BUTTER FAT AND COCOANUT OIL IN MARGARINE 123 STANDARDS FOR MALT VINEGAR. BY A. CHASTON CHAPMAN, F.I.C. (Read at the Meeting, March 6, 1912.) DESPITE the numerous attempts which have been made to define malt vinegar and to lay down analytical limits for its composition, the present state of affairs in regard to that condiment can scarcely be described as satisfactory. It is clearly a much simpler matter to say what malt vinegar should be in terms of the materials used or the methods adopted in its production than to lay down chemical standards of composition. The latest pronouncement in this country appears to be contained in a letter addressed by the Assistant-Secretary of the Local Government Board to the Secretary of the London and Country Vinegar Brewers’ Association. In this it is stated that malt vinegar ‘( is derived wholly from malted barley or wholly from cereals, the starch of which has been saccharified by the diastase of malt.” Under the general definition of ‘( vinegar,” moreover, it is to be “ a liquid derived wholly from alcoholic and acetous fermentations; it shall contain not less than 4 grammes of acetic acid (CH,COOH) in 100 cubic centiinetres of vinegar ; it shall not contain124 CHAPMAN : STANDARDS FOR MALT VINEGAR arsenic in amounts exceeding 0.0143 milligramme in 100 cubic centimetres of vinegar, nor sulphuric or other mineral acid, lead, or copper ; nor sball it contain any foreign substance or colouring matter except caramel." On June 26, 1906, the United States Department of Agriculture issued a circular (No.19) giving definitions of various kinds of vinegar. I n this circular malt vinegar is defined as '( a product made by the alcoholic and subsequent acetous fermentations, without distillation, of an infusion of barley malt or cereals, whose starch has been converted by malt, is dextro-rotatory, and contains, in one hundred (100) cubic centimetres (20" C.), not less than four (4) grammes of acetic acid, not less than two (2) grammes of solids, and not less than two-tenths (0.2) gramme of ash; and the water-soluble ash from one hundred (100) cubic centimetres (20" C.) of the vinegar contains not less than nine (9) milligrammes of phosphoric acid (P205), and requires not less than four (4) cubic centimetres of decinormal acid to neutrttlise its alkalinity." Some time ago a certain firm of vinegar manufacturers, finding that their vinegar, although made strictly in accordance with the requirements of the United States Department of Agriculture-that is to say, wholly from barley malt and cereals, without the use of any sugar whatever-did not comply with the analytical require- ments of the Department, asked me, if I could, to explain the apparent discrepancy. On referring to the above definition, it will be seen that malt vinegar must be, among other things, dextro-rotatory ; and presumably any vinegar which did not comply with the definition in this respect would not be admitted into the United States under the description of " malt vinegar." Now, it so happened that, whilst the vinegar manufactured by the firm in question sometimes had a dextro-rotatory action on polarised light, it was very much more frequently appreciably lavo- rotatory.Thus, twelve samples which 1 examined, and which represented in the aggregate almost the whole of the vinegarmat that time in stock, were all laevo- rotatory, the rotations varying from -0.56" to -0.76' when examined in a tube 200 mm. long, using sodium light. Inasmuch as it was absolutely certain that no sugar of any description had been used in the manufacture of this vinegar, it became necessary to explain this Iaevo-rotation. I t was clear that certain laevo-rotatory constituents had been extracted from the grain during the manufacture of the vinegar, and a moment's consideration sufficed to show that these constituents could only be the proteins or certain of the products of their hydrolysis, such as the proteoses, peptones, and amino-acids. I t may be pointed out here that the vinegar in question contained an average of 0.085 per cent.of nitrogen, equivalent to 0.53 per cent. of proteins. I t is, of course, very well known that the naturally-occurring proteins are almost invariably strongly laevo-rotatory, and the same applies to many of the products of their hydrolysis, particularly the proteoses and peptones, the specific3 rotatory powers of these substances for sodium light varying from - 50" to - 80" in a 200 mm. tube, using sodium light. Asparagine, which occurs in malt wort, is slightly laevo-rotatory, and certain other of the amino-acids and amides are slightly dextro-rotatory. The important point, however, is that the more complex products of hydrolysis are those which are most strongly laevo-rotatory. I t is clear that, if it were possible so thoroughly to ferment away the carbohydrate constituents of malt wort as to leave practically nothing undecomposed, the protein matters presentCHAPMAN : STANDARDS FOK MALT VINEGAR 125 might quite well cause the resulting wash to exhibit a lzvo-rotatory action, and my first experiments were directed to ascertaining whether such was the case.It is, of course, the practice of vinegar manufacturers to carry out the fermentations of their worts in the presence of diastase, siiice by this means the malto-dextrins and other comparatively stable carbohydrates are converted into fermentable substances, and so the maximum amount of alcohol, and subsequently of acetic acid, is obtained.I n the experiments described below I have therefore carried out the fermentations of the worts in question in the presence of active diastase-that is to say, the worts were not boiled prior to the introduction of the yeast. I n the first place, two worts having the same sp. gr. (1070') were prepared from the same malt, and were pitched with a small quantity of ordinary distillers' yeast, the temperature being kept within the ordinary limits of practice. The fermentations were started on December 4, and five days later the sp. gr. of each had fallen below 1000". The following results show the optical activities in a 200 mm. tube, using sodium light, of these worts on three different dates: No.1. No. 2. December 9 ... ... ... -0.40"' - 0.080 ,, 11 ... ... ... - 1.00" - 0.70" ,, 13 ... ... . . . -1.00" - 0.80" Two further experiments were then made with worts of rather lower sp. gr. (1050'), and employing the yeast actually used in the vinegar factory in question in place of the distillers' yeast used in the preceding experiment. The following results were obtained : No. I. No. 2. December I1 ... ... ... +0*54" - ,, 13 ... ... ... +0*04" + 0.20" ,, 14 ... ... -0.44" - 0.04" I t will be scarcely necessary to point out that these worts were not examined polarimetrically until their density showed that almost the whole of the carbohydrate matter had disappeared, and for that reason no considerable plus rotations are shown. These results show very clearly that, with a suficiently perfect fermentation, worts prepared from malt alone are capable of yielding liquids which are appreciably 1 zvo-rot at ory.I n the next place, experiments were directed to proving that the Izvo-rotation of these worts was in fact due to the presence of proteins and their products of hydrolysis. If such were the case, it should be possible, by the removal of these substances, to convert a lzvo-rotatory liquid into either an inactive or a dextro- rotatory one. I t is well known that, by the combined use, under proper conditions, of aqueous solutions of mercuric acetate and phosphotungstic acid, both the proteins and the majority of the products of their hydrolysis may be removed from organic liquids without effecting any change in the carbohydrates present.The mercuric acetate solution used in the following experiments consisted of a saturated aqueous solution of the salt; the phosphotungstic acid solution consisted of a 25 per cent. solution of the substance in water without the addition of mineral acid. I n the first place, the mercuric acetate and the phosphotungstic solutions were added to some126 CHAPMAN STANDARDS FOR, MALT VINEGAR fermented malt wort made in the laboratory, and having a rotation in a 200 mm. tube of -0.45". After filtering and making the requisite correction for the dilution due to the volume of the reagents eniployed, the liquid was Iound to have a rotation of +0*46". Similar experiments were then made with five samples of lzvo-rotatory vinegar, with the following results, the numbers under (a) representing the rotation of the vinegar prior to the addition of the reagents, and those under ( b ) the rotation after filtration : i 4 ( b) No.1 ... ... ... ... -0.63" + 0.02" ,, 2 ... ... ... ... - 0.60" inactive ,, 3 ... ... ... ... - 0.65" + 0*08" ,, 4 ... ... ... ... -0.63" -+ 0.13" ,, 5 ... ... ... ... -0.72" + 0.06" These results entirely bear out those obtained with the malt wort itself, and show that the lzvo-rotation of this vinegar is, in fact, due to the presence of proteins and the products of their hydrolysis. During the process of fermentation the amino- acids, and to a less extent the proteoses and peptones, will have been utilised by the yeast for its nutrition, and the residual nitrogen in the vinegar must consist largely of proteins and of their more complex hydrolytic products--that is to say, of those substances which exhibit the greatest degree of lmo-rotation.I may perhaps add that I have made experiments with the mercuric acetate and phosphotungstic acid solutions in the presence of a small quantity of lzvulose, and have found that when used under proper conditions these reagents bring about no change whatever in the rotation of that carbohydrate. This is borne out by experiments which have recently been made by Neuberg and Ishida (Biochem. Zeitsch., 1911, 37, 142- 169), who call attention to the errors which may result in the polarimetric estimation of sugar in natural products owing to the presence of lzvo-rotatory proteins and amino-acids. These authors have also made use of mercuric acetate and phosphotungstic acid, and have recorded a great many results to show that these reagents have no effect on the rotation of the carbohydrates present.The above results show very clearly that a genuine malt vinegar-that is to say, a vinegar prepared entirely from malt and cereals whose starch has been converted by diastase -need not necessarily be dextro-rotatory, but may be either lzvo-rotatory or optically inactive. As further showing the fallacy of insisting on dextro-rotation as an indication of genuineness, it is only necessary to point out that the unfermentable matter in many samples of commercial invert sugar is optically inactive, aad that even if a considerable proportion of invert sugar were used in the manufacture of the vinegar, the latter would not necessarily be lzvo-rotatory.It would appear, there- fore, that unless the United States Department of Agriculture modify their existing regulation, considerable injustice may be done, not only to the home producer, but also to vinegar manufacturers in other countries, whose inalt vinegar may be optically inactive or slightly lzvo-rotatory, and who may wish to introduce it into the United States. Whilst many samples of malt vinegar are undoubtedly dextro- rotatory, to insist upon this analytical requirement would be tantamount to exclud- ing from the United States the vinegar of every manufacturer who is successful inCHAPMAN : STANDARDS FOR MALT VINEGAR 127 very thoroughly fermenting away the carbohydrate constituents of his worts, and would practically amount to putting a premium on bad working, to say nothing of tempting dishonest manufacturers to make their vinegar comply with the require- ments by the appropriate additions.Perhaps the simplest way of manufacturing a malt vinegar to insure its satisfying the United States authorities in respect of its rotation would be to eniploy in its manufacture a proportion of coinmercial glucose. The requirements of the United States Department of Agriculture in respect of the proportion of soluble phosphoric acid in the ash of malt vinegar affords a further example of the inadvisability of putting forward standards for manufactured products, the composition of which must in the nature of things depend somewhat considerably on the precise methods adopted in their manufacture.The amount of phosphoric acid passing from the grain into the wort, as also that left in the soluble conditioii when the resulting vinegar is incinerated, must depend very largely on the mineral composition of the water used for mashing. Although this is sufficiently obvious, it may perhaps be of interest to record the following experiments which illustrate this point. Two different malts were taken, and were mashed (a) with distilled water, (b) with water containing about 20 grains per gallon of total dissolved solid matter, and (c) with a very hard water cortaining more than 100 grains per gallon of calcium sulphate. The mashes were made under precisely the same conditions of time and temperature, and were, in fact, treated in the same way throughout.At the end of the mashing period of one and aquarter hours the mashes were filtered, and the total phosphoric acid determined in the worts in the usual way. MALT A. Phosphoric Acid (P,O,). Distilled water ... . . . 46.56 grains per gallon. Water containing 20 grains of total dissolved solid matter ... 42.44 ,, ,* Very hard water ... ... 30.44 ,, 1 , Distilled water ... . . . 44.77 grains per gallon. Water containing 20 grains of total dissolved solid matter ... 37.61 ,, 9 , Very hard water ... ... 26.88 ,, 9 , MAL'r B. Phosphoric Acid (P,O,). I may add that in the preparation of the above worts the same proportions of malt and water were used in all cases. I have at different times examined several samples of genuine malt vinegar made with very hard water, and have found that when ignited they yielded ashes which contained almost the whole of the phosphoric acid in the insoluble condition-that is to say, as tribasic calcium phosphate. On extracting these ashes with water, the resulting solutions were practically free from phosphoric acid, and were almost neutral. I think I have shown that the United States definittion of malt vinegar requires amendment in at least two respects, and have incidentally provided yet another example of the dangers of setting up official standardi for the composition of manufactured foodstuffs. My best thanks are due to my assistant, Mr. R. L. Collctt, for help in connection with this investigation.