l H 1 nl 1.. E‘ ‘ M 1W ‘| 1 I .1 | 1‘ t I! ‘ \ ‘l \ L» «l L‘ WI "IN—As +1 (1)0003 {SOLATION AND IDENTSFECATION OF A STRASN OF BACILLUS SUBTILjé CAUSING PICKLE SPOQLAGE Thesis for the Degree cf M. S. MICHIGAN STATE COLLEGE Arnaid Lesmr Demain 1950 This is to certify that the thesis entitled The Isolation and Identification of 8 Strain of W M Causing Pickle Spoilage presented by Arnold L. Danain has been accepted towards fulfillment of the requirements for Master of Scienceaegree inmogy & Public Health fixézz/g/ ' Major professor Date May 3, 1950. v 63,. 1.... . .27; "" ’- ‘4; vs... . V 4‘ .v ‘ "‘ nu. .' J‘ ISOLATION AND IDENTIFICATION OF A STRAIN OF BACILLUS SUBTILIS CAUSING PICKLE SPOILAGE BY Arnold Lester Demain A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of TMSTER OF SCIENCE Department of Bacteriology and Public Health 1950 THESIS ACKNOWLEDGMENT The author wishes to express his appreciation to Doctor.F. W} Fabian, Research Professor of Bacteriology and Public Health, for his patient and willing guidance throughout this work. ********** **#****# *#**** ##1## 13* * FMP1flru) .r.-."| ".P-‘o? . 3%.. TABLE OF CONTENTS Page IBITRODIICTIOBIOOOOOOOO00......OOOOOOOOOOOIOOOOOOOOOCO l PRESENT INVESTIGATION.............................. 2 LITERATURE REVIEW}................................. 3 EXPERIMENTAL PROCEDURE............................. IO RESULTSs........................................... 16 DISCUSSION......................................... 19 SUMMARY............................................ 23 CONCLUSIONS........................................ 24 PUEFEREITCTE‘SOOOO.0.000.000...OOOOOOOOOOOOOOOOOOOOO... 32 TABLES II III IV V a. VI LIST OF TABLES RECORD OF TANK CONTAINING SPOILED SALT STOCK PICKLES FRO?l DELATi/IIAFLEOO.00.00.00.0000000000000.00.. CHENICAL AND BACTERIOLOGICAL ANALYSIS OF FOUR SAN- PLES OF SPOILED PROCESSED DILLS FRO” NICHIGAN...... CHENICAL AND BACTERIOLOGICAL ANALYSIS OF TWO SAM- PLES OF SPCILED SALT STOCK AND TWO SANPLES OF PROCESSED DILLS MADE FROM SPOILED SALT STOCK FROM DELAWARE........................................... NICROSCOPIC EXAMINATION CF BACTERIA ISOLATED FROM SPOILED PICKLECOOOCCOOOOOOOOOOOOOOCOCOCOOOOUOOO0.. CULTFRAL CHARACTERISTICS OF BACTERIA ISOLATED FRON SPOILED PICKLES ON NUTRIENT ACAR................... CULTURAL CHARACTERISTICS OF BACTERIA ISOLATED FROM SPOILED PICKLES IN NUTRIENT BROTH.................. PHYSIOLOGICAL REACTIONS OF BACTERIA ISOLATED FROM SPOILED PICKLmOOCCOOOOCOOOCCOOOOO00.00.000.00..... PAGE 25 26 27 29 30 31 ISOLATION AND IDENTIFICATION OF A STRAIN OF BACILLVS SUBTILIS CAUSING PICKLE SPOILAGE INTRODUCTION Spoiled pickles are a problem that has faced the industry for many . years. The spoilage of salt stock pickles comes from six months to a year after the cucumbers have been placed in tanks for salting. In the case of processed dills, softening of the pickle is also a problem. 'Workers who have investigated the problem have attributed softening to the decomposition of pectic materials in the middle lamella of the cucumber -- the change of insoluble protopectin to soluble pectin by the action of bacterial enzymes. PRESENT INVESTIGATION The present investigation has to do with two different lots of pickles. The first batch arrived from the state of Delaware and con- tained two jars of soft salt stock pickles and two jars of processed dills made from soft salt stock. The receiving stations for the origi- nal cucumbers were within 25 miles of the salting station. The cucum- bars were trucked to the salting station within two to three hours after being received. They were placed into a 30 degree brine and held . until the acidity had reached 0.5 percent determined as lactic acid. At this point, the salt was raised to 50 degrees salometer within the next five days. The cucumbers were held at this salinity. Specific data for salinity, acidity and temperature from one of the spoiled tanks is shown in Table I. The soft processed dills came from the State of Michigan. They arrived in four jars, each pair representing a different batch. One batch was processed on November 29th, and the other on December 2nd. Each run contained approximately 45 barrels with two spoiled and 43 good barrels. The process consisted of three freshening tanks, each holding 15 barrels. The dill brine came from one large tank; the same brine was used for all 45 barrels. LITERATURE REVIEW Van Tieghem (1879) considered that Bacillus amylobacter was re- sponsible for the decomposition of vegetable tissue. The action of Bacillus mesentericus vulgatus was studied by Vignal (1889). He found that the organism secreted a ferment which dissolves the middle lamell of potato tubers without dissolving the cell walls, thereby dissociating the cells. Aderhold (1889) reported that Bacterium coli was the cause of pickle softening. Kossowicz (1908) reported that the Bacillus mesentericus group, not Bacillus coli, was responsible for softening. The pectase enzyme produced by Bacillus carotovorus and other soft rot organisms was studied by Jones (1909). This enzyme was capable of softening carrots very quickly by dissolving out the middle lamella, leaving the cells free. Rahn (1913) reported the factors involved in salt stock spoilage. He concluded that acid would inhibit very quickly the growth of pro- teolytic bacteria while salt alone could not always do this. LeFevre (1919) concluded that cucumber softening was caused by many organisms including the cellulose destroyers, the soft-rots, and the spore-bearing aerobes. The only organisms isolated that were cap- able of living in high salt concentrations were Bacillus vulgatus and Bacillus mesentericus fuscus. In the relation of salt to the TABLE I RECORD OF TANK CONTAINING SPOILED SALT STOCK PICKLES FROM DELAWARE 1 2 BRINE AIR PICKLES SALT DATE SALINITY ACIDITY TELEP. TET-EP. ADDED ADDED top bottom 0F. °F. in lbs. in lbs. July 6 3260 500 7 23976 3100 6 4322 500 9 26 37 1.1 76 74 10 27 35 1.3 65 67 12 33 36 2.1 66 69 13 53 36 3.1 75 65 14 30 37 4.1 74 67 15 29 35 5.6 75 76 1200 16 42 43 5.7_ 72 76 1000 17 47 49 5.6 74 76 19 51 52 5.7 76 76 20 39 51 5.6 77 67 150 21 43 . 50 5.6 77 60 200 22 51 51 5.6 76 64 23 50 51 5.7 79 60 24 46 50 5.8 79 76 150 26 52 51 5.9 74 76 27 53 55 5.6 76 65 26 52 53 5.7 76 62 29 52 52 5.6 79 63 30 52 52 5.6 76 60 31 52 52 6.0 76 60 Aug. 2 51 51 5.6 76 77 3 ‘ 50 52 5.9 76 79 4 47 50 5.9 76 76 5 49 50 5.9 75 76 50 6 46 50 5.6 72 64 50 7 50 51 5.6 75 66 9 53 53 5.6 74 69 10 52 53 5.7 73 66 50 11 52 52 5.5 75 72 12 52 52 5.7 75 74 13 52 52 5.6 74 74 14 52 53 5.7 74 74 33.5 1 aExpressed in salometer degrees. ‘Expressed in grains determined as lactic acid. Continued next page -4- TABLE I - Continued DATE SALINI 771 A61 DI n2 BRINE AIR PICKLES SALT TEI-IP. TEMP. ADDED ADDED top bottom. OF. OF. in lbs. in lbs. Aug. 16 51 51 5.6 72 66 17 52 52 5.7 73 70 16 53 53 5.6 74 70 19 50 52 5.9 73 70 20 51 51 5.7 73 70 21 46 50 6.3 73 66 150 23 52 52 6.1 73 70 24 52 52 6.0 74 72 25 53 53 5.6 74 72 ' 26 53 53 5.6 75 77 27 53 53 5.8 76 76 26 53 53 5.6 60 63 50 30 53 53 5.3 76 79 Sept. 1 53 53 5.6 72 66 100 3 53 53 5.7 71 72 16 54 54 5.1 24 51 54 74 , 75 50 Oct. 11 51 54 5.3 66 72 100 26 55 55 5.2 Nov. 12 54 55 5.7 30 52 55 5.2 50 46 200 Dec. 17 56 56 5.5 50 50 500 1 Expressed in salometer degrees. Expressed in grains determined as lactic acid. -5- preservation of cucumbers, the critical point was regarded as between seven and eight percent. Dill pickle spoilage was investigated by Joslyn (1928). He con- cluded that softening was due to a bacterial cause. He noticed that in a brine of pH 3.00 - 5.10, slipperiness increased. Since this pH was lower than that tolerated by Bacillus vugatus in LeFevre's work, Joslyn.suggested that enzymes secreted by this organism may act at a lower pH than that tolerated by the organism itself. Lesly and Cruess (1928) studied dill pickle spoilage and found that high initial acidity favored softening and that brine from spoiled pickles had a higher acidity and lower pH than.brines of normal pickles. They suspected that the softening may be due to the high acidity which would cause the splitting of hemicelluloses or pectin. They also ob- served that softening did not all occur during fermentation but increas- ed with length of storage. The larger pickles were less prone to become soft than smaller ones. Fabian, Bryan, and Etchells (1932) studied sections of normal cured cucumber pickles and pickles in different stages of decomposition. They observed certain structural differences between the various lots of pickles. The most obvious histological change that had occurred in the soft pickles as compared to the normal pickles was in the intercellular spaces where there was a conspicuous lack of cementing material after the pickles had started to soften. This condition progressively increas- ed until there was no longer any material left in the middle 1amella.. After what was assumed to be the pectic material had disappeared, the cell wall was attacked until finally all the parenchymatous cells were gone. The most resistant parts of the cucumber were the seeds and vas- cular bundles. Flax rotting was studied by Trevethick, Robinson, and Snyder (1926). They reported that the Bacillus mesentericus-megatherium group caused retting by dissolving out the pectic substances. The optimum.pH was found to be seven. Hof (1935) studied bacterial life in strong brines. Pure cultures of ”normal" bacteria originating from a saltless environment tolerated only a relatively low amount of salt in their culture liquid. Taking into account that bacteria as occurring in nature may adapt themselves better to a life under abnormal conditions than pure cultures, enrich- ment cultures were made for various bacterial groups in media with in- creasing percentages of salt and with inoculum.of garden soil. Under these conditions, bacteria which attacked pectin.under aerobic condi- tions grew in solutions containing up to 18 percent salt in comparison with a pure culture that grew only up to a six percent salt concentra- tion. Fabian and.Johnson (1938) studied the bacteriological, zymological, histological and chemical changes in spoiled pickles. They found that the Bacillus mesentericus fuscus group produces a protopectinase which can cause pickle spoilage rapidly. This protopectinase becomes active after the first day's growth of the organism, and under favorable conditions, reaches its maximum activity in six days. The histological and chemical studies proved that the softening of pickles was due to a change in the insoluble protopectin to soluble pectin by bacterial enzymes. Also acids, heat, and enzymes were found to increase the sus- ceptibility of the pickles to softening, either immediately as by cook- ing, or over a long period of time with a weak acid. Faville and Fabian (1948) found that bacteriophage races which lysed Lactobacillus plantarum.cultures was isolated from soil where the cucumbers had grown but not from water or from genuine dill pickle brines in which spoilage had occurred six to eight months previously. In studying the antibiotic effect of aerobic sporogenic bacteria common- ly found in soil and in fermenting cucumber vats, it was found that these organisms were capable of producing substances which inhibited greatly the growth of Lactobacillus plantarum, the.0rganism chiefly re- sponsible for cucumber fermentation. The two organisms capable of this action, Bacillus mesentericus fuscus and Bacillus vulgatus, were also able to elaborate pectin-hydrolyzing enzymes. In Eh studies, it was found that the six species of aerobic spore formers from spoiled pickles reduced the oxidation-reduction potential of the medium.to a consider- able degree while the 1actobacilli had little effect on the potential. When the NaCl content of the medium was increased, the time required by the organism to reduce the potential of the medium to a level at which multiplication could take place was also increased. The effect of lactic acid was similar and when both agents were added together, the results -8- were additive. This indicated that the salt tolerance of these organ- isms decreases with increasing concentrations of lactic acid. Most of the spoilage organisms could be induced to grow in significantly higher concentrations of salt if the oxidation-reduction potential of the medium'was reduced to the minimum level established by the organism.dur- ing normal growth. Fabian and Faville (1949) isolated and identified a mold, Oospora lactis, which was the causative agent in two cases of pickle spoilage. In one case, the organism developed during the freshening process of salt stock when the period of freshening was too long. In the other case, the organism caused the damage in processed dill pickles due to the use of dirty barrels. -9- EXPERIMENTAL PROCEDURE Upon arrival, the jars of salt stock and process dills were analyzed bacteriologically and chemically. The bacteriological procedure con- sisted of plate counts on three media. Tryptone-glucose-yeast extract agar (TGYE) was used to grow and differentiate peptonizers, acid bacteria and inert bacteria. Peptonizers were distinguished by a clear zone around the colony; acid bacteria by turning the medium.yellow; and inert bacteria by leaving the medium purple. The total count was also deter- mined by this medium. Its composition is as follows: Tryptone lO gramS' Yeast extract 5 grams Beef extract 3 grams ’ HPO4 1 gram G ucose 1 gram Agar 15 grams Dist. water 1000 ml. Brom—cresol-purple 2 ml. of 1.3% solution 1 ml. sterile skim milk per plate added when plates are poured. Potato dextrose agar (PDA) acidified to a pH of 3.5 with tartaric acid 'was used for yeast counts. Its composition is as follows: Infusion from potato 200 grams Dextrose 20 grams Agar 15 grams Dist. water 1000 m1. Tomato juice agar was used to determine the count of lactic acid bacteria. Its composition is as follows: Tomato juice (400 cc) 20 grams Peptone 10 grams Peptonized milk 10 grams Agar 11 grams Dist. water p 1000 ml. -10.. The TGYE and tomato juice agar plates were incubated for two days at 52° C. while the potato dextrose agar plates were incubated at room temperature for five days. The chemical analysis consisted of acid, salt, and pH determina- tions. The acid was determined with 0.1666 N NaCH and two drops of a five percent alcoholic solution of phenol-thphalein. The direct read- ing was in percent acetic acid which, when multiplied by ten, gives grains of acetic acid. The salt was determined with AgNO3 solution using 0.5 percent of a 95 percent alcoholic solution of dichloro- fluoroscein. This read directly as percent salt. The pH was deter- mined electrometrically by a Beckman pH meter. After the plates were counted, typical organisms were fished and put through a purification procedure. This procedure consisted of streaking the cultures and fishing isolated colonies seven times. The purification was carried out on beef extract agar, then.transferred to beef extract agar slants. The bacterial cultures were then put through identification tests as outlined in "Aerobic Hesophilic Sporeforming Bacteria" by Smith, Gordon and Clark. Microscopic Examination pfkbacteria Gram stains of 24 hour cultures incubated at 55° C. on beef ex- tract agar were made on the 16 bacterial cultures. Dacroscopic Examination of bacteria Growth on nutrient agar was observed on slants incubated at 35° C. for-four days. Growth in nutrient broth was observed after incubation 81: 35° C. for four days. -11- Physiological Examination.of bactefia Casein hydrolysis was shown on milk agar plates prepared by mixing equal quantities of sterile skim milk and sterile two percent agar, both cooled to 450 C. before mixing. After solidifying, cultures were streaked on the plates and observed for clearing of the casein. The ability to grow at pH 6.0 was determined on slants of nutrient agar. The reduction of nitrates to nitrites was made on cultures grown in nutrient broth plus 0.1 percent KNO for four days. To five m1. of 5 the culture was added 0.5 ml. of a one percent solution of KI, and after mixing, one drop of concentrated H2804. If nitrates were formed a blue color was the result. If there was much nitrite, a heavy blue precipitate resulted and gas was evolved. The production of urease was demonstrated by the growing of cul- tures on slopes of nutrient agar and testing for urease after five days. The growth was washed off with two m1. of distilled water and divided equally between two clean test tubes. A drop of phenol red indicator was added to each and the reaction brought to pH 7.0 by a few drops of very dilute HCl or NaOH. Approximately 0.02 gm. of crystalline urea was mixed with the suspension in one tube and the other was kept as a control. If urease was present the suspension with the urea became very alkaline in a few minutes. The hydrolysis of starch was determined by streaking plates of nutrient agar containing one perCent potato starch that had been added -12- after filtering. After incubating for one day, the plates were flooded with 95 percent alcohol. If the starch remained unchanged the medium became white and opaque but if it was hydrolyzed a translucent zone appeared around the growth. For the fermentation studies an ammoniacal medium.was used. The formula was as follows: IBI4H2 P04 1 .0 gram KCl 0.2 gram l'gSO4 0.2 gram Agar 13.0 gram Dist. water 1000.0 ml. The pH was adjusted to 7.0 and 12 m1. of a 0.04 percent solution of brompcresol-purple was added as the indicator. The solution of the test carbohydrate was then sterilized separately by filtration through a Seitz filter. The basal medium was autoclaved and the filtered car- bohydrate solution added aseptically to it (one gram carbohydrate in 25 ml. distilled water added to 175 ml. basal medium resulting in a 0.5 percent concentration of carbohydrate). Five ml. were pipetted into each tube and slants were made. These were streaked and stabbed in the butt and incubated at 35° 0. Observations were made at 3, 8, l5 and 21 days. The utilization of citrate was demonstrated on the following medium: Na citrate 2.0 grams NH4N03 2.0 grams KHZ P04 0.5 gram Agar 13.0 grams Tap water 1000.0 ml. 0.04% Phenol red soln. 10.0 ml. pH adjusted to 6.8 before addition of indicator -13- These slants were observed for growth and reaction after four days of incubation. The'Voges-Proskauer reaction was demonstrated on the following medium: Proteose-peptone 7.0 grams Glucose 5.0 grams NaCl 5.0 grams Dist. water 1000.0 m1. Five m1. portions in 18 mm. tubes were inoculated and incubated at 320 C. for two and four days. The presence of acetylmethylcarbinol was demonstrated by the appearance of a red color after the addition of an equal volume of a 40 percent NaOH solution and a few milligrams of creatine and shaking. Gelatin hydrolysis was determined on plates of nutrient agar con- taining 0.6 percent gelatin. These were streaked and incubated at 350 C. for one day. This is slight modification of the procedure of Smith, Gordon and Clark. The plates were then covered with nine ml. of the following test solution: Dist. water 100 ml. Conc. HCl 20 ml. HgCl2 15 grams This reagent formed a white opaque precipitate with the unchanged gela- tin and left a clear zone where the gelatin.was hydrolyzed. The results of all identification tests were checked with Bergey's Vanual of Determinative Bacteriology, sixth edition. The isolates were placed in groups resembling certain species and one member from each -14- group was selected to determine the protopectolytic properties of each group. The media used was that suggested by Fabian and Johnson in 1938. Beet Su,ar Molasses medium 5 100 parts beet molasses 100 parts monobasic ammonium phosphate 10 parts urea Diluted to 5° Brix with tap water The organisms selected to represent the groups were inoculated into flasks containing 330 ml. of medium. Into another flask was inoculated the organism Bacillus mesentericus fuscus, a known pectolytic organism. A control flask was also incubated with no inoculum. The flasks were incubated for six days at 32° C. and then filtered through Buchner funnels to remove sediment. The filtered media were then placed in sterile jars, each jar containing three intact salt stock pickles fresh- ened to 20 degrees salometer. The mediunlwas layered with toluene to prevent bacterial action and the jars incubated at 32° 0. They were ob- served for signs of spoilage at l, 4, 6, 13 and 44 days. -15.. RESULTS The results of the chemical and bacteriological analyses of the four samples of processed dills (samples 1, 2, 3 and 4) are given in Table II. From these four samples, the following organisms were iso- lated: From TGYE agar W4.....raised dull inert colony W5.....colony appearing as sporeformer W6.....sma11 colony appearing as sporeformer with clear zone "W7.....pinpoint glistening colony From Tomato juice agar W8.....large round dull colony W9.....large round dull colony (duller than W8) W10....colony appearing as sporeformer W11....small glistening colony Analysis of the four samples of spoiled salt stock (samples 5, 6, 7 and 8) gave the results shown in Table III. The following organisms were isolated: From TGYE agar L1T....from sample 5 L2T....from sample 6 L4T....from sample 8 _]_6.. 00.. C... From Tomato juice agar L4JS...from sample 8 L4JL...from.sample 8 The results of microscopic, macroscopic and physiological examina- tions of the isolates are presented in Tables IV, Va and b, and VI re- spectively. The results given in Table VI are those observed at the final reading. During purification, isolate W6 was separated into two organisms -- W6b and W6p. Also two other isolates -- W12 and W13 -- were separated. 0n comparison of all identification data with Bergey's Manual, the isolates were placed into four separate groups. Each group, with the organism it most closely resembles, is shown in the following: Group Isolates Organism I. “01, LlT, L4T, L4Js Bacillus cereus (var. mycoides) I I . W4, W5 , WGb , W6p, W7, W8, W9, W12, W13, L4JL Bacillus subtilis III. L2T Bacilis pumilis IV. W00 Doubtful. may be a B, subtilis- Rf pumilis intermediate After growth for six days in Beet Melasses medium, growth appeared as follows: Group I...Slight ring on surface. Group II...Heavy wrinkled dry growth on surface. -17- Group III...Light membraneous growth on surface. Group IV...Light membraneous growth on surface. 3. mesentericus fuscus...Heavy wrinkled greasy growth on surface. Control...Clear. The results of each group on freshened intact salt stock are as follows: Group 1 day 4 days 6 days 13 days 44 days I. - - - - - II. - - soft soft soft III. - - - - - IV. - - - - - v, - - - soft soft VI. - - - - - -18- DISCUSSION Considering the Uichigan processed dills first, the chemical analy- sis shows a definite lack of sufficient acidity. The highest acidity of any of the four samples was 4.0 grains and one sample was as low as 2.5 grains. The minimum for process dills should be six grains. A re- cent survey of.American and Canadian pickle packers showed an average of 8.5 grains used in the manufacture of processed dills. This lack of acidity was probably the cause of the large bacterial populations growa ing in the samples as shown by the TGYE agar results. 0f the eleven isolates from these processed dills, nine were found to be of the group that showed pecthrhydrolyzing activity which is the reason why the pickles became soft. 0f the isolates obtained from the Delaware samples, only one showed pectin-hydrolyzing activity. It originally was isolated from sample eight which contained processed dills made from soft salt stock. This same sample, six months later, contained nothing more than a mushy liquid since the cucumbers completely disintegrated. 0n the other hand, the samples of salt stock still retain their shape, even though they were soft. This indicates the possibility that the organisms that caused the spoilage of the salt stock may have been killed or the enzyme inactivated or both after the pectin breakdown occurred in the salt stock. This may have been the reason for the lack of success in isolat- ing the causative organism from the salt stock samples. Another possi- bility is that the salt stock was softened by another organism and the -19.. causative organism isolated in this work was picked up during the freshening process. Examination of the salting data of the spoiled tank shows that for five days after the tank was closed and eight days after it was started, salinities below 400 were present in parts of the vat. This is significant, as spoilage may easily result in such loW'salt concen- trations when.temperatures of 750 F. and above are reached. These first few days are the most important days of the salting procedure. If pectin-hydrolyzing enzymes are elaborated during these days, there is a very good chance of the cucumbers becoming soft over a period of time since it requires a long time for a small amount of enzyme to work. many pickle packers use an.acidity ofsix grains determined as acetic acid in manufacturing processed dills. This has been accepted as the minimum for this type of product. However, the fact that the causative organism was so active in sample eight which had an acidity of 6.5 grains sheds new light on the problem of spoilage. Possible solu- tions are raising the acidity or pasteurization of processed dills. Some packers pasteurize this product but there are many who do not. The accepted procedure of 71.10 C. (1600 F.) for 20 minutes should be enough to inactivate the destructive enzyme. Although the specific enzyme hydrolyzing the pectic materials of the cucumber has not been purified and analyzed, the deactivation times and temperatures for pectin-hydro- lyzing enzymes of other sources have been determined by other workers. From.their studies, it seems reasonable to assume that the pasteurization -20- temperature and time used in the industry today is sufficient to de- activate the enzyme. Further work is needed along these lines. The fact that a strain of Bacillus subtilis showed pectin-hydro- lyzing activity in this work is not as startling as it may seam. The organism has been.used in the flax retting industry where it is known to hydrolyze pectic materials between the fibers. Previous workers have shown that it does not spoil pickles, but it must be remembered that the classification of the Bacillus group has been a confused one. Organisms called B. subtilis in the past may not be B. subtilis at all (according to present day classification). For instance, in 1948, Faville and Fabian found Bacillus vulgatus to be capable of pectinphydrolysis, howh ever, today this organism is regarded as a stage in the growth of B. subtilis. The organism called B, subtilis in this paper checks well with the sixth edition of Bergey‘s manual in all features examined ex- cept the lactose and arabinose fermentations. There are four main types of spoiled pickles encountered in the industry. They are classified as follows: (a) The mushy type which usually starts out as a slippery pickle, called a "slip", and continues until the whole piCkle is mushy. (b) The cheesy type which is not slippery but crumbles through- out when pressure is applied. (c) The "watery" type in which the inside is watery while the outside skin is still intact. This is not found with the -21- black spine pickling variety but in the thick skinned, white spine, slicing type of pickle and is undoubtedly the same as the cheesy type (b) except the skin is so thick that it withstands the hydrolysis longer. Even- tually it will break down. (d) Loss of firmness or turgidity. A flabby pickle due to physiological factors rather than biochemical changes as in the three above mentioned types. The type encountered in this work was the cheesy variety. This is the most frequent type of spoilage found. It is not known whether these types are caused by different factors or are merely different stages in the softening of a pickle. -22- SUUHRHY Two samples of soft salt stock and two samples of processed dills were received from Delaware and four samples of processed dills were received from a Michigan packer. All were of the "cheesy" type of spoilage. The eight samples were analyzed chemically and bacteriologi- cally. Typical organisms were fished, purified and identified. The organisms isolated resembled Bacillus cereus var. mycoides, Bacillus pumilis and Bacillus subtilis. The only group showing pectin-hydro- lyzing action on freshened intact salt stock pickles was the one re- sembling B, subtilis. cor-mmrsxopg l. The probable reason for spoilage of the Nichigan process dills was the low acidity. The four samples contained acidities of 2.5, 3.0, 3.0 and 4.0 grains. According to a recent survey the average for the industry is 8.5 grains. 2. As for the Delaware salt stock, spoilage probably resulted from the lack of sufficient salt concentrations in parts of the tanks during the first few days of salting. A 40° salinity is advisable in hot climates and going below this is dangerous. The fact that the causa- tive organism thrived in an acidity of 6.5 grains in the processed dills shows how potent it is. Possible solutions are raising the acidity or pasteurization to deactivate the enzyme. -24- TABLE II HEHICAL AND BACTERIOLOGICAL ANALYSIS OF FOUR SAMPLES 0F SPOILED PROCESSED DILLS FRO? NICHIGAN Chemical Analysis Sample NaCl Acidity as acetic pH product 1. 3.9% 4.0 grains 3.45 proc. dills 2. 5.2% 2.5 grains 3.50 proc. dills 3. 5.4% 3.0 grains 3.55 proc. dills 4. 5.3% 3.0 grains 3.55 proc. dills Bacteriological Analysis (in organisms per ml. brine) Sample _ TGYE PDA Tomato Juice agar l. spreaders 297,000 280,000 2. 9,400,000 (inert) 185,000 10,100,000 3. 7,300,000 (inert) 144,000 5,600,000 4. 15,300,000 (inert) 237,000 spreaders -25- TABLE III CHEVICAL AND BACTERIOLOGICAL AKALYSIS OF TWO SANPLES OF SPOILED SALT STOCK AND TWO SAHPLES O? PROCESSED DILLS MADE FROM SPOILED SALT STOCK FRO” DBLAJARE Chemical Analysis Sample NaCl Acidity as acetic pH product 5. 18. % 2.0 grains 3.5 salt stock 6. 18.8% 2.0 grains 3.6 salt stock 7. 8.8% 6.5 grains 3.1 proc. dills 8. 8.8% 6.5 grains 3.2 proc. dills Bacteriological Analysis (in organisms per m1. brine) Sample TGYE PDA Tomato Juice agar 5. 1190 inert; 180 pept. 4118 0 6. 1360 inert; 170 pept. 5900 18,500 7. 0 0 O 8. 330 peptonizers 2850 n 3,000 -25- TABLE IV XICROSCOPIC EXANINATION OF BACTERIA ISOLATED FRO! SPOILED PICK ES W4 Gram variable rods; occurring singly, in pairs and short chains; round ends; lu x 2.3 - 3.0u; spores central, subterminal; some spores spherical l.lu in diameter, some oval 1.0u x l.8u; spor- angia not definitely swollen. we Gram - rods; occurring singly, in pairs and short chains; round ends; lu x 2.3 - 3.0u; spores central, subterminal; some spores spherical 1.lu in diameter, some oval 1.0u x l.8u; sporangia not definitely swollen. WBb Gram - rods; occurring singly, in pairs and short chains; round ends; 1.0 x 2.3 - 3.0u; spores central, subterminal; some spores spherical 1.1u in diameter, some oval 1.0u x l.8u; spor- angia not definitely swollen. 'W6p Gram variable rods; occurring singly, in pairs and short chains; round ends; 1u x 2.3 - 3.0u; spores central, subterminal; some spores spherical 1.1u in diameter, some oval 1.0u x l.8u; spor- angia not definitely swollen. W7 Gram variable rods; occurring singly, in pairs and short chains; round ends; lu x 2.3 - 3.0u; spores central, subterminal; some spores Spherical 1.1u in diameter, some oval 1.0u x l.8u; spor- angia not definitely swollen. W8 Gram - rods; occurring singly and in pairs; round ends; 1.0u x 3.5u; spores few, subterminal; oval 1.0u x l.4u; sporangia not definitely swollen. Some long chains noted. W9 Gram - rods; occurring singly and in pairs; round ends; 1.0u x 2.7u; few spores, subterminal; oval 1.0u x 1.4u; sporangia not definitely swollen. WHO Gram - rods; occurring singly and in pairs; round ends; 1.0 x 2.3 - 3.3u; spores subterminal; oval 1.0u x 1.5a; sporangia not definitely swollen. DUI Gram - rods; occurring in long and short chains, in pairs and singly; round ends; 1.3u x 4.5u; spores very prominent, sub- terminal; oval l.3u x 1.6 - 2.2u, spherical 1.3u in diameter; sporangia not definitely swollen. Continued next page -27- TABLE IV - Continued “HZ Gram - rods; occurring singly and in pairs; round ends; 1.1u x 3.3u; spores central, subterminal; oval 1.1u x l.4u, cylindri- cal, diameter 1.1u; sporangia not definitely swollen. W13 Gram - rods; occurring singly and in pairs; round ends; l.0u x 3.3u; spores few, central, subterminal; cylindrical, diameter 1.0u; sporangia not definitely swollen. LIT Gram - rods; occurring in long and short chains, in pairs and singly; round ends; 1.3u x 4.5u; spores very prominent, sub- terminal; oval l.3u x 1.6 - 2.2u, spherical 1.3u in diameter; sporangia not definitely swollen. L2T Gram - rods; occurring singly and in pairs; round ends; 1.1u a 3.3u; spores central, subterminal; oval 1.1u x l.4u, cylin- drical, diameter 1.1u; sporangia not definitely swollen. L4T Gram - rods; occurring in long and short chains, in pairs and singly; round ends; l.3u x 4.5u; spores very prominent, sub- terminal; oval l.3u x 1.6 - 2.2u, spherical 1.3u in diameter; sporangia not definitely swollen. L4JS Gram - rods; occurring in long and short chains, in pairs and singly; round ends; 1.3u x 4.5u; spores very prominent, sub- terminal; oval l.3u x 1.6 - 2.2u, spherical l.3u diameter; sporangia not definitely swollen. L4JL Gram - rods; occurring singly; round ends; 0.8u x 2.3u; few spores, subterminal; oval 0.8u x 1.1u; sporangia not definitely swollen. 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S - designates slight positive reaction. -31- 1“Results shown in table are those observed at final reading. Fermentation results apply to production of acidity. PHYSIOLOGICAL REACTIONS OF BACTERIA ISOLATED PROV SPOILED PICKLES‘ Fermentation Reactions1 m In no :1 :3 m5:; g,o:3 Iioti<5 g g o o o 3 8 8 o '3 o '3 3 a fimfisfi 434300me lei o no In to +> :1 £1 0 m 49 In L O s: 0H ”KMPO 00 mc>m3