma- tion achieved when air is substituted for nitrogen some risk remains. In these experiments, softening was seen in two instances with the soft pickles found directly under the purger outlet. One tank had a few soft pickles (<50 bu) and another had soft ends (<5 bu). Compared to the total number of bushels involved (W18,000) the loss is negligible. Industry representatives have also reported finding an occasional tank with a few bushels of soft pickles directly 56 57 under the purger outlet. However, no one has reported a serious loss. The relationship of CO levels in the brines and 2 bloater control remains obscure. Originally, Etchells et 31. (9) recommended that CO2 concentrations be maintained below 20 mg/100 ml of brine to control bloater formation in controlled fermentations. Fleming et_al. (14) reported that maintenance of CO2 concentrations below 50% of satura- tion was necessary to ensure against bloating. However, even with CO2 concentrations as high as 90 mg/100 ml of brine, essentially no bloaters were found in the commercial tanks studies herein. The temperature of these tanks was 70i2°F (m21°C), and most brine strength was near 25° salometer so there would be about 120 mg C02/100 m1 brine at saturation. Therefore, CO levels in excess of 75% of 2 saturation occurred without any noticeable increase in bloaters. Laboratory scale fermentations using air as the purging gas presents a different perspective from commercial tanks. The purging unit is located in the bottom of the container allowing direct contact of the air with the cucumbers. It is also possible for gas bubbles to become entrapped in the cucumber mass. When very high purging 58 rates, approximately 500 times that used in commercial tanks (100 ml air/min), were used the fermentations were not of the nature of typical lactic fermentations. Little acid was produced, the pickles were for the most part very soft and an "off" odor was observed. Yeast populations were not well controlled even though the fermentations were continually exposed to ultra-Violet light, and high popula- tions of aerobic bacteria were present. Either or both of their microbial groups were probably utilizing any acid produced. This high purging rate produced 100% cure (opaque tissue) in the cucumbers after only 7 days. When using lower purging rates (20 ml air/min), approximately 100 times the rate used in commercial tanks, extensive softening was still found in many instances. Initial high concentrations of acetic acid (0.06%) and salt 35° salometer tended to retard softening but also retarded the start of lactic fermentation. Softening was completely eliminated when the cucum- bers were prewashed in hypochlorite solution as in the con- trolled fermentation process, when 0.035% potassium sorbate was added to the fermentation, and when the cucumbers were blanched before brining. These results indicated that the causative organism(s) of softening was located on the skin H .4-..__ ._._A. 59 or in the tissues of the cucumber. After removal, rein- fection did not readily occur since brine made from wash- ings of untreated cucumbers did not produce any softening in cucumbers which had been previously blanched. Softening was also severely curtailed or totally eliminated if nitrogen was used as a purging gas for the first 24-48 h. It is likely that the causative organisms could not survive the anaerobic environment for this long of a period. Using pectinolytic activity as an indicator, attempts to culture an organism(s) Capable of causing softening from the brines were unsuccessful. Inoculation of fresh brines with samples of brine which contained high pectinase activity was also unsuccessful. Organisms iso- lated from liquid withdrawn from the inside of a soft cucumber with an intact skin using a sterile hypodermic syringe produced no significant pectinase actiVity. Neither did they cause any softening of blanched prebrined cucumbers. The results of these experiments are consistent with the findings that molds growing in brined cucumber tissues are primarily responsible for softening of pickles from air—purged fermentations. When cucumbers were surface- treated with hypochlorite, sliced and plated on agar, mold 60 growth was consistently seen on Slices of brined cucumbers which were air-purged and conversely not seen in nitrogen- purged cucumbers. When thin sections of pickles were examined microscopically, mold hyphae was visible in every instance when the section was taken from a soft pickle or soft spot in the pickle. No hyphae was observed in firm tissues. Molds which were cultured from sections of hypo- chlorite treated cucumbers and inoculated into pasteurized brined cucumbers resulted in softening of the cucumber tissues. All three genera of molds isolated from cucumber slices have been previously found to be associated with pickling cucumbers (10). The softening of pickles in these experiments was not correlated with pectinolytic enzyme activities in brines. Thus, extensive softening was observed in some fermentations in which negligible enzyme activity was found and vice versa. Therefore, the softening observed after 2 weeks of air-purging was primarily the result of enzymatic activities produced by the molds in pickle tissues. Further softening would occur if pickles were stored in brines with significant pectinase activities (3,7). It is possible that some of the enzyme(s) in the brine was 61 produced by microorganisms growing in the brine, on the surface of the cucumbers or in the pores of the polyethylene sparger. This may have been the source of enzyme in those fermentations in which no soft pickles were found. Mold .growth on the spargers was observed and, in some cases, resulted in partial plugging of the pores. However, no pectinase activities developed in pasteurized aerated cucum- ber brines inoculated with brines from fermentations in which enzymatic activity was increasing. No comprehensive studies have been reported of softening enzyme(s) levels in commercial fermentations which have been air—purged. No activity was found in the brine from the one tank which contained a few bushels of soft pickles in a localized area under the sidearm outlet. The soft pickles observed in air-purged commercial tanks have remained localized in a small area of the tank during sev- eral months storage (19). In addition, not all pickles in a given area are soft and some pickles have only soft spots. Data from these experiments indicates that the first 24-48 h after brining is a critical period when air is used as the purging gas. Moldsgrowing in tissue would be protected to some extent from inhibitory effects of the brine during this period. Also, there may be considerable 62 air dissolved in fresh tissues. Large cucumbers were found to contain 45-50 ul gas/gram in the tissues at brining. The gas contained about 23% oxygen (15). However, the amount of oxygen in the tissues at brining would vary with the way the fruit had been handled. Those from the central areas of large masses of cucumbers would probably have used all the oxygen in respiration, while those near the surface would have high amounts. This variable could be important in commercial fermentations. Fresh cucumbers in the tOp few feet of large tanks are frequently not covered with brine for several hours before the brining Operation is completed. No soft pickles were found when nitrogen was used for purging for 2 or more days after brining before start- ing continuous aeration. It is possible that careful limitation of the air flow rate and purging on an inter- mittent schedulewould eliminate the sporatic softening problems. These results and those of Potts and Fleming (16) Show that dissolved oxygen usually disappears from brines rapidly when the air supply is shut off. Although the oxygen demand is variable, it is usually maximal during the first 3 days of fermentation. Fleming 33 31, (12) found no softening of pickles fermented in five 1 gal pails and 63 air—purged on an intermittent schedule (2 h/day). In experiments designed to purge commercial fermentations at varying rates with air, difficulties with the air compres- sors and electrical supply, caused frequent shut-off of the air supply for variable and unknown periods of time during the night (6). In these nine tanks, although the CO2 levels were not well controlled, there were no significant dif- ferences found in the number of bloaters compared to control tanks of nitrogen-purged pickles and no softening was observed. CONCLUSIONS Air can be used successfully to replace nitrogen as a purging gas in commercial fermentations. When air is used as a purging gas, a limited amount of softening of the salt-stock may occur. Softening may be prevented by keeping the fermentation anaerobic for the first 48 h or by pretreatment of the cucumbers to destroy surface microbes. Molds growing in the cucumbers are the cause of softening. 64 9. BIBLIOGRAPHY Bell, T. A., and J. L. Etchells. 1955. A method for testing cucumber salt—stock brine for softening activ- Bell, T. A., and J. L. Etchells. 1961. Influence of salt (NaCl) on pectinolytic softening of cucumbers. J. Food Sci. 26:84—90. Bell, T. A., J. L. Etchells, and I. D. Jones. 1950. Softening of commercial cucumber salt—stock in relation to polygalacturonase activity. Food Technol. 4:157-163. Buescher, R. W., J. M. Hudson, and J. R. Adams. 1979. Inhibition of polygalacturonase softening of cucumber pickles by calcium chloride. J. Food Sci. 44(6):0000. Costilow, R. N., C. L. Bedford, D. Mingus, and D. Black 1977. Purging of natural salt—stock pickle fermenta- tions to reduce bloater damage. J. Food Sci. 42:234-240. Costilow, R. N., K. Gates, and C. L. Bedford. 1980 Air—purging of commercial salt-stock pickle fermenta— tions. (Submitted) Demain, A. L., and H. J. Phaff. 1957. Softening of cucumbers during curing. Agr. Food Chem. 5:60—64. Etchells, J. L., T. A. Bell, R. N. Costilow, C. E. Hood, and T. E. Anderson. 1973. Influence of temperature and humidity on microbial, enzymatic and physical changes of stored, pickling cucumbers. Appl. Microbiol. 26:943-950. Etchells, J. L., T. A. Bell, H. P. Fleming, R. E. Kelling, and R. L. Thompson. 1973. Suggested 65 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 66 procedures for the controlled fermentation of commer- cially brined pickling cucumbers - The use of starter cultures and reduction of carbon dioxide accumulation. Pickle Pack. Sci. 3:4-14. Etchells, J. L., T. A. Bell, R. J. Monroe, P. M. Masley, and A. L. Demain. 1958. POpulation and softening enzyme activity of filamentous fungi on flowers,' ovaries, and fruit of pickling cucumbers. Appl. Micro— biol. 4:103—110. Etchells, J. L., A. F. Borg, and T. A. Bell. 1968. Bloater formation by gas forming lactic acid bacteria in cucumber fermentations. Appl. Microbiol.‘ 44:1029. Fleming, H. P., J. L. Etchells, R. L. Thompson, and T. A. Bell. 1975. Purging of C02 from cucumber brines to reduce bloater damage. J. Food Sci. 49:1304. Fleming, H. P., R. L. Thompson, J. L. Etchells, R. E. Kelling, and T. A. Bell. 1973. Bloater formation in brined cucumbers fermented by Lactobacillus plantarum. J. Food Sci. 34:499. Fleming, H. P., R. L. Thompson, and R. J. Monroe. 1978. Susceptibility of pickling cucumbers to bloater damage by carbonation. J. Food Sci. 42:892. Jorge, F. 1978. Gas diffusion from cucumber fruits. North Carolina State University, Raleigh, NC (M.S. Thesis). Potts, E. A., and H. P. Fleming. 1979. Changes in dis- solved oxygen and microflora during fermentation of aerated, brined cucumbers. J. Food Sci. 44:429. Rogers, P. 1979. Private communication. The H. J. Heinz Co., Lakeview, MI. Shoup, J. L., J. J. Cuniglio, and B. F. George. 1976. Relationship between fermentation time and bloater-type damage of brined cucumbers. J. Food Sci. 44:1335. Switzer, R. G. 1980. Private communication. Western Food Products Co., Inc., La Junta, CO. "I7'111'1111'1111111I