2 THE ANALYST. NOTES ON THE ALCOHOLIC FERMENTATION OF MILK-SUGAR, BY P. VIETH, PH.D., F.C.S. Read at the Meeting, December 8th,, 1886, A PAPER on (6 The Composition of Mares' Milk and Koumiss," whioh I read before this Society a t the November meeting, 1885, gave rise to a oonversation on the alooholio fermentation of milk-sugar, in the course of which it wm remarked that there exists some difficulty to start alcoholic fermentation in solutions of milk-sugar. I n order to convince myself of this fact, I made the following simple experiment : 5 per cent. solutions of cane-sugar and of milk-sugar were made, and German yeast added fo each of them, in the proportion of 1 gramme to 100 C.C. The solutions were kept loosely covered side by side in a moderately warm place, the temperature of which varied from 16" to 28OC.Samples-5 c.c.-were taken daily, evaporated on the steam bath, dried in the air bath at 100QC., and weighed. The cane-sugar solutions were on the evening of the first day already in active fermentation, and the solids diminished rapidly during the early part of the experiment, viz., about at the rate of 30 per cent. per day, the residue of 5 C.C. of the solution weighing, when freshly made, 02574, after one day *1674, after two days -1150, after three days *0787. I n the solution of milk- sugar, alcoholic fermentation was not perceptible; there wa8, however, also a slight diminution of solids. The solutions contained solids :- I. Series. 11. Series. Cane-Sugar. Milk-Sugar. Cane-Sugar. Milk-Sugar. When fresh . . 5.14 4-96 5.36 5.16 4 days old .. 1.18 4.90 1.38 5-00 8 99 99 . . *62 4-80 -64 4.96 12 1, 9 , . L *52 4.66 058 4.90 From these figures it appears that within twelve days the solids of the cane-sugar solution were reduced to about one-tenth of the original amount, while those of the milk-sugar solution diminished by about 6 per cent. only. I have never tried the experiment to keep a plain solution of milk-sugar, without or with yeast, added to it, for a longer period, and see if alcoholic fermentation would have set in, but done so with a preparation of milk containing as much milk-sugar and as little of the other solid constituents as possible-whey. Milk set in Bhallow pans will turn sour and thick in two or three days, if kept at mean temperature. I f the milk is then slowly heated the precipitated casein is contracted and forms lumps of tough curd, while whey is separated, which, provided poor skim milk had been used, will be almost clear.Whey prepared in this manner in November, 1885, was bottled immediately after having separated and cooled down, the bottles corked and wired and removed to a cool vault. The whey was analysed when it was six and twelve months old; after six months already it was found t o be highly effervescent. The whey contained :- 6 Months Old. 12 Months Old, Total Solids . . 6.28 5.46 Lactic Acid . . 1.73 2-58 Alcohol .. *63 -93 In May last I repeated the experiment, and at the same time put aside isome wheyTHE ANALYET. 3 prepared of sweet milk by means of rennet. The two kinds of whey were analysed when fresh and six months old.The sour whey again was found highly effervescent when six months old, but the whey prepared by means of rennet contained a small quantity of gas only. The results of analysis were as follows :- $our Whey. Sweet Whej. Fresh, 6 Months Old. Fresh. 6 Months Old, Total Solids . . 7-51 5.88 6-84: 6.24 Lactic Acid . . *65 2.38 - 1-41 Alcohol .. - -53 c -20 These experiments show that spontaneous alcoholic fermentation sets in if whey is kept for a considerable length of time, and, in the ms0 of sour whey, develops to a very marked degree. I expected that the fermented whey would act as a powerful ferment on milk-sugar solution, and in order to try it made two 5 per c?ent. solutions of milk-sugar containing twelve months old fermented whey, the one in the proportion of ten and the other of twenty per cent.The solutions were kept loosely covered at a temperature varying from 16” to 28”C., and the solids determined daily. Instead of a decrease, I, during the early part of the experiment, found an increase in solids, due, no doubt, to the evaporation of alcohol introduced with the fermented whey; after a time the solids very slightly diminished. The percentages of solids found mere as follows :- 5 per cent. solution of milk-sugar+ 10 per cent. old whey. Newly prepared . . 5.06 5-40 5 9 8 J J . . 5.12 5 9 2 99 11 ,? . . 5.02 5-06 20 per cent. old whey. After 4 days . . 5.08 5.54 In my paper ‘‘ On Mares’ Milk and Koumiss,” I have mentioned that mares’ milk spontaneously undergoes not only lactic but also alcoholic fermentation, sometimes within a very short time ; I have seen mares’ milk, milked at S a.m., frothing with carbonic acid gas at 6 p.m.of the same day. I n the manufacture of koumiss alcoholic fermentation is supported or effected by the addition of koumiss, which is well fermented. It is well known that a beverage, similar to and having all the properties of mares’ milk koumiss, can be prepared of cows’ milk. During the last few years attention has fre- quently been drawn to a new preparation, consisting of fermented cows’ milk, and known under the name of Kef3.r.” I n this case alcoholic fermentation is started in the milk by the addition of a ferment called kefir grains. These grains contain, according to Dr. Kern, of Moskow, a bacillus, to which he gave the name of Diospora Caucasica, and yeast cells, apparently a modified form of Saccharomyces Cerevisia. Milk, to which kefir grains have been added, and which is subjected to a certain treatment, undergoes alcoholic as well as lactic fermentation within an exceedingly short time.After one day the alcohol four d in kefir amounted to -3, after two to three days to 08, after five days to 1 per cent. I dare say every Public Analyst has made the experience that milk samples kept for some length of time get fermented, developing gaseous products. That the solids in such samples decrease to a considerable degree has frequently been discussed. Some4 THE ANALYST. years ago I brought before the Socisty* some observations relating to the speedy diminu- tion of solids in milk, which is kept in small quantities a t mean temperature, and allowing the free access of air to it.I then suggested that this diminution in solids must be attributed to alcoholic fermentation, and am of the same opinion still. One might think of a decrease caused by the formation of di-lactic acid on drying up sour milk, but supposing the whole of the milk-sugar be converted in lactic acid-which is highly improbable-and the whole of the lactic acid in di-lactic acid, which is impossible under the circumstances-then a loss could be caused only equal to a 4 5 to *50 per cent, in milk containing 4.5 to 5.0 per cent. milk-sugar. I have repeated the experiment of keeping small quantities of milk in platinum dishes exposed to the air in a moderately warm place-about 25" C.-for varying periods of time, and compared them as to the amount of solids with other samples of the same milk kept in a stoppered bottle, but otherwise under the same conditions.At the same time I tried to find out what eEict the addition of yeast-1 gramme to 100 c.c.-would have upon the milk under those conditions. The experiment, for certain reasons, was made with very poor sweet skim-milk. The following results were obtained :- Solids in skim-milk kept in stoppered bottles. Fresh ... ... 9-80 10.08 (Lactic Acid *15) ,, S ,, ... 9.32 9.53 ( ,, 1.16) ,, 11 ,, ... 9-32 9.31 ( ,, 1-21> Without yeast. With yeast. After 4 days ... 9.48 9.70 ( ,, 1.01) Solids in skim-milk kept in platinum dishes. 4 days ... 8.80 9.04 8 ?I ... 8.34 8.40 11 9 , ...8.30 8.10 With regard to the samples taken from the bottles, it must be remarked that particles of precipitated casein stuck to the glass, and could not be removed, not even by violent shaking, a circumstance which accounts for the relatively great difference between the milk when fresh and four days old. On reviewing the facts which have been brought forward we see, on the one hand, the difficulty of starting alcoholic fermentation in pure solutions OF milk-sugar as well as in milk by means of added yeast ; on the other hand, alcoholic fermentation readily developed in milk by a certain ferment ; further, alcoholic fermentation setting in spontaneously in whey and milk, if kept for some considerable length of time ; and a very marked diminution of solids within a very short time, which seems to be at- tributable to alcoholic fermentation, if milk is kept in small quantities exposed to the air in a moderately warm place.I do not think I need apologise for bringing these observations under your notice, as they have a direct bearing on the much-dhcussed question of analysing old samples of milk, but I have to apologise for leaving the question a t a stage where a more close investigation ought to commence. In common with many analysts engaged in practical work, I do not always find time to further investigate into questions, the clearing up of which is very desirable, Without yeast. With yeast. Otherwise, the results want no commentary. * THE ANALYST, 1882, p. 213.THE ANALYST. 5 DISCUSSION. Mr. HEHNER said there was an article in the market something of the nature of a ‘‘ bland,” but it was perfectly transparent, and he understood it was the whey of the milk fermented.It contained no acid, and he should like to ask how it had fermented. Mr. KINGZETT regretted that ha had not heard the early part of Dr. Vieth’s communication, but he thought that a brief description of some results which he had obtained with reference to the fermentation of milk and milk-sugar some time ago might have some general interest for the members. The experiments in question formed part of an exhaustive investigation, made with a special object, and were as follows :- A quantity of fresh milk was taken, and the milk-sugar present in it was determined by first of all diluting 10 C.C. with 90 C.C.of water, and then estimating it by the well-known process which makes use of Fehling’s solution volumetrically. It contained 40422 grammes milk-sugar in each 100 C.C. It was also experimentally proved to be quite free from lactic acid. 50 C.C. of this milk was diluted with 50 C.C. of water, and the mixture placed in a flask loosely covered with filter paper, on July 7th. On July 20th, the mixture was gently warmed, to complete coagulation, and was then filtered, the filtrate being made up to a known volume by washing the coagulnm with warm water, after which the sugar present in the filtrate, and the acidity thereof, were respectively determined. Gugar originally Sugar present Difference due to present. at end. fermentation. Final Acidity. 2.2 1 1 grammes.None. 2 *2 1 1 grammes. 28 C.C. 2 NaHO. A microscopic examination of the fermented mixture revealed the presence of the bacterium Zuctis in great numbers, and the bacteilium termo was also seen to be present in a very active state. As the fermentation of 2-211 grammes of milk-sugar should yield, if entirely fermented into lactic acid, the same weight of that substance, whereas the acidity of the fermented mixture was only equal to 0.253 gramme of that Substance, it was concluded that the lactic acid had itself been resolved by subsequent changes into acetic, butyric and propionic acids, and that, finally, these acids had been split up by oxidation into carbonic anhydride and water ; moreover, as the fermentation was conducted fortuitously, it doubtless was not of a pure character. Experiment 2.This was made with another sample of milk, containing 3.88 grammes milk-sugar in each 100 C.C. ; the mixture being allowed to stand from August 4th to August 13th. Sugar originally Sugar present Difference due to present. a t end. fermentation, Final acidity. I n this experiment the quantity of lactic acid found is greater in proportion to the sugar employed, and, doubtless, this result was due to the fact that the milk was not allowed to stand so long as in the previous experiment. I f the whole of the milk- sugar which had disappeared had been changed into lactic acid, and none of this had been destroyed by subsequent chemical changes, there would have been obtained 096 gramme lactic acid, instead of 0.3375, actually calculated to be present.This experiment was conducted as follows :-Some 245 C.C. of fresh milk was inoculated with 5 C.C. of very sour milk; then the acidity and the milk-sugar of the inoculated mixture were carefully determined ; after which two quantities of milk 50 C.C. each were respectively diluted with 60 C.C. water, and allowed to stand from August 20th to August 24th. 1.940 0.980 -960 37.6 C.C. NaHO Experiment 3. Sugar originally Sugar present Difference due to Original Acidity present. a t end. fcrmentation. acidity. at end. A. 2.155 1.675 -510 0.5 C.C. 44.5 C.C. i$ NaHO B. 2.185 2.654 -53Q 0.5 C.C. 45.7 C.C. $ NaTlO6 THE ANALYST. The results of the examination of the two mixtures are seen to be thoroughly and mutually confirmatory in character. Taking A, the loss of the milk-sugar could produce a maximum of -510 gramme lactic acid, whereas the acidity of the ultimate mixture was equal to ,4005 gramme, leaving apparently only -1095 gramme to be accounted for.It is, however, to be noted that the approximation of the theoretical and practical quantities of lactic acid is much greater than in either of the previous experi- ments, and doubtless the reason is that the mixtures were only allowed t o stand over four days. In order to test the supposition that alcohol might also be found amongst the products, thus accounting for the difference, one half of each mixture A and B was subjected to distillation, and the specific gravity of each distillate after being made up to the original volume, was determined and found to be respectively -99977 and -99973. Alcohol was thus proved to be present in each fermented mixture, in more than sufficient amount to fully account for the deficiency in lactic acid produced. The explanation is this : the alcohol which was found present was all derived from the fermentation of lactose ; whereas a small proportion of the total acidity of mixtures A and B was derived from butyric and other soluble fatty acids, which resulted from the rancidity of the cream or fat contained in the milk, thus swdling the amount of acid which is, as explained, not derived solely from the lactose.Either, then, the 6acterium Zactis performs the chemical changes whereby lactose is resolved both into alcohol and lactic acid, or it was accompanied in these experiments with other forms of microscopic life, which fermented a part of the lactose into alcohol and carbonic anhydride. Dr. VIETH, in reply to Mr. Hehner, said there was no impossibility about having L preparation which might contain whey, and might ferment a t tho same time, but he did not think it was suggested that the preparation referred to consisted entirely of whey. What had principally struck him was, on the one hand, the great difficulty of starting fermentation of milk-sugar by adding yeast as the fermenting power, and, on the other hand, the quick fermentation by adding some other ferment,