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(23) Neuberg, O., and Reinfurth, E., Ibid., 89,365 (1918) (24) Robison, R., and Morgan, W. T. J., B i o c h a . J., 22, 1277 (1928). (25) Warburg, O., “Uber den Stoffwechsel der Tumoren,” Berlin, 1926. I
VOL. 27, NO. 9
(26) Warburg, O., “Uber die katalytischen Wirkungen der lebendigen Substanz,” Berlin, 1928. (27) Warburg, O., and Christian, W., Biochem. Z.,254, 438 (1932).
RECEIVED April 27, 1935.
Application of Oxidation-Reduction Potential to Brewing Control
.
The theory of the oxidation-reduction systems and their potentials has recently been applied to brewing control work and the preliminary results appear to justify a more intense study. Certain factors influencing the flavor and keeping quality of beer, which have been little understood, may thereby find an explanation. Outstanding are the influence of aeration during the various stages of the brewing process, the propagation of microorganisms, and the effect of oxidizing and reducing reactions on beer flavor. The oxidation-reduction potential is
generally expressed by the symbol, rH (the negative logarithm of the pressure of the reducing hydrogen present in the solution). The oxidizing power of a solution is the greater the higher its potential, and the reducing power is greater the lower its potential. An electrometric and a colorimetric method are available for the determination of rH, providing means for a methodical study of the oxidation factor wherever it occurs during the entire brewing process. Its application is described in the study of aeration, of the so-called light-taste, and of yeast turbidities.
F. P. SIEBEL, JR., ARD E. SINGRUENJ’ French wineries recognize the effect of the oxygen in the air on the wine five decades the Siebel Institute of Technology, aroma and are now investigating the brewing industry Chicago, Ill. oxidation-reduction potential as a has more and more turned to science possible means of controlling aerafor assistance in the solution of its tion. It also finds application in sewage treatment and in the practical problems. The advancements in physical, colloidal, study of the biological condition of soils. and biochemistry, in particular, have contributed a great De Clerck in France ( 1 ) and iMendlik in Holland (2) were deal to the better understanding of purely empirical experithe first to study the merits of the oxidation-reduction potenence and rule-of-thumb methods. I n many cases brewing tial for brewing control work. Although their investigations scientists have traced the causes of frequent disturbances are still in the experimental stage, the preliminary results a p and, based on this knowledge, have been successful in devising pear to be deserving of attention. means to remedy faulty conditions and eliminate them for the future. Theory of Oxidation-Reduction Systems The application of the theory of hydrogen-ion concentration to malting and brewing has become a well-known, comUnder oxidation, originally all those reactions were classiparatively simple, and effective method for controlling procfied in which oxygen combined with another substance to essing methods. Sumerous other problems of physicoform a new compound. I n the meantime, however, a better chemical character, however, have remained unsolved. It understanding of intermolecular arrangement has taught us is only during the past year that European brewing scientists that atoms consist of an inner, positively charged nucleus have put another modern theory-that of the oxidation-re(protons) around which particles with negative charges duction systems-to work, to study into some of the hereto(electrons) revolve. Atoms which lose a n electron in the fore least understood factors influencing the flavor and the course of a chemical reaction gain a positive charge. keeping quality of the beer. These include all oxidation and For instance, an increase in valence constitutes a gain of a reduction reactions which take place during beer production. positive charge-in other words, an oxidation. Since no Outstanding in this respect are the influence of aeration, the electron can exist by itself, it will be bound by another subpropagation of microorganisms, and the effect of oxidation stance which is thereby reduced. It is evident, therefore, and reduction on the beer flavor. that neither oxidation nor reduction can take place alone but Other industries are already making practical use of the oxithat both reactions always are concurrent and consist in the dation-reduction potential. I n the dairy industry the detransfer of electrons from the oxidized to the reduced subgree and, to a certain extent, the nature of milk infections are stance, If we consider the gain of a positive electric charge determined b y means of suitable oxidation-reduction indicaas oxidation, although no oxygen may be involved at all in the tors; it is also claimed that the suitability of milk for certain reaction, and the gain of a negative electric charge as reductypes of cheese can be predicted by this method. tion, the modern definition of oxidation would be “loss of electrons.” 1 Present address, 542 Arlington Place, Chicago, Ill.
URING the past
.
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VOL. 27, NO. 9
during fermentation, the system cysteine-cystine which has been found in the barley proteins, those systems present in the yeast, and others such as the system succinic acid-fumaric acid. The oxidation-reduction potential offer;. means for a methodical study of the oxidation factor, wherever it occurs during the malting and brewing process. Since oxidation is one of the most vital functions of all living cells, it covers much territory and is deserving of attention. AERSTIOS. One of the outstanding problems which should find a t least part of its solution through this study is the much-discussed question of aeration. This refers to aeration of the wort during boiling, cooling, and fermentation and of the beer when transferred and particularly during the filling operations. ' There is no doubt t'hat during the y a r i o u s stages of the brewing process the oxygen of the air exert's a definite influence on the flavor as 4
DIOXIDESTOR4GE TANKS FILTER1 % .4 BREWERY
CARBON .4ND
@
ing the entire oxidation-reduction scale and every hydrogen-ion c o n c e n t r a t i o n a n d , if properly handled, does not influence the system. The danger of polarization of the electrode, hoxever, is great, and minute traces of oxygen (air) cause variations in the result. In the colorimetric method numerous dyes are suitable as indicators in specific rH ranges. These organic compounds are colored in oxidized condition and decolorize during reduction; they have the remarkable advantage of being colorless in their reduced form. Added in minute quantities to a solution, they indicate an oxidation-reduction equilibrium, and the intensity of coloration serves as measurement for the r H of the solution. However, it must be emphasized that the indicators, as such, affect the oxidation-reduction equilibrium of the solution to be tested very strongly; therefore it is imperative that as little as possible of the dye be added, since otherwise they may change the r H of the solution. In this particular point the measurement of the oxidation-reduction potential differs distinctly from the otherwise similar determination of the hydrogen-ion concentration. I n brewing control the indicator method is the most practical, particularly when running test series. Methylene blue appears to have given the best results in wort and beer. Although a titration method would be desirable, the development of such a procedure will meet with great difficulties because of the scarcity of reversible oxidation-reduction systems. T o permit more accurate measurements of the r H in wort and beer, it will be necessary to improve and refine the methods now in use. I n the colorimetric as well as in the electrometric method, it is of utmost importance that all contact with air be avoided in taking the samples, as well as during the tests.
Application The oxidation-reduction systems to be considered in the brewery and malt house include the reactions of the sugars
hfASH
TUB.4SD
KETTLE I V 4
BREWERY
well as on the keeping qualities of the finished beer; however, the nature of this effect is much contested. It is well known that the breakage in the kettle is affected by aeration, but it is exceedingly difficult to trace the specific influence of the individual constituents and their reaction compounds on taste, foam formation, and keeping quality. I n this connection, n-e must consider the hop resins which so readily oxidize and which have such a distinct effect on the aromatic properties of the finished beer. The oxidationreduction theory may become of assistance in determining and controlling the nature of the oxidation products of the bitter substances and thereby insure a better uniformity of hop flavor in the beer. LIGHTTASTE. Beer, in general, has a very low reducing power which, however, becomes considerably accelerated when the beer is exposed to light. The time required for reduction is shortened by exposure to sunlight from several days to less than half an hour, and to a lesser extent by artificial light. The detrimental effect of sunlight on beer flavor may, therefore, be due to a n oxidation process caused by certain light rays and may probably be prevented by assuring a
i
SEPTEMBER, 1933
INDLSTRIAI, AND ENGINEERING CHEMISTRY
proper oxidation-reduction equilibrium in the beer before bottling. De Clerck, who conducted a series of experinients to inrestigate this phenomenon, found that beers redured rapidly by sunlight' had a distinct "light' taste" while check samples Tvhich had reduced slo~vlyin the dark were absolutely free from it. On the ot'her hand, light did not change the taste when reduction had been prevented by aeration. Beer which was partly reduced by standing for some time in the ljarrel or hottle was much more susceptible to light taste than beer which had heen filled up recently. These results were obtained by determining the reducing action of the beer on niethylene hlue. Becauqe of the high sensitivity of beer to air, the oxygen of which disturbs the once-established balance of the oxidation-reduction systems, it seems that every contact of the finished beer with air should be avoided; this is particularly true during the filling operation. Replacement of the air in this process by carbon dioxide gas can only prolong the keeping qualities of the heer.
Yeast Turbidities Biological tests have indicated that for the developnient of each microorganism a specific r H range represents the most favorable conditions. Considered from this point of v i e y the heretofore accepted classification between aGrobic and anaerobic microorganisms finds a different explanation. Experiments have been successful to grow so-called anaerobic bacteria in the presence of air after t'he r H of the nutrient medium was properly adjusted.
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Yeasts growonlyat a rather high r H (above 13)-that is, in well-aerated beers. Otherwise propagation stops and the yeast cells present precipitate, in the majority of cases forming only a minute sediment. The keeping quality of a beer, particularly where yeast turbidities are to be expected, depends therefore on the fact that reduction of the beer takes place to an r H below the range of the respectire yeast strain before propagation can start. Compared with this factor, the clegree of infect'ion is of minor influence. Assuming from the results obtained so far that the propagation of inicroiirganisms has a definite relationship to the rH, the control of this factor will be an effective means for combating yeast turhidities. Low fermentable sugar content of the beer and a limited contact of the beer with air during processing will minimize returns caused by yeast turbidities. The occasional use of minute quantities of an as sulfurous acid, t'o prevent yeast grom-th finds its explanation in the reducing action of the chemicals rather t,han in their germicidal effect, which is imperative in the concentrations used. Bacteria seem to be less sensitive to the rH, but undouljteclly further studies will also bring about a better underqtanding of their living conditions.
Literature Cited (1) De Clerck, J., TVochschr. Brazc., 51, 196-200. 204-7, 37s-81 (1934). ( 2 ) Mendlik, F., Ihid., 51, 305-7 (1934). RECEIVED .ipril 27, 1933
Reduction of Nitrobenzene with Dextrose in Alkaline Solution N1CHOL;IS OPOLONICK
V
OHL first carried out the reduction of
iiitrohenzene to aniline in alkaline solution (ii), and later a process of reducing nitrobenzene with molasses or sawdust was patented ( 2 ) . In recent years an exhaustive study of the influence of experimental conditions on the nature and yields of the reaction products fornied in the reduction of nitrobenzene has been in progress in the laboratory of Fordham University ( i ) . The present investigation deals with the yields of azoxybenzene, azohenzene, and aniline under varied conditions. I t has been found that the yields of reduction products are materially influenced by temperature, relative concentration of the reactants, hydroxyl-ion concentration, and the extent of dilution. The hydroxyl-ion concentration was varied in some of the experiments by substitution of sodium carbonate or calcium hydroxide for sodium hydroxide.
Experimental Procedure The reductions were carried out in a three-neck flask equipped with a reflux condenser and a stirrer which could be driven at 600 revolutions per minute. The third neck of the flask served for introduction of the glucose. At the start the sodium hydroxide solution of the desired concentration was run into the flask, along with I/a mole of
Fordham UniFeeraity-,Sew York, N. l-.
nitrobenzene-i. e., 34.2 cc. or 41 grams (the amount used in a11 experiments). With the temperature of the hath at' 55" t o 60" C., the dextrose in solid form was introduced in small portions over a 1-hour period, and then a reaction temperature of 100" C. \Tas maintained for 2 hours. The process was carried out with continuous stirring. At the end of the operation, the aniline and unreduced nitrobenzene were separated from the nonvolatile azoxghenzene by steam distillation. The aniline was separated from the nitrobenzene by steam distillation after the former had been converted to hydrochloride. It was found that, with azobenzene present, there mas no unchanged nitrobenzene. The azobenzene and azoxybenzene were purified by recrystallization from methyl alcohol, and the aniline was converted to acetanilide. Boiling points or melting points, as well as the usual comparison with authentic samples, were made on all three products. It was observed in the case of strongly concentrated caustic solutions that the reduction proceeded with the greatest velocity in direct sunlight; accordingly all experiments were performed approximately under the same light conditions. Preliminary experiments showed that, when the temperature \vas held at 55" to 60" C. for 1 hour and then brought to 100' C. for 2 hours, maximum yields were obtained. These temperature conditions were maintained in all experiments. In the study of the effect of the concentration of sodium hy-
