DOCTORAL DISSERTATION SERIES THE triPOfcT/MCL Of BfOTMs MACt/t title M b PAtoWHt/IIC M/D (N cm .uMOLL r m a u th o r m m _________________________________________________________ u SMML-MMU_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ UNIVERSITY M CM dh DEGREE ILL- SfA Jl CPU.. DATE / 9$2 j PUBLICATION _ NO, H5820 45M s UNI' 1 UNIVERSITY MICROFILMS —% A U U A D D ft D ki If U IA A U THE IMPORTANCE OP 3I0TIN, NIACIN AND PANTOTHENIC ACID IN CUCUMBER FERMENTATION By Samuel Rosen 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 OP PHILOSOPHY Department of Bacteriology and Public Health Year 1952 ACKNOWLEDGMENTS The writer wishes te express his sincere appreci­ ation te Dr. F« W. Fabian Ter his enceuragement and help­ ful suggestions given during the course of this study. Also, the writer is Indebted te Roland C. Fulde and Dr. Hilliard Plvnlok for their instruction in the use of certain pieces of laboratory equipment and te Ralph N. Cestilew for his guidance in the study of brine yeasts. Further­ more. grateful acknowledgment is made te the H. V. Madison Pickle Company of Mason. Michigan and to the Department of Horticulture of Michigan State College for generously supplying encumbers. Appreciation is also given to Dr. C. A. Hoppert and to Dr. H. J. Stafseth for suggestions in the writing of this thesis. TABLE OP CONTENTS Page GENERAL INTRODUCTION .................................. 1 PART I THE MICROORGANISMS OP THE CUCUMBER FERMENTATION . 2 Introduction................ • • • •• .......... 3 Review of the Literature* • • • • • • • • • • • 3 Laotlo F omentation • • • • • • Hydrogen Fomentation • • • • • Yeast Fomentation* • • • • • • Composite Microbial Population* • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 4 Experimental* ........... • • • • • • • • • • • 6 5 Microbiological Analysis of Cucumber Fomentation* • • • • • • • • • • • • • • • 6 Identification Studies* • • • • • • • • • • • 9 Identification of Acid-producing Baoterla • 15 Identification of Aerobaoter and Related Organisms • • • • • • • • • • • • 16 Identification of Yeasts* • • • • ..... 17 Discussion* • • • • • • • • • • • • • • • • • • II A VITAMIN STUDY OP THE CUCUMBER FERMENTATION* • • Introduction* • • • ........ 20 23 • • • • • • • • • 24 Review of the Literature* • • • • • • • • • * • 25 Vitamin Requirements of Laotic Acid Bacteria* Bletln, Niacin and Pantothenlo Acid Content of Cucumbers, Pickles and Cabbage • • • • • Microbial Synthesis and Destruction of Vitamins* • • • • • • • • • • • • • • • • • Experimental• • • • . • • • • • • • • • • • • • Vitamin Requirements of Lactobacillus piantarum Isolated from Cucumber F o m e n t ail on • • • • • * * • • • • • • • • • Determination of Blotln, Niacin, and Pantothenlo Acid in Cucumber Juice• • • • • Effeot of Aerebacter cloacae and Various Yeasts on the Vitamin Content of Cucumber Juice* • • • • • • • • • • • • • * 25 28 29 32 32 38 4° TABLE OP CONTENTS CONT. Pago PART Vitamin Analysis During Cucumber Fermentation* • • • • • • • • • • • • • • • • Discussion* 1*6 ......... • • 47 III ROLE OF BIOTIN IN CUCUMBER FERMENTATION........... 53 Introduction* • • • • • • • • • • • • • • • • • • 54 Review or the Literature* • • • • • • • • • • • • 5 4 .Hi story of Biotin • • • • • • • • • • • • • • • 54 Bietln Requirements for Microorganisms* • • • • 55 Antibietins • • • • • • • • • • • • • • • • • • 5 5 Substitute* f6r ,Biotin......................... $6 Functions of Biotin • • • • • • • • • • • • * • 5.9 Experimental* • • • • • • • • • • • • • • • • • • 6 0 Blotln Utilisation by L* piantarum in Cucumber Juice* • • • • • • • • • • • • • • • 6 0 Blotln Utilization by L • pi ant arum in Blotln Assay Medium • • • • • • • • • • • • • 6 0 Tween 80 as a Blotln Active Material for L« piantarum* • • • • • • • • • • • • • • • • 6 2 UtTllsation of the Biotin Aotivo Fraction of Tween 80 by L* piantarum • • • • • • • • • 6 3 Effect of L* planTarum on fche Biotin Content of Assay""taealum Containing Only Biotin and a Combination of Blotln and Tween 80* • • • • 6k Blotln Requirements of Aerobacter cloacae • • • 6o Effect of Aerobacter cloacae on the Blotln Activity of Blotln Assay Medium Containing Varying Concentrations of Blotln and Tween 80 68 Effect of A* cloacae on the Blotln Active Substances in Varying Dilutions of Cucumber Juice. • • • • • • • • • • • • • • • 6 9 Discussion. • • • • • • • • • • • • • • • • • • • 7 0 GENERAL D I S C U S S I O N .......... 74 SUMMART.................................................... 76 CONCLUSIONS....................... BIBLI0GRAPH7 78 . • 79 LIST OP FIGURES PIgure 1 Page Apparatus used for sampling brine from fermenting cucumbers* • • • • • • • • • • • 2 Numbers of organisms per ml in 30° salometer brine • • • 3 4 5 .......... • • • • • 13 Vitamin responses of Lactobacilli isolated from cucumber fermentation compared with Lactobacillus arablnosus 17-5 • • • • • • • 37 Effect of A* cloaoae and various yeasts on Effeot of A* cloaoae and various yeasts 8 9 43 on the niaoTn content of cucumber juice* * * * 7 12 Numbers of organisms per ml in 30° salometer brine • • • • • • • • • • • • • • the biotTn content of cucumber juice** * * 6 7 44 Effeot of A* cloaoae and various yeasts on the pantothenic acid content of cucumber juice • • • • • • • • • • • • • 45 Effeot of L* piantarum on the blotln content of cucumber juice ............ 6l Effect of L* pi antarum on the content of the biotTn aciive fraction of Tween 80 in blotln assay medium* • • • • • • • • • • 65 LIST OP TABLES Table 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Page Total acid, pH, and organisms per ml during cucumber fermentation salted at 30° salometer (Crock A ^ ) ........................ 10 Total acid, pH, and organisms per ml during cucumber fermentation salted at 30° salometer (Crook ) • • • • • • • • • • • • * 11 List of numbered Isolates from cucumber fermentation.............. lL|_ Biochemical reactions of Aerobaoter and related organisms* • * • • • • • • • • • • • • 18 Morphological, cultural, and biochemical characteristics of yeasts Isolated during cucumber fermentation* .......... 19 Vitamin requirements of various species of lactic acid baoterla (after Snell)* • * * * * 27 Blotln requirements of L* pi ant arum and L* arablnosus 17-5 * 7 . 3 4 Niacin requirements of L* piantarum and L* arablnosus 17-5 • • • • • • • • • • • • • • 35 Pantothenic acid requirements of L* piantarum and L* arablnosus 17-5 • • • • • • • • • • * • 36 Vitamin content per ml of cucumber juice of different varieties of cucumbers obtained from various sources • • • • • • • • • • • • • 39 Effect of Aerobacter cloaoae on the vitamin content of cucumber juice* • • • • • • • • • • i|l Effeot of Hansenula subpelllculosa isolated from laboratory cucumber ferment; at Ions on the vitamin content of cucumber juice* * * * * 42 Effect of various yeasts isolated from commercial cucumber fermentations on the vitamin content of cucumber juice* • • • • • • 42 Total acid, organisms per ml, and vitamin content per ml brine of cucumber fermen­ tation (30 3) (Crook kr>) • • • • • • • • • • 48 LIST OF TABLES CONT Table 15 16 17 18 19 20 21 22 23 Pago Total acid, organisms per ml, and vitamin content per ml brine of cucumber fermen­ tation (30° S) (Crock Bg ) • • • • • • • • • • I4.9 Dilution factors for cucumbers fermenting in crooks A2 w d ............... 50 Vitamin content per ml of cucumber Juice of cucumbers at the beginning of a fermen­ tation and after 20 days • • • • • • • • • • * 50 Vitamin content per ml of cucumbers and brine at the beginning of a fermentation and after 20 d a y s * ............................... 5l Effect of L* piantarum on the biotin content in blotln assay medium • • • • • • • • • • • * 62 Bffeet of Tween 80 as a biotin active material for L • pi ant arum and L . arablnosus 17-5* • • • 63 Utilization of biotin by L* piantarum from assay medium containing"”only blotln and a combination of biotin and Tween 80 * * * * * * 66 Summarized data for the utilization of biotin active components from various sources by L • pianta rum, • • • • • • * • • • • • • • * 67 Biotin requirements of Aerobacter cloacae* * * * 68 2lj. Utilization of biotin active substances by A* cloaoae in biotin assay medium containing biotin and Tween 8 0 . • • • • • • * 25 Utilization of blotln active substances by A* cloacae in varying dilutions of cucumber Juice • • • • • • • • • • • * • • • • 69 70 GENERAL INTRODUCTION The cucumber fermentation, as it is carried en in many parts ef the world, is uncontrolled. Cucumbers are picked frem the field, placed in a vat, cevered with brine, and allewed te ferment* The microorganisms involved in the fermentation come frem the surfaoe ef the cucumbers. Need­ less te say, a vast variety ef erganisms are introduced into the fermentation* Many types ef mioreerganisms are prevented from multiplying due te the high salt concen­ tration ef the brine* Seme ef the more important groups ef erganisms which persist in a cucumber fermentation are the Lactobacilli* Aorobaotor and related erganisms, and yeasts* It is a well established fact that the Laotobaollll are the most vital group of microorganisms in a cucumber fermentation* It is also well known that many Laotobaollll are rather fastidious in their .vitamin requirements* The purpose ef this study was te demonstrate that the Lacto­ bacilli frem ououmber fermentations required certain vitamins, and te show hew these vitamins were made avail­ able for this group ef organisms* It was also intended te Investigate thoroughly the role ef any specific vitamin or vitamins in the oueumber fermentation which appeared te be orltleal for the Laotobaollll* PART I THE MICROORGANISMS OF THE CUCUMBER FERMENTATION 3 INTRODUCTION In the fermentation ef cucumbers fer salt stack, three types ef microbial fermentations are generally recognized. These are the laotle fermentation, the hydrogen fermentation, and the carbon dioxide fomentation produced by Laoto­ baollll, Aerobacter. and yeasts respootlTely (1*1). The primary objective of this particular study was te further investigate those groups of organisms, REVIEW OP THE LITERATURE Laotlo Fomentation, - Aderhold (2), In 1899, and Kessewloz (57), in 1909, wore among the first workers to study the flora of aetive cucumber fomentations. Their work, however, was concerned with spoilage baoteria, Rahn (85), In 1913, reported that at the height of a fer­ mentation, 50,000,000 to 200,000,000 acid-forming baoteria were present. Brown (10), in 1916, found that the acid- forming baoteria wore short rods or oocol and facultative anaerobes, Le Fevre (61*, 65, 66), In 1919, 1920, and 1922, shewed that cucumbers Introduced laotlo aoid-preduolng baoteria as well as other organisms. He found the optimum temperature for this group of organisms te be 30* C, Fabian et al (1*2), In 1932, studied the populations of acldproduolng organisms In 30* and 1*0° salometer brines. They found the number of aeid-preducing organisms to be greater and to roach a maximum sooner In the 30* salometer brine. i4. The maximum number ef aola-preducers was 195*000*000 per ml In the 30* salometer brine* which was attained after 6 days. The maximum In the I4.0* salometer brine was li|l,000,000 per ml* which was reached after 13 days* In 1935* Vahlteloh* Haurand* and Perry (105) hollered that organisms of tho Lactobacillus type were the chief acld-producers* However* they indicated that the following organisms might also bo present: Louconostoo, yeasts* and other unclassified organisms* Btehells and Jones (38)* in 1914.6* identified all of I4.9 cultures of acid-producing baotoria isolated from fermentation of salt-stock cucumbers te be Lactobacillus pi ant arum* Hydrogen Fermentation* - Veldhuls and Btehells (106)* in 1939* demonstrated that hydrogen was evolved from cucumber fermentations* Etohells* Fabian* and Jones (36)* in 19kS» Indicated that Aerobacter developed during the cucumber fermentation and were responsible for the production of hydrogen* They found Aerobacter to occur in 20*, 14.0* and 60° salometer brines in southern areas of tho United States* However* they stipulated that in 20* and I4.0* brine this fermentation might not ocour* Yeast Fermentation* - Etchells (32)* in 19i|l» showed that yeasts were involved In cucumber fermentations* In 1950* Etohells and Bell (33) found that the predominating genera of yeasts in southern cucumber fermentations wares Torulopsla. Brottanomyoos* Hansonula* and Zygosaooharoacrces* 5 They found that Torulopsia and Hansenula appeared in high numbers within twenty days while the other two genera did not appear until lata in the fermentation. In the following year Btehells, Coatilow and Boll (35) studied yeast popu­ lations in northern brining areas. They found that tho predominating genera wore: Brottanomyces, Torulaapora. Torulopsia, and Hansenula. Film yeasts on cucumber brines hare also been reported. Mrak and Benar (73) and Btehells and Bell (34.) have pointed out tho predominating genus to bo Dobaryomyoos. Composite Microbial Population. - Etohells and Jones (37) hare shown fermentation trends in 20°, 40*# and 60° salometer brines. In 20* salometer brine, the acid-forming baoteria reached a high point in about four days; in 4.0* salometer brine the peak was not reached until nine days. The total count was lower in the 40* brine than in the 20* brine. At 60° salometer, the acid-forming baoteria appeared in low numbers and gradually disappeared. Aorobaotor appeared in similar fashion as the lactic acid organisms. The lower the salt concentration, the quicker the onset of these bacteria and the higher their count. Tho Aorobaotor fermentation was not too active or prolonged at 20° or 40* salometer. At 60* salometer, these erganisms declined at first and became active after eight days. Toasts occurred in fermentations at all levels ef salt concentration. A high salt oenoentratlon tended to hold 6 back the rate but not the quantity at which the yeasts developed* EXPERT MENTAL Mloroblological Analysis ef Cucumber Fermentation Two euoumbor fermentations wore sot up in the labor­ atory in five-gallon crooks* A bent glass tube was placed in each crock so that a sample of brine could bo withdrawn from tho approximate geometric center of the crock* Tho part of the glass tube which extended into tho center of tho crock was protected by a hollow wooden baffle through which numerous holes had boon drilled to permit an inter­ change of the brine (Figure 1)* Number two cucumbers (throe to four inches in length) wore then plaoed in the orock and salted aooerdlng to the 30° salometer schedule* To prevent an excessive accumulation of surfaoe yeasts, an ultraviolet lamp was placed about six inches from the surface of tho brine and kept on throughout the fermentation except when samples wore being withdrawn* Samples of brine wore withdrawn into a Mason jar of ono-plnt capacity* Smaller samples wore then taken from the Mason jar in order to determine tho amount and kinds of ml ore organisms present* Samples wore taken every day for the first six days and thereafter every two or throe days for twenty days* The brine remaining in the Mason jar was returned to tho crook by positive nitrogen pressure* Hollow wooden baffle Pint Mason jar Figure 1. Apparatus used for sampling brine from fermenting cucumbers* *A dap ted from Faville, L. W., and Fabian, F. W #, The influence of bacteriophage, antibiotics, and Eh on the lactic fermentation of cucumbers, Tech. Bull. 217 > Michigan State College, 1 914-9» and Fulde, R. C., Unpublished thesis* 6 Total a d d , calculated as laotlo acid and tho pH were determined oaoh time a sample was removed Tor mlereblelogical analysis* Te Isolate and oeumt the Laotobaollll, samples of brine were plated in an agar medium containing Campbells* V-8 togotable juice as the main constituent* An indicator, brem ore sol green, was added te this medium ibk) • The plates were ineubated at reen temperature for two te three days and only the aold-preducing erganisms were oeunted* The yeasts were isolated and counted by plating in dextrose agar te whleh fire percent NaCl and three ml ef fire peroent tartarlo aold had been added per 100 ml ef medium* Counts were made after four days incubation at room temperature* The isolation and determination ef numbers ef Aoro­ baotor and related erganisms was aoeomplished in a some­ what different manner* Varying dilutions of the samples ef brine were placed in triplicate into tubes containing lauryl sulfate tryptese broth* The tubes were Incubated at room temperature and observed for the formation of gas afct tho ond ef hours* The most probable number ef Aorobaotor and related erganisms was determined from tables which appear in "Water Bacteriology" by Prescott, Winslow, and MoGrady (81|_) • Te isolate Aerobacter, material frem the highest dilution shewing gas in the lauryl sulfate tryptese tubes was streaked ante eesln-methylene blue agar* 9 The L t o f b a c i l l i , yeasts, and Aerebaoter erganisms were iselated by seleoting eolenies typical ef those groups ef organisms* The erganisms were purified by repeated platings in their respeotive Isolation media* The Laotebaeilli were maintained on laetese motility medium; the yeasts were maintained on V-8 agar slants; the Aorobaotor and related organisms were maintained in laotose broth and on nutrient agar slants* Tho numbers of the various groups of mloroorganlsms are presented in Tables 1 and 2 and in Figures 2 and 3* The Laotobaollll which wore subsequently found to belong to tho piant arum speelos could not be detected at first, but grow rapidly for the first few days* They persisted in high numbers for about one week and then there was a gradual decline* The Aorobaotor and related erganisms wore present at the start of the fermentation, grew very well for the first two days, and then declined rapidly* The yeasts were also present initially* Their numbers rose gradually reaching a high point in about eight days* mien there was a gradual decline, but they persisted in comparatively high numbers* 1 dontlfloatlon Studies Cultural, biochemical, and morphologioal studios wore made on these organisms* a number* Each organism studied was assigned Table 3 gives a key to eaoh of the isolates* The day the organism was removed from the fermentation is also 10 indicated* TABLE 1 TOTAL ACID, pH, A*D OROAHISMS PER ML DCJHTMG CUCUMBER FERMEHTATION SALTED AT 30# SALOMETER (CROCK Ax ) Day Pareant acid (Calculated as laotlo aoid) pH L ao tabacil lua niantarum Aerebaeter an«L related erganisms Yeast 0 0*007 6.17 0 147.000 2.900 1 0*023 5*81 50,000 430,000 6,000 2 0*12 5.23 1 ,200,000 430,000 21,400 3 0*46 3.80 145.000,000 220,000 129,000 4 0.77 3.56 41.400,000 920 120,000 5 0*95 3.47 52,000,000 2,500 87,000 6 0.77 3.42 80,000,000 2,500 63.000 8 0.77 3.35 22,200,000 2,500 250,000 10 0.84 3.31 6,800,000 2,500 64,000 12 0.93 3.31 1 .310,000 0 44,000 14 1*01 3.30 640,000 0 32,000 17 1.01 3.30 94.000 43,000 85,000 20 1.02 3.29 86,000 14.700 29,000 11 TABLE 2 TOTAL ACID, pH, AND ORGANISMS PER ML DURING CUCUMBER FERMENTATION SALTED AT 30* SALOMETER (CROCK B± ) Day Percent acid (Calculated as laotlo aOld) pH Laotobaollluo plant arum Aerebacter and related erganisms Yeast 0 0*015 6*61 0 92,000 830 1 0*038 5 .5 5 10,100 9 2 0 ,0 0 0 8,300 2 0*112 5 .4 1 880,000 9*200,000 18,400 3 0*20 5. o i 5» o o o ,o o o 9 2 0 ,0 0 0 12,000 4 0*31 4 .2 4 21, 500,000 92,000 9*400 5 0 .3 1 3.80 26, 700,000 2,200 74*000 6 0*40 3.68 53*000,000 25*000 160,000 8 0.514- 3 .5 3 40, 000,000 2,500 670,000 10 0*66 3 .5 0 20, 400,000 240 104*000 12 0.72 3 .4 3 6, 500,000 4*300 42,000 1*4- 0 .7 7 3 .4 4 2, 090,000 4*300 5o rooo 17 0.80 3 .4 1 1 * 9 0 0 ,0 0 0 2,500 58,000 20 0 .7 7 3 .4 1 2, 270.000 4*300 84*000 8 Crock A 7 Logarithm of count per Lactobacillus piantarum 6 5 Yeasts 4 Aerobacter and related organisms •— • 3 2 0 5 10 15 Days Figure 2<, Numbers of organisms per ml in 30° salometer brine 20 8 Crock Logarithm of count per 7 Laotobaoillus piantarum 6 Yeasta 5 4 ierobacter and related organisms 3 _________________________ i o 5 i 1 -■ 10 15 20 Days Figure 3* Numbers of organisms per ml in 30° salometer brine 13 14 TABLE 3 LIST OF NUMBERED ISOLATES FROM CUCUMBER FERMENTATION Aeld-foxving baotorla Day No* isolated Aorobaotor and related t p g « No* Day No* Day isolated Isolated 600 2 407 2 439 1 701 12 601 3 408 2 44L 2 703 12 602 5 409 2 442 2 701*. 12 603 5 410 3 443 2 706 0 604 6 411 3 ki.i. 3 707 1 605 6 412 3 445 3 709 2 609 3 420 5 446 3 711 3 610 k 421 5 447 4 713 4 611 5 422 5 448 4 714 5 612 6 423 6 449 4 715 8 613 8 424 6 451 5 716 17 61^ 10 425 6 452 5 717 20 615 12 426 8 454 6 718 0 616 17 427 8 455 6 719 1 617 20 428 8 456 8 720 2 621 k 429 10 457 8 723 6 623 6 430 10 459 8 724 7 624 8 431 10 461 10 725 8 625 9 432 17 462 10 726 9 433 17 463 10 728 11 434 17 464 11 729 13 437 0 465 12 470 13 No* Day isolated 15 Identification ef Acid-producing Bacteria* - Nineteen isolates of acid-forming baoteria were maintained on laotose motility medium* Transfers were made te sterile ououmber juice diluted 1:2 and Incubated for 14.8 hours at 30° C. Gram stains were made ef the cultures at the end of 2l|. and 4.8 hours* After 48 hours, transfers were made in duplicate to the following media: nutrient broth, nutrient agar slants, litmus milk, and into purple broth baao containing the following carbohydrates: arabinose, xylose, dextrose, fructose, galaotose, mazmose, sucrose, laotose, maltose, raffinose, dextrin, mannitel, and starch* The tubes were Incubated at 30° C for one te two woeks* The Gram stain showed that all nineteen Isolates were Gram-positive, short, stubby rods with rounded ends* Growth in nutrient broth and on nutrient agar slants was sparse* In litmus milk, the organisms produced an acid curd by the end of seren days* All of the isolates produced acid from the carbohydrates tested except staroh* There was no change in the tubes containing starch although growth was observed* None ef the erganisms produced gas in any of the tubes* According to Bergey* s Manual of Determinative Bacteriology, those organisms were identified as Lacto­ bacillus pi ant arum (9)* 16 Identification of Aerobacter and Related Organisms# Forty-five organisms Isolated from oosln-mothylene blue agar were maintained in laetose broth and on nutrient agar slants* A Gram stain was made from a 2Ji-hour laotose broth culture Inoabated at 35* C* Each of tho pure cultures was streaked onto eesin-methylene blue agar In order to study further their cultural characteristics, Indol* methyl rod* Veges-proskauer, and oltrato tests woro performed* Gelatin llquefaction and glyoerel fermen­ tation tests wore employed for speoles differentiation* All ef the organisms produeed gas in laotose broth* The Gram stain showed all ef the Jj.5 isolates te be small Gram-negative rods* Colonies on eesin-methylene blue agar exhibited the following characteristics after J4.8 hours of Incubation at 35® Cs Sixes Two to five mm in diameter* Colors Light pink to orange to brown* Centers: Dark centers were observed in many colonies* Confluence: Colonies tended to run together* Elevation: Colonies were considerably raised and markedly convex* Metallic sheen: A metallic sheen was observed on rare occasions* The results ef the indol, methyl red* Voges-Preskauer* oltrate* gelatin llquefaotlen and glycerol fermentation 17 tests are presented In Table if* According te Bergey* s Manual of Determinative Bacter­ iology 29 organisms were identified as belonging te the genus Aorobaotor (9)* These erganisms were lndol negative* methyl red negative* Voges-Proskauer positive* and citrate positive* Twenty-one ef those 29 erganisms liquefied gelatin but did not ferment glycerol* Hence* these organ­ isms were identified as Aorobaotor cloaoae* The remaining eight Aorobaotor erganisms oeuld net definitely be ealled Aorobaotor aerogenes since these liquefied gelatin as well as fermented glycerol* One organism (No* 459) was identi­ fied as belonging to the genus Esoherlohla* 15 erganisms remained unclassified* The remaining They oeuld* however* be grouped as intermediate conforms* Identification ef Yeasts* - Twenty-one yeast isolates were maintained on V-8 agar slants* This medium was pre­ pared according to Etohells and Bell (33)* Three-week old cultures from the V-8 agar slants were examined mloreseopioally* Cultural characteristics were studied using dextrose agar and dextrose broth* The yeasts were further studied by determining their ability to ferment the follow­ ing sugars: dextrose* maltose* galaotose* laotose* and sucrose* A nitrate assimilation test as described by Etohells and Bell (33) was also employed* The merphelogioal* cultural* and biochemical charac­ teristics ef the yeasts are presented in Table 5* 18 TABLE i*. BIOCHEMICAL REACTIONS OP AEROBACTER AND RELATED ORGANISMS Isolate no* Indel «e eo k o i I4.O8 — — — k - 0 9 m o 411 412 420 ll? l i |2 2 423 42k 425 426 Methyl red 4- — — — — +• ♦ 4* + — — — “ • f t • — 429 430 431 432 433 434 437 439 441 442 443 i.i.i. 445 446 — — — — — — — — ♦ •• •» — + — VesgesProskauer 4+ + — ♦ + 4- +■ + 44« + + + + 4- + + + 4+■ - + 4- — + +- mm Citrate • 444— 4* + +• 1*9 451 kS2 if? 456 457 439 461 462 463 464 465 470 «• - — — — — + — — — 4* - — — ■* “ + 4* + + + + — + + + 4* 44^ 4mm 4- 4^ 4«. 444444444* 44444444- 4* + +■ 4* 44444444444* + Glycerol 44* 4* 4•1 44+ 4+ 4* + m + 4- 4* 4- — - — + 4* 444* 444444444444- + f t Gelatin 444> 4+ 444444444* - m negative test; + m positive test — ♦ + 4444» • + 444«• 4* 4— — - 4- TABLE 5 MORPHOLOGICAL, CULTURAL, AND BIOCHEMICAL CHARACTERISTICS OP YEASTS ISOLATED DURING CUCUMBER FERMENTATION no, 701 703 10k 706 707 709 711 713 I lk 715 716 717 718 719 720 723 12k 725 726 728 729 Dextrose agar growth Microaoopic picture Dextrose broth growth Rough spores Copulation tube visible Large, pleomorphic cells, non-sperulating Rough spores Hat-shaped spores Round cells Round cells White White White White rough, dull, abundant White smooth, glistening, abundant Rod, smooth, glistening, abundant Red, smooth, glistening, abundant Film Ring Red ring Red ring Hat-shaped spores Round cells Small, pleomorphic cell: $ non-sporulating Small, pleomorphic cell: t non-sporulating Cells and fragments, non-sporulating Hat-shaped spores Hat-shaped spores Hat-shaped spores Hat-shaped spores and fragments containing hat-shaped spores Same as 723 Small, pleomorphic celli 1 non-sporulating Small, oval colls, nonsporulatlng Same as 723 Hat-ahaned meres White, smooth, glistening, abundant Red, smooth, glistening abundant White, smooth, dull, abundant Red ring Ring White, smooth, dull, abundant Ring rough, dull, abundant smooth, glistening, abundant smooth, glistening, abundant Heavy film Ring Ring White, smooth, glistening, abundant White, White, White, White, smooth, smooth, smooth, smooth, glistening, glistening, glistening, glistening, abundant abundant abundant abundant White, smooth, glistening, abundant White, smooth, glistening, abundant White, smooth, glistening, abundant no ring White, smooth, glistening, abundant Whit*. noath. ffliifeninff. Abundint T„ n.t- _____________Sugara farmentad_______________ Hitrata M Daitraaa Maltaaa Galaotaae Laotaaa Sucroaa aaalallatira 701 703 70S. 706 +4» + 707 709 711 713 + • • 714 715 716 717 718 719 720 723 aa + Debaryomyces sp* f. rose! aa • - + - •> * - ■a - «■ • * + - Debaryomyces so* ♦ • a. ft ft • - - • ft • - a. +• + ft + • - + ft H. subpelliculesa fihodeterula ap* Rbocleterula sp. Sansenul'a sp* aa • m m m ft Rhodeterula an* «* aa •a am m - - - • - - - * ft - * + 72k 725 726 728 729 m _ m - • + . a. ft m - - ft •> ft “ ft • • tm + + ft “ + + ft «, ft + ft ft m ft ft ■V m ft m ft • ft ft ft ee + ft ft ft “ • + ft + * positive test - * negative test » s organism net classified * « H. subpelliculesa subpelliculesa Ha subpelliculesa * Hansenula sp* • T. holmll lansenula sp* H» subpelliculesa 20 According to Etchells and Bell (33), Lodder (70), and Stalling Dekker (99)# the yeasts were classified as Indicated In Table 5>* Of the 21 Isolates, eight were placed In the genus Hansenula, The remaining Isolates were classified as follows: Rhedoterula (3 isolates), Debaryomyoes (2 Isolates), Torulospora rose! (1 Isolate), and Torulopsis holmll (1 isolate)* Six isolates could not be elasslfled* DISCUSSION Although scientists are readily tempted to look for a constant sot of data, one must be extremely cautious In analyzing data obtained from a cucumber fermentation* The following six points are suggested as uncontrollable factors which may lead to different results* 1* The microorganisms introduced into the fermen­ tation vary according to the type of soil and weather conditions under which the cucumbers were grown* 2* During fermentation there may be a large variation In temperature* Even in the same area, fermentations started a few days apart may have a great temperature difference affecting the various stages of the fermentation* 3* A tank may be contaminated with chemicals, scum, filth, etc* if* The salt concentration could easily vary from one to two percont* This variation would affoot the numbers and kinds of mloroorganlsms present during a fermentation* 21 5. saltors* The method of rilling a tank varies with different Some may take several days to fill a tank whdreas others may fill their tank In one day* 6* The variety of cucumber Introduced Into the fer­ mentation may have different skins* nutrient materials* water content* etc* In spite of the difficulty of dealing with an uncon­ trolled fermentation certain trends were noted* The aoId-producing organisms apparently belong to one species* vis** Lactobacillus piantarum. They appeared In very high numbers and reaohed the peak of their aotlvlty between three and eight days* The Aerobaoter fermentation was significant In that It disappeared as quickly as It appeared* The peak of this fermentation preceded that of the laotlc fermentation* It seemed that the acid produced by L . pi ant arum could decrease the number of Aerobaoter* Perhaps these organisms benefited the fermentation by oreatlng conditions suitable for L* pi ant arum* They might have oreated lower oxygen tension conditions or synthesised vital nutrient substances* The identification of many of these organisms as Aerobaoter cloacae confirmed the work of Etchells* Fabian* and Jones (36)* The yeasts appeared in slightly lower numbers In these ououmber fermentations than reported previously* This may have been due to the fact that the incubation period of four days was not long enough to detect the tiny yeast* Terulopsls carollnlana* 22 It should bo pointed out that no quantitative char­ acterization of the different yeasts was attempted* Isolated colonies were picked at random* The faot that Hanaonula appeared so frequently In Table £ la not an indioatlon that this yeast occurred to that extent during the actual cucumber fermentation* PART II ▲ VITAMIN STUDY OP THE CUCUMBER FERMENTATION 2k INTRODUCTION During the Initial stages of a cucumber fermentation, the acid-producing baoterla appear In great numbers In spite of the faot that only a few of these organisms are Introduced Into the fermentation* Obviously the environ­ mental conditions must permit these organisms to dominate the early part of the fermentation* It has been explained that the aold-preduoing baoterla tolerate the high salt concentration which Is Inhibitory to many ether types of organisms* It has also been said that the pH of the brine is optimum for the acid-produoers* It was the purpose of this particular work to study another possible reason for the early appearance of aoidpreducing organisms* This study was concerned with the nutrition of these bacteria* Since It has been reported that many Laotebaollll require certain vitamins, It was first decided to establish a few vitamin requirements for L* plant arum isolated from cucumber fermentations* Blotln, niacin, and pantothenic acid were the vitamins selected for study since it has been shown that these vitamins are needed as growth fmotors for many laotlc acid bacteria* After determining the vitamin requirements for this organism, cucumber juice would be analysed for these vitamins* The effect of non-aold-preducing organisms Isolated from cucumber fermentations on the vitamin oontent of cucumber juice would also be studied* 25 The vitamin content during an actual fermentation would also be determined whioh weuld demonstrate the net amount ef vitamins available fer microorganisms* REVIEW OF THE LITERATURE Vitamin Requirements of Laotio Acid Baoterla* - There is confusion in the literature concerning the nomenclature of lactic acid baoterla* Bergey* s Manual of Determinative Baeterlolegy (9 ) lists many laotic aoid baoterla to be synonemous• The literature eoncerned with the nutrition of these organisms dlsousses them as individual species and indioates different nutritional requirements fer organisms listed as identical in Bergey* In fact, Campbell and Hucker (14) found that cultures of Lactobacillus plan-* tarum obtained from various sources differed markedly in their riboflavin requirements* Some cultures grew well in the absence of riboflavin* while others failed to grow regardless of the amount ef riboflavin added to the medium* One strain* L* pi an tarum var* rudensl a would net grow in a riboflavin-free medium but produced increasing amounts of aold with additional amounts of riboflavin* The confusion in nomenclature of laotic acid organisms has not stepped the enormous progress made in their nutri­ tional studies* Sandferd (87) h*s indicated that the earliest nutritional studies ef microorganisms were made with yeasts* It was noted that yeasts failed to grow when small Ineeula were used* It was stated that Wlldlers 26 believed the ability of yeasts to grow to be governed by the presence of water soluble nutrient material occurring In large inooula* Thiamine was perhaps the first pure vitamin to be shown to be required by microorganisms* This was shown in 1935 with the mold Phyoomyoes blakesleamua * Earlier in 1930, Williams and Roehm (110) had demonstrated that antineurltle vitamin preparations would stimulate the growth of yeasts* A few years later work was begun on the vitamin re­ quirements of laotlo acid baoterla* In 193d, Snell and Strong (95) found that laotlo aold baoterla may or may not require riboflavin, depending on the species tested* In 1938 and 1939, Snell, Strong, and Petersen (96, 97) reported that pantothenic and nicotinic acid were required by certain laotic acid baoterla* The following year, Snell and Peterson (93) confirmed the werk of Mueller that pyrldoxlne was required* In 19^4-0 , thiamine was shown to be required by oertain heterefermentative laotlo aold baoterla by Wood ot al (116)* In 1941, Mitchell at al (77) using a purified preparation of folio acid from spinach demonstrated that this vitamin stimulated Lactobacillus easel, Lactobacillus delbrueckll, and rats* Chattaway ot al (15), in 1942, noted that p-aminobenzeic acid would stimulate L* easel* Biotin was found to be required for Lactobacillus easel, in 1943, by Shull et al (90), At this time, studies were broadened to determine if substances resembling the vitamins in chemical structure 27 influenced microbiological aotlvity. Keser (55). in 1941* showed that nicotinic acid and nicotinamide varied in their effect on various microorganisms. Snoll and Rannefeld (9U-), in 19ij-5» found that pyrldexamine and pyrldexal woro more offootire than pyridoxino for most microorganisms tested* Studies woro completed by several workers on numerous Xn 19k2, Behonea ot al (7 ) species of laotic aoid bacteria. found many laotic aoid baotoria to require pyridoxino. They also showed that other laotlo aold organisms did not require this vitamin. In 19*4-5. Choldolln ot al (16) listed 33 strains of laotlo aoid baotoria requiring pantothenic acid. Xn 1945>» Snoll (92) summarised the vitamin requirements of certain laotlo aold baotoria. niose requirements may bo soon in Table 6. TABLE 6 VITAMIN REQUIEEMENTS OP VARIOUS SPECIES OP LACTIC ACID BACTERIA (AFTER SNELL) ew.l L* del* bruockll 3 Riboflavin + ♦ - Pantothonio aold + ♦ + VI t w i n Nicotinic aoid + Biotln + + Pyridoxino + •* Thiamine — - Folio aold + + L. for- L • arab- Leuoonostoo mentum lnosus mesontoroldes 36 p-60 17-5 - + + + + + + - - - - - - +■ + >r vitamin required; - = vitamin not required 28 In 19l|.5* Peterson end Petersen (81) stated that blotln, nicotinic acid, pantothenic acid, and riboflavin were the nest frequently reported vitamins required by microorganisms, each being required by about 50 different organisms* There is a constant search fer additional nutritional factors. In 19if6, Guirard et al (I4.6 ) showed that aoetate and certain lipids could stimulate certain laotic acid bacteria* It was suggested by these workers that aoetate might play an important role in lipid metabolism* In 1947* Shankman et al (88) indicated that many vitamins considered essential would net be required if the organisms were inoubated over a longer period ef time* Blotln, Wiacln and Pantothenic Acid Content of Cucum­ bers, Pickles and Cabbage* - There is a paucity of infor­ mation on the vitamin content of cucumbers and pickles* Slightly more data is available on the vitamin content of cabbage* Per this reason and the faot that the fermen­ tation ef cabbage is similar to that of cucumbers, a liter­ ature search was made on the vitamin content of both vegetables* The research staff ef the H* J* Heinz Company (i|-7) reported less than one mg nicotinic aold per 100 g fer cueumbers and cabbage* Cheldelin and Williams (17) geve the vitamin content ef cabbage as fellows: 29 Blotln 0 #002l4. rag/100 g cabbage HIaoin 0,21 mg/100 g cabbage Pantothenic aold 0,18 mg/100 g cabbage Elvehjem (30) reported the vitamin content of cabbage to be 0*3 mg niacin per 100 g and 0,18 mg pantothenic acid per 100 g, Camillo, Hoppert, and Fabian (13)# in a atudy of fresh and fermented cucumbers shewed that the type of soil in which oucumbers were cultivated had little effect on their vitamin content. They also showed that certain water soluble vitamins (ascorbic aold, thiamine, and riboflavin) in pickles decreased after a fermentation, whereas the vitamin A value Increased# Lampen e_t al (60), In a study of free and bound blotln found that vegetables contained a water extractable form of blotln whereas yeasts, animal products, seeds, and nuts had most of their blotln in bound form. This is particu­ larly significant in food fermentations because one may more readily understand how vitamins are made available fer bacteria during a fermentation. Microbial Synthesis and Destruction of Vitamins, The vast majority of publications Is concerned with vitamin synthesis by microorganisms. However, in this study, vitamin destruction by baoterla and yeasts was of primary interest* In 1930, MoElroy and Gross (61) reported that ribo­ flavin, pyridoxino, thiamine, and pantothenie aold formed 30 in the rumina of ah#op and cows fed diets low in those vitamins* In 1933* Williams et al (109) reported that Aspergillus nlger could synthesize pantdthenlc acid. In 1938* Almqulst at al (4) showed that vitamin K was produced by organisms isolated from fish meal* Xn the same year it was demonstrated by Abdel-Salaam and Leong (1) that thiamine was synthesised by baoterla isolated from the intestine of the rat. In 1939* Silverman and Workman (91) proved that proplonlo aold baoterla could be adapted to synthesize thiamine* Evans et al (39)* In the same year, Indicated that certain strains of Corynobaoterlum diphtherias would synthesize pantothenic aoid if nicotinic aold* plmellc acid and beta-alanlne were present in the medium* In 19*4-0, it was shown by MoElroy and Jukes (72) that blotln was produced in the rumen of the oew* The following year, it was demon­ strated by Landy ana Dickon (62) that many microorganisms would synthesize blotln in an amino acld-glucose medium* In 1942, Eakln and Eakin (27) reported that blotln was produced by Aspergillus nlger in a medium containing plmellc aold* In the same year, Nielson ejb al (79) pointed out that blotln oould be synthesized in the intestines of rats* Mitchell and Isbell (76), in 1942* showed that intestinal baoterla were capable of synthesizing many B vitamins especially blotln and pyridoxino. Nicotinic and panto­ thenic acids were synthesized slightly* They also pointed out that a diet containing laotose, favoring the production of aold, had a marked Influence on the amounts ef vitamins synthesized* An aeld-formlng diet made pantothenic aoid more 31 available while pyridoxino and thiamine were made more available by a non-acid-forming diet. In 1942# Burkholder and MoVelgjh (12) showed that blotln, niacin, riboflavin, and thiamine were synthesized by Bacterium aero genes and Esoheriohla coll# Xn 191+2* Thompson (103) demonstrated that many baoterla Including Aerobaoter aerogenes were capable of synthesizing blotln, niacin, pantothenic aold, and other B vitamins. This worker found that the vitamin content of the bacterial cell remained constant. Any excess vitamin produced was wexcreted" into the medium# Xn 1911.3# Rogosa (86) indicated that riboflavin was syn­ thesized by certain lactose fermenting yeasts. Xn 1944# Miller (74) reported that Escherichia coll could synthesize folic acid and blotln. In the same year, Lewis et al (67) found that Toruleppsls utllls was able to convert fermentable substrates into many B vitamins except blotln. Although bacteria are most frequently given credit for synthesis of vitamins, a few workers have pointed out that baoterla are also capable of destroying vitamins# Kendall and Chinn (50), in 1938# and Esselen and Puller (31), in 1939# showed that ascorbic acid was oxidized by many intestinal bacteria# In 1943# Allison (3) demon­ strated that certain soil bacteria were capable of destroy­ ing nicotinic acid. Xn the same year, Mirick (75) found that p-amlnobenzolc aold was oxidized to carbon dioxide, water and ammonia by soil organisms. In 1944# Poster (43) discovered that Pseudomonas oxidized riboflavin to luml- 32 chrome* In 1944* Koser and Baird (54) found that Pseudo- monaa and Serratla species when grown In a medium ualng only nlootlnic aold aa a carbon aource, would deoreaae the content ef thla vitamin* If nlcotlnio acid was replaced by glucose, these organiama grew and synthesized thla vitamin* EXPERIMENTAL Vitamin Requirements of Lactebaclllus piantarum Isolated from Cucumber Fermentatlon Ten organisms isolated from different fermentations were aeleoted for study* The well known assay organism, Lactobacillus arablnosus 17-5* was run concurrently with the other Lactobacilli* Since this organism was known te require blotln, niaoln, and pantothenic acid, it served aa a check for the materials and methods of the determination* The media used to determine the vitamin requirements of these organisms were the dehydrated assay media manu­ factured by Dlfco Laboratories, Inc* The inoculum was prepared by inoculating 10 ml of sterile, centrifuged cucumber juice diluted 1:2 with dis­ tilled water* The cucumber juice waa contained In small centrifuge tubes* After Inoculation, the tubes were in­ cubated for I4.8 hours at room temperature* They were then centrifuged in order to separate the cells from the nutrient medium* The supernatant was poured off and the cells were resuspended in 100 ml of sterile physiological saline* 33 One drop per tube of this suspension was used as the inoculum. Varying amounts of biotin, niaoln and pantothenic acid were dispensed into test tubes. The concentrations used were suggested by a paper of Krueger and Peterson (£8)* who determined what these vitamin requirements were for a strain ef Laotobaoillus pentosus. The respective assay medium was added to the tubes containing the vitamin solu­ tions. Eaeh tube contained a final volume of 10 ml. The tubes were sterilised for only eight minutes at 15 pounds pressure. Incubation was carried out for 72 hours at 28° C. The amount of growth was determined by titrating with 0.1 W HaOH. The results are presented in Tables 7* 8, and 9 and are summarized in Figure lj.. As expected, L. arablnosus 17-5, represented by the solid line in Figure 4, responded to increasing amounts of vitamins. The Laotobaollll Isolated from cucumber fermen­ tations responded in a similar fashion. The top dotted line represents the most active, whereas the bottom dotted line represents the least active of the ten Isolates tested. These data definitely indicated that Lactobacilli of the cucumber fermentation required blotln, niaoln, and pantothenic acid when the determination was made by the previously described method* BIOTIN REQUIREMENTS OP L. PLANTAHUM AND L. ARABINOSPS 17-5 Blotln (aiosAO ml) Ml 0.1 N aold produced L. arabinosus 17-5 (control) Numbered Isolates of L. piantarum 600 602 60it 609 611 613 615 617 621 625 0 1 .0 0.5 0.3 0.8 0.1 0.8 04 0.7 0 .2 0.5 0.9 0*2 6.3 3.8 it.6 lt«6 lt.7 lt.1 lt.3 44 4.5 5.4 4.0 0 ,1*. 8 .2 5.9 6.7 6 .6 6.3 6 .2 6.5 6.3 6.8 7.8 6 .0 0*6 9.9 7.0 8 .0 7.6 8.8 7.7 8 .0 7.8 8.3 9.2 6.9 0*8 11.1 7.7 8.9 8.9 9.5 8 .6 9.0 8 .6 8.5 10.5 7.9 1 .0 12.1 8 .6 10.0 10.3 10.1 94 9.2 9.2 10.4 11.3 8.4 1 .2 13.0 9.7 10.7 10.9 11.2 10.3 10.2 10.3 11.4 1 2 .4 9.3 1.4 13.2 10.1 11.7 11.1 12.9 11.2 12.1 11.3 13.4 9.8 1 .6 14.5 10.8 12.0 12.2 12.7 12.7 12.5 12.3 13.6 10.2 • 12.3 - s determination not mad# * TABLE 8 HIACIN REQUIREMENTS OF L. PLANTAHPM AND L. ARABINOSUS 17-5 Niaoln (mog/10 ml) Ml 0.1 N aold produced L. arabinosus 17-5 (oentrol) 600 602 0 0.6 04 0.1* 0.1* 0.1* 0.1* 0.1* 0.1* 0.2 54 3.0 3.6 3.0 3.7 34 3.1 0.1* 7.9 5.3 6.6 6.1 7.2 5.8 0.6 10.3 8.1* 8.0 8.7 8.8 0*8 114 9.9 9.7 1.0 12.1 11.3 11.2 11.3 1.2 12.3 12.0 12.3 11.8 14 13.3 12.7 1.6 13.2 12.7 Nunborod Isolates of L. plantarum 611 601* 609 615 617 613 621 625 0.1* 0.5 0.1* 3.2 3.9 54 3.7 5.9 6.0 6.5 8.9 5.8 8.3 8.7 7.0 9.3 10.9 8.0 10.1* 10.1* 10.2 10.1 10.1* 11.1 12.1 9.6 12.3 11.6 11.2 U.l* 12.0 12.1* 10.1* 13.1 12.3 12.6 12.2 12.9 12.8 11.0 12.3 12.1* 1 3 4 12.9 12.9 12.1* 13.8 13.3 11.2 13.2 12.5 12.6 13.5 12.3 13.3 11.2 13.1 134 U) vn TABLE 9 PANTOTHENIC ACID REQUIREMENTS OP L. PLANTAHPM AND L. ARABINOSUS 17-5 Pantothenic aold (mcg/10 ml) L. arabinosus 17-5 (control) 600 Ml 0.1 N aoid produced Numbered isolates of L. plantarum 602 611 609 604 615 617 613 621 625 0 04 0.3 0.3 0.3 0.1 0.2 0.4 0.4 0.2 0.3 0 .3 0.05 8.9 14 4*5 3.4 5.8 5.6 5.4 5.3 3.2 5.2 5.0 0.10 11.8 4.0 7.4 8 .4 7.5 7.3 7.7 7.2 8.2 8.6 5.5 0.15 13.2 4.7 10.3 104 10.2 9*7 10.3 9.5 9.8 10.4 7.5 0.20 4.3 7.4 11.7 1. ; 11.5 11.9 11.9 11.4 11.2 12.2 9.6 0.25 4.7 11.4 12.7 12.3 12.2 12.4 12.7 12.7 12.2 13.4 12.3 0.30 16.0 13.5 13.3 12.6 13.1 13.6 13.2 13.8 13.8 14.0 13.0 0.35 15.8 14.7 4 4 4t0 13.8 4.7 14.2 14.9 14.6 13.3 040 16.5 14.9 4.9 4*1 4.1 15.1 14.8 14.6 i5.o 13.5 14.8 ijj o i Ool N aold l — — L# arabinosua £• Pl&ntarum from cucumber fermentation _J 0.5 1.0 Mmcg biotinAO ml 1.5 0.5 1.0 McS niacin/10 ml 1.5 0.1 I 0.2 I 10 0.3 0.4 Meg pantothenic acid/10 ml Figure i|e Vitamin responses of Lactobacilli isolated from cucumber fermentation compared with Lactobacillus arabinosus 17-5 01 38 Petermlnation of Blotln, Niaoln, and Pantothenic Acid In Cucumber Julco It was then decided to determine the content of these vitamins In cucumber juice. biological means. The assay was made by micro­ The assay organism, L • arabinosus 17-5, was used for the assay of all three vitamins. The media used were the same as those used for the determination of the vitamin requirements, viz*, Dlfoo's dehydrated assay media* The method of assay was similar to that desorlbed In "Methods of Vitamin Assay" (5)« Several varieties of cucumbers obtained from different sources were analyzed* The cucumber juice was extracted by macerating the suoumbers In a Waring Blendor* The ground cucumbers and aooumulated liquid were then pressed through cheesecloth* Appropriate dilutions of this preparation were made and assayed* Since It was desired to determine the vitamin content ;hat would be available for microorganisms, no attempt was lade to free any combined vitamins. It was not necessary ;o filter or adjust the pH of the cucumber juice since >nly 0*01 ml of cucumber juice could bring about a response corresponding to the standard curves. The results are given In Table 10* It m ay be noted that all of the cucumbers tested con­ tained a rich supply of the three vitamins studied. The ixtreme values for the vitamin content of cucumber juice 39 TABLE 10 VITAMIN CONTENT PER ML OP CUCUMBER JUICE OF DIFFERENT VARIETIES OF CUCUMBERS OBTAINED FROM VARIOUS SOURCES Variety or cuoumber Source Blotln (mmog) Vitamin content Niacin Pantothenic aold (nog) (mog) 17*3 5.05 2.^2 MR-17 b 33.0 4.62 2.13 MR-17 o 16*2 2.90 2.24 produeer a+ 5.20 1.83 2.05 Producer a+ 8.85 2.80 1.45 Produoor b 2.61 1.05 SR-6 a 3.37 1.45 Model a 12.2 2.96 1.70 Darls a 10.8 2.63 1.84 Torkstato b 9.13 3.05 1.61 s 7.00 3.04 1.85 d 6.75 2.26 2.85 o 3 o Largo rlpo Largo long groon slleer 0 a ! MR-17 llt.6 6.65 ^Variety not known +0btalned on different day* a H. V. Madison Pleklo Co., Mason, Ml eta.* b Dept. or Horticulture, Ml obi gan Stato Collogo o Food Storos, Michigan Stato Collogo d Market Basket, East Lansing, Ml oh* . ko were as follows: Blotln 5«20 to 33*0 ramcg/ml Niacin 1*03 to 5*05 mcg/ml Pantothenic acid 1*05 to 2*1+2 mcg/ml Xn general, there was nodifference In the vitamin content of the different varieties of cucumbers* However, MR-17 cucumbers, obtained from three different sources, contained more blotln than any of the other varieties tested* Bffect of Aerobaoter oloaoae and Various Yeasts on the Vltastln Content of Cucumber Juice Since a review of the literature and data presented earlier In this study Indicated that A* cloacae and various yeasts were present during the cucumber ferment tatlon, It was decided to determine what effect these organisms had on the vitamin content of cucumber juice* The vitamins seleoted fer study were blotln, niacin, and pantothenic aoid beoause In a previous experiment these vitamins wore proven to be essential fer L • plant arum Isolated from cucumber fermentations* Vine Isolates Aerobaoter cloacae were seleoted for study* of Three yeast isolates** identified as Hansenula subpelliculesa as well as the following yeasts obtained from Northern Regional Research Laboratory were also tested: Hansenula aubpelll•isolafe* Nos. 1*26, 1*27. 1*28, 1*29, 1*32. 1*33, 1*31*. 1*1*9, 1*57 **I**lat« *oa. 707, 718, 729 ia oulost RY-135, Torulopals oarellnlana RY-ll|.7» Torulopals roael HY-8, Brettanomyoea voraatllia Y-146, Zygoaaocharomycea ap. A YS-590, and Torulopala holmli FPL-Y-307• These yeaata war* Isolated by Etchells from oemmeroial cucumber brines. The teat organisma war# Inoculated Into 10 ml of cuoumbar juice diluted 1:2. Two tubas containing only euoumbar juloa wara usad aa a oentrol In each determination. Aftar 30 hours lnoub&tion* the microbiological activity In tha cuoumbar juloa was stoppad by autoclavlng. Appropriate dilutions wara nada and the vitamin assay waa done as previously described* The results are given In Tables 11* 12* and 13* and are presented graphically In Figures 5# 6, and 7. TABLE 11 EFFECT OF^ A E R O B A C T E R ^ 0H TH® Vitamin Trial Ha. 1 . . . . , . Average of nine Isolates Unineculafced inoculated (oentrol) Trial Ho. 2 Average of two isolates (N£.# ^ & k57j uninooulated inoculated (central) Blotln (mmog/ml) 5.6 2.0 3.50 1.02 Hlaoln (mag/ml) 1.3 1.7 1.55 1.50 0.95 0.85 0.76 Pantothenlo 0.87 aold (reog/ml) TABLE 12 EFFECT OF HANSENULA SUBPELLICULOSA ISOLATED FROM laboratory 75TISUMMER A.H 6Wg ~on the vitamin COFT ENT OF CUCUMBER JUICE ■' n .'I'laxa. ,1j J .J. ,a.-. Trial No. 1 Trial No. 2 ATorage or throe isolates Average of two Isolates (Nos. 707. 718. 729) (Nes. 707. 718) Unlnoeulated Inoculate* Unlnoeulated Inoculated (control) (control) Bietin (mmcg/ml) If.56 2.57 3.5 2.80 Niacin (meg/ml) 1.85 1.75 1.55 1.53 pantothenic 0.97 acid (mcg/ml) 1.19 0.85 1.07 TABLE 13 EFFECT OF VARIOUS YEASTS ISOLATED FROM COMMERCIAL CUCUMBER FERMENTATIONS ON THE VITAMIN CONTENT OF CUCUMBER JUICE Yeast Bietin (saeg/al) Vitamin VIaelm Pantothenic a d d (meg/ml) (mog/ml) Control (unlneoulated cucumber juice) 3.95 1.61 1.17 Hansenula subnellleulesa o CO. <\l 1.37 1.39 Torulonsls oarellnlaaia 1.90 1.15 1.11 Terulensis resel 1.35 00 CVI . H l.llf Brettamemyoea ▼ersatllls 3*90 1.30 1.36 3.70 1.67 1.07 1.16 1.01 1.32 Zygesacoharemyces Torulonsls holmll sd. A Mmcg Biotin Figure £<> juice Effect of A„ cloacae and various yeasts on the biotin cucumber a & W ti

& o P o cr © content of o © Zo 8p» A To holmil ■ Unlnoeulated cucumber juice Figure 6* □ Inoculated cucumber juice Effect of A. cloacae and various yeasts on the niacin content of cucumber juice | Unlnoeulated cucumber juice Figure 7. Q Inoculated cucumber juice Effect of A. cloacae and various yeasts on the pantothenic acid content of cucumber juice k* From Figure 5 It may be seen that most of the yeasts and A* oleaoae markedly levered the blotln content of euoumber Juice* Figure 6 reveals that the niacin content was net altered appreciably by A* oleaoae and the various yeasts* Figure 7 Illustrates that pantothenic a d d was barely utilized by any ef the organisms* Xn fact, most of the yeasts appeared to synthesise this vitamin* Vitamin Analysis During Cucumber Fermentation Cucumber f o m entations were set up in the laboratory aa described earlier In this study* Samples were taken for total acid, microbial numbers and vitamin content* The mlereblelogloal analysis was carried out as previously described* The vitamin analysis was accomplished by pipetting samples ef brine Into volumetric flasks* Enough brine was added so that when the contents of the flask were brought to volume, there would be a It5 dilution of the brine* As soon as the brine was pipetted about three times as much distilled water was added to the flasks* flasks were then auteclaved for 15 minutes at 121° C* The When an assay was to be made the contents of the flask were brought to volume* Appropriate dilutions were made and the assay was accomplished as described In previous seotlons ef this study* The cucumbers were analyzed for vitamins at the be­ ginning ef the formentation and after 20 days* The volume of the cucumbers and brine were measured at the end of the k-7 fermentation in order to d e t e m i n e whether vitamins were synthesised or utilized* The vitamins determined were 0 biotin, nlaoln and pantothenic acid* The cucumbers in crook A 2 were of the producer variety whereas those in crock B2 were of the MR-17 variety* The results are presented in Tables II4. to 18* Tables H 4. and 15 illustrate the rapid entry of biotin* niacin and pantothenic acid from the cucumbers into the brine during a fermentation* Table 17 indicates that the cucumbers furnished much of the vitamins for the fermen­ tation* However* the vitamin content of the cucumbers after 20 days was not depleted* From Table 18 the micro­ biological effect on vitamins during a fermentation may be noted* The pantothenic acid content was more than doubled in both crocks* The niacin content remained unchanged* There was a slight Increase in blotin in crock A2 811(1 a marked decrease in this vitamin in crock B2 * DISCUSSION The need for blotin* niacin and pantothenic acid was clearly established for lactic acid bacteria Isolated from cucumber fermentations (Figure I4.) • The vitamin content of cucumbers (Table 10) indicated that sufficient blotin, niacin and pantothenic acid was supplied for these organ­ isms (oompare with Figure If.)• Although most of the non- acid—producing organisms of the cucumber fermentation lADUu Xtf TOTAL ACID, ORGANISMS PER ML, AND VITAMIN CONTENT PER ML BRINE OP CUCUMBER FERMENTATION (30° S) (CROCK A2 ) aaaaaaaaagaasitBri'Tij'yui.j 1 UBrssssssaaEssesssarsa .a Day 0 P*ro«nt aoid (as laotlo acid) _ _ _ _______ Orgtnlras par 1 L. piantarum Aarobactar Yaaats - 0 25,000 0 1. .resaega!" i.i.btj.:, ■:t 1 Vitamin par al brine Blotin Niacin Pantothenic acid (meg) (mog) (meg) 0.330 - 0.0835 m 1 0.017 1,400 290,000 35 2 0.020 15,000 2,290,000 0 2.69 0.650 0.61)5 3 0.061 7,700,000 29,000 0 3.17 0,820 1.00 k 0.070 9,000,000 430 0 3.66 0.840 1.22 5 0.13 12 ,400,000 4,300 0 4.27 0.670 0.800 6 0.20 28,700,000 1,470 4,000 3.69 1.06 0.991 8 0.28 8 ,500,000 43,000 80,000 3.38 1.12 0.963 10 0.34 3,600,000 4,300 230,000 3.93 1.11 0.813 12 0.38 1,800,000 1.470 70,000 4.63 0.983 1.08 34 0.43 730,000 920 220,000 4.50 0.923 1.17 17 0.S4 300,000 430 300,000 4.30 0.980 0.987 20 0.69 300,000 1,470 800,000 2.89 0.938 1.27 datarmination net made - 0.324 TOTAL ACID, ORGANISMS PER ML, AND VITAMIN CONTENT PER ML BRINE OP CUCUMBER FERMENTATION (30° S) (CROCK Bg) Day laotio acid) £• piantarum Ml Aerooacter Teeata iSletin Hlaeln (meg) 0 - 0 1^,700 90 0,071 (■eg) - P&ntothanlo i (meg) 0.0181) 1 0,018 40,000 220,000 6,000 3.12 0.217 0.356 2 0.038 12, 000,000 14.30,000 25,000 5.30 0.523 0.880 3 0.15 45, 000,000 920,000 1, 210,000 5.75 0.631 0,870 4 0,28 33,000,000 36,000 850,000 I). 57 0.91)6 1.52 5 0.37 30, 000,000 14.30,000 760,000 5.91) 0.525 l.l!) 28, 700,000 250,000 600,000 6.57 0.1)1)2 1.08 6 8 0.54 1,000,000 250,000 110,000 6.35 0.1)75 1.1)7 10 0.54 450,000 71*., 000 65,000 6.10 0.508 1.29 12 0.54 300,000 2,500 550,000 5.88 0.520 1.1)7 14 0.46 200,000 430 700,000 5.78 0.600 1.1)3 17 0.43 45,000 920 1)60,000 k.ko 0.828 1.37 20 0.43 60,000 21|0 120,000 3.97 0.683 1.60 • = determination net made 50 TABLE 16 DILUTION FACTORS FOR CUCUMBERS FERMENTING IN CROCKS Ag AND Bg Total volume (cucumbers + brine) Volume of brine Volume of cucumbers Dilution factor for cucumbers Crook * 2 Crook B 2 11,900 ml 12,600 ml 5,300 ml 5,500 ml 6,600 ml 7,100 ml 11.900 _ T .80* 6,600 12 t600 * 1.78* 7, 100 The vitamin content of the cucumbers* when divided by this factor, represented the potential vitamin content per ml of brine* These values were taken as the actual vitamin content at the beginning ef the fermentation* These values are given in Table 18. TABLE 17 VITAMIN CONTENT PER ML OF CUCUMBER JUICE OF CUCUMBERS AT THE BEGINNING OF A FERMENTATION AND AFTER 20 DATS Crook B 2 Beginning After 20 days 16.20 l\) • o o Crook A2 Beginning After 20 days Bietin (mrnog) 8.85 2.72 Niacin (meg) 2.80 0.750 2*90 0.683 1.33 2 .214- 1.54 Pantothenic 2 .214acid (meg) 51 TABLE 18 VITAMIN CONTENT PER ML OF CUCUMBERS AND BRINE AT THE BEGINNING OF A FERMENTATION AND AFTER 20 DAYS Crock A£ Beginning After 20 days Crock B 2 Beginning After 20 days Blotin (mmcg) 1I-.90 5.61 9.10 5.97 Nlaoln (meg) 1.50 1*69 1.63 l.Si Pantothenic 1*24 aoid (meg) 2*60 1.26 3.11* reduced the blotin level (Figure 5) It appeared that enough blotln remained to assure adequate growth ef the Lactobacilli* The other two vitamins did not appear to be critical since their level 6 and 7)« remained unchanged or was Increased (Figures The action of the yeasts may easily be explained* Since most yeasts require blotin, one would expeot a reduction of this vitamin by these organisms* However, the faot that A* cloacae reduced the blotin content of cucumber juice was rather surprising (Figure 5)* This finding was studied further and the results are presented In the following seotion of this study* Regardless of the microbiological activity, there seemed to be In the brine an abundant quantity of blotin, nlaoln, and pantothenic acid within 2i\. hours after brining (Tables lij. and 15) • The faot that pantothenic acid was synthesized (Table 18) during a cucumber fermentation was to be expected when one considers that brine yeasts syn- 52 thesised this vitamin (Figure 7)* The Tact that blotin vras synthesised in one orock, but reduced in another (Table 18) was difficult to explain* It may be that where the bLotln content was initially low, one may expect an increase due to some organism of the cucumber fermen­ tation which has not yet been isolated. It is conceivable that there may have been types of microorganisms which appeared in one fermentation and not in another depending on the availability of nutrients in the brine* PART XII ROLE OP BIOTIN IN CUCUMBER FERMENTATION Sh INTRODUCTION The previous sections of this study indicated that biotin could be a critical vitamin during a cucumber fermentation* This vitamin was markedly reduced by most of the organisms occurring in a cucumber fermentation* In one fermentation studied* biotin was largely reduced* whereas the other vitamins remained constant or were syn­ thesized* It was noted that blotin exhibited the largest variation in concentration among the different varieties of cucumbers studied* This particular study attempted to demonstrate the Importance ef blotin during a cucumber fermentation* REVIEW OP THE LITERATURE History of Blotin. - Eddy (29) has given an account of the recognition of this vitamin* The first indication of the existence of biotin was given by Wildlers in 1901 who stated that bios contained a multiplicity of growth factors for yeast* In 1927* Boas described an injury to rats caused by feeding raw egg white* This injury could be prevented by feeding rats other foods* In 1939* Georgy called the factor capable of preventing raw egg white Injury, vitamin H. In 1933, Allison reported a growth factor for Hhlzoblum which he called ooenzyme R* In 1935, the bios lib fraction of Wildlers was orystalllzed from *g£ yolk by Kogl which he oalled biotin* Minute quantities 55 of* this substance were stimulatory for yeasts* In 19^0, Georgy and co-workers demonstrated that vitamin H, coenzyme R» and Kogl's blotin were Identical* Blotin Requirements for Microorganisms * - The blotin requirements of lactic acid bacteria have been discussed in a previous section of this study* Burkholder (11), Kogl and Tennis (53) » end Lockheed and Landerkln (69), have Indicated the blotin requirements of yeasts* Burk­ holder showed that of 38 yeasts tested, 36 required blotin* West and Wilson (107) and Wilson and Wilson (III4.) have demonstrated that many strains of Hhlzobla require biotin* Peterson et al (80), Lantpen and Peterson (61), and Snell and Williams (98) have tested several species of Clostridia and found that they required blotin* Du Vlgneaud at al (26) have proven blotin to stimulate the diphtheria baoillus* Hot tie et al (lj.9) Indicated the need of hemolytic streptooocol for this vitamin* Bohonos and Subba-Row (8) tested 33 strains of pneumooooci and found that they required biotin* Requirements of a variety of other microorganisms are given In a review by Knight (51)* Antlblotins* - The antiblotlns Include avldin and a list of compounds related in chemical structure to blotin* In 19i{-0, Eakln, Snell, and Williams (28) reported the iso­ lation of avldin from raw egg white* Avldin was described as a protein capable of inactivating blotin* Landy et al (6 3 ) demonstrated that avldin was an antlblotln to micro­ 56 organisms which required biotin but would be ineffective in preventing growth of organisms that did not require biotin* An excessive amount of blotin added to the medium could overcome the effect of avldin* Xn 19U4* Dlttmer and du Vlgneaud (20) reported that desthloblotln* blotin sulfone* and other compounds related structurally to blotin possessed antlblotln activity* Lilly and Leonlan (68) showed that the antlblotln effects of desthloblotin could be overcome by additional amounts of biotin* Willi sms at al (108) have published an up to date list ef antlblotlns* Substitutes for Blotin* - Many vitamers of blotin are listed by Williams ejb al (108)* Desthloblotin* previously discussed as an antlblotln was found to stimulate certain organisms. Melville et al (73) found this compound to be active for certain yeasts and some bacteria* They also noted that this compound was inactivated by avldin* Stokes and Gunness (100 ) found biotin methyl ester to be active for Lactobacillus case!* These workers in a later publi­ cation (101 ) showed that synthetic blotin gave responses identical to those of natural blotin* d-blotln was the active form of blotin* They believed that Although they found that large amounts of 1 -biotin had biological activity* they believed that this Isomer was contaminated with dblotln* Many workers have shown oxyblotln (o-heterobiotin) to be active for mloroorganlsms and animals. Pilgrim et al 57 (82), Duschinsky et al (25>), Kruager and Peterson (59)* and Wlnnlok e_t al (115) have shown t.hls compound to be aotlve tor various microorganisms* The latter workers have demonstrated that this compound Is Inhibited by avldin* Hofmann e£ al (i+8 ) proved oxybiotin to have blotin activity for the rat and chiok* Recently, much emphasis has been placed on the sub­ stitution of lipids for blotin* Perhaps the first indi­ cation that lipids might substitute for blotin was in 1932 when Evans and Lepkovsky (I4.O) noted that rats could utilize certain unsaturated fatty acids In place of vitamin B* In I9I4.O* Cohen and Mueller (18) found that oleic acid could stimulate colony development of Corynebaoterlum dlphtheriae* In 1944-5* Dubos (21) began a series of experiments in an attempt to obtain a better medium for the growth of tubercle bacilli* He first noted that phospholipids and long chain fatty acid esters of polyhydric alcohols (e%g*, Tween 80) permitted submerged and rapid growth of tubercle bacilli* Xn 1914-6 , Dubos (22) found that fatty acids In themselves were toxic whereas esterlfled fatty acids were non-toxic* Xn the same year, Davis and Dubos (19) found Tween 60 to be toxio to small lnocula, but they were able to show that this substance contained small amounts of unesterlfled fatty acids* The first explanation for this activity of Tween 80 was given in 1914-6 by Dubos and Davis (214.)* They claimed that the wetting action of the Tweens prevented pellicle formation 58 and that the nutrients or the medium were In oleser contact with the bacterial cell* In 1914.7* Dubos (23) also suggested that fatty acids* supplied by certain Tweens* functioned In the metabolism of the tubercle bacillus* He also suggested that serum albumin be added to the medium to offset the toxic effect of unesterlfled fatty acids* Kodlcek and Worden (52) hare shown that fatty acids Inhibited the growth of Lactobacillus helvltlous, while the methyl esters of those fatty acids did not show any Inhibitory effect* In I9I4.6 * Williams and Fleger (111) feund that oleic acid would stimulate the growth of Lactobacillus easel In a blot infree medium* In 1914.7* Trager (IOI4.) discovered a fat-soluble material from plasma having the biological aetlvltles of biotin* He showed that this material was not Inactivated by avldin* He did not believe this material to be a fatty acid fraction of plasma since this fraction was non-saponIflable and Insoluble in acetone. In 19l|.7* Williams and Fleger (112) found that oleates* especially Tween 80* were excellent substitutes for biotin In stimulating growth of L* easel* In 1914-7* Axelrod et al (6 ) demonstrated that vaoeenlc acid had biotin activity for higher animals* In 19VT* Williams ejfc al (113) showed that Tween 80 was better than elelc acid as a biotin substitute for several lactic acid bacteria* They suggested that biotin catalyzes the synthesis of oleic acid because certain organisms require this fatty acid even In the presence of blotin* 59 Functions of Blotin. - It has already been indicated that biotin prevents egg white injury to animals and serves as a growth factor for many microorganisms. It has been postulated by Williams ejb al (113) that one function of blotin is to catalyze the formation of oleicacid. was given further support by This Gavin and McHenry (i+5) who demonstrated that excessive blotin fed to rats would cause fatty livers and an Increase in body fat to that animal• Petter and Slvehjem (83 ) believed blotin to have three functions: 1. Aid in synthesis of oleic acid* 2* Aid in synthesis of aspartic acid* 3. Catalyze the beta oarboxylatlon of pyruvic acid to aspartate* Several other workers have indicated an interrelationship between biotin and aspartic acid. Koser at al (56), in 191^2, found, that aspartic acid served as a partial sub­ stitute for blotin for Torula creamorls* Stokes et al (102), in 19i(.7» showed that several lactic acid bacteria when supplied with blotin would synthesize aspartic acid. There have been indications that blotin may also serve as part of an enzyme system which functions in the carboxylatlon of pyruvic acid* Shive and Rogers (89) have demonstrated that blotin is Involved in the biosynthesis of oxalacetlo and alpha-ketoglutaric acid from pyruvic acid* 60 EXPERIMENTAL Blotin Utilization by L . piantarum In Cucumber Juice Since it was established that L. plantarum required blotin9 It was believed that L. piantarum could reduce the blotin content of euoumber juice* The procedure for this experiment was the same as that used for the effeefe of Aerobaoter and yeasts on the vitamin content of cuoumbiNr Juice* Three Isolates (Nos* 602, 609, 611) of L • piantarum were selected for study* Since It was difficult to visually detect growth of L • pi ant arum In cucumber juice, the number of organisms was determined by plating on V-8 agar* The values for the effect of all three isolates of L. plantarum on the blotin content of cucumber juice were very close* An average ef those values is given in Figure 8# A very slight reduction of the biotin content was observed* This was rather surprising In view of the faot that this organism required blotin* It was therefore decided to ddterralne whether L* plantarum could diminish the blotin content In a medium which contained pure blotin* Blotin Utilization by L . plantarum In Biotin Assay Medium This experiment was set up In a similar fashion to the previous experiment except that measured amounts of biotin added to Its respective assay medium were used Instead of cucumber juice* Each time the experiment was performed. Mracg Biotin 5 HHm Uninoculated cucumber juice □ Cucumber juice after JO hours incubation containing 106,000,000 cells/ml Figure 8. Effect of L. plantarum on the biotin content of cucumber juice 62 the blotin from the control tube was assayed by the usual procedure* The results are given In Table 19* TABLE 19 EFFECT OF L. PLANTARUM OF THE BIOTIN CONTENT IN BlOflNASSAY MEDIUM Blotin in unlnoeulated assay medium (mmog/ml) Blotin in Inoculated assay medium (mmcg/ml) 4.42 0.065 3.60 0.070 0.153 0.0070 Unlike cucumber juice, the blotin In the assay medium, which contained only biotin as a "biotin-activen material, was reduced considerably. This led to the opinion that the biotin activity of cucumber juice might be due to a variety of substances* Tween 80 As a Blotin Active Material for L* plantarum Tween 60 has been reported to be an excellent biotin active material (112, 113). To prove that this substance could stimulate the growth of L • plantarum varying amounts were added to blotin assay medium* The experiment was first done using one Isolate of L* plantarum and L* arabinosus 17—5. The experiment was repeated using three isolates of L* plantarum and L* arabinosus 17-5. The tubes were Incubated at 30° C for 72 hours. Growth 63 was determined by titrating for the amount of acid pro­ duced* The results appear in Table 20 and show clearly that Tween 80 could serve as an excellent blotin substitute for L • plantarum. TABLE 20 EFFECT OF TWEEN 80 AS A BIOTIN ACTIVE MATERIAL FOR L. PLANTARUM AND L. ARABINOSUS 17-5 Mg Tween 80/ 10 ml Ml 0*1 N acid produced per 10 ml medium Trial 1 Trial 2 L. arabinosus 17-5 (control) L. L. plantarum 611 arabinosus 17-5 (control) L. plantarum (VI 609 611 0.5 0.5 0.3 0*1 it.it it.5 3.it 3.3 2.3 3,1 0*5 9.6 9.2 8.3 5.7 3.9 5.8 7.5 5.1 7.7 12.0 5.0 I5.it 9.7 lit.3 8.5 5.9 9.0 16.7 10.7 16.3 8.1 5.2 9.5 H O « 12*8 O 1.6 o • o H 2.5 O . H 0 . H 602 Utilisation of the Blotin Active Fraction of Tween 80 by L* plantarum An attempt was made to explain the fact that L « plan­ tarum would markedly deplete the blotin in the assay medium, but would hardly diminish the blotin content in cucumber juice* It was believed that Tween 80 could support the growth ef L • plantarum without much of its blotin active substanoe being utilised* 614. The procedure Tor this determination was the sane aa that used in previous experiments of this study. Tween 80 was added to blotin assay medium In concentrations of 0*5 mg and 0*05 mg per ml. These amounts were subsequently found to be equivalent to 0*303 nmcg and 0*035 rameg of blotin respeotlvely• The results are given In Figure 9 and It may be noted that when Tween 80 was used In the blotin assay medium, the medium still peaeesaed considerable blotin activity after 30 hours lnowbatlon with L. plantarum* Effect of L * pi»r*tarum on the Blotin Content of Assay Medium Containing Only Blotin and a Combination of Blotin and Tween 80 Xt was still possible that the biotin was not lowered by Laotobaollll In eucumoer juice because these organisms did not grow as well In this medium as they did In the blotin assay medium* Xt has been shown that Tween 80 "resists" reduction of its biotin active fraction (Figure 9). However, Tween 80 did not appear to support growth as well as blotin as judged by titration values given In Tables 7 and 20. It was, therefore, decided to culture L* plantarum In the assay medium containing blotin and In the assay medium containing a combination of biotin sued Tween 80. was determined by measuring the acid produced. were Incubated for 30 hours at 30° C. given In Table 21* Growth The tubes The results are Mmog Biotin 65 Uninoculated assay medium Assay medium after 30 hours incubation Figure 9® Effect of L» plantarum on the content of the biotin active fraction of Tween 80 in biotin assay medium i 66 TABLE 21 UTILIZATION OP BIOTIN BY L. PLANTARUM PROM ASSAY MEDIUM CONTAINING ONLY BIOTIN AH d A <56MBINATION OP BIOTIN AND TWEEN 80 Ml acid produced per 10 ml medium Unlnoeulated. Blotin (mmog/ml) Inoculated* Blotin (mracg/ml) Blotin In assay medium 17*9 3.60 0.070 Blotin and Tween 80 (0.1 mg/ml) In assay medium 18.5 2.73 0.80 Blotin and Tween 80 (0.1 mg/ml) In assay medium 1 8 .14. 2 .I4.5 0.75 Thus, It was Indicated that whan pure blotin was availabla far L# plantarum, It was markedly reduced. When Tween 80 was available as the blotin active material for this organism there was not as sharp a reduction In the blotin content. This simulated the results obtained In Figure 8 where It was shown that L. plantarum hardly affected the blotin oontent of cucumber juice. A summary ef the results presented In this section Is given in Table 22* Bietin Requlresents of Aerebacter oleaoae Earlier In this study It was shown that A. cloacae deoreased the blotin oontent of cucumber juice (Figure 5). It was therefore assumed that this organism might require blotin. This was unusual since similar organisms have been 67 TABLE 22 SUMMARIZED DATA FOR THE UTILIZATION OF BIOTIH ACTIVE COMPONENTS FROM VARIOUS SOURCES BY L. PLAHTAHUM Uninoculated* Blotin (mmog/ml) Inoculated Biotln (mmog/ml) Cucumber juice 4.10 3.79 Cucumber juice 3.10 2.84 Blotln in assay medium 4.42 0.065 Blotin in assay medium 3.60 0.070 Blotln in assay medium 0.153 0.0070 Tween 80 (0.5 mg/ml) in assay medium 0.303 0*225 Tween 80 (0.05 mg/ml) in assay medium 0.035 0.019 Blotln and Tween 00 (0.1 mg/ml) in assay medium 2.73 0.80 Blotin and Tween 80 (0.1 mg/ml) in assay medium 2.45 0.75 Substances containing blotln aotlve materials reported to synthesis* this vitamin (8l, 103) . A simple experiment was performed in order to determine if this organism neede'd biotin. Two Isolates (Nos. 449 and 457) were selected for study. The organismswere inoculated into blotln assay medium containing no biotln and varying concentrations of blotin. Growth was observed visually and the results are indicated in Table 23» Obviously, this organism did not require biotin. On 68 TABLE 23 BIOTIN REQUIREMENTS OP ABROBACTER CLOACAE Blotin (mmcg/ml) 0 Isolate Nos. of Aorobaoter cloacae W5 " 1|57 - (uninoculated) - 0 ++++ ++++ 0 . 1 0 ++++ ++++ O H • ++++ • * no growth; ++++ = heavy, turbid, frothy growth the oontrary, It eould probably synthesize blotln from this medium* Effect of Aerobaoter cloacae on the Blotln Activity of Blotln Assay Medium Containing Varying Concentrations of Blotln and Twoon 80 Although A . cloacae depleted the blotln content of cucumber juice (Figure f>)» the preceding experiment Indi­ cated that this organism might synthesize this vitamin from fr the assay medium. The organism was grown in blotin assay medium containing varying concentrations of blotln and Tween 80 and in the assay medium containing no added nu­ trient . The tubes were incubated for 30 hours at 30® C and assays were made as previously described. The results are given In Table 214.* The results Indicated that A. cloacae was capable of synthesising blotln when no blotln or very small amounts 69 TABLE 21*. UTILIZATION OP BIOTIN ACTIVE SUBSTANCES BY A. CLOACAE IN BIOTIN ASSAY MEDIUM CONTAINING BIOTIN ANlS TV/Ek'ff T O Uninooulated* Blotln (mmog/ml) Inoculated* Blotln (xtmcg/ml) Blotln 0 0.169 Blotln 0*061*. 0.131*. Blotln 3.35 1*72 Tween 80 (0*05 mg/ml) 0.035 0.101 Tween 60 (0*5 mg/ml) 0.303 0.382 of tills nutrlllte was present* When a large amount of blotln was present, an amount equal to a 1:2 dilution of oueumber juice, a partial reduction was evident* Effoot of A* oloaoae on the Blotln Active Substances In Varying Dilutions of Cuoumber Juice The preceding experiment Illustrated that A* cleaoae was capable of synthesizing blotln In the assay medium If no blotln or If small amounts of blotln active substances were present* Therefore, It was considered possible that If cuoumber juice were diluted so that only a small amount of blotln was present, synthesis of this vitamin might occur* This experiment was performed using 1:2 and 1:20 dilutions of cucumber juice* The procedure was the same as that used in preceding seotlons* sented in Table 25# The results are pre­ 70 TABLE 25 UTILIZATION OP BIOTIN ACTIVE SUBSTANCES BY A. CLOACAE IN VARYING DILUTIONS OP CUCUMBER JUICE Uninoculated. Blotln (mmog/ml) Inoculated. Biotin (mmog/ml) lr2 3.84 1.27 lr20 0.384 0.070 dilution of cucumber juice No synthesis of blotln occurred at either dilution of cuoumber juice. At a lower concentration of cucumber Juice, a greater percentage of biotin was diminished* DISCUSSION It was believed that biotin might be a critical factor for L* plant arum during a cucumber fermentation. However, it was shown that when L • pi ant a rum grew in cucumber Juice, the biotin oontent was barely diminished (Figure 8)* When L • pi ant arum grew in an assay medium containing pure biotin, the oontent of this vitamin was markedly depleted (Table 19)* Cucumber juice must therefore contain compounds oapable of substituting for blotln Instead of or in additlonto this vitamin. The biological activity of this substance or sub~ stances was hardly affeoted by the growth of L. pi ant arum, an organism requiring blotin. It was found that Tween 80 had blotln activity (Table 20) and when L. pi ant arum grew in the assay medium containing Tween 80, there was only a moderate 71 reduction of the blotln activity (Figure 9)» This behavior might have been explained that when pure blotln was used, growth would be greater, and consequently more blotln would be utilized* However, from Table 21 It may be noted that acid production was the same In tubes containing blotln, and In tubes containing a combination of blotln and Tween 80* The blotln activity In the tube containing only blotln was reduced about 50 fold* The blotln activity In the tubes containing the combination of blotln and Tween 80 was reduced less than four fold* Thus Tween 80 seemed te not only have the effect of being a blotln substitute, but was also capable of resisting changes to Its blotln active fraction when subjected to bacterial growth* An explanation for this behavior may be In the supposition that the con­ stituents of Tween 80 and not blotln are required for the growth of this organism* Tween 80 Is a polyoxyethylene derivative of sorbltan mono-oleate. Xt Is suggested that L • piantarum is capable ef producing a lipase to split off oleic acid from Tween 80* Xt Is believed that oleic acid Is the blotln active fraction of this compound (111, 113) but If net presented to the organism in an esterifled form (as In Tween 80) It Is somewhat toxic (22, 23, 52)* Xt has been suggested that blotln serves as a catalyst for the production ef essential fatty acids (113)* There­ fore, It Is expected that If blotln were present and not the essential fatty acids, that the blotin might be util­ ized In the formation ef these substances, and thus, the 72 blotin level would be markedly decreased* On the other hand, If an organism is supplied with its essential nutrients in a different form it is possible that propor­ tionately less of those nutrients are utilised* Hence, when Tween 80 was used as a blotln substitute, the organ­ ism might have to produce a lipase as it was multiplying* Growth would be dependent on the amount of Tween 80 avail­ able and the capability of the organism to utilize this compound* It would be of Interest to show more conclusively that elelc acid is synthesized when blotln is present* It is suggested that lectio acid organisms be grown in an assay medium containing blotin* The oleic acid content would be determined before and after growth* This could be done roughly at first by determining the iodine number* If the iodine number increased, the experiment would be repeated on a larger scale and oleic acid would be iso­ lated, identified and its concentration determined* The role of Aerobaoter cloacae and its effect on blotin levels in cucumber juice and in the assay medium proved to be of interest* It was observed that A. cloacae reduced the blotln level of cucumber juice (Figure !>)• However, it was shown that this organism did not require blotln (Table 23)• In fact, this organism was capable of synthesizing this vitamin from an assay medium containing little or no blotin (Table 21±) * If blotln was added in an amount equal to that in a ls2 dilution of cucumber juice. 73 the blotln oontent was lowered. The reason Tor this decrease may be due to the production of* antibiotins which this organism may produce from blotln. Many substances similar In structure to blotln exert an antiblotln effect (20 , 108 ). In dilute cucumber juice, A. cloacae did not synthe­ size blotln. On the contrary, a greater percentage of this vitamin was reduced more in 1*20 cuoumber juice than in 1*2 cuoumber juice. The synthefcls of this vitamin did not occur In dilute cucumber juice probably because the available materials from which this synthesis might occur was also diluted. The fact that a greater utilization of this vitamin occurred in the more dilute cuoumber juice indi­ cated that materials having blotin activity may have served as a source of energy for this organism. GENERAL DISCUSSION The curing of salt stock cucumbers is a complicated process* Cucumbers are placed in a vat, covered with brine, and in a few weeks many biological and chemical changes have taken place* Lactic acid and other products have been formed as the result of microbial activity in the brine* Besides lactic acid bacteria, Aerobacter and yeasts have left their influence on the fermentation* In addition to these groups of organisms, many other types of biological activity may have taken place* The moat complicated group of microorganisms studied so far appear to be the yeasts* Many genera of these organisms appeared during the fermen­ tation* The Aerobacter, common soil-type bacteria, appeared only at the beginning of a 30° saloraeter fermentation* Although these organisms may have an Important bearing on the lactic acid bacteria, they probably do not have any effect on the curing process* It is extremely interesting that L* piantarum was able to assume a dominant role in the fermentation since this organism was present in small numbers at the beginning of the fermentation* Many factors probably contributed to the growth of this organism. The high salt concentration, the low pH and the moderate temperature are probably optimum for the lactic acid bacteria when in competition with other 75 microorganisms. Xt was demonstrated that this organism required blotln, niacin, and pantothenic acid. Cucumbers were shown to contain a large quantity of these vitamins* These vitamins were available quickly due to a withdrawal of the water soluble nutrients of the cucumbers when the brine was added. The vitamin content was high enough within 2i+. hours to support excellent growth. remained high for at least 20 days. The content At this point the important lactic fermentation was In its last stages. Therefore, It appeared as If cucumbers were able to furnish some of the essential nutrients required by L. piantarum. An Important consideration may be drawn at this point. In this study, three common growth factors (blotln, niacin, and pantothenic acid) were selected for study. Many other growth factors are probably required by L. piantarum which undoubtedly are contained in cucumber juice. growth factors have been Isolated from foods. Many accessory Since L. piantarum probably requires many vitamins It is suggested that cucumbers may contain vitamins which to date have not been characterized. Further evidence of the probability that cuoumber juice contains a variety of growth factors was indicated by the behavior of L. piantarum in cucumber juice. It was found that this organism hardly depleted the blotln content of cucumber juice although It required this vitamin. This suggested that cucumber juice contained other factors which oould substitute for blotln. SUMMARY 1* A study was made of the kinds and numbers of microorganisms present during the cucumber fermentation when salted at 30° salometar. All of the acid-producing bacteria studied were identified as Lactobacillus plantarum* These organisms were found in great numbers; the peak of their activity was between three and eight days after the fermentation began* Aerobacter were isolated and many were Identified as A* cloacae. Their activity lasted for a brief period* and the peak of activity proceded that of the laotlc fermentation* Varieties of yeasts were found and their numbers Increased slowly and steadily and sub­ sequently declined gradually* 2* made* A vitamin study of the cucumber fermentation was Xt was found that L* pi ant arum required blotin* niacin* and pantothenic acid* Cucumber juice of various varieties of cucumbers was analyzed for these vitamins and found tocontain the following amounts: Blotln 5.20 to 33.0 mmog/ml Niacin 1*83 to 5*05 mcg/ml Pantothenic acid 1*05 to 2*42 mcg/ml The effect of non-acid-producing organisms isolated from cucumber fementations on the vitamin content of cucumber juice was studied* Xt was found that the niacin content remained unchanged* the blotln content was decreased* and 77 the pantotb.en.lc acid content was Increased, The vitamin oontent or a cucumber fermentation was followed, Xt was demonstrated that an abundant supply of biotin, niacin, and pantothenic acid was available within brining. hours after After fermenting 20 days, the vitamin content of cucumbers was decreased. However, the vitamin content of the pickles and brine remained unchanged for niacin, in­ creased for pantothenic acid, and results were variable for the change in blotln content* 3* The role of blotln in the nutrition of L« plantarum and A, cloacae was further studied, Xt was found that when L , plantarum grew in cucumber juice, the blotln content was reduced slightly. When L , plantarum grew in an assay medium containing blotln, the content of this vitamin was markedly depleted. Tween 80 was found to serve as a blotln substitute and appeared to "resist" reduction of blotin when subjected to bacterial activity. It was demonstrated that A, cloacae did not require t blotln. This organism was shown to synthesize this vitamin if grown in an assay medium containing small amounts of blotin or no blotin. This organism lowered the blotln level when this vitamin was present in high concentrations. When grown in cuoumber juice, A, cloacae reduced the blotln content* CONCLUSIONS 1* The lactic, hydrogen, and carbon dioxide fer­ mentations produced by L. plantarum, Aerobacter and yeasts respectively were of Importance In a 30° salometer cucumber fermentation under laboratory conditions* 2* Blotln, niacin, and pantothenic acid, shown to be required by L* plantarum, were not considered critical vitamins in a cucumber fermentation because cucumbers were found to contain an adequate amount of these vitamins, which were made available within 2l± hours after brining* 3* Studies on the blotln nutrition of L • plantarum and A, cloacae indicated that cucumber juice probably contains one or more compounds capable of substituting for blotln which may be lipoidal in nature* I BIBLIOGRAPHY Abdel-Sal asm, A* and Leeng, P. C. Synthesis ef vitamin B-» bj intestinal baotarla ef rat* Blochem. J., 32:955. 1938. Aderheld, B. Uatarsuehuagaa dbar da a Elnaauran van Pruobtan uad Gamusan. X. 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