_ . ~‘BACTERIOLOGICAL; ZYMOLOGICAL- AND CYTOLOGICAL STUDIES OF THECAL‘SE OF SOFT CUCL‘MBER F‘ICKLES Thais-for {he Degreé of M. S. ' Edwin A; Johnson ..H , 1937 . . r Ii. \ » I. u.. .. ,‘ .v. . .II. . q. ‘ , .~YP~ WI; TH £33.55 BACTERIOLOGICAL, ZYMOLOGIGAL.AND CYTOLOGICAL STUDIES OF THE CAUSE OF SOFT CUCUMBER PICKIES A.THESIS Submitted to the Faculty of the Michigan State College in partial fulfillment of the requirements for the degree of Master of Science by ‘1: 2"} Edwin A‘ffigggmson March. 1937 ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the help and constructive criticism of Dr. F. W. Fabian. Associate Professor of Bacteriology, under whose supervision the work was done. I also wish to express not gratitude to Dr. R. deZeeuw, Associate Professor of Botany. for his invaluable assistance with the cytological work. and to Professor C. D. Ball of the Chemistry Department for his help and interest on the chemical aspect of the problem. 1035083 II III VI VII VIII XI XII TABLE OF CONTENTS Introduction Literature Review Description of Organism Bacteriological Studies Zymological Studies Cytological Studies 1. Introduction 2, Review of Literature 3. Experimental Work 1|». Discussion of Results Practical Application Sumary of Results Conclusions Tables Drawings Bibliography BACTERIOLOGICLL, ZYMOLOGICAL AND GYTOLOGICAL STUDIES OF THE CAUSE 0? SOFT CUCUMBER PICKLES INTRODUCTION Every year the pickle industry experiences considerable losses due to the spoilage of pickles. Some years the losses are greater than in other years. As a rule the spoilage does not take place until the following spring or summer. There are instances, however, where spoilage is noted within several week after the pickles have been salted. Sometimes the whole tank spoils and again only certain areas or layers in a tank spoils;_ The spoilage is progressive. The first sign of spoilage appears on the skin of the preserved cucumber and is known as a "slip" due to the fact that the skin of the pickle is slippery and when taken into the hand and rubbed between the fingers, the skin is easily removed. It usually appears in spots on the skin but in a short time the entire skin may become slippery and.is .easily removed. The second stage of spoilage is when the deeper cells of the cucumber become involved and the entire pickle becomes soft. Cucumbers in which spoilage has progressed to this stage are known as "mushy" cucumbers. Greater losses occur in genuine dill pickles than in salt stock. This is due to the lower salt concentration used in curing genuine dill pickles than in salt stock. In a dry, hot year more pickles spoil than in a cool, dry year. These differences have been observed frequently during the past six years. Some years as high as 50 per cent of the genuine dill pickles packed have spoiled. In addition to the spoilage occurring in the fermented cucumbers, it frequently happens that‘softening occurs in the -2... finiShed product after it has been bottled and marketed. An exhaustive review of the literature discloses the interesting fact that all previous investigators have assumed that softening of cucumber pidkles has been due to the decompo- sition of the pectic materials present in them.without having demonstrated this to be the case. Reasoning.g nriori they have assumed that since other types of softening in vegetables and fruits have been shown to be due to a dissolution of the pectic materials that such was the case when cucumber pickles softened. In fact some of the previous work on the causes of softening in vegetables has not clearly demonstrated the nature of the changes taking place. The object of this work was to study the various agents causing spoilage, the different types of spoilage found in pickles, and to determine the nature of the changes taking place within the spoiled pickles. -3- LITERATURE REVIEW The cytolytic action of organisms on plant tissues has been observed and studied for a great many years. As early as 1879, Van Tieghem (1879) considered the decomposition of vegetable tissue was due to a single poly- morphous species of bacillus which he called.Bacillu§ gmylobacter. Today, this name is regarded as a class of bacteria rather than a single species. Vignal (1889) studied the action of Bacilkug mesentericgs vulgatus on potato tubers. He records that this organism secretes a ferment capable of dissolving the middle lamella without dis- solving the cell walls, thereby dissociating the cells. One of the earliest researches on pickle softening was con- ducted by Aderhold (1889). He isolated spore forming rods and acterium coli, but attached.no significance to the former. He reported.the Bacterium ggli as the cause of softening. Upon the request of a salting station, Kossowicz (1908) carried out researches on the softening of pickles. He concluded that the Bacillug mesentericgg group was somehow connected with soft pickles and not aggillg§.ggli as previously reported by Aderhold. Jones (1909) studied very extensively the pectase enzyme as produced.by'figgillp§.ggggtggggg§ and other soft rot organisms. He determined the Optimum.cultural conditions for the production of the enzyme, and also the effect on it of various outside influences such as heat. acids, alkalies, and long storing. The enzyme was studied by various methods. One method which was very -h- satisfactory consisted of treating with such germicides as chloro- form, thymol or phenol. These germicides were added in small enough amounts to just inhibit the growth of the organism and still not impair the enzyme. The enzyme as produced.by Bacillus caroto- .zgggg was capable of softening carrots very quickly by dissolving out the middle lamella, leaving the cells free. One of the earliest pieces of work on spoilage of pickles in America was carried on by Rahn (1913). He recorded various contrib- uting factors in the spoilage of salt stock. Acid was found to very Quickly inhibit the growth of proteolytic bacteria while salt alone could not be depended upon to do this. Softening was found usually occurring then when the acid content of the pickles became low or the pickles were left protruding out of the brine. LeFevre (1919) studied the ability of about fifty organisms to attack vegetables. Of these, sixteen were more or less capable of softening the cucumber. The only ones capable of growing in a high salt concentration, however, were Bacillus vulgatug which reached six per cent and Eacillus mesenteriggs fgsggs which grew in four per cent. most of them failed to grow above one per cent salt. Only four out of the 16 organisms were able to grow in a pH range of seven to four. These included gggillug‘yplggiug,'Eggillug carotovoggs,'Bacillus lgctis agrgggngs, and.Baci;;g§ cereus. Further inoculations Showed.Eggilgugunggg£g§_capable of growing at pH 3.8. He concluded that softening in cucumbers was caused by a wide range of organisms including the soft-rots, the cellulose destroyers, and the spore4bearing aerobes. In the relation of salt to the preservation of cucumbers, the critical point was regarded as between seven and eight per cent. -5- Joslyn (1928) investigated the spoilage in dill pickles. He came to the conclusion that softening was definitely due to a bacterial cause, but noticed that slipperiness increased in a brine of pH 3.00 - 3.10. This was lower than the pH tolerated by Bacillus vulgatus as investigated by LeFevre. Joslyn suggested that enzymes secreted by this bacteria may be active at a lower pH than that tolerated by the organism itself. Studies on spoilage of dill pickles were conducted by Lesly and Cruess (1928). They found that high initial acidity favored softening and that brine from spoiled pickles had a higher acidity and a lower pH than brines of normal pickles. hr. Lesly suspected that the softening might be due to the high acidity which would cause the splitting of hemi-celluloses or pectin. They also observed that softening did not all occur during fermentation but increased with length of storage. The larger pickles were less prone to become soft than the smaller ones. Flax retting was studied by TrevethiCk, Robinson, and Snyder (1928). They found that the Bacillus mesenterigus - megaterium group caused retting by'dissolvingeeut the pectic substances. The optimum pH was found to be seven. Bacterial life in strong brines was investigated quite fully by Hof (1935). He found that 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, enrichment cultures were made for various bacterial groups in media with increasing percentages of salt and with garden soil as inoculum. -6- Under these conditions, bacteria which attackgpectin under aerobic conditions grew up to eighteen per cent salt in comparison with a pure culture that only grew up to a six per cent salt concen- tration. DESCRIPTION OF ORGANISM The spoilage organism used in this study was isolated from a barrel of salted pickles that had.become soft. This organism was proved.the causal agent in softening by inocup lating a culture of it back into normal firm cucumbers which were softened by it in a very short time. It was necessary that a pure culture be obtained for pure culture studies. Several attempts were made to single cell this organism'but without success. Therefore. it was purified by plating out approximately fifteen times. This final culture was tested on pickles and found to be as active in causing spoilage as the original cunture. This organism was found to have the following character- istics: Beds: 0.5 to l by 1| to 6 micronspccurring. singly. in pairs, and short chains. Spores: Central 1.0 by 1.5 microns. Gram.positive, motile. Gelatin Stab: Liquefaction napiform. .Agar colonies: Irregular, rough, contoured. Agar slant: Spreading, membranous, wrinkled, light yellow in old.cu1ture. Broth: Heavy growth on tOp. clear solution. Litmus milk: Rennet curd, peptonization. Potato: Luxuriant wrinkling growth. Carrot: Luxuriant wrinkled growth. Indol not formed. Nitrates reduced. Ammonia formed. Acid in dextrose, sucrose, maltose, mannose, and mannitol. No action on lactose. raffinose. galactose, arabinose, rhamnoee, xylose, trehalose, adonitol, dulcitol, inositol, sorbitol, inulin, dextrin. starch. cellulose. Starch is not hydrolyzed. Blood serum liquefied in two days. Aerobic, chultative. Optimum temperature 37°C. The foregoing characteristics showed that it was a spore fermer and therefore in the 18m group. It appeared to fit in quite well with the Essillue W - seam group. The following members of this group were assembled and their characteristics determined and compared with the unknown: We Insane. Bacillus W. Essillne W fume. Mills: Wm. Esalllna same. and Bacillus W- The results of this comparison showed only one organism to correspond. with the unknown in all salient points. There appeared only two small differences between the two organisms. There was a slight difference of surface growth on the broth cultures and a difference of eight days in the. liquefaction of blood serum. The organism to which the unknown corresponded very closely was M W m. This organism was found to have the ability to cause pickle spoilage equally as well as the unknown. The unknown was concluded to be _____1.1._Bacill s W fascia. or a close variant of it. -9- BACTERIOLOGICAL STUDIES The first question that arose in connection with this organism was concerned with its salt and acid tolerance. Table I shows these figures for salt. acetic acid, and lactic acid calculated as acetic acid. This organism was found to grow quite readily in as high as nine per cent salt. Since this was a much higher value than obtained by other workers, several other members of this group were tested and their critical salt tolerance found to be exactly the same. The organism tested were hauillus Means. Bacillus W. heaillns We e er c fusuus. B__illusae W. The higher results were attributed to the medium used for their growth. The medium used in growing the organism for all the exper- imental work was made of 100 parts beet molasses. 10 parts monobasic ammonium phosphate and 10 parts urea diluted with tap water to a Brix of five degrees. The final pH was about six. The best broth was adjusted to pH seven when the acid and salt tolerance were determined, but otherwise it was left at pH six. It can be seen that this medium is much more representative of the actual conditions found. for instance, in a pickle brine. The pickle brine contains carbohydrates, mineral salts, and other water or salt extractable substances which is a condition very similar to that found in the beet residues plus added salts. This medium could almost be termed an enrichment medium in that it more closely resembles actual working conditions than nutrient broth consisting of peptone, meat extract and salt which is generally used. The very fast rate of growth and the large amount -10.. of enzyme promiced by the organism in this medium as compared with.the nutrient broth would seem.to verify this assumption. 'Upon repeated inoculation from.the nine per cent salt in best sugar broth to higher percentages of salt in best sugar broth. it was feund.that all the above organisms would give a scanty growth as high as 11 per cent salt concentration. The acid tolerance was found to be in agreement with other workers. The organism isolated in this study was found to be capable of growth in 0.15 per cent acetic acid and 0.2 per cent lactic acid calculated as acetic acid.upon the initial inocula- tions. By repeated inoculation from the concentration producing growth to a.higher concentration. it was made to grow in a 0.2 per cent acetic acid and a 0.3 per cent lactic acid calculated as acetic acid. -11- ZYMOLOGICAL STUDIES In the present work, it was early found that the softening of pickles was not necessarily an invasion of the tissue by the organism but that it was strictly an enzymatic decomposition of pectic materials in the tissue. Also that this enzymatic action was capable of acting in the absence of the causal organisms and had an entirely different tolerance of salt and acids as compared to the organism that produced it. The enzyme.solution used in this experimental work was pre- pared by inoculating two liters of the previously described‘beet molasses medium.in a six liter flask with 10 c.c. of broth culture. These flasks were incubated for twelve days at 3700. During this time there developed a oneehalf inch layer of bacterial growth on the surface of the media leaving the mediumtperfectly clear. Any'flaak which was cloudy at the end of twelve days was discarded as this was found to be due to contamination. The clear liquid was siphoned off from.the bacterial surface growth and.preserved with 0.2 per cent sodium'benzoate plus a layer of toluene over the surface. This method of preserving gave very satisfactory results without any noticeable deleterious effects.on the action of the enzyme. This enzyme solution.ihen placed in the ice box was kept for a relatively long period of time without any appreciable loss of potency. Twelve liters of this solution that had been stored in the ice box for one year showed a relative activity of approximately three-fourths that of the freshly prepared enzyme solution. The activity of the enzyme solution -12- was greatly enhanced by permitting it to act at a relatively high temperature such as 37°C. This enzyme solution behaved similar to any other enzyme solution in that it was destroyed by'boiling, and its activity increased with temperature up to a certain limit and its rate of deactivation increased with the temperature above 37°C. The effect of salt. acetic acid and.lactic acid upon the activity of the enzyme solution was studied in an effom.to contrast these results with those of the same agents upon the organism producing the enzyme. It was hoped that these results would shed some light on the problem of pickle spoilage. Tables II and III give the results of these experiments. The acid and salt tolerance of the enzymic solution was compared with the growth of the organism was almost reversed. The growth of the organism.was stopped at 11 per cent salt and at 0.2 per cent acetic acid and 0.3 per cent lactic acid calculated as acetic acid. In contrast to this, the action of the enzyme solution was stopped.by two per cent salt and.by 1.1 per cent acetic acid or 0.7 per cent lactic acid calculated as acetic acid. Salt apparently had little effect upon the enzymes until a two per cent concentration was reached since the effect of a combination of acid and salt was practically the same as acid - alone. However, at a two per cent concentration the salt effect predominated over the acid effect as the results were the same as with salt alone. Cytological Studies -13- INTRODUCTION .A study of the cytologic changes brought about by the action of the enzyme solution as produced.by the gggillug mesenterigug group of bacteria was deemed necessary for the completion of this work. It has been generally assumed that soft pickles were due to the dissolution of the middle lamella without adequate proof. The object of this study has'been to determine and.prove the exact cause of softening in pickles as produced.by this group of organisms. Pickles that had been treated in a great variety of ways were also studied. -1)4- REVIEW OF LITEEATUIE The close association of pectic materials with the cell walls and middle lamella of plants has long been noticed. One of the first workers in this field was Frémy (18146-1881), who investigated the structure and. chemical constitution of cells. He dissolved out the cell walls with Schweizer's reagent and identified the framework left by use of alkalies and acids. He put forward the suggestion that there was an insoluble pectose which changed to a water soluble pectin upon the ripening of fruits. Closely following Frémy, Mangin (1888-18914) published a series of researches on pectic materials. He used in his earlier work such basic stains as phenosafranin. methylene blue. Bismarck brown, Victoria blue and others for staining pectic substances. For cellulose in neutral solution. he used acid green. acid brown, nigrosin, indulin. the croceins, the ponceanx, Congo-red. azo-blue. and benzopurpurin. By using double stainings, he obtained instruc- tive data on the constitution of cells. Later, he employed ruthenium red. a compound investigated by Joly (1890-92) along with applications of chemical reagents to confirm the results obtained by staining. He believed as did Frénw that the ripening of fruits was accompanied by a change of the insoluble pectose to the soluble pectin. Also, he believed the lining of the air spaces and middle lamella were composed of calcium pectate. Allen (1901) investigated the structure of several woody tissues by using the stains and methods of Mangin. He believed that the pectic materials in the middle lamella did not act as a -15- cement,but were formed by a secretion of pectic material from the cell walls which acted as a plastic material in order to allow the cell walls to expand or contract during growth. Then after growth was complete, it was drawn back into the cell walls as insoluble protopectin. It is known that no stain is absolutely specific for any tissue. Therefore. to gain a clearer idea of the staining capacities of different tissues, Mehta (1925) conducted tests showing the relative specificity of each stain. She isolated alpha. beta. gamma cellulose, alpha. beta, gamma oxycellulose, hydrocellulose. mannan, galactan. pectin, amylocellulose, hemi- cellulose, gums, starch, lichenin and chiten in as pure a state as possible. Their intensity of staining with several different staining reagents was then determined. Ruthenium red which is generally thougit of as specific for pectic substances and their derivatives was also found to stain alpha. beta, gammaoxycelluloses, hemicelluloses, gums, galactans, free lignin, mannan and amylocellu- lose. However. this stain approaches the nearest to being specific for pectic substances as any stain yet found. Allen subsequently studied pitch pine by use of her stains and specific extracting agents. She was able to interpret her results better by using the knowledge obtained from.her pure staining experiments. Carre and Horne (1927) studied the microscopical changes occurring in apples and other plant tissues by using a combination of staining and chemical methods. The changes observed by using ruthenium red were correlated with changes produced by using specific —16. chemical extractions. By this method, they were able to definitely follow the changes produced in the pectic substances during senes- cence of fruits. The latest conception of cell walls as reviewed by Anderson (1935) pictures them as composed of a skeletal framework composed of cellulose. During cell division, a protoplasmic plate, known as the‘ggllgplatg. is formed between the daughter nuclei. There seems to be a difference of Opinion as to the exact method by which this first cell wall or middle lamella is formed‘but it is known to consist of pectic material. Alongside this pectic material. between the protoplasts of the adjacent cells. a layer of cellulose contain- ing pectic material is added.by each protoplast. This is called the primary wall or cambial wall and is different from the secondary wall in that it can undergo changes of growth and reversible changes in thickness. The secondary wall is formed from all the subsequent cell wall material deposited.within.the primary wall. and is usually added as definite lamellae. 'When stained with ruthenium red, this middle lamella composed of almost pure pectic material shows under the stain as a fine distinct line of red.between the cells. The cell walls show a light purplish color while the intercellular spaces may show as a.bright red if deposited pectic material is present. EXPERIMIWTAL WORK Pickles that had been treated in a great variety of ways were studied. Sections were made of fresh cucumbers before they were salted (See Section.A) as well as cucumbers that had been fermentedin salt brine (See Section B) and dill brine (See Section H). In addition to these. sections were made of cucumbers that had been softened by bacterial enzymes (See Section C), by acids such as hydrochloric (See Section D); acetic (See Section E) and lactic (See Section.F), and finally of pickles that had.been cooked.three times and preserved in acetic acid (See Section G). In this way pickles which.had‘been subjected to various chemical. physical, and biological agents were obtained in order to study the effect of these agents upon their pectic content. The technique of obtaining very thin sections of cucumber tissue was that of the paraffin method. For our purpose, the desired sections of pickles were cut about one-fourth inch thick and placed immediately into 50% alcdhol. Every six hours, the strength of the alcohol was increased successively from 50 to 70 to 95 per cent until finally the sections were in absolute alcohol and completely dehydrated. Following this. the alcohol was gradually replaced by xylol. Paraffin was then added. a few shavings at a time, until the solution was saturated” The xylol was allowed to slowly'evaporate by placing the opened'bottles in an oven at 55°C, which left the sections imbedded in pure paraffin. The sections were then cast in paraffin blocks, trimmed. and cut into sections 12 microns thick with a microtome. The resulting very thin sections were fixed on glass slides by means of Meyer's fixative -1 g- and two per cent formalin. The sections on the slide were prepared for staining by successively treating with xylol to remove the paraffin, absolute alcohol to remove the xylol, 70 per cent alcohol to partially remove the alcohol and water to remove all the alcohol. This rendered the section in such a condition that it could be treated with water soluble agents. It was very essential that comparable results be obtained in this study. Therefore, as a control on the results obtained by staining with ruthenium red. a specific extracting agent for pectic substances was used. By treating similar tissue from the same plant on the glass slide with 0.5 per cent warm ammonium oxalate and subse- quently staining with ruthenium red, a definite comparison could be made .with the untreated tissue. It was thought advisable to check these results by using an entirely different method for extracting the pectic material. - A solution of N/30 HCl was added to normal sections followed by a warm solution of N/lO NaDH. These sections were also checked with ruthenium red. It was necessary that all the sections be treated alike with the stain to insure comparable results. Consequently, all the sections were placed in a beaker of the ruthenium red (1:5000) and heated four times to approximately 70 - 80°C for five minutes. The slides were then placed in Coplin jars and successively treated with 70 per cent alcohol. 95 per cent alcohol. absolute alcohol, xylol and mounted with neutral balsam. -19- DISCUSSION OF RESULTS The main point of interest in this study was centered around the results obtained in the pickles softened by bacterial enzymes (See Section C). These sections showed up very plainly that it was the pectic material between the cells that was being dissolved away. Partially softened sections offered a very interesting study. The larger cells in the middle of these sections still showed pectic substances present as indicated by the ruthenium.red, while the smaller cells nearer the outside of the section and the epidermal cells showed the absence of pectic material. These sections compared very well with sections that had been extracted for pectic substances. The middle lamella of normal sections as stained by ruthenium red was hardly perceptible due to the closeness of the cells to each other. It could be observed quite prominently though in the intercellular spaces. The cells walls were a blue-green color which was attributed to the cellulose. The protoplasmic contents were in a normal condition with the nucleus showing clearly. The sections of salted pickles gave a very distinct middle lamella between the cells as the cells were pushed further apart. This could.be accounted for'by a slight hydrolysis of pectin rendering it more hydrophilic. Mangin and other workers, in order to make their sections show the middle lamella more clearly with this stain. treated themywith acidpalcohol before staining. Evidently salting and the subsequent fermentation has the same effect according to the results secured.here. The protoplasmic contents of the cells were plasmolyzed in all but the normal pickles due to the previous -20- drastic salt treatment. It was observed in all the sections that the epidermal cells and the cells nearer the outside showed a higher pectic content than the larger cells in the center. This no doubt was due to their relative size. The seeds apparently contained a large amount of pectin as shown by this stain. This is perhaps due to the small size of the cells. The sections extracted for pectic substances and the sections acted upon by - bacterial enzymes showed no evidence of pectic materials as indicated by this stain with the exception of the partially soft- ened pickles or partially extracted sections and these showed a gradation. The pickles that had been put up in 2.2 per cent hydrochloric acid calculated as acetic acid were very soft (See Section D). The softness in this case was due to the action of the strong acid and not to a bacterial cause. When these sections were observed under the microscope. a large amount of pectic material was observed which was probably due to a splitting out of the protopectin from the cell walls combined with a hydrOphilic effect. There was noticed also a purplish red concentration of color adjacent to the cell walls which was considered to be the protopectin in the process of being split out. This is a type of softening produced by the hydrolysis of the insoluble protopectin from the cell wall to form a soluble pectin by an acid and is basically a structural softening produced by a partial breakdown of the cell walls to a more soluble substance. This is a very extreme case of acid hydrolysis but it gave us a means of comparison for the action of weaker acids. The evidence of pectic material in normal pickles or cucumbers is less than that found in the salt stock or processed pickles. The acetic _2]_- acid and lactic acid show a slight hydrolysis as compared to the normal pickle. This hydrolysis becomes quite pronounced as evidenced.by sections of two year old pickles (See Sections E and.F). No definite difference between the action of lactic acid and acetic acid could be observed. Cooked pickles showed a pronounced.hydrolysie in addition to a wrinkled appearance of the cells, (See Section G). One of the cooked pickles which had become soft was examined and the pectin found to have been dissolved out. It is thought that this preliminary hydrolysis of the cell walls by acids is a step in softening by ren- dering the pectin more susceptible to enzymic action. -22- PRACTICAL.APPLICATIONS It will be seen, therefore, that softening in pickles may be due to one of several causes or a combination of them. For example, cucumbers that had not been properly salted thereby permitting the mesenteric group of bacteria to grow sufficiently to produce enough pectic enzymes to start the decomposition of the pectic materials might not show evidences of softening for several months due to the slow action of the enzyme in the presence of salt and acid. .Again these enzymes might be present but temporarily held in abeyance due to the acid content but when the acid was reduced by Mycoderma would start acting again. The action would.be slow but after a year or so would cause slips or softening. Another possibility is that the protopectase produced.by the bacteria would reduce the protopectin to pectin where it would be more easily hydrolized by the acids; or the pectase might carry the hydrolysis to pectic acid.where acid.hydrolysis would be more easily accomplished. It is evident from.these results that the mesentericus group of bacteria are capable of secreting prot0pectase, pectase and pectinase since there is no evidence of any of the three forms of pectin left as judged by ruthenium.red. Another condition that might result is after the pickles have been salted and ready for processing, they might be left in the freshening water long enough for bacteria to grow and start the softening process. These pickles are then placed in acid during the finishing process and finally stored in acid. If they were left in acid sufficiently long. the action of the acid on the protopectin plus the action of the'bacterial enzyme on all the -23- pectic substances would most certainly weaken the structure of the pickle. There is no fundamental reason why genuine dill pickles should not keep almost indefinitely. Genuine dill pickles which were made in 193“ and after six weeks' fermentation in a closed barrel were packed in glass jars in the original brine. These dill pickles are as firm in texture today (1937) after three years as the day they were placed in the Jars. There is no reason why genuine dill pickles properly handled and packed should not keep for several years at least. Yet the industry has trouble in keeping them from.August or September until early summer of the following year, a period of six to nine months. The acidity and salt concentration are such that they do not preclude the growth and action on the mesentericus group under certain conditions. I These experiments also emphasize the inadvisability of cooking pickles. Cooking causes a certain amount of disintegration of the cellular structure depending upon how long cooking is continued and the degree of heat applied. Placing the cooked pickles in an acid solution tends to further disintegrate the cell structure. Finally there is the action of the acid itself. These experiments demonstrate that if the acids are permitted to act long enough on pickles, they will cause a certain amount of cellular deterioration. This is evidenced'by the loosening of the protOpectin from the cellu- lose. This does not necessarily result in a slippery pickle at once but if permitted to act over any length of time results in a soft pickle. Pickles that have undergone acid deterioration retain their normal shape in the container but when you attempt to pick them up they crumble between your fingers since the adhesive material -21; has disappeared or been reduced. The eXperiments on enzymes indicate that if any enzymes are introduced into the finished product that they would almost surely spoil because the acid content would only tend to slow them up but would not stop them. If two to three per cent salt were left in them. this possibly would stop or greatly reduce their action. 1. -25- SUMMARY OF RESULTS Bacteriological An organism was isolated from.a barrel of spoiled pickles which corresponded in all essential details to Eagillug mesentericus fggggs, This organism.when inoculated into pickles was capable of causing "slips" inside of six to twelve hours and "mushy" pickles within twelve to twenty-four hours The salt tolerance of this organism was higher than that formerly reported by other investigators. It grew readily in concentrations of salt up to and including nine per cent. It could.be induced to grow in salt concentrations up to and including eleven per cent It was shown that the type of medium.had an important bearing on the salt tolerance of not only this organism but also upon organisms in this same group. It was demonstrated.that not only this organism but also stock cultures of'g, gulgatus, 3;. mesenteziggs, 5- mesentezigig M. and E. W. grow in higher concentrations of salt in a special beet molasses medium than has been reported by other workers for nutrient broth” In nutrient broth.the limit of salt tolerance was six per cent while in a special beet molasses medium.they grew in eleven per cent salt. The limit of acid tolerance for 3.- W M was 0.15 per cent acetic acid and.0.20 per cent lactic acid upon the initial inoculation, but it could.be induced to grow up to 0.20 per cent acetic and 0.3 per cent lactic acid. 10. -26- Zymological It was demonstrated that all the members of the mesentericus- megatherium.group with which we worked produced protopectinese, pectase and pectinase as Judged.by cytological studies on the decomposed pickles. The optimum.temperature for the action of these enzymes is 37°C, Above this temperature there was a gradual diminution in action until the enzymes were finally inactivated. The salt tolerance of the enzymes was not as great as that of the organisms which produced them. The action of the enzymes was inhibited by a two per cent concentration of salt whereas the organisms grew in eleven per cent salt. The inhibiting action may have been due to a "salting out“ effect. The enzyme solution showed a marked resistance to the acids as compared to the organisms which produced.them. The enzymes were capable of causing slippery pickles in one per cent acetic acid and 0.6 per cent lactic acid within five days. Salt in combination with acids apparently has little effect upon the enzymes until a two per cent concentration is reached since the results secured with a combination of the two were practically the same as with acid alone.' However, at a two per cent concentration the salt effect predominates over the acid effect since the results were the same as with salt alone. 11. 12. 13. 11+. 15. 16. -27- Cytological Sections of normal pickles stained.with ruthenium red showed the presence of pectic materials in the middle lamella. Sections of pickles that had cured in salt and dill brines showed no diminution of pectic substances as compared to normal pickles when stained with ruthenium red'but a larger amount. It is believed.that this is due to a change in the pectic materials during the fermentation of the pickles rendering themnpre hydrophilic. Sections of pickles that had become"slippery” or "mushy" by the growth of bacteria of the mesentericus-megatherium. group in a solution in which.they were placed showed a dimimution of the pectic materials in the case of the "slippery” pickles and its complete absence in the case of the ”mushy” pickles. Likewise, sections of pickles placed in an.enzyme solution in which the bacteria had been excluded showed the complete absence of pectic materials when stained with ruthenium.red. Sections of pickles which had been extracted with ammonium oxalate, a specific extracting agent for pectic substances, when stained with.ruthenium red showed the complete absence of pectin and was similar in all respects with the sections acted upon by enzymes. - The pickles which.had.been put up in 2.2 per cent hydrochloric acid and had.become soft were found to have a superabundance of pectic material due to the hydrolysis of the protopectin in the cell walls to a more soluble form of pectin. Sections -28- of pickles which had been put up in acetic and lactic acid and held for two years were found to have undergone a structural weakening. as indicated by ruthenium red but not to such a marked degree as those in hydrochloric acid. 17. Cooked pickles put up in acetic acid were examined and found to wr/n/r/ec/ appearance have undergone a structural weakening plus a partial m of the cells. -29- CONCLUSIONS The results of the experiments would indicate that there are at least two types of softening commonly occurring in pickles. l. Softening due to enzymes which completely dissolve the pectic materials present in the middle lamella of the cucumber cells. 2. Softening due to acid hydrolysis which loosens the proto- pectin from the cellulose of the cell walls thereby causing a weakening of the cell structure. TABLE 1 The Effect of Sodium Chloride. Acetic, and lactic Acids upon.Bacillus mesentericus fuscus +odium Chloride Lactic.Acid Per cent __gime - Time 505-11111! ours a 3 Per Cent Hours Day Chloride 2 hrs hr 3 Lda Lactic hrs rs 3 733 ' cid * 6 ‘e + + + ‘L‘ 7 + * * * 0.05 + + + + 8 + +. + + 0.10 + + + + 9 ' + + + 0.15 - + + + 10 ' ‘ ‘ ' 0,20 - - +. t 11 - - - - 0 1 '25 - - - ‘ 2 - - - - 0.30 C -I- - u- 9 inoculated 0.20 in, into 10 - - + -+ oculated 10 inoculated into 0.211. - + + into 11 - - - + 0.25 inp oculetedi 11 inoculated 1 t 0 0 _ _ - into 12 - - - - n o '3 + 0.30 in. Acetic Acid oculated into 0.3 - - - - Per Cent Acetic Acid 0-05 + + + + 0.10 - + + + 0.15 - - - + Oew "' "' " - 0.25 - - - " 0.30 " " .‘ " Gel-5 inocu" lated into 0.20 " - " + 0.20 inocu- lated into 0.25 - - - - * Calculated as acetic acid - a no growth +8 growth TABLE III Acid Plus One Per Cent The Effect of Sodium Chloride in Combination with Acetic and Lactic Acids on the Activity of Bacterial Enzymes Dissolving Pectic Substances in Pickles Acid Plus Two Per Cent Ch ri Sodium Chloride 21:. us 4 5 ._ Control M I! H M Control I! M M M Per cent Per cent acetic acid acetic acidf added ‘ added 0.1 S M I ii 0.1 - - - - 0.2 S M M M 0.2 - - - - 0.3 S M M H 0.3 - - - - 0. S M I! H 0. - - - - 0.5 S S u M 0.5 - - - - 0.6 s s s u 0.6 .. - - - 0.7 - S S S 0.7 - - - - 0.8 - S S S 0.9 - s s s 1.0 - - - S Per cent Per cent lactic‘aci 1actic'acid added added 0.1 S M I M 0.1 - - - - 0.2 S M M M 0.2 - - - .. 0.3 s M n u 0.3 - '- e- .— 0. S S S n 0. - - - - Oe5 " S S S 0.5 - - - - 0.6 - - s s 0.6 - - - .. 0.7 c— .. "u- - 0.7 I «- - - .- * Calculated as acetic acid - = no action 3 " 8111313017 M " Section A Section B Section C Section D KEY TO DRAWINGS Drawings from.Histologic Sections Stained with Ruthenium.Red Viewed with Diffused Light Magnified 720x Section of a normal pickle before salting Section of a normal pickle after salting Section of a salted pickle acted.upon by enzymes Section of a pickle put up in 2.2 per cent hydrochloric acid. (Two years old) p - pectic material stained red (when present) s = intercellular spaces 0. w. = cell wall of parenchymatous cells n = nucleus I-C'. asecnou c . . sec-nos o . all! u .l .- ‘C‘.i\tt:m¥...\\ , . i . I v. . I . . . . . . . ._ . . . . . . 17 | bin.. 1.. u , - . - n - . so . . . .. .. .n...: . . - . . -. .. . . . . . a... . .‘...¢ .. . ,. o.... . . . u. _ 1:, ....~ . . 1 ... . , .e .2 . . It, k f:r~tEiIrR.—L. .... nus-ll!!! thefdii. Section E Section F Section G Section B KEY TO DRAWINGS Section of a pickle put up in 2.5 per cent acetic acid. (Two years old) Section of a pickle put up in 2.” per cent lactic acid. (Two years old) Section of a pickle that had.been cooked three times and put up in acetic acid. (Two years old) Section of a genuine dill pickle." ’ pectic material stained red (when present) ‘6 I ' intercellular spaces c. w. = cell wall of parenchymatous cells n = nucleus .‘mr . t. ° SCC'HON Q. ‘ °S€CT\ON G° °§£CT\ON H ° BIBLIOGRAPHY Aderhold, R. (1899) Untersuchuagen fiber das Einsauern von Fruchten und Gemusen. Cent. fur Bakt. 5 (2): 511. Allen, C. E. (1901) On the Origin and Nature of the Middle Lamella. Chicago Bot. Gaz. L: 1. Anderson, D. B. (1935) The Structure of the Walls of the Higher Plants. Bot. Rev.. J. : 52. Carre, M. H., and Horns, A. S. (1927) An Investigation of the Behavior of Pectic Materials in Apples and other Plant Tissues. Ann. of Bot.. It]. : 193 . Frémy, M. E. (18113) Memoirs sur la maturation des fruits. Annales de Chem. et de Physical. £13; (3) : l (1859) Recherches chimiques sur la composition des cellules. J. de Pharm. et de Chim. 35 (3) : 81. 15 (3) :5. (1876) Methods générale d'analyse du tissu des végétanx. Compt. rend., £1 : 1136. and Urbaine. (1881) Etudes chimiques sur la squelette des végétaux. I bid.. 153 z 926. Hof, T. Joly, A. Jones, L. B. Joslyn, M. A. Kossowicz, A. (1891) (1892) Btude historique et critique sur la presence des composes pectiques dans les tissue des végétaux. J. de Botanique, 5 : 1100 and 1410.6 : 13. (1892) (1893) Propriétés st- réations des composes pectiques. ’ Ibid'.... 5; : 207 and 235, 1 : 37 and 121 and 325. (189+) Sur un essai de classification des mucilages. Bull. (19 le,Soc. Bot. de France EL. (1935) Investigations Concerning Bacterial Life in Strong Brines. Extrait du Recueil des Travaux botaniques néerlandais 3_2_ 3155- (1890) (1892) Compose’s anmoniacaux derives du- sesquichlorure du ruthénium. Compt. rend., in : 969, m : 1299. (1909) Pectinase, the Cytolytic Enzyme produced by W W and certain other Soft- rot Organisms. N. Y. Ag. Exp. Sta. Bull. No 11. Geneva. Tech. (1928) Some Observations on the Softening of Dill Pickles. Fruit Products J. a (s) : 19, 5 (9) : 16. (1908) Bacteriologische Untersuchungen fiber das Weichwerden eingesauerter Gurken. Zeit. fur Landwirtschaftliche's Versuchiwesen in Oesterreich. .11: .. ~ LeFevre, E. (1919) Salt Tolerance of Certain Rot Organisms. Absts. of Bact. 3 : 3-11» Lesly, B. E. and Cruess, w. v. (1928) The Effect of Acidity on the Softening of Dill Pickles. The Fruit Products J. and Am. Ving. Ind. 1 (10) 12. Mangin, L. (18118) Sur la constitution de la membrane des végétaux. Compt. rend., 191 : 11114 (1889) Sur la presence des composes pectiques dans 1es végétaux. Ibid.. .129. : 579. (1890) Sur la substance intercellulaire. I_bid.. 119 3 295 and 6W. (1890) Sur les reactifs colorants des substances fundamentales de la membrane. I bid” ill 3 120. (1893) Sur 1'emploi de ruthenium en anatomie végétale. I bid., 116 : 653. Mehta, M. M. (l925) Histolog of Cell Wall Constituents. Biochem. J. 19 z 979 Hahn, 0. (1913) Bacteriological Studies of brine pickles. The Canner and Dried Fruit Packer. Nov. 20 and 27. Trevethick, A.,Robinson, B. B., Snyder, R. M. (1928) Studies in Flax Betting. Mich. Agr. Exp. Sta. Tech. Bull. 95 Van Tieghem, P. (1879) Sur la fermentation de la cellulose. Compt. rend., 85, : 205. Vignal, W. (1889) Contribution a l'etude des bactériacées. Paris. .3 I- Y .r . L 0 .1- . E A .- S U . - nu a. U a...”- .. e. » \_ _.( J I; a... . .w. I . 'fiafi. eWA \IWV \’ . 9 .l “v: . e... n. .. a». r . . ... . or. - . 1;. a. A... . a . .1 . I a: