THE AVAILABILITY OF THE ESSENTIAL VITAMIITS AMD AMINO ACIDS FOR LACTOBACILLUS HLANTARUM IN CUCUMBER FERMENTATIONS By Ralph Norman Cojitilow 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 DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health 1953 ProQ uest Number: 10008285 All rights reserved INFORM ATION TO ALL USERS The quality o f this reproduction is dependent upon the quality o f the copy submitted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQ uest 10008285 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 TEE AVAILABILITY OF THE ESSENTIAL VITAMINS AND AMINO ACIDS FOR LACTOBACILLUS PLANTARUM IN CUCUMBER FERMENTATIONS By Ralph. Norman Costilow AN ABSTRACT 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 DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health Year Approved 1953 Ralph Norman Costilow 1 Lactobacillus plantarum. which is responsible for the desirable acid fermentation of cucumbers for salt stock, is known to require a numter of vitamins and amino acids for growth* Therefore, these essen­ tial nutrients must be available in cucumber brines for a desirable fer­ mentation to occur. If the concentration of any one or more of these nutrients was low enough to limit the growth of L. plantarum very little acid production would be expected. This study included investigations of the vitamin and amino acid requirements of L. plantarum isolates from cucumber fermentations and the availability in cucumber brines of those found essential* Ten commercial and three laboratory fermentations were characterized for microbiological activity as a basis for the nutrition study* The vitamin and amino acid requirements of L. plantarum were determined by titrating the acid produced by various isolates of this organism in lots of semi-synthetic or synthetic media deficient in the individual nutrients. Microbiological assay techniques were employed to determine the available concentrations in cucumber brines of the vitamins and amino acids found to be required by L. plantarum. Under commercial conditions the acid-forming bacteria attained their maximum activity in the first 3 to 6 days and declined throughout the re­ mainder of the fermentations period studied. Yeast populations declined during the first few days and then multiplied rapidly reaching their maximum in from 10 to 20 days after brining. The coliform organisms played no significant role in these fermentations. The microbiological activity in laboratory fermentations was similar except for the yeast picture; yeast activity was variable in these brines. The principal acid-forming Ralph No naan Costilow 2 organism was L. plantarum, and the most active yeast was Torulopsis holmii, However, in the laboratory fermentations and in one commercial tank Torulaspora rosei was predominant in the yeast flora* 1. plantarum isolates from cucumber fermentations were shown to require the vitamins— -biotin, niacin, and pantothenic acid; and the amino acids— leucine, isoleucine, valine, glutamic acid, cystine, trypto­ phane, and threonine in the basal medium used. The strains tested were variable in their requirements for p^-aminobenzoic acid, alanine, phenyl­ alanine, arginine, and tyrosine. Biotin, niacin, and pantothenic acid became available in cucumber brines very rapidly and the concentrations were relatively constant after the first 5 to 7 hays. No great influence of fermentation on the concen­ trations of these vitamins was evident. Although the amino acids were considerably slower in reaching their maximum concentrations in the brine than the vitamins, in most fermentations they were present in sufficient concant rat ions within 24 hours to support the growth of L. plantarum* Tryptophane was the only essential amino acid which was consistently affected by the fermentation. The level of this amino acid was markedly reduced in the brines at the same period when the yeast activity was greatest, and was also reduced by pure cultures of these microorganisms and by L. plantarum in control brine* Leucine, isoleucine, and valine were also rendered less available by the growth of brine yeasts in con­ trol brine and available cystine and glutamic acid concentrations were greatly lowered by the growth of a coliform isolate from cucumber fermen­ tations* Ralph Uonnan Costilow 3 The available concentrations of all of the nutrients studied were influenced by the size of the cucumbers; brines containing smaller cu­ cumbers were richer* The results indicated that the vitamins and amino acids were not limiting to L. plantarum in cucumber fermentations under these condi­ tions* However, in fermentations where Aerobacter activity is prevalent during the first few days, cystine concentrations might well be reduced to a critical level* Also, it was indicated that excessive yeast activ­ ity early in the fermentation might result in very low tryptophane con­ centrations. TABLE OF CONTENTS Page LIST OF FIGURES.............. iv LIST OF T A B L E S .......................................... vi ACKNOT&EDCMSNTS.......................................... ix INTRODUCTION............................................ 1 REVIEW OF LITERATURE..................................... 3 Microbiology of Cucumber Fermentations............. . 3 Vitamin and Amino Acid Requirements of 6 L. plantarum •. Nutrients Essential for L. plantarum in Cucumbers, Cucum­ ber Brines, and P i c k l e s .......................... 14 Effect of Brine Microorganisms on Vitamins and Anino Acids 16 EXPSRIMENTAL PROCEDURES AID METHODS........................ Fermentations Studied and Methods of Sampling ....... IS IS Chemical and Bacteriological Methods ................. 19 Microbiological Assays for Vitamins and Amino Acids 20 * . RESULTS.................................................. Microbiology of Fermentations ...................... Vitamin and Amino Acid Requirements of L. plantarum .. 21 21 50 Essential Vitamins and Amino Acids for L. plantarujftin Brines of Commercial Cucumber Fermentations....... 59 Essential Vitamins and Amino Acids for L* plantarum in Laboratory Fermentations and Non-fermented Control Brines.......................................... 69 The Effect of Various Microorganisms on the Vitamin and Amino Acid Content of Cucumber B r i n e s ............. 82 iii TABLE OF CONTENTS (Cont) Page DISCUSSION.................................... 91 SUMMARY........................... 95 BIBLIOGRAPHY............................... .9S LIST OF FIGURES Figure No* 1* 2. 3* 4. 5* 6* 7* 5. 9* 10. 11# 12. Page Average fermentations by acid-forming bacteria and yeasts in 10 commercial tanks of cucumbers................ 22 Average changes in acid and salt concentrations during the fermentation of 10 commercial tanks of cucumbers • 3^ Populations of various microorganisms in three fermenta­ tions under laboratory conditions.................. 35 Changes in acid and salt concentrations during the fer­ mentation of three lots of cucumbers in the laboratory 4o Estimated sequence of various yeast species in the fer­ mentation of cucumbers under commercial conditions . • 46 The response of L. plantarum 17-5 and of two strains of L. plantarum isolated from cucumber fermentations to L-leucine, EEL-isoleucine, and EL-valine............. 57 The response of L. plantarum 17-5 sad o f two strains of h ? Pl&atarum isolated from cucumber fermentations to L-tryptophane, L-glutamic acid, and L-cystine....... $8 Niacin, pantothenic acid, and biotin concentrations in the brines of two commercial cucumber fermentations* • 62 Leucine, isoleucine, and valine concentrations in the brines of two commercial cucumber fermentations. . . . 66 Tryptophane, glutamic acid, and cystine concentrations in the brines of two commercial cucumber fermentations 67 Comparison of the rates of diffusion and the maximum con­ centrations attained of niacin, pantothenic acid, and bio tin from three different sizes of cucumbers in non­ fermented brines ............................. 73 Comparison of the rates of diffusion and the maximum con­ centrations attained of leucine, isoleucine, and valine from three different sizes of cucumbers in non-fermented brines.......................................... J8 V LIST OF FIGURES (Cont) Figure Page Ho* 13. 14. 15* 16* 17* Comparison of the rates of diffusion and the maximum con­ centrations attained of tryptophane, glutamic acid, and cystine from three different sizes of cucumbers in non­ fermented brines................................. 79 Comparison of tryptophane concentrations and yeast activ­ ity in three laboratory fermentations............... Si The effect of various microorganisms on the available niacin, pantothenic acid, and biotin content of cucum­ ber brines.................................... . 8b The effect of various microorganisms on the available leucine, isoleucine, and valine content of cucumber brines.......................................... 85 The effect of various microorganisms on the available tryptophane, glutamic acid, and cystine content of cucumber brines................ ................. 86 LIST OF TABLES Table Ho. 1# 2. 3* b, 5* 6. 7* 8. 9» 10. 11. 12. 13. Page The amino acid requirements of Lactobacillus arabinosus 17-5 a-s noted by various workers................... Percent acid and salt and the populations of various groups of microorganisms in tank No. 13........... 10 23 Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 14.......... . . 2^ Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 21.......... 25 Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 22.......... 26 Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 23* . . . . . . 27 . Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 2b.......... 28 Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 3 ^ . ......... 29 Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 33........ . 30 Percent acid and salt and the populations of various groups of microorganisms intank No. 3^............. 31 Percent acid and salt and the populations of various groups of microorganisms in tank Ho. 35.............. 3^ Acid and salt concentrations and the populations of various groups of microorganisms in laboratory fermenta­ tion FQ 1........................................ 36 Acid and salt concentrations and the populations of various groups of microorganisms in laboratory fermenta­ tion FG 2......................................... 37 vii LIST OF TABLES (Cont) Table No. Page 14. Acid and salt concentrations and the populations of var­ ious groups of microorganisms in laboratory fermentation EC 3............................................. 3^ 15. Distribution of yeast isolates as to tanks and fermenta­ tion age * .................. . .................. *+3 16. Distribution of the various species of yeasts according to the fermentation from which they were isolated. . . *+5 17* Distribution of yeast species according to the time of fermentation when isolated ........................ 47 18. Yeast isolates from laboratory fermentations .......... 49 19. The effect of the omission of various vitamins from a synthetic medium on the acid produced by various strains of L. plantarum........................... 51 20. Response of L. plantarum to biotin, niacin, and panto­ thenate......................... 52 21. The effect of the omission of various amino acids from a synthetic medium on the acid produced by various strains of L. plantarum. ................................. 5I+ 22. Response of L. plantarum to six amino acids noted to be essential for this species........................ 56 23* Comparison of the concentrations of the amino acids and the acid produced by L. plantarum (L-15) in a complete basal medium to their concentrations and the acid pro­ duced in assay media............................. 59 24. Niacin, pantothenic acid, and biotin content of brines during the commercial fermentation of cucumbers. . . . 6l 25* Leucine, isoleucine, and valine content of brines during the commercial fermentations of cucumbers........... 64 26. Tryptophane, glutamic acid, and cystine content of brines during the commercial fermentation of cucumbers. . . . 65 viii LIST OF TABLES (Cont) Table No. 27- 28>. Page A comparison of the niacin, hiotin, and pantothenic acid levels in non-fermented and fermented brines covering cucumbers of three different sizes................ 7^ A comparison of the leucine, isoleucine, and valine con­ centrations in non-fermented and fermented brines cover­ ing cucumbers of three different sizes............ 76 29. A comparison of the tryptophane, glutamic acid, and cys­ tine concentrations in non-fermented and fermented brines covering cucumbers of three different sizes * . 30. Effect of various microorganisms on the vitamin and amino acid content of non-fermented cucumber brine....... 77 SJ ACKNOWLEDGMENTS The author is very appreciative of the counsel of Dr. P. W. Fabian* Dr. Fabian’s comprehensive knowledge of the practical and scientific as­ pects of cucumber fermentations was an invaluable asset to this study* The advise and instruction in microbiological assay techniques by Dr. R. W. Luecke and Miss Sally Wade of the Department of Agricultural Chem­ istry and by Dr. Samuel Rosen of the Department of Bacteriology and Public Health, Michigan State College were also of much value. The generosity and cooperation of the H. W. Madison Co. and of their repre­ sentative at the Mason, Michigan salting station, Mr. Ben Weaver, is also gratefully acknowledged* THE AVAILABILITY (KF THE ESSENTIAL VITAMINS AND AMINO ACIDS FOR LACTOBACILLUS PLANTARUM IN CUCUMBER FERMENTATIONS INTRODUCTION A large percentage of the cucumbers harvested yearly is preserved in the form of salt stock pickles until such time as they are needed. To prepare salt stock the cucumbers are placed in a large tank and cover­ ed with a brine ranging from 22° to 4o° salometer (5*5 to 10.5 percent salt) depending on the salter and the climate. The brine strength is increased gradually over a period of several weeks until a salt concen­ tration of 14.5 to IS.5 percent is reached. The osmotic influence of the brine solution withdraws the juice from the cucumbers which in turn supplies the nutrients necessary for fermentation. In a desirable fer­ mentation much of the carbohydrate material in the cucumber juice is converted to lactic acid by lactic acid bacteria. The resulting acid and the salt produces an excellent medium which preserved the salt stock for extended periods of time— as long as three or four years. The lactic fermentation of cucumbers for salt stock is dependent on the rapid utilization of sugars with acid production by the ls„ctobacilli. Many of the microorganisms introduced into the brine with the cucumbers are inhibited by the relatively high salt concentration. How­ ever, there are some haloduric and halophilic organisms which may com­ pete with the acid-fonners for the sugar available* The rapid growth of such organisms results in an undesirable type of fermentation. 2 Lactobacillus plantarum is the organism commonly responsible for the acid fermentation. This species is notably fastidious in its nutri­ tional requirements and is used in many microbiological assays for vitamins and amino acids* While there is no doubt that the nutritive elements in most brines are adequate to support the growth of this or­ ganism, it is possible that one or more elements may approach the crit­ ical level. Thus, in some cases an abnormally low level of proper nutrients may delay the development of L* plantarum to such an extent that an undesirable fermentation is obtained. It is also possible that the acid fermentation might be initiated in all fermentations more rapidly with less danger of spoilage organisms growing if only small amounts of some critical element were supplied initially. Besides the acid fermentation by Lactobacilli, definite gaseous fermentations due to Aerobacter and to many species of yeasts have been described. These organisms also may have a bearing on the nutritive elements available in the brine. In this work several commercial and laboratory fermentations have been studied throughout the acid fermentation period to determine the organisms active in them and the nutritive elements available to these organisms. The vitamins and amino acids found to be essential for L. plantarum have been considered in the nutrition study. REVIEW OF LITERATURE Microbiology of Cucumber Fermentations That Lactobacillus plantarum is the principal species involved in the acid fermentation of salt stock cucumbers has been well established by Etchells and Jones (l6) and Rosen and Fabian (51) • Ehe former work­ ers isolated and studied *+9 strains of acid producing organisms from small scale commercial fermentations. All strains were found to answer the description of the species L. plantarum. Nineteen isolates from laboratory fermentations ranging from 2 to 20 days in fermentation age were identified by Rosen and Fabian as L. plantarum. The rate of growth, total population attained, and the amount of acid produced by these organisms probably depends on a great number of factors. In studying the effect of various concentrations of salt, Fabian et_ al. (IS) observed that the population of acid producing oro ganisms reached a peak earlier and was higher in a 30 salometer brine (S percent salt) than in a Uo° salometer (lO.b percent salt) and the percent titratable acidity increased much more rapidly in the former. Etchells and Jones (15) a^d Jones and Etchells (29) using 20° (5*3 per­ cent salt) and. ^0° salometer brines obtained similar results. They reported that the titratable acidity reached a maximum of about 0.7 percent in 8 to 9 days in the lower brine concentration and attained a maximum of only 0.^ percent in the higher salt concentration after a period of 12 to 13 days. 1+ Other factors which have been investigated as to their possible influence on fermentation are bacteriophage, antibiotics, and changes in Eh (21). It was demonstrated by these workers that in certain cases bacteriophage from the soil, antibiotics produced by microorganisms, and/or low Eh values due to the growth of certain types of organisms may result in little or no lactic fermentation. The next most prevalent and pronounced phase of the cucumber fer­ mentation is that of the yeasts. The work of Etchells and his co-work- ers is notable in this field (11, 13)* In fermentations under Southern conditions (11), the first and most prominent yeast activity was found to be predominantly that of a highly fermentative, non-sporulating yeast which was classified as a new species; Torulopsis carol iniana^. This yeast became active in b to 5 days from the start of the fermenta­ tion, attained maximum populations of over one-million per ml in from 10 to 15 days, and died off very rapidly in 20 to 30 days. Following the Torulopsis. another non-sporulating species predominated; this yeast was also named as a new species— Brettanomyces versatilis^. Although this yeast never attained the high populations of the Torulopsis. it was found consistently throughout the remainder of the fermentation period up to 100 days at which time the study was concluded. Two other yeasts which were believed to play some role in the fermentation were Hansenula subpelliculosa and an unnamed species of Zygosaccharomyces. In a study of the Northern fermentations over a 3 year period, Etchells et_ al. (13) found a somewhat similar pattern. The largest variation noted from the Southern fermentations was that Torulopsis holmii dominated the early 5 fermentation period in place of Torulopsis caroliniana. However, the overall pattern of yeast fermentation in the northern brines was quite similar to that observed in the South* Torulaspora rosei^was believed to be active in a number of the fermentations but not all* In Rosen and Fabian*s work (51) with laboratory fermentations, no attempt was made to follow the predominating yeasts occurring in the brines since isolated colonies were picked at random. However, three species of yeasts were obtained that were found by Etchells et^ al. (13) to be common to Northern cucumber fermentations; viz.. Hansenula subpelliculosa, Torulaspora rosei. and Torulopsis holmii* A hydrogen fermentation due to Aerobacter which occurs soon after brining has been definitely established in commercial cucumber fermenta^tions in the South (l4, 15)* Eighteen of 20 isolates from such fermen­ tations were identified by Etchells et_ al* (l4) as Aerobacter cloacae and the other two were considered as varieties. These authors noted that Aerobacter activity occurred most consistently in brines of 60° salometer (15*8 percent salt), while they were active in only some of the brines having an initial concentration of 20° or Uo° salometer. However, the hydrogen fermentation was observed earlier in the brines of lower salt concentration than in the higher. Rosen and Fabian (51) noted an early Aerobacter fermentation in two laboratory experiments in L/ The Dutch authorities, Lodder and Xreger-Van Rij (M-O), have recla,ssified this yeast as Torulopsis lactis-condensi. S/as Torulopsis versatilis. and i / as Saccharomyces rosei. However, since this new classification scheme has not come into wide acceptance at this time, to avoid confusion, the original names will be used in this manuscript* 6 which the "brines had initial salt concentrations of 30° salometer* one instance a maximum population of ah out 5 x 10'* Per ^ in 2 days* In was reached These populations declined rapidly in hoth instances and were practically insignificant in from b to 5 days after "brining* Twenty-nine isolations were made at random of these organisms, and all proved to belong to the genus Aerobacter* Twenty-one isolates were identified as Aerobacter cloacae* In addition to the above types of fermentations, luxuriant films due to oxidative yeasts are always found where the brining tanks are not exposed to the direct rays of the sun and the surface is not disturbed* These yeasts have been studied extensively by Mrak and Bonar (Ub) and Etchells and Bell (12). In both studies, the predominating yeast grow­ ing on the surface of salt stock fermentations was found to belong to the genus Debaryomyces* Etchells and Bell (12) classified this yeast Debaryomyces membranaefaciens var. Hollandicus* Vitamin and Amino Acid Requirements of 1* plantarum Great progress has been made in the last two decades in the study of the nutritional requirements of microorganisms and their application to microbiological assay techniques for various vitamins and amino acids* The lactic acid bacteria have been the subject of many investigations due to their requirements for many of these nutrients. The species L. plantarum is one of the lactic acid organisms used most commonly in microbiological assays. This review will be restricted 7 l/ . to the requirements of this organism and its synonyms—7 Vitamin requirements. In a comprehensive review of the relation of bacteria to vitamins and other growth factors, Peterson and Peterson (*+7) noted that hiotin, niacin, pantothenic acid, and riboflavin were the most frequently reported as essential for microorganisms. All of these vitamins except riboflavin have been established as essential for Jl* Pl&fttarum. Thus, Snell and his co-workers (bO, 6l, 62) have demon­ strated the requirements of L. arabinosus 17-5 ^or nicotinic acid, panto­ thenic acid, and bio tin, Cheldelin et^ al (9) demonstrated that 33 strains of lactic acid bacteria including one labeled as L. plantarum. two as L. arabinosus. and two as L. pentosus required pantothenic acid* Rosen and Fabian (51) observed that 10 isolates of L. plantarum from cucumber fermentation all required biotin, niacin, and pantothenic acid as did L. arabinosus 17-5- Similar results were reported by Kreuger and Peterson (32) for L. pentosus 12^2, By increasing the concentrations of the vitamins and amino acids several fold over those commonly used and lengthening the incubation time up to about J days, Shankman et^ a l , (56) obtained greatly different results than those previously noted. Thus, it was observed that biotin, pantothenic acid, and niacin were not essential for L. arabinosus 17-5 when a 7-day incubation period was used but were necessary for maximum i r The 6th edition of Bergey* s Manual (5) lists 19 probable synonyms of this species including Streptobacterium plantarum. Lactobacil­ lus arabinosus. Lactobacillus pentosus. Lactobacillus brassicae. and Bacillus cucumeris fermentatae. Therefore, studies of the nutrient requirements of bacteria designated by these names are included in this review. s acid production during a short incubation period. With L. pentosus 12*4— 2 and L* hrassicae (80*4-1) nicotinic acid was still noted as essential after 7 days incubation and the acid produced in the absence of either hiotin or pantothenic acid was less than one-half that in the control* There are conflicting reports in the requirements of L. plantarum for p-aminobenzoic acid. Snell and Mitchell (5^) noted that this vita­ min was non-essential for L. arabinosus 17-5 an& f°r L. pentosus. while Isbell (37) an& Lewis (35) observed it to be essential for L. arabinosus. Krueger and Peterson (3^) found g^aminobenzoic acid to be stimulatory for L. pentosus when specially prepared casein was used. However, in the concentrated synthetic medium of Shankman et^ al. (56) the absence of this vitamin had no apparent affect on the acid production of either L. arabinosus 17-5 or B* pentosus 12*4— 2. Snell and Strong (59) noted that riboflavin was not required by L. arabinosus 17-5* B. pentosus 12*4—2, nor by L. brassicae (80*4-1). Similar results were obtained by Campbell and Hucker (8) with cultures labeled L. arabinosus P-17-5* Bacillus cucumeris fermentatae L-25, and two strains identified as L. plantarum. However, two other cultures labeled k* Plantarum would not grow in the medium regardless of the riboflavin content, and Lactobacillus plantarum var. rudensis was noted to require this vitamin. Bohonos at al. (3, *4-) reported that pyridoxine was not required by either L. arabinosus or L. pentosus but was synthesized by them* However, Shankman et_ al. (56) noted that both L. arabinosus 17-5 snd L. brassicae (80*4-1) were stimulated by pyridoxine when short incubation periods were used. Ho stimulation of L. pentosus was observed. 9 Neither thiamine or folic acid were noted ho he essential for L. arabinosus 17-5 Shankman et al (5^) or Baumgarten et_ al_ (l) . How­ ever, the latter workers have noted some stimulation by folic acid. L. pentosus did not require these two vitamins (3^, 5&)• Snell and Wright (62) observed that pyridoxine, thiamine, and riboflavin stimulated the growth of L. arabinosus 17-5 during the first few hours of incubation. Therefore, the addition of these vitamins to assay media for this organism was considered desirable. Amino acid requirements. The findings of the various workers on the amino acid requirements of L. plantarum are still more conflicting than on the essential vitamins. Starting with the report of Molier (*+5) who reported that Streptobacterium plantarum 10S required glutamic acid, leucine, aspartic acid and valine for growth in a chemically defined medium, practically every investigation has resulted in different obser­ vations as to the essential amino acids for this organism. Even on the strain of L. arabinosus 17-5 there have been considerable differences in the observations made in various laboratories. The results obtained in five investigations of the requirements of this strain are summarized in Table 1. The number of amino acids noted to be required by L. arabinosus 17-5 varied from 10 (2*0 to 5 (10) > but five amino acids (viz. glutamic acid, valine, leucine, isoleucine, and tryptophane) were noted to be essential in all five investigations. Cystine was found to be essential in all instances except when a greatly enriched medium was used (10). Even in this medium some of the data indicate that there was some 10 TABLE 1 The amino acid requirements of Lactobacillus arabinosus 17-5 as noted by various workers Dunn al. (10) Kuiken et ad. (33) Glutamic acid + Lyman Hegsted (2*0 ©t al. (^3) Shankman (55) + + + + Valine + + + + + Leucine + + + + + + + + + Isoleucine Tryptophane + + Cystine (or cysteine) + + + Methionine + - + - Arginine - - + - Phenylalanine t Tyrosine + Threonine Lysine et — + 2/ + - - - + - - 4" + - - - - + - - - 1/ A + indicates that the amino acidwas notedto be - that it was not essential. essential, and a 2/ -(The results of some experiments indicated these amino acids were required while in other instances they were not. 11 stimulation of acid production when cysteine was added. Riesen £t al. (*4-9), also noted that cystine was not absolutely essential for L. arabinosus 17-5 but was stimulatory* Such variations in results indicate that either variations occur­ red in the cultures or that differences in the methods of determining the amino acid requirements influenced the response of the organism. Strains of L. arabinosus which did not require tyrosine and others which did not require tryptophane were developed by James (28) and by Wright and Skeggs (71) respectively by growing on media deficient in these amino acids. Dunn et^ al. (10) have demonstrated that six different cultures of L. arabinosus 17-5 carried for more than two years on three different media all had the same amino acid requirements* Lyman e£ al* (*4-3) have conclusively demonstrated a relationship between the composition of the medium used for testing and the amino acids found to be essential. Thus, these workers observed that threonine, lysine, and alanine were essential for L. arabinosus 17-5 when pyridoxine was omitted, but not essential in its presence. Arginine, phenylalanine, and tyrosine were noted to be essential in the presence of pyridoxine if COg was not available, but not required in the presence of both vita­ min B£ and C02* However, pyridoxine was not required by this organism when all these amino acids were present. The requirements for valine, leucine, isoleucine, tryptophane, cystine, and glutamic acid were un­ changed by the presence of pyridoxine or of both CO^ and pyridoxine* Stokes and Gunness (6*4-) noted that pyridoxamine in a basal medium would eliminate the requirements of L. arabinosus 17-5» h* co>sei and L. delbruckii for lysine, threonine, and alanine but pyridoxine would not. 12 However, if a basal medium containing pyridoxine was sterilised at 15 pounds for 30 minutes similar results were obtained as when pyridoxamine was used. It was suggested that pyridoxamine may be formed dur­ ing the sterilizing process when pyridoxin© is present. These observa­ tions might well account for many of the conflicting results obtained in the various investigations. Impurities in individual amino acid preparations are another source of variation in results. Hegsted and Wardwell (26) have shown that some commercial samples of synthetic DL-leucine contained appreciable amounts of isoleucine. A similar observation was made on one commercial batch of L-leucine in this investigation (unpublished data). That there may be considerable variation between strains of the same species was demonstrated by Dunn et_ al. (10). Thus, one strain of Leuconostoc me sent ero ide s was found to require 15 amino acids while an­ other strain required only two. In this same report, it was noted that L. pentosus and L. brassicae had identical amino acid requirements; glutamic acid, valine, isoleucine, leucine and cysteine were essential for both. L. arabinosus 17-5, however, did not require cysteine, but required tryptophane and in some instances methionine and arginine. L. piant arum (strain Ho. 800U from the American Type Culture Collection) would not grow in the enriched medium used by Dunn et al. (10). Krueger and Peterson (32) reported valine, leucine, isoleucine, glutamic acid, and phenylalanine as essential for L. pentosus 124-2. Cystine, threonine, and alanine stimulated growth, but the omission of tryptophane from the basal medium had no effect on this strain. 13 In most instances, only the naturally occurring isomers (the In­ forms) have been found to be effective in promoting growth of L. plantarum. Thus, Kuiken et al. (33) have shown that only the isomers having the L-configuration of leucine, isoleucine, and valine were capable of promoting growth of L. arabinosus 17-5* Hegsted (25) obtained similar results except that some response to ^-leucine was noted after long in­ cubation periods. However, this response enough to influence the results of assays was not believed to be great for this vitaminwhen DL-leu- cine was used to prepare the standard curves# Snell (57) Greene and Black (22) noted that while only the In­ form of tryptophane was active with L. arabinosus. both indole and anthranilic acid would replace tryptophane, but that occurring substances could be readily removed extraction. from these two naturally samplesby ether Snell (57) observed that L. pentosus would not utilize either indole or anthranilic acid in place of tryptophane# Microbiological assays for glutamic acid using L. arabinosus 17-5 are not specific as glutamine has been shown to produce a greater growth response of this organism than glutamic acid (23, 36* ^1) • The D-form of glutamic acid was noted to have some activity in the absence of the naturally occurring isomer (4l). Pollack and Lindner (^) observed that glutamine would also replace glutamic acid for L. pentosus# Riesen et_ al. (4g) noted that L-cysteine had about the same activity as L-cystine for L. pentosus 12^2. Other sulfur containing compounds were also found to have some activity# ih Nutrients Essential for L. piantarum in Cucumbers. Cucumber Brines and Pickles Until recently, the only essential nutrient for the acid fermenta­ tion of cucumbers which had been investigated as such was reducing sugar# Thus, Fabian and Wickerham (20) noted that the addition of sucrose late in the fermentation resulted in an increase in the number of acid pro­ ducing bacteria# However, Jones et al# (30) Veldhuis et al. (67) demonstrated that while the addition of sucrose brought about an increase in the population of acid-forming bacteria, the titratable acidity formed in such brines was no higher than that in control lots# served in both dill and salt stock fermentations. This was ob­ Yeast populations were also noted to be higher in fermentations to which sucrose was added. From these results it would appear that some other factor was limiting the amount of acidity formed# Vitamins. Practically no information was available as to the con­ centrations of biotin, niacin and pantothenic acid in cucumbers and in the brines of fermenting cucumbers until the recent report of Rosen and Fabian (51) • to their importance to this investigation the results obtained by these workers are briefly summarized as follows: 1# In a study of the biotin, pantothenic acid and niacin available to L* arabinosus 17-5 the extracts of six different varieties of cucumbers the following observations were made I a. Considerable variation occurred in the vitamin concentra­ tion in the juice from different cucumbers but no signifi­ cant difference between varieties of cucumbers was evident# b. Biotin concentrations ranged from 5*2 to 33*0 njng/ml, niacin from I .83 to $.05 jpg/til and pantothenic acid from 1.05 to 2*42 jig/ml of cucumber juice# 2. The available concentrations of these three vitamins in the brines of two laboratory fermentations were studied through the first 20 days of fermentation* It was noted that the concentra­ tion of all three reached their maximum in the first 5 to 6 days, and did not greatly change thereafter* Sufficient vitamins were available throughout the fermentation to support the growth of L. plantarum* 3* The vitamin content of the cucumbers was greatly diminished after 20 days in brine. While no effort was made to extract the bound forms of these vit­ amins, the concentrations observed by Rosen and Fabian are those of importance to cucumber fermentations. According to the work of Lampen et al. (34), most vegetables have a water extractable form of biotin so the concentrations of this vitamin observed by Rosen and Fabian are probably close to the total concentrations. In a compilation of nu­ trient data for various foodsby the Heinz Co. ( f O ) the concentration of nicotinic acid in cucumbers is given as 0.2 mg* per lOOg (2 jug per g) this compares favorably with the data noted above* Amino acids. No information was found in the literature on the amino acid composition of cucumbers or pickles. However, Camillio et al. (7) noted the crude protein content of various cucumbers to vary from 0.7 to 1.4 percent, and this was reduced significantly after fer­ mentation for salt stock or dill pickles. In the nutritive charts pre­ pared by the Heinz Co. (70) the average total protein content of fresh cucumbers is given as 1.1 percent, while that for fresh cucumber pickles is 0.8 percent and for dill pickles is 0.7 percent. 16 Effect of Brine Microorganisms on Vitamins and Amino Acids While there has been considerable work on the synthesis of vitamins and some reports on the destruction of vitamins by microorganisms, most of these reports are not pertinent to this study as the activity of var­ ious species and groups of microorganisms are greatly different. This review is restricted to those investigations of microorganisms which are closely related to those found in cucumber fermentations. Since L. plantarum requires niacin, bio tin, and pantothenic acid for growth, it would be expected to lower the concentrations of these vitamins in the growth medium. Rosen and Fabian (52) noted, however, that while the biotin content of an assay medium was lowered greatly, the concentration of biotin in cucumber juice was not greatly affected by L. plantarum. Since it was found that Tween 80 had a biotin sparing action in the assay medium, it was suggested that cucumber juice may have a lipoidal substance present which is capable of substituting for biotin. The relationship of oleic acid and of Tween 80 (an esterified form of oleic acid) to biotin requirements of lactic acid organisms has been observed by Kodicek and Worden (3l)» Williams and Fieger (69), and Williams et_ al. (68). A number of investigators have noted that the coliform group of organisms are capable of synthesizing vitamins (6, 44, 66). However, Rosen and Fabian (51* 52) found that Aerobacter cloacae from cucumber fermentations would also destroy bio tin in cucumber juice and other media if a substantial quantity of biotin is present initially. This 17 organism synthesized this vitamin in the same medium when no hiotin was added. No appreciable effect of Aerobacter cloacae was noted on the niacin and pantothenic acid content of cucumber juice (51) - Various species of yeasts vary greatly in their vitamin require­ ments and in their ability to synthesize vitamins* In a study of 18 species of osnophilic yeasts, all belonging to the sub-genus Zygosaccharmyces. Lochhead and Landerkin (38) noted that all strains required bio­ tin and some required pantothenate for growth. Rogosa (50) noted that ll4 strains of lactose fermenting yeasts required nicotinic acid or nicotinamide. Torulopsis utilis was noted by Lewis ejfc al. (37) to synthesize significant quantities of biotin, niacin, and pantothenic acid in media such as fruit and prone juice, and molasses. Rosen and Fabian (51) studied the effect of six species of yeasts isolated from cucumber fermentations on the biotin, niacin and panto­ thenic acid content of cucumber juice. The growth of four species (viz. Kansenula subpelliculosa. Torulopsis caroliniana. Torulopsis holmii. and Torulospora rosei) resulted in a marked decrease in the biotin level. The niacin and pantothenic acid concentrations were not greatly affected although some increase in the concentration of the latter was noted with three yeast species. No information was found concerning the effect of brine microorgan­ isms on the various amino acids. However, since L. plantarum has been shown to require several amino acids for growth this organism must de­ plete the concentration of these amino acids in the growth medium. EXPERIMENTAL PROCEDURES AND METHODS Fermentat ions Studied and Methods of Sampling A total of 13 fermentations were studied— 10 commercial and 3 the laboratory. The 10 tanks of fermenting cucumbers under commercial conditions were located at the salting station of the H. W. Madison Co. at Mason, Michigan* All fermentations were brined within a 20 day per­ iod during the month of August 1952. The commercial tanks were of the regular wooden variety and ranged in size from J00 to 1100 bushels capac­ ity. Five tanks were filled with size No. 1 cucumbers and the other five were filled with a mixture of sizes N o 1s. 2 and 3 cucumbers. The 3 laboratory experiments were carried out in 5“Sallon crocks over which an ultraviolet light was used to prevent growth of film yeasts. The same variety of cucumbers, Davis, grown on the same plot of ground was used in filling all three crocks. Crock FC 1 contained size No. 1 cucum­ bers, crock FC 2 contained size No. 2 and crock FC 3 w &s filled with No. 3 size cucumbers. Samples of the brine were taken daily from all fermentations for a period of 15 days and then at increasingly greater intervals. samples of the laboratory brines were taken The last 36 days after the cucumbers were salted and from the commercial brines at from 51 to 71 days, depend­ ing on the tank, after the cucumbers were salted. The sampling technique used for the large tanks was essentially that recommended by Etchells and Jones (17) except that only three per­ forations (about 2mm in diamter) were made in the 3/l6-inch (inside 19 diameter) stainless steel tube. These perforations were spaced IS inches apart, starting at the sealed end of the tube. 6 feet long and the tanks about 7 to S feet deep. The tuhe was Thus, a composite sample was obtained including brine froip various depths of the tank. The laboratory brines were sampled in much the same manner except that a glass tube was used and the open end moved slowly up from the bottom to the top as the sample was being withdrawn. On removal from the tanks the brine samples were immediately re­ turned to the laboratory and chemical and bacteriological analysis made. The samples were then held at ~12>.5°C (0°F) until time was available for the nutritional studies which were made later. Qhemical and Bacteriological Methods Brine sub samples were titrated with standard NaOK to determine tit ratable acidity and with standard AglJO^ using dichlorofluorscein as an indicator to determine percent salt. The V-8 agar with brom cresol green indicator devised by Fabian et al. (19) was used in the enumeration and isolation of acid-producing bacteria, and dextrose agar acidified with 5 ^ acid per 100 ml of medium for yeasts. of 5 percent tartaric Lauryl-tryptose broth tubes were inoculated in triplicate with various dilutions of the samples to deter­ mine members of the coliform group. All incubation was done at 30° C* Three days incubation was allowed for the acid-formers, two days for the coliforms, and five days for the yeasts. 20 Microbiological Assays for Vitamins and Amino Acids This study was concerned with the vitamins and amino acids avail­ able in cucumber brines for the growth of L. plantarum. Therefore, the brine samples were not treated to release the bound vitamins or hydro­ lyzed to free amino acids from protein material prior to running the microbiological assays. The brine samples were removed from the freezer and allowed to thaw in a refrigerator at about 4.9° C just prior to running the assays. The acid in measured portions of the brine samples from fermenting cu­ cumbers was neutralized with N NaOH and the samples made up to volume with distilled water to give either a 1:5 o r 1:10 dilution as desired. This solution was stored in the refrigerator under toluene. Further dilutions were made at the time of the individual assays from this ori­ ginal as needed. The dehydrated media prepared by Difco Laboratories, Inc. were used to assay the samples for biotin, niacin and pantothenic acid. 17-5 was used as the test organism. L. plantarum The techniques employed in the assays were essentially the same as those described in "Methods of Vitamin Assay" (65). Assays for leucine, isoleucine, valine, and glutamic acid were run as outlined by Sauberlich and Baumann (5*0 using medium I and L. plantarntw 17-5 as the test organism. Difco* s dehydrated medium was used for tryptophane assays with L. plantarum 17-5* The method of Lyman et_ al. (*^) as modified by Sarkar et_ al. (53) was used for the determination of cys­ tine. Leuc. me sent ero ides P-60 was the test organism used in this deter­ mination. The individual amino acids used were obtained from either Nutritional Biochemicals Corp., Merck and Co., or Ffanstiehl Chemical Co. RESULTS Microbiology of Fermentations Sequence of organisms in the fermentations. With a few exceptions, the ten commercial fermentations studied were found to have similar pat­ terns with respect to acid-forming organisms, yeasts, and coliforms. The general pattern of acid-forming bacteria and yeast activity in the fermentations studied is illustrated in Fig. 1. This represents the average populations of the 10 fermentations at each time interval. The complete results of this study are given in Tables 2-11. In general the acid-forming bacteria count was found to rise sharply within 1 to 3 days after brining; reaching a peak within 5 to 6 days. The total numbers of these organisms declined rapidly for the following 5 to 10 days, and then continued to decline at a slower rate for the re­ mainder of the fermentation period studied. However, the maximum popu­ lations obtained in the various brines varied from 5*5 x 10^ (Table 6) to 1.29 x 10^ per ml (Table . Six of the ten fermentations had maxg imum populations ranging from 3 x 10 g to 6 x 10 per ml. In contrast to the acid-producing bacteria, the yeasts were gener­ ally found to decline in number during the first few days after brining, started rapid growth after about 5 days, and reaohed their maximum pop­ ulation in from 10 to 20 days of fermentation. steady decline in yeast population. Thereafter, there was a The maximum populations attained r were quite variable, ranging from 1.57 x 10 g to 2.6 x 10 per ml in tank 1^ (Table 3)* per ml in tank 23 (Table 6) 22 s a m -*> 05 cd o irt © ’’d a cd cd © a +3 § cd f*Q a *rH s CM • 01 £ u o © Vi1 ,o a nd 3 •rH o o P cd o >B V o © © o •H cd +> -cdP r H a ■cdH © H © Vi O u © e! o o u o © rH > © cd • c-1 -O 0> co GO Log. of IT) count per m l. • jO ro ?5J0 •H P*4 CM 23 TABLE 2 P e rc e n t a c i d and s a l t and th e p o p u la tio n s o f v a r io u s group s o f m ic ro ­ org an ism s i n ta n k No* 13 » Percent salt Percent acid as lactic 0 2/ 1 2 6.25 7.70 6.85 0.059 0.267 0.798 95*0 620.0 540.0 1*7 0.2 0.1 4,300,000 1,500 4,300 3 4 5 6.65 s.30 7-75 0.435 0.368 0.383 610.0 134.0 50.0 0.5 - 430 24 250 6 7 8 s. 50 8.55 s. 25 0*390 0.345 0.443 11.9 16.3 53-0 1.0 1.0 5-6 * 43 43 9 10 11 8 .4o s.50 8.70 0.480 0.510 0.510 44.0 34.0 10.6 7-5 3-6 4 .6 * * * 12 17 it 8.55 8.80 s.70 0.495 0.525 0.525 15.2 21,0 5-7 3-3 2-3 0.9 * * * 15 17 19 9*10 8.90 9.30 0.503 0.615 0*555 5-4 8 .8 13.2 l4 .o i4o.o - ♦ * * 21 24 29 9*55 9*70 10.00 0.533 0.465 0.503 16.4 15.1 4 .6 310.0 51.0 4.2 * * * 36 43 50 10.20 11.15 10.90 0.435 0.443 0.420 4.1 0-9 0.3 2.0 3*o 1.7 57 64 11.20 11.00 0.450 0.437 0 .14 0.004 2.7 Timedays 1/ 2/ Acidformers x 10°/ml Yeasts x lO^/ml Coliforms No./ml - — Tank No. 13 contained 718 bushels of No* 1 size cucumbers* The zero time sample was taken eight to ten hours after covering the cucumbers with brine. * = Less than 10/ml. - = No determination made. 24 TABLE 3 Percent acid and salt and the populations of various groups of microorganisms in tank No. l W Percent salt Percent acid as lactic 11*10 b. 60 0.00s 0.00s 0.1 7*50 0.015 0.3 *** 9,200 3 i+ 5 S.95 s.4o 0.045 0.135 0 .21S *•3 1.0 125.0 360.0 *** - 25,000 9,200 250 b 7 s S.40 9.95 0.30S 360.0 0.210 10*90 0.255 95*0 SO.O **** **** **** *** ** ***** ***** s .90 s .50 9-20 0.375 0.405 0.1 0.5 0.360 ***** ***** ***** 13 i4 9.65 9-65 0.330 0.405 10.15 15 17 19 21 Timedays 0 2/ 1 2 9 10 11 12 2k 28 9.10 * 6S .0 2S.0 14.5 Yeasts x 103/ml ** 12.0 - Coliforms No./ml 43,000 920,000 5.4 29.0 0.345 2. S 2.5 119.0 ***** ***** ***** 9-50 9-b5 9-bO 0.450 0.42S 0.495 2.S 3*4 1*3 590*0 2,600.0 680.0 ***** ***** ***** 9.35 10.95 11.05 0.4SS 0.390 0.465 0.72 310.0 250.0 ***** ***** - 10.70 0.44 O .25 49 10.75 11.10 0.54S 0.525 0.563 5b 10. SO 0.555 35 42 Acidformers x 10^/ml 1.4S 0.64 54o .o 93*0 1.04 23.0 mm 0.70 1.3 mm 0.60 - 0.1 - Tank No. contained 706 bushels of No. 1 size cucumbers. The first sample was taken one to three hours after the cucumbers were covered with brine* * = Less than 10 million/ml* ** = Less than 10 thousand/ml. *** = Less than 1 thousand/ml* **** = Less than 100/ml. ***** = Less than 10/ml. = No determination made* 2/ 25 TABLE 4 P e r c e n t a c i d and s a l t and th e p o p u la tio n s o f v a r io u s group s o f m ic ro ­ o rg an ism s i n ta n k Ho. 21 1 / Timedays o £7 i Percent acid as lactic Acidformers x 10°/ml S. 60 7-95 7-95 0.008 0.128 3 b 5 6.60 S .10 0.323 0.375 7.95 0.368 6 7 8 S.20 8.80 s .25 0.420 0.405 118.0 .3 9 s 87.0 p 0.263 0 Yeasts x 10^/ml 1,290.0 1,060.0 520.0 540.0 l?*3 1 920 5*6 215*0 850.0 Coliforms No. ./ml 0.1 * 4,300 43,000 * * * 920 ** * 0.49 5*0 5*9 430 43 43 25 *** 250 *** *** 8.35 0.323 8 .3O 8.3O 0.428 0.443 14.7 15.0 8.45 8.96 0.450 0.465 lb 8.45 0.495 17.1 22.0 20.0 1,290.0 *** 15 17 19 8.80 9.00 9-10 0.488 680.0 830.0 350.0 *** *** 0.510 7*2 12.0 11.4 21 24 30 9.10 9*55 9.90 0.548 0.525 0.518 11.2 8.1 2.6 79*0 24.0 6.8 37 44 51 9*90 9*90 10.20 0.525 0.525 0.570 0.28 0.19 0.028 0.30 0.9 0.1 - 58 10.20 0.533 0.10 0.8 — 9 10 n 12 13 1j 2/ Percent salt 0.510 19*0 480.0 — 25 *** *** *** *** - Tank No. 21 contained 71? "bushels of No. 1 size cucumbers. The first sample was taken six to eight hours after the cucumbers were covered with brine. * = Less than100/ml. ** — Less than1,000/ml* *** = Less than10/ml. — No determination made. 26 TABLE 5 Percent . groups of microacid and salt and the populations of various 1 0rgani sms in tank No. 22 y Percent salt Percent acid as lactict 0 2/ 1 2 9.35 4.25 8.00 0.015 0.173 0.195 1-7 440.0 490.0 3 .0 4.0 0.5 3 4 5 8.60 8.30 8.70 0.248 0.315 0.360 4oo.o 249.0 118.0 * 6 7 8 8.60 s.30 8.30 0.360 0.353 0.413 137.0 38.0 40.0 * * 280.0 9 10 n 8.40 8.20 8.50 0.428 0.420 0.428 48.0 11.2 13.8 64.0 320.0 12 1? 14 9.10 9.25 8.60 0.435 0.390 0.435 12.2 1.9 2.2 2,400.0 123.0 1,360.0 15 17 19 8.45 9.00 9.20 0.533 0.518 0.510 2.6 4.1 5.4 1,170.0 500.0 23.0 ** ** 21 2k 28 9-50 10.10 9.85 0.510 0.480 0.443 6.6 6.3 4.8 44.0 4.9 0.9 ** ** 35 42 49 10.50 10.80 10.80 0.375 0.383 0.390 0.93 0.45 0.34 0.5 0.2 3.2 mm - 56 63 11.40 10.85 0.390 0.375 0.13 0.22 1.0 0.21 - Timedays 1J 2/ * ** - Acidformers x 10°/ml Teasts x lO^/ml - * Coliforms No ./ml 9,200,000 2,500,000 43,000 2,500 ** 250 43 25 ** ** ** ** ** ** ** Tank No. 22 contained 702bushels of No. 1cucumbers. The first sample was taken five to eight hours after the cucumbers were covered with brine. = Less than 100/ml. = Less than 10/ml. = No determination made. 27 TABLE 6 P e r c e n t a c id and s a l t and t h e p o p u la tio n s o f v a r io u s groups o f m ic ro ­ o rg an ism s i n ta n k N o. 23 If Timedays 0 J/ * ** 10.68 0.034 0.045 1.4 b.3 1.6 0.062 24.3 1.2 2.0 4, 300 1+3,000 0.117 O.I89 55.0 54.0 37.0 0.7 * 430 920 43 * * ★ 11.95 10.95 3 4 5 10.50 10.70 7 s 2/ Percent acid as lactic 1 2 6 l/ 2j Percent salt 11.30 0.230 Acidformers x lO^/rnl 11.05 10.35 0.199 10.90 0.203 41.0 39*0 7*6 0.276 9 10.45 0.300 17.O 10 11 10.70 O .293 6.4 10.60 0.300 8.8 12 10.50 13 14 0.323 0.330 0.315 7.0 10.60 11.15 15 17 19 10.90 11.10 0.315 0.308 0.423 21 10.85 10.40 0.525 2b 29 11.00 0.57S 36 b3 50 10.80 0.63S 11.00 11.30 0.565 0.576 10.45 0.623 If Yeasts x 103/ml Coliforms No./ml 920 ^3 25 ** 3*9 9*7 157*0 250 ** ** 2.6 3.12 104.0 25 ** ** 1.24 0.64 0.07 89.0 6.0 125.0 ** *♦ ** 2.29 0.83 1.67 95*0 74.0 6*7 ** i| i* ** 0.84 0.k7 0.39 15*7 — 2.3 - 67.0 3.6 r, 11.30 2.3 0.041 0.593 57 64 11.50 0.003 2.4 o. 615 11.45 0.600 0.0003 1.0 71 Tank 23 contained 701 bushels of No. 1 size cucumbers. The first sample was taken eight to ten hours after the cucumbers were covered with “brine. Two-thirds of a barrel of vinegar and a barrel of brine from an ac­ tively fermenting tank were added to tank 23 between the 17th and 19th days of sampling. — Less than 100/ml. — Less than 10/ml = No determination made. 28 TABLE 7 P e rc e n t a c i d and s a l t and th e p o p u la tio n s o f v a r io u s groups o f m ic ro ­ o rg an ism s i n ta n k No. 2 4 1 / TimePercent salt days 0 5/ 13.60 1 12.SO 2 9.90 0.027 0.039 0.032 Acidformers x lO^/ml 0.68 0.99 2.2 Yeasts x 103/ml Coliforms No./ml 2,500 4.2 3*0 0.4 25,000 9,200 3 4 5 9.so 9.70 S.SO 0.075 0.143 0.209 35.0 73-0 0.24 * 0.53 9,200 ** 15 6 7 11.30 9*70 9.70 0.201 0.228 0.24S Sl.O 57-0 46.0 3*3 0.43 102.0 92 43 25 9 10 11 9.65 0.240 0.255 37.0 9.b5 9.45 0.270 19.0 27.O 10.7 4.0 79*0 25 15 ** 12 13 14 9.70 9.50 9.70 0.300 0.345 0.354 26.4 S.5 8.7 — 240.0 25 ** ** 15 17 19 9.50 9.50 9.4o 0.39S 0.443 210.0 300.0 0.495 y 3-1 5.1 2.1 770.0 ** ** ** 21 24 9.90 9.55 10.75 o.4so 0 .5S5 0.555 0.26 0.26 0.93 160.0 ll4.o 240.0 ** ** ** 10.60 0.570 0.54s 0.563 0.091 0.020 114.0 0.015 16.0 s 29 36 43 50 11.10 11.15 29.0 17.6 ** — -- 6.0 0.023 0.570 — 12.SO 0.54s 0.015 5*7 0.009 11. *4) 3*1 0.525 Tank 2k contained J12 bushels of sizes No*s. 2 and 3 cucumbers. 57 64 71 YJ 2] Percent acid as lactic 10.95 The first sample was taken eight to ten hours after the cucumbers were covered with brine. One-third of a barrel of vinegar and one barrel of brine from an ac­ tively fermenting tank were added to tank 24 between the 17th and 19th days of sampling. * = Less than 100/ml. ** = Less than 10/ml. - = No determination made 29 TABLE 8 P e rc e n t a c i d an d s a l t and th e p o p u la tio n s o f v a r io u s groups o f m ic ro ­ o rg an ism s i n ta n k No. 32 » Timedays Percent salt 0 2/ i4.4o 1 2 10.10 8.60 3 Ac idformer s x 10°/ml 0.002 0.002 Yeasts x 10^/ml 0.1 0.2 0.2 Coliforms No./ml 4,300 2,900 0.045 0.013 S.55 7.90 7.75 0 .0^5 0 .2*4-8 0.285 0.120 236.0 3U0.O * * * 2,500 8.4o 8.45 3.45 0.293 0.330 0.375 162.0 58.0 _ ♦ * ** ** ** n S.75 8.40 s .70 0.420 0.480 0.480 35.0 19*5 16.3 * ★ * ** ** ** 12 s .90 0.480 13 lft 8.65 s.90 0.555 0.570 19.2 I8.3 18.1 * 0.2 4.0 ** ** ** 15 17 19 S .95 0.585 0.615 0.623 19.7 8.9 11.2 5.5 10.0 ** ** ** 23 30 37 9.50 9.75 9.75 0.653 44 51 10.00 10.00 0.b90 ft 5 6 7 8 9 10 u Percent acid as lactic 0.008 0.015 9.20 9.30 0.668 0.630 0.690 81.0 165.0 1.4 0.98 1.1 350.0 235-0 17.6 0. *4-3 0.46 11.2 l4.l 4,300 ft3 ** — •m Tank No. 32 contained 1100 "bushels of No's. 2 and 3 sizes cucumbers, The first sample was taken eight to ten hours after covering the cucumbers with "brine. * = Less than 100/ml. ** = Less than 10/ml. - = No determination made. g/ 30 TABLE 9 Percent acid and salt and the populations of various groups of microorganisms in tank No. 33 y Timedays Percent salt Percent acid as lactic Acidformers x 10°/ral 0 1 2 10.60 9-25 9-55 3 4 5 7*85 6.95 S. 60 6 7-50 7.40 7.60 0.263 204.0 0*300 230.0 12S .0 s .30 8.50 S. 60 0.323 7 s 9 10 11 12 13 14 15 17 19 22 26 33 0.003 0.015 0.023 0.03S 0.12S 0.075 0.323 0.006 0.016 0.015 0.44 94.0 25.1 0.360 0.360 54.0 45.0 9.6 8.55 s.SO S. 60 0.413 13.0 S. 60 9.00 s. 70 0.510 9.05 9.4o 9.75 Yeasts x 103/ml Coliforms Ho./ml 1.0 430 2,500 * 2,500 0.1 1,500 ** ** 74 92 0.3 ** ** 0.2 2.1 1.4 0.450 6.6 O .72 13.I 0.45S 9.8 16.5 0.430 52.0 0.518 6.9 S.l 7-2 0.555 0.54S 0.54s 3.4 1*9 2.2 *** *** *** *** *** *** *** *** *** 79-0 180.0 *** *** *** 520.0 *** 14S.0 64.0 - 9.S5 4l.O 0.578 2.5 — 10.20 1.3 0.555 1-9 54 0.67 10.15 0.3 0.563 1j Tank 33 contained 1153’ bushels of sises No's* 2 and 3 cucumber s. * = Less than 1,000/ml* 40 ^7 ** *** - = Less than 100/ml* = Less than 10/ral* = No determination made* 31 TABLE 10 P e rc e n t a c i d and s a l t and th e p o p u la tio n s o f v a r io u s gro u p s o f m ic ro ­ organism s i n tank: Ho. J>k Timedays 2/ Percent acid as lactic Ac idformer s x 10°/ml Yeasts x 103/ml Coliforms Ho./ml 0.008 0.030 0.128 0.1 0.2 0.4 1.4 175.0 1.0 6.25 0.218 0.263 0.27S 280.0 440.0 460.0 0.18 0.10 0.20 25 * * 6 6.60 0.270 0.40 7 g 6.40 6-95 0.293 0.33S 231.0 157.0 88.0 0.55 * * * 9 7.SO 0.405 0.450 0.503 31.0 28.9 24.0 28.0 7.4 131.0 150^0 430.0 550.0 0 *r 1 2 Q.SO 3 4 5 6.30 6.10 7.00 6.10 10 11 7.85 S.00 12 S .10 8.60 0.495 8.80 0.495 7.5 5.3 1.3 15 17 19 8.95 8.80 8.90 0.51S 0.525 0.533 4.8 5.S 3*8 21 24 30 9.40 9.so 10.15 0.503 0.510 0.548 4.3 37 tii 51 10.00 10.00 10.00 0.563 0.563 0.585 58 10.25 0.585 13 l4 l/ Percent salt 0.510 0.50 — 187.0 183.0 256.0 340.0 l4,000 92,000 2,500 * * * * ♦ * * * * * * - 2.8 1.7 200.0 0.97 0.54 0.19 13.4 10.3 1.2 - 0.064 1.1 - — Tank Ho. jk contained 1149 "bushels of sizes No* s. 2 and 3 cucumbers. The first sample was taken six to eight hours after the cucumbers were covered with brine. * = Less than 10/ml. - = Ho determination made. 32 TABLE 11 P e r c e n t a c i d and s a l t and th e p o p u la tio n s o f v a r io u s groups o f m ic ro ­ o rg an ism s i n ta n k No. 35 =- Timedays Percent salt Percent acid as lactic Acidformers x 10 /ml Yeasts x lO^/ml Coliforms No./ml * 92,000 1.0 0.2 14,700 348.0 1,230.0 670.0 * * 0.8 4,300 4,300 430 470.0 173.0 93*0 9.0 56.0 35.0 920 250 25 0.413 0.420 0.435 15.0 79-0 4.4 2.6 220.0 25 ** ** 7.90 0.503 0.473 0.473 2.8 2.9 2.5 230.0 690.0 220.0 ** ** 4c* 19 s.15 S. 20 s.45 0.503 0.480 0.495 2.8 1.1 1.4 230.0 207.0 34o.o ** ** ** 21 23 27 9.15 9*00 9.60 0.488 0.495 0.503 0.64 1.1 1.2 260.0 280.0 230.0 ** ** - 34 4i 9.65 9.70 4g 10.30 0.488 0.458 0.480 1.3 1.0 0.l4 97-0 10.7 0.7 0 2.1 1 2 5*20 6.50 o.oos 0.023 5*85 0.045 3 4 5-95 5-45 6.20 0.188 0.263 0.248 6 7 s 7*30 6.SO 6.SO 0.248 0.345 0.405 9 7-30 7.75 7.90 12 13 14 s.15 s.05 15 5 10 li 17 0.19 0.21 9.6 _ 25,000 - 0.428 o.i4 10.50 1-5 55 l/ Tank No. 35 contained 1125 bushels of sizes No* s. 2 and 3 cucumbers. 2j The first sample was taken eight to ten hours after the cucumbers were covered with brine. * = Less than 1,000/ml. ** = Less than 10/ml. — No determination made. 33 Although some large variations may he noted in the populations of colifomas present initially in the various brines, these organisms dis­ appeared from all 10 fermentations rapidly. None were found after 13 Only in 5 of the 10 tanks studied was there any increase in the days. population of coliforms and these increases were not large. Thus, it is believed that the coliforms played no significant part in these fer­ mentations. Rather, it is believed that the coliforms entered the brines on the cucumbers, found conditions unfavorable for growth and died. The percentages of salt and of titratable acidity, expressed as percent lactic acid, at various stages in the commercial fermentations are given in Tables 2-11. No evident correlation exists between the total titratable acidity formed and the maximum populations of acidformers found in the various brines. However, in the cases of tanks 23 and 24 where quite low populations of the acid producing bacteria were noted, vinegar and brine from an actively fermenting tank were added between the 17th and 19th day of fermentation. Thus, the measure­ ments of the total acidity is not an accurate picture in these two in­ stances. The initial salt concentration in these two tanks was exces­ sively high and this explains possibly the low maximum populations of acid-forming bacteria present, as well as the slow formation of acid* The trend lines for the 10 fermentations indicating the changes in acid and salt concentrations are shown in Pig. 2. The population of acid-formers, yeasts, and coliforms in three laboratory fermentations are presented in Pig. 3 and. in- Tables 12, and l4. 13, The curves representing the acid-forming bacteria counts are 3^ io Percent salt CVJ a> CD rCl d •iH u d * d a> CO CO fl o >rl +> c3 d +> d CD O d p u g O CO o 3 o d +-> +3 rH rH «3 aj CO ♦H rc ^ TTt d d •I— 1 o c d © S o o d o d •H d © cd SlO S d d d CD «tH > * cu • W) '•rt fa CD to ro CVJ Percent acid as lactic O 35 8 FC I Size No.! Cucumbers 7 6 5 4 3 2 FC 2 Size No. 2 Cucumbers 8 7 6 5( 4 • —• Acid-Forming Bacteria o— © Yeasts ▲— ▲ Coliforms 3 2 ' 8 FC 3 Size No. 3 Cucumbers 7 6 5 4 3 X A 2 10 20 HO UR S . 3» 20 25 DAYS 30 35 Populations of various microorganisms in three fermen­ tations under laboratory conditions. 36 TABLE 12 A c id and s a l t c o n c e n tr a tio n s and th e p o p u la tio n s o f v a r io u s g ro u p s o f m ic ro o rg a n is m s i n l a b o r a t o r y f e r m e n t a t io n FC 1 . Time Percent salt Percent acid as lactic Acidformers x 10°/ml Yeasts x 103/ml 0.000 0*000 * * ** ** 129.0 Coliforms Ho. /ml Hours 0 1 9-7 3*7 7-5 125-0 25,000 25,000 9*200 25,000 0.001 31.0 4,300 0.05 430.0 9.4 9,200 4,300 5 .9 0.003 0.005 0.003 2 3 4 5-3 6.95 7.10 7-50 0.015 0.105 0.21S 0.300 24.3 63.0 56.0 5 6 7 3 7*20 7.60 7.Ho 7.30 0.300 o.Hss 0.5H0 0.605 16.9 2.4 3 .H 3 -H 9 10 11 12 3.05 s.05 s .25 3.45 0.645 0.b43 0. 668 0.663 4.1 7-7 0.40 2.0 3.25 s .10 0.630 0.632 3.20 0.632 0.632 1.1 1.9 0.79 3 6 12 6.6 — 340.0 Days 1 13 i4 15 17 9.15 9.10 9.20 9.70 19 22 29 36 * ** *** - 10*40 = = = = 0.632 0.630 0.573 0.543 Less than 1,000/ml* Less than 10,000/ml* Less than 10/ml* I\To determination made* 0.30 0.063 0.023 ** 0.011 6.7 15.0 320.0 32.0 1,12 0.0 42,0 23.0 64.0 19.O 54.0 *** *** *** ♦** #** *** *** 190.0 39.O 7.0 *** 9.0 23.0 350.0 33-0 *** *** — — 37 TABLE 13 A c id and s a l t c o n c e n tr a tio n s and th e p o p u la tio n s o f v a r io u s groups o f m ic ro o rg a n is m s i n l a b o r a t o r y f e r m e n t a t io n EC 2 . Time Hours 0 1 Percent salt Percent acid as lactic Acidformsrs x 10 /ml 0.002 * * ** * 0 .0 0 3 0.001 5 .4 5 6 .3 0 6 .7 0 7 .2 0 0 . 01s 0 .1 4 3 0 .3 4 5 0 . 39s 0 .0 3 0 1 0 3 .0 5 6 7 8 7 .0 0 7 .5 0 7 *75 7 .4 o 0 .4 5 0 0 .4 8 8 0 .5 5 5 0 .6 1 5 9 .5 7 .7 1 .6 3 .1 9 10 11 12 7 .7 0 7 . SO s . 25 s . 30 O .63S 0 .6 7 5 0 .6 7 5 0 .6 7 5 0 .9 5 0 . 3s 0 .0 5 0 0 .S 5 13 l4 15 17 s . 30 S . 75 s . 90 9 .3 0 O .69O 0 .6 3 0 0 .6 5 3 0 .5 5 5 19 22 29 36 9 .3 0 9 .3 0 9 .7 0 1 0 .3 5 0 .6 0 0 0 .5 3 3 0 . 63s 0 .5 6 3 9-S3 9 *3 5 8 .3 0 7 .2 0 6 .2 5 0 .0 0 0 0 .0 0 0 0 .0 0 0 2 3 4 3 6 12 Days 1 * = Less than 1,000/ml. ** = Less than10,000/ml. *** s=Less than10/ml. - = No determination made. Yeasts x 103/ml Conforms No. /ml SS.O 240 113.0 2,400 8 4 0 .0 1 9 3*0 1 5 9 -0 4 ,3 0 0 9 #200 9,200 1 ,5 0 0 920 111.0 580.0 60.0 12.2 5 4 .0 l4 .0 *** 210.0 200.0 **# *** *** *** 2 5 0 .0 1 , 6 0 0 .0 2 3 0 .0 2 3 0 .0 *** *** ** + *** 0 .1 6 1 .0 0 .4 6 1 .3 2 8 0 .0 74o . o 1 2 S .0 *** *** *** 0 .1 2 0 .2 5 ** ** 7 9 *0 9 7 *0 3 4 o .o 5 3 *0 *** 5ft * * 7 5 0 .0 210.0 — ~ TABLE l 4 A c id and s a l t c o n c e n tr a tio n s an d th e p o p u la tio n s o f v a r io u s gro u p s o f m ic ro o rg a n is m s i n l a b o r a t o r y f e r m e n t a t io n EC 3 Time Hours 0 1 3 6 12 Percent salt Percent acid as lactic Acidformers s lO^/ml 9-65 9-35 S.90 S. 20 7-35 0.000 0.000 0.000 0.000 0.002 * * * * * 6.35 0.003 Yeasts x 10^/ml 1*3 4.2 3-3 2.0 Coliforms Ho./ml 430 920 240 91 36 0.70 Lays 1 2 6.SO 7.00 7.10 0.053 0.0S3 0.150 0.001 0.029 10.6 67.0 0.21S 50.0 S4.0 0.195 0.24S 44.0 7 3 7-15 7-65 7*50 s. 25 21.0 110.0 470.0 0.270 13-5 1,360.0 9 10 11 12 s.05 S.20 7.35 7.90 0.305 0.375 0 .45S 0.495 s.o 1,S30.0 5.3 0.S1 530.0 122.0 112.0 13 0.540 0.94 0.493 0.525 0.510 i.4o 71.0 15 17 7.75 s .50 s. 30 S.35 1.72 2.45 1,220.0 190.0 19 22 29 36 9.^5 9.20 9.55 10.35 0.4SS 1.06 0.570 0 .5S5 0.563 0.20 47.0 75.0 124.0 ** 56.0 - 3 4 5 6 ik * = Less than 1,000/ml» ** = Less than 10/ml. - = Ho determination made. 1.15 0.03 o.oo4 110.0 650.0 430 250 920 55.0 43 0.34 •• ** ** %* ** ** ** ** ** ** ** ** ** ** ** 39 very similar to those obtained in the commercial fermentations studied, and are in close agreement with the findings of Rosen and Fabian (51) in the study of two laboratory fermentations. However, the maximum pop­ ulations of acid-forming bacteria attained in these lots as well as in those reported on by Rosen and Fabian were considerably lower than the maximum counts noted in the commercial fermentations. No significant difference was noted in the acid fermentations of sizes No. 1 (crock EC 1) and 2 (crock FC 2) cucumbers but in both of these crocks the acidformer s started rapid growth about one day earlier than with the £To. 3* s ( crock FC 3)• The total titratable acidity correlated with the acid-forming bac­ teria in that the rapid development of acid started more slowly with the large size (No. 3) than with the two smaller sizes. Also, the maximum acidity was not as great as in the crock containing large cucumbers (Fig. 4). The greatest difference in the fermentations in the laboratory as compared to those under commercial conditions was in the yeast fermen­ tation. While a rather definite yeast phase of the fermentation was evident in the commercial tanks, as outlined previously, the yeast pop­ ulations in the laboratory experiments were quite variable and, on the whole, were at a fairly high level throughout the fermentation period studied. Pronounced differences were evident in the yeast species re­ sponsible for the fermentations and will be discussed in detail later. The coliform populations were even more insignificant in the labor­ atory fermentations than they were in the commercial. The highest count observed was 2.5 x 10^ per ml (Table 12) and that was in a sample taken ko FC t Size No.l Cucumbers 8 _^ acid Percent soft as lac tic FC 2 Size No. 2 Cucumbers Percent • — • % Acid as Lactic 0 - - 0 % S a lt 8- . FC 3 Size N o.3 Cucumbers -8 20 5 10 15 20 25 30 35 HOURS Fig. Changes in acid and salt concentrations during the fermen­ tation of three lots of cucumbers in the laboratory. 41 immediately after covering the cucumbers with brine* No coliforms were found to be present after the fifth day of fermentation in any of the three crocks# Identification of acid-forming organisms* Since previous work by Etchells and Jones (l6) and Rosen and Fabian (51) has quite definitely established that L. piantarum was the princip&l acid-forming organism in cucumber fermentations, only a few random isolations were made of these bacteria. However, of 20 isolates obtained by picking isolated colonies from the V-S agar plates only 16 were definitely identified as L. plantarum according to the description of this species in Bergey*s manual (5)* fhe other four isolates were gram-positive cocci similar to Leuconostoc mesenteroides but failed to produce slimy growth on sucrose-gelatin agar or CO^ in dextrose broth as determined by use of the Eldridge tube technique. Yeasts from commercial fermentations. Since marked variations in the various yeast species active in different cucumber fermentations have been noted by Etchells et al. (11, 13)» it was necessary to isolate and screen several of these organisms from each tank throughout the fer­ mentations in order to characterize their activity. A total of 279 isolations were made from the commercial brines by picking isolated colonies from the highest dilutions showing growth on the acidified dextrose agar onto vegetable juice sporulation medium!^. The distribution l / T h i s medium was prepared as described by Etchells and Bell (11) ex­ cept that the V-S juice was filtered through a single layer of cheese cloth in a Buchner funnel in order to prevent excessive foaming in the tube and wetting of the cotton plugs. 42 o f th e s e i s o l a t e s i n T a b le in r e la tio n to th e ta n k s and f e r m e n t a t io n age i s g iv e n 15. All 279 isolates were screened by microscopic examination of the vegetable juice agar slant cultures after 2 to 4 weeks incubation at room temperature. On the basis of distinctive morphological character­ istics and the general appearance of slant growth, the isolates were broken down into five groups; viz.. Group 1, 123 isolates of asporogenic yeasts similar to Torulopsis holmii; Group 2, S4 isolates with morpholog­ ical characteristics of Torulaspora rosei; Group 3* IS sporulating cul­ tures with hat-shaped spores and pleomorphic cells similar to Hansenula subpelliculosa; Group 4, 8 cultures similar to Brettanomyces versatilis; and, Group 5* comprised the remaining 46 isolates which were a group of miscellaneous yeasts. Complete identification studies using the methods described by Etchells and Bell (11) and the classification systems of StellingDekker (63), Lodder (39), a-nd Bedford (2) were made on 25 of the isolates taken at random in Group 1, 42 of those in Group 2, and on all of those in Groups 3, 5* All 25 isolates of Group 1 which were studied to completion were positively identified as Torulopsis holmii. Thus, it was not believed necessary to run the physiological tests on the re­ maining 98 isolates of this group. The probability is that alll23 iso­ lates were of the same species and will be referred to as such for the purposes of this paper. All except one of the 42 cultures of Group 2 were identified as Torulaspora rosei. and the one isolate not so classi­ fied was a non-sporulating yeast with similar cell morphology but dif­ ferent sugar fermentaions than noted for this species. On the basis of ^3 TABLE 15 Distribution of yeast isolates as to tanks and fermentation age No. of isolates obtained from various tanks l4 21 22 24 34 32 33 23 Time days 13 0-4 5 2 0 0 4 3 7 1 0 1 5-8 7 3 3 1 3 8 0 0 5 6 9-12 7 4 5 7 6 2 1 3 6 6 13-16 3 10 7 7 4 1 5 4 3 5 17-20 1 3 3 3 2 5 5 2 3 4 21-24 2 3 3 2 4 3 0 2 2 3 26-30 l 2 0 2 1 1 2 2 1 1 33-37 1 l 1 1 2 1 2 2 1 1 40-44 2 l 1 0 1 1 1 1 1 1 *7-51 1 l 1 1 1 l 1 1 1 1 5^-53 1 2 3 2 2 2 0 1 1 1 63-71 0 0 0 3 3 4 0 0 0 0 31 32 27 27 33 32 24 19 24 30 TOTAL No. GRAND TOTAL 35 279 44 these results, 83 of the 84 isolates in Group 2 will "be referred to as Torulaspora rosei. The 18 sporulating cultures in Group 3 were identi­ fied as Hansenula subpelliculosa. and the 8 isolates in Group 4 plus 8 more from Group 5 were identified as Brettanomyces versatilis. The 39 remaining isolates were broken down into several groups, but were not completely identified and will be referred to as miscellaneous yeasts. Based on the total 279 isolates from the 10 commercial fermentations, Torulopsis holmii represented 44.1 percent; Torulaspora rosei. 29.8 percent; Hansenula subpel1iculosa. 6.4 percent; Brettanomyces versatilis. 5.7 percent; and the miscellaneous group accounted for the remaining l4.0 percent. The occurrence of the two predominating species in the various fer­ mentations was of particular interest, Table 16. ’.fhile both yeasts were found in all 10 fermentations, Torulopsis holmii was apparently in great predominance in 7 of the tanks, and in the other 3 tanks (No*s. 13, 23, 24) Torulaspora rosei appeared to predominate. Based on only k those isolates from brines having a population of over 1 x 10 per ml the predominance of Torulopsis holmii in the seven tanks was even more pronounced, and in tanks Ho* s. 13 and 24 the number of isolates of the two major species were not greatly different. However, in tank 23, Torulaspora rosei was apparently responsible for the major yeast fermeatation. Prom the percentage of isolates obtained at various stages in the fermentation there appeared to be a significant sequence of the various yeasts. This is illustrated in Fig. 5; based on average data given in Table 17. The first few days after brining a miscellaneous group of 45 TABLE 16 Distribution of the various species of yeasts according to the fermentation from which they were isolated Tank No. Total No. of Isolates u Torul­ opsis holmii Percent of total isolates Torula­ Hansenula Brettanomyces subpell­ spora versatilis iculosa rosei 16.1 0.0 Misc. (a) (b) 31 4 9-7 35*5 50.0 (a) (b) 32 20 62*5 so.o 50.0 25.O 29.0 0.0 0.0 20.0 0.0 3*1 0.0 21 (a) (b) 27 15 4S.2 SO.O 18.5 20.0 0.0 0.0 11.1 0.0 22.2 0.0 22 (a) (b) 27 22.2 25.O 0.0 0.0 14.8 0.0 18.5 16 44.4 6S .7 (a) (b) 33 12 3.0 3*0 8.3 0.0 12.1 0.0 (a) (b) 32 IS 18.8 0.0 6.3 6.3 38.S 75*3 91-7 40.7 55* 6 6.1 0.0 0.0 5-6 (a) (b) 2k 11 53.3 100.0 16.7 0.0 0.0 0.0 0.0 0.0 25.0 (a) 73.9 15.8 Cb) 19 13 92.3 7.7 0.0 0.0 0.0 0.0 (a) (b) 2k 17 62.5 25.0 82.4 5*9 0.0 0.0 0.0 9.4 11.7 (a) (b) 30 24 63.3 75*0 20.0 12.5 0.0 0.0 0.0 0.0 16.7 12.5 279 44.1 29.8 6.4 5.7 i4.o 68.7 26.0 0.6 0.0 *.7 13 i4 23 2k 32 33 34 35 G-rand (a) Totals(b) u 150 28.1 3.1 9*7 0.0 9.4 0.0 6.3 0.0 5.3 0.0 The values of (a) are based on the total number of isolates studied during the entire fermentation, while the (b) values represent only those isolates obtained from brine samples having a population of 10,000 per ml* or above. Fig, © ® 001 P ercent o f isolates p er tim e in te rv a l Estimated sequence of various yeast species in the fermentation of cucumbers under commercial conditions* ke 47 TABLE 17 Distribution of yeast species according to the time of fermentation when isolated Torul­ opsis Bolmii Time days 1-4 No, . iii 1 4.8 No* fo 1 Ho. 4 16 No. 1° 32 68.1 17-20 No. 1° 21-24 26-30 5-8 2.7 Torula­ Hansenula spora subpelliculosa rosei 4 Brettano­ myces versatilis 19.00 23.8 1 4.8 11 30.6 10 0 0 5 27.8 Misc. 10 l4 Total No. 21 47.6 36 38.9 20 42.6 3 6.4 1 2.1 7 47 1.49 l4 0 0 0 0 1 2.1 47 29.8 23 74.2 8 25.$ 0 0 0 0 0 0 31 No. t 19 82.6 4 17.4 0 0 0 0 0 0 23 No. fo 8 61.5 5 0 0 0 0 13 38.5 0 0 No. $ 7 53*g 0 0 1 1 30.8 13 7.7 7*7 40-44 No. i 7 70 2 20 0 0 1 10 0 0 10 47-51 No. $ 5 3 30 0 0 1 10 1 10 10 50 No. 1° 6 40 15 26.7 0 0 2 20 13.3 No. dP 1 16.7 16.7 0 0 3 50 1 16.6 9-12 13-16 33-37 54-58 63-64 1/ 34.0 3 4 4 1 Percentage of total number of isolates in each age group 6 48 yeasts apparently predominated* Daring the most active phase of yeast fermentation the predominating yeast was Torulopsis holmii, which was replaced by Brettanomyces versatilis* Torulaspora rosei was found fre­ quently throughout the fermentations. As noted above, this sequence does not necessarily occur in all cucumber fermentations but is merely an overall estimate of the general yeast activity in these 10 tanks. Yeasts from laboratory fermentations. A total of 109 isolates of yeasts from the three laboratory fermentations were studied. However, 22 of these were found to be film-forming types and probably are not active in the sub-surface fermentation. Such yeasts are found growing luxuriantly on tanks which are protected from the direct rays of the sun (12) but are rarely found in brines from unprotected tanks. Heavy films did not form on the laboratory brines as they were kept under ultraviolet irradiation. Nevertheless, a little scum did form occasion­ ally around the edges of the crocks where the brine surface was protected from the ultraviolet light, and this was thought to be the source of these 22 isolates. For this reason, only the 87 non-film-forming isolates will be considered here. Of these 87 cultures, 53 or 66.7 percent were found to be Torula­ spora rosei; l4 (16.1 percent) were Hansenula subpellieulosa. and 15 (17.2 percent) were unclassified. The unclassified group represented a number of different species and will be referred to here as miscellan­ eous yeasts. The distribution of these isolates as to the source is given in Table IS. TABLE IS Y e a s t i s o l a t e s fro m la b o r a t o r y fe r m e n ta tio n s Crock Ho. Torulaspora rosei No. * FC 1 IS Sl.S 0 0.0 4 18.2 22 FC 2 13 44.s s 27.6 s 27.6 29 FC 3 27 75-0 6 16.7 3 S.3 36 Grand Totals 5S 66.7 l4 16.1 15 17.2 87 Hansenula subpel1iculo sa No. i Miscellaneous No. $ ' Total No. In all three lots the principal yeast appeared to1 be Torulaspora rosei. Thi s was in marked contrast to the commercial fermentations whe: Torulopsis holmii predominated, in all instances but three and was iso­ lated from every fermentation. Hot a single isolate of this yeast was obtained from the laboratory fermentations. Identification of coliform organisms. Twenty isolates of members of this group were obtained by streaking eosin-methylene blue agar plates from positive lauryl-tryptose broth tubes and then picking isolated col­ onies from the agar plates onto nutrient agar slants. All cultures proved to be gram-negative, non-spore forming, aerobic or facultative anaerobic, short rods, and produced acid and gas in lactose broth. Therefore, they were members of the coliform group. Further identifica­ tion was not made as the significance of this group in the fermentations studied was very doubtful. 50 Vitamin and. Amino Acid Requirements of L. plant arum The vitamin and amino acid requirements of L. plant arum 17-5 have "been the subject of many investigations, and the requirements of this strain have been well established* The results of investigations on other strains of L. piantarum from various sources, however, have been quite variable* With the exception of the work by Rosen and Fabian (51)* dem­ onstrated that 10 isolates of L. piantarum from cucumber fermentations required biotin, niacin, and pantothenic acid, no study of the vitamin and amino acid nutrition of isolates from this source was found in the literature. Thus, four isolates obtained from four different commercial cucumber fermentations were screened for their vitamin and amino acid requirements in comparison to L. plantarum 17-5* Vitamin requirements of L. plantarum* The medium used to test for vitamin requirements was made up from individual ingredients according to the formula given for the pantothenate assay medium of Difco Labor­ atories, Inc. except that 0.002g per liter of calcium pantothenate was added. Individual vitamins were omitted from various lots of the medium to test for the effect of their omission on the five strains of L. plantarum. The same procedures for the preparation of the inoculum and for in­ oculation and incubation of the cultures were followed as in the vitamin assays except that an incubation temperature of 30° C was used instead of 37°C. 51 As in the work of Rosen and Fabian (51)* all strains of L. plantarum tested were noted to require biotin, niacin, and pantothenic acid (Table 19). In addition, both riboflavin and p-aminobenzoic acid were essential for two strains, L-20 and L-15- Strains 17-5 * L-10, and L-5 were not appreciably affected by the omission of riboflavin from the medium, but isolate L-10 required p-aminobenzoic acid and 17-5 a-ad I*-5 were greatly stimulated by this vitamin. Pyridoxine and thiamine were not essential for any of the 5 strains tested but the former was stimulatory for strain L-5TABLE 19 The effect of the omission of various vitamins from a synthetic medium on the acid produced by various strains of L. plantarum Itfone L-10 L-5 15-50 13.05 13.60 12.90 13.70 0.20 0.10 0.4-0 1 .60^ 0.10 Niacin O .30 0.30 0.45 Pantothenate 0.20 0.00 Q*Q5. 0.00 0.00 l4.20 0.50 o.4o 9.50 14.00 4.95 OsSfi 0.50 0.50 7.20 Pyridoxine 11.60 11.60 12.10 11.30 b.40 Thiamine 13.05 12.25 13.15 12.20 12.65 Riboflavin p-ami nobenzoic acid C\J Biotin 0 • L-15 r*”' L-20 0 • 0 17-5 O Vitamin omitted Ml NaOH (ca. 0.1N) to titrate acid produced by various strains All results are underlined where the amount of NaOH necessary to titrate the acid produced was less than one-half that of the con­ trol- 52 The response of the L. plantarum isolates from cucumber brines and of strain 17-5 to various concentrations of biotin, niacin, and panto­ thenic acid was tested in the same manner as in the preparation of stand­ ard curves for vitamin assays. The results are given in Table 20. The response of the isolates from cucumber fermentations to biotin and niacin was quite similar to that of the 17-5 strain. However, in the case of pantothenate the 17-5 culture was noted to produce much more acid in the presence of comparatively smaller concentrations than the other four strains. These results are comparable to those obtained by Rosen and Fabian (51)* TABLE 20 Response of L. plantarum to biotin, niacin, and pantothenate ■Vitamin Biotin Niacin Pantothenate Concen­ tration /10 ml 0.0 0.2 0.8 1.4 2.0 mjog iqug rrjug mug njug 0.0 0.2 0.8 1.4 2.0 pig pig pig pig pig 0.0 pig 0.05 P-S Ml NaOH (ca. 0.1N) to titrate acid produced by various strains L-20 L-10 L-15 L-5 17-5 1.60 4.95 11.85 12.20 0.10 1.80 b.10 7.50 7.90 0.30 0.30 5-*+5 11.10 12.80 13.30 3.80 9.20 11.55 9-70 0.20 b.35 0.10 pig 11.70 0.25 pig 15.30 16.80 0.40 pig 0.30 2.35 6.85 9.30 10.10 0.10 2.20 5.95 8.40 9.50 0.40 4.20 10.20 11.00 12.05 0.30 0.45 4.55 10.85 11.95 13.60 13.10 0.20 3.55 10.20 12.55 13-65 0.00 0.05 0.00 0.00 0.55 1.65 5.90 13.00 0.60 I .70 0.50 1.50 5.50 0.50 1.90 7.00 13.40 3.90 9-65 11.50 13.05 12.80 b.80 11.20 53 •Arc!no &cAd- requirements of L. plantarum. The basal medium I pro­ posed by Sauberlich and Baumann (54) for microbiological assays using L. plantarum 17-5 a-s the assay organism was used as the basal medium to determine the amino acid requirements of these isolates. The five strains of L* plantarum were screened for amino acid requirements in the same manner as for vitamins. The effect of the omission of the various amino acids from the basal medium on the acid produced by those various strains is given in Table 21. The five L. plantarum cultures were constant in their re­ quirements for tryptophane, leucine, isoleucine, valine, glutamic acid, cystine, and threonine. Phenylalanine and tyrosine were required by three strains (L-20, L-15. and L-10), arginine by one (L-5), and alanine was noted to be very stimulatory to the four isolates from cu­ cumber fermentations. 17-5. Alanine also appeared slightly stimulatory for Some stimulation of all five strains was noted with histidine. The other amino acids did not greatly influence the acid produced by these cultures. By variations in the basal medium used for testing, other workers have shown that the requirements of L. plantarum 17-5 “ay be reduced to include only five amino acids; viz. tryptophane, leucine, isoleucine, valine, and glutamic acid. However, cystine has been noted to be es­ sential except in a very enriched medium (10). Since the amino acid requirements of the isolates from cucumber fermentations appeared quite similar to the 17-5 culture, except for variations among individual strains, the above six essential amino acids were selected for further study. 54 TABLE 21 The effect of the omission of various amino acids from a synthetic medium on the acid produced by various strains of L. plantarum Amino acid omitted 11.95 9.65 9.75 9.90 10.05 L-1 ryptophane l!> o• o None Ml NaOH (ca. 0.1N) to titrate acid produced by various strains L-10 L-20 l -5 L-15 17-5 0.00 0.00 0.00 0.00 L-leucine 1^ 1 - 0.00 1.30 1.20 hJSL DL-isoleucine U 50 1.30 2-30 2.75 2.60 DL-valine 0.80 0.70 0.65 0.70 0.40 L-glutamic acid 0.80 0.80 0.70 Q.S.5, 0.60 L-cystine 0.00 0.00 0.00 0.00 0.00 L-asparagine 10.70 8.50 8.4o 8.70 7.55 L-lysine 11.Uo 8.55 9.50 s.75 10.60 ls25. 2.90 2.00 DL-threonine 2-55 DL-alanine 7.75 3.20 3.75 DL-methionine 12.15 8.65 9.35 9.40 8.20 DL-phenylalanine 11.20 0.40 0.40 0.35 8.4o L-arginine 11.85 8.20 s.05 7.90 0-75 L-tyrosine 10.40 0.40 O.fe Q.Uo 8.70 L-histidine 7.90 7.75 7.50 7.50 8.00 DL-serine 11.75 8.90 9.55 9.35 8.20 L-proline 11.30 9.00 9.45 9.05 7.95 Glycine 11.90 9.50 9.95 10.20 9.80 \J 4-85. The results are underlined in all instances where the titration value was less than one-half that of the control# 55 The response of the five strains of L^. plantarum to graded amounts of these six amino acids is given in Table 22^/, These data are shown in Figs. 6 and 7 for the 17-5 strain and for the strains showing the greatest and the lowest response of the four isolates from cucumber fermentations. The type of curves obtained were similar for all five cultures tested* With leucine, isoleucine, tryptophane, and cystine the concentra­ tions employed were sufficient to approach the maximum response in the complete basal medium (Table 23). £he decline in the total acid pro­ duced by the L. plantarum isolates when the concentration of k-cystine was increased from 70 jog per ml to 100 jig per ml is of doubtful signi­ ficance since the concentration of L;-cystine was 200 jig per ml in the complete basal medium. It is possible that the pH of the medium was changed significantly when sufficient L-cystine solution to attain a concentration of 100 jag per ml was added directly to the tubed medium, as HC1 is used for solution of this amino acid. Thus, the rate of growth and acid production by L. plantarum would be affected# With L-valine and L-glutamic acid it was evident that higher con­ centrations than those tested would be required to attain maximum acid production by L. plantarum. L/ All response tests were made in the various media used for the as­ say of each particular amino acid except in the case of cystine. Medium I of Sauberlich and Baumann (54) was used in this instance. 56 TABLE 22 Response of L. plantarum to six amino acids noted to "be essential for this species Ml NaOH (ca. 0.1N) to titrate acid produced by various strains L-10 L-15 L-20 17-5 Amino acid 10 ml L-leucine 0.0 10.0 30.0 60.0 100.0 8.10 10.45 0.0 10.0 4.40 1.30 2.05 60.0 100.0 6.30 8 .9O 11.60 3.75 5.70 7.90 0.0 10.0 0.80 0.70 1.90 1.40 2.60 3.90 6.10 DL-isoleucine 30.0 DL-valine 30.0 60.0 100.0 Lr tryptophane 0.0 2.0 5-0 8.0 12.0 L-glutamic acid 0.0 10.0 30.0 60.0 100.0 L-cystine 0.0 5-0 10.0 20.0 30.0 50.0 70.0 100.0 1.40 2.80 0.0 1.30 1.90 2.10 5.35 3.30 5.25 8.40 5.50 8.85 5 .so 8.90 2.90 3.70 5.05 6.80 9.00 2.75 3.^5 3.30 3*95 6.15 8.80 3.95 0.65 1.50 2.90 4.b5 6.75 1.20 2.00 3-30 4.80 6.65 8.60 L-5 1.35 2.40 4.45 7.10 9 .so 2.60 3.^5 4.85 6.50 8.50 0.70 1.50 2.80 4.50 6.50 0.40 1*30 0.00 2.45 4.50 0.00 2.60 4.45 6.60 0.00 0.00 2.30 2.45 4.50 6.15 7.90 4.80 6.60 8.25 0.80 1.85 3.50 4.80 5.60 0.80 0.70 0.85 1.70 1.55 3.00 4.55 5.70 1.80 3.20 5-15 0.60 1.25 2.40 3.S5 6.55 5.25 0.80 5.30 6.20 8.80 9.70 0.30 0.35 1.55 3.10 4.60 5.20 7-85 9.80 9.00 o.4o 1.70 3.85 4.85 o.4o 1.65 3.10 4.30 5.90 s .05 8.30 9.15 8.90 9-35 9.70 0.00 2.40 4.45 6.25 8.10 11.30 11.70 10.80 3.20 4.70 6.MO 1.90 3.60 4.60 5.so 7.^5 9.10 8.45 5.25 7.95 1.90 4.60 6.45 7.70 5.80 57 \\ \\ \ \ \\ £ ir O $ W \ £ o> CL ^ No< \\ \\ N\ C -J 04 Q O' =\ Pi O C o Z3 a> o OJ cr> 00 a> c 4> OJ V£> *rH PH o 00 OJ ML N a O H (co. ./ N ) to t i t r a t e a c id p ro d u c e d 5B a> a. a> c to >% 0 1 cr> o«C o e o a> o. o o o 6 o •1n CF> OJ cn cvj v Cl CD C O a o +C-L >% XL a I _J cn «yD o 00 CJ M i N a O H fca. I N ) t o titra te a c id produced 59 TABLE 23 Comparison of the concentration of the amino acids and the acid produced "by L. plantarum (L-15) in a complete basal medium to their concentrations and the acid produced in assay mediai/ Basal medium ;ug/ml ml NaOH^ Amino acid Level where maximum response was observed ml NaOH^/ ng/ml 10 8.85 L-leucine 200 9.75 L- isoleucine 2oo2/ 9.75 5^ 9.00 L-valine 200^/ 9-75 5^ 6.75 80 9.75 1.2 8.25 L-glutamic acid 800 9*75 L-cystine 200 9*75 L-tryptophane l/ 2/ 2/ 10 7.0 5*70 9 .so Compiled from data given in Tables 21 and 22. Ml of NaOH (ca. 0.1N) to titrate acid produced in 10 ml of medium. Double this concentration of the DL form was used, but the L-form is the only active fraction. Essential Vitamins and Amino Acids for L . plantarum in Brines of Commercial Cucumber Fermentations It has been established that L. plantarum isolates from cucumber fermentations require at least three vitamins and six amino acids to produce a significant quantity of acid. Therefore, the presence of these nutrients in cucumber brines are essential for the acid fermenta­ tion. The concentrations, are also important as the amount of acid produced by L. plantarum is limited when the level of any one of these vitamins or amino acids is too low. Go As a basis for the study of the nutrients essential for L. plantarum in the brines, 10 commercial fermentations were studied in detail as to their microbiological activity— reported previously. Five of these 10 tanks contained size Uo. 1 cucumbers and the other 5 contained mixed sizes of No’s. 2 and 3* for this study. Three of each of these two groups were selected The six fermentations chosen included both the high and the low extremes of acid forming bacteria and yeast populations noted in the various fermentations, Tables 2-11. Microbiological assays for the available vitamins and amino acids were run on brine samples taken at short intervals during the active acid fermentation— about the first 20 days of fermentation. A few brine samples taken later in the fermentation were assayed to test for any major changes in the concentration of the available nutrients due to continued yeast activity or other factors. Vitamins in commercial fermentations. The complete results of the study of the niacin, pantothenic acid, and biotin content of the brines are given in Table 2 b , and the results of two representative fermenta­ tions are shown in Fig. 8. The general trend of the concentrations of these vitamins with time were quite similar in all six tanks. Thus, the maximum concentration of the vitamins in the brine was reached in from 5 to 7 days and a slight decrease in the levels was generally noted thereafter. There was a significant difference in the concentrations of the three vitamins in the brines from tanks with the small size cucumbers (size Uo* i) as compared to those with the larger sizes (itfo1s. 2 and 3)* 61 TABLE 24 Niacin, pantothenic acid, and hiotin content of hrines during the commercial fermentation of cucumbers* Time from ■brining Niacin 0-10 hrs* 1 day 2 3 5 7 10 ik Tanks filled with size No* 1 cucumbers 14 21 23 jug/ml jug/ml hft’/ W 0.282 0.635 0.653 1A 15 0.613 0.733 1.233 1.490 1.310 2.880 1.202 2.120 2.480 2.410 1.519 2.102 1.420 2.3I*0 1.230 1.790 1.248 1.858 1.615 1 / 1*743 _ 23-24 1.335 — 1.364 30-36 1*735 — — 37-41 1.663 — 55-58 1.335 1.655 64 1.360 Pantothenic acid _ 0.774 0*738 0-10 hrs-> 0.684 1 day 1.478 0.861 — 2 1.293 1.809 2.370 1.305 3 1.600 2.140 2.085 5 — 2.321 2.810 7 2.620 10 2.27*+ 1.719 — 14 2.53S 1.575 2.152 2.394 1.9S3 19 — U 2.044 1.680 23-24 2.136 30-36 1.675 — — 2.180 37-^1 1.996 1.596 55-58 1.508 — 64 mug /ml nsug/ml Biotin 5.84 15-20 0-10 hrs* 11.75 12.72 17.70 1 day 10.99 — 12.00 2 16.70 16.33 3 13.00 16.99 19.74 5 — 14.75 19.26 7 — 14.16 19.7S 10 13.94 l4 17.90 18.30 13.72 18.20 19 i/ 17.50 14.18 23-24 13.32 17.40 30.36 — 19.26 37-41 12.13 18.40 55-58 64 11.57 x j Day varies with the individual tanks* * = Less than 0.02 jig/ml. - = No determination made. — — — — — k _ Tanks filled with sizes No* 2 and No. 3 cucumbers 24 33 35._ jag/ml jug/ml jug/ml 0.128 O.Obl 0.17S 0.310 0.55S 0.354 — 0.493 1.182 0.725 0.723 1.548 0.970 0.95S I .676 0.992 1.366 1.206 1.533 1.090 0.975 O.78I 1.273 0.979 — 1.008 0.808 0.894 O .896 0.842 — 0.921 0*763 0.811 - - 0.167 0.446 - 0.969 1.320 1.395 1.615 1.S75 1.95S 1.570 1.411 1.1+33 1.407 “HS/111! O.856 1.512 3.509 4.758 4.835 6.540 5*165 5.200 5.053 4.750 - 4.492 4.487 ♦ 0.632 1.268 1.435 1.435 1.438 1.603 1.618 1.400 1-337 1.294 1.128 mug/ml 1.860 4.604 5.S35 6.895 7.670 6.325 7.395 7.550 7.313 5-962 5.865 5.145 0.158 0.444 0.488 0.843 1.505 — I .838 I.8O3 — mug/ml 0.452 1.5S0 2.280 3.200 5.500 - 6.160 b.240 — - 2.5 niacin per ml. 2.0 >o 1.0 2 .5 2.0 Or ju g pantothenic odd per mi jug 0 .5 1.0 - • — •-T a n k 14 Size N o.I Cucumbers 0.5 -o , - o - - o - T a n k 3 3 Sizes Nos. 2 and 3 Cucumbers ml. 20 biotin 0 Mju g per 15 5 -a 0 5 F ig , S. 10 T IM E IN 15 DAYS 20 25 N i a c i n , p a n to th e n ic a c id , and " b io tin c o n c e n tr a tio n s in th e b r in e s o f two com m ercial cucumber fe r m e n ta ­ t io n s . 63 The most striking difference may he noted in the bio tin levels; the bio tin concentrations in the brines from tanks filled with No* 1 cucum­ bers are from 2 to h times as high as those in brines covering the larger cucumbers* However, it should be noted that in all instances the vitamin levels at one day after brining were well above those re­ quired by L. plantarum for rapid acid production in the assay medium* No obvious correlation existed between the vitamin levels in the brines and the microbiological activity* The decline in vitamin con­ tent which was observed in brines after the first 10 to 19 days of fer­ mentation occurred at the same time that the yeast activity was greatest in all these brines* However, this may well have been due to a number of factors. Amino acids in commercial fermentations* The concentrations in the commercial brines of the six amino acids studied are given in Tables 25 and 26 and those in two representative fermentations are shown in Figs. 9 and 10* In general, the maximum concentrations of all the amino acids were reached more slowly than were those of the vitamins; 10 to 19 days being required for the amino acids as compared to 5 to 7 days for the vitamins* This was probably due to the relatively low solubility in brine of protein material and of the amino acids themselves as compared to the three vitamins* The results of the study of the levels of leucine, isoleucine, and valine were quite comparable over all six fermentations, and is well illustrated in Fig* 9* As with the vitamins the brines from the tanks containing the smaller cucumbers were in practically all instances 64 TABLE 25 Leucine, isoleucine, and valine content of brines during the commercial fermentation of cucumbers Time from brining Tanks ; filled with size No. 1 cucumbers 21 i4 .. ,.?2.... p&lpi. Leucine 0-10 hrs.► 1 day 46.30 114.70 2 — 3 5 191.00 10 250.00 238.50 19 30-36 55-64 194.70 1/ 268.10 271.60 Isoleucine 0-10 hrs. 1 day 2 3 5 10 19 If 30-36 55-64 ?aline 0-10 hrs.» 1 day 2 3 5 10 19 30-36 55-64 5.45 42.10 — 103.90 108.00 117.80 111.20 145.20 147.20 2b. 58 77.50 25.90 26.20 8.30 43.80 - 47.10 109.40 126.20 208.00 24.50 - 121.50 153.00 215.00 228.50 284.00 263.60 * * — 22.90 57.50 102.90 103.90 144.00 142.40 6.90 13.80 — 133.00 77-40 150.00 105.00 154.50 166.OO 188.50 174.50 i f 170.10 178.10 Tanks filled with sizes No. 2 and No. 3 cucumbers 24 33 35 jug/ml hg/ml 331.00 318.50 - 12.90 24.08 54.20 62*90 io4.bo 181.00 184.50 - - 12.78 27.20 59.80 70.70 123.15 211.00 214.00 182.05 - 16^.30 * Day varies with the individual tanks. * = Less than 5*0>ug/nil. - = No determination made* 85.00 125.00 I85.OO IS7.50 218.40 205.80 ♦ * — 6.25 36.70 — 94.00 133*50 172.50 181.30 208.60 202.80 * 16.20 - 19.00 19.10 44.10 101.10 105.90 80.20 80.60 98.60 120.80 105.40 117.20 112.50 * * - 53.50 86.10 126.to 133.50 129.50 128.30 * 22.25 63.30 96.50 117.00 140.50 136.00 116.00 b.kO 30.75 50.00 89.50 152.60 231.00 256.00 - * 15*38 27.50 50.00 88.50 120.00 133.60 — * 15*05 28.4o 54.30 93.70 139.80 153*80 - 65 TABLE 26 Tryptophane, glutamic acid, and cystine content of "brines during the commercial fermentation of cucumbers 3?ime from brining Tanks fili*ed with size No. 1 cucumbers 21 2V 14 1 Tryptophane mw— 0-10 hrs. 1 day . M S / ™ 1- Tanks filled with sizes No. 2 and No. 5 cucumbers gT 35 35 ns/mL /lg/ml ng/ml — * 5.70 11.75 — 2.15 4.36 — 5.22 11.61 3 5 17.60 11.69 14.12 24.30 15.50 16.25 9.00 10 32.10 29.60 1 / 19 y .SO 19*20 17.20 33.46 29.55 24.10 is.25 6.62 21.75 2.94 7.1S 10.10 is. 90 26.05 50.00 100.00 119.00 2 19 30-36 55-64 Glutamic acid 0-10 hrs. 10.35 1 day 47.60 — 2 248.00 3 207.20 5 10 179.70 19 i/ 93.60 30-36 y 152.so 55-64 l4i.S0 Cystine 0-10 hrs. 1 day 2 3 5 10 19 30-36 55-64 * 7.05 — 16.22 11.52 14.02 11.74 y1/ 15.91 14.20 — 43.40 SI.20 110.40 100.00 151.20 i44.4o * * — 4.7s 6.45 6.76 5.68 1.77 • - 1.92 — 10.28 * 4.38 17.05 27.50 35-00 39.00 31.10 9.06 24.24 5.20 7.70 15.40 13.40 — 56.60 - 213.40 76.40 56.50 110.40 336.00 326.00 203.20 106.60 70.60 62.60 - i4i.6o 122.40 146.80 128.20 * l.SS 6.80 s.56 15.24 IS. 80 16.74 12.62 10.92 1J Day varies with the individual tanks. * = Less than 1.0 /pg/ml. - = No determination made. * * — 2.54 5.96 8.92 7.50 * 3.01 3.76 10.56 19.90 32. 3U.9O — 5.05 26.20 45.30 81.00 159*40 253.15 259.20 — - * * 3.^5 2.65 — 3.60 7.50 7.84 10.04 12.80 17.22 11.06 14.04 3.18 7.88 16.88 14.77 15.33 - — 350 o £ 150 100 - 50 240 5. 160 -o 120 m • Tonk 14 Size No. I Cucumbers _o— ©-Tank 3 3 Sizes Nos. 2 and 3 Cucumbers 40 240 o ^ 160 •5 120 80 40 0 F ig . 9 - 5 10 T IM E IN 15 DAYS 20 25 L e u c in e , i s o le u c in e , and v a l i n e c o n c e n tr a tio n s i n th e "brines o f two com m ercial cucumber f e r m e n ta tio n s . 35 30 25 20 !5 10 5 0 350 300 Gr 250 ZOO 150 100 50 -•-Tank (4 Size No. I Cucumbers -o --o -T a n k 33 Sizes Nos. 2 and 3 Cucumbers 0 j ______________I_____________ i_____________ i____________ 24 20 16 12 8 4 0 5 g. 10. 10 TIME IN 15 DAYS 20 T ry p to p h a n e , g lu ta m ic a c id , and c y s tin e c o n c e n tra ­ t io n s i n th e b r in e s o f two com m ercial cucumber f e r m e n ta tio n s . 68 richer in leucine, isoleucine, and valine throughout the fermentation period studied. The concentrations of isoleucine in tanks 23 and 24 and of valine in tank 24 were still relatively low one day after brining— less than 5*0 ^ig per ml. However, since 5*0 P S P©r ^1 'of the L-form of these two amino acids and 10 p g per ml of L^-leucine were sufficient for rapid acid production by L. plantarum in the assay media, these three amino acids did not appear to be limiting. Ho influence of the size of the cucumbers on the concentration of tryptophane in the brines was evident since the same levels were ap­ proached in all six fermentations (Table 26 and Pig. 10). However, in two fermentations, tanks l4 and 24, great reductions in the tryptophane content occurred during the fermentation. Some reduction in the levels of this amino acid also occurred at about the same time in tanks 21, 23, and 35* As the reduction in tryptophane occurred at the same period when yeast activity was greatest in these brines, it is indicated that these microorganisms may either utilize or destroy large quantities of this amino acid. Considerable variation was noted in the concentrations of glutamic acid present in the brines of the six different fermentations (Table 26). For example, considerable reduction in the glutamic acid content of brines from tanks Ho1s. 21, 24, and 35 were noted 10 to 19 days after the beginning of the fermentations but similar reductions were not ob­ served in the brines of the other three tanks. Also, there were large differences in the maximum concentrations of this amino acid attained in the various tanks. 69 From the standpoint of the levels of the various amino acids re­ quired for rapid acid formation by L. piantarum, the study of the cys­ tine content of these brines was of the most interest. 4 to 7 Thus, while per ml of L^-cystine was found to be required for optimum acid production, in 3 of the fermentations studied (Tanks 23, 24, and 35) the concentration of this amino acid was below 5*0 jug per ml three days after brining. low at 19 days. The cystine content of these brines was still relatively However, the levels of cystine in these three fermen­ tations approached those of the other three as the fermentation proceeded. Ho relationship was evident between cucumber size and the cystine content of the brine. Essential Vitamins and Amino Acids for L. piantarum in Laboratory Fermentations and Hon-f ermented Control Brines It is important for the acid fermentation of cucumbers to be initi­ ated very soon after brining. This decreases the possibility of unde­ sirable groups of organisms utilizing a large quantity of the available sugar resulting in low final acidity. Therefore, it is quite important that the nutrients essential for L. plantarum become available in the brine rapidly. This study was concerned with the rate of exodus of these essential nutrients from cucumbers of various sizes in carefully controlled laboratory experiments. Rosen and Fabian (51) have shown that yeasts and colifonn organisms may influence the concentrations of some vitamins in brine during the fer­ mentation of cucumbers. similarly influenced. It is also reasonable that the amino acids may be Therefore, two lots of each size of cucumbers were 70 brined; one lot was allowed to ferment while the fermentation of the other lot was inhibited by the use of preservatives. By comparison of these fermented and non-fermented lots, it was possible to note any marked effect of the brine organisms on the content of these essential nutrients during the fermentations* Three bushels of cucumbers were obtained through the courtesy of the H* W. Madison Co. at Mason, Michigan; 1 bushel of each of 3 sizes — No. 1, No* 2, and No. 3* These cucumbers were all of the Davis variety and were grown on the same plot of ground. Each bushel was broken down into two lots and one lot used for the non-ferment ed control and the other for a laboratory fermentation* The non-fermented controls were brined in quart fruit jars; 11 jars of each size. Ten size No. 1 cucumbers per jar were brined in lot C 1, 5 size No. 21s per jar in lot C 2, and 2 size No. 3*s per jar in lot G 3* After determining the weight of the cucumbers in each jar, they were covered with a 3&° salometer (10 percent salt) brine at the ratio of 1.8 ml brine to Ig or cucumbers. This ratio was necessary to completely immerse the cucumbers in the brine. Five ml of toluene and 5 ml of chloroform were added to each jar to prevent fermentation and the jars sealed with regular screw type lids* Composite samples were taken of each lot by removing 5 ^ from each jar and pooling the samples from all 11 jars. of brine The samples were tested for microbiological activity, and for acid and salt concen­ trations. No organisms were found in any of the samples. samples were frozen until the nutritive stuay was made. All brine 71 To prevent depletion of the brine in the jars, 5 ^1 °f a 3^° salometer brine was added to each jar when samples were removed for the first 5 days. None was added thereafter. The fermented lots (PC 1, PC 2, and FC 3) were brined in 5—gallon crocks. Nine kilograms of each size cucumbers were weighed, placed in separate crocks, and covered with 9 liters of 3&° brine. Sufficient salt was added within the first 2 days after brining to make the salt concentration about equal to that of the non-fermented controls after equalization— about 27° salometer. The brine strength was increased about 2° salometer per week in the fermented lots. Sampling of these laboratory fermentations was carried out as des­ cribed earlier. As fairly large samples were taken, at each interval, about 100 ml, and much evaporation occurred, 3^° salometer brine was added as needed to maintain the original brine levels in the crocks. Vitamins in non-fermented controls and in laboratory fermentations. In general the results of the vitamin study on the laboratory fermentations and the non-fermented control brines were similar, Table 27* The concentrations of the 3 vitamins in the 3 non-fermented control lots (Cl, C2, and C 3) are shown in Fig. 11. The differences in the vitamin levels which were noted in the coiamercial brines from tanks with different sizes of cucumbers were even more pronounced in these experiments. Thus, the brines covering the No. 1 cucumbers had the greatest and those on the No. 3 size the least concentration of vitamins. As in the study of the commercial brines, the largest differences were noted in the biotin levels, Lot C 2 brine contained only about one-half the amount of biotin that was found in 72 TABLE 27 Time from brining Niacin 0 hr. 1 3 6 12 1 day 2 3 5 7 15 29 Size C 1 ;ag/ml * * 0.124 0.255 0.599 0.980 1.243 1.28S 1.277 1-299 1.305 1.241 Pantothenic acid ** 0 hr. 1 0.033 0.145 3 6 0.365 0 • H A comparison of the niacin, biotin, and pantothenic acid levels in non— fermented and fermented brines covering cucumbers of three different sizes FC 1 * 0.063 0.131 0.283 0.604 1.062 1.4l4 I.69S 1.764 1.436 1.692 1.782 12 1 day 2 3 5 7 15 29 2.960 4.910 b.850 s. 350 10.045 9.770 11.180 10.880 11.025 11.100 0.052 0.890 1.825 2.525 6.700 6.36O 6.860 12.740 6.560 5.770 0.760 0.726 0.789 ** ** m&l™1 *** 0.094 0.460 0.624 o.54o 0.752 isbs/p i 0.090 0.343 0.938 2.170 2.790 2.320 13.SOO 13.060 12.900 7.310 12.670 10.470 1.023 0.120 0.204 0.808 1.778 1.307 1.342 1.787 0.097 0.3SO 0.913 ws/®1 *** 0.064 0.653 1.554 2.580 3-335 5.490 5.980 6.525 6.600 6.S35 6.465 1.706 2.065 0.209 0.342 0.531 0.750 O .836 ** 1 * * * - 0.910 mug/ml - 1.875 1.348 ngig/ml 0.3IO 0.648 ** ** 0.084 0.320 0.571 0.691 O.S36 0.877 2.018 2.067 0.826 1.192 Biotin 0 hr. 1 3 6 1-732 l.6l4 1.324 1.542 0.161 2.166 1 day 2 3 5 7 15 1.152 1.690 1.055 1.02S 1.040 0.969 ng/ml * * * - 1.168 1.328 1.248 0.422 0.950 29 0.605 O .836 O .992 jug/ml * * 0.074 0.154 0.456 0.972 1.202 2.024 2.278 2.465 0.093 0.117 0.579 1-385 1.420 jug/ml * * * 0.139 0.353 Size Ho. 3 f c 317 C 3 ** ** 0.018 0.074 0.149 0.243 0.379 0.496 0.605 0.746 O .856 O.9O8 12 m Size No. 2 EC 2 C 2 *** 0.037 0.148 0.473 0.774 1.510 1.605 2.495 2.595 3.1^5 3.2U0 0.012 0.035 0.092 0.433 0.586 0.798 1.034 1.160 1.210 1.515 *** *** 0.034 0 .13s 0.380 1.125 2.090 2.005 3.085 3.235 3.235 3-585 The 0 series were the non-fermented control samples and the EC series the laboratory fermentations* * = Less than 0*05 <*ig/ml of niacin. ** = Less than 0*02 jag/ml of pantothenic acid. *** = Less than 0.025 iqug/ml of biotin. =s No determination made. mi. 1.4 1.2 0.6 ▲ 1.4 -o--- acid per mi. jug niacin per 1.0 jug pantothenic 1.0 0.8 0.6 0 .4 ▲ 0.2 • • C l . Size No. I Cucumbers —©■—o—C2. Size No. 2 Cucumbers - A r - ^ C 3 . Size No. 3 Cucumbers 6 - Mjug biotin per ml. 12 HO U R S Fig. li. DAYS Comparison of the rates of diffusion and the maximum concentrations attained of niacin, pantothenic acid, and hiotin from three different sizes of cucumbers in non-fermented brines. 7^ Cl brine, and C 3 only about one-fourth that of the C 1 brine. The maximum concentrations of all the vitamins studied were in most instances attained in from 5 to 7 days after brining. The lots with the larger sizes required a longer time than those with smaller sizes of cucumbers. This would be expected due to the ratio of the surface area to volume in the different sizes. The ratio of cucumbers to brine in the fermented lots was 1:1 while that in the non-fermented lots was 1:1.S. Therefore, the nutrients in the brine would be expected to be more concentrated in fermented brines than in the non-feimented unless these nutrients were utilized or de­ stroyed by the brine organisms or the products of their metabolism. The relative concentrations of niacin and pantothenic acid in the two groups of controls were about what might be predicted from the cucumber-brine ratios* However, the biotin content of two of the fermented brines was apparently influenced by some other factors, particularly during the first day. The biotin level in the PC 1 lot was only about one-fourth that of the C 1 lot and in the FC 2 lot only slightly over one-half that of the C 2 lot after one day. The biotin content of the FC 3 lot was slightly lower than that in C 3 for 12 hours, but was higher than that in the C 3 at one day. However, two days after brining the biotin levels in all three fermenting brines were significantly higher than in the nonf ermenting. The principal differences observed in the microbiological activity in the three laboratory fermentations during the first day were in the yeast and coliform populations. The populations of these two groups of organisms were noted to be much higher over this period in the FC 1 and 75 FC 2 brines than in that from the FC 3 lot* Both groups were observed to decrease in numbers in the FC 1 and FC 2 brines after one day. Ac­ cording to the work of Rosen and Fabian (51), either one or both of these groups of brine organisms may utilize biotin and, thus, might be responsible for the relatively low levels noted in the FC 1 and FC 2 brines during the first day after brining. Amino acids in non-fermenting and fermenting control brines. The results of the study of the amino acids concentrations in the non-fermented controls and fermented laboratory brines are given in Tables 2 8 and 29. Since these were only a few instances where the results obtained with the fermented brines were noted to be very dissimilar to those of the non-fermented brines, only the concentrations of the amino acids in the latter lots are presented in Figs. 12 and 13* A definite relationship was evident between the size of the cucum­ bers and the concentration of all six of the amino acids in the brines. Thus, fermentations of small sized cucumbers would have a higher poten­ tial of both the vitamins and amino acids which are essential for L. piantarum than would those of large sized cucumbers. The time required for the brines to attain the maximum concentra­ tions of the amino acids was much longer than it was with the vitamins. Fifteen to 29 days were required for the amino acid levels to reach their maximum. The cystine content of the brines of all 6 lots was noted to continue to show a significant increase at 29 days. The concentrations of leucine, isoleucine, valine, glutamic acid, and cystine observed in the three fermented brines as compared to those 76 TABLE 28 A comparison of the leucine, isoleucine, and valine concentrations in non-fermented and fermented "brines covering cucumbers of three different sizes Time from brining L-leucine 0 hr. 1 3 6 12 1 day 2 3 5 7 15 29 Size No. 1 0 1 FC 1 ng/ml ng/ml * * * * * 5-55 8.60 11.50 207-00 275.30 Size No. 2 FC 2 C 2 )i§/mL ★ * * * ♦ * 5.65 6.65 17.65 15.75 57.10 40.77 81.13 126.20 110.27 165.00 205**4) 130.67 265.70 142.33 233.70 155.50 214* 00 274.50 167.50 230.20 ** ** ** 4.60 11.43 39.75 ** *>1 * ** 3.25 10.60 ** ** ** ** 7.93 33.00 64.13 29.90 b5 *60 120*10 152.33 178.00 I85.IO 21.70 68.90 167.40 200.40 235.6O 213.30 L-isoleucine 0 hr. ** ** 1 ** 3 5.60 6 12 18.05 42.80 1 day 78.50 2 97.00 3 104.0 5 106.10 7 122.45 15 29 126.10 117.30 15S.55 15^.45 L-valine 0 hr. 1 3 6 12 1 day 2 3 5 7 15 ** ** ** 5.45 14.08 46.80 111.70 136.80 155.75 i 4o .3o 181.40 ** ** ** 6.88 21.75 50.25 89.20 107.00 116.20 122.50 131.60 88.95 110.50 130.75 25.^8 51. b7 69.64 77.57 81.04 93.20 93.75 85.50 108.00 142.50 125.75 128.25 .0 s .so 29.23 ** ** ** 2.68 10.08 39.50 57-60 73-80 85.63 92.25 101.50 103*05 107.50 131.60 172.50 150.35 153.25 ♦* ** ** 3 11 81.35 Size No. "5 7 n m ng/ml pg/wk ♦ * * * ♦ * * 5.20 10.85 5.50 23.20 20.80 72.70 43.10 58.60 79.30 122.80 82*30 135.60 95.00 167*30 120.00 130.87 I87.5O ** ** ** ** 5.50 11.52 23.70 32.30 47.37 59.54 71.37 79.00 ** ** ** ** 4.93 13.25 29.50 39.30 54.00 61.10 73.00 79.70 ** ** ** ** ** 12.48 40.00 46.95 68.05 74.55 96.25 108.35 ** HeHe ** ** H*He 16.45 52.00 62.60 84.00 92.00 116.50 134.40 137.10 177.75 The”c series were the non-fermented control samples and the FC 1/ series the laboratory fermentations* * = Less than 5*0 jig/ml. ** = Less than 2*5 ^lg/ml. 29 77 TABLE 29 A comparison of the tryptophane, glutamic acid, and cystine concentrations in non-fermented and fermented hrines covering cucumbers of three different sizes Time from Size Ho, 1 ... SiJelfo. 2 brining Cl FO 1 C 2______ EC 2 Tryptophane jug/ml ,n&/ml ■agZgi jWS/“l. * * 0 hr. * * * * ♦ * 1 * * * 0.52 3 6 1.69 0.85 1.02 0.55 12 4 .7s 3.69 2.51 2.76 1 day 6.06 11.71 11.75 9.75 2 18. IS 10.61 19.54 13.40 23.78 25.30 l4.4s 15.20 3 30.02 28.7^ 19.92 16.26 5 21.24 8.28 24.92 31-30 7 23.87 15 16.91 33-72 9.05 29 31.82 11.26 24.60 7 .3 s Glutamic acid ** 0 hr. ** 1 ** 3 6 10.37 ** ** ** 13.35 30.10 20.60 63.50 104.00 115.SO 55.70 116.70 12 1 day 2 3 5 7 132.50 149.60 15 182.60 29 203.00 Cystine 0 hr. 1 3 6 12 1 day 2 3 5 7 15 29 1/ * * * 0.76 1.84 3.^7 10.35 13.16 lM3 17.79 19-22 22.80 l46.4o i64.oo 1H0.30 130.20 118.90 * * * 0.65 1.80 4.08 13.61 19.52 20.56 19.89 1 7 .7 * + 18.8*+ ** ** ** 5.15 16.30 36.55 61.50 81.10 95.60 107.10 129.20 137.00 * * * * 1.19 2.86 7.60 10.20 13.44 14.69 18.15 20.05 ** ** ** 6.10 12.95 36.10 92.20 116.20 134.00 168.00 138.40 136.00 * * * 0.55 1.89 5.37 9.04 14.07 12.92 19*02 14.73 16.32 Size No. 5 ~C 3 FC 31/ jasfr11* ♦ ♦ * 1.35 3.22 6.08 8.54 12.15 13.47 16.00 17.05 ** ** ** ** 10.80 21.65 39.20 48.50 64.20 75.50 101.90 111.80 * * * * 0.68 1.56 3.50 5.53 8.96 11.14 16.10 17.49 jig/ml * * ♦ * 0.61 4.17 11.56 10.74 7-73 3.65 5.03 6.57 ** ** ** ** 6.20 22.10 47.90 65.20 105.50 109.00 128.20 136.10 * ♦ * ♦ 0.70 2.38 5.85 7.06 11.56 12.91 13.65 14.70 The C series were the non-fermented control samples and the EC series the laboratory fermentations. * = Less than 0.5 ** = Less than 5*0 >ig/ml. 7 160 o 120 80 40 0 120 100 80 60 p * i / •- - • C l . Size No.l Cucumbers /d - o - - o - C 2. Size No.2 Cucumbers 40 20 C 3. Size No. 3 Cucumbers -J______1_____ I ______L____ _J______I 0 140 120 100 O ____ ______ 80 60 40 O' 20 0 j_ 10 20 5 10 15 20 25 30 j HOURS DAYS 12. Comparison of the rates of diffusion and the maximum concentrations attained of leucine, isoleucine, anc valine from three different sizes of cucumbers in non-fermented brines. 79 © 20 Or C240 120 24 20 v CX 16 * 12 s Ch . Size No. I Cucumbers . 0 — o - 0 2 . Size N o .2 Cucumbers 8 - i - - A - C 3 . Size N o .3 Cucumbers .V 25 30 35 20 DAYS HOURS Comparison of the rates of diffusion and the maximum concentrations attained of tryptophane, glutamic acid, and cystine from three different sizes of cu­ cumbers in non-fermented brines* 10 15 so in similar lots of the non-fermented "brines showed no evident influence of the fermentation except during the first 12 hours or 1 day* After that time the concentrations of these five amino acids in the ferment­ ing "brines were higher in every instance than in the comparable lot of non-fermenting brine. Conversely, just the opposite relationship was observed in the samples taken 12 hours after brining* This is the same type of relationship observed in the biotin concentrations of these brines. The tryptophane content of the brines was markedly influenced by the fermentation. For the first few days the tryptophane levels in the fermenting brines was comparable to those of the non-fermenting brines. However, a marked reduction in the concentration of this amino acid was noted at 5 days in lot FC 3» 7 days in FC 1, and at 15 days in FC 2. On comparison of the microbiological activity in these brines with the tryptophane levels, it was noted that yeasts were very active in all 3 lots at the same period of time that the reduction in tryptophane occurred. The trends of yeast populations are compared to the trypto­ phane levels in the brines in Fig. lU. In the FC 1 lot the tryptophane level dropped at two different times and in both instances the yeast activity was noted to increase. Ulhile increases in yeast numbers were noted in both FC 2 and FC 3 lots prior to a decrease in tryptophane, this occurred within the first 7 days after brining when the concentrsution of the amino acids in the non-fermented control brines were still increasing. This indicates that the yeasts were quite active in the destruction of tryptophane in these three laboratory fermentations. \ — ►Tryptophane Concentration Population mL -o - -o -Y e a s t per o 25 population per mi 30 jjg Log • of yeast tryptophane 20 30 25 20 -3 15 TIME F ig . 1*+. IN 20 DAYS 25 30 Com parison o f try p to p h a n e c o n c e n tr a tio n s and y e a s t a c t i v i t y i n th r e e la b o r a t o r y fe r m e n ta tio n s . 82 The coliform organisms had completely disappeared from these fer­ mentations by the fifth day and the peak of the acid—forming bacteria populations had been passed in both the FC 1 and FC 2 lots before any reduction in tryptophane was noted* Therefore, these organisms probably did not contribute greatly to the destruction of tryptophane* The Effect of Various Microorganisms on the Vitamin and Amino Acid Content of Cucumber Brines The study of commercial and laboratory fermentations indicated that the levels of some of the vitamins and amino acids in the brine may be influenced by the microflora present. Thus, a survey was made of 2 species of bacteria and K yeast species that have been found to be most active in cucumber fermentations (viz. L. plantarum* a coliform organism, Torulopsis holmii. Torulaspora rosei. Torulopsis caroliniana* and Kansenula subpel1iculosa) as to their effect on the concentrations of these nutrients in brine. Large lots of the non-fermented brines described in the previous section w e r e obtained when the cucumbers were removed from them. brines were frozen and held for use in this study. These The brine was pre­ pared by filtering through cheese cloth to remove dirt particles. After thorough mixing, it was dispensed in 5^0 ml Erlenmyer flasks, 250 ml per flask, and sterilized at 15 pounds pressure (121°C) for 20 minutes. The bacteria and yeast cultures to be tested were grown in microinoculum broth (Difco) for 2 b hours, centrifuged, and resuspended in five ml of sterile isotonic saline. One drop of this suspension was used to inoculate a flask of brine. Each organism was inoculated into S3 one flask of the sterile "brine* One flask was not inoculated, and served as the control* After five days incubation at 30°C, heavy growth was noted in all of the inoculated flasks as evidenced by turbidity and (or) sediment* Since several days were required to run all the microbiological assays, the flasks of brine were autoclaved at 15 pounds pressure for 10 minutes to prevent further activity. The results of the vitamin and amino acid studies on these lots may be noted in Figs. 15, 16, and 17, and Table 30. It is readily ap­ parent that the bacteria and yeasts tested had little or no effect on the niacin content of the brine. The niacin concentrations of the var­ ious inoculated lots of brine varied less than 5 percent from that of the uninoculated control. Thus, they were not believed to be signifi­ cant* However, L. plantarum produced a significant reduction in both the pantothenic acid and biotin content of the brine. The coliform or­ ganism and the four yeasts failed to lower the pantothenic acid level, but all of these organisms except Hansenula subpelliculosa reduced the biotin content by considerably more than 10 percent. The Hansenula species 3.owered the biotin level by almost 10 percent. A synthesis of pantothenic acid was indicated in the brines inocu­ lated with Hansenula subpelliculosa and Torulaspora rosei. These results were in general agreement with the studies made by Rosen and Fabian (51) on the effect of these microorganisms on the niacin, pantothenic acid, and biotin content of cucumber juice. However, Zk Uninoculated control plantarum Coliform » Torulopsis holmii mmmmmmmm Torulaspora rosei “ ■^■“ “ “ Torulopsis caroliniana ■^■■“ ■^“ “ ■^“ “ ■■“ ■“ ■^ Hansenula su b p e llic u lo s a *i™,^ ^ ™ ^ " ^ " ^ ^ " " ™ ,™ '"i,',B™ ---- 1---- 1---- 1_ _ _ _ _ _ i_ _ _ _ _ _ t____ l____ i_____ i_____ i_____ i ____ t____ i_ _ _ _ _ i_ _ _ _ _ _ 0 0 .2 0.4 0.6 0.8 10 1.2 14 MQ niacin per ml Uninoculated control l_ .p la n to ru m ^ ^ ^ ^ ^ ^ ^ « " " « C Q lifo rm * 1^ " ^ * 11^ ^ ^ ^ ^ 11^ * 1 Torulopsis ^1011™ !*^“ “ ^ ^ ^ ^ ^ “ “ “ ^ “ “ “ “ ^ “ “ “ ■“ Torulaspora rose i “ “ “ ■“ “ “ “ ^ ^ ^ “ ^^™™“ “ Torulopsis c a ro lin ia n a "^ "'^ ^ ^ “ “ ^ ^ ^ “ ,l^,^ “ Hansenula s u b p e l l i c u l o s a ^ i i . 0 I 0.2 i J i 0.4 l l 0.6 i_ _ _ _ _ I_ _ _ _ _ 1_ _ _ _ _ I_ _ _ _ _ I_ _ _ _ 1_ _ _ _ _ i_ _ _ _ L 0.8 1.0 1.2 1.4 1.6 pg pantothenic acid per ml Uninoculated ■“ ^ “ ^ ^ “ L. plantarum — Col i f or m “ control Torulopsis holmii Torulaspora ^ “ ■“ Torulopsis caroliniana■^■l^ ^ ^ ™ ,,ll,■ ■■“ ^ ■ ^ “ ■ Hansenulo i 0 ----------Fig* 15* ro s e i^ ^ ^ " " " 1^ ^ ^ ^ - " t i 2 i 4 subpel lic u lo s a “ “ “ “ “ ^ ^ “ “ “ i i_____ i------1----- i----- 1----- 1----- 1----- 1----- 6 8 Mjug biotin per ml 10 12 14 The effect of various microorganisms on the available niacin, pantothenic acid, and biotin content of cucumber brines. 85 Uninoculated control L . p lo n t o r u m ^ ^ ^ ^ ^ v iK — Coliform ^ Torulopsis holmii — * — ■ Torulaspora rosei » Torulopsis carol iniana Hansenula su b p e llicu lo s a“ B™ ^^™ ^^“ --------------- 1-------------- 1--------------- 1------------- !-------------- 1____ J 0 40 i i 80 120 jug leucine per ml 1_________I 160 I 200 "■■■■"■■■» Uninoculated control \_m plantarum ^ Coliform — ■— Torulopsis holm ii mmKmm^^m Torulaspora rosei Torulopsis caroliniana Hansenula subpelliculosa J_______ I________ L ______ I_______ L_______ I_______ I_______ I_______ I_______ I________ I________I__ 0 20 40 60 80 100 120 pg isoleucine per ml Uninoculated c o n t r o l" ■ ‘^ “ aM“ " B" Bl^ ^ “ , i, i|,B1" plantarum — Coliform — ^ Tor ul opsi s holmii. Torulaspora rosei —— Torulopsis c a r o l i n i a n a * " ^ H a n s e n u la t I I 20 0 Fi^“* l6. I 40 S U b p e l l i C U lO SO " ^ m m I______ l______ I______ I------------1-----------1-----------1------------ 1-----------1---------- 60 80 jjq valine p er ml 100 120 140 The effect of various microorganisms on the available leucine, isoleucine, and valine content of cucumber brines. 86 Uninoculated L. plantarum q 0 jjf0rm — i ^ Torulopsis holmii Torulaspora rosei Torulopsis caroliniana Hansenula subpelliculosa _____________ 1____________ i _________ _ j ____________ I____________ l_________ 0 5 10 15 20 jug tryptophane per ml 25 Uninoculated control L. plantarum —i ^— Co l i f o r m^ — Torulopsis holmii “ "■■■l™Bi— Torulaspora rosei i— Torulopsis c a r o l i n i a n a ^ Hansenula s u b p e llic u lo s a - - " * 1" ^ ^ ^ " ^ ^ ^ " 1^ ______ i______ i______ I ______ I______ 1-- o 40 — I------ 1------ 1 ------ 1------ 1------ L 80 120 160 jug glutamic acid per ml 200 Uninoculated control “ ™ l . p la n ta ru m ^ ■ ^ “ ,l^™ ,^ ^ ■ ,,™ Coliform Torulopsis h OI m i i “ 1i1^ Toruiaspora rosei Torulopsis ca ro lin ian a i 0 i |.o Fig. 17. Hansenula subpelliculosa i i < i i--- 1 ---- 1---- 1 ---- 1 ---- 1 ---- 1---2.0 3.0 4.0 5.0 6.0 70 jug cystine per ml The effect of various microorganisms on the available trypto­ phane, glutamic acid, and cystine content of cucumber brines. 37 0 CMJ / —' / “ • 0 d v ^ '- ' rt •H 49 •a 0 Gd rH d * VO • • LTVvO o d rt 0 o •H 49 a CD 0 o •rt d 49 O ra -H 0 *a 0J § •rl a a 0 <+H £1 CO s o ■a GO s rg >ad ^GO *H. O CD 49 ij -O ^o i—i o ® H a o• * 0 /- v / —^ 0 d >—r V_- 0 d LTV LTV d GO d v O CM O GO CM O • * • * • • -d CM Gd VO l— • vo • l*"V LTV rH r— LTV LTV CM O • dGd rH • d ■ —' GO • vD Is— lo GO rH d * o> # GO OG • rH CM rH CTs • GO GO rH CO o o GO • rH Gt~\ • G— O rH i—1 • Gd O • crv i i — CM o o o 0 d vo o• • -O r — CM O LTVVO VO vo UTN GO • LTV CO O CM # d CM © d • crv JD 8 d* O LTV rH CO o CM f> ft *H (D 49 P a 0 o o +9 •H *----. rH <+h 0 d 9 EH •H Ft a d •H e St 0 0 +» d •H > a p o a d o 0 +> 8 ft d 0 co a •fd Ft O •H a 0 a o •H Ft 0 > O 1— • -d rH rH a* « - p tyO © •a a ^ +» •iH > C/5 a 0 * »J0 p 0 f ! rH *H S § 0 o o CM O GO LTV o> CO vO GO ■O CT\ LTV d* vO Ov GO OV LTV urv o co d O a 0 I S3 rH O -H S W O ■"-- •H a cs a o •H GO 0 0 d 0 a a ft LO •H § 0 vO d •H o co 4 tn o 0 GO * .s a +> *-— o *H PQ g ft o •H +3 4g ft -S 0 O £ o o o CM CM LT\ 1— rH • crv o CM • CM i—1 r-~ o o s 1— • cr CM d « o cn lo go CM * CO o •rH a 0 G*~\ LO GO CM rH o rH CM r CM • rH G— O CM ■ —t irv CM r Cjd 49 *fH • GO O 49 S3 0 CM CJV • c o CM • c o ro GO rH CO o VO CM CM ft as p — * rH rH O CM d CM • rH LTV GO CM «H o +» o 0 «H — 0 pH «H O -H ■ —i a s rH a o O d EH 0 a tn o •rH CD P4 w 0 H rH 0 « a a N -' f-i O Eh 01 ol •H 0 rH s. +9 49 rH P —1 0 a 1 -9 0 o CM GO 4 Q> rH GO a OG 0 a • • a 0 © LTV l o a <+H •aH a 0 o » O +> O o a 0 a a o a o 0 o 49 O o g 0 a g a a d o o o a LTV 0 o a d I — 0 o• 4> Vt 0 CM1 0 d a a 0 0 CM 0 d rH +9 rH d 4 0 ■3 9 •I a o to ao pq 0 <44 d O a dd 0 tn a 49 4h VO m • 0 Vi ft d a 0 •rt 0 d o> d a d £ a 49 d 0 0 0 409 d 49 d 49 GO a 0 0 a *H a •H rH o £ •H 0 a O 49 a •H 0 CM rH •rl 0 a 49 VO O d © a >1 49 a a •H 0 0 rH o rH >> a a © •rH 0 d tn a ft a •H •rH >s K 0 tn a © ft O LTV P •rl 4o9 © 0 o a O g rl o 0 a 3 49 p a tn o tn a 0 o 0 d EH £ 0 0 a t—i d 0 49 0 ,— . tn 0 3 EH d V-O s 7^ «n 88 a much greater reduction in the hiotin content of the "brine used in these experiments was noted with L. piantarum than in the diluted cucum­ ber juice as tested "by these workers. The extent of growth and acid production in the two different media as well as the difference in the strains tested are variable factors which may account for this signifi­ cant difference in bio tin utilization by L_. plantarum. The results of this experiment were quite similar with regard to the influence of the various microorganisms on the leucine, isoleucine, valine and tryptophane content of the cucumber brine (Figs. 16 and 17 and Table 30)* Thus, L. piantarum and the four yeasts tested were noted to lower the levels of all these amino acids, although the decreases in valine and tryptophane contents due to Hansenula subpel1iculosa were too small to be significant. The coliform isolate failed to affect the concentrations of these four amino acids to a significant extent. In the case of glutamic acid, about the opposite relationship was noted. The coliform isolate lowered the glutamic acid content by more than 50 percent while the yeasts failed to decrease the level signifi­ cantly. In fact, the growth of Torulaspora rosei and Hansenula subpel- liculosa resulted in an increase in the available glutamic acid. Only a slight decrease was noted due to L_. plantarum. The concentrations of all of the amino acids except cystine in the uninoculated control lots of brine were approximately what were expected on the basis of the assays made on the non-fermented brine samples in the previous section. lower. The cystine content, however, was considerably The only plausable explanation for this was that much of the cystine was destroyed or rendered unavailable for Leuc. mesenteroides P-60 89 by the sterilizing process. Similar observations have been made by Hiesen et_ al. (I+9) and Sarkar et a1 . (53) on the cystine content of basal media. The former workers demonstrated that the effect of ster­ ilization reduced the availability of cystine to Leuc. mesenteroides about 40 percent but did not affect its availability to Lactobacillus casei. Sarkar et^ al. (53) noted that the loss in test material was proportional to that in the standard and, thus, did not result in in­ accuracies in cystine assays. To be sure that the differences in cystine concentrations noted in the various inoculated lots of brine was not the result of the ster­ ilization process the experiment was repeated for this amino acid. The brine used in this experiment was also from the non-fermented control lots. It was filtered through cheese cloth and dispensed in 16mm tubes, 10 ml per tube. One tube was saved as an unheated control and kept un­ der toluene in the refrigerator. The other tubes were sterilized at 15 pounds pressure for 10 minutes, cooled, and inoculated with the various microorganisms. The preparation of the inoculum and the inoculations were carried out in the same manner as in the foregoing experiment ex­ cept that the initial cell suspension was diluted 1-25 before making the drop inoculations. After five days incubation at 30°C, microbiological assays for cystine were made immediately to prevent the necessity of resteriliza­ tion. The results of this experiment are given as the (b) values for cystine in Table 30* On comparing the heated with the unheated controls, it was evident that the sterilizing process reduced the available cystine content of 90 the brine markedly. However, the results obtained on the influence of the various isolates on the cystine content were quite consistent in both trials. Thus, it was believed that the reduction in cystine was incidental to this experiment. The average values for the two trials are shown in Fig. 17* Both L. plantaruia and the coliform isolate reduced the available cystine con­ tent of the brine markedly— by about 30 percent and 50 percent respective­ ly. The growth of Torulopsis holmii resulted in an increase in the a- vailable cystine, while the other three yeasts failed to influence the concentration of this amino acid to a significant extent. It should be pointed out, that the utilization of cystine by the coliform group could be quite important in cucumber fermentations. This group of organisms have been noted to be active in some fermentations the first few days after brining and it was at this period that the cys­ tine concentrations in a few of the fermenting brines studied were noted to be relatively low. LISCUSSIOU The acid fermentation of cucumbers for salt stock is dependent on the growth of the common lactic acid organism, L. piantarum. These re­ sults agree with those of Etchells and Jones (lb) and Rosen and Fabian (5b)* However, cucumber fermentations are not that simple since a num­ ber of yeast species have been shown to be active in both commercial and laboratory fermentations. As was noted by Etchells et, al. (13) » Torulopsis holmii was the yeast species found to be most prevalent in the early part of most commercial fermentations studied. Torulaspora rosei. however, was dominant in one commercial tank and in three labor­ atory fermentations. This shows that even under similar conditions the yeast flora may be significantly different. Yifo.ile no significant Aerobacter activity was demonstrated in these experiments, Etchells et_ al. (14) and Rosen and Fabian (51) have noted these organisms to be quite active in commercial fermentations in the South and in laboratory fermentations respectively. Therefore, a wider survey of commercial fermentations in the Horth for Aerobacter activity is indicated. The vitamin and amino acid requirements of L. piantarum isolates from cucumber fermentations were found to be similar to those for the well known 17-5 strain. The requirements of this species for biotin, niacin, and pantothenic acid and for leucine, isoleucine, valine, glu­ tamic acid and tryptophane noted in this investigation are in agreement with the reports by other workers (10, 24, 33, 43, 55). Cystine has 92 also been observed to be essential for this organism in this work and in that by other workers (24, 33* 43, 55)* Conversely, this amino acid was found by Dumet al. (10) to be non-essential for L. plantarum 17-5 in a greatly enriched medium. lYhile threonine was found to be essential for all strains tested including 17-5 i*1 the basal medium used in these experiments, other investigators (43, 64) have definitely established that L. plantarum 17-5 does not require threonine in different media. Thus, it appears that the requirements for these two amino acids depends on the basal medium used for testing. Some variations were noted among the isolates tested in respect to riboflavin and p-aminobenzoic acid and to phenylalanine, tyrosine and arginine requirements. Previous reports have also been conflicting as to the requirements of this species for these nutrients. However, since the basal medium was constant in these experiments the variations noted must be due to differences in strains rather than in the test medium. Similar variations between strains of the same species have been demonstrated by Dunn at al. (10) and Shankman et_ al. (56). Since L. plantarum from cucumber fermentations requires a number of vitamins and amino acids for growth and acid production these essen­ tial nutrients must be available in cucumber brines in sufficient quantities for this organism if a desirable acid fermentation occurs. This has been found to be true in this investigation and in that of Rosen and Fabian (51) in the case of bio tin, niacin, and pantothenic acid. These vitamins are extracted rapidly from the cucumbers by the brine and apparently reach a point oi equalization in about 5 days. 93 While yeasts and bacteria which were isolated from the cucumber fermentations markedly reduced the biotin content of the brine when grown in pure culture, similar reductions were not observed in the cu­ cumber fermentations. This is due possibly to a buffer action of the cucumbers with more biotin diffusing out as it is being utilised. As might be expected due to their relatively low solubility, the amino acids required longer to attain their maximum concentration in the brine. Nevertheless, all six essential amino acids were present in sufficient concentrations in most instances to support the growth of L. plantarum within 24 hours and were not apparently limiting for the growth of this species in any fermentation studied. It should be noted that very little yeast activity occurred in these brines in the first week and practically no Aerobacter fermentation was evident and this may have greatly influenced the results obtained with at least two of the amino acids. Thus, it was demonstrated that marked reductions occurred consistently in the tryptophane levels of the brine when yeast activity was greatest and that similar reductions were ob­ tained when pure cultures of the predominating yeasts were grown in sterile brine. If such activity occurred early in the fermentation as demonstrated in some instances by Etchells et al. (14), this amino acid might be reduced to a critical level for the growth of L. plantarum. Also, much activity of coliform organisms could possibly result in crit­ ical concentrations of cystine as these bacteria were shown in tnese studies to reduce the concentration of this amino acid in brine greatly when grown in pure culture. Although, the other essential amino acids were affected by one or more groups of brine microorganisms their 94 concentrations in the "brine were relatively high in comparison to tryp­ tophane and cystine and, thus, would not "be expected to "become limiting. A definite relationship was evident "between the concentrations of all the vitamins and amino acids studied and the size of the cucumbers in the brine. Thus, the brines covering smaller cucumbers contained higher concentrations of these nutrients than those covering comparable cucumbers of larger sizes when the ratio of brine to cucumbers was con­ stant. Therefore, larger cucumbers must contain less of these nutrients per unit weight or there is less complete equalization of the nutrients between the brine and cucumbers with the larger sizes than in the case of the smaller. However, Rosen and Fabian (51) have shown large differ­ ences in vitamin concentrations in cucumbers of the same size. A study of these nutrients in cucumbers of various sizes and varieties would be of much interest. SUMMARY A study of the vitamin and amino acid requirements of L. plantarum isolated from cucumber fermentations and the availability in cucumber brines of those found essential has been presented* Ten commercial and three laboratory fermentations were characterized for microbiological activity as a basis for this nutrition study. Under commercial conditions the acid-forming bacteria attained their maximum activity in the first 3 to 6 days and declined throughout the remainder of the fermentation period studied* Yeast populations de­ clined during the first few days and then multiplied rapidly reaching their maximum in from 10 to 20 days after brining* The coliform organ­ isms played no significant role in these fermentations. The microbio­ logical activity in laboratory fermentations was similar except for the yeast picture; yeast activity was variable in these brines. The principal acid-forming organism was L. plantarum* and the most active yeast was Torulopsis holmii. However, in the laboratory fermen­ tations and in one commercial tank Torulaspora rosei was predominant in the yeast flora* Hansenula subpelliculosa and Brettanomyces versatilis were also present in the fermentations* The vitamin and amino acid requirements of L. plantarum isolates from cucumber fermentations were very similar to those for L. plantarum 17-5* Thus, the vitamins— biotin, niacin and pantothenic acid; and the amino acids— leucine, isoleucine, valine, glutamic acid, cystine, trypto­ phane, and threonine were essential for this species in the basal media 96 used. Riboflavin was essential for two strains and ^aminobensoic acid for three strains from cucumber fermentations. Alanine, phenylalanine, arginine, and tyrosine were either stimulatory or essential for one or more of the isolates* Biotin, niacin, and pantothenic acid became available in cucumber brines very rapidly, reached their maximum concentrations in 5 to 7 days, and the levels remained relatively constant thereafter. influence of fermentation on these vitamins was evident. No great However, the biotin concentration in control brine was lowered considerably by the growth of the various brine organisms in pure culture. Although the amino acids were considerably slower in reaching their maximum concentrations in the brine than the vitamins, in most fermentations they were present in sufficient concentrations within 24 hours to support the growth of L. plantarum. Cystine was found to diffuse more slowly than the other five amino acids from the cucumbers into the brine* Tryptophane was the only amino acid studied which was consistently affected by the fermentation* The level of this amino acid was markedly reduced in the brines at the same period when the yeast activity was greatest, and was also reduced by pure cultures of these microorganisms and by L. plantarum in control brine* Leucine, isoleucine, and valine were also rendered less available in the control brine by the growth of brine yeasts but were not measurably affected in the cucumber fermenta­ tions studied. Available cystine and glutamic acid concentrations were greatly lowered by the growth of a coliform isolate from cucumber fermen­ tations. 97 The available concentrations of all of these essential nutrients for L. plantarum were influenced by the size of the cucumbers; brines containing smaller cucumbers were richer. 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