THE INSTITUTE OF BREWING RESEARCH SCHEME

SYSTEM OF WORT ANALYSIS. IV. THE ESTIMATION OF THE TRUE PROTEIN NITROGEN OF WORTS AND BEERS

By L. R. Bishop, Ph.D., F.RJ.C.

(Received by the Secretary 21/7/44)

by him from the yeast of top-fermentation brewery in Edinburgh. There is not yet any recognized subdivision of the species S. cerevisiae Hansen, so for the time being yeast 6470 must be left here without any further label. In due course, numerous strains of this species will be recognized each with slight, but technically important, differences in characterization. Figures to illustrating this paper are drawings copied mechanically from photographs; they preserve the scale but bring outthe characteristic features more clearly and enable significant cell forms to be brought together in small area. The writer is indebted to Dr. C. G. C. Chesters of the Botany Department of Birmingham Univer sity for advice on this matter.Institute of Brewing Research Laboratories, University of Birmingham.19th April, 1944.A method is given in which wort or beer is heated with sufficient sodium nitrite and acetic acid to deaminate the nitrogen compounds present. On cooling precipitate forms which, the experiments suggest, represents the true protein* present, since it appears to correspond to the albumin and globulin of barley extracts and to include the nitrogen compounds of wort which are coagulable by boiling. It is possible that the amount of this fraction bears relation to non-biological haze, while subsequent paper suggests an important effect on fermentation. The results indicate that boiling conditions play an important part in controlling the amount, as also may the amount of tannin in hops and fermentation conditions.Many writers have pointed out that the nitrogen compounds of wort, although they are recognized as important, represent only small proportion of the total solids—usually some 4-5 per cent.; while other writers have claimed in turn that it is not the total quantity of nitrogenous matter that is important but the "quality" of those nitrogen compounds. One interpretation of "quality" would be that some particular nitrogen compound (or group of compounds), although constituting only small proportion of the whole, is of distinctive function and importance. If this be so, then the nitrogen factor to be considered would appear to represent an important quality factor among the nitrogen compounds of wort. For in the first place the quantity present is small, since, as protein, it ranges from-some 0,4 per cent, of the total" solids in an unboiled sweet wort down to vanishingly small amounts in hopped wort and fermented beer (0,02 per cent, or less). Yet, despite the small amounts present, there are indications that this fraction is of special importance in brewing—for instance evidence is presented in subsequent paper showing an important effect! on fermentation in conditions of nitrogen shortage.For reasons which are discussed in detail in Section II the fraction estimated has been named the "true protein nitrogen," and appears to consist of the most complex nitrogen fractions present.

* The bulk of the nitrogen compounds of wort consist of protein derivatives (proteoses, peptones,polypeptides and amino-acids); that is they are built up of the same units (amino-acids) but are ofsmaller size than the true proteins, with which alone this paper is concerned.For easy reference the method of estimation is given in Section and the experimental basis in Section II.SECTION IMethod of Estimation of True Protein NitrogenThe method of estimation depends on the deamination of the nitrogen compounds present by heating with acetic acid and sodium nitrite, followed by cooling, when the particular fraction is insoluble and- is precipitated. It is then estimated either by the Kjeldahl method or by turbidity. The details were as follows:Method for True Protein Estimation.Four 110 ml. lots of wort or beer are centrifuged preferably at 5000 r.p.m. for 20 minutes using "lucite" cups and an "angle" head on an "International" centrifuge. Alternatively 100 ml. conical glass oil vessels may be used, as for the estimation itself, in an ordinary centrifuge at 2000-3000 r.p.m., but they are not so effective. The bright wort is poured off, bulked and filtered through Whatman No. 42 or 50 filter paper to remove any remaining suspended matter.The specific gravity of the wort (or the original gravity of the beer) is determined. One hundred ml. portions are measured into each of four 100 ml. conical oil vessels. *1,25 ml. of glacial acetic acid is added to each and stirred in. This is followed by 1,5 ml of 30 per cent, sodium nitrite droppedin gradually with stirring. The four vessels are then placed (in a wire basket) in rapidly boiling water bath. One drop of capryl alcohol is added to each. After 20 minutes the vessels are taken out, cooled in running water and allowed to stand one hour at 55-60° F. They are then centrifuged. Surface particles of precipitate are detached and about 0,01 grm of kieselguhr added in 1-2 ml of water. (The kieselguhr must have been purified by boiling in HC1, washing and drying, even if listed as "acid-washed.") The vessels are re-centrifuged, the clear liquid poured off and ml. of water is used to wash the inside of the vessel and to stir up the precipitate which is again centrifuged down. After pouring off the clear liquid, the washing is repeated. The precipitate is then transferred to Kjeldahl flask with the help of little dilute soda. The contents of the four vessels are bulked in pairs to provide duplicates.It is possible to use single 100 ml. quantities (in duplicate) and to .estimate the nitrogen of the precipitate by micro-Kjeldahl. Another possibility is to heat 10 ml. quantities of wort with the appropriate proportion of reagents in test-tubes and, after cooling, to estimate the amount ofturbidity formed in the mm. or 2-5 mm. cells of the Pulfrich nephelometer. In this way comparative estimations can be made quickly on long series of worts and beers. If turbidity estimation is used, then an alternative suggested by the present work is to use trichloracetic acid [10 ml. of freshly prepared 25 per cent, (w/v) solution per 100 ml.] and to measure the increase in turbidity. The gelatinous nature of this precipitate in this case is an advantage. *The precipitate is light and is easily disturbed in an ordinary centrifuge tube, but it can be retained successfully in the narrow tip of conical tube. This shape is also effective for washing the precipitate.

SECTION IIExperimental Basis of the Method, and Consideration of the Literature As result of recent work in physical chemistry the true proteins may be defined 'approximately as compounds with molecular weight of 17,000 or more and built up from amino-acids. That is they contain the linked residues of' some 150 amino-acids or, more frequently, twice or more times this number (see this Journ., 1943, i68). The nitrogenous constituents in many extracts consist of such proteins together with, in the main, relatively simple nitrogenous constituents with molecular weights of the order of one hundredth of that of protein. As result, the differentiation of proteins from other nitrogenous constituents is comparatively simple in most protein-containing liquids: such as milk, blood, egg-white or extracts of ungerminated seeds, both because of the relatively large proportion of protein and of the relative freedom from nearprotein compounds; so that earlier methods of "protein" estimation have not been based on as precise distinction in molecular size as that made above. Wort on the other hand is the extract of germinated "seed," and, as the. result of enzymic breakdown of insoluble protein, large proportion of the nitrogen compounds consists of complex material approaching, but not reaching, proteins in molecular size, while large proportion of the remainder consists of relatively simple nitrogenous compounds. Apart from these, liowever, there also appears to be in wort relatively small quantity of protein in the true sense of this word. It is with this small fraction of the total nitrogen that the present study is concerned, and it will be clear that the estimation of these small amounts of true protein in the presence of relatively large amounts of near-protein compounds represents difficult problem. Another difficulty in dealing with wort and beer proteins is that of nomenclature. It will be recalled that, in his original work on barley and malt, Osborne described one protein, an albumin, which he called "leucosin," soluble in water and coagulated on heating solution to temperature of 56° to 60° C. (133° to 140° F.). He also described globulin, "edestin," which was dissolved in dilute salt solutions and was not readily coagulated by boiling (J. Amer. Chem. Soc.,1895, 17, 539). Schjerning subsequently pointed out that in the brewing process the albumin would naturally be coagulated on boiling and that, if any true protein remained in brewers' wort, it would be the globulin, as, in general, plant globulins tend not to be precipitated on boiling. Therefore he suspected this to be the cause of brewing troubles such as haze formation. While the sugges-lation of Schjerning is considered to be a good one, number of experimenters have criticized his methods of estimating the nitrogen fractions of wort.In recent years the idea that globulin may be the cause of haze in brewing has been taken up again by workers including Hartong {Wock. Brau., 1937, 54, 33; this Journ., 1937, 121). In Hartong's method he salts out fraction, which he calls globulin (or more recently globulose) by adding 29 per cent of saturated solution of ammonium sulphate, and then by saturating with ammonium sulphate he obtains further fraction which he calls albumin.This view would seem, however, rather too simple. For instance, Horace Brown has precipitated, by saturation of cold water extract of malt with ammonium sulphate, fractions which the amino index showed to be well below protein size (malt peptones I and II of Brown—Trans. Guin. Res. Lab.,1903-1906). There is also evidence that the "albumin" fraction of Hartong contains non-protein constituents, for Helm (this Journ., 1939, 80) found that the second ammonium sulphate fraction contained 32-8 per cent, of pentose which he assumed to be in the form of pentosans.

This was followed by the finding of Hopkins, Amphlett and Berridge (this Journ., 1941,108) that, on electrophoresis of unboiled sweet wort, the insoluble fraction which separated contained nucleic acid, as also did the fraction which coagulated on heating. In turn this might explain Helm's discovery of pentoses in the second ammonium sulphate precipitate since nucleic acid contains the pentose sugar ribose or desoxyribose, as well as phosphoric acid and nitrogen bases. difficulty in this inter pretation is that the ash content of Helm's preparations was low indicating absence of phosphoric acid, and other work summarized in review by I. A. Preece (this Journ., 1940, 38) suggests that pentosans are present as such in wort in appreciable quantities. Osborne and Campbell (J. Amer. Chem. Soc,1900,22,379) state that the albumin prepared from whole wheat extracts contained no phosphorus and therefore no nucleic acid; whereas from wheat germ they prepared nucleic acid and found that it precipitated with the albumin on coagulation and at the same time affected the coagulation tempera ture somewhat. Apparently therefore nucleic acid is liberated also from the barley germ in malting and mashing and associates with the albumin fraction. In hopped wort, besides nucleic, other complex acids, chiefly tannic, melanoidic and the resin acids, will also be present and will modify the behaviour of the protein fractions: as may be instanced in the precipitation of protein tannates on cooling and on chilling. This offers possible explanati6n of the difference between the results of Hartong (loc.cit.) and Helm (loc.cit.) using the precipitate from 30 per cent, saturated ammonium sulphate solution. Hartong found this did not coagulate on boiling and Helm found that it did. It is possible that Hartong's precipitate contained only globulin, while Helm's contained some albumin also because of modifying factors, such as proportions of complex acids, in their respective worts. In any case the albumin should be distinguished by coagulation at tempera tures below boiling. Variation in the proportion of complex acids also offers an explanation why De Clerck (Bull. Assqc. Anc. Etud. Ecole. Sup. de Brass. Louvain, 1939, 39, 117) found that Hartong's precipitations with 30 per cent, saturated ammonium sulphate gave uncertain indications of the chill-haze-forming tendency of different beers,, although with one type of beer the indications were good. These were results with lager beers. In British beers the problem is com plicated by the proteins added in finings. With the modified behaviour of the proteins in the presence of complex acids it is not easy to distinguish the albumin and globulin. It would seem that, other than the criterion of coagulability and noncoagulability, no sharp distinction can be drawn between them, for the chemical com position of the two. classes, as far as it is known, is not sharply distinct, nor is the distinction of solubility in water and in salt solutions. (R. M. Ferry, E. J. Cohn and E. S. Newman, J. Amer. Chem. Soc, 1936,58, 2370.)Another criterion 'distinguishing proteins is the molecular weight. One of the best methods of studying the molecular size of protein compounds is by the use of the ultracentrifuge; which, by centrifuging at extremely high speed, sorts out compounds in solution according to their molecular size, because the larger the molecule the more quickly it moves to the outside under centrifugal force. This method is not available in most laboratories, but has been used for protein fractions from barley, malt and wort in the laboratory of Prof. Svedberg at Upsala. There is paper in English on the results by 0. Quensel and T. Svedberg (Compt. rendu. Carls. Lab., 22,441) and fuller account in German by H. Lundin (Woch. Brau., 1938, pp. 241, 249 and 259; this Journ., 1938, 476). The results show that the proteins which they separated from salt solution extract of barley are notsharply distinguished in molecular size, but there appears to be, as well as material below protein in

size, preponderance of protein with molecular weight of 35,000 and indications of fifth as much of another with molecular weight around 70,000. Hartong and Helm assume that the latter is the globulin, although direct evidence is not available in the paper. An interfering substance was found in electrophoretic experiments quoted by Lundin which produced the indefinite molecular weight results and it is possible that nucleic acid was the cause of this blurring. Also since the saturated ammonium sulphate used for precipitating the proteins might well precipitate proteoses, it would seem desirable to repeat the ultracentrifugal studies using protein fractions precipitated by series of different concen trations of ammonium sulphate. The molecular weights found were in the protein region and afford little justification for Hartong's suggestion {Amer. Brewer, 1939, 72, 37; this Journ., 1940, 83) that the name globulin should be changed to globulose. Equally it is clear that Osborne's early guess is not correct, for the globulin is not identical with the edestin of hemp seed, which has molecular weight of 300,000.To summarize the position as it may be regarded at present there appear to be in wort two nitrogenous substances of sufficiently high molecular weight to be regarded as proteins. One of these proteins is not readily coagulated on boiling. Alternative names for this are globulin (Osborne), noncoagulable protein (Hopkins, Amphlett and Berridge) and globulose (Hartong). The other protein fraction is readily coagulated on heating the solution below the boiling point. It was designated albumin by Osborne and coagulable protein by Hopkins, Amphlett and Berridge. Precipitated from wort it would contain also nucleic acid as the latter showed, and precipitated by full saturation with ammonium sulphate (as by Helm and Hartong) it might be expected to containproteoses as well as non-protein constituents. This is the view taken on the individuality and nomenclature of the two proteins separately. Together they could be "described as "the proteins" or "the true proteins." However, both these terms have been used to convey other meanings and are ambiguous. "The proteins" is frequently used to include the whole of the nitrogen compounds of wort or other biological material; while "true protein" (as determined by precipitation with copper hydroxide) is misleading when applied towort. However, the term "true protein" is adopted as the best available to define the range which the present estimation seeks to cover.ExperimentalThe suggestion that the amount of "true protein" in worts and beers might play an important part in affecting haze indicated the need for suitable method of estimation. In previous work on extracts of barley and malt the true protein was separated by trichloracetic acid precipitation (this Journ.,1929, 316). This method was not entirely suitable for worts and beers, because the precipitate formed with trichloracetic acid at 2-3 per cent, concentration is gelatinous and cannot be removed by centrifuging. However, it was later found that, if wort treated with trichloracetic acid at per cent concentration were allowed to stand overnight, the precipitate coagulated and could be separated. There still remained the difficulty that, as the concentration of trichloracetic acid was increased, more nitrogen was precipitated and it was difficult to know where to draw the line between protein and protein derivatives. Tests were also made with acidic substances with large molecules which would combine preferentially with the protein and cause precipitation. Fairly satisfactory results were obtained with low concentrations of phosphotungstic and tannic acids and by acidic dyes, such as Biebrich Scarlet, but again it was difficult to obtain specific end-point which sharply distinguished the true protein from other nitrogenous constituents of wort.

The genesis of the present method lay in the observation that sometimes in the Van Slyke method for the determination of amino-nitrogen slight precipitate was formed. Tests were therefore made by heating worts with low concentrations of hydrochloric acid and sodium nitrite sufficientto deaminate the nitrogenous substances present. It was observed that white precipitate settled out in the cold from unhopped worts, but from normal hopped worts only slight precipitate, or none was formed. Cooling to 40° F. or adding alcohol appeared to precipitate only slight additional amounts of material. Reasons are given later why it is considered that this precipitate represents the true protein of wort. The precipitate offered the alternatives of estimation by turbidity measurements or Kjeldahl nitrogen determinations. For most of this work the latter method was used, but, if the method is found desirable for use in breweries, it may often be sufficient to compare turbidities. Before either type of estimation, it is, however, necessary for correct result to remove all traces of suspended protein matter from wort or beer. In the present work this was effected by high speed centrifuging (5000 r.p.m.) using artificial resin (lucite) cups and an "angle" head on an International centrifuge. It is difficult to choose satisfactory alternative, for, although worts can be filtered bright throughfilter pad of the Seitz type, this is not satisfactory, since the pad would absorb some of the fraction sought. As mentioned, it is possible, at least with some worts, to centrifuge bright in conical oil vessels. In the earlier method 3ml of conc. HCl and ml of 30 per cent, sodium nitrite to 180 ml. of wort were used in process similar to the final one. The results obtained by this method were later found to be too low, as the concentration of hydrochloric add was too high, but some of the figures givenbelow were determined in this way. They serve for comparative purposes and the results obtained were confirmed by the later method. It appeared that the precipitate had maximum insolubility in the region of pH 4 and that at more acid pH an appreciable proportion was dissolved. As the results were of interest study was next made of the best proportions of acid and of nitrite, of times and conditions of heating, using the measurement of turbidity as test. When the amount ofhydrochloric acid was reduced sufficiently to avoid loss with most worts the quantity of acid was found to be so small that the required pa was not reached in exceptional worts of high buffering power. Consequently acetic acid was then used and, after tests for optimum conditions, the method was worked out which is given in Section I.Earlier Results—Effect of Boiling and Hopping The first observations, by the earlier method, showed that the amount of true protein present depended largely upon the extent to which the wort had been boiled. For instance wort from malt 9 which had been repeatedly sterilized gave 0-11 mgrm of true protein nitrogen per 100 ml. at1025° sp.gr., while fresh mash of the same malt after one short boiling gave 1-24 mgrms of complete protein nitrogen or over ten times as much.Also it was found that although boiled sweet brewery worts contained significant amounts of true protein, hopped worts contained only traces or none, e.g.

So it appeared that hops and boiling reduced the amount of true protein. Both these findings were confirmed by subsequent work using the improved method of estimation.True Protein Estimation of Barley ProteinsThe early results suggested that the nitrous acid did in fact estimate only true proteins in wort; but for more direct proof attention was directed to barley extracts in which the true proteins are known to constitute much larger proportion of the soluble nitrogen compounds present, and in which the proteins can be readily precipitated by trichloracetic acid, which is fairly specific protein precipitant. For these tests 120 grm sample of finely ground barley "B" 1940 was extracted with 800 ml. of distilled water with shaking for half hour and the clear centrifuged extract made to 1025 ml. Another 120 grm lot was similarly extracted with per cent potassium sulphate solution and the clearextract made to 1025 ml. 400 ml. lots of each extract were heated at 82° C for 45 minutes, cooled, centrifuged and made to 410 ml. There were thus four solutions, all of which contained the simpler nitrogen compounds of barley. The potassium sulphate extract should contain in addition the albumin and globulin. The distilled water extract should contain albumin together with a little globulin dissolved by the salts in barley. The heated distilled water extract should contain only portion of the small amount of globulin originally present. Thus the four variations in nature and amount of protein were available and on each of these comparative estimations were made of the nitrogen precipitated by trichloracetic acid at per cent, concentration and by the action of nitrous acid, using the later method for true protein. The results are given in Table I. From the table it will be seen that the amounts shown by the two methods of estimation in each extract are closely related to one another over wide range, so that it seems likely that they are precipitating similar material from each extract. Furthermore the differences found between the different extracts follow the expected variation in the protein amounts, which argues for the protein nature of the precipitates.In this series of studies the amino-index (ratio of free amino to total nitrogen) has been used as the main guide to the complexity of the substance under examination, but it is. impossible to deduce the complexity of the nitrous acid precipitate from its amino-index since the free amino nitrogen is removed in the method of estimation. However, since the material precipitated is clearly similar to that precipitated by trichloracetic acid, it seems safe to conclude for this reason also that it is the most complex fraction, because the trichloracetic precipitate has been found to have low amino index (4 per cent.) indicating that it consists mainly of true protein. The nitrous acid precipitate was also shown to consist almost exclusively of protein as largescale preparation from wort contained (moisture and ash free) 14-3 per cent, of nitrogen, and it is probable that adsorbed colouring matters and deamination made the figure slightly below the 15-16 per cent N of pure protein.Another way in which the identity of the material precipitated could be checked was by cross precipitation by the two methods. For this test the per cent, potassium sulphate extract of another barley was used. One portion of 150 ml. was precipitated by heating with sodium nitrite and acetic acid. The precipitate was separated by centrifuging and the nitrogen content determined (after washing). The clear liquid was evacuated to remove nitrous acid and then precipitated by trichloracetic acid to give final concentration of per cent, and the nitrogen content of this precipitate estimated after washing with per cent, trichloracetic acid.

Another portion of the barley extract was first precipitated by trichloracetic acid at per cent, concentration and the nitrogen content of this precipitate determined. The clear liquid remaining from this precipitation was then heated with sodium nitrite and, after cooling, the precipitate separated, washed and the nitrogen content estimated. The results showed again that both precipitation methods removed similar amounts of nitrogen from 100 ml. of the original barley extract, for, out of total of 491 mgrms. the true protein method precipitated 201 mgrms. and the trichloraceticprecipitated 225mgrms. Besides being similar in amount of nitrogen, these two methods must have precipitated almost identical material because, after the true protein estimation, trichloracetic acid treatment only precipitated further 20 mgrms of nitrogen; while, after trichloracetic acid precipitation, the true protein method only precipitated l-7mgrms. of nitrogen. Although similar therefore the two methods are not exactly identical and it will be noted that the nitrous acid precipitated amounts of nitrogen slightly less than those by the trichloracetic acid. part of this difference should be due to deamination, but it is possible that the trichloracetic precipitationalso brings down some proteose as well. It is already recognized that trichloracetic acid is fairly specific protein precipitant (Luck, J. Biol. Chetn., 1928,77, 1) and since nitrous acid precipitates, in the main, identical material it must therefore also precipitate protein. However it was of interest to test the method on solution of purified protein. Many proteins are completely coagulated on boiling and so are not suitable. For the present tests edestin (B.D.H.) was dissolved in warm sodium chloride solution and recrystallized. About 1 grm of the moist, washed precipitate was dissolved in 1700 ml of 20 per cent, sodium chloride and comparative estimations were made of total nitrogen content and of nitrogen precipitated by per cent, trichloracetic acid and by the true protein method. The three methods gave similar results, the trichloracetic add method precipitated 91 per cent, and the true protein method 97 per cent, of the nitrogen present. The results suggest that proteins are precipitated by the present technique but it is also important to seek evidence that near protein compounds are not precipitated under the same conditions, although it seems unlikely that, at least in the near future, method will be found that will exactly distinguish protein from near-protein compounds, if proteins are defined as compounds with molecular weight of 17,000 and above. One possible test of the method is to use the proteoses and peptones from animal sources. However, although obtained more easily and abundantly, exactly-defined individuals of this complexity have not been isolated, that is with the exception of the abnormal, anti-bacterial substance gramicidin and the phosphorus-containing peptone from casein digests. Seven commercial peptones were

tested at the rate of 5 grms per litre and the following percentages of the total nitrogen were precipitated (see p. 231).It appeared that the first two of these preparations contained significant amounts of true protein. Fractional precipitation by alcohol did not remove it. However, precipitation with trichloracetic acid removed the fraction and the peptones so purified showed in dilute solution no precipitation on treatment with nitrous acid. The average complexity of the remaining purified peptones was shown by determining the ratio of free amino to total nitrogen, which gave 9-3 per cent, for Witte peptone and 9-4 per cent, for flesh peptone. A further line of argument for the specificity of the deamination technique is that provided by the extremely small amounts, or absence of, nitrogen precipitated from some worts by this method, although worts are known to contain considerable proportion of their nitrogen in a form approaching protein in molecular size. When these results had indicated the protein nature of the material precipitated by the nitrous acid treatment, study was made of the amounts present in worts under different conditions. In considering the results it should be remembered that small proportion of the deaminated protein escapes precipitation.

Estimations of the solubility of the five times washed precipitates suggested solubility of the nitrous acid precipitate in water equivalent to 0,2 mgrm of nitrogen per 100 ml. and the solubility of the trichloracetic acid precipitate in per cent, trichloracetic acid gave a closely similar figure. Results from Different WortsThe results obtained by the later method for true protein with 18 worts of the set under examination in this series of papers are given in Table II. The results are given as illustrations, not as characteristic figures,for they record the amounts present at the time of analysis in worts preserved by sterilization. While this amount may depend in part on the particular malt, it will be realized from what has been written before that it depends also on how often the wort has been boiled to sterilize it, as is shown in the Table by the letter (N) indicating analysis, on new mash after sterilizing boil or the letter (O) indicating wort which has been sterilized number of times before analysis, and it will be seen that these latter results are lower than those for fresh mashes.A direct comparison of amounts in worts from new and old mashes of the same malt confirmed that the effect of repeated boning is to reduce the amount of true protein present. The results (again as mgrms of true protein nitrogen at sp.gr. of 1025°) were:

Effect of Mashing Conditions on Amount of True ProteinIt was necessary to test whether, in addition to the effect from boiling, variations in mashing condition also might influence the amount of true protein. This was tested by making four laboratory mashes of malt. In two of these the standard amount of 50 grms. of malt was mashed in 360 ml of water, which in one case was distilled water and in the other case brewing liquor. Two other mashes were made in the same volumes of distilled water, but using 150grms. of malt instead of 50. After one hour they were all cooled and the first two made to 515 ml. and filtered and the second pair to 545 ml. followed by filtration. The resulting worts were centrifuged bright, adjusted to 1025° sp.gr. and the total soluble nitrogen and true protein nitrogen were determined 200 ml. of each wort was then boiled for 10 minutes, made to the original weight with distilled water and the resulting precipitates centrifuged, washed and the nitrogen contents determined. On the clear liquids the permanently soluble nitrogen was determined and the amount of true protein nitrogen. The results given in Table III suggest thefollowing points:(1) The amounts of total and permanently soluble nitrogen are greater (at the same sp.gr.) with the more concentrated mashes, according to the well-known effect. (2) Comparing the four mashing conditions, the amounts of true protein in the four unboiled worts are similar to one another but slightly higher for the more concentrated mashes. The same applies to the amounts in the four boiled worts and also to the amounts of coagulated nitrogen. So that, although the amounts are slightly higher for the more concentrated mashes, the mash concentration does not appear to have an important influence on the amount of true protein extracted. Equally the presence of salts in the brewing water appears to have had little effect in this respect.(3) The amounts of coagulated nitrogen reflect the difference between the true protein nitrogenin the unboiled and boiled worts, suggesting strongly that the coagulable nitrogen constitutes portion of that estimated by the deamination technique, as would be expected if this measures true protein. This is supported by the results given earlier, showing reduction in the amount of true protein after repeated boiling.

Effect of TanninThe results given earlier suggest that protein coagulation during boiling in brewery copper would reduce the amount of true protein, but it is also possible that the tannin of hops has further precipitating action. This was indicated by model experiments with commercial tannin purified by solution in ethyl acetate and fractional precipitation by benzene 600 ml. portions of wort at 1043,660 sp.gr. were treated in the cold with this tannin in concentrations ranging from to 67mgrms. per 100 ml. The precipitates were removed and the true protein estimation applied to the clear worts, with the results given in Table IV. These figures show that increasing amounts of tannin cause, at first, rapid and thereafter gradual, reduction in the amount of true protein nitrogen.

It seemed likely that hops would havesimilar effect. To test this an experimental mash from wort at 1040° sp.gr. was dividedinto portions and boiled for one hour, one portion with no hops, one with hops at therate of 1,6 grm. per litre and one at 7,5 grms per litre (corresponding approximately to0, ½ and 2 1/2 lb of hops per barrel). After boiling the worts were made approximatelyto the original volume, strained, centrifuged bright and then analysed for true protein and total nitrogen. The results, calculated to 1040,00 sp.gr., are given in Table V. Theyshow the expected reduction in true protein as result of the action of the hops.

Further experiments indicated that time of boiling, degree of agitation in boiling, andboiling under pressure also might affect the amount of true protein remaining in the wort.Effect of Fermentation on the Amount of True Protein

It is well known that continued shaking of protein solution will result in coagulationof the protein and recent physico-chemical work summarized by the writer (this Journ.,1943,168) has indicated how this may happen by spreading the protein on the surface of bubble with the reactive groups facing in wards to the solution so that when twobubbles come together the reactive groups of the two adjacent films meet and link up to

produce an insoluble film. From this it is clear that similar conditions arise in fermentation when innumerable bubbles collect together in the head, and indeed the resulting protein films have been photographed (this Journ., 1938, 69, Fig. 9). Consequently it might be expected that fermentation would cause decline in the amount of true protein in wort, and this was found in experimental

brewery tests. Two experiments using the later method of estimation gave the following result:It follows that fermentation conditions may have an influence on the amount of true protein in the unfined beer.

Brewery TestsThe general picture presented by estimations with the deamination technique is thus of special nitrogen fraction of the wort which is small in relation to the total amount of nitrogen in the wort and is reduced by boiling, so that variation might be anticipated between different breweries as result of details of plant and conditions in the boiling, hop-back and cooling stages. This protein fraction is further reduced in amount by precipitation with tannin, which may vary in brewery practice with the hop rate and with the percentage in the hops, while fermentation still further reduces the amount.

These findings were checked by analyzing samples from breweries taken after mashing, after boiling (from the refrigerators) and after fermentation (before the addition of finings). Unlike those in Table II the present samples were analysed directly, without previous sterilization, and so give more correct picture of the amounts present. Results are given in Table VI from four breweries (A, B, and D). is the Institute's Experimental Brewery. With Brewery three fermentations were studied, AI is an "all malt brew. All is the corresponding malt with per cent, flaked barley, 6 per cent, malted oats and per cent, invert sugar. While AIII is the malt with percent, invert sugar. As the different fractions were at widely different gravities they have all been calculated for comparison to sp.gr. of 1040,0o. It must be remembered in consequence that the proportion contributed by the second and third mash tuns and coppers is smaller than that suggested by the figures in this form. The results were also calculated according to the proportions in the final brew contributed by the first, second and third worts and the true protein then calculated as percentage on the total soluble nitrogen with the following results

It would appear from this that in some beers traces of true protein may survive.AcknowledgmentsGrateful acknowledgment is made to the breweries who have kindly supplied samples and to Miss V. J. Kemp and Miss B. D. Hadley, who have successively assisted in the work. Summary 1. When the nitrogen compounds of wort or beer are deaminated by heating with, nitrous acid, nitrogen compound is pre cipitated on cooling which amounts to some per cent, of the total

nitrogen of unboiled wort, some per cent, of the nitrogen of hopped wort and 0-2 per cent, or less of the nitrogen of fermented beer.2. Tests by the deamination method on extracts from barley show that the amounts of nitrogen compounds precipitated are closely similar to the corresponding amounts precipitated by trichloracetic acid at concentration of 4-5 per cent. While, in wort, the coagulable nitrogen is included in the fraction precipitated by deamination. These and further tests suggest that the amount of nitrogen precipitated by this method is measure of the "true protein" present—that is the nitrogen in the form of complex protein alone, not that in the form of proteoses, peptones or other protein fragments of smaller size which constitute the bulk of the nitrogen compounds in wort.3. The results of the tests suggest how variations in the amount of true protein mayarise between brews and breweries as the result of variations in the boiling conditions,hops, hop rate and fermentation conditions.