INFLUENCE OF SUGAR, AMINO ACIDS, AND CORN STEEP LIQUOR ON LACTIC ACID PRODUCTION AND THE MICROBIOLOGIC ACTIVITY OF CUCUMBER FERMENTATION By Hussein Sadek Ragheb AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Sciences in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1956 H. S. Ragheb Studies were made in 1953 195^ under semicommereial and laboratory conditions of the influence of the addition of sugar, addition of cystine and tryptophane, and addition of corn steep liquor on the production of lactic acid, the microbiologic activity and the availability of certain vitamins and amino acids in cucumber fermentations. Under the conditions of these experiments, acid production was not increased by the addition of sugar or amino acids. Under ideal conditions Lactobacillus plantarum was capable of producing only so much lactic acid irrespective of the amount of sugar present in the brine. acid formation. Corn steep liquor enhanced It also Increased faster and greater utilization of sugars in the brine. No significant effect of added sugar or added cystine and tryptophane on the total and acid-forming bacterial populations and yeasts was observed. the 7 ^ Addition of sugar on day, however, caused a rapid increase in the yeast population which was followed by a rapid decline when the sugars in the brine were depleted. Addition of corn steep liquor stimulated the bacterial flora resulting in rather high numbers observed under both semicommereial and laboratory conditions. Conversely yeasts were lower, especially after the active fermentation had ceased. With the exception of tryptophane, the vitamins and amino acids required for acid production by L^ plantarum, H. 8. Ragheb were not affected by the different treatments. Addition of cystine and tryptophane, or corn steep liquor showed that tryptophane was not likely to be a limiting factor for L« plantarum. The possibility that corn steep liquor might contain a substance or substances, other than those examined, which stimulated the acid-forming bacteria when its concentration was depleted in the brine, was indicated. INFLUENCE OF SUGAR, AMINO ACIDS, AND CORN STEEP LIQUOR ON LACTIC ACID PRODUCTION AND THE MICROBIOLOGIC ACTIVITY OF CUCUMBER FERMENTATION By Hussein Sadek Ragheb A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Sciences in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1956 ProQuest Number: 10008668 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008668 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 Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 ACKNOWLEDGMENTS The author wishes to thank Dr. F. W. Fabian for his guidance in various aspects of this study. Acknow­ ledgments are also made to Dr. R. N. Costilow and Dr. S. Rosen for their advice in demonstrating different techniques. The cooperation of the H. W. Madison Co. at Mason, Michigan, is greatly appreciated. TABLE OP CONTENTS Pag© INTRODUCTION . .................................. REVIEW OP L I T E R A T U R E ............................... Influence of sugar ©n lactic acid formation 1 1+ • • • Influence of sugar on the microbial population of cucumber fermentation ............. 7 Corn ........... 6 Influence of corn steep liquor on cucumber fermentation • « » • , . « • * • • • • • • • • 9 steep liquor • • • » « • • • • Bacterial changes during fermentation .......... 10 Yeast changes during f e r mentation.............. 12 Coliform changes during fermentation 13 Biochemical changes in fermenting cucumbers Microbiology of cucumber fermentation • . . 1? .......... 16 Acid-forming organisms during cucumber fermentation . . . . . . Yeast fermentation........ . ............. G-aseous fermentation ............ 16 17 18 Bloater f o r m a t i o n ................... 19 Biotin, niacin, and pantothenic acid in cucumber fermentation .......... 20 Amino acids in cucumber fermentation............ 23 EXPERIMENTAL M E T H O D S ............................... 26 Semicommercial fermentations studied and methods of sampling • • • • « • • ........ . . . . . . 26 1953 Experiments • • . . * • • * • . . . . • 26 Chemical and bacteriological analyses « . * • • • 29 1951+ experiments......................... 30 TABLE OP CONTENTS - Continued Laboratory fermentations studied .......... » 31 Microbiological assays for vitamins and aminoacids* • 3k- ............................... . ............. 36 RESULTS I - Influence of added sugar on cucumberfermentations II - Influence of added amino acids on cucumber fermentations ............................... 3& 80 III - Influence of added corn steep liquor on cticumber fermentations • • • • « . ......... 92 IV - Vitamins and amino acids in semicommercial fermentations ....................... lOlj. D I S C U S S I O N ................... 115 SUMMARY 121 BIBLIOGRAPHY ........................................... 125 LIST OP FIGURES FIGURE 1* 2. 3* Ij.* 5* 6* 7* PAGE Influence of addition of sugar to brines of semicommercial cucumber fermentations on lactic acid ................. * and acid-forming bacteria 58 Influence of addition of sugar to brines of semi­ commercial cucumber fermentations on per cent invert sugar • • ............................... 59 Influence of addition of sugar to brines of semi­ commercial cucumber fermentations on yeasts • • . • 69 Influence of addition of sugar, addition of cystine and tryptophane, and addition of corn steep liquor to brines of laboratory cucumber fermentations on lactic acid and L* plantarum........... 79 Influence of addition of cystine and tryptophane to brines of semicommercial cucumber fermentations on 3a ctic acid and acid-forming bacteria • • • • * • • 89 Influence of addition of cystine and tryptophane, and addition of corn steep liquor to brines of semicommercial cucumber fermentations on yeasts • • . . 90 Influence of addition of cystine and tryptophane, and addition of corn steep liquor to brines of semi­ commercial cucumber fermentations on per cent invert sugar .................. 91 8* Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on lactic acid and acid-forming bacteria ................. 103 9* Influence of addition of sugar to brines of semi­ commercial cuoumber fermentations on tryptophane • • 108 10* Influence of addition of cystine and tryptophane to brines of semicommercial cucumber fermentations on . . . • * • • tryptophane • 109 11* Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on tryptophane ............. 110 LIST OP TABLES TABLE PAGE 1# Effect of different treatments on lactic acid formation and pH measurements In brines of semi­ commercial cucumber salt stock (1953 Fermentations)• 3& 2* Perceeht salt In brines of semicommereial cucumber salt stock (1953 Fermentations) • • • • • • • • • • 3. lj.0 Effect of sugar and corn steep liquor treatments 6 n lactic acid formation and pH measurements in brines of semicommercial cucumber salt stock (19514Fermentations) • • • • lj.1 Ij.. Per cent salt in brines of semi commercial cucumber salt stock (195I|- Fermentations) • • • . . ......... 14-3 5# Difference in brine acidity during the first week between similar lots of semicommercial cucumber fermentations prepared in two different seasons (1953 suig- 19514- Fermentations) ..................... I4I4. 6* Effect of different treatments on lactic acid formation in brines of laboratory cucumber fermentations (Third Flask Experiment) 7* Effect of different treatments on lactic acid formation in brines of laboratory cucumber fermentations (Fourth Flask Experiment) • • • • • « 53 8# Per cent reducing sugars in brines of laboratory cucumber fermentations (Fourth Flask Experiment) • •55 9* Per cent reducing sugarsin brines of semicommercial cucumber fermentations (1953 Fermentations) . . . • 56 10* Effect of the sugar treatments on total and acidforming bacteria counts in brines of semicommercial cucumber fermentations (1953 Fermentations) • . . • 60 11. Difference in the log. of acid-forming bacteria counts when different sugar additions were applied to semicommercial cucumber fermentations (1953 Fermentations) 61 12® Difference in the log. of acid-forming bacteria counts in brines of semi commercial cucumber fermentations during the first week (1953 Fermentations) ........ 62 LIST OF TABLES - Continued 13* Effect of the addition of sugar on total and acidforming bacteria counts in brines of semicommercial cucumber fermentations (1951|- Fermentations) • • . . • 63 lit* Difference in the log. of acid-forming bacteria counts in brines of semicbmmercial cucumber fermen­ tations prepared in two different seasons (1953 and 195U- Fermentations) • • • • • • • • • ............. • 6 lj. 15 • Effect of the sugar treatments on L„ plant arum counts in brines of laboratory cucumber fermentations (Third Flask Experiment) • • • • . • • • • • • • • • • 6 5 16, Effect of the sugar treatments on L. pi ant arum counts in brines of laboratory cucumber fermentations (Fourth Flask E x p e r i m e n t ) ........................... 66 17. Effect of different sugar treatments on yeast popula­ tions in brines of semi commercial cucumber fermenta­ tions (1953 Fermentations) • • • • • • • • • • • • • 7 0 l8 # Difference in the log* of yeast counts of semi­ commercial cucumber fermentations during the first week (1953 Fermentations) ......................... 72 19» Effect of sugar on yeast populations in brines of semicommereial cucumber fermentations (19511Fermentations) ............................. . * * 7 3 20* Difference in the log. of yeast counts in brines of similar lots of semi commercial cucumber fermentations prepared in two different seasons (1953 and 19514Fermentations) ........ * ...................... 7 I4. 21. Effect of different sugar treatments on coliform populations in brines of semicommercial cucumber fermentations (1953 and 19514- Fermentations) . . . • . 78 22. Effect of the cystine and tryptophane treatments on total and acid-forming bacteria populations in brines of semicommercial cucumber fermentations (1953 Fermentations) • • • • ........ . • • • . • ♦ . . ♦ ♦ 8 3 23. Effect of the cystine and tryptophane treatments on yeast and coliform populations in brines of semicom­ mercial cucumber fermentations (1953 Fermentations) • 8I4. LIST OF TABLES - Continued 214.* Effect of the cystine and tryptophane, and the corn steep liquor treatments on L. plantarum counts In brines of laboratory cucumber fermentations (Third Flask Experiment) .................... . • • . . • • 8 5 25* Effect of the cystine and tryptophane, and the corn steep liquor treatments on L. plantarum populations in brines of laboratory cucumber fermentations (Fourth Flask Experiment) ................................. 86 26. Effect of corn steep liquor treatments on total and acid-forming bacterial populations in brines of semi­ commercial cucumber fermentations (1953 and 19514Fermentations) • • • • • ..................... • • • 9 6 27* Difference in log. of acid-forming bacteria counts when different corn steep liquor treatments were applied to semicommercial cucumber fermentations (1953 and 195k- Fermentations)........................... . . . 9 7 28. Effect of corn steep liquor treatments on yeasts and coliform populations in brines of semicommercial cucumber fermentations (1953 and 19514- Fermentations) .101 29* Difference in the log. of yeast counts when different corn steep liquor treatments were applied to semicommercial cucumber fermentations (1953 and 19514Fermentations) . . . . . . . • • • • • .102 30. A comparison of the tryptophane, cystine and valine concentrations in brines of different semicommercial cuclimber fermentations (1953 Fermentations) * • • • 111 31. A comparison of leucine, isoleucine and glutamic acid concentrations in brines of different semicommercial cucumber fermentations (1953 Fermentations) * • • • 112 32. A comparison of biotin, niacin and pantothenic acid concentrations in brines of different semicommercial cucumber fermentations (1953 Fermentations) • • • • 113 1 INTRODUCTION The commercial manufacture of cucumber pickles generally involves three principal processes* The first is the pro­ duction of salt stock by salting fresh cucumbers in sodium chloride brines of suitable concentrations* The second is the processing or de-salting of the salt stock to withdraw the excess salt, and the third is the finishing of the de­ salted stock into the many kinds of dill, sweet, and kosher pickles and pickle products. After the cucumbers are harvested at the proper stage of maturity they are inspected for defects and graded for size* They are then placed in vats and covered with brine ranging in concentration from 2 2 ° to 1^0 ° salometer depending on climate and other environmental conditions* In general, there are two methods for adding brine to cucumbers, although there are many variations of these methods. In one method the initial brine concentration is 30° salometer (nearly 8.0 per cent NaCl); and sufficient salt is added to raise the brine two degrees salometer each week until a final reading of 50° (13*1 per cent NaCl) is reached; and one de­ gree each week until 60° salometer or higher is reached. In the second procedure a brine containing ij.0 ° salometer (1 0 .6 per cent NaCl) is used, and the salt concentration of the brine 2 is increased two degrees each week until it reaches 5 0 °, and thereafter one degree each week until a final reading of 6 0 ° salometer or higher is obtained* When the cucumbers are surrounded by the brine they lose water by osmosis* Dissolved in this water are sugars, soluble proteins, vitamins, minerals, and other substances which are used as food by the lactic acid bacteria and many other organ­ isms found in the brine* As the dissolved nutrients are with­ drawn, there is a penetration of the salt into the cucumbers* Thus the brine is diluted, and under these circumstances, the salt concentration of the brine, unless corrected, may be­ come so reduced that spoilage type of microorganisms may grow and result in an undesirable fermentation* Consequently, numerous investigations have stressed the importance of care­ fully controlled salt concentrations of the brine* In the normal fermentation that takes place in cucumber stocks, the fermentable carbohydrates, largely reducing sugars, are almost completely converted into organic acids* The principal acid formed is lactic. Most commercial manu­ facturers consider the development of an appreciable brine acidity necessary for curing cucumbers and for maintaining a desirable prime color during the subsequent storage of the stock until it is used. Furthermore, it is the opinion of many that the acid formation should start promptly after brining and be maintained at a rapid rate to prevent the growth of undesirable organisms* 3 As Lactobacillus plantarum is the organism mainly respon­ sible for the acid formation (7 , 2 0 ), rapid utilization of sugars by it is desirable* Several investigations have sug­ gested the addition of sugar to the brine in order to acceler­ ate the acid formation, or favor the production of greater amounts of it* In this work, semicommercial and laboratory fermentations have been studied with the object of investigating the effect of different treatments of cucumber salt stocks on the forma­ tion of acid by the lactic acid bacteria* k. REVIEW OP LITERATURE Influence of Sugar on Lactic Acid Formation Several investigators have recommended the addition of sugar to salt stock cucumber brines with the object of ac­ celerating and producing more lactic acid* however, do not favor it* Other workers The following is a review of the literature found on the subject. As early as 1899 Aderhold (1) claimed that the addi­ tion of 0 *0 5 to 0 * 1 per cent dextrose to cucumber brine resulted in faster and more acid production than in the case where sugar was not added* In 1920 (3 6 ) and 1922 (37) LePevre showed that the addition of a readily fermentable sugar to pickle brine at the beginning of the salting process resulted in more active fermentation. Consequently higher and more prolonged acidity occurred* In 1929 Joslyn (3^) reported that the use of two to five pounds of dextrose per barrel of dill pickles acceler­ ated the fermentation and decreased spoilage by producing more lactic acid* In 1932 Pabian _et al. (22) investigated the addition of destrose or sucrose when the salt concentration was 6 6 ° salometer and after the active fermentation had ceased. They indicated that the added sugar seemed to be beneficial 5 when pickles are to be stored Tor any length of time. There were sufficient acid-producing bacteria to cause an increase in acidity. In a similar experiment, carried out in the laboratory, addition of one per cent sucrose increased the acidity from 0 .3 0 to 0 .8 5 per cent. In 1935 Fabian and Wickerham (25) studied the curing process in the production of genuine dill pickles. They showed that the addition of cane sugar at the beginning of the fermentation accelerated the curing process and decreased spoilage of pickles. In 1937 Fellers ejt al. (28) conducted experiments on cucumber salt stock and other pickled vegetables. Dextrose, in concentrations of one and two per cent of the weight of the vegetable, was added before fermentation started, to brined vegetables, ranging from 5 to 15 per cent salt. The addition of sugar materially increased the titratable acid­ ity which, in turn, decreased bacterial spoilage and improved the quality and texture of the pickled vegetable. The most striking results were obtained with dill and salt stock cucumber pickles. In the same year Campbell (6 ) also recommended the addition of one to two per cent glucose or any other cheap fermentable sugar in the curing of salt stock if it is de­ sired to produce a higher percentage of acidity* In 1938 Fabian and Johnson (2lj.) indicated that the influence of sugar addition to dill pickles or cucumber salt 6 stock depended upon two factorss the amount of sugar already present in the cucumber, and the bacterial flora of the brine* In 1939 Fellers (27) investigated the addition of dex­ trose to fermenting genuine dill pickles and cucumber salt stock* For three pickling seasons, the results showed that the addition of sugar accelerated the fermentation* It also resulted in a slightly increased final acidity and improved the keeping quality of the product* Other advantages cited for addition of sugar were better control of slippery pickles and bloaters* The added sugar might have been beneficial in supplementing low sugar cucumbers* However, Jones ejb al. (33) observed no beneficial re­ sults from the sugar treatment in the manufacturing of genuine dill pickles. When three and one half pounds of sugar was added at the start of fermentation, the amount of lactic acid increased over the control lots by 0*1 per cent* When the same amount of sucrose was added on the 7 th day of fermenta­ tion, less acid was observed. In both cases, however, the variations were not much greater than those that would nor­ mally be expected with the same treatment* In 19ij-l Veldhuis and co-workers (55 ) showed that in four curing seasons, the addition of sugar to salt stock or dill brines at the start of fermentation, or after 10 days, or in small amounts at short intervals throughout the fermen­ tation, caused no appreciable change in acidity as compared with the control* 7 Influence of Sugar on the Microbial Population of Cucumber Fermentation Many workers have investigated the influence of sugar addition on the bacterial flora of cucumber fermentation# In 1932 Fabian et. al# (22) studied the bacteriologic changes when the sugar was added to cucumber salt stock at 6 6 ° salometer. The results showed that the number of acid- producing bacteria immediately increased after sugar addition despite the high salt concentration# In 1935 Fabian and Wickerham (25>) showed that the addi­ tion of sugar at the beginning of fermentation increased the number of weak acid-producing bacteria* The low pH levels in the early stages of fermentation enhanced the growth of the acid-forming bacteria and enabled them to outgrow any spoilage types of microorganisms# In 1937 Fabian (21) explained that the beneficial ef­ fect of sugar addition was due to presence of an adequate supply of energy source for all organisms in the brine, which enhanced acid production and protected the pickles from spoilage# In addition, the studies of Fellers et al. in 1937 (28) and those of Fellers in 1939 (27) showed that the sugar stimulated lactobacilli and consequently Increased acidity. Also, the results of Fabian and Johnson (2k) agreed with those of Fellers (27, 28), i.e., salt inhibited 8 the growth of pectin decomposers whereas sugar stimulated the acid-producing organisms. In 191^-0 Jones _et al* (33) reported that by adding sucrose to fermenting dills, the population of acid-forming bacteria markedly increased with no appreciable rise in final acidity* Also, the yeast population was somewhat higher when sugar was added* Furthermore, Veldhuis et al* (55) indicated that the addition of sugar to brines at the start of the curing process of salt stock or dills, resulted in a condition that favored an increase in microbial population. This increase appeared in some cases to be only that of the acid-forming bacteria, whereas in other cases, only yeasts generally increased. Still in other cases, greater numbers of both types of organisms were observed. There was no increase in acid production, however, even when the acid-producing bacteria showed a sig­ nificant increase in numbers as a result of sugar addition. When the experiment was carried out on a commercial basis, the addition of sugar did not affect the numbers of the acidforming bacteria, whereas yeasts increased rapidly. Corn Steep Liquor Corn steep liquor or corn steep water is a nutrient ex­ tract obtained during the manufacture of starch and other corn products. During the processing of corn, it is steeped for 9 approximately two days in warm water containing a small quan­ tity of sulfur dioxide to extract the soluble materials. steep water is concentrated to approximately 12 Baume. The Batches of corn steep liquor, made by several manufacturers, have dif­ ferent^ compositions* In general, corn steep liquor contains, on a dry weight basis, 12 to 27 per cent lactic acid, 7 *i+ to 7 * 8 per cent total nitrogen, 2 *6 to 3 *3 per cent amino nitrogen, l.f? to II4. per cent reducing sugars (calculated as glucose), and 18 to 20 per cent ash (2)* In addition, corn steep liquor appears to contain adequate quantities of K ^ P O ^ and MgS0^#7H20 (29)* Corn steep liquor has been incorporated in several media used for the industrial manufacture of various organic acids and antibiotics. Influence of Corn Steep Liquor on Cucumber Fermentation Since corn steep liquor is a cheap industrial waste product, it could be used, if beneficial, on a commercial basis in cucumber fermentations. Very little information, however, is available on its use for such a purpose. A patent by Poliak (I4.3 ) in 19lj.l, recommended the addi­ tion of one ounce of corn steep liquor per gallon of brine for making genuine dill pickles. marized as follows: Its advantages may be sum­ 10 (1) The lactic acid formation started earlier and reached a higher maximum when compared with control batches* For example, when corn steep liquor was added at the start of the fermentation of genuine dill pickles, the acidity reached 0*7 per cent after four days* Without steep water, the same acidity level was reached in 8 days. In addition, after 30 days of fermentation, the acidity reached 1*[|_ per cent in the cases where corn steep liquor was added, whereas in control batches it decreased to 0*6 per cent* Further­ more , the treatment of cucumber fermentations with corn steep liquor showed more acid formation when compared with the addition of sugar* (2) The formation of surface yeast scum did not occur in contrast to parallel batches which were not treated* Consequently, spoilage did not occur in the barrels which were treated with corn steep liquor. (3) Addition of corn steep liquor improved the quality of the product and eliminated all faulty fermenta­ tions. Bacterial Changes During Fermentation In 1913 Rahn (i|i|.) studied the fermentation of cucumber under commercial conditions in Michigan and reported that the total number of bacteria at the beginning of fermentation was 11 about 3 5 0 per ml, but readied 2 0 0 ,0 0 0 ,0 0 0 per ml during active fermentation* Xn 1932 Fabian e_t al. (22) showed an increase in the total number of bacteria during the first 2ip hours after the cucumbers were placed in either 3 0 ° or I4.0 0 salometer brine. A gradual decrease then occurred when the brine concentration increased to 50° salometer. While the total number of bac­ teria decreased due to suppression of the peptonizing bacteria, there was a gradual increase in the acid-producing organisms. Furthermore, the number of the acid-producing bacteria was greater and reached a maximum sooner in 3 0 ° salometer brine than in the lj.00. They claimed that the following factors influenced the number of the acid-producing bacteria: con­ centration of salt, presence of available food, and acidity produced by the bacteria themselves. In 19lp3 results obtained by Etchells et al. (19) under commercial conditions agreed with those of Fabian ejfc aJL. (22). When comparing initial brine concentrations of 20°, 1+0° and 6 0 ° salometer, they reported that the highest populations occurred in the 20° brines. started earlier* Also, fermentation at 20° brine However, the duration of fermentation was shorter than in the case of 1^.0° brine. When the brine salt concentration was 6 0 ° salometer, the acid-forming organisms were markedly inhibited and, therefore, the population pro­ gressively decreased. 12 In 1953 Rosen and Fabian (2j.f>) investigated the popula­ tion changes in cucumber salt stock prepared in five-gallon crocks with an initial salt concentration of 3 0 ° salometer. They showed that the acid-forming bacteria did not appear until 2l|. hours after the start of fermentation. Then, they persisted in large numbers for about one week, after which there was a gradual decline. In the same year Costilow and Pabian (8 ) found that the acid-forming bacteria, principally Lactobacillus piantarum, grew rapidly within 2ij. hours after brining under both commer­ cial and laboratory conditions. The organisms attained their maximum population in about three to five days, declined rapidly for the next five days, and then continued to decline at a slower rate for the rest of the examination period which was over f?0 days. Yeast Changes During Fermentation Different environmental conditions affect the yeast population present during cucumber fermentations. For example, It has been shown (19) that the population of yeasts reached a higher maximum at 60° brines than at 1^.0° or 20°. Further­ more, the yeast fermentation started earlier at 2 0 ° than at l±0° or 6 0 ° brines. However, Etchells ejt al. (17) indicated that the dif­ ferent salt concentrations did not materially influence the number of yeasts found in the brine. 13 In 1953 Costilow and Fabian (8 ) found that, under com­ mercial conditions (1 0 tanks examined), the yeast population declined for the first three to five days before initiating rapid growth* The peak populations were reached between 10 and 20 days after brining* noted thereafter* A steady decline in numbers was In the laboratory experiments however, the yeast numbers were relatively high throughout the period of examination* Yet Rosen and Fabian (1|5), in experiments carried out in five-gallon crocks at 3 0 ° salometer, did not observe the decrease in yeast population at the beginning of fermentation. The yeasts which were present initially gradually increased in number and reached a peak in about 8 days, after which a gradual decline occurred* Coliform Changes During Fermentation Population changes of the coliform organisms during fermentation of cucumber salt stocks are Influenced by several factors. When studying the effect of different salt concen­ trations on these organisms Etehells et al* (19) found that in 2 0 ° brines the rapid onset of acid fermentation cut short the activity of Aerobacter because they were unable to tolerate the increasing acid content of the brine. Their numbers declined rapidly after two days and reached a level of 1000 cells per ml on the i|-th day of fermentation. In 1^0° brine the Aerobacter fermentation started promptly and remained 1k active for about five days with counts above 1000 per ml* On the 8 th day a sharp decline occurred and the count was 1000 organisms per ml. In 60° initial brine concentration a different picture was observed. The number of Aerobacter organisms constantly declined during the first 6 days, and remained more or less stationary for a short interval. Then a fermentation started, covering an active period from about the 11th to the l8 th day. A gradual decrease was observed thereafter. In another study Etchells et al. (17) reported typical hydrogen fermentation at 60° salometer salt treatment. At 1*.0 ° brine, hydrogen fermentation may or may not result, where­ as in the 20° treatment it was generally absent. In general, the fermentation at the higher salt concentrations resulted in larger quantities of evolved gas. On the other hand, when Costilow e_t al. (8 ) studied the Aerobacter fermentation under laboratory and commercial conditions in Michigan no significant activity of the coli­ form bacteria was noted. They indicated that coliforms, when entering the brines on the cucumbers, must have found the conditions unfavorable for their growth and consequently died. The results by Rosen et, al. (l\$) obtained under labora­ tory conditions, agree with those of Costilow et al. (8 ). Aerobacter and related organisms at the start of fermentation grew rapidly for 2l± hours, then declined sharply for the rest of the examination period. 15 Biochemical Changes in Fermenting Cucumbers Early work by Rahn (Ijij.) showed that, under commercial conditions, the acid content of cucumber brines varied from 0 .6 to 1 . 2 per cent at the end of active fermentation. Xn 1916 Brown (i|) observed the formation of lactic and acetic acids in the ratio of 2 to 1. Other acids, e.g., propionic, butyric, and benzoic, occurred in traces. In 1926 Tanner (50) claimed that brine acidity resulted from volatile acids, rather than lactic. Today it is well known that several factors affect acid formation during cucumber fermentation. Bacteriophage from soil, antibiotics produced by micro­ organisms and low Eh value due to the growth of certain types of organisms may result in little or no lactic acid forma­ tion (26). The initial salt concentration of brines also have been mentioned as an influencing factor. LeFevre (3 6 , 37) and Jones (30) showed more acid formation at high salt concen­ tration when compared with weaker brines. For example, the average acidity was 0 . 8 per cent when the initial salt con­ centration varied from 5 to 7 per cent, whereas acidity reached 2 .I4. per cent with Initial salt concentration of 8 to 10 per cent. However, Fabian ^et al. (22) reported that the titratable acidity was greater and reached a maximum sooner in the 3 0 ° than in the I4.O0 salometer brine. 16 Results by Etchells and Jones (19) and Jones and Etchells (32) showed that the titratable acidity peached a maximum of about 0*7 per cent in 8 to 9 days when the initial brine con­ centration was 20° salometer. In I4.O0 salometer brine, a maximum acidity of only 0 •J4. per cent occurred after a period of 12 to 13 days* In 19lj-9 and 1950 Pederson ejt _al* (1^.0, I4-I) investigated the effect of the initial brine concentration and tempera­ ture on acid formation* Their results showed more acid pro­ duction in the lower brine concentrations than in the higher concentrations* At 1|5°F, fermentation was limited and the curing process was slow. The initial rate of acid formation was greater at 97 °F, but the extent of fermentation was usually greater at 75° and 8 6 °F* Microbiology of Cucumber Fermentation It is well known that the character of any cucumber fermentation Is dependent upon the type and number of organ­ isms present and their effect during the course of fermen­ tation* Acid-forming organisms during cucumber fermentation* The first systematic study of the microflora of cucumber fer­ mentations was done by Fabian e_t ai* (22)* They showed that the flora consisted chiefly of weak acid formers. 17 In 1933 Wustenfeld (57) divided the lactic acid bacteria found in cucumber fermentation into the genera: Bacillus» Streptococcus. and Pedecoccus. In 1935 Vahlteich et. al* (53) were the first to conduct bacteriologic studies of cucumber fermentation under commer­ cial conditions. They believed that the formation of acid during fermentation was due to Lactobacillus cucumeris and two species of the genus Leuconostoc* Several other workers (7> 8 * 20) considered Lactobacillus plantarum (Orla Jensen), Bergey ejfc al., the predominantly active organism In salt stock cucumber fermentation. However, studies by Pederson (I|-0 , Lj.1) showed that one or more of the following organisms might also play an active role in these fermentations: Leuconostoc mesenteroides, Streptococcus feacalis, Pediococcus cerevisiae♦ and Lacto­ bacillus brevis. However, his studies were conducted under laboratory conditions and at a low salt concentration and, therefore, are not typical of commercial cucumber fermentations. Yeast fermentation. A secondary fermentation which occurs in fermenting cucumbers is caused by the action of subsurface yeasts. Several workers have mentioned true yeasts in con­ nection with cucumber fermentation. Recently population studies (8 , 1 2 , 1 3 , 1 6 , 1|5 ) showed that a part of the typical fermentation of cucumbers was brought about by yeasts, such as Torulopsis caroliniana, Torulopsis rosei, Torulopsis 18 holmii, Brettanomyces versatilis * Hansenula subpelliculosa, and Zygosaccharomyces* Furthermore, film producing yeasts might grow on top of brines especially when protected from the sun light (llj., 15, 22)* Etchells .et al* (11\.9 15) have identified Debaromyces membranaefaciens var. Hollandicus* Zygosaccharomyces Halomembranis * Hansenula anomala * Bndomycopsis ohmeri* Candida krusei, and Pichia alcoholophilia, as species of scum yeast, under commercial conditions, and in salt concentration ranging from 5 to 19 per cent* Endomycopsis ohmeri var* minor was associated with film formation under laboratory conditions* Gaseous fermentation* Gaseous fermentations have been frequently observed in cucumber salt stock in the southern areas of the United States. In 1939 Veldhuis and Etchells (5^1-) reported a definite correlation between C02 production and the presence of typi­ cal yeast fermentation* In l|-0o and 60° salometer brines, two distinct phases of gas evolution were observed (17)* The first phase was brought about by the Aerobacter group with gases evolved, similar in composition with respect to H2 and C02* The second phase was brought about by yeasts, and the gases evolved considered chiefly of CO^* Recent studies showed that Aerobacter, especially A* cloacae, was the major species that caused the formation of gas (17, 19$ kS)• 19 Bloater Formation In commercial production or cucumber salt stock, forma­ tion of bloaters (or hollow cucumbers). is considered undesir­ able* Several investigators have carried out extensive studies on the reasons for production of bloaters. and Etchells (5^4-) and Etchells In 1939 Veldhuis al. (17) found marked simi­ larity between the gases formed in hollow cucumbers collected from the top and interior of the vats. In 19lj-l Etchells et, al. (1 8 ) indicated that the chemical analyses in sweet pickle stock showed the production of CO£ and alcohol, whereas non­ volatile acids were absent. Therefore, yeasts were considered responsible for the gaseous fermentation resulting in the production of bloaters. Other studies carried out by Et­ chells and Jones in 19l|-3 (19) and Etchells and Bell in 1950 (13) incriminated Aerobacter and yeasts in the formation of bloaters. It would thus appear, that Aerobacter and yeasts are in general associated with the production of bloaters in cucumber fermentations. Other studies dealing with the addition of sugar to cucumber fermentations have indicated marked increase in the percentage bloaters formed as a result of this treatment (1 8 , 55). 20 Biotin, Niacin, and Pantothenic Acid in Cucumber Fermentation It is well known that the lactic acid group of organisms is responsible for the acid fermentation in cucumber salt stocks* L. plantarum is considered by many workers to be one of the most active organisms during cucumber fermentation. This species is quite fastidious in its nutritional require­ ments and is used in many microbiological assays for vitamins and amino acids. Since biotin, niacin, and pantothenic acid were shown to be required by many lactic acid bacteria (I4.2 ), they were selected for this investigation. The following re­ view of literature will deal only with these vitamins. Until recently, practically no information could be found as to the concentrations of biotin, niacin, and panto­ thenic acid in fresh cucumbers and cucumber pickles. The nutritional data published in 1950 by H. J. Heinz Company (5 0 ) indicated 0.2 mg per 100 gm of nicotinic acid in cucum­ bers. In 1953 Rosen and Fabian (ij.5) studied the amounts of biotin, niacin, and pantothenic acid available for the lactic acid bacteria during cucumber fermentation. When comparing the vitamin requirements of 10 isolates of L. plant arum from cucumber fermentations with the well known L. arabinosus 17-5, they established its need for biotin, niacin and panto­ thenic acid. Furthermore, they analyzed samples of cucumber juice obtained from 6 different varieties of cucumbers, and reported that biotin concentration varied from 5*2 to 33 21 mmcg/ml; niacin from 1 *8 3 to 5 * 0 5 meg/ml; whereas pantothenic acid showed a concentration of 1.05> to 2 .14.2 mcg/ml. In addi­ tion, they investigated the effect of different brine micro­ organisms (9 isolates of A. cloacae and 9 isolates of yeast) on the utilization or synthesis of these vitamins. Aerobacter cloacae markedly lowered the biotin content of the cucumber juice. Hiacin was not appreciably altered, whereas panto­ thenic acid was not utilized by any of the organisms tested. In fact, most of the yeastcs appeared to synthesize pantothenic acid. In two laboratory fermentations, the maximum concen­ trations of the three vitamins in the brine were reached in the first 5 to 6 days, and did not greatly change thereafter. They concluded that regardless of microbiological activity there seemed to be an abundant supply of these three vitamins for the growth of L. pi ant arum. The same workers then investigated the role of biotin in the nutrition of L. plantarum, and Aerobacter cloacae isolated from cucumber fermentation (I4.6 )• They showed that L. plant arum reduced the bio tin content slightly when grown in cucumber juice, whereas it was markedly depleted when the organisms were grown in an assay medium containing biotin. They concluded that the cucumber juice probably contains one or more substances, lipoidal in nature, which are capable of substituting for biotin in supporting the growth of L. plant arum. Furthermore, A. cloacae can synthesize biotin in 22 an assay medium if it is present in only minute amounts. If, however, substantial amounts of blotin are initially present, a marked reduction of it occurs* In the same year, Costilow and Fabian (11) identified four isolates, obtained from four different commercial cucum­ ber fermentations, as L. plantarum. They studied the require­ ments of these isolates for biotin, niacin and pantothenic acid. Their results agreed with those of Rosen and Fabian (1+5) • All strains of L* plantarum examined required biotin, niacin, and pantothenic acid. In another investigation (9) by the same workers on the rate of diffusion and concentra­ tions of these vitamins during cucumber fermentation, six commercial and several laboratory fermentations were studied. In commercial salt3stock fermentations, the vitamins diffused rapidly from the cucumbers after brining and reached their maximum concentrations in from $ fo 7 days, with no great changes after that time even when tested 61+ days after salting* However, in all instances examined, the vitamin levels were well above those required by L. plantarum. When the effect of some brine microorganisms, L. plantarum* a coliform, and four isolates of yeast, on the vitamin levels in brine was studied, they reported that bacteria and yeast had no effect on the niacin content (10). Only L. p lantarum caused a significant reduction in pantothenic acid, but biotin levels were lowered by both bacterial cultures and 23 all four yeasts• In addition, they observed some synthesis of pantothenic acid by Torulaspora rosei and Hansenula sub* pelliculosa. Amino Acids in Cucumber Fermentation Extensive studies have been carried out on the amino acid requirements of L. plantarum (52)* However, before 1953 no information was found available on the amino acids content of cucumbers and cucumber pickles* In 19ij-l Camillo et al* (5 ) reported that the protein content of fresh cucum­ bers varied from 0.7 to 1 *24. per cent* When the fresh cucum­ bers were made into salt stock, the protein content was de­ creased from l.ij.27 per cent in fresh cucumbers to 1 .0 3 8 per­ cent in the salt stock. The nutritional data by the H* J* Heinz Company (58) indicates 1.1 per cent total protein in fresh cucumbers; 0 . 8 per cent for fresh cucumber pickles; and 0 . 7 per cent for dill pickles* In 1953 Costilow and Fabian (11) were the first to study the amino acids content of pickle brine during fermen­ tation. They investigated the amino acid requirements of four isolates of L* plantarum obtained from commercial fer­ mentations and showed that leucine, isoleucine, valine, glu­ tamic acid, tryptophane, cystine and threonine were necessary for growth. Since other studies (3 8 , 2+9) have established that L. plantarum 17-5 did not require threonine in certain 2k media, Costilow and Fabian (11) indicated that the requirements of L* plantarum for cystine and threonine depends on the basal medium used for testing* In another study on the amino acids available for the growth of L. plantarum in brine, six com­ mercial and six laboratory fermentations were examined (9 )* The amino acids reached their maximum concentrations in 10 to 19 days in the commercial fermentations, and in 15 to 20 days in the laboratory experiments* In all cases examined, a reduction of tryptophane occurred during fermentation about the same time that yeast activity was greatest. Therefore, yeasts may either utilize or destroy large quantities of this amino acid. On the other hand, coliforms and acid-forming bacteria did not contribute greatly to the destruction of tryptophane* Cystine showed a low concentration level even after 19 days of fermentation. This reduction which occurred in the commercial fermentations, disappeared as fermentation proceeded. The concentrations of leucine, isoleucine, valine and glutamic acid were affected by one or more of the brine microorganisms. However, in all instances, there was an abundant supply of them for the growth of L* plantarum. Thus, the great activity of yeasts and/or the coliform organisms, especially at the beginning of the fermentation, might result in critical concentrations of tryptophane and cystine for the growth of L. plantarum. All the other amino acids are not thought to be limiting factors. In the same 25 year Costilow and Fabian (10) extended their work on the effect of brine microorganisms on the amino acids content of cucumber brines. They observed that yeasts and L. plantarum reduced to varying degrees the concentrations of leucine, isoleucine, valine and tryptophane. showed no effect. The coliform organisms On the other hand, the levels of glutamic acid and cystine were greatly lowered by the coliform organ­ isms, whereas yeast had no such effect. In fact synthesis of glutamic acid and cystine by yeasts was noted. 26 EXPERIMENTAL METHODS Semicommercial Fermentations Studied and Methods of Sampling The first experiment began August 3, 1953, at the salting station of the H. W. Madison Company, Mason, Michigan® Twelve barrels were filled with size 2 cucumbers of the variety MR-17 (about 2|. 1 / 2 bushels of cucumbers per barrel)® The cucumbers used in all barrels were taken from the same field, to minimize variations as much as possible. A 30° salometer schedule (7*9 per cent NaCl) was followed in salting, i.e., the initial salt concentration was 3 0 ° salometer which was raised two degrees every week to 1+5 °, and one degree weekly till the end of the experiment, September 13, 1953* The barrels were left outdoors. The following additions, in terms of per cent in these and all subsequent experiments, are based on the weight of cucumbers and brine: 1953 Experiments Barrel 1: nothing added (control). Barrel 2: 0.5$ sucrose added at the time of salting. Barrel 3: 0*5% sucrose added on the 7th day of fer­ mentation. Barrel ij.: 1.0$ sucrose added at the time of salting. 27 Barrel 5* 1*0$ sucrose added on the 7th day of fermentation* Barrel 6 : 1*0$ sucrose divided into three equal parts: 1 /3 added at the time of salting9 l/ 3 added on the 7 th day# and l/ 3 added on the li^th day. In this way, the influence of the addition of different concentrations of sucrose to the cucumber salt stock was in­ vestigated as to its effect when added at different times* Similarly, the influence of adding tryptophane and cystine to fermenting cucumbers was studied, and barrels 7 and 8 were treated as follows: Barrel 7: 0*0025$ tryptophane, and 0*0025$ cystine added at the time of salting* Barrel 8 : the same amounts of tryptophane and cystine added on the 7 th day of fermentation* Furthermore, to investigate the effect of the combined treatment of sugar, tryptophane and cystine, a barrel was prepared as follows: Barrel 9: 0*0025$ tryptophane, 0*0025$ cystine, and 1 *0 $ sucrose, added at the time of salting* The effect of adding other materials, besides sugar and amino acids to cucumber salt stock, was also studied. material chosen in this work was corn steep liquor* ingly, the following barrels were prepared: The Accord­ 28 Barrel 10: one. ounce;of corn steep liquor1 for every gallon of brine added at the time of salting. Barrel 11: the same amount of corn steep liquor added on the 7 th day of fermentation to study its influence when added after active fermenta­ tion had started. Barrel 12: corn steep liquor in the same concentration as in barrels 10 and 1 1 , and also 1 ,0 $ sucrose added as follows: 1 /3 added at the time of salting, 1 /3 on the 7 th day, and 1 /3 on the ll^th day of fermentation* This bar­ rel was included to study the effect of the combined treatment of corn steep liquor and sucrose. The corn steep liquor used in these experiments had the following composition: Substance Per dent Substance Dry substance 5^,5 - 57.5 SO2 (avg,) Reducing sugars (max,) Acid as lactic (min,) 5.0 Per0 cent 0*05 protein (total) I|S - 50 2 0 *0 pH (max*) 4*2 A stainless steel tube, 30 inches long and 3/16 inches inside diameter and sealed at one end with holes spaced 9 .5 ■*"Com steep liquor obtained from Corn Products Refinery Co,, Argo, Illinois, 29 inches apart starting at the sealed end, was used in sampling* Samples were taken through a hole in the head of each barrel by inserting the tube and then syphoning the brine into ster­ ile sample tubes# In this way it was felt that a composite and representative sample was obtained# During the first week samples were taken every day to follow the fermentation closely. During the second week samples were taken every two days except barrels 1, 8, 11 and 12, from which the samples were taken on four successive days after the specific treatment# During the $r& week sampling was made every three days except the control barrel and bar­ rels 6 and 12 where sugar or corn steep liquor was added on the li^th day* In these barrels samples were taken for four consecutive days after the specific treatment* During the ipth, 5th, and 6th weeks, samples were taken every four days# On removal of the samples from the barrels, they were immediately transferred to the laboratory where chemical and bacteriological analyses, were made, after which they were frozen at - 1 8 *5 ° C until the microbiological assays were carried out# Chemical and Bacteriological Analyses (1) The brine samples were titrated with standard NaOH solution to determine the titratable acidity which was expressed as grams lactic acid per 100 ml of brine• 30 (2) The salt concentration was determined by titration with standard AgNO^ using dichlorofluroescein as indicator (3 5 )* (3) Total sugars in the brine samples were determined by the Munson and Walker Method (39)* The results were expressed as grams invert sugar per 100 mL of brine. (i|.) The pH of the brine was determined using a CENCO glass electrode titration-pH meter. (5) Total bacteria count was made using dextrose tryp tone ag ar • (6 ) Total yeast count was made on dextrose agar acidified with 5 * 0 ml of five per cent tartaric acid per 100 ml of the medium. (7) Acid-forming bacteria count was made using V- 8 medium (2 3 ) • (8 ) Total coliform count was made using single strength lauryl tryptose broth inoculated in triplicate with various dilutions of the brine samples. All plates and tubes were incubated at 30°C for all microbial analyses. Three days incubation was allowed for the total bacteria count and for the acid formers, two days for the coliforms, and seven days for the yeast. 19f>il Experiments A second set of experiments was started on July 30$ 195>lj., under the same conditions and at the same place as the first one. This time, only six barrels were put down. 31 The cucumbers used were of size 2 of the variety SR-6 . The same salting schedule as in the 1953 experiments was followed. The cucumbers and brine were treated in the following manners Barrels 1 and 2 : no treatment (control). Barrels 3and Ij.: 0.5 per cent sucrose added to each barrel on the 7 th day of fermentation. Barrels 5 and 6 : corn steep liquor, one ounce per gallon of brine, and one per cent sucrose divided into three equal parts: l/ 3 added at salting, l/ 3 on the 7 th day, and 1 /3 on the lij.th day of fer­ mentation for each barrel. The above experiments were carried out as checks on the 1953 experiments which showed a slight increase in the lactic acid produced in the barrels with the above mentioned treatments over the control barrel. Thus, it was felt necessary to in­ vestigate again the influence of the specific treatments on lactic acid formation.» The chemical and bacteriological analyses were carried out, using the same methods as in the former experiments, except that determinations of reducing sugars and the micro­ biological assays were not made. Laboratory Fermentations Studied A third set of experiments was carried out in the labora­ tory with the object of investigating the effect of the addi­ tion of sugar, amino acids and corn steep liquor on the salt 32 stock under* ideal conditions* Eighteen flasks were prepared in the following manners The cucumbers were washed thoroughly and the juice was extracted* Hundred-ml portions of the cucumber juice were then placed in 500-ml Erlenmeyer flasks. ml of one per cent 3STaCl was added* To each flask 100 This made the final con­ centration of NaCl in the flasks 0*5 per cent. Sucrose, amino acids, or corn steep liquor, according to the specific treat­ ment, were added to the flasks* at 15 lbs* for 15 minutes. The flasks were sterilized A pure culture of L* plantarum 17-5 was first activated by inoculation into microinoculum broth, then centrifuged and the bacterial cells were suspended in physiological salt solution. Each flask was then inoculated with 0*5 ml of the bacterial saline suspension. were incubated at 30° C. All flasks According to Bergeyfs Manual of Determinative Bacteriology (3), these conditions are consid­ ered'- to be the optimum for the growth of L. plantarum* Samples were taken periodically from the flasks for deter­ mination of percentage acid formed, and the lactic acid bac­ teria count* The period of the experiment was 25 days* The specific treatments were: flasks (1 , 2 , 3 , I4.): no treatment (control)* 3 flasks ( 5 9 6 , 7) : 0.5 percent sucrose added* 3 flasks (8 , 99 1 0 ) : 1 * 0 per cent sucrose added. 2 flasks (1 1 , 1 2 ) : 0.0025 per cent tryptophane and 0 *0 0 2 5 per cent cystine added* 33 2 flasks (13, lij.) i 0.0025 per cent tryptophane, 0.0025 per cent cystine and 1 «Q per cent sucrose added. 2 flasks (1 5 , 1 6 ) 0 .8 2 1 per cent corn steep liquor added. 2 flasks (17, 1 8 ) 0 .8 2 1 per cent corn steep liquor and 1 *0 per cent sucrose added. It should be noted that the concentration of corn steep liquor used was the same as that for the barrel experiments. A fourth set of experiments, designed the same as the third one, was carried out in the laboratory to study the relation between the reducing sugars, the amount of acid formed and the lactic acid bacteria count. The following additions were made: 2 flasks (1 , 2 ) : no treatment (control). 2 flasks (3 , k) : 0 . 5 per cent sucrose was added. 2 flasks (5 , 6 ) : 1 .0 per cent sucrose was added. 2 flasks (7 , 8 ) : 0 .0 0 2 5 per cent tryptophane and 0 .0 0 2 5 per cent cystine added. 2 flasks (9 , 1 0 ): 0.0025 per cent tryptophane, 0.0025 per cent cystine and 1 .0 per cent sucrose added. 2 flasks (1 1 , 1 2 ): 0 .8 2 1 per cent corn steep liquor added. 2 flasks (13, iWs 0.821 per cent corn steep liquor and 1 * 0 per cent sucrose added. 3k Determinations of the amount of acid formed and the lactic acid bacteria count were carried out periodically following the same methods as previously described. The re­ ducing sugars were determined by the Munson and Walker Method (39) , at the start and at the end of the experiment* The ex­ periment lasted 35 days# Microbiological Assays for Vitamins and Amino Acids In the first set of experiments made in 1953# it was of interest to study the effect of the different materials added to the cucumber brines on the amino acids and vitamins avail­ able for the growth of Lactobacillus plantarum. Consequently, treatment of the brine samples to release the vitamins, or to free the amino acids from the protein material prior to running the microbiological assays, was unnecessary* The brine samples were removed from the freezer and allowed to thaw at ij.*5 °C just prior to running the assay* They were then stored in the refrigerator under toluene. At the time of assaying, proper dilutions were made from the original brine samples as needed. The methods em­ ployed in the assays were essentially the same as those described in "Methods of Vitamin Assays" (5l)* The assays for valine and glutamic acid were carried out following the method described by Sauberlich and Baumann (Ij.7), using medium I and L* plantarum 17-5 as the test organism*. The dehydrated 35 media prepared by Difco Laboratories, Inc., were used to assay the samples Pei* biotin, pantothenic acid, niacin, leucine, isoleucine, tryptophane and cystine. L. plantarum 17-5 was the test organism used for the assays of biotin, niacin, pantothenic acid and tryptophane. Leuconostoc mesenteroides (strain P-60) was used for cystine, leucine and isoleucine assays. 36 RESULTS I — Influence of Added Sugar on Cucumber Fermentations It is well known that two identical cucumber fermentations might show some variations in the amount of lactic acid formed as well as in many other aspects of the fermentation. Before presenting the effects of the sugar treatments on lactic acid formation, it is more appropriate to study the variations nor­ mally present in brines of similar lots. Table 3 shows that the highest value of variation in brine acidity between in­ dividual fermentations, which received similar treatments in the 1951+ experiment, is 0.10 per cent. Greater variations in brine acidity, however, might appear if comparisons are made between similar treatments prepared In 1953 and 195^* It is noted that three control fermentations were prepared in this investigation. However, other cucumber salt stocks did not receive any treatment until the 7 th day of fermenta­ tion, e.g., barrels 3> ?> 6 and 11 in 1953 > and barrels 3 and lj. in 195^* Therefore, lactic acid formation in brines of these barrels could be considered similar to that of the control fermentations only during the first week after brining. "When comparison is made between these barrels and the control barrels, the greatest difference observed was 0*10 per cent (Table 5)* Therefore, it is logical to assume 37 that, under the conditions of these experiments, if a speci­ fic treatment resulted in an increase greater than 0 .1 0 per cent lactic acid the difference should be considered sig­ nificant. Effect of 0.5 per cent sucrose on lactic acid formation. Table 1 indicates that, in the 1953 experiments, the amount of lactic acid formed in the brine in barrels 1 , 2 and 3 gen­ erally increased after salting, reached a maximum in from 10 to 21 days, with little change thereafter* In individual experiments it seemed as if the addition of sugar might have resulted in a slight increase in acidity as compared with the case where no sugar was added. For example, in barrels 2 and 3 , brine acidity reached a level of 0 ,6 per cent on the 11th day. The same level of acidity in the control lot was attained after 18 days of fermentation. Yet, this ap­ parent rise in acidity over the control fermentation did not exceed 0,10 per cent. Consequently, it could not be attri­ buted to the specific treatment. On the other hand, it was noted that the amount of lactic acid formed after 13 days in barrel 3 , was 0 .6 7 per cent as compared to 0 ,5 2 per cent in the control* Also, brine acidity reached a level of 0.72 per cent after three weeks and remained slightly higher than the control for the rest of the examination period. Thus, it was deemed of interest to repeat the experiment in which sugar was added after active fermentation had started, and to check 38 TABLE 1 EFFECT OF DIFFERENT TREATMENTS ON LACTIC ACID FORMATION AND pH MEASUREMENTS IN BRINES OF SEMI COMMERCIAL CUCUMBER SALT STOCK 1953 Fermentations Time Barrels filled with size 2 cucumbers After of the variety MR-17 Brining --------------- -----------------------------------------1 2 3 k 5 8 A pH pH A pH pH A pH A A pH A 8t r 0 -3 hrs. ■ 1 day 0 *0 1 2 days 0 . 1 5 0 .3 0 3 0 .3 0 k * 5 6 0 .3 7 7 0 .3 7 — 8 9 o .if-5 — 10 11 0 .5 2 0 .5 2 13 0 *5 2 15 16 17 18 0 .6 0 21 0 .6 0 0 .6 0 25 29 0 .6 0 33 0 *6 0 37 0 *6 0 il-1 7 .6 7 .2 5 .9 5 .0 i^.6 l f .5 lf .5 •* 0 .0 1 0 .0 7 0 .3 0 0 .3 0 0 .2 7 0 .3 7 0 .3 7 7 .8 7 .2 6 .3 If.7 lf .5 lf .5 lf .5 if .if — k~.$ 0.1*5 lf .3 mm lf .5 lf.5 if .if 0 .6 0 0 .6 0 0 .6 0 if.3 if.3 if.2 ■» « l f .1 if.l l+ .l ». if . 2 lf .1 lf .1 0 .6 7 0 .6 7 0 .6 0 0 .6 7 0 .6 0 0 .6 0 0 .6 7 if. 2 if. 2 if.O if.l lf .1 if.l *1 •8* 0 .0 1 0 .0 7 0 .3 0 0 .3 0 - o*l|.5 0.1*5 o.lj.8 0 .5 2 0 .5 2 0 .6 0 0.67 0 .6 0 7 .9 7 .3 6 .7 3 .8 lf.3 - lf.3 if.if if.itlf.3 lf.3 if* 3 lf.3 lf.3 * 0 .0 1 0 .0 7 0 .3 0 — 0 .3 lf 0 .3 7 0 .3 7 o .lf3 ifTif «V» - 0 .5 2 0 .3 2 0 .6 0 if .if if.3 lf.3 _ * — if.2 if • 2 if.l if.l if.l if.l if.l lf.3 lf .3 if.3 — — 0 .6 7 0 .7 2 0 .6 7 0 .7 0 0 .7 2 0 .7 2 0 .7 3 7 .8 7 .1 6 .1 if.8 0 .7 3 0 .6 0 0 .6 0 0 .6 2 0 .6 0 0 .6 0 0 .6 7 * • if.2 lf .2 if.l if.l if . 1 if.l if.l * 0 .0 1 0 .0 7 0 .2 1 0 .2 3 0 .3 3 0 .3 3 0 .3 3 o .lf3 0 .3 2 o .if3 o .lf3 o .if3 0 .5 2 - 0 .6 0 0 .6 0 0 .6 0 0 .3 2 0 .6 0 0 .6 0 0 .6 0 7 .9 7 .2 6 .1 if.9 lf.6 if.3 if.6 if.6 lf .5 if*3 if .3 if .3 lf .3 lf.3 — - if. 2 if .3 ifil if.l if.l lf .2 if .2 *• 0 .0 1 0 .0 7 0 .2 2 0 .3 0 0.IJ.3 0 .if 2 - 0 .3 7 0.1f5 0 .5 2 0 .6 0 0 .6 0 0 .6 7 0 .6 7 0 .6 7 0 .6 7 0 .6 0 0 .6 0 0 .6 7 0 .6 0 0 .6 0 0 .6 0 - 9 no determination made * = less than 0*01 per cent lactic acid A = per cent lactic acid Barrel Barrel Barrel Barrel Barrel Barrel 1 = Control, nothing added. 2 =0.5$ sucrose, added at the time of salting. 3 =0.5% sucrose, added on the 7th day of fermentation. k *1.0# sucrose, added at the time of salting* 5 -1*0% sucrose, added on the 7th day of fermentation. 6 = 1.0# sucrose, equally divided into 3 portions; 1/3 added at the time of salting, 1/3 on the 7th day, and 1/3 on the Hj-th day of fermentation* 7 .9 7 . if 6 . if if.7 lf .5 lf .5 if.5 - if .3 if.3 lf .3 if.3 lf .3 lf .2 if.2 if.2 lf .2 lf .2 if.2 if.O if.1 if.l if.l 39 TABLE 1 - Continued Time After Brining Barrels filled with size 2 cucumbers of the variety MR-17 8 A 0 - 3 hrs. -* 4* 1 day 2 days 0.07 3 4 5 6 7 8 9 10 11 13 15 16 17 0.15 0 .3 0 0 .3 0 0.37 0 .1*0 0.37 o.l*5 0 .5 2 0 .6 0 _ pH 7.8 7.3 6.5 5.3 4 .6 4-5 '4.5 4.5 A ■il­ ls0.05 0 .2 2 0 .3 0 0 .3 0 O.J4.5 O.Il2 0.1)5 4.5 0.1)5 • 0.1)5 lj-.il- o.l)5 — 4*3 4.2 0 .6 0 _ _ _ 18 0 .7 5 21 25 29 33 37 lj-1 0.67 0.60 0 .6 0 0.60 0.60 0.60 4 .2 I4-.2 4* X 4 .x 4 .x 4 .x 0 .6 0 0 .5 2 0 .6 0 0.60 0.60 0 .5 2 4.1 0.52 7.9 7.2 6.3 4-7 4-5 4.4 4-5 4.3 1).3 1 )4 l).l) 4.5 1)«3 l).3 — 1).2 1).2 1).X 1).X 1).2 1)«2 1).X A •a « 0.07 0 .3 0 0.37 0.1)5 0.1)5 0 .5 2 — 0 .5 2 0 .5 2 0 .6 2 — _ 0 .6 5 0 .7 5 0.67 0.67 0.60 0.67 0.65 pH 7.9 7.0 6.3 1+.7 A 12 11 10 pH pH A pH * 7.8 a 7.X 6.5 6 .2 0.07 6 .0 1).9 0 .2 2 i).7 4-5 0.33 l).5 i ) 4 0.31) 4-5 1).5 0.37 4«5 l).l) 0.37 1).5 0.52 l).l) 1)«3 0.1)5 4*3 0 .5 2 4.1) l).l) 0 .6 0 4.4 l).l) 0.60 4.3 0.67 4.3 l)-3 0.67 4.3 1).3 0.75 4*2 4« 2 0.67 4 .x 4 -3 0.75 4*3 1).2 0.67 4 .x 1)»2 0.67 4 .x 4.2 0.75 4 .x A 6 .2 •a 0.X5 0 .2 2 0.37 1).5 0 .1)8 l).l) 0.52 i ) 4 0.1)5 4.3 0 .6 0 — i).3 0 .6 0 I).3 0.67 1).2 _ — — _ 1).2 0.75 1).2 0.67 1).X 0.75 l).x 0.75 l).x 0.67 l).x 0.75 4 .x 0.67 * 0.07 0 .3 0 0.34 0 .3 0 0 .43 0 .4 5 0 .5 2 0 .5 2 0 .6 0 0 .6 0 0 .6 0 0 .7 5 0 .7 5 0.75 0.75 0.67 0.75 0.75 0.75 0.7X 0.75 pH 7.4 6 .8 6 .2 4.8 4.5 4.5 4.5 4.3 4*4 4.4 4.4 4 .4 4.3 4.3 4.3 4.3 4° 3 4.2 4.2 4.2 4 .x 4 .x 4 .x Barrel 7 = 0*002?$ cystine, and 0.0025$ tryptophane, added at the time of salting* Barrel 8 = 0*>0025$ cystine, and 0.0025$ tryptophane, added on the 7th day of fermentation* Barrel 9 = 0.0025$ cystine, 0.0025$ tryptophane, and 1.0$ sucrose, added at the time of salting. Barrel 10= corn steep liquor (1 oz/gallon of brine), added at the time of salting. Barrel 11= corn steep liquor (1 oz/gallon of brine), added on the 7th day of fermentation. Barrel 12= corn steep liquor (1 oz/gallon of brine), and 1.0$ sucrose was equally divided into three portionsJ 1/3 added at the time of salting, l/3 on the 7th day, and l/3 on the ll*th day of fermentation. ko TA B LE Z PER CENT SALT IN BRINES OF SEMI COMMERCIAL CUCUMBER SALT STOCK 1953 Fermentations Time No* of Barrel After ______________________________ Brining 1 2 6 3 5 k 17 18 21 25 29 33 37 9.1 8.1 7.7 7.9 7.3 7.6 7.9 • 8*8 8.6 _ 8.9 9.1 9.5 — .. 9.6 9.6 9.8 10 .k 10.8 10.8 10.7 9.1 9.2 9.7 _ — 9.9 10.1 10.1 10.6 10.9 10.9 10.9 o. 16 13-5 9.6 7.9 74 7.5 7.2 7.7 7.1 aCO . CO rH 0-3 hrs. 1 day 2 days 3 k 5 6 7 8 9 10 11 13 15 9.5 8.1 7.3 7.3 7.3 7.2 7.3 8.5 84 8.7 8.7 8.8 9.6 - 94 9.6 9.9 10.0 10.6 10.6 10.6 13.1 13.0 12.9 8.9 9.6 8.8 8.5 8.7 8.5 7.5 7 4 7.3 7.1 74 7 4 7.6 7.6 7.1 7.6 7.6 7.3 7.5 7.5 7.6 8.3 8.3 84 8.5 8.3 — 8.2 8.5 8.8 9.0 8.6 9.0 8.5 8.6 8.9 9.0 8 4 - ' 9»9 — 9.5 9.2 9.6 94 10.0 9.6 10.0 10.0 10.1 10.1 10.1 1 0 .3 10.3 10.6 10.6 10.7 10.6 10.7 10.6 11.0 10.8 10.7 7 8 9 10 11 12 1 3 .2 13.9 13.3 13.2 12.5 4 # 2 8.7 8.1 7.1 7.6 7.7 74 7.5 8.8 8.k 8.8 9.5 8.6 7.3 7.5 7.7 7.7 7.5 8.8 9.0 9.1 9.2 9.2 9.6 8.3 8.1 8.6 8.7 8.9 8.8 8.7 7.8 7.3 7.2 7.3 7.1 74 8.0 8.3 8.3 8.7 8.3 8.3 — - - - - 9.2 9.6 10.1 10.0 10.9 10.8 10.9 9.5 9.9 10.2 9.1* 8.3 7.3 7.2 7.1 7.3 7.5 • 84 — — 8.0 8.7 7.9 7.1 7.3 74 7.1 74 - 8.3 - 9.8 9.6 9.3 10.0 9.5 9.2 10.3 9.8 9.9 10.5 10.1 10.2 11.2 10 4 10.7 ■l*Ir. 1 10.7 11.0 11.1 10.8 H . 5 104 n.i 11.2 11.7 Q.k 8.6 7.5 7.5 74 7.2 74 7.9 8.5 8.6 8.6 8.9 8.8 9.2 9.5 94 94 10.5 10.2 10.7 10.8 10.9 41 TABLE 3 EFFECT OF SCJGAR A M CORN STEEP LIQUOR TREATMENTS ON LACTIC ACID FORMATION AND pH MEASUREMENTS IN BRINES OF SEMI COMMERCIAL CUCUMBER SALT STOCK 1954- Fermentations Time arrels filled with size 2 cucumbers After of the variety SR-6 Brining ......... ..... .................................... 1 2 pH A A pH A pH A pH M2 d l-2 M1 0-3 hr s. * 2 days0.05> 0 . 21) 3 0.3l) 4 o4 o 5 7 o •1(4 A 9 12 7.7 5 4 t4.il l).l li.i li.o 18 0.39 li.o 0 .1)1 0.22 0.28 0.52 0.56 0.50 0.58 0.69 0.62 0.51 0.58 - 0.61). I4..0 O.I4.8 0.59 3.9 0.53 3.8 0 .6 0 3.9 0 .6 5 3.8 0.62 3.8 0 .5 I4 3.9 0.58 li.o - - 0.59 *540 .0 6 0 0.02 0 .2 1 0 .0 6 0 .3 0 0.10 0 .3 7 0 .0 5 0 . 1 4 o»56 0.07 0 .5 7 0 . 0 6 0 .6 1 0 .0 6 0 .5 8 0.03 0 .6 1 0.07 0 .6 2 0 . 0 1 0.61). 0.07 0 .6 1 0.01 0 .6 8 0 .6 6 0.09 0 . 6 3 «. 7 .6 5 .5 1|.5 li.i I4-.3 li.o li.o : 3 .9 I4.0 3 .9 3 .8 3 .9 3 .8 3 .8 3 .9 3 .8 3 .8 ft 0 .0 5 0 .3 1 0 .3 5 0.1)0 0.1)5 0!*53 0 .5 8 0 .6 k 0 .5 8 0 .6 0 0 .6 5 0 .5 7 0 . 6)4. O .6 4 0 .6 li 0.61) 7 . 9 54 5.1+ 0 .0 5 0 .2 6 l ) . l 0 .3 2 l ) . l 0 .3 8 1)..0 0.1)1) 1 .0 " .o .5 i) l i . i 0 .5 7 li.o 0 .6 2 li.o 0 .5 8 3 .9 0 .6 0 3 . 9 0 .6 3 3 . 9 0 .6 0 3 .8 0 .6 2 i).0 0 .6 6 3 .8 0 .6 5 3 .9 0 .6 3 - - 0 .0 1 0 .1 0 0 .0 5 0 .0 3 0 .0 1 0 *0 3 0 .0 1 0 .0 3 0 0 .0 1 0 .0 3 0 .0 7 0 .0 3 0 .0 4 0 .0 2 0 .0 1 0 0 .0 3 0 .0 1 0 .0 3 0 .0 3 - = — A = M = d = no determination made, less than 0*01$ lactic acid* per cent lactic acid* average acidity values* difference in acidity between specific treatment and the control. ^M-j-M = difference in acidity between and M2. Barrels 1, 2 = control, nothing added. Barrels 3, 4- = 0*5$ sucrose added on the 7th day of fermentation. «. 0 .0 9 0 .0 3 0 .0 5 0 0 .0 2 0 .0 2 0 .0 8 0 .0 8 0 . 0 21 25 29 33 37 41 0.30 7.7 ft 5.6 0.05 1+.1+ O .2 3 1+.2 0 . 3 1 ll.lt- 0.35 CO . 045 I4 .1 0.62 li.o 0.56 li.o 0.61 3.9 0.62 3.9 0.63 3.9 0.88 3.9 0.59 Il.O 0.514- 3.8 0.55 3.8 •ft 0.05 d>ij.% 1-m 2 TABLE 3 - Continued Barrels filled, with size 2 cucumbers of the variety SR-6 Time After Brining 6 5 A 0-3 hrs. 2 days 3 k 5 7 9 12 Ik 16 18 21 25 29 33 37 kl a 0.07 0.22 0.33 0.35 O.lt-3 0.59 0.62 0.38 0.71 0.73 0.71*. 0.73 0.73 0.69 0.70 0.70 pH A pH M3 7.6 S.k ■Si- 7.5 5.1 1+-3 ■Si- 0.08 0.21*. 3.9 ii-.O 3.9 3.9 3.8 3.9 3.8 3.8 3.9 3.8 0.33 0.1).l 0.59 0.61 0.58 0.73 0.72 0.73 0.75 0.70 0.70 0.70 0.70 li- .i lj.« o 4 .0 li.O 3-9 l+.o 3.9 3*9 3.8 3.8 3.9 3.8 3.8 0.09 0.27 0.31 0.31 0.l(.0 0.60 0.61 0.59 0.75 0.70 0.72 0.76 0.68 0.71 0.70 0.70 li. o li.i 4 .0 3.8 X -M 3 d5-6 _ 0.02 0.05 0.02 0 . 0I4. 0 .3 2 0 .0 3 0.01 0.01 0.01 o.oi}0 .0 5 0.01 0.01 0.02 0 0.11 0.02 0.05 - 0.02 0.01 D», = difference in acidity between M1-M3 0 .0 3 0 .0 5 0.02 0 0 0.12 0.12 0.13 0.16 0.12 0.11 and 3YU* J Barrels 5, 6 = corn steep liquor (1 oz/gallon of brine), and 1*0$ sucrose was equally divided into 3 portions; l/3 added at the time of salting, l/3 on the 7th day, and 1/3 on the ll|_th day of fermentation. 1*3 TABLE 4 PER CENT SALT IN BRINES OP SEMICOMMERCIAL CUCUMBER SALT STOCK 1951* Fermentations 0-3 hr s • 2 days 3 1* 5 7 9 12 % 16 18 21 25 29 33 37 1*1 u o • o & Time After Brining 1 2 3 Barrel 1* 13*0 8.3 7.1* 7.5 7.5 7.7 8*1 8.k 8.8 12.8 7.7 7.5 7.1* 7.2 7.1* 8.2 8.7 8.3 12.8 7.7 7.5 7.1* 7.2 7.1* 8.2 8.7 8.3 » - • - 9.3 9.0 9.6 9.8 9.5 9.6 10.1* 9.1 9.7 9.9 9.5 9.8 9.9 10.2 9.5 9.8 9.1+ 9.5 9.8 10.8 10.8 9.3 9.0 9.6 9.8 9.5 9.9 io.5 13.3 7.2 7.1 7.5 7.5 7.7 8.0 8.6 8.1* 5 13.5 8.1* 7.9 7.1* 7.5 7.7 8.1* 8.8 8.7 9.1 9.1 9.8 9.8 9.5 9.6 9.8 10.9 6 13.3 9.2 8.1* 8.1* 7.6 7.8 8.1 8.k 8.8 9.0 8.9 9.5 9.5 9.7 9.8 9.6 10.5 kk TABLE 5 DIFFERENCE IN BRINE ACIDITY DURING THE FIRST WEEK BETWEEN SIMILAR LOTS OF SEMICOMMERCIAL CUCUMBER FERMENTATIONS PREPARED IN TWO DIFFERENT SEASONS 1953 and 1951+ Fermentations Time 1953 Fermentations 1951+ Fermentations N®. ©f Barrel After " Brining 1 A 3 A 5 A 8 A 11 A ^1 1 A 2 A 3 A k A D2 D3 2 days 0.15 0.07 0.07 0.05 0.07 0.10 0 . 0 5 0 . 0 5 0 . 0 6 0 . 0 5 0.01 0.10 3 0.30 0.30 0.21 0.22 0.22 0.09 0.21+ 0.22 0.21 0 . 3 1 0.10 0.10 k 0.30 0.30 0.25 0.30 0.33 0 . 0 8 0.31+ 0 . 2 8 0.30 0.35 0.07 0.10 5 - - 0.33 0 . 3 0 0.31+ 0.01+ 6 0.37 0.1+5 0.35 o.i+5 0.37 0.10 7* 0.37 0.1+5 0.35 0.1+2 0.37 0.10 0.1+0 0 . 3 0 0.37 0 .1+0 0.10 0.10 - -- - - - - - 0.1*1+ 0.39 0.1+1+ o.i|-5 0.06 0.10 - = no determination,'.: made, # = determinations made before each barrel received its specific treatment* A — per cent lactic acid* D-i = maximum difference in acidity between barrels 1, 3, 5> 8 and 11 in 1953* D P = maximum difference in acidity between barrels 1, 2, 3 and 1+ in 1951+. Do = maximum difference in acidity between the 1953* and the 1951+ fermentati ons• k5 again the influence of the treatment. Consequently in 1954, control barrels 1 and 2 and two fermentations where sugar was added on the 7th day after brining (barrels 3 and 4) were prepared. in Table 3. The results of this experiment are included In general, brine acidity increased after salt­ ing and reached a maximum of about 0.6 per cent approximately on the 16th day of fermentation. occurred. Thereafter, small changes These results are in close agreement with the gener­ al trend of acidity changes obtained before, i.e., in the 1953 experiment. Also, the average brine acidity of the sugar treatments did not significantly increase over that of the no-sugar treatments. Results in Table 3 show that the maximum difference in acidity, i.e., * between both treatments was 0.09 per cent on the 9th day. This value is still within the limit of variations that occurred be­ tween individual barrels of similar treatments. Therefore, addition of 0.5 per cent sucrose at the time of salting, or on the 7th day of fermentation had no effect in inducing faster or greater acid formation than in the case where no sugar was added. Furthermore, to clarify this matter, a laboratory experi­ ment was carried out in which very favorable conditions were created for the growth of L. plantarum. The complete results of this experiment are given in Table 6. It is evident from these results that the average brine acidity values of both the sugar and no—sugar treatments were almost identical 1*6 throughout the examination period* acidity over the control, i*e*, The highest Increase of * was on^ 0*02 per cent* The flask experiment was repeated again in the same man­ ner, except that two flasks were used for each treatment and that the reducing sugars of the brine were determined at the start and at the end of the experiment, i*e., after 35 days* Results in Table 7 confirm the previous findings* Therefore, addition of 0*5 per cent sucrose at the time of salting did not result in more acid formation even under very favorable conditions* It should be noted that the total titratable acidity values were somewhat lower in the l|_th experiment than those observed in the 3rd experiment. This might be attributed to the fact that different varieties of cucumbers were used in each experiment since the cucumbers were supplied by local distributors. The reducing sugar contents expressed as per cent invert sugar, are recorded in Table 8* As a re­ sult of added sugar, the invert sugar values were found to be higher in flasks 3 and ij. at the start and at the end of the experiment than the corresponding values obtained in the control flasks* However, the amounts of reducing sugars utilized by the organisms after 35 days were almost the same in all four flasks. This would indicate that the added sugar was not converted into acid. It also indicates that L* plant arum can only use a certain amount of sugar and produces a certain amount of acid irrespective of the amount of sugar present* 1+7 Under normal conditions, the cucumbers probably have suffi­ cient sugar present to meet the needs of L. plant arum. Effect of 1.0 per cent sucrose on lactic acid formationo Table 1 shows that in barrels 1| and no significant increase in acidity occurred as a result of adding sucrose• At any given time the slight increase in brine acidity over the control ndver exceeded 0.10 per cent. Only in barrel increase of 0.13 per cent acid on the 18th day. was there an This dif­ ference did not persist for any considerable time and there­ fore, is considered insignificant. Also, addition of sugar on the 7th day of fermentation did not cause an appreciable increase in acidity on the following days. It is true that the percentage lactic acid slightly increased from 0.35 per cent on the 7th day to a level of 0.52 per cent on the 9th day after the sugar treatment. But the total titratable acidity decreased to 0.1}-5> per cent on the 10th day and showed the same value on the 13th day. These fluctuations in acid­ ity are not significant inasmuch as the control fermentation showed a similar increase during the same period of time. They are probably due to experimental error and/or normal variations between samples and microflora. Inasmuch as no appreciable difference in brine acidity was observed to be induced by the addition of 1.0 per cent sucrose either at the start or after seven days, it seemed desirable to approach the study in another manner. It was k& thought that, by adding 1*0 per cent sucrose in small quantities at intervals over an extended period of time, the added sugar might be converted more efficiently into acid, thereby resulting in the formation of an increased quantity of lactic acid* Accordingly, barrel 6 was prepared and 1*0 per cent sucrose of the weight of cucumbers and brine was divided into three equal portions, 1 / 3 added at the time of salting, l/ 3 on the 7th day, and 1/3 on the llj.th day. When the first por­ tion of sugar was added, the amount of lactic acid formed after 6 days was O.lj.2 per cent as compared to 0*37 per cent in the control* When the second portion of sugar was added, the brine acidity was 0 * 6 per cent on the 1 3 th day as compared to 0*52 per cent in the control. Therefore, the increase in acidity was 0 * 0 5 per cent as a result of the first addition of sugar and 0 * 0 8 per cent as a result of the second addition* These differences are insignificant as greater differences might occur between duplicate treatments within one lot. Similarly, addition of the third portion of sugar on the li}.th day did not result in an appreciable rise in acid production. Therefore, the addition of 1.0 per cent sucrose in small quantities at seven-day intervals throughout the fermentation showed no significant effect on the amount of lactic acid formed. When the fermentations were carried out in flasks under optimum conditions, similar results were obtained (Pig. !{., k-9 part A). The average acidity values of the sugar and no«*sugar treatments were almost the same (Tables 6 and 7). Determina­ tion of the reducing sugars at the start and at the end of the experiment (Table 8) indicates that sugar utilization by I** plant arum was not enhanced as a result of the treatment# On the contrary, the amounts of reducing sugars utilized in flasks 5 and 6 were even slightly lower than those of the control flasks# These small differences however, could be considered in the range of experimental error# Since no significant increase in brine acidity occurred as a result of addition of sugar, in neither the 1953 nor the 195k- experiments, the average acidity values of all sugar treatments are graphically presented in Fig# 1# A series of observations was also made on the changes occurring in contents of the reducing sugars of brines of the 1953 fermentations (Table 9). In Fig. 2, part B, the curve representing the control fermentation indicates a fairly rapid increase in reducing sugar after salting# This was mostly due to leaching of the naturally occurring sugars from the cucumbers into the brine. The reducing sugars reached a maximum about the 7th day, then decreased rapidly and could not be detected on the 25th day of fermentation. This decrease is most likely caused by the utilization of sugar by the brine microorganisms during fermentation* The curves representing the contents of reducing sugars of brines, to which 0.5 per cent sucrose was added either at the time of 50 salting or on the 7th day, are somewhat different. When sugar was added at the time of salting, the concentration of the reducing sugars at the beginning of the fermentation was 1*2£ per cent, whereas no sugar could be detected in the control barrel. As fermentation progressed, the reducing sugars in­ creased at first, and, like the control, reached a peak about the 6th day. However, greater amounts of reducing sugars were present, i.e., 1*697 per cent as compared to 1.008 per cent for the control. A rapid decline then occurred until the concentration was almost the same as that of the control, i.e., 0 . 3 8 6 per cent for barrel 2 and 0.33& Ver cent for the control on the 13th day. In barrel 3* the concen­ trations of reducing sugar were similar to that of the con­ trol during the first week of fermentation. Since 0.5 per cent sucrose was added on the 7th day, the content of reducing sugars continued to increase and reached a peak about the 9th day, then declined sharply to a value of O.ij.69 per cent on the 13th day. Therefore, in both barrels 2 and 3 greater amounts of reducing sugars were utilized by the brine micro­ organisms than in the control fermentation. Yet, Table 1 shows that this added sugar was not converted into acid since the titratable acidity did not appreciably increase. When the amount of added sugar was increased to 1.0 per cent, similar observations were made (Fig. 2, part A). Apparently, then, sugar was not utilized mainly by the acidforming bacteria. 51 C O CM rH rH O O O O I rH ° o o * o o o s • • • •t« O O rH rH rH C O O v D CM C O ifc 0• O« O •H •O • O O r l i—I rH O ['«-1_f\-ZhO o < $ CO C O O v£) C A O H H v D <3j ^ •• ••• O o O rl H H A—HD O H O Sh CQ O eg 0 vO O 1S\< £ « O • O O • H H • H C O v O IS- C A O so O O O H d • •H A Eh O _d- H eg >> £ O -P *H H H *H • P O - P t i eg • O £ o eg O H eg •rl e a Sh eg P 0 0 .d C to ■ P - P 0 eg o U © © *d 0 0 d > O 0 0 eg d H a. II II II II H 1 # O CQ X 0 eg H 0 0 2 H eg > H eg > >** -P •H d *H O 0 »>> P •H d •H O eg 0 60 eg Sh 0 > eg 0 60 eg Sh 0 > eg II CM JSJ 0 0 2 • CM co S s d £ eg d eg H H S f! © 0 & -P 0 rO s a 0 0 £ -p 0 d !>s ■P -P •H d •r-t O eg 'd •H O eg d •H 5 0 O jc! 0 d 0 0 O d 0 !h 0

-P d •rl O d i—i ai > >» -p •H O d cd cd > > Ns +3 -P •rl d *H d d •H O •rH O O cd cd cd •rH © © © 50 50 50 cd cd ai S h S h © © S h S h © v£> 1 A C - * O O s O O O H H © > t> O O H H H • 01 ^ oi 3 at>3i c o 50 > © cd cd cd II II II II a nO -d a a N- • -d a • J-T\ a d § C O d d cd rH • N- • ' v£> s d £ © rH © rH © © rH *k «k «k ©01 © © o d d Si o © © d d d a a. • 01 o O'd >3 • Si Sh © rH -P +3 «v S h a Is -p IS -p © © © £ © © Is +3 © d d d d 43 -P 43 *H d d © d © © © £! © © +3 t>3 ♦rH *H d •H O d •rl O d •rH O d •rH O cd cd © © d •H d *H S h •rl •rH © © O d © Sh © O d •rH © © O O u II II II CM_d-vOCO rH rH rH rH © © S h © Si © S h © •H d II •H d P •rl d II II II P U\ -d" I rH S i rH H GOlANi —Ii —Ii —Ii —I oi ai oi oi nO S 1 rH s CM CM rH rH O O CMCM O• O• CO• GO• oooo © 3 >3 Sh Sh OO OO Oo d P a © d d -p43 © ^ Ch ©,d o d rH • d p i r a M dd d oi oi oi oi ©©© © i —IrH i —Ii —I pt, |3Hfx, 53 a i rH CM O O O I • •o • oo d 3 a I I Eh 00 SE=! PP O O H EH vO js_crs-co ••• • oooo 03 S h PI © © »H l3^ 03 fO a •H 'H *H CA O I H f^CMA • vO cd cd rH H i—l a O o a w O Sr © ra © Sh > cd © Sh Sh © cd !» Pi a O -P H H H *H o a cd • O *H cd o a O •H rH cd p! .p 0 Sh P i bO © a -P -P © cd o a XA ra •v a ra O © i— 1 S •% >> -P *H t3^ a a P! a pi cd cd a pi pi © © £ -p & -p © a a >5 a © O d © Sh © O Pi © Jh © Sh a ^ V J . PI "L A O o • • O O H cd © II II CM a © «H ai rH a p cd * © •H bO © • a © a a cd o ra ra poo O Pi © © •H • a © a • a a cd © a bo a p j cd •rl a a © •H bO > !>s a •H a •H O cd © Sh © •P •H O cd Sh rH a © © -P •H a •H O cd a CA a rH • ti •H O cd sjs • ♦ • • oooo H fczs Ph W CM C M IS- O X A _zt-is-oooo CQ Eh o • oooo o § P CM fAOACM'LA rococo • • • © CM sh Pi O O• O• O• O• oooo I H cd fH cd 0 « • U\1S • « • « : £ -da • • • • ■H i rH U \ l A H CM O O O O cd > oooo Ms -P t • • •• £ CA N - l A CA a n -o o C O I • • •• £ oooo CM I A N - CA _rj“ N - c O c o c o ; cd i—1 r U d rH cd > O CO r © d i—I cd > f Ms > * 1 +3 -p •H » •H d d •H •H O O cd cd • fcfl d G © > cd II © bO cd G © > cd r—l rH a G © © is -P © a G © © *3 -p © A G © © £ -P © a Ms -P •H d •H •H d •H •p •H d •H O cd © &Q cd Ph © > cd a. o ,d • Ms >3 Cd r H G G •P -P «v ^ G O ia t a d cm cm a * O O r*» -P **H - H i—1 i—1 © © G G O o G G O o •k «v © © © © -p -p G G oo ra •H -H •H d O cd O cd G •H G •H G •H © O G © G © 5 j>* o o "L A IA rH rH CM CM CM CM O O CO CO O O • • • • O O O O II II II II CM _d~ O r l i—1 CO rH •v rH A N - CA i—I i—1 ra oo ra ra jsj ra 1 a r‘ *1 r-H im 1 ra oo ra ra i rH cd cd cd cd i—1 i—1 rH p a vO a ^ i rH i rH a P a a p * G O d a* © • o o a a nH O cd Na o o © ^ -p -p d o O O Ms vO a G cd a -P O d * oo a . P L d a II II r rH Sh G cd cd a a • d G cd S? "LA a d G P P .d ra CO «i P p4 Ah ?H 0 ia d CM rH rH o o d O CM Ai CM nO On £ P Jh A f-t 0 Ah O 0 • o • <0 • O d ON CM O rH O 'LA S3 nO CO 0 CO d ra CM OO CO I S h • bO o ra co rH at On CO bOn xt rH O d On CO H O On rH O o d SO CM 'LA CA ON nO O On o 0 CA On O' # d o S3 $ O S3 0 XI p ra ra © P ® g ra n P Sh P O 0t r H 2 bOP p P 0W2 cd S h © Or Ph at o Ti p S h bO ra CA O 0 bO o o 0 0 0 o P n ra Ph a « co O 0 0 ra ra rH rH 6 0 ON \A * CM On O o CM ■m P e XA •3 © 03 P 56 CM r— I "LA | O « o t ca # $ $ S - CM CM CM 0 0 G O 00 OOcacA H ' O 'O N | i— I S — _ r j - C M •• O O H O CA CM 3jeP d - IS-0 0 \ A * J 3 cd X* 1 CM d " ' 0 c o O' O o o p Sh 0 O, p Sh p > S3 rH C A 1 A 1 A C A rH rH rH rH CM MS 1 0 a o o CQ P 09 • rH f3 P o 3 xt 0 Ph l—1 « P Sh 0 > J3 P 0 ra xj a cd 3 a Sh Jh O T A •H -d h P O 0 « S3 O •H a a Sh 0 0 4h P P 0 ra ra O 0 £ P II II 1 £ 57 Effect of sugar on total and acid-forming bacteria counts* In addition to the study of the effect of sugar on brine acid­ ity* tpbservations were also made on the total and acid-forming bacteria counts (Tables 10 and 13)* To determine if there is a difference in bacteria counts as a result of addition of sugar, it was necessary at first to study the differences that normally occur between duplicate lots. Thus in 1953» it was possible to compare barrels 1, 3* 5» 8 and 11, only during the first week of fermentation since these barrels did not receive any additions during this period. Maximum differences in the acid-producing bacteria counts, expressed as log. of count, are recorded in Table 12. difference observed was 1.2. The greatest In 195J+* comparison in the same manner between individual barrels within one treatment indicates that the greatest variation, i.e., 1.1 (Table 13). or was Since variations between individual barrels might be greater if comparison was made between similar treatments performed in different seasons, a study of the differences occurring between the 1953 and the 195^- fermen­ tations was carried out. Results in Table 114. indicate that the largest difference in the log. of acid-forming bacteria counts was 1*L}_* Thus, it was safe to assume that if a speci­ fic treatment resulted in a difference of 1.5 (3 2 times greater) or more over the control lot, the difference is sig­ nificant. 5S 'IN 0*01 o ci id3d lN O O O q 00 o 3 0 ‘9 0 3 o cd o IO o o rO ro I-d ao X A CM MD O O CA C\J r H a O rH r H rH EFFECT O Eh O • CA vO Sh d s ,d at erf d •H Cj-j «H EH <*{ U PQ • CM O - v O MD _ 4 0 O A - r H CM rH O l A A - O O O O O X A l t r O O X A o CM \ A CM O O I - 4 4 X A CM rH CAO H O O O O X A CM o o O O O o o o * « * * * # • O I vO XA i—I r H O O O O O O O O A N O O C M O O bO | ••••••• OOOOOOO O Eh 125 I \ D jCAcA rH rH rH l A rH O O O O A X A rH O O O O O 61 TABLE 11 DIFFERENCE IN THE LOG-* OF ACID-FORMING BACTERIA COUNTS WHEN DIFFERENT SUGAR ADDITIONS WERE APPLIED TO SEMICOMMERCIAL CUCUMBER FERMENTATIONS 1953 Fermentations Time After Brining No. of Barrel 1 2 6 3 5 log. Dl-if log. log. log. D l-2 log. .Dn log. d 1-6 1-3 D i -5 0-3 hr s. (3.0) 1 day k*k 2 days 6*k 6.8 3 7.8 k 7.2 5 6 7.3 7.0 7 8 6.6 9 _ 10 11 8.3 6.0 13 5.8 15 16 17 18 5.5 21 5.5 k*6 25 3.0 29 3.3 33 (3.0) 37 3.2 lj-1 3.0 k.3 6.3 8.1 8.2 7.9 8.0 7.7 7.7 _ 7.3 7.3 6.3 _ 5*1 k*8 if.O 3.0 3.0 3.0 3.0 0 (3.0) 0 (3.0) 0 (3.0) 0 (3.0) 0 0.1 i|..0 0 .k ii-.ij. 0 k*6 0.2 if.if 0 0.1 5.7 0.7 5.7 0.7 6.2 0.2 6.3 0.1 8.1 1.3 0 1.3 6.9 0.1 8.1 1.3 6.8 0 .k 7.9 0.1 7.9 0.1 7.2 0.6 8.1 1.3 8.2 1.0 0 0.7 7.6 o . i f 7.6 O.if 7.2 0.7 7.9 0.6 7.6 0.3 7.0 0.3 8.0 1.7 0.7 7.3 0.3 7.1 0.1 6.9 0.1 7.6 0.6 — _ 7.2 6.3 7.3 0.8 6.6 0 7.6 1.0 7.1 0.5 1.1 7.k mm — 7.2 7.6 6.7 0.8 6.1 0.2 1.0 7.5 1.2 7.2 0.9 7.1 1.3 7.1 1.1 7.3 1.3 6.1 0.1 6.7 0.7 9 5.6 0.2 6.3 0.5 6*3 0.5 0.5 5.8 _ — — •> — 6.2 5.k O.if. 5.7 0.2 5.3 0.2 5.3 0.2 5.3 0.2 1.3 5.1 O.if if.6 0.9 k*3 1.2 5.0 0.5 0 0.6 if.2 O.if k.3 0.3 k.3 0.3 if.6 1.0 1.2 if.O 3.7 0.7 0 3*5 0.5 0 (3.0) 0.3 3*6 0.3 0.3 3*k 0.1 3.3 0 3.0 0 (3.0) 0 3.3 0.3 (3.0) 0 0.1 0.2 0.1 (3.0) 0.2 3.0 0.2 3.3 3.3 - = no determination made* D = difference in the log. of acid-forming bacteria count between the specific treatment and the control, log, = logarithm of acid-forming bacteria count. 62 TABLE 12 DIFFERENCE IN THE LOG-. OP ACID-FORMING BACTERIA COUNTS IN BRINES OF SEMI COMMERCIAL CUCUMBER FERMENTATIONS DURING THE FIRST WEEK 1953 Fermentations Time After Brining 0-3 hrs. No. of Barrel 1 log. 3 log. 5 log. 8 log. 11 log. D (3.0) (3.0) (3.0) (3.0) (3.0) 0 i|-.3 5.0 1.0 1 day 1+.0 2 days 6.1+ 5.7 6.2 6.1+ 6.9 1.2 3 6.8 6.9 6.8 7.9 7.9 1.1 k 7.8 7.9 7.2 8.0 7.8 0.8 5 7.2 7.6 7.2 8.3 7.8 1.1 6 7.3 7.9 7.0 7.9 7.7 0.9 7# 7.0 7.1 6.9 7.8 7.3 0.9 # = counts were made before applying the specific treatment. D = maximum difference between barrels 1, 3* 5> 8 and 11. (3*0)= less than 1000 organisms per ml of brine. The log. of count was considered equal to 3*0 when comparative differences were studied. logo= logarithm of acid-forming bacteria count. 63 GA P O ' H LA L A P « « • • * P O 1-io o O O r H O O O r H O O P O H CO OQ QO O CM L A P P * «0 °ALA • #C\JlT\OCOcOCOlAH'Lf\HvO «••**•••«• « • ~ d bO O i— 1 ^ -d CVI p OO O o o o o o o O O O O 1-to OA ONONCXJCOOvOajQOHNOf^O^o ° ••*•••»•««•••»• i a °o °o oo ['■'-co a - f^-p p LALA-d oA o a on rr! O O O O' C M CM n o H GA O P rH 0 y o1 0 0 O 0CO0 •0•0•0•0« 0• 0 • 0•cA'oH o O o o dOO NPH O O o O O •O o o O• •'OOO CO M 0 0 r|H *H - P X ) bO . -P ° £ g 0 y bo H © ® 0 £> £ o£ 0 O P II > P +=> a _S S h - d © $ e 0 o •H O b O r Q ^ o £ P o 0 d I rH ©O CoD p £ © 0 *H *£ £ © Xj 0 O fA1 © rH IS -O 0 © it •£ O rH £ O O II » o 1^ bD d £ p p 1— 1 r H <+H 0 0 ^ P P P C M P p o o bo XJ P P o H II II II II H c Eh p P P bO o 1— 1 6k TABLE 34 DIFFERENCE IN THE LOG. OF ACID-FORMING BACTERIA COUNTS IN BRINES OF SEMICOMMERCIAL CUCUMBER FERMENTATIONS PREPARED IN TWO DIFFERENT SEASONS 1953 and. ±9$k Fermentations ETo. of Barrel Time After Brining 0-3 hr s. 2 days 3 k 5 7 8 9 18 21 25 29 33 37 41 1(53) log . i(5ip) 2(51+) log. log. (3.0) 6.k 6.8 7.8 7.2 7.0 (3.0) 8.3 6.9 8.3 8.1 7.7 6.6 5*5 5.5 1+.6 3*0 3*3 (3*0) 3.2 7 .5 6 .7 6 .3 5*6 1+.3 1+-3 3.6 3.1+ D1 (3.0) 5.9 6.9 8.2 8.0 7.8 0 .5 0 .1 0 .5 0 .9 0 .8 — — 8.0 6.9 6.1). 6.0 1+.1+ i+.O 3.6 (3.0) 1*4 1*4 0.9 i.i 0 1 .4 1 .0 0.6 04 3(53) 3(51+) log. log. l+(5l+) log . (3 .0 ) 6.8 7.6 8.7 8.5 8.3 8.2 8.1 6.9 6.5 5.5 4*2 i+.i+ 1+.1+ (3.0) 5.9 (3.0) 5.7 6.9 7.9 7.6 7.3 7.3 7.6 5.7 5.1 4.2 3.5 34 (3 .0 ) 3 .0 3 .6 8 .0 8.7 8.2 7.8 8.0 7.6 6.1 5.7 5.0 k-3 3.9 3.3 (3.0) D2 0 1.1 1.1 0.8 0.9 1.0 0.9 0 .5 1.2 1.4 1.3 0.8 1.0 l.k 0.6 - = no determination made, log.- logarithm of acid-forming bacteria counts. Dn= maximum difference in log. of acid-forming bacteria count between barrels 1 (53)* 1(54)* 2(54). DP~ maximum difference in log. of acid-forming bacteria count between barrels 3(53)* 3(5i|)* ^(5^)* (3.0)= less than 1000 organisms per ml of brine. The log. of count was considered equal to 3*0 when comparative differences were studied. 65 -• bO O-dOxOaO cr\ 3 oo 0 CM>-’ Lr\_dcvj O O Q O N O #» + • « • O O O H o CM tr\-d-drH rH CM rH xO w O l A O o * V H « O r^co • * o CO O OOO nO _d O *© •O oO •l T •N O• I S O O H C V I O rH O'LTYLA r H p H C\J r H C^rH l-r\ 0-d- O O O O OOO © rOxO I A O O O CM O CM r H l A O rH \ A C O CM • bO o o^ax O ooooo•ood•,o-d• 44 © © o rH rH • O co ox co t^-xo _d Ph to • o

~ $ •H EH n o 4 O O O O O x D O O G G oo bO Q 9 9 o © W •H 5 TZi CO I C M v O H (X- LT\ O i— I i— i C M ©

> oa I O aj T3 bO O co i —i cd o a CA "LA OA M<3 O K OQ -P £ £ O O 67 In the 1953 fermentations, when 0*5 or 1*0 per cent sugar was added at the time of salting, or on the 7th day, or in small portions at 7-day intervals throughout the fer­ mentation, there was no indication of an appreciable increase in the acid-forming bacterial population (Table 11)* Slight variations in counts, which did not exceed the range of dif­ ferences that might occur between individual barrels, were observed. Thus, in barrel 5 the acid-producing bacteria counts were 8*1 x 10^/ml on the 7th day, whereas the control L barrel showed a population of 10 x 10 /ml* After adding 1*0 per cent sugar, the counts on the 9th day were 5 x 10^/ml £ and If. x 10 /ml for barrels 5 and 1 respectively. No signifi­ cant difference as a result of the treatment is observed. Also, in barrel 6, addition of the second portion of sugar on the 7th day of fermentation caused no significant increase £ in counts over that of the control, i.e., 27 x 10 /ml as compared to If. x 10 /ml on the 9th day. When the third portion of sugar was added, no increase in population occurred. On the contrary, the counts continued to decrease gradually for the rest of the examination period. Also in 195^4-* the maximum difference in the log. of acid-forming bacteria counts between the sugar and no-sugar treatments, i.e., D^, did not exceed 1*1, which is not a significant difference (Table 13). Since no difference in the acid-forming bacteria counts was observed between the different concentrations of sugar, 68 the average counts for all sugar treatments under semicommer­ cial conditions, are presented in Pig# 1# It is evident from this graph that the population of these organisms in general, increased rapidly after brining and reached a peak within 5 to 6 days* During the second week of fermentation the num­ bers remained somewhat high, but declined during the third week with very low population observed after JLpl days* The third and fourth laboratory experiments carried out under the best of conditions indicate no significant effect on the numbers of L# plant arum as a result of adding different amounts of sugar (Tables 15 and 1 6 )* In one instance, there was a significant increase in the population of this organ­ ism over the control, e.g., flask 9 showed a population of 6 6 0*10 x 10 /ml on the 2 5 th day as compared to 0*01 x 10 /ml in the control (Table 15) • However, this was not true for flasks 8 and 10 which received the same treatment as flask 9* The average counts of L# plantarum for all the sugar treatments as well as the control fermentations, carried out in the third laboratory experiment, is shown in Pig# ij., part A* There was no need for determining the total bacterial count in the laboratory experiments since the flasks were inoculated with L. plantarum only. Effect of sugar on yeast populations* A study of the differences in yeast populations between similar barrels 69 A 9 .0 8 *0 _ _ A Sugar treatm ent at 7 day intervals _ A Sugar treatments on 7th day (avg. of 3) i - *1 1 1 J 7.0 ML. 5.0 PER 4 .0 - COUNT 6.0 3.0 A /: * LOG. OF 2 .0 Control (avg. of 3 ) n Sugar treatments at time of salting (avg. of 2 ) 10 Figure 3* 15 20 T IM E 25 30 IN DAYS 35 40 45 50 Influence of addition of sugar to brines of semicommercial cucumber fermentations on yeasts* 70 TABLE 17 EFFECT OF DIFFERENT SUGAR TREATMENTS ON YEAST POPULATIONS1 IN BRINES OF SEMICOMMERCIAL FERMENTATIONS 1953 Fermentations No. of Barrel 1 3.4 3.0 0-3 hrs. 1 day 3-1 1.5 2 days 2.8 0.7 0.2 2.3 3 4 (0.1) (2.0) 2.0 0.1 5 2.0 6 0.1 3.0 1.0 7 2.0 0.1 8 0.4 2.6 9 3.0 1.0 10 150.0 11 5.1 250.0 5.3 13 5.8 750.0 15 221.0 16 5*3 380.0 5.5 17 250.0 18 5-3 5.0 120.0 21 5.0 120.0 23 7-0 3.8 29 4.2 17.5 33 22 •6 4.3 37 23-2 41 4.3 log 2 lOgq 0.80 0.40 (0.10) (Q.lO) 0.60 0.10 0.10 0.40 — 12.50 — 10.10 88.0 51.0 — - 12.8 7-0 5.4 4.5 76.0 6.7 10.6 i°g2 2.9 2.6 (2.0) (2.0) 2.7 2.0 2.0 2.6 C\J 1 H Time After Brining 0.5 0.5 0.8 0.3 0.7 0 0 0.4 — 4.0 — 1.4 — 4.0 4.9 4.7 — 1.1 0.4 1.1 — - 4.1 3.8 3-7 3.6 4.2 3.8 4.0 - 1.2 1.2 1.3 0.2 0 0.5 0.3 3 dl-3 3.0 1.0 1.0 3.0 2.4 0.3 (0 .1) (2.0) 0.4 2.6 0.2 2.3 (0.1) (2.0) (0.1) (2.0) 4.0 10.0 6.8 6900.0 6.2 1800.0 2200.0 6.3 4.6 46.0 4.4 28.0 — — 4.2 17.5 20.0 4.3 3.4 3.1 4.2 19.8 4.6 40.0 11.0 4.0 3.8 6.5 — - 0.4 0.1 0.4 0.3 0.6 0.3 0 1.0 2.0 4.2 3*2 1.2 0.7 1.4 — — 1.1 0.7 1.6 0.4 0.4 0.3 0.5 counts xlO^/ml = no determination made = logarithm of yeast count per ml of brine ^1-2 dx ^ = difference between logq* log2 * - difference between logpi log^* (2*0) * less than 100 organisms per ml of brine. The log. of ”1’" count was considered equal to 2.0 when comparative differences were studied. 71 TABLE 17 - Continued Time After Brining Ho. of Barrel 4 0-3 hrs,. 3.0 1 day 28*0 2 days 11 .0 10.0 3 4 7*4 0.1 5 0.1 6 0.1 7 — 8 0.5 9 — 10 79.0 11 83*0 13 132.0 13 _ 16 17 13.4 18 70.0 21 27.3 25 10.8 29 32.0 33 20.6 37 41.0 41 i0S4 dl-4 3*44.4 4.0 4.0 '3.8 2.0 2.0 2.0 0 1.3 1.2 1.7 1.8 0 0 1.0 — — 2.6 — 4.8 4.9 3.1 0 — 0.3 0.4 0.7 3 3.9 1.3 1.9 (0.10) (0.10) (0.10) (0.10) (0.10) 7.7 38000.0 3500.0 100.0 140.0 880.0 1?§5 dl-5 6 3.5 3.1 3.2 (2.0) (2.0) (2.0) (2.0) (2.0) 3.8 7.5 6.5 5.0 5.1 5.9 0.1 0 0.4 0.3 0 0 0 1.0 1.8 4.9 3.5 0.1 0.2 0.1 — — 1.5 1.5 9.0 0.1 0.4 0.2 1.5 3.5 65.0 100.0 125.0 800.0 500.0 1000.0 21000.0 11000.0 88.0 30.0 90.0 45.0 160.0 19.0 11.0 — _ — 4.1 4.8 4.4 4.0 4.7 4.3 4.6 1.2 0.2 0.6 0.2 0.3 0 0.3 — 130.0 61.0 90.0 123.0 57.0 23.0 25.0 d1_Z|_ = difference Between log-^ log^. d^ ^ = difference Between log1 , log^. df difference — — 5.1 4.7 4.9 5.0 4.7 4.3 4.3 0.2 0.3 0.1 1.2 0.5 0 0 Between log-p lo&6’ l°s6 dl-6 3.1 3.1 3.9 2.0 2.6 2.3 3.1 3.5 4.8 5.0 5.0 5.9 5.6 6.0 7.3 7.0 4.9 4.4 4.9 4.6 5.2 4.2 4.0 0.3 0 1.1 0.3 0.6 0.3 1.1 0.5 2.8 2.4 2.0 0.8 0.3 0.2 2.0 1.5 0.4 0.6 0.1 0.8 1.0 0.1 0.3 72 TABLE 18 DIFFERENCE IN THE LOG. OF YEAST COUNTS IN BRINES OF SEMICOMMERCIAL CUCUMBER FERMENTATIONS DURING THE FIRST WEEK 1953 Fermentations No. of Barrel Time After Brining 1 log. 3 log. 3 log. 8 log. 11 log. D 0-3 hrs. 3*4 3.0 3.5 3*9 3.3 0.9 1 day 3.1 3.0; 3.1 3.4 3.1 0.4 2 days 2.8 2.4 3.2 3.6 2.3 1.4 3 2.3 (2.0) (2.0) (2.0) 2.0 0.3 4 (2.0) 2.6 (2.0) (2.0) 2.0 0.6 5 2.0 2.3 (2.0) (2.0) 2.0 0.3 6 2.0 (2.0) (2.0) (2.0) 3.2 1.2 7* 3.0 (2.0) (2.0) (2.0) 3.2 1.2 ♦Counts were made before applying the specific treat­ ment . log. = logarithm of yeast count. D = maximum differences in the log. of yeast count be­ tween barrels 1, 3* 3? 8, and 11. (2.0) = counts less than 100 organisms per ml of brine. The log. of count was considered equal to 2.0 when comparative differences were studied. 73 CO CO 0 0 1 m d 60 o rH Ph O CQ PQ Ssq ^3 * ro s K N CM 0 0 C JH 0 0 CO • • • • i—1O O O rHMOCMr—f C^-M-O>r-HLr\-Q“OJO-l£)CM00U0 O. . . . . . . . . . • • • • • • a ooooooooooooooooo cQOcDrHOOOOOOinLnmoOOvQcOLn'XS mcMCMmm^M>cDO^Lr\npmm^M-Mo o o o o o o ih ia ^ ^ o o o o o o o o o o o o ^ O O H O O O O O O O C O iD ^ O O O ^ H CD O O O M" O4- KM-T\ O O O OCM rH O K A m K\ H P3 CQ PQ d m 60 o 1 —1 CQ s3 o *H -P cd -P S3 CD a Ph CD Ph Lf\ iD 00 O i—I rH O O (T' Lf\ i—I m CO GO C T' mCM0jmmmLf\vDOvDU'\M' 0"K\m m m L fM A tN O iA v O O O O O O O O O H O J O CD Ph Ph l^ O O H m 0 PQ i—I d o • o- CM i rH M " CM CM O O O O O O d S3 0 0 i—1 m ^ CO 60 O rH O d 0 d • S3 S3 0 0 £ 0 0 £ -P 0 0 o o -p 0 H [N 0 0 0 0 * 0 U fN CO i—i d -H m 60 0 Ph 0 d g d a *rH d •rH •H d TO S it It B 0 Ph 0 d d CM 1 1 rH d o 1— 1 H O O O C O ifl • • • • • • 0 - 0 LIACO CD | • • • • • ! M * CM CM CM CM m M " LC \ LT \ 0 - M * y O rH «CM 60 O rH M-i d i d m d O O O O o 1 1 S3 0 o Ph o 0 P-l * g co £ d i—I O- CD 0~ • • • • LT\ M- d 0 o d S3 i— I L fN Ph CD -Q " Q ~ 0 ^ O O 0 *h d P h-p s3 d • S3 CD -P -P 0 0 d CQ f3 £o £ O OOOO o o o o o 0 - (—I <—I i—I O - CD CM 1^0000^ 1 —I I • • • • • ! CO O O rH rH l A rH 00 O' •Q- m o o o o • • • • o - cm o in LALA^-lA EH PQ 0 Ph 0 rH CM 60 CQ Ph Ph s3 1— t 0 03 O PQ 60 d dn m -P 0 d ^ 60 d H ^ C \ lO O O O 4 O I M > O '0 0 rH O O O CM rH CM rH O O m m VO o CO *' rH 60 O c— 1 d § d o S3 OOO OOO i— i • CM 60 60 O O rH r—1 rH 60 O (—1 o o cd cd mcM cD o m * • • • • • I • • • * • ! m cm cm cm cm m 4 - m m in in 0-0 o o oo oo CD <—1 i— I i— I r H m o M " CM O O O H o o o o o M * CM o o O O C n ^ tc D rH CM C M o PQ o in in r H d o it It M ~ i— I CM Ph Ph> d ctf d rH m i cm m ^ t m o c o o c m -O' oo i—i m c n m O r H o II o 10 CQ rH H rH CM CM CM m m M - l (0 1 *0 Ph S3 0 0 -H s -p s3 •H d -H eh <3 Ph o o o o • • • • m rH 60 cdoocm « • • • mM'-ct-M* d CQ o (D O •H O Ph o « 0 S3 d 0 O CM & -P 60 0 S3 Sh • o 0 0 O o 60 P> 0 0 o •H O h> -P O 1—1 i—i S3 0 0 d 0 O i—1 S3 i—i 0 Ph o d C7* 0 S 0 EH 0 d a \ 0 m Ph CD d d o 0 d d d -p rH -P -P • 0 d *rH 60 O -P o 0 Ph •H S3 S3 i—i d CQ -p rH lo g . O £3 M M EH << EH CQ J sj £3 PQ O M EH PQ <3 Ph d 03 * -1 d P h PQ O PQ pq d d pq Eh O ft 21 5ft* 5f-if 23 if* *5fC 29 *5ft 5 f t * 33 5ft5fC •ftsf 37 sft* 5 f t 5 + t 41 3 4 5 6 1 2 740 740 1180 920 430 92 430 630 2200 1180 740 920 230 9200 9200 430 5ft* sftSft 24 2500 240 430 sftSft s f : S f ! 5 f ! I f : 24 24 15 sftsft SfSf sftsft *5ft sfSf sfsft sftsf: Sft5ft sftsft SftSft _ __ sfsf *Sf 5ft5|C sftSft sftsft sfSf sfsf sfsf sfSf 5ftsft SftSft sfSf SfSf sftSft Sftsft sfsf 5fesft s f s f s f ; i f 5ft5ft _ _ _ sftsft *Sf sf:sf: 5ft5ft _ — *sf *if sf* sftsf: sftSft Sf*f sfsf Sfif sfSf SfSf SfSf SftSf: 5ftsft sftaft sfSf Sft5|s sfsft sft* — sfSf *s ft *sft sf!sft — _ sfSf sfSf Sfsf Sftsft *Sft sftsft sfsf SfSf ♦5f sftSft SftSf 5ftsft SfSf SfSf sftsft Sfsf *Sft sftSft Sfsf s f S f s f : s f t S f S f SftSft Sf:sft S f s f S fSf S f * S f t s f t sftSft Sfts*t S fSf s f S f s f s f Sftsft Sftsft Sftsis s fSf S f S f *Sf SftSft Sftsft sftsft 1No. per ml of brine. - = no determination made. ** = less than 10 organisms per ml. 3 4 0 00 c\) 10 rcNd oj CVJ K\d 1 430 — sfSf sfsf SfSf — — Sfsf 9200 4300 sfSf sfSf _ Sfsf sfsf sfSf — — *Sf sfsf — — SfSf SfSf sfSf sfSf sfSf SfSf SfSf ♦^ sfSf — — sfsf sfSf SfSf Sfsf Sfsf SfSf Sfsf 79 L.plantarum counts Acidity Control (avg. of 4 ) o — ——o Sugar treatments (avg. of 6 ) 0 .8 0 ML. Control (avg. of 4 ) - 11.0 COUNT LOG. OF PERCENT LACTIC o ———o Cystine a tryptophane treatments (avg. of 4) PER ACID 0 .6 0 0 .8 0 Control (avg. of 4 ) *« ■o Corn steep liquor treatments (avg. of 4) .4 0 r 0 .8 0 5 Figure 4. 20 T IM E 25 30 IN DAYS 35 40 45 Influence of addition of sugar, addition of cystine and tryptophane, and addition of corn steep liquor to brines of laboratory cucumber fermentations on lactic acid and L . plantarum. 80 II - Influence of Added Amino Acids on Cucumber Fermentations Studies by Costilow and Fabian (10) showed that cystine and tryptophane might be reduced during cucumber fermentation to a critical level for the growth of L# plantarum. There­ fore, the effect of adding these amino acids to cucumber salt stock was investigated# In 1953* barrel 7 was prepared by adding the amino acids at the time of salting# Barrel 8 was prepared in the same manner except that cystine and trypto­ phane were added on the 7th day of fermentation. In this way, addition of amino acids at the start and during the active fermentation period was studied# It was then thought that the combined treatment of amino acids and 1#0 per cent sucrose might have an effect on brine acidity as well as on the micro­ biologic activity. Consequently, barrel 9 was prepared by adding both the amino acids and sugar at the time of salting# Based on the above studies (10), a concentration of 0*0025 per cent of each amino acid was used in this investigation# These concentrations should be more than sufficient to pro­ vide the brine microorganisms with plentiful amounts of these amino acids# Effect of amino acids on lactic acid formation# Results of the effect of the addition of amino acids on lactic acid formation are found in Table 1#. It was shown earlier in this study that a variation greater than 0#10 per cent in acid 81 formation between different treatments should be considered significant* This value was also used in these studies as a basis for comparison between the barrels receiving cystine and tryptophane and the control barrels. In all fermentations examined the amount of acid formed in brines receiving cystine and tryptophane was not appreciably greater than that in the control fermentation. It is true that sometimes an increase of about 0.15 per cent acid over that of the control occurred as in barrel 7 on the 3rd and18th days. However, this and similar increase in acidity did not last for a considerable period of time, and consequently is of no importance. flask experiments confirmed these results* The It is evident from Tables 6 and 7 that under ideal conditions the difference in brine acidity between all the cystine and tryptophane treat­ ments examined and the control fermentations, were very small / and negligible. Therefore, it was not surprising to find that under laboratory conditions, the cystine and tryptophane treatments did not result in more utilization of reducing sugars by L* plantarum (Table 8). Fig. I*., part valuejs of all the B, and Fig. 5> show the average acidity cystine andtryptophane treatments examined under laboratory and semi commercial conditions respectively. In 195>3* the reducing sugar contents of brines of the cystine and tryptophane fermentations were also studied. Table 9 and Fig. 7, part A show that in both barrels 7 and 8, 82 the reducing sugar concentrations increased after brining, reached a maximum about the 6th day, thendeclined to a level less than O.Olj.5 per cent invert sugar after 25 days* These results are similar to those obtained in the control fermen­ tation; indidating no faster or greater utilization of re­ ducing sugars as a result of these treatments* When the two amino acids were added in combination with sugar at the time of salting (barrel 9)* the concentrations of reducing sugars were considerably higher during the first week. However, they decreased until their level was comparable to that of the control, i.e., 0.5l5 P©** cent invert sugar in barrel 9 as compared to 0.33& P©** cent in the control, on the 1 3 th day. In general since no greater amounts of acid were formed in all these fermentations, one can only conclude that trypto­ phane and cystine under the conditions of these experiments, are not limiting factors in acid production. Effect of cystine and tryptophane on microbial popula­ tions. The total bacterial population, the number of acid formers, the number of yeasts and coliforms for each speci­ fic treatment are presented in Tables 22 and 23. Pig. i|_, part B, and Pig. 5 represent the average acid-forming bacteria counts under laboratory and semicommercial conditions. The average yeast numbers are also presented in Pig 6, part B. It is evident from these studies that no significant rise in the total and acid-forming bacteria populations, as well 83 CVJ E L A d 00 L A E ON I I— I d PCS 4 i—I i — Ii—Ii—(CO CO CO CO * | CO O O i IO O O O KN ,H OJ O O O OCA • • • • • • • ! • * i — ICO •• d 0 -P o 0J rO 4 bD 4 •H C A bD o 00 E ••••••••I O CD H PA CA CA CAPA CA PA CA O O O H P A 4 E C 0 E 0 0 E E E 4- 4 - O i —I I 1 O 4- o O O O J O O O O * • • • • • • O O LAO E O O ^ HOCOO E OJ 1 —I O Pq CA I p ) H O < j pq o EH i-q C4 pq EH O Eh o o ^5 O CQ CQ Eh OJ pq l-d pq EH C Q 4 <3$ pq EH P ; Ph h pq eh Ph ^ pq w pq pq fe pq H 4 E ri K M Ph C Q O s I— I H oj O • O O 1—I( —I O OOOJOJOOO • I • • • • • * • ( 00 LA 00 O O • • O L A 00O O O O CO OJ CQ u 4 O Ph •H -p a j -p bD 1—I pq pq K \ 1 —I OJ • r—I vQ 00 • • • • • ) O O O L A r H O O O O O O O 00 O O LA O O O O O O O 00 PA 1 —I la4o j4pacooo oco 4 * 1 —I 1 —I 1 —I O O O 1 —1 0 0 0 rH 1 OPA4CAOPACAc0OE00C04OCA004OrH00O Eh 1 d *H O aj O Eh o 0 -p *H • d P aj 4 bD 4 o o rH O PA4OEC000E E E E E E E E lA lA lA 4 4 papa II pq 0 ti E O OJ o O O O O O O O O O O O O O h o oo cD LA l A O O H H O O LA C A E PA O O O O bD O rH O O p A lA L A O la lA lA lA lA O O O O O O O O O O 00 rH O 00 O 4 - L A O E P A H 1 —I OJ o P d 1 —) h LAO O LA LA PA4" 4" 1 —I < —I c a c o l a o o o o o o o o o o l a e e o j o o o o o • • • • • » • • • • • • • • • • • • • • • L f\ o O O O J O O J lA lA O O O O O O O O O O O O O O E rH E E E O J O O E O J rH P> El O I —II —I X E 4- rH LA OJ O I i—H s E bD pq 4 P EH H Ph Eh pq CQ EH O i —I • • • • PA • * PA 4" LA LA l A 4 I • PA LA O O O O 1—I1—Io o o o o O rH o o o o o o O O PAPAlD 00 PAPA O PA PA 4- O O PA O o ' o ' E PA4- L A E E E E E E O O la OJ 1 —I i—I 1 —I 1 —I E O I H O O O O O O O O J rH O O O O O O • • • • • • • • I • I la la 4 pa pa pa pa pa pH O -p d d o * O 1—I ^0 0* aj\ d -H O aJ d O S 0 rH -P O 4 pq O rH 4O O O JO O O O O J ♦ • • O O O LA O LA OJ 4 w OJ L A O OJ OJ e p i EH pq I H rH • • • 1 OJ OJ O O O O O O O O O Eh O EH pq Pq pq pq O LA LA EOLALAOOOLA OJ OOO00OLALAO I E OJ E O CO OJ rH bD u 0 ^ 0 p •rH E H EH 4 d *H d *H d pq to d ,4 aj d PA I H O OJ i—I rH 1 —I 4 OJ E H O O O O OJ E - 1 —I 1 —I O O O O O O IO J r H O O O O O O O O w w w cq g 0 o d eh 0 I d d •h 0 y rH aj -p o d ajp 11 11 11 aj d OJ PA 4 d o y aj x H P aj P H aj bD d d d0 •H ■H P ay d aj -p 1 -4 Eh LA O E 00 CA O H PA LA CO H lACA PA E H 1 —I 1 —t 1 —I 1 —I 1 —I OJOJ OJ PA PA 4 P h CQ aj *H 0 d d d 0 -P •H -P y d aj d O 4 0 bO rH 4 d o d Eh 3 -p Eh d d •H o o d Eh y c6 P3 4 Pi EH ElO PH B pq d 0 O i —I d 0 0 Ph 0 O 4- H O O O O 00 0 Ph pq EH o s & PA 4- O O O rH O rH O O d Ph O pq O H OJ LA4 4 O 00 1—fO OJLAPAOOJOOO • • » * • • • • O O OJ e 4* pa o O «-H CO E O E 00 rH rH M Eh pq E E O o 00 I pq CQ *| • • • • • • • ( PA LA LA OJ 3 H O 0 -P r4 a P 0 I s d 4 -p •H 0 aj O 0 0 aj £ d -p P Eh 0 o d y H -P eh bD •H o o O 0 aj •rH ait • • • • KACODI • KA KA KA C\1 • • o 125 • OJ OJ KA o o o o KA p - O t>- KD o j | • • •* I* * * *** • • • cj- LfA • • -d* l£) 00 KA r-l rH • « • • • • d" KA KA p - cj- p - t * * * * * * * * * * * * ************ O O KA* KA OJ O p * CA ca rH a \ o >H K A (—I M bO •r-( -p aj -P KA C\J (D P3 pq «! eh CQ H KA o I —I X LT\ O O O O O O O O O O O H H H H H M / A O O O O O O O Q O O O • • • • • • • • • • • • • • • • • 00 KA LfA O O O O O O O l G O O O O C O O L f A ^ t O 4* 4* ........... 0 - 0 1 t>OJ OJ rH OJ K A C\J i— I i— I O J ca O H H O O O O O bD Q • • • • • • • • KA I KA OJ Of OJ OJ OJ OJ OJ O [>- 0 \ o bD P H P 0 -P •H *H H Eh ^ P pq • KA OJ LfALfACA l 0 CA L0 L0 EH O | • • • * • • p* UA LfA P* p- p- • • * p- ^ » p- o• o• o• o* * * * * I* | * * * * * * * * * * o o o o * * ** * * * * * * * * * * * OJ O - LfA rH C A P" rH i— I a oooo \ LfA LfA LD i— I i — I rH i— 1 i— 1 >H KA 0 1 -1 r H O O O O O O O X p (L) CD Y = yeast populations C = coliform populations log* = * logarithm of yeastcounts 4 - C\J U N O 1—I O O O O O O O O O O O OJ i D O O O O O O O J-O I— 1 LfA LfA 00 ^ C A LfA LfA rH rH KA KA • • • • • • • • • • CO CQ IH l>5 h Ctf 0j KA O I rH OJ K A ^j- LfA CO O - 00 C A O rH KA U A CO rH U A C A K A O - r H rH i— I i— I i— IrH OJ OJ OJ K A K A P* - « no determination made ** = less than 10 organisms /ml (0.001) = less than 100 organisms /ml bD o 85 TABLE 24 EEFECT OE THE CYSTINE AND TRYPTOPHANE, AND THE CORN STEEP LIQUOR TREATMENTS ON L. plantarum COUNTS IN BRINES OE LABORATORY CUCUMBER FERMENTATIONS Third Flask Experiment: No . of Flask Time After Brining 11 No. 12 log. .8.1 0-5 hrs. 128.0 2 days 2010.0 9.5 6 1450.0 9.1 11 96.0 7.9 1.0 6.0 17 0.006 5.7 25 14 15 No. log. No. log. No. log. 115.0 1840.0 2250.0 95.0 1.1 0.007 8.0 9.2 9.5 7.9 6.0 5.8 125-0 2110.0 5 1220.0 116.0 8.5 0.48 8.0 9.5 9.0 8.0 6.9 4.6 112.0 1670.0 1200.0 78.0 7.0 0.49 8.0 9.2 9.0 7.8 6.8 4.6 16 15 No. log. No. 0-5 hrs. 126.0 2 days 2470.0 1450.0 6 11 195.0 12.5 17 8.7 25 8.1 9.5 9.1 8.2' 7.0 5-9 128.0 2540.0 950.0 160.0 15.0 8.9 18 17 log. No. log. No. log. 8.1 9.5 8.9 8.2 7-1 5*9 117.0 1510.0 880.0 195.0 52.0 10.4 8.0 9.1 8.9 8.2 7-5 7.0 128.0 5110.0 870.0 181.0 57.0 8.1 8.1 9-4 8.9 8.2 7-5 6.9 No* = counts x 106/ml Flasks 11, Flasks 15, Flasks 15, Flasks 17, 12 = 0.0025% cystine, 0.0025% tryptophane, added 14 = 0.0025% cystine, 0.0025% tryptophane, 1.0% sucrose, added 16 = 0.821% corn steep liquor, added 18 = 0.821% corn steep liquor, 1.0% sucrose, added 86 TABLE 25 EFFECT OF THE CYSTINE AND TRYPTOPHANE, AND THE CORN STEEP LIQUOR TREATMENTS ON L.//plant arum POPULATIONS IN BRINES OF LABORATORY CUCUMBER FERMENTATIONS Fourth Flask Experiment of Flask Nc 7 No. 88.0 0-5 hrs. 1 day 2tL00.0 5 days 194-0.0 4-.0 9 0.002 55 8 log 7.9 9-5 9.2 6.6 5-5 No. 11 No. 0^5 hrs. 100.0 1 day 2860.0 5 days 554-0.0 270.0 9 0.15 55 log 90.0 2010.0 2200.0 15.5 0.005 10 9 7.9 9.5 9.5 7.1 5.6 No. 90.0 1600.0 1800.0 1.1 0.005 12 log No. log 7.9 9.2 9.2 6.0 5.6 85.0 194-0.0 1700.0 0.2 0.004 7.9 9.2 9.2 5.5 5.6 14 15 log No. log No. log No. log 8.0 9.49.5 8.4 5.1 88.0 2650.0 2750.0 510.0 0.20 7.9 9.49.48.4 5.5 100.0 5000.0 2500.0 78.0 — 8.0 9.49.2 7.8 — 92.0 5180.0 2550.0 124.0 7.9 9.5 9.5 8.0 4.9 o • o \D H Time After Bnmng - - no determination made No, = counts xlO^/ml Flasks 7, 8 Flasks 9, 10 = 0.0025% cystine, 0.0025 tryptophane, added = 0.0025% cystine, 0.0025% tryptophane, 1.0% sugar, added Flasks 11, 12 = 0.821% corn steep liquor, added Flasks 15, 14- = 0.821% corn steep liquor, 1.0% sugar, added 87 as yeast numbers, was noted* At one time or another a dif­ ference of 1*5 or even more in the log* of counts of the respective organisms occurred. For example, the acid-forming bacteria showed differences of this magnitude on the following days* after 21 days in barrel 7, on the 11th day in barrel 8, and on the 3rd day in barrel 9 (Table 22). Also, comparison between Tables 17 and 23 shows that the log. of yeast counts increased by 1.7 over the control in barrel 9 after 9 days. In all these cases, however, the differences were not consis­ tent and did not last for a considerable period of time. Under very favorable conditions, results in Tables 2lj. and 25 indicate no effect as a result of these treatments on L. plantarum counts. In general, Fig. 5 shows that the acid-forming bacteria increased after brining, reached a peak about the 1+th day, maintained high population levels -until about the 8th day, then declined gradually for the rest of the examination period. The total bacterial counts closely resembled those of the acid formers. Conversely, yeast counts generally decreased after brining, remained low during the first week of fermen­ tation, then increased to a maximum about the 15th day with little change thereafter (Fig. 6, part B). These results are somewhat different from those of Costilow and Fabian (9), who found that,under commercial conditions, yeasts gradually de­ clined in numbers after reaching a maximum between 10 and 20 88 days# This gradual decrease in yeast count was not observed in this investigation* Under laboratory conditions, however, Costilow and Fabian (9) reported a fairly high level of yeast population throughout the fermentation period* Thus, the changes in yeast numbers observed in this study under semi­ commercial conditions might very well represent the differ­ ences in conditions between the fermentations in crocks and commercial tanks* As to coliform population, an increase in numbers was generally observed on the first day of fermentation (Table 23)* The numbers then declined rapidly and very low popula­ tions were present on the l|.th day* Since large differences in coliform counts existed between individual barrels of similar treatments as previously reported, no significant effect of the addition of cystine and tryptophane alone or in combination with sugar is indicated. Figure 5 . Influence of addition of cystine and tryptophane brines of seraicommercial cucumber fermentations lactic acid and acid-forming bacteria. to on &9 1W M3d IN H O O 3 0 9 0 3 OIOV 0 I1 0 V 3 ±N30U3d 90 &— •— & Corn steep liquor at salting a— — + Corn steep liquor 8 sugar at 7 day intervals (avg. of 3) LOG. OF COUNT PER ML. + Corn steep liquor on 7th day B 7.0 Control (avg. of 3) — o Cystine 8 tryptophane treatments (avg. of 3) 6.0 5.0 4 .0 3.0 2.0 0 Figure 6. 15 20 25 30 35 T IM E IN DAYS 40 45 50 Influence of addition of cystine and tryptophane, and addition of corn steep liquor to brines of semicomraercial cucumber fermentations on yeasts. 91 2.5 - A 2.0 ^ 9 .... .MM. a Q SUGAR ———a at salting Cystine 8 tryptophane on 7 th day o _____0 Cystine, tryptophane f 1.5 9 Cystine 8 tryptophane \ 8 sugar at salting o 1.0 PERCENT INVERT 0.5 0 2.5 B o " o Corn steep liquor at salting A — — —A Corn steep liquor on 7th day 2.0 + — •— t- Corn steep liquor 8 sugar at 7 day intervals 1.5 1.0 0 .5 0 5 Figure 7. 10 15 20 T IM E 25 30 IN DAYS 35 40 45 50 Influence of addition of cystine and trypto­ phane, and addition of corn steep liquor to brines of semicommercial cucumber fermenta­ tions on per cent invert sugar. 92 XII — Influence of Added Corn Steep Liquor on Cucumber Fermentation Effect of corn steep liquor on lactic acid formation# The effect of addition of corn steep liquor to cucumber salt stock on lactic acid production was investigated along with the studies of sugar, cystine and tryptophane treatments* In 1953* barrels 10 and 11 showed the effect of the corn steep liquor when added at the time of salting and on the 7th day of fermentation respectively. Addition of corn steep liquor in combination with 1.0 per cent sucrose at 7-day intervals was also studied by preparing barrel 12. Results in Table 1 show that during the first two weeks of fermentation, no significant difference in brine acidity was observed between the corn steep liquor treated fermenta­ tions and the control. From the 15th day to the end of the examination period, however, acidity was somewhat higher than that of the control. found to be 0*15 Pe** cent. The maximum increase in acidity was It is true that this difference might occur between similar fermentations within one treat­ ment. Yet, in all corn steep liquor treatments examined, this slight increase of acidity occurred consistently and lasted for a considerable period of time. of corn steep liquor was reinvestigated. In 1951}., the effect Barreljs 5 and 6 were prepared in the same way as barrel 12 in 1953* of this experiment are recorded in Table 2. Results Here again, the 93 amount of lactic acid formed during the first two weeks of fermentation in general did not appreciably differ from that of the control* On the other hand, after 18 days of fermen­ tation acidity was higher than in the control by 0*12 per cent* Thereafter, the total titratable acidity remained al­ ways higher by at least 0*1 per cent* It should be mentioned here that the changes in pH levels (Tables 1 and 3), and salt concentrations (Tables 2 and 1|_) in both 1953 and 1951+ experiments were similar in each case to that of the control* steep liquor was observed* No buffering action of corn In addition, the initial pH in brines where corn steep liquor was used, showed no signifi­ cant difference that might be attributed to the acidity of the corn steep liquor itself. The average acidity values of all corn steep liquor treatments prepared in 1953 and 1951+ are presented in Fig. 8* The laboratory experiments were then carried out to check the above findings. Table 6 shows that addition of corn steep liquor, alone or in combination with sugar, resulted in a significant rise in acidity on the 11th day and there­ after* For example, on the 25th day acidity increased over the control by 0*12 per cent (for flasks 15, 16); and by 0.19 per cent (for flasks 17, 18). These values are considered significant since they exceeded by far the variations found among replicate determinations. Fig. i+, part C, represents 9lj- the average acidity values obtained when corn steep liquor was used in the laboratory experiment# When the flask experiment was repeated (Table 7), the increase in brine acidity was even greater and more pronounced throughout the whole examination period# One can only conclude, then, that the increase in brine acidity observed under semicommercial conditions is signifi­ cant# Results obtained with the reducing sugars content in fermenting brines under laboratory conditions indicate that when corn steep liquor was added, L. plantarum was able to utilize more of the naturally occurring sugars in the cucum­ bers as compared to the control (Table 8)# Thus the amount of invert sugar utilized in flasks 11 and 12 was 1*279 per cent as compared to 0#902 per cent (avg# of flasks 1, 2) in the control. The combined treatment of corn steep liquor and 1.0 per cent sucrose showed almost the same effect. There­ fore, utilization of reducing sugars remained the same as when corn steep liquor only was used. Apparently, then, the added sugar was not utilized by L. plantarum. In 19^3 and under semicommercial conditions, the reducing sugars of brines of all three fermentations examined (barrels 10, 11, 12), rapidly increased after brining and reached a maximum similar to that of the control approximately after 6 days (Fig. 7, part A)# decreased. Thereafter, the reducing sugars rapidly For example, in barrel 10 no invert sugar was 95 detected on the 9th day as compared to 0*7 per cent in the control (Table 9)* A similar correlation was observed in barrel 11 on the 11th day* When corn steep liquor was ap­ plied in combination with 1*0 per cent sucrose, the reducing sugars decreased rapidly even after adding the second portion of sugar* However, the acidity remained the same in all corn steep liquor treatments* Therefore, the added sugar was probably utilized mainly by yeasts* The increase in acidity over that of the control when corn steep liquor was added is mostly due to greater utilization of the carbohydrates in the cucumbers by the acid-forming bacteria* Effect of corn steep liquor on total and acid-forming bacteria* The populations of total and acid-forming bacteria observed during the fermentation of cucumber salt stock which received the corn steep liquor treatments in 1953 and 195^4- are given in Table 26* A variation of more than 1*5 in the log. of the bacterial numbers was considered significant here as previously* The average populations of acid-forming bacteria in five corn steep liquor barrels and three control barrels prepared in 1953 and 195ii is illustrated in Fig. 8. It is evident from this figure that there was no significant difference during the first two weeks of fermentation between the corn steep liquor treatments and the control* In general, the popula­ tions rapidly increased after brining and reached a maximum 96 1 o a N— •* \ no CO o 03 I —I d o H Pd X •H -P pq EH O C/2 <4 -p O EH u Eh d d CD pq I O—1 0 H EH pq a§ d L A ON i— 1 o s Pq Ph i pq i—I 0 \ NO O L A pq Ph (H O P3 X EH ^ o -r13 3 ° eh i-q 00 o pq Eh pq cq H pq EH O tz; O l-q EH u u pq £5 ft; pq m ss d i —! a \ cO O ONI i— ) o o X t23 O O « • O 00 O KN i—1 < —1 | 1 o o • 1C* 1 rH ON KN CM i— 1 1 CO O 4* CM CM 00 A CM KN d• 1 • 1 • 1 • • • • • • • A-0CJ KN rH H O O CM o CO i—1 i—4 rH A O o O O O O LA A A • 1 • 1 • 1 • • • • • • • A I—1 4* A -00 KN CM rH o o i—1 ■ —l CM CM i—1 o o CM O O O O O • 1 • • • • I • 1 • 1 o LA NO LA NO rH LA w ON NO 4" ON CO OJ rH 1 —1 1 ON O O O O la la LA O O • 1 m 1 • 1 • • • • • • • LA 00 o COONOOKNHOO 1 —1 rH i—1 CM i— 1 o o CM O O O O o • • • • | • I * 1 • 1 o A NO 00 cO A CM V-/ ON 00 CM A i— 1 CM rH i— 1 1 O lA O O O M A o O • 1 • t • 1 • • » • • • • ON CO NO NO 00 O KNO O 4<— 1 i— I CM 1— t i—1 o (—1 O NO o o o o o o o o o o O O • • • • • • • * • • • • OOL0NLALALAOLAOOLALA AoOrHNOACOANOCM rH CM a -o L A O IO O O O O O O O O O OHIAO^EOEOIAOKN EH O O 00 L f\ UN LfN LT\ LA CM rH rH rH eh pq *4 cq pq O O • 1 • 1 rH I AKN IH CM rH KN rH i—I O A-A-LAO LA LA LA O O 4" i—l i—1 d - < —1 • • • # • • • • • • 1 1 nOCM nO nO O H O O O O r H rH CM CM i—1 o • o O • f rH CM O • i— I i—I CM rH LON • O • O rH 4 " LON O CM O O LON i—I i—I i—I i—I * • • • • • « O l T n cm o o o o rH rH 03 Ph Pq EH O P I CO o Pq d o iH -p S O’H M Ph d 0 Ph fH 0 i-qpq Pq i—i pq Pq CO CO £ h KN L A EH o £h H K Eh \ NO O i— 1 rH X Eh LON O LfN O CM NO 4 - KN LfN rH CM rH O OO CN4-OA-OOOOA-GO W CO O-CN^D CM H H LON LTN i —Ii—Ii —Io o o o C O K N O O O O O O O O O C M LfN NO O 4 " 4 * CM O O ^ O O O lT M I M N O E E i—I (—I i—| O LO NCM rH i—I CM rH H H CM 4- oo • • * • • • • • • • • • •I •I i •I •t I • • • • • • O O o o o H O • I O O I —II —II —I ON 1----1 rH KN OOO O «4 O nq ■) Ph Ph O O Ph Eh O O r H O O O O O O O O O L A O •H -P d <4 —I I O I— I 02 O r 1 pq Ph x Ph Eh pq P fctO hd 0 0 H 0 -p d •H 4 oJ ^ lon no determination made total bacteria counts cO i—1 o o LA O • 1 • • o rH 00 CM O rH o /T \ 0 O C O C A O r H C M r O N 4 - L A N D C > - C O r H L A C N K N [ > - r H O H H H H H H H H H C M C M C M AKN 4- I EH CD ■ ■ rH pq pq Fd CO Pd o •H -P Eh 03 CO Is; o te; h W EH O EH pq 0 0 oo oo 00 d o o > > > rH d OJ d • 1 1 • o o pq Ph o> Ph I • o O i—J • • 1 O rH O O LA • o * | | I o • 1 1 O > G ' ' d ‘ rH OJ ON •I • * • * | • | « | | KN CO [>-00 00 00 > LA d UN • ,_) l_r\ o o o o h h o j o • I I I • 1 LAcO KN rH > 0 OJ 1 • • • • • • • O O rH OJ rH OJ OJ • 1 1 OJ KN *| • > > I . . . • . ON OJ KN LA O O ON 00 • | • • • • • • • > cO cO CD cO l a l a CD d o CO|>[>KNd' • i— I OJ O N o n d~ i—I cD cD > > c 0 CO CD CD U N lA • | • • • • • • • ON rH *=3 S! H O cd o Pd H Eh ^ o Pd LTN O ON > K N cD KN CO CO CO CO > > > CD CD CD L A d •I • • • * | *1 *11 1 J OJ rH 1 rH d • • OJ rH • CO OJ K N { > 00 O N CO 00 KN • • • • • • • • • | KNd- CO > 0 0 CO [ > > E>[>[>[> CO > • I f i—I KN KNcD d" • ••••*• d K N KN LfN LfN iH 00 rH 00 • • • • • • » i—I i—I i—I OJ i—( C\J i—f KN d O 00 OJ O i—Ii—IrH <—IO > [>[>c0 > > C O * | LfN LfN LfN, LfN * o 523 rH rH CQ J Pd O rH pq *H -P C -P CD LfN rH • • • COd'KN • • • | O O O r H O O O O d CO LfN CO CO CO LfN rH O o o o O OOHOJOJOJH LfN d- rH rH rH rH K N O CD CO CO CO CD LfN LfN LfN d KN KN • | | 00 OJ rH OJ > 0 d • • • • • • • i —I i—I O * | * | • | • | | O LTN • • • • • • • tf pd a> i —J rH O O N O N O O CO I > K N O O CO O N • • • • • • • • • • • • I K N LfN CD [ > > [ > [ > [ > [ > > CO CO O • I • II KN • • • • • • • Ph made Fd hd O rH o o , | pq fed !=> Pd o ^ o CD hd eh r-H LA -p Eh pq K\ • • * • I o • 1 • 1 • 1 1 • > > 00 > • • • • • KN KNUN OJ O O CO • • • • • • • CD C O v O LfN LfN LfN d P^ eh Pd o !25 Pd Pd O Pd Pd gp Ph h Ph Hi i—1 O d- d- 00 00 • • • • • • • • I OJ K N O KN d t> > [> C0 CO w H rN) d cd ctl t J“ d KN I ^ OJ K N d Q £> *| CDKN O 00 LfN LfN cO O KN O determination O cD 00 OJ KNI-H • D = difference in log. of acid-forming 'bac­ terial counts between the control and the specific treatment 97 OJ • I • I • I I • • • • • • • COCDCO LfN LfN LfN d- KN KN KN KN v— y K N CD > 0 0 0 > 0 rHOJ K N d * K N CO> 0 0 rH LfN C ^ K N I > r H i— | i— Ii— I rH r—Ii— I r— I rH i— IOJ OJ OJ K N KN d~ O d 98 of about 100 x l o Vml in from 3 to 5 days. Table 26 also shows that the time of adding corn steep liquor during fer­ mentation was not an influencing factor. Xn all corn steep liquor treatments, however, it was noted that the numbers of acid—formers were higher than those of the control on the 29th day and thereafter. Table 27 shows that except for bar­ rels 10 and 11 after Ij.1 days, the log. of counts of these organisms was higher than that of the control by at least 1*7 (about 50 times greater). Even in barrels 10 and 11, the numbers of acid-forming bacteria were about 32 times greater than those of the control after 1^.1 days. It is true that in these two examinations the observed difference did not exceed the limit of normal variation that might be found between similar treatments. However, some significance should be given to it since the largest difference in the log. of acid-forming bacteria between duplicate determinations pre­ pared in one season was always smaller than this, i.e., 1.2 (Tables*13 and 27). The over-all total bacterial changes during fermentation resembled those of the acid formers. In general the popula­ tion increased after brining and reached a peak in from 3 to 6 days. The numbers then declined rapidly to the end of the examination period. Between 29 and lj.1 days, however, the total bacterial numbers maintained a higher level than that of the control. A corresponding increase in acid-forming bacteria has been shown before* 99 Effect or corn steep liquor on yeasts and conforms* Results of the studies on population changes of yeasts during fermentation of 3 barrels in 1953 9-n.d 2 barrels in 1951j-* are presented in Table 28 and Pig. 6, part A. It is evident from these results that in all corn steep liquor treatments, the yeast population increased to a maximum during the second week of fermentation# A rapid decrease in numbers then oc­ curred until the level was generally lower than that of the control on the 18th day. During the rest of the fermentation period that followed, the yeast numbers remained lower than those of the control. In only two instances during this period, however, namely in barrels 11 and 12 after 29 days, was there no significant difference between the corn steep liquor treatments and the control (Table 29)* As a matter of fact, yeasts were even slightly lower in numbers than in the control. This is of doubtful significance, however, and is most likely due to experimental error. There is some evidence that combining sugar with corn steep liquor might have some effect on the yeast population. For example, yeast numbers in barrel 12 increased from 2.5 x 10-Vtfil on the 7th day to 130 x 10^/ml on the 9th day. (Table 28). The time of adding the corn steep liquor did not greatly in­ fluence yeast population. It was noted only that yeast numbers were higher than in the control between the first 3 to 5 days of fermentation when corn steep liquor was added at the time of salting. No such difference, however, was observed when 100 corn steep liquor was added, in small portions throughout the fermentation* This was probably due to the fact that considerably smaller amounts of corn steep liquor and sugar were added at the time of salting. The coliform populations varied considerably from one barrel to another. There is no indication that addition of corn steep liquor, alone or in combination with sugar, had any effect on the numbers of coliforms present during fermentation. In general coliforms disappeared after 6 days. Apparently unfavorable conditions in the brine inhibited them. Similar to the treatments with sugar, cystine and tryptophane, the coliforms played no significant part when corn steep liquor was applied. 101 0 o H Eh CQ a o •H -P 0 -P a 0 B 0 Pq § OJ o w O pq H o H EH pq H O pQ CQ Ph PQ PQ is; PQ H Eh Cd CQ pq S25£3 o o o S23 I * * |* O h d* * KN 1 O C A OJ * KN •O’ d- * 1* * 1 1* * 1* 1* * * * * * * 1* * * * * * | OJ O O C A L A L A OJ K N C A KN d" NO 1 • rH OJ rH rH O O | LA rH >H KN o o o o• A O t N C O I i • • • 1 —1 rH rH O O o • • o o 1 • o o NO O 0 0 * 1 • 1 1 1 —1 0 0 NO ( —1 C A CA | 1 i —! PQ § & PQ O t O NO d" LA CA i— 1 cq I £5 X o Ph o o ••••• Pi 0 CQ O -P O Pi NO a a a> o Ph as PQ OJ i— 1 <+h K N d" O K N * KN A - * d- H- [>- o U rH o \ 0 pH KN O o * * * * * ***** * * * * * * * * * * * * * 1* t* * * * I* * * * * -P *H CQ * i— i cti O 0 O 1>S OJ d* O O LfN vD 0Q (M H O (^O Lf\O O O O # • • • * # • • • • • • O O O O r - iO v Q O J O O O C O OJ rH K N O KN o I —I X I CO I —II —i rH CQ a o •H 4 3 0 -P a 0 LA KN 00 o o o o O O rH t —I i—I O O OJ rH O O KN CXD CO­ i —Ii—Io o o M II • ■ • • • O pH I —1 a o o Ss; rH i— 1 0 Pq o o o * * I* I* * * * * * * * * * LfN LfN K N * OJ OJ d" * * * * * * * * >H KN 0 i—| KN OJ i—I i—I i—! NO CO rH O O O • • • • • • • • • • • • I O o d" o • OJ rH o O O O rH rH O rH rH O OJ LA I ** ** * * * * * * I* * o I I 1 —1 rH k n la o kn rH rH o • t « • • OJ rH rH H KN O O r H O O O O O O • • • • • • • • Q- OJ O - O OJ LA Co rH H K N 4 CD LAKN rH o • o KN o 1i • LA OJ i o • d- OJ l 00 « OJ LfNi—I CO I I • • • rH OJ rH o no oo • • • rH rH rH o o o OJ M hO Ph Pi CQ kn EH 3 Pn > rC} aS 0 as 0 H a -p a 'H H CO 0 g *0 CQ 0 -H a pi OJ 1- 1 KN LA CA i— 1 I rH a d* o o o a Ph H O O Ph o rH i —I O O Eh CQ -P K N d" L A CO C N 00 C A O rH OJ K N d" L A NO A - 00 rH L A C O KN ( A H i— trH i— 1 rH i— I i— I i— Ii— I i— 1OJ OJ OJ K N K N d “ 102 VD I LfN * t O O KN • • • VO LA VD KN OVQ rH VO • 1 • 1 1 • 1 * 1 ! 1 1 • • rH O O rH OJ O CM fd -Q- OJ -Q- O - 0 OJ OJ OJ rH 0 d r(Q I EH 0 h 0 t Q M O S < EH O PQ id o £ a 'd VO O KN VO O-D-CNJ LA L A LA L A -d d* -d • • I •••• rH CD u o d o -p 0 —10 0 rd A 00 d Fh ^ 0 i—I I i —I Ph CD pq PQ A O OJ ^ •0 0 • CM 0 ED 0 O O rH H -P CNJ [>~OJ L A K N O ( A d K N O L A O V0 d H O O O O rH O CNJ O OJ OJ OJ o O O O rH OJ D - C A K N O O C A d K N O r H O L A CA CAOOLAO O -O' LA LA d NN K N O J OJ CNJ • • • • • • • • • • • • I * | • LA O • KN • • | O C O H O J H • • • • • OJ O OJ OJ OJ 0 O d 0 II o • • • • • • • OJ OJ OJ OJ K N OJ d • • • • • | KN d > L A L A d • | • • • • ( KN O rH rH OJ OJ O I A fd 0 0 0 0 Fh 0A A 0 d 0 •H A 0 A d 0 a Fh 0 Ph Eh CQ O < Eh PQ Ph fd PQ Ph m O id Ph o o id PQ Pd PQ PQ H 1 —1 1 rH fd (—1 rH rH • O O LA KN • • O O O K N rH KN O • • • • O • OJ OJ 4- K N O J OJ • • • • | *| O rH O O O rH rH o O • CNJ OJ O • • • • O • K N NN • I K N K N CNI OJ OJ OJ K N K N CNJ K N 4* L A O r— 1 1 rH fd KN LA CA rH PQ £s OJ OJ d rH VD 00 O - d • • • • • • • • | O O rH CNJ CNJ CNJ CNJ rH rH • ] d CO • t rH O C A O CO I • • • • ( OJ rH CNJ O O KN rH O d K N KN K N KN * 1 1 • • O • t>CM O d OJ 0 - 0 • | • I *1 *11 * * * l CNJ O rH OJ CM rH OJ KNOJ * • CNJ OJ O rH • | • • OJ CNJ OJ rH K N • • • OJ OJ CM CQ Is; Eh O i —I H (s; VO K N OJ d VD 00 C N 4" • • » • • • • • ! K N K N d d d d d d KN KN KN *1 *1 *1 *11 LA d d KN rH K N O * * * l kn knkn O OJ O • • • OJ c m OJ PQ § Eh £d 5 o A •H o •H a 0 ED 0 • tQ O Phi—I 0 1—I0 O Fh 0 0 0A d d rd d o A A o o •H a d d 0 0 d 0d -H 0 £ A a o A t Fh d 0 0 A Fh •° s 0 0A d A A d d 0 -H o 0 d 0 »H EH n o -p o d 1 O d o 0 -P o O CQ O 0 t> -H -P Q o cd • 0 § pd d> PQ o Ph d> Ph O -H CO pj O 0 Fh tcJ ED 0 d 0 Fh 0 P c H rH O K N O J rH vO 00 • j • • • • • • ! lA K N KN KN KN OJ OJ I *111 —IFhtH * 1 LA 0 0KN L A KN O • • • * * O 00 OJ KN KN • • • LA L A L A L A LA L A KN d • • 4- d 0 Q 0 dA oa 11 n 0 0 1 fd u h> h> ,0 0 0 KN 1 r-( CM K N d q L A VD O - 00 C A O H OJ K N ^ L A VD O - C O rH L A ( A KN O - rH t— I i— Ii— I1 — I1 — i <— I <— I 1 — 11 — I CM OJ CM KN KN -O' Figure g. Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on lactic acid and acid-forming bacteria. 103 "IW 83d 1 N O O O JO '901 a 19V 0I19VT ±N30d3d lOlj. IV - Vitamins and Amino Acids in Semi commercial Fermentations It is well known that a prompt and appreciable acid pro­ duction in fermenting brines is desirable. This decreases the possibility of undesirable types of microorganisms util­ izing a large quantity of the available sugar in the brine which would result in a low final acidity. Since L, plantarum Is considered by many workers as one of the major groups of organisms responsible for acid formation, the presence of plentiful amounts of the essential vitamins and amino acids is important. Therefore, it was interesting in this study to Investigate the changes that occur in these vitamins and amino acids and which might be a result of the addition of sugar, cystine and tryptophane, or corn steep liquor. The complete results of the different fermentations prepared in 1953 &re shown in Tables 31 through 33* With the exception of tryptophane, the brine contents of the vitamins: niacin and pantothenic acid; and the amino acids: biotin, leucine, isoleucine, cystine, glutamic acid, and valine, in general gradually increased after brining. This was due to leaching of the different nutrients from the cucumbers into the brine. After reaching a maximum, rather high concentration levels were maintained until the end of the examination period* Thus all three vitamins and five amino acids appear not to ■{50 limiting factors for the growth of acid—forming bacteria. Significant amounts are probably present to satisfy the 105 growth, requirements of L* plantarum. Xt was also observed that the general trend of changes in the levels of these vitamins and amino acids was the same for all the different treatments when applied to the different cucumber barrels* Addition of different amounts of sugar at different times throughout the fermentation, showed no significant difference that can be attributed to the specific treatment* High con­ centrations were generally observed when corn steep liquor was applied, with no effect as a result of combining it with sugar* Brines in barrels 7, 8 and 9 were richer in cystine content as compared to the control (Table 31)* Undoubtedly, this was a result of adding 0 *0 0 2 5 per cent of cystine to each of these barrels* From the standpoint of tryptophane, the change in con­ centrations, especially at the end of the examination period, was quite different* In general, tryptophane increased gradu­ ally after salting as a result of withdrawing nutrients from the cucumber by osmosis* Except for the corn steep liquor treatments, the tryptophane concentrations after reaching a maximum, markedly decreased about the 11th day and continued to decrease thereafter. For example, a rapid decline in the tryptophane concentrations occurred between the 11th and 2 5 th day in the control barrel as well as in the fermentations where sugar was applied at the time of salting (Fig. 9)* When sugar was added on the 7th day, or at 7-day intervals, a drop in 106 tryptophane levels occurred even earlier, namely on the 9th. day* Comparison between Tables 28 and 30 show that trypto­ phane decreased when yeast activity was greatest* Thus, in barrel 3 the tryptophane concentration decreased from 18 jag/ml on the 6th day to 11*5 jig/ml on the 9th day* During the same period, yeast population increased from less than 100 organisms per ml to 6900 x 10^/ml* Similarly, in barrel 5* tryptophane decreased from 19*7 jig/ml to 7•!+ jig/ml, while yeast numbers increased from less than 100 cells per ml to 38000 x 10^/ml* phane by yeasts* This indicates a rapid utilization of trypto­ A similar findingfwas reported by Costilow and Fabian (9)* in cucumber salt stock under laboratory and commercial conditions* A good illustration of the tryptophane decline occurred in barrel 8 (Fig. 10)* Following the addition of tryptophane ini combination with cystine on the 7th day, the tryptophane concentrations increased from 1 5 *6 fig/ml on the 6th day to 44*6 jig/ml on the 9th day. However, a rapid drop occurred and the tryptophane levels decreased to 15.1 Jig/ml on the 25 th day. In general, the tryptophane concentrations in barrels 7, 8 and 9, were somewhat higher at the end of the examination period than in the control. This indicates that the added tryptophane was not completely utilized by the brine microorganisms. Since no greater amounts of acid for­ mation occurred as a result of these treatments, it can be concluded that sufficient tryptophane was present in the cucum­ bers for the growth of the acid-forming population* 107 When corn steep liquor was added, tryptophane concentra­ tions generally increased after brining, and reached a maxi­ mum similar to that of the control (Fig. 11). With the ex­ ception of the combined treatment of corn steep liquor and sugar, the tryptophane levels in all other corn steep liquor treatments did not show a rapid decline on the 11th day as the control* A slight decrease was observed only at the end of the examination period* Fig. 11 is a good illustration of the difference in the tryptophane levels during fermen­ tation of the corn steep liquor treatments as compared to the control. Evidently, considerably smaller amounts of tryptophane were utilized by the brine microorganisms. This correlates with the fact that lower yeast populations were found after 18 days when corn steep liquor was applied. On the other hand, the acid-forming bacteria showed a marked increase in numbers over the control. It has been previously reported that more and faster utilization of reducing sugar occurred when corn steep liquor was applied under laboratory and semi commercial conditions. Apparently, corn steep liquor enhanced the utilization of the available sugar in the brine resulting in the production of greater amounts of acid. When corn steep liquor was applied in combination with sugar, the decline in the tryptophane levels was greater at the end of the examination period than in the case where 108 ▲ Sugar treatments on 7th day 25.0 A Sugar treatment at 7 day intervals 20.0 ML. 5.0 JllG. TRYPTOPHANE 10.0 PER 15.0 • Control 3 0 .0 o Sugar treatments at salting 25.0 20.0 15.0 10.0 5.0 5 20 25 30 T IM E IN DAYS 0 Figure 9 . 35 40 45 Influence of addition of sugar to brines of semi commercial cucumber fermentations on tryptophane. 109 Cystine 8 tryptophane at salting Cystine 8 tryptophane on 7th day Cystine, tryptophane 8 sugar at salting ML. TRYPTOPHANE 4 0 .0 3 0 .0 JUG. 4 5 .0 PER 5 0 .0 20.0 3 5 .0 25.0 -+ 10.0 5.0 ^ O 5 10 Figure 10* 15 20 25 30 35 T IM E IN DAYS 40 45 Influence of addition of cystine and tryptophane to brines of semi­ commercial cucumber fermentations on tryptophane. 50 110 — ML. 30.0 - 2 5 .0 - C ontrol -° Corn steep l i q u o r at s a l t i n g ■A Corn steep l iq u o r on 7 t h day ■+ Corn steep l iq u o r 7 day i n t e r v a l s Q sugar at 20.0 s r i." ' (5.0 _ i ____ JLLG. TRYPTOPHANE 35.0 PER A' + ’• 10.0 ft' 5.0 \h 10 15 20 TIME Figure 1 1 . 25 30 35 40 45 IN DAYS Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on tryptophane. 50 Ill 00 KM AvD 0 0 0 4 0 n4 a o o k n CNJ 0 -0 • I O 00 KN 4 KN O - CNJ 11 i— I r——i OJ i— 1 rH CD H LAcD 4 k n i—i k n i—i • rH O O knh LTN A ON K N O ON KN 4 • • * O ' O - 00 4 KN r—i (X) • • • • • • • • O H O N H A 4 0 0 C O L A rH CNJ CNJ i— | i— | i—1 iH K N L fN L fN O O K N O O O O L A L A cD O N A H C D 4 i—I i—I i—i f —I OJ C\J rH i—I H J -lA O O O O rH O O KN LfNO O CNJ CNJ O • * • * • • • • • CNJ KN 4 00 o KN 14 Ld pq 0 u a d 0 P 0} P Pi CD hD • O LA L A O LA KN O h- O O ' * CNJ i—I i—I i—I * a O oo 4 CNJ O 4 A ^ 0 4 * 4 * 4 L A 00 O K N O OJ 00 4 * 4 - ON • * • • • KNKNONCNI 4 LfN O O LTN O O O KNcD 4 O K M N 00 a a a a * O O O LAO KN O - K N A A KN KN KN K N L A cD LA rH CO O 00 O CNJ O KN O ' CNJ 4 L A O LA LA CO LA 00 KN CO t O A O N K N C A O -44LA LA L A O A rH KN CO H C A K N i—I ON ,—| KN LA LA LA k n h h L A A K Nc OO- ONL AHC O KN KN KN KN CNJ rH rH a H 00 H A O • • • • • • • * a NNcO CD CO o 00 LfN CNJ O '' a O' 0 - 0 - 0 0 KNH- o kn rH U"N L A 4 h h h ^ • 4 L A O LA L A O A LA O ' 4 CO * * • • * 00 CD 4 - CA OLA KN 4 LA LA rH i— I i— t ON 00 CNJ KN K N r H CO L A CNJ ♦ A i —I4 LA00 ^ • * • • 00 KNcO CD CNI CNJ CNJ i —Ii —J Ht * a a a a 00 0 - 0 4 rH O rH A Ph 0 EH NN • CD C O O O O O r lO O O KN O O n cD CD i—! i—I 4 i—i • , • * • • • • • • O- o £ O O O LfNrH CNJ O O- LA Lf\ 0 0 rH C NJ LTNKN O 0 0 LIN cQ pq o •rH 4 LA L A O LA O'CD 4 LA CD O N O N L A rH 4 LTN00 4HHININ4NN CTn 0 Ph CNJ ^f- A O LA 4 O CNJ • O ' KN LfN CO L A O O O ' 00 CNJ O 4 * • * • * O n J - o i—I 00 CO LA 4 [>_ LfN LTNKN KN 4 • • • • • • • • • O LA LA A O ' KN ON CA * • • • 4 ON A - O N ON ON CNJ CNJ O rH CNJ 4 * 4 * k n LTNOKNLTN o~ 4 O O O - C A i—| rH O rH i—I 0 - 0 LAO O LAO O 4 LfN i—I O - t$N LTN LTN 4 Ph CD * LTN O O N O C M N l A H C N J rH H rH KN LTN LA LA LA LA CO rH O ON P 00 O O ' O LfN 4• 4 • rH « • • 00 KN 00 O ' CNJ CM CM 00 ON O • 4 ON O A • KN A 0 ON CNJ O O 4 CO A O 4 O H CNJ KN LTN LT\ KN LTN KN i—I a ON rH CD KN CM CM CM A ON ON i—| O d }> L A P cD CM CM A o o a O O 4 O H O J O lD ^ ^ rH rH CNJ OJ CD O H O LAKNcD O O O 4 O 4 0 0 O • KN O O O O O O O • • • • • • • • .—1 CNJ 0 0 i—I cD o i—I rH i—I i—I lt\ cQ i—I a a a LA L A O O KN KN O - KN • • • • K N O nO n ON rH O-ON A i—I ON O a kn a a A K N O LTN L fN O O O L fN O LfN 0 0 LA CD LfN CO CO 4 CO KNO O a a O cD l a A LA ON cO * * * • * * • • • CNJ A CD LfN 4 0 0 LfN LfN i—I i—I i—I CNJ CNJ i—I A a 4 A i—I I —II —I rCj f4 o p 0 Pi •H P 0 tJ 8 KNt A Ph eh O 4 CD O ' i—I LfN KN i—I rH CNJ K N 4 o Ph >> ,d H LA O rH O rd rd p P A A Ph KN I A O H KN a LA O a 0 KN I A O O KN ON KN i—I i —I P's d CA LA rH A 9 a rd ON L A P A 4 bD A a & d nd hO O 0 0 0 0 0 0 CQ Ph f>* A d a 00 4 A CO N N A O A fcO d 0 0 •H a a p •H <+H *H EH Ph PQ a * 0 PH I —\ I —I LA • o 4 rH LA • a oo rH CO d •H =C rH O L A O A O ' ON KN A A H O ^ O rH A KN « • • • 00 KN rH ON A A rH O ^ O O O O O O O O CO CD COCO O - C A H rH O • • * • • • • • • O CNJ i—I LfN i —I LfN LfN NO CD i—I i—1 rH CNJ i—) 0 CD LA O - O 'C D O [A 4 4 d •H 11 LA L A O O 00 A 4 KN • • • • 4 0 0 0 0 0 0 i—1 i —I 112 O O lD C V J O UN LfNKN CD O CNJ * • • • • CNJ CNKNcO O t>-CD CNJ i— J CNJ OJ KN 4 4 o a o o o o # i —! a pq O a EH oo § Pq pq 4 o EH P> o I P! pq EH f-q pq 4 <4 pq # o pq pq Pi 4 o o o H 0 Fh Fh 0 03 0 o •H -P 0 \ 0 pq bD =L 4 o -P D- # o 0 Pi 0 s 0 Ph LfN # ON pi pq CQ Eh Pi pq pq pq 4 o * a a a a a a a a KN a a a a i —Ji —Ii —I a O rH CO LfN O CX) OK CO OJ OJ OJ OJ Ph rH CM L fN O 00 rH CD O Ph O O Szj CM # a a a a LfN OK rH O K N O - 00 CM CM CM OJ KN o O O O O L fN O O i—I CQ H pq # O O O 00 UN U N rH CO * 0 KN 4 CM O K U N rH CM 4 O 1 a a a O O O rH L fN O UN rH a a a a KN 00 OK O KN 4 k n 4 U N O O LfN OJ O - UN OK a a * a O O o o O U N O LfN a a a a Ok o j CD KN KN II— II— I KN CQ U N O J UN 4 4 UN rH rH i— 1 4 •H O 0 0 0 •H o 0 O •H 0 0 •H 0 0 O i—1-P 0 o 0 0 CQ 4 i—1•H bD 0 0 4 4 4 O O O o UN • KN 00 o o o L f N O LfN • • • CD L O O K U N 4 KN 4 4 4 O i— 1 cD O OJ U N U N O a a a a 4 0 0 0 0 4 * • • • [>-00 K N O J 0 - 4 4- UN rH 4 ( —1 4 O a a a a O UNO O CM CD LfN U N a a a i— J i—14 0 0 0 bD bD bD = 1 CD O O OJ UN 4 0 - 0 0 CM CM 00 LfN 4 4 i— 1 4 i— 1 * * a KN CD OJ OK KN 4 KN KN I — O CD O O LfN UN CD CD i—1 i—1i—1 O O O - LfN LfN OJ CD 4 * LfN QK CM CM CM OJ •H C Q C Q 0 Fh P“a 0 Pi at 0 4 rH KN 1 I OJ O K LfN rH O OJ 4 a 4 0 - 0 O UN OJ CM O K cD a a K N rH rH OK 4 KN KN CM * 4 4 O K 4 CD O4 kn 4 4 i— 1 i— 1 rH * * a o o o o * O LfNO CM LfN 00 • • • • O K CD CD CM 4 kn 4 4 i— t i— J rH O o ] * a O O * * * a a OK CJK OK NN UN 4 KN 4 rH i—I i—f * a * LfN KN CO KN UN 4 rH rH rH 4i a CM UN O - O - * a a a 1 —II —Ji—I U N O OKcD CM LfN CD KN a Ph Ph 4 PI a * 0 4 - 0 0 UN 4 - O - O a a i —I<—Ii —J a 0 4 - 0 0 O CO K N O # * O O O KN 00 O CD LfN a a a * * CD CM O LfN 0 - 4 * CD LfN rH CM OJ CM CQ * • O KNOO o U N K N O - cD CD L fN O O CD L fN O O # • 4 CM LfN LfN O k O-CM OJ rH OJ KNKN CD LfN OJ O LfN 4 * LfN CM CM CM OJ KN LfN • * a a O rH OK OK O H O LfN p a a • a i —Ii —Ii —J i —Ji—Ii—I • OK KN CD CM OJ 4 L f N O OJ CM CM OJ £q o • O -C O U N O LfNO -KNcD a a o O OJ O O CM 00 CD 4 - 0 -0 O O rH L fN O O a o * 0 - 0 0 KN LfN rH K N O O - U N LfN rH r— I i— I i— I o 4 oo i>- a • O O O OJ C^OJ O - * • LfN LfN CD KN UN KN LfN LfN a « 4- 0 0O - C 00 0 U N O OKOJ O - 4 - LfN 00 rH CM OJ CM # • • O• too tOcD • • « • OJ CM OJ OJ n pq H Ph o 5, (1914-6). 30. Jones, I. D. Salting of cucumbers. 22, 858-861, (19 I4.O). 31* ___________ , Etchells, J. L., Veerhoff, 0., and Veldhuis, M. L. Observations oh bloater formation in cucumber fermentations. Fruit Prod. J., 20, 202-206; 219-220, (19I|-1). 32. ^__________ , and Etchells, J. L* Physical and chemical changes in cucumber fermentation. Food Indust., 15. Ind. Eng. Chem., 62- 61}., ( 19^3 ). 33. , Veldhuis, M. K., Etchells, J. L., and Veerhoff, 0. Chemical and bacteriological changes in dill pickle brines during fermentation. Food Research, £, 533-5k7, (1921.0). 3k* Joslyn, M. A. Some observations on the softening of dill pickles. Fruit Prod. J., Q, (ho. 8), 19-21; (No. 9), 16-17, (1929). 3 $* Kolthoff, I. M., Lauer, W. M., and Lunde, C. J. The use of dichlorofluorescein as an adsorption indicator for the argentometrie titration of chlorides. J. Am. Chem. Soc., 5l» 3273, (1929). 128 36. LeFevre, E. Pickle processing investigations. Canner, £0, 230-232, (1920). The --- 37. _______ ♦ Bacteriology of sauerkraut and pickles. Chemical Age. 30, 2^, (1922). 38. Lyman, C. M., Moseley, 0., Wood, S., Butler, B., and Hale, F. Some chemical factors which influence the amino acid requirements of lactic acid bacteria. J. Biol. Chem.. 167, 177-187, (1914-7). 39. Official Methods of Analysis of the Association of Official Agricultural Chemists. 7th ed., 506, (1950). 140 . Pederson, C. S., and Albury, M. N. Effect of temperature upon bacteriological and chemical changes in fermenting cucumbers. New York State Agr. Expt. Sta., Tech. Bull., Mfc* (1950) . If-1. ______________, and Ward, L. The effect of salt upon bacteriological and chemical changes in fermenting cucumbers. Hew York Agr. Expt. Sta., Tech. Bull., 2 8 8 , (1914-9). Peterson, W. H., and Peterson, M. J* Relation of bacteria to vitamins and other growth factors. Bact. Rev., ^g, 14-9, (1914-5). k3. Poliak, A. Process of pickling, curing, and preserving fruits and vegetables. IJ. S. Patent Ho. 2322880, (19l|.l)* Rahn, 0. Bacteriological studies of brined pickles. The Canner, 37, (No. 20), ljl^-14-5; (Ho. 21), l±3-}+59 (1913)* k5- Rosen, S., and Fabian, F. W. The importance of biotin, niacin, and pantothenic acid in cucumber fermentation. Food Technol., 7., 2[|Ij-2i|7, (1953). Ij.6. _____ _____ fermentation. _• Hoi© of biotin in cucumber Food Technol., 7.* 21^.2-21^, (1953). Sauberlich, H. E., and Baumann, C. A. The effect of dietary protein upon amino acid excretion by rats and mice. J. Biol. Chem., 166, ipl7-l4-28, (19l|-6). Jj.8. Stefaniak, J. J., Gailey, F. B., Brown, C. S., and Johnson, M. J. Pilot plant equipment for submerged production of penicillin. Ind. Eng. Chem., £8, 666, (19M>). 12% ^4-9• Stokes, J. L#, and Gunness, M. Pyridoxamine and the synthesis of amino acids by lactobacilli. Science. 101, I+3-lf5, (191|3). ------ 50. Tanner, F. W. Pickle fermentation# 123, (19i}-0)• 51* The Association of Vitamin Chemists, Inc., Methods of Vitamin Assay, 2nd Ed., Interscience Publishers, Inc., New York, (1951). 52. Tittsler, R. P., Pederson, C. S., Snell, E. E., Hendlin, D., and Niven, C. P., Jr. Symposium on the lactic acid bacteria. Bact. Rev., 16, 227, (1952). 53* Vahlteich, H. Haurand, C. H., and Perry, Or. A. Modern science apply itself to cucumber salting. Pood Indust., 7, 331+-336, (1935)* Canning Age. 7. 121— ----~ 5i+. Veldhuis, M. K., and Etchells, J. L. Gaseous products of cucumber pickle fermentations. Food Research, 1^, 621-630, (1939)• 55* ,Jones,I.D.,and Veerhoff, 0. Influence of sugar addition to brines in pickle fermentation. Pood Indust., 13» (No. 10), 51-56; (No. 11), 14.8 -5 0 , (191+lTI 56. Wooster, H. A., and Blanck, P. C. Nutritional data, H. J. Heinz Co., Pittsburgh, Pa., 2nd ed., (1950). 57. WustenfeId, H., and Kreipe, H. Versuch uber das Weichwerden der Sauern Gurken. Deutsche Essigindustrie, 27, 77-81, (1933).