November. 1928
INDUSTRIAL AND ENGIXEERING CHEMISTR P
1187
Gas Production in the Making of Sauerkraut’ L. M. Preuss, W. H. Peterson, and E. B. Fred DEPARTM~STS OF AGRICULTURAL CEZMISTRY A N D AGRICULTURAL BACTERIOLOGY, UNIVERSITY OF WISCONSIN, MADISON, WIS.
I
N T H E production of sauerkraut the first visible evidence that fermentation is taking place is the evolution of gas and the formation of clumps of pearly white foam on the surface of the cabbage juice. The foam usually appears in from 1to 3 days after filling the vat. At the end of 10 days it darkens, gradually disappears, and is followed by a film of mycoderm, which covers the entire surface of the liquid. A careful reading of the literature shows that no one has analyzed the gas evolved in an actual sauerkraut fermentation. Textbooks and reference works2 state that the gas consists of carbon dioxide, hydrogen, and methane. These statements are based on the work of Conrad,’ who did not analyze the gas produced from fermenting sauerkraut, but analyzed the gas produced by a microorganism which he had isolated from sauerkraut. He inoculated this organism into a sterilized cabbage decoction to which had been added 3 per cent glucose and 0.5 per cent sodium chloride. The gas evolved from this medium consisted of 85 per cent carbon dioxide, 24 per cent hydrogen, and 3 per cent methane. To assume that this fermentation is identical with that which takes place in a sauerkraut vat seems unwarranted. The environment in regard to temperature, oxygen supply, and composition of the medium and the interrelations of a mixed flora such as occur in a sauerkraut vat could not have existed in Conrad’s experiment. It is now generally recognized that the organism which Conrad isolated from sauerkraut, Bact. bmssicae acidae, is not *~g organism typical of the flora in ~ a u e r k r a u t . ~ ~Conrad’s was a motile, Gram-negative rod, which, among other products, formed carbon dioxide, hydrogen, and methane. Most of the organisms which have been isolated from sauerkraut in this laboratoryl0Pl1also do not possess the characteristics shown by Conrad’s organism. The work reported in this paper was undertaken for the purpose of obtaining data on the amount and composition of the gas produced in sauerkraut fermentation. Additional work must be done before a final conclusion can be reached regarding the gases formed under various conditions of fermentation, but the data here presented are a distinct step in that direction.
cutting a hole in the top. Two 13-inch (23-cm.) standard companion flanges were bolted together around this opening, which was closed with a standard square-head screw cap. An outlet for the gas was made by replacing the screw cap in the drain opening of the barrel with a gas cock. The barrel was coated with paraffin on the inside, and tested with a manometer to prove it free from leaks. Before Experiment I1 was begun, a flanged opening was made in the side of the barrel about half way between the top and bottom. This opening was closed with a two-hole rubber stopper. A piece of glass tubing, closed a t one elid with a rubber tube and pinchcock, was inserted through one hole. From this a sample of sauerkraut juice was drawn off for determining titratable acid and number of bacteria. Through the other hole of the stopper a thermometer was inserted to record the temperature of the sauerkraut. In order to prevent any sulfur compounds such as hydrogen sulfide from passing through the gas meter and causing corrosion, the gas was passed through a gas train before being measured by the meter. The gas train consisted of an empty safety bottle, a second bottle containing approximately 0.2 N iodine solution, a third containing distilled water (to dissolve any iodine carried over by the gas), and a fourth containing 5 per cent lead acetate and 1.5 per cent glacial acetic acid. I n none of the experiments was precipitate of lead sulfide formed. Figure 1 shows the apparatus. Although the gas measured by the meter during the first days of the fermentation was not the actual gas evolved, the
Apparatus and Methods ~ ~ P P A R A T u s - A ~ opening in a 58-gallon (219.8liter) all metal barrel, through which to fill the barrel and to remove the sauerkraut, was made by Figure 1-Apparatus for S t u d y i n g Gases Formed by F e r m e n t a t i o n of Sauerkraut A-Gas train: I-empty safety bottle. 2-bottle containing 100 cc. of approximately 0.2 N iodine solution; 3-bottle containini 100 cc. of distilled water; 4-bottle containing tion, Mddison, Wis. 100 cc. of 5 per cent lead acetate and 1.5 cc. glacial acetic acid. 2 Dukes, “The Bacteriology of Foods,” p. 162, H. K. B-Wet-test gas meter. Lewit & eo.,Ltd., London, 1925. C-Gas collection buret. 0 Fuhrman, “Einfuhrung in die Grundlagen der technischen Mykologie,” p. 442, Gustav Fiscber, Jena, 1926. meter reading was exactly equal to it, since an equivalent volGreaves, “Agricultural Bacteriology,” p. 412, Lea and Febiger, ume of air was driven out of the barrel by the evolved gas. Philadelphia and New York, 1922. Toward the middle of the fermentation period the metered gas a Henneberg, “Das Sauerkraut (Sauerkohl),” p. 20,, Institut fiir Garunnsnererbe. Berlin. 1916. ~. contained little or none of the air present in the barrel at the 8 Marshall, “Microbiology,” p. 570, P. Blakiston’s Son & Co., Philabeginning of the fermentation. delphia, 1921. FILLING OF BARREL-shredded cabbage mixed with about 7 Arch. R y g . , 29, 56 (1897). 8 Wehmer, Cmfr. B o k f . Parasitenk., I1 .4bt., 10, 625 (1903); 14, 682 2.5 per cent sodium chloride was put into the barrel. At in(1903). tervals it was packed by the use of a heavy wooden stamper. 9 Butjagin. Ibid., I1 Abt., 11, 540 (1904). A clean cheesecloth was placed over the surface of the cabbage 1.J. Bid. Chem., 39, 347 (1919); I b i d . , 48, 385 (1921). and a wooden cover, in sections, placed on top of the cloth. J . Agr. Research. 34, 79 (1927). Received June 2,1928.
Published with the permission
of the Director of the Wisconsin Agricultural Experiment Sta-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
1188
Cross-boards were next laid on the wooden cover, and stones were piled in to weigh down the cabbage. With the cabbage and stones in place, there stdl remained about 10 inches (25.4 cm.) space in the barrel. GAS CoLLEcT1oN-h the first three experiments the gas sample for analysis was collected from a glass Y located a t the end of the gas train. (Figure 1) In Experiment IV the gas sample was collected directly from the barrel. In all cases the gas was collected over mercury in a 185-cc. gas buret by means of slight suction. At the end of the experiment, when the gas consisted almost entirely of carbon dioxide, it was necessary to take large samples of gas, a liter or more in order to have enough gas left after the carbon dioxide had been absorbed to determine if hydrogen or methane were present. GASANALYSIS-The gas in the collection buret was transferred to a Burrell gas apparatus and analyzed for carbon dioxide, oxygen, hydrogen, and methane. AcIDITY-Acidity was determined by boiling 10 cc. of the kraut juice for a few seconds to expel carbon dioxide and then titrating with 0.1 N sodium hydroxide to the phenolphthalein end point. BACTERIAL CouNTs-In Experiment IV the number of bacteria were determined by the direct count and by plating on glucose yeast-water agar. The plates were incubated for 6 days a t 28" C. before counting. of Gas during t h e F e r m e n t a t i o n of Sauerkraut GAS (760MM. 0' C.) ANALYSIS Total Rate per hour COz 0 2 Liters Liters % b y uol. % b y uol.
Table I-Evolution
TIME Hours 0 45 65 88
112 136 161 185 209 233 260
0.00 2.0s 15,35 39.66 78.87 124.11 172.20 192.24 199.21 210.00 212.27,
0:05
0.67 1.05 1.63 1.88 1.92 0.83 0.29 0.45 0.08
i:3 9.0 40.5 76.8 93.2 97.2 98.0 98.3 98.6 98.6
19:s 17.6 8.8 3.8 0.9
..
0:4 0:3
Experimental Work
EXPERIMENT I-The barrel was filled with 300 pounds (136 kg.) of cut cabbage taken directly from the field. From Table I it can be seen that during the first 45 hours about 2 liters of gas were evolved. The gas evolution increased rapidly and steadily until, a t the end of 161 hours, 172 liters had been given off. From this time on, the gas evolution decreased slowly, ceasing a t 260 hours when 212.3 liters of gas had been evolved. The composition of the gas was practically constant after 192 liters had been given off. The gas a t this time consisted of 98 per cent carbon dioxide and 0.3 per cent oxygen. Since the small, daily gas samples showed that hydrogen and methane were absent, a large sample, 1395 cc., was taken to see if these gases were present and had remained undetected in the smaller samples. After removing the carbon dioxide and oxygen from this large sample, the residual gas (21.3 cc.) decreased in volume 1.8 cc. when burned with air, but no carbon dioxide resulted from the combustion. The diminution in volume suggests that a trace of hydrogen was present. The barrel was opened 20 days after filling. The sauerkraut was uniformly whitish yellow and very raw. The temperature of the cabbage when removed from the barrel was 6.1" C., and the acidity 0.26 per cent calculated as lactic acid. Owing to the low temperature of the cabbage, bacterial activity must have been limited, as shown by the small amount of acid and gas formed compared with that of the following experiments. EXPERIMENT 11-In this experiment 300 pounds (136 kg.) of shredded field cabbage heated with steam to 20' C. were placed in the barrel. Table I1 and Figure 2 show the results of this experiment.
Vol. 20, No. 11
Table 11-Composition of Gas Evolved f r o m Barrel EXPERIMENT I1 EXPERIMENT I11 coz 0 2 Time COz 0
Time
Hours
% b y vol.
% b y vol.
30:4 88.9 96.6 97.1 98.9 98.4 98.6 98.7 99.6
12.7 1.9 0.4 0.5 0.4 0.3 0 3 0.3
0
63 87 111 135 185 231 279 303 423 a
...
...
Hours
... 41
1
% b y uol. % b y uel.
65 89 115 233 305
... ... ...
53:5 96.8 98.5 99.3 99.5 98.80
.. ..
..
7:3 0.5 0.1 0:09 0.20
.... ..
Gas taken directly from barrel.
After 63 hours of incubation a rapid and steady evolution of gas was reached. This continued up to the 135th hour, when 214.2 liters of gas had been produced. At this point the evolution of gas became less rapid, but remained steady as shown by the straight line on the graph. After 185 hours, when 227.9 liters of gas had been evolved, the composition was practically constant and consisted of about 98.5 per cent of carbon dioxide and 0.3 per cent of oxygen. No hydrogen or methane could be found even in samples as large as 350 cc. Gas evolution ceased a t the end of 423 hours. The total amount of gas produced was 328.9 liters. The sauerkraut, removed from the barrel a t the end of 24 days of fermentation, was dark-colored in spots, acid in taste, but still raw. The temperature of the sauerkraut when taken out of the barrel was 17.0' C. and the acidity 1.90 per cent. Although gas production did not begin any earlier, more gas and a higher acidity were obtained than in Experiment I. Acid and gas production are quite parallel, especially during the first six days. As the temperature dropped from 17.5" to 10.5"C., the gas and acid production decreased markedly, the acid production more so than the gas. As the temperature again increased to 20" C., there was an increase in acidity. When the temperature is low the activity of the microorganisms is diminished and there is a corresponding decrease in the acid and gas production. When the temperature rises, the bacteria become more active and gas and acid production increases. EXPERIMENT 111-Cabbage that had been kept in storage for several weeks was used in this and the following experiments. After removal of the outside leaves, the heads were washed thoroughly in running Madison drinking water before they were cored and shredded. The 306 pounds (138.8 kg.) of shredded cabbage, previous to being put into the barrel, were treated with steam to bring the temperature up to 20" C. The barrel containing the cabbage was kept a t a relatively constant temperature. Table 111-Gas
TIME Hours
a n d Bacterial D e t e r m i n a t i o n s during F e r m e n t a t i o n of Washed Cabbage a t 25-28' C. GAS ANALYSIS BACTERIAL COUNTS c02 0 2 Plate Direct % b y uol. % b y vol. Milliqn! fier Million! fier cc. juice
cc. j u i c e
40 fiA
As can be seen from Figure 3 and Table 111,the evolution of gas began soon after the cabbage was placed in the barrel, and was vigorous until the end of 115 hours, a t which time 262.7 liters had been produced. From then on the formation of gas was not great, ceasing after 305 hours. The total amount of gas was 301.4 liters. The last sample contained 98.8 per cent carbon dioxide and 0.2 per cent oxygen. Three large samples of gas (1193, 1206, and 1043 cc.) were collected and analyzed. Although the last two samples showed both diminution in volume (1.5 and 2.3 cc., respectively) and traces of carbon dioxide after combustion with air,
IAVD CXTRIAL A,VD E,VGILYEERI,VG CHE-MIXTRY
Sovember, I928
the first sample showed neither. If hydrogen and methane are produced in the formation of sauerkraut, it would appear that larger quantities than mere traces should be present. It is difficult to draw a definite conclusion as to the presence of traces of hydrogen and methane in the large gas samples because of the possible interference due to mustard oils, alcohol, and other volatile compounds given off in the fermentation. I t seems certain, however, that, if present a t all, the quantities of hydrogen and methane are insignificant. When gassing had ceased, the temperature of the incubation room was raised t o see if a secondary fermentation could
Figure ?--Experiment
I1
1189
cent in the next 24 hours, but the gas production began to decrease. The gas evolution was not very great after 64 hours, although the amount of acid increased slowly. At the end of 141 hours the evolution of gas ceased; a t this time 414.7 liters of gas had been evolved. As in previous experiments, the gas and acid formation followed each other quite consistently. When the barrel was opened after 11 days of fermentation,
Figure 3-Experiment
I11
Figure 4-Experiment
I\'
Temperature, Acidity, and Gas a t Various T i m e s during F e r m e n t a t i o n of Sauerkraut
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be produced. The temperature of the sauerkraut ranged between 21" and 24" C., and about 59 more liters of gas were given off in 10 days. When the barrel was opened on the 23rd day. the temperature of the sauerkraut was 24.3" C. and the acidity 2.24 per cent. The sauerkraut mas still somewhat raw, but the flavor was distinctly superior to that of the sauerkraut made in the preceding two experiments. Evidently the washing of the raw cabbage resulted in a better flavored sauerkraut. EXPERIMENT IT'-The purpose of this experiment was to obtain more data concerning the effect of higher temperatures (25" to 28" C.) on the evolution of gas. Stored cabbage was used. The heads were washed before being cored and shredded. Steam was passed into the shredded cabbage until it reached 25' C., and then 300 pounds (136 kg.) were packed into the barrel. All gas samples were taken directly from the barrel outlet. Numbers of bacteria a t different times during the fermentation were determined both by plate and by direct count methods. A chemical analysis of the sauerkraut was made a t the end of the experiment. From Figure 4 it can be seen that this experiment mas characterized by an exceedingly quick and vigorous fermentation. At the end of 40 hours 258.5 liters of gas had been evolved-about the same quantity that was evolved a t the end of 115 hours in Experiment 111. All the residual air in the barrel had been displaced a t the end of the second day, as is shown by the fact that the gas sample (1740 cc.) consisted of almost 100 per cent carbon dioxide. The acidity a t the end of 40 hours had reached 0.89 per cent, and the bacterial counts were correspondingly high. The acidity increased 0.5 per
numerous pink areas were found in the sauerkraut. These pink areas consisted for the most part of the thick, coarse cabbage shreds. The sauerkraut was still somewhat raw, but had a good flavor. The acidity was 2.1 per cent, and the temperature was 24.4" C. when the sauerkraut was removed from the barrel. Analysis of the sauerkraut juice gave the following fermentation products: 0.63 per cent volatile acids (as acetic); 1.51 per cent non-volatile acids (as lactic); and 0.28 per cent alcohol (as ethyl). It was found, on analysis, that the salt content and titratable acidity of the juice from the red sauerkraut was different from that of the juice from the white, thus: Red Juice
Per cent Salt content (as NaCIJ Titratable acidity (as lactic)
3 49 2.00
White Juice Per cent 1.86 1.76
Fred and Peterson'? have shown that pink sauerkraut may be the result of yeast growth. Stained mounts showed that yeasts were present in the sauerkraut juice during this experiment. The high temperature, rapid rate of acid formation, and a relatively high salt content seem to favor the growth of the pink yeast. To say what is the causative agent in the production of the gas in sauerkraut is difficult, because the fermentation is the result of the combined action of many organisms. Wehmers concluded that gas formation was due to the activity of alcohol-forming yeasts, and that acid production was caused by 12
J . Bacf., 7,257 (1922).
ILYDUSTRIAL A,VD EXGISEERING CHEMISTRY
1190
an organism, Bacterium brassicae, which did not form any gas. Hennebergs states that the carbon dioxide production may a t first be due to the respiration of the plant cells, but later the yeasts play a more important role. Peterson, Fred, and their a ~ s o c i a t e s ~ ~have J ~ J ~isolated a large number of mannitolforming bacteria from sauerkraut. These bacteria convert 20 to 25 per cent of the glucose and other sugars into ethyl alcohol and slightly higher percentages of the sugars into carbon dioxide. It is therefore not necessary to have yeasts in sauerkraut in order to have gas and alcohol production. A fact clearly seen in all the graphs is the close parallelism of gas and acid production, especially in the earlier stages of the experiments. Since acid production in sauerkraut is attributed to bacterial activity, the logical conclusion can be reached that gas production is also a bacterial activity. Further proof for this conclusion lies in the fact that with the rapid increase in gas and acidity in Experiment IV there was a simultaneous rapid increase in the number of bacteria. I n order to determine how many of these bacteria produced gas, 15 colonies from the plates of each sample of juice were picked into tubes of sterile, glucose yeast-water medium; in all 105 colonies were picked. The tubes were sealed with sterile melted vaseline incubated a t 28"C., and observed from day to day for turbidity and gas formation. If the vaseline plug was pushed u p they were recorded as gas-positive. If the vaseline remained a t the surface of the liquid and there was no apparent leakage of gas, they were callqd negative. After 8 days of 18 14
J . Biol. Chem., 41, 273 (1920). Ibid., 64,643 (1926).
Vol. 20, s o . 11
incubation all the cultures which showed no gas formation were reinoculated into fresh, sterile medium, plugged as before, and incubated a t 28" C. for 5 days. Twenty per cent of the 105 cultures showed gas formation. Three-fourths of those cultures showing gas were obtained from the samples taken within 4 days after the cabbage was packed in the barrel. Even a higher percentage of gas-forming bacteria was reported by Priem, Peterson, and Fred" in commercial sauerkraut which had been fermented a t temperatures below 10.5" C. These investigators found that 70 per cent of the bacteria produced gas. I n the light of these data it seems justifiable to conclude that bacteria are the primary agents responsible for the production of the gas in sauerkraut fermentation. Summary
1-The gas evolved during the formation of sauerkraut is almost 100 per cent carbon dioxide. 2-Most of the gas formed is given off within 40 to 160 hours after the cabbage is packed into the container. 3-Since there is a very close relationship between gas, acidity, and numbers of bacteria, the conclusion is reached that the gas production is due t o . bacterial activity and not to yeast growth or plant-cell respiration. 4-The fermentation of cabbage is much quicker at higher temperatures than a t lower temperatures. +Washing the cabbage before cutting and packing has an apparently favorable effect upon the flavor of the sauerkraut.
Dufton Distilling Column for Preparation of Absolute Alcohol' W. A. Noyes UNlVERSITY OR ILLINOIS, U R B A N A .
I
T HAS been shown that, with a suitable apparatus, very
nearly absolute alcohol may be obtained by means of calcium chloride.2 A simplified apparatus, including a Dufton distilling ~ o l u m n ,has ~ now been devised. The alcohol is heated in a 15-liter copper boiler, A , by means of a steam coil of galvanized iron pipe, B , 18 mm. in diameter, using steam under a pressure of 6 kg. per sq. cm. (90 pounds). The distillation is controlled by a valve which regulates the rate a t which the condensed water runs away. Description of Apparatus
The Dufton column consists of an inner brass tube, C, closced a t both ends, and of 21 mm. outside diameter. Around this is wound an annealed copper rod, D, 6.5 mm. in diameter, leaving the successive spirals 2 cm. apart. The spiral is inserted in a copper tube of 35 mm. inside diameter and fits so closely that it was necessary to file flat places on the outside of the spiral a t frequent intervals to permit the solution of calcium chloride to flow downward more freely. The column is 140 cm. long. It is soldered to the bottom of a brass reservoir, E , 10 cm. in diameter and 30 cm. long. A short piece of brass tubing, E , 20 mm. in diameter, passing through the bottom of the reservoir, delivers the vapors of alcohol, coming from the top of the column, immediately beneath the center of a conical, perforated diaphragm, upon 1 Received
2 3
June 13, 1928. Noyes, J Am. Chem. SOC.,46, 857 (1923). Dufton, J . SOC.Chem Ind., S8, 45 (1919).
ILL.
which the anhydrous calcium chloride was placed. A short tube a t the side of the reservoir a t its bottom is connected by means of rubber tubing and a short U-tube, G, with a similar tube a t the top of the column. This makes it possible to see the character of the solution of calcium chloride running from the bottom of the reservoir down the column. A ooil of small copper tubing, H , in the top of t h e r e s e r v o i r , through which water is allowed to run very slowly, causing the condensation of a small amount of alcohol, controls the downward flow of the solution. From the top of the reservoir a tube of block tin connects with a worm, I , for the condensation of the alcohol. Operation Put in the boiler 1500 cc. of alcohol recovered from the last distillation; distil 500 cc. and return this through the reservoir a t the top. Put 10 liters of ordinary alcohol into the boiler, and 800 grams of calcium chloride into the reservoir a t the
