Addition of Sugar, Amino Acids and Corn Steep Liquor to Cucumber Fermentation1

Addition of Sugar, Amino Acids and Corn Steep Liquor to Cucumber Fermentation1 H. S. RAGHEB AND FR W. FABIAN Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication December 28, 1956 The development of appreciable brine acidity in cucumber salt stock is necessary for curing the cucumbers and maintaining a desirable color during subsequent storage of stock. Several investigators (Le Fevre, 192,1922; Joslyn, 1929; Fabian et al., 1932; Fellers et al., 1937; Fellers, 1939) recommended the addition of sugar to cucumber fermentations with the object of stimulating the acid forming bacteria and producing more lactic acid. However, Fabian and Wickerham, (1935) showed that the addition of 1 per cent sucrose to barrels of genuine dills caused an initial increase of acid forming bacteria but not an increase in final acidity. Other studies by Jones et al. (194) and Veldhuis et al. (1941) reported that the addition of sugar at the start of fermentation, or after 7 to 1 days, or in small amounts at short intervals throughout the fermentation, did not appreciably increase acid production even when the acid forming bacteria showed a significant rise in numbers. Lactobacillus plantarum is considered to be mainly responsible for acid production in cucumber fermentations (Etchells et al., 1946; Costilow et al., 1956), therefore, the presence of plentiful amounts of essential vitamins and amino acids for its growth is of great importance. Several workers (Rosen and Fabian, 1953; Costilow and Fabian, 1953a) have demonstrated that, during cucumber fermentation, the essential vitamins are present in sufficient amounts to satisfy the needs of L. plantarum. However, Costilow and Fabian (1953a, b) reported that great coliform and yeast activity caused a decrease in cystine and tryptophane levels respectively. They indicated that these two amino acids seem to be reduced during cucumber fermentation to a critical level for the growth of L. plantarum. Since corn steep liquor is an inexpensive industrial by-product, it could be used, if beneficial, on a commercial basis in cucumber fermentation. Little information, however, is available on its application for such a purpose. A patent by Pollack (1941) recommended the addition of one ounce corn steep liquor for every gallon of brine when making genuine dill pickles. The corn steep liquor treated barrels showed 1.4 per 1 Journal Articles Nos. 1876-77, Michigan Agrictultural Experiment Station. 253 cent lactic acid after 3 days, whereas, it was.6 per cent in control batches. Consequently, the object of this study was to investigate the influence in cucumber fermentation of the addition of sugar, of cystine and tryptophane, and of corn steep liquor on the microbiological activity and acid formation by lactic acid bacteria. EXPERIMENTAL METHODS Semicommercial Fermentations In 1953 and 1954, 2 barrels were prepared outdoors at the salting station of the H. W. Madison Co., at Mason, Michigan. Cucumbers size 22 of the varieties MR-17 and SR-6 were used in 1953 and 1954 respectively. A 3 salometer schedule was followed in salting. The different concentrations of sucrose, and cystine and tryptophane used in these experiments were determined on the basis of the weight of cucumbers and brine in each respective barrel. Corn steep liquor was added according to the volume of brine. The following treatments were carried out: (A) Sucrose treatments. Four barrels received.5 per cent sucrose which was added at the time of salting to 1 barrel and on the 7th day to the others. Each of 3 barrels was given 1. per cent sucrose. The sugar was added at the time of salting to the first barrel and on the 7th day to the second barrel. The third barrel received /% the amount of sugar at the time of salting, on the 7th day, and 13 on the 14th day. In this way, the influence of the addition of different concentrations of sucrose when added at different times during fermentation was investigated. (B) Cystine-tryptophane treatments. Each of 2 barrels received.25 per cent cystine and.25 per cent tryptophane which were added at the time of salting to 1 barrel and on the 7th day to the other. To investigate the effect of the combined treatment of the amino acids and sucrose, 3 barrels were prepared by adding.25 per cent cystine,.25 per cent tryptophane, and 1. per cent sucrose at the time of salting. Based on the studies of Costilow and Fabian (1953a) these con- 2 Size 2 cucumbers refers to cucumbers 3 to 4 in. long and 118 to 1% in. in diameter. Downloaded from http://aem.asm.org/ on August 22, 218 by guest

254 H. S. RAGHEB AND F. W. FABIAN [VOL. 5 centrations of cystine and tryptophane were considered more than sufficient to provide the brine microorganisms with plentiful amounts of these amino acids. (C) Corn steep liquor treatments. Two barrels received corn steep liquor (1. oz for every gal of brine) which was added at the time of salting to 1 barrel and on the 7th day to the other. The combined treatment of corn steep liquor, in the same concentration, and 1. per cent sucrose was studied by preparing 3 barrels. The corn steep liquor and sugar were divided into 3 equal portions and % added at the time of salting, % on the 7th day, and 1 on the 14th day. (D) No treatment. Three barrels were prepared from the same cucumbers at the same time as controls. Method of Sampling A stainless steel tube 3 in. long and Yl6 in. in the inside diameter with holes spaced 9.5 in. apart was used for sampling. Samples were taken through a hole made in the head of each barrel by inserting the tube and syphoning the brine into sterile sample tubes. During the first week, samples were taken every day. As the fermentation progressed, the periods of examination were as indicated in the graphs. On removal of the brine samples from the barrels, they were immediately transferred to the laboratory where chemical and bacteriological analyses were made, after which they were frozen at - 18.5 C until the amino acid and vitamin assays were made. Chemical and Bacteriological Analyses The following determinations were carried out: (1) Total titratable acidity: by titration with standard NaOH expressed as g of lactic acid per 1 ml of brine. (2) Salt concentration: following the method of Kolthoff et al. (1929). (3) Total sugar: by the Munson and Walker method as outlined in Official Methods of Analysis of the Association of Official Agricultural Chemist (195). The results were expressed as per cent invert sugar. (4) ph of the brine: Cenco ph meter. (5) Total bacteria count: on dextrose tryptone agar medium. (6) Lactic acid bacteria count: on V-8 medium devised by Fabian et al. (1953). (7) Yeast count: on dextrose agar acidified with 5 ml of 5 per cent tartaric acid per 1 ml of medium. (8) Coliform count: using single strength lauryl tryptose broth inoculated in triplicate with various dilutions of the brine samples. All plates and tubes were incubated at 3 C for all microbial analyses. Three days' incubation were allowed for the total and lactic acid bacteria counts; 2 days for coliform and 7 days for yeast. Amino Acid and Vitamin Assays The brine samples were removed from the freezer and allowed to thaw at 4.5 C just prior to running the assays, then stored in the refrigerator under toluene. Assays for valine and glutamic acid were carried out following the method of Sauberlich and Baumann (1946) using medium I. The dehydrated media prepared by Difco Laboratories, Inc., were used to assay for biotin, niacin, pantothenic acid, valine, glutamic acid, and tryptophane. Streptococcus aquinus strain P-6 (Leuconostoc mesenteroides) was used for the assays of cystine, leucine, and isoleucine. Laboratory Experiments under Ideal Conditions An experiment was designed to study sugar utilization and acid production by L. plantarum under ideal conditions. In this way it was hoped to show either the advisability or futility of the addition of sugar; of cystine and tryptophane; and of corn steep liquor to cucumber fermentation. For this purpose 32 flasks were prepared in the following manner: The cucumbers were washed thoroughly and the juice was extracted. One hundred ml portions of the cucumber juice were placed in 5 ml Erlenmeyer flasks. To each flask 1 ml of 1. per cent NaCl was added, which made a final salt concentration of.5 per cent after which sucrose, cystine, and tryptophane, or corn steep liquor, according to the specific treatment, were added. The flasks were sterilized at 15 lb for 15 min. Each flask was inoculated with.5 ml of an active pure culture of L. plantarum strain 17-5 suspended in physiological salt solution. All flasks were incubated at 3 C. The experiment covered a period of 35 days. Specifically the following additions were made: 5 flasks,.5 per cent sucrose; 5 flasks, 1. per cent sucrose; 4 flasks,.25 per cent cystine,.25 per cent tryptophane; 4 flasks,.25 per cent cystine,.25 per cent tryptophane and 1. per cent sucrose; 4 flasks,.82 per cent corn steep liquor; 4 flasks,.82 per cent corn steep liquor and 1. per cent sucrose; and 6 flasks, no treatment (control). It should be noted that the concentration of corn steep liquor used here is the same as that used under semicommercial conditions. Samples were taken periodically to determine the amount of acid formed and L. plantarum counts. Two flasks from each specific treatment were examined for the total sugar (expressed as invert sugar) in the brine at the start and at the end of the experiment. RESULTS Sugar Treatments (1) Effect of sugar on lactic acid formation. Before presenting the effect of the different sugar treatments, a Downloaded from http://aem.asm.org/ on August 22, 218 by guest

1957] LACTIC ACID BACTERIA IN FERMENTATION 255 study was made on the variations in lactic acid formation that are normally present in brines of similar lots. Comparison between similar fermentations prepared in 1953 and 1954 showed that the greatest difference in brine acidity was.1 per cent. Therefore, it was assumed that, under semicommercial conditions, if a specific treatment resulted in an increase greater than.1 per cent lactic acid, the difference should be considered significant. In figure 1 are shown the average acidity values of all sugar treatments under semicommercial conditions. These results indicate no significant difference in brine acidity between the sugar and nonsugar treatments. In general, the amount of lactic acid increased from its lowest level at the time of salting to a maximum of.6 per cent after fermentation for 15 to 2 days. It should be mentioned here that the changes in ph levels and salt concentrations were similar in both the sugar and nonsugar treatments. The laboratory experiments confirmed the previous results. Figure 4 shows that acidity in both the sugar and nonsugar treatments was almost identical. (2) Effect of sugar on total and lactic acid bacteria counts. Comparison between similar fermentations prepared in 1953 and 1954 indicated that the largest difference in lactic acid bacteria, expressed as log of count, was 1.4. Thus, it was safe to assume that differences of 1.5 (32 times greater) or more in the log of count of these organisms are significant. Data compiled under semicommercial conditions showed no indication of an appreciable increase in lactic acid bacteria numbers as a result of sugar addition. Only slight differences were observed. These small variations, however, never exceeded the range of differences that might occur between individual barrels within one treatment. The average lactic acid bacteria counts of all sugar treatments under semicommercial conditions.7 a.6 o.5 <.4 z.3 w a..2 A- f - o - 46 ~~~~ IQs AS Acidity Acid-formers are presented in figure 1. Their populations generally increased after salting and reached a peak within 5 to 6 days. During the second week, the numbers remained somewhat high, but declined during the third week with very low numbers observed after 41 days. The total bacteria numbers showed a similar trend during fermentation. The counts resembled that of lactic acid formers with no significant increase in numbers when sugar was added. Determination of total sugar as invert sugar in brines of the 1953 experiment was carried out to correlate the changes in lactic acid bacteria populations with the available sugar in the brine. Table 1 indicates that in the control fermentation a rapid increase in the brine sugar contents occurred after salting. This was mostly due to leaching of the naturally occurring sugars from the cucumbers into the brine. The total sugar content, expressed as invert sugar, reached a maximum about the 7th day and could not be detected on the 25th day. This decline was most likely due to the utilization of sugar by the brine microorganisms. In the barrels where sucrose was added, greater amounts of invert sugar were detected in the brine at the start of fermentation. However, the levels of invert sugar decreased to a level comparable to that of the control indicating greater utilization of sugars by the brine microorganisms. Yet, as is shown in figure 1, the amounts of lactic acid formed were similar to that of the control. Apparently then, the added sucrose was mainly utilized by organisms other than lactic acid bacteria. Under laboratory conditions, no significant differences in L. plantarum counts were observed between the sugar and nonsugar treatments. Furthermore, the amounts of sugar utilized by L. plantarum were similar (table 2). The added sucrose was not converted into lactic acid. This would indicate that L. plantarum can - 4Io._-._- Control (avg.of 3) A O --o A--6 Sugar treatments (avg. of 7) ~~~~~ 1. 9. a w 8. al 1-- z 7 : 6. 5. Downloaded from http://aem.asm.org/ on August 22, 218 by guest.1 4. If Ia III 3. 5 1 15 2 25 3 35 4 45 FIG. 1. Influence of addition of sugar in brines of semicommercial cucumber fermentations on lactic acid and acid forming bacteria.

use only a certain amount of sugar and produce a certain amount of acid irrespective of the amount of sugar present in the brine. (3) Effect of sugar on yeast and coliform populations. Comparison between similar barrels prepared in 1953 and 1954 showed that a variation of 2.1 in the log of yeast count might normally occur. Accordingly, a difference greater than 2.1 (approximately 1-fold) was considered significant. In figure 2, part B, it is shown that addition of sugar j z D IL -J 7. *- Control (avg. of 3). O Sugar treatments at time of salting (avg. of 2) 6.- 4. ^= l = ^ Ze _t j A e f 2... - _ 5 1 15 2 25 3 35 4 H. S. RAGHEB AND F. W. FABIAN I 45 5 FIG. 2. Influence of addition of sugar to brines of semicommercial cucumber fermentation on yeasts. 4 4-J at the time of salting caused no significant increase in yeast population over the control. However, when.5 or 1. per cent was added on the 7th day, the yeast counts immediately increased (figure 2, part A). The log of yeast count always exceeded 2.1. The numbers then rapidly declined until they reached a level similar to that of the control. When sugar was divided into three equal portions and added at different times throughout the fermentation, only the second addition of sugar on the 7th day resulted in a significant rise in yeast population over the control (figure 2, part A). In contrast to the lactic acid bacteria, yeasts, which had generally decreased in numbers during the first few days after salting, started rapid growth after about 7 days and reached their maximum population in from 1 to 15 days. As to coliforms, they disappeared from all barrels after 5 days. It is believed that they played no significant part in these fermentations. Cystine and Tryptophane Treatments Effect of added cystine and tryptophane on lactic acid production. Since a variation greater than.1 per cent acid between different treatments was previously considered significant, this variation value was also used here as basis for comparison between the specific treatments and the control. In all barrels receiving cystine and tryptophane, alone or in combination with sucrose, brine acidity was not appreciably higher than the control. The average acidity values of all cystine and tryptophane treatments are graphically presented in figure 3. Since no greater amounts of acid were formed, the addition of cystine and tryptophane, under the conditions of these experiments, was of no value in increasing lactic acid production. In table 1 it is shown that the utilization of sugar by the brine microorganisms was similar to that of the con- -j w a. [VOL. 5 Downloaded from http://aem.asm.org/ on August 22, 218 by guest I-- z IJ cr. w. z 3 IL. U CD -i FIG. 3. Influence of addition of cystine and tryptophane to brines of semicommercial cucumber fermentations on lactic acid and acid forming bacteria.

1957] LACTIC ACID BACTERIA IN FERMENTATION 257 trol. Since brine acidity was not appreciably affected, the cystine-tryptophane treatments did not stimulate the conversion of the naturally occurring sugars in the cucumbers to lactic acid. The previous results were confirmed under laboratory 1.4-1.2 1..8 < 1.4 I-1.2 _ 1. O.8 r. f L. plontorum counts. I Acid ity * Control (avg. of 4) o---o Sugar treatments (avg. of 6) *-C Control (ovg. of 4) ---o Cystine a tryptophone treatments (avg. of 4) A B <.V j -i 1. z XCL 9. 7. U. 5. *-A.6 *~ --- Control (avg. of 4) ~~~~~~~~~~~~3.J - 4- ^ 1.4 - --- o---o Corn steep liquor - 11. treatments (avg. of 4) 1.2 - -- - 9. 1. ( - 7..8so - 5. M% j.6 /53. 5 1 15 2 25 3 35 4 45 FIG. 4. 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 Lactobacillus plantarum. Time after Alow, I -f% conditions; the cystine-tryptophane treatments showed no effect on the total and lactic acid bacteria counts as well as yeasts. Figures 3 and 4 present the average lactic acid bacteria populations of all cystine and tryptophane treatments under semicommercial and laboratory conditions respectively. Figure 5, part B, shows the average yeast numbers. The coliform populations varied considerably from one barrel to another. There was no indication that the cystine tryptophane treatments had any significant effect on the number of coliforms during fermentation. In general, coliforms disappeared from all fermentations after 6 days. Apparently, unfavorable conditions in the brine inhibited them. Corn Steep Liquor Treatments (1) Effect of added corn steep liquor on lactic acid production. When corn steep liquor was added to the barrels, brine acidity was similar to that of the control during the first 2 weeks of fermentation. Thereafter, acidity was slightly higher. Although there is still the possibility that variations of such magnitude might occur between duplicate fermentations, yet, in all barrels receiving corn steep liquor, lactic acid production was considerably higher than the control. Furthermore, this slight increase of acidity lasted for a considerable period of time. Figure 6 presents the average acidity values of all corn steep liquor treatments examined. The invert sugar contents of the different brines and consequently the total sugar levels showed a rapid increase after salting. After reaching a maximum, the levels decreased at a more rapid rate than in the control. For example, when corn steep liquor was added at the time of salting, or on the 7th day, no invert sugar was detected on the 9th day as compared to.7 per cent for the control (table 1). Also, when corn steep TABLE 1. Per cent total sugar* in brines at semicommercial cucumber fermentations Per Cent Invert Sugart Treatments Brining No.5%.5% 1.% 1.% 1.% Amino Amino Amino Corn steep Corn steep treatment Sucrose at Sucrose on Sucrose at Sucrose on Sucrose acids at acids on acids an liquor at liquor on suor (control) salting 7th day salting 7th day (divided) salting 7th day salting salting 7th day (divided Downloaded from http://aem.asm.org/ on August 22, 218 by guest -3 hr t 1.254 t 1.191 t.673 t t t t t.687 2 days.331 1.269.395 1.73.436.82.318.318 1.862.45.423.782 6 days 1.8 1.697 1.13 1.964.882 1.234.963.927 2..96 1.37 1.132 8 days.821 1.329 1.637 1.81 2.144 1.359.621.714 1.835.327.668.732 9 days.777 1.3 1.29 1.529 2.24.791.593.782 1.316.327.165.418 11 days.725.615.512.968.646.418.52.427.712 1 t.268 13 days.336.386.469.624 _.422.361.295.515-15 days.91.47.317.55.19 -.35 25 days.151.114.241.25.136 33 days.165.138.173.29 t t.147 41 days 4.56.62.8.69 t 1.122 t * Total sugar expressed as grams invert sugar per 1 ml of brine. t Average of two determinations. t Less than.45 g invert sugar per 1 ml of brine.

258 liquor was added in combination with 1. per cent sucrose, the invert sugar levels generally decreased during the fermentation indicating its rapid utilization by the brine microorganisms. However, the brine acidity was similar but not greater than where only corn steep liquor was added without sugar. Therefore, the added sucrose was not converted to lactic acid. One can conclude then that the slight increase in brine acidity observed over the control in all corn steep liquor treatments was probably due to greater utiliza- TABLE 2. Per cent total sugar* in brines of laboratory cucumber fermentations H. S. RAGHEB AND F. W. FABIAN Per Cent Invert Sugart Treatments Time No Amino Corn treat-.5% 1.%Amn acids Cor steep ment Su u cd and con- crose crose cs u1- liquor liquoran asn trol crose crose O hr 1. 284 1.926 2.23 1.294 2.434 1.334 2.252 35 days. 381.944 1.358.341 1.379 t.899 Sugar utilized. 93. 982. 872. 953 1. 55 1. 289 1. 353 * Average of two determinations. t Total sugar expressed as grams invert sugar per 1 ml of brine. t Less than.45 g invert sugar per 1 ml of brine. It was considered equal to.45 g when the amount of sugar utilized was calculated. -i cr. w z L. -i B *- - Control (avg. of 3) 7. - o-.-o Cystine a tryptophone treatments (avg. of 3) 6. 5. 5t 4., 3. * <@~~ 2. p2 o 5 1 15 2 25 3 35 4 45 5 FIG. 5. Influence of addition of cystine and tryptophane, and addition of corn steep liquor to brines of semicommercial cucumber fermentations on yeasts. [VOL. 5 tion of the carbohydrates in the cucumbers by lactic acid bacteria. The laboratory experiments agreed with the previous results. Figure 4 shows addition of corn steep liquor, alone or in combination with 1. per cent sucrose, resulted in high acidity levels on the 11th day and thereafter. This increase in acidity is significant since it exceeded by far the variations found between duplicate determinations. The total sugar contents showed that L. plantarum was able to utilize more of the naturally occurring sugar in the cucumbers when corn steep liquor was added to the flasks. Table 2 indicates that the amount of invert sugar utilized was 1.3 per cent in the corn steep liquor treated flasks, whereas, only.9 per cent was observed in the control. The combined treatment of corn steep liquor and sucrose showed almost the same results. Thus, the added sucrose was not utilized by L. plantarum even in the presence of corn steep liquor. (2) Effect of added corn steep liquor on microbiologic activity. The average populations of lactic acid bacteria in 5 corn steep liquor treated barrels and 3 controls is illustrated in figure 6. It is evident from this graph that there was no significant difference during the first 2 weeks of fermentation between the corn steep liquor treatments and the control. However, their numbers were higher than those of the control on the 29th day and thereafter. The log of count of these organisms was higher than the control by at least 1.7 (about 5 times greater). The over-all total bacterial changes during fermentation resembled those of the acid formers. Under laboratory conditions, L. plantarum counts were higher than the control about the 15th day and thereafter (figure 4). Population changes of yeasts during fermentation of 5 barrels of cucumbers supplemented with corn steep liquor are shown in figure 5, part A. In general, yeasts reached a peak during the second week of fermentation but rapidly declined to a level lower than the control about the 18th day and maintained lower levels during the rest of the examination period. There was some evidence that combining sucrose with corn steep liquor might have some effect on yeast population. Their numbers significantly increased when the treatment was made on the 7th day. As to coliforms, the corn steep liquor treatments had no effect on their numbers during fermentation. Availability of Vitamins and Amino Acids In general, the vitamins; biotin, niacin, pantothenic acid; and the amino acids; leucine, isoleucine, cystine, valine, and glutamic acid gradually increased after brining. This was mostly due to withdrawing of the nutrients from the cucumbers by osmosis. After reach- Downloaded from http://aem.asm.org/ on August 22, 218 by guest

1957] LACTIC ACID BACTERIA IN FERMENTATION 259 ing a maximum, they maintained high levels to the end of the examination period. Tryptophane was the only amino acid affected by fermentation. Its levels reached a peak in from 6 to 1 days, then markedly declined. This trend of changes was observed in all sugar and cystine-tryptophane treatments (figures 7 and 8). A good illustration of the tryptophane decline occurred when cystine and tryptophane were added on the 7th day (figure 8). Since no greater amounts of acid were formed as a result of these treatments, tryptophane might have been a limiting factor. However, it was noted that the presence of plentiful amounts of cystine and tryptophane during the active fermentation period was of no value in enhancing brine acidity. When corn steep liquor was added alone, tryptophane decreased slightly at the end of the examination period as compared with a large decline in case of the control (figure 9). Evidently, smaller amounts of tryptophane were utilized by the brine microorganisms. This correlates with the results that lower yeast populations were found after 18 days. About the same time, lactic acid bacteria maintained somewhat higher numbers than in the control. It has been previously reported that greater utilization of sugar was observed when corn steep liquor was added. Apparently then, the added corn steep liquor enhanced the utilization of the available sugar in the brine resulting in the production of greater amounts of lactic acid. When the combined treatment of corn steep liquor and sucrose was applied, the decline in tryptophane levels was greater at the end of the examination period than in the cases where corn steep liquor was added alone. However, a similar increase in acidity occurred. This would indicate that tryptophane was mostly utilized by yeasts and not by lactic acid bacteria..8.7 _ o 5.6 ~~~ a..5, ff.4 a..2_'i DISCUSSION In this investigation, addition of.5 or 1. per cent sucrose did not accelerate acid formation. The increased acid production reported by many workers was probably only slight variations that normally occur between individual fermentations. Total and lactic acid bacterial populations were not appreciably affected by the different sugar treatments. Normal variations in bacterial populations are probably responsible for discrepancies in the results of other studies. Under presumably optimum conditions, the utilization of total sugar was not enhanced by increasing the sugar concentration in the brine. As a matter of fact, L. plantarum strain 17-5 was not able to utilize all the naturally occurring carbohydrates in the cucumbers. The carbohydrate level available in the brine was not a limiting factor for the growth of these organisms. No appreciable effect on yeast numbers was observed when sugar was added at the time of salting. Apparently yeasts, which were introduced into the barrels by adhering to the cucumbers, were becoming adjusted to environmental conditions. As fermentation progressed, their adjustment was most likely the influencing factor on their numbers. The rapid increase in yeast population which was noted when sugar was added on the 7th day was probably due to the presence of a rich source of carbohydrates which they immediately utilized. When the sugar in the brine was considerably lowered, a corresponding decline in yeast numbers occurred. Addition of cystine and tryptophane showed no effect on total titratable acidity and microbiologic activity of cucumber fermentations. Addition of corn steep liquor to the cucumber salt stock barrels enhanced the production of lactic acid during the third week of fermentation and thereafter. _-5_ 1. - _ 9. i ity Acid-formers 8. -- A-A Control (avg.of 3) - Corn steep liquor 7. Z treatments (avg. of 5) o 6 E 5. Downloaded from http://aem.asm.org/ on August 22, 218 by guest 5 1 15 2 25 3 35 4 45 FIG. 6. Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on lactic acid and acid forming bacteria.

26 During the same period, high population of lactic acid bacteria were present, whereas, yeast numbers were low. With the exception of tryptophane, studies on the vitamins and amino acids which are essential for the j *v.v : 5. a. o C z 4 a. 3. a. 1-25. :A2.1 15. 1. 5. 5 1 15 2 25 3 35 4 45 FIG. 7. Influence of addition of sugar to brines of semi commerical cucumber fermentations on tryptophane. O O Cystine a tryptophone 5. at salting i^ Cystine a tryptophone X 45 (L 4.- w x 43 5. - oj *-.* Control --o Sugar treatments at salting H. S. RAGHEB AND F. W. FABIAN on 7th day Cystins, tryptophone a kl 4 j\ + sugar at solting A -. ~~~~~~~4.% 4~~~~~~~~~~*te -I~~~~~~-z a I I a I 25\4 IL~~~ ' [VOL. 5 growth of L. plantarum indicated than an abundant supply of these nutrients was present at all times during fermentation. However, Costilow and Fabian (1953a) reported that cystine might possibly be reduced to a critical level under certain conditions. Since Aerobacter fermentation in this study was insignificant, no such effect on the cystine levels was observed. In all sugar and cystine-tryptophane treatments, tryptophane levels declined after the second week of fermentation. Thus, it seemed at first that tryptophane might have an important role in acid production. However, addition of corn steep liquor in combination with sugar stimulated acid formation, whereas, tryptophane levels were greatly lowered during the fermentation. Therefore, under the conditions of these experiments, tryptophane was not limiting for the production of lactic acid. Apparently, greater yeast activity was responsible for this reduction. Furthermore, in all barrels examined, tryptophane levels declined when yeast activity was greatest. It was probably utilized or destroyed by yeasts. These results agree with those of Rosen and Fabian (1953) and Costilow and Fabian (1953a, b). Results have also demonstrated that in all corn steep liquor treatments, there was a rapid utilization of sugar by the brine microorganisms. Thus, corn steep liquor might contain a substance or substances, other than those examined, which enhanced the utilization of the naturally occurring sugars in the cucumbers. Another possibility might be that corn steep liquor provides an unknown factor (Heimbuch et al., 1956) when its concentration declines in the brine to a critical level. Several workers have shown that corn steep liquor Downloaded from http://aem.asm.org/ on August 22, 218 by guest 15.! P 1.Oj 5. 5 1 IS5 2 25 3 35 4 45 5 FIG. 8. Influence of addition of cystine and tryptophane to brines of semicommercial cucumber fermentations on tryptophane. 2 25 3 35 4 45 FIG. 9. Influence of addition of corn steep liquor to brines of semicommercial cucumber fermentations on tryptophane.

1957] LACTIC ACID BACTERIA IN FERMENTATION 261 is a good source of soluble protein derivatives and minerals as well as growth promoting substances (Bowden and Peterson, 1946; Stefaniak et al., 1946). Since arginine, alanine, phenylalanine, and tyrosine have been shown sometimes to be stimulatory or essential for L. plantarum (Lyman et al., 1947), further investigations along these lines would be of great interest. SUMMARY Studies were made during two seasons under semicommercial and laboratory conditions of the influence of the addition of sugar, the addition of cystine and tryptophane, and the 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 fermentation. Under the conditions of these experiments, acid production was not stimulated by the addition of sugar or cystine and tryptophane. Corn steep liquor, however, enhanced acid formation. It also increased the utilization of sugar in the brine. Under ideal conditions, Lactobacillus plantarum strain 17-5 was capable of producing only a certain amount of lactic acid irrespective of the amount of sugar or cystine and tryptophane in the brine. No lasting effect was observed on the total and lactic acid bacteria populations in all sugar and cystinetryptophane treatments. Addition of corn steep liquor, however, stimulated the bacterial flora. Only the addition of sugar on the 7th day of fermentation caused an immediate increase in yeast population which was followed by a rapid decline when the total sugar in the brine was considerably lowered. Tryptophane levels consistently decreased when yeast activity was greatest. The possibility that corn steep liquor might contain a substance or substances, other than those examined, which stimulated lactic acid production is indicated. REFERENCES BOWDEN, J. P. AND PETERSON, W. H. 1946 The role of corn steep liquor in the production of penicillin. Arch. Biochem., 9, 387. COSTILOW, R. N., COUGHLIN, F. M., ROBACH, D. L., AND RAGHEB, H. S. 1956 A study of the acid-forming bacteria from cucumber fermentations in Michigan. Food Research, 21, 27-33. COSTILOW, R. N. AND FABIAN, F. W. 1953a Availability of essential vitamins and amino acids for Lactobacillus plantarum in cucumber fermentations. Appl. Microbiol., 1, 32-326. COSTILOW, R. N. AND FABIAN, F. W. 1953b Effect of various microorganisms on the vitamin and amino acid content of cucumber brines. Appl. Microbiol., 1, 327-329. ETCHELLS, J. L. AND JONES, I. D. 1946 Characteristics of lactic acid bacteria from commerical cucumber fermentations. J. Bacteriol., 52, 593-599. FABIAN, F. W., BRYAN, C. S., AND ETCHELLS, J. L. 1932 Experimental work in cucumber fermentation. Michigan Agr. Expt. Sta., Tech Bull., 126. FABIAN, F. W., FULDE, R. C., AND MERRICK, J. E. 1953 A new medium for determining lactobacilli. 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