Nov.. 1916 THE JOURNAL OF INDUSTRI.4L AND ENGINEERING

Nov.. 1916

T H E J O U R N A L OF INDUSTRI.4L A N D ENGINEERING C H E M I S T R Y

be avoided a n d t h e y are not in a n y way objectionable. T h e y sometimes startle a canner who sees t h e m for t h e first time a n d the chemist inspecting his product should be able t o explain them. K i t h some products, patches of iron sulfide, apparently in colloidal form a n d sometimes mixed with particles of food, adhere t o t h e sides of t h e can. These patches of iron sulfide are not frequent on t h e whole but have been found in considerable numbers. They vary in size and sometimes their thickness is so great t h a t they become mixed with t h e food in their immediate vicinity and are objected t o b y buyers who do not understand their nature. When large, a n d especially when they become mixed with t h e food, these patches are unsightly b u t t h e amount of iron present is really exceedingly small. T h e cause of these patches is only partially understood. They always form like the spots just referred t o on pea cans on the portion of t h e can t h a t is uppermost after processing and cooling, which therefore does not come in contact with t h e food. On storage, t h e spots gradually become less conspicuous. T h e conditions leading t o their formation are not entirely understood, b u t i t has been determined t h a t t h e y are less numerous and smaller as t h e can is fuller and freer from oxygen. Before opening a can of food, i t is often of value t o determine t h e vacuum in t h e can b y means of a suitably equipped gauge. This is a matter which has no significance in itself b u t may be of value when considered in connection with other data. T h e amount of tin (or perhaps soluble tin) and iron in acid fruits may be significant when considered in connection with other d a t a . On t h e other hand, with vegetables, such as asparagus, string beans and pumpkin, whose action on t h e t i n is believed t o be due t o amino bodies, .there is no relation between t h e amount of t i n in t h e food and t h e amount of hydrogen in t h e gas content of t h e can. We are just beginning t o understand this subject and all d a t a bearing on it is of value. I t m a y sometimes be of interest t o determine t h e composition of t h e gas in a can with bulged ends. When t h e can is sealed t h e air is not entirely removed. Some carbon dioxide is formed in processing and some may be present in t h e food before processing, especially if it is not sterilized very promptly. Hydrogen is formed b y the action of fruit acids on t h e metal of the container. It has been pointed out b y Baker’ t h a t hydrogen is not found in t h e gases of t h e can until oxygen has disappeared. T h e composition of t h e gas present, therefore, m a y sometimes serve t o confirm d a t a obtained b y other methods of examination. XATIONAL CANNZRS ASSOCIATION, WASHINGTON, D. C.

-

THE CHEMICAL COMPOSITION OF COMMERCIAL GLUCOSE AND ITS DIGESTIBILITY By J . A . WESEHERAND G. L. TELLER Received June 29, 1916

T h e t e r m commercial glucose has been applied t o a product obtained b y the action of certain catalytic agents upon refined starch, until t h e starch has lost 1

Original Contributions, 8th Inlevn. C O I Z RA~p .p l . Chem., 18, 45.

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its identity as such a n d has been converted into a series of products, consisting of d-glucose, maltose and different forms of dextrin. The dilute liquid thus produced is clarified b y passing over boneblack a n d is t h e n carefully evaporated, either t o a solid substance or t o a syrupy condition known as glucose syrup. T h e glucose t h u s produced in this country is made entirely from corn starch and, for this reason, t h e syrup is designated as corn syrup. It consists of about onefifth its weight of water, very minute traces of nitrogenous bodies, a very small amount of ash a n d a group oE carbohydrate bodies, consisting of dextrose, maltose and probably several forms of dextrin, and paralleling closely t h e bodies of a similar nature obtained b y t h e action of malt diastase and other diastases upon starch. There is apparently a bbrder line between t h e bodies commonly known as dextrin and, t h a t known as maltose, which has led t o great confusion in t h e minds of t h e chemists who have investigated this subject, largely because of the difficulties attendant upon t h e separation of such bodies from maltose on t h e one hand, and t h e different forms of dextrin, on t h e other, a n d t h e consequent confusion with these bodies and with mixtures of them. This confusion of ideas concerning glucose syrup has been participated in b y a large number of investigators, extending in time over a period of 2 0 or 30 years, a n d has led t o t h e introduction into t h e literature of such terms as “gallisin,” “isomaltose,” “malto-dextrin” a n d others. T h e voluminous literature on this subject appears t o have been reviewed completely b y various writers without coming t o a n y definite conclusions as t o results. T h e latest edition of such a n authoritative work as Allen’s“Commercia1 Organic Analysis” (4th Ed., 1701. I , I ~ I Z ) , dismisses t h e matter with t h e following brief summary: “Gallisin as hitherto obtained is not a definite compound, and i t appears advisable only t o retain the term as synonymous with unfermentable matter. The whole question of the structure of starch, the nature of the various dextrins and of isomaltose still remains a vexed question in carbohydrate chemistry and the utmost confusion exists as regards the subject. [The reader is referred t o Ling’.; article on Starch in Sykes’ ‘TextBook of Brewing’ (1g07).”] A review of t h e earlier literature is quite fully set forth in t h e third edition of the same work, published in 1898, and in Brown’s “Handbook of Sugar Analysis’’ (1912),under t h e title of Iso-maltose. A clear and concise statement of what appears t o be t h e general understanding of these terms, as they are used, is embodied in t h e following definitions, as given in t h e “Century Dictionary and Encyclopedia:” “Gallisin, in chemistry, a substance analogous t o dextrin, obtained by fermenting with yeast a solution of commercial glucose or starch sugar and adding t o the residual liquid absolute alcohol in excess. Gallisin is precipitated as a white powder, of faintly sweetish taste, hygroscopic, dextrogyrate, incapable of fermentation and yielding dextrose by prolonged heating with dilute sulfuric acid. Probably identical with iso-maltose, CIZH z z O ~(Sadtler, ~. “Handbook of Industrial Chemistry,” p. I 78.) “Iso-maltose, a substance formed together with maltose from starch on diastatic digestion. It has been produced synthetically from dextrose, does not ferment and is isomeric with maltose, C12H2?011. “Malto-dextrin, a variety OF dextrine or starch-gum, produced in ‘mashing’ brewers’ malt as an intermrdiate product between

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T H E J O C K S , 1 L OF I S D I ‘ S T R I A L A N D EArGILTEERISG C H E M I S T R Y

starch and fermentable glucose. Its claim t o be considered a distinct substance is not clearly established.”

T h e presence OE so-called gallisin or iso-maltose in glucose has been attributed b y some authors t o a reversion process brought about by the action of dilute acid upon dextrose during t h e process of manufacture. This has led t o an apparent misconception of t h e significance of its presence and has brought in question t h e value of this portion of glucose as an article of food. One recent writer has gone so far as t o state t h a t this body is not only unfermentable. b u t is not hydrolyzable by enzymes,’ while Allen states:? “ B y heating with dilute sulfuric acid for some hours, gallisin yields a large proportion of dextrose, b u t its complete conversion has not so far been effected.’’ I t has been t h e purpose of t h e work upon Tvhich this article is based t o clear. u p some of these points, especially with reference t o the assumed presence of an unfermentable body which is not hydrolyzable by enzymes a n d which consequently would not serve as an article of food. SAMPLES C S E D Ih- E X P E R I M E S T S

I n carrying out this work we have made about 600 comparative fermentation tests upon glucoses, starches. foodstuffs and various substances used in control experiments and have supplemented many of these by more or less complete analytical determinations t o show t h e nature of t h e residual products after the fermentation has been completed. I t should be borne in mind in this connection t h a t yeast used for fermentation is necessarily of a more or less variable character, as are also t h e various diastatic enzymes available for investigations of this kind. T h i l e recognizing t h a t there are more or less differences in glucose of different manufacture and also in different lots of glucose of the same manufacturer, we have confined our work largely t o a few lots of glucose! purchased in t h e market.3 and apparently suitable for this purpose because of fulfilling the conditions esliibited b y t h e glucoses experimented upon by those who have claimed t h e presence of the unfermentable body known as “gallisin“ or “iso-maltose.” The solids in t h e samples examined varied from 79.8 t o 83 per cent. ASH-The ash of glucose, while minute in amount, is always present. I t s source is partly t h e grain from which t h e starch is derived, and partly certain ingredients used in the manufacture of glucose. For these reasons, t h e composition as well as the amount of ash will necessarily vary. Phosphoric acid is present in small amounts and r e r y probably comes from t h e grain. T h e same is t r u e of minute traces of potash and possibly also of t h e lime. Soda is added during the process of manufacture. Traces of iron are accidentally present. The full analysis of the ash (amounting t o 0.34 per cent) of one sample of t h e glucose is given below. Geoffrey Martin, “Industrial Organic Chemistry,” 1912. “Commercial Organic Analysis,’’ 1 (18981, 361. T h e various lots of glucose used were purchased from Bunte Brothers, Chicago, Ill., and were such as they use regularly in the manufacture of high-grade confections. They represent the commercial product of two different glucose manufacturers. 1

PERCESTAGE AXALYSIS OF , 0.15 Calcium Oxid ) 0.03 Trace . Trace

.

v01. 8,NO. 11

ASH OF GLIJCOSS Sulfur Trioxide (soo).. 0,O-i

THE

0.03 Carbon 0.09 Chlorine (Cl). , . . , . . . . Trace(a) ( a ) In the ash, as burned, no chlorine was obtained, in spite of t h e fact t h a t the ash was distinctly alkaline. When, however, t h e glucose is burned with the addition of chlorine-free caustic soda, chlorine is found to the extent of 0.16 per cent.

.

The above ash was absolutely white and contained no traces of copper or other poisonous metals. We made a T-ery extensive examination of glucose for arsenic and were unable t o detect the presence of a n y trace of this material. Sulfites, which during t h e early history of the manufacture of glucose were used for whitening t h e product, are now no longer used, a n d no trace of t h e m was found in the glucose examined. N I T R O G E K O T S BODIES-lvhile starch used in t h e manufacture of glucose is freed as far as possible from nitrogenous matter, it is practically impossible t o remove the last traces, so t h a t very minute amounts of these bodies stili remain in t h e finished product. X careful examination of t h e glucose examined for nitrogenous bodies gave t h e following results (percentages) : Ammonia and Amino Nitrogen.. . , . . . . . . . . . . . . . . . . . 0.00012 Protein Nitrogen.. . . . . , . Equivalent to Protein (

C;ZRBoHTDRATES-The results of the work of various investigators during the past 2 0 years has been t o establish in glucose t h e presence of dextrose, maltose and dextrins. I n our own experiments %e have determined the presence of these bodies b y methods somewhat different from those used by others. Our fermentation tests were conducted substantially as follows: A glass bottle of about joo cc. capacity is used t o contain the fermenting mass, and during the process of fermentation is immersed in water in a metal tank having a false bottom and suitable for keeping the fermentation bottle a t any desired temperature. The temperature ordinarily used was 3 j o C. The material to be fermented was dissolved in water so that the amount of water present was zoo cc. The gas developed was collected in a large measuring jar, about 4 in. in diameter atid 16 in. high, suitable for collecting 2,000 or more cc. of gas. The top of this jar was connected by glass and rubber coiincctions, with a bottle containing the fermenting liquid., Before the fermentation, the collecting jar was filled with water to the zero mark, or rather slightly above, t o provide for the first escape of water, which always takes place to release tension. As fermentation progresses, the water escapes from an opening in the bottom of the jar and passes into a small control bottle of about 4 oz. capacity through a glass tube which extends t o near the bottom of the small bottle. I t then escapes from the top of the small bottle through another glass tube, thus providing a trap t o protect against the entering of air from the outside aucl to allow for absorption of the gas by water in thc collecting jar near the close of the experiment. Used in this way the apparatus is suitable for fermenting z to 8 g. of sugar, and collecting the gas produced so t h a t it may be measured with a considerable degree of accuracy. In these experiments 5 g. of sugar were ordinarily used. The amount of gas produced varies from day to day, depending upon the special lot of yeast used for thc iermentation. The yeast used was the ordinary compresseti yeast as it is sold largely in the markets throughout the country for the use of bakers in the manufacture of bread: the amount of such yeast was large, to produce a rapid fermentation, generally 15 g. The variation is ordinarily greater with different makes of compressed yeast than with the same make as it is

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used from day to day. There are some remarkable differences in this respect, which indicate that the results of fermentation are within certain limits due to the special kinds of yeast present, or to certain kinds of bacteria which appear always to accompany the yeast in greater or less amount The amount of gas ordinarily produced from 5 g. of cane sugar, when 15 g. of compressed yeast were used, varied from 1050 to 1100 cc., and reached its maximum amount in from z 1 2 to 3 hours.

The relative activity of t h e yeast used on different days can be determined b y making a series of experiments on fermentation with pure cane sugar. When t h e work is carefully done, very concordant results can be obtained and this method can be used with a considerable degree of accuracy in estimating t h e a m o u n t of fermentable sugars in a sample of material, calculating from the amount of gas produced from t h e sample a n d from t h e cane sugar used in control, t h e equivalent amount of cane or other fermentable sugars which t h e sample contains. It is found t h a t pure dextrose gives substa'ntially 9 5 per cent of t h e amount of gas t h a t will be obtained b y t h e same amount of cane sugar fermented under t h e s a m e conditions. This is as we would expect from t h e molecular equivalents of these two sugars. Following out this method of fermentation on glucose, we found t h a t a certain a m o u n t of glucose is always fermentable a n d a still larger a m o u n t is not fermentable until it has been converted into some other form of carbohydrates b y t h e use of diastase, either from malt extract or from some other source. I n some series of experiments, gas was obtained on t h e original glucose, equivalent t o 28 t o 33 per cent calculated as cane sugar. The results of two series of these determinations are shown in Table I . TABLE1 Glucose A: 79.7 Per cent Solids Glucose B: 81.7 Per cent Solids Reducing sugars were in all cases determined by Fehling's solution weighing the resulting cuprous oxide in a Gooch crucible and calculatini the results according t o the Tables of Munson and Walker (Bureau of Chemistry, U. S. Dept. of Agric , Bull. 107). A B Total Gas Produced from 5 grams Cane Sugar.. . . . . . . . 1040 cc. 1100 cc. Total Gas Produced from 10 grams Glucose.. . . . . . . . . . 690 cc. 720 cc. to Cane Sugar.. . . . . . . . . . . . . . . . . . . 3 3 . 8 % 32.7% GAS EQUIVALENT: to Dextrose.. .................... 35.5 34.6 UNFERMENTED REDUCING SUGARS:

FERMENTED REDUCING SUGARS'Calcu

Calculated as Maltose APPARENTDEXTROSE. Calculated from gas and reduction. APPARENT MALTOSE Calculated from gas and reduction APPARENT DEXTRINS(by difference).

43.7

. . . . . . . . . . . . 11.7 22.9

. . . . . . . . . . . . . . . .44.8

47.1 17.2 16.4 47.7

Glucose B, containing 81.y per cent total solids, gave as much gas as would cane sugar, equivalent to 32.7 per cent of the weight of glucose taken. The unfermented residue contained reducing sugar equivalent to 8.2 per cent of dextrose. The original reducing sugar in the sample, calculated as dextrose, was 34.6 per cent; deducting the amount of unfermented from this leaves a difference of 26.4 per cent dextrose, which was fermented. According to our premises, as stated, if this had been all dextrose, we should have had a production of gas equivalent to substantially 26.4 per cent of dextrose, but we find from our experiments that we have a gas production equivalent to considerably more than this. On the other hand, if we had calculated all our reducing sugars as maltose we should have had 61.6 per cent maltose in the original sample, and 14.5 per cent maltose by reduction after fermentation. This would leave of fermented

IO11

sugars, calculated as maltose, 47.1 per cent, which is much higher than we have found by fermentation. Following out a simple arithmetical calculation, based upon these two series of figures, we can calculate the proportion of maltose and dextrose in the fermented sugars. Thus if the reducing sugars had been all maltose we should have found 20.7 per cent more than if they were all dextrose. Since a considerable part of the fermented reducing sugars of the glucose is maltose and the remainder is dextrose and on taking our gas equivalent of dextrose in comparison with dextrose we have a higher amount of fermenting sugar present than if we take it as cane sugar or maltose, it is well to take the mean between the apparent amount of sugar as dextrose (34.6 per cent), and the apparent amount of sugar a5 cane sugar or maltose (32.7 per cent), which gives 33.6 per cent. On this basis, if the fermented sugar had been all dextrose we should have had 7.2 per cent less than we found by fermentation. By using the correct proportion,' then, of the amount of reducing sugars present we have 20.7 : 7 . 2 : : 47.1 : x , which would give 16.4 per cent of maltose. The difference between 16.4 per cent, the apparent maltose, and 33.6 per cent, the total fermented sugars, is 17.2 per cent, as the amount of dextrose fermented. Deducting the amount of fermentable sugars found from the total carbohydrates in the glucose, as previously determined, we find the dextrin to amount to 47.7 per cent. Working in the same manner, we find the dextrose insample A to be 11.7 per cent, maltose 2 2 . 9 per cent, and dextrin 44.8 per cent.

I t is t r u e t h a t if we h a d used another compressed yeast we would have obtained results a little different f r o m these, b u t i n all cases we found t h e results t o show a large proportion of dextrose t o t h e maltose, a n d yet t h e maltose was present in considerable quantity. This is a method which could readily be p u t into practice for factory control, a n d in our opinion it would be very satisfactory for t h e purpose if t h e proper compressed yeast were used a n d blank experiments conducted with glucose of known quality. A few slight corrections might be introduced, b u t for our experimental purposes, t h e y were unimportant. THE

FERMEKTATION

OF

GLUCOSE

AFTER

DIGESTIKG

W I T H A COLD W A T E R E X T R A C T O F MALT FOR T H E PURPOSE

O F CONVERTING DEXTRINS I K T O SUGARS

T h e malt used for this purpose was a good quality of distillers' malt, selected because of its being rich i n diastase. I n preparing t h e cold water extract for use the practice was t o grind zoo g. of t h e malt moderately fine a n d digest i t in 1000 cc. of cold water for a period of 2 t o 6 hrs. T h e mixture was t h e n filtered a n d t h e clear water extract t a k e n for use. T h e amount of solids extracted in this way naturally varies t o some extent, depending upon time of extraction a n d am0un.t of stirring during t h e time of extraction. Glucose was, treated with extracts of this kind under various conditions, at different temperatures, a n d for different lengths of time, a n d t h e n subjected t o fermentation with compressed yeast. T h e results of such fermentation were found t o depend upon t h e temperature of 1 This proportion is based upon the reasoning that if the sugar were all maltose i t would be 47.1 per cent, which is 20.7 per cent above the dextrose figure, but we find it b y fermentation t o be 33.6 per cent which is 7.2 per cent above the dextrose figure. It follows from this t h a t the maltose must be 7.2/20.7 of 47.1 per cent.

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Vol. 8, NO. I I

mashing a n d upon t h e kind of yeast used in t h e fermentation, as is shown in Table 2 .

carbohydrates, which consist almost wholly of starch. T o show a comparison between t h e carbohydrates TABLE2-cC. GASPRODUCED B Y 15 GRAMS OF COMPRESSED YEAST, ACTING of these severa1 foods a n d glucose, t h e y were mashed UNDER THE S A M & CONDITIONS ON 5 G. O F CANE SUGAR OR GLUCOSE malt extract as described above, a n d subseWHICH HAD BEEN DIGESTEDFOR 2 HRS. WITH A COLD WATER EXTRACT with OP MALT (50 Cc.) quently fermented. First a complete analysis of t h e Temp. M a l t Glucose & T,emp. Malt Glucose & Temp. Cane GluC. Ext. M a l t Ext. C . Ext. M a l t Ext. C. Yeast Sugar cose Eoodstuffs was made a n d t h e a m o u n t of carbohydrates 40 320 1200 40 220 1100 45 A 1100 1060 determined in the usual manner (by difference), after 50 300 1220 50 200 1140 45 B 1000 940 60 290 1160 60 200 C 1080 1060 980 45 subtracting from t h e total weight of material t h e T h e most favorable temperature for t h e action of amounts of moisture, ash, protein, fat a n d crude t h e malt extract was 50' C. When using compressed fiber. Then t h e same q u a n t i t y of each material was yeast from different manufacturers t h e amount of mashed a t a temperature of 50' C . for 2 hrs. with a gas produced from t h e glucose mashed with malt ex- properly prepared solution of cold water extract of tract is closely related t o t h e comparative amount of malt, cooled t o t h e proper temperature, 1 5 g. of yeast gas obtained with t h e same yeast acting upon cane added, a n d t h e gas resulting from t h e fermentation sugar. This difference in brands of compressed yeast collected a n d measured. T h e corn meal, Quaker is such as we ha.ve found in a large number of gas Oats, rice a n d Cream of Wheat were well cooked, fermentation tests extending over a period of several probably more thoroughly t h a n t h e y would ordinarily years, i n studying the comparative strength of yeast be as prepared for consumption on t h e table. T h e Corn Flakes a n d Shredded Wheat are breakfast foods in bread-making, T h u s it is a general rule t h a t Yeast €3, when acting on sugar under conditions i n prepared for immediate consumption a n d required these experiments, will produce less gas t h a n will no further cooking. Irish and sweet potatoes were Yeasts A a n d C. The probable cause lies in t h e by- baked in t h e usual manner a n d t h e edible portion products which are always produced when yeast carefully dried at a low temperature a n d powdered. acts upon sugar a n d in a complex body like compressed The same was done with white bread. T h e results of these experiments are given in Table yeast may be brought about b y t h e yeast cells them3, together with t h e a m o u n t of carbohydrates selves or by certain forms of bacteria which accompany t h e yeast.' TABLE 3 - G 4 ~ PRODUCED BY FERMENTING M A L T E D CARBOHYDRATBS O F As has been already s t a t e d , where yeast acts upon CEREAL FOODS COMPkRED WITH MALTEDGLCCOSE Per Cc. Gas pure cane sugar a n d upon pure dextrose, t h e amount cent for 5 g. Total Cc Gas Carboof gas produced during t h e fermentation with like hydrates Carbohy. for 5 g. of in Material drates Material quantities of t h e same compressed yeast is closely 1170 Corn Meal, well cooked.. . , . . , , , . . , 78.67 920 1143 760 proportional t o t h e amount of sugar present. Some Quaker Oats, well cooked.. , . , . . . . , , , 66.55 1140 870 Rice, well cooked.. . . . . . . . . . . . . . . . . . 76.30 things have been found in our experiments which have Cream of Wheat, well cooked., . , . . , , 75.50 1165 880 1043 840 Corn Flakes.. . . . . . . . . . . . , . . . , . , , . 81.52 shown t h a t where certain starches, or starch products, Shredded Wheat.. . .. . . , . . . . , , . , , , , , 75 .25 1030 780 1095 945 are acted upon by malt a n d t h e result of t h e mashing Baked Potatoes, dried.. . . . . . . . , , , . . !6.30 ,

,

,

is subjected t o fermentation, a larger quantity of gas will be obtained t h a a is directly proportional t o t h e amount of sugar which we should expect t o be produced from t h e starch present; t h u s in treating t h e prepared starch known as Mazam, with malt extract, a considerably larger proportion of gas is obtained t h a n we should expect from t h e amount of starch used; assuming t h a t t h e usual amount of dextrose was eventually obtained from t h e starch b y t h e combined influence of t h e malt extract a n d t h e maltase secreted b y t h e yeast itself. Mazam is a starchy preparation obtained b y hydrolyzing starch much t h e same as b y gelatinizing it with boiling water, except t h a t in t h e Mazam t h e starch is subsequently dried a n d flaked. T h e a m o u n t of gas produced from t h e dry starch present in Mazam has been found in several experiments t o correspond t o as high as 1 2 2 t o 1 2 8 per cent of t h e * starch used, calculating on the basis of pure dextrose, t o which t h e starch must be eventually converted before fermentation. As is well known: most of t h e cereal foods, corn meal, Quaker Oats, rice, Cream of Wheat, etc., and materials baked from t h e m , like bread, are rich in 1

I t may be mentioned incidentally that while B gives the least amount

of gas in fermentation tests, i t often, in fact, generally, gives the best re-

sults in bread-making, where its influence in softening the gluten of the flour appears to be especially beneficial. This also is probably due to bj-products produced during fermentation.

White Bread, Vienna, dried.. . , , . , . . , I 5.70 White Bread, Pullman, dried.. , , , . . . , 75.40 Bran Bread, dried.. . . , . . . . . . , . , , , , . 78.56 80.00 Glucose ...........................

840 820 640 880

1110 1090 815 1100

found on analysis a n d t h e total gas calculated t o j g. of dry carbohydrates. It will be seen t h a t t h e results obtained from glucose compare favorably with those obtained f r o m several cereal foods a n d potatoec, used, indicating t h a t t h e dry matter of glucose yields substaiitially as much food material as many of these cereal foods. especially when allowance is made for t h e increased sugar content due t o t h e hydration of t h e starch of t h e cereal foods. What has been found t o be t r u e of Mazam with regard t o a n unexpectedly large proportion of gas production has been found t o be t r u e in a like manner of other starches and similar carbohydrates present in foodstuffs. Accordingly, t o determine t o what extent t h e glucose was converted into fermentable bodies, it was necessary t o determine t h e amount of unfermented matters still present in t h e fermented solution as well as t h e nature of these bodies. After fermentation t h e fermented liquor was made u p t o a definite volume, filtered a n d a n aliquot part taken for determination of t h e total solids and t h e reducing sugars. a n d in some instances for various other determinations. Typical results i n determinations of this kind, using malt evtract as t h e source of t h e diastase

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TABLE 4 large q u a n t i t y of unfermented matter. T h e other (1) Glucose, 10 g., digested in incubator over night a t 45’ C., with 50 cc. cold water extract of malt, then fermented with 15 g. yeast in comportion was analyzed a n d calculated back t o t h e original parison with 50 cc. of the malt extract and with 5 g. of cane sugar. 1000 cc., a n d also t o t h e original glucose GLUCOSEFERYEXTATION IN TRIPLICATEvolume of Cane Malt Glucose and with t h e results shown in Table 5 . The material Solids in the glucose i9.770 Sugar Extract M a l t Extract precipitated b y alcohol was taken u p b y water a n d 260 cc. 1980 cc. Total Gas Formation.. . . . . . . . . . . . . 1040 cc. . . . . . . . 1720 cc. Deduct Gas of Blank . . . . . . . . . . . . . . . . . . . . . . . . . 8 2 . 7 Per cent TABLE ~--UNFERXESTED Gas equivalent t o C a RESIDUES (PERCENTAGES) FROM 10 LBS. OF Unfermented Solids (corr. for Malt Extract). . . . . . . . . . . . . . 1 5 . 4 GLUCOSG 2.5 UNFERMENTED REDUCING SUGARS: Calculated as Dextrose. PRECIPITATED IN SOLUTION Calculated as Maltose. . 4.4 BY ALCOHOL IN ALCOHOL Polarization of fermented solution made to 250 cc., 200 mm. tube, In t h e Calc. In the Calc. 6’ Sugar scale. 600 grams to 1000 grams to (2) Fermentation of 6 g. of glucose after digesting over night a t S O o C. syrup glucose syrup glucose with 50 cc. of cold water extract of malt and sufficient water to make 100 obtained taken obtained taken cc. Fermented with 15 g. yeast in comparison with 5 g. of cane sugar Total Solids ..................... 42.95 35.00 7.73 5.68 and with 50 cc. malt extract solution; all in duplicate. Ash.. .......................... 1.58 0.21 1.12 0.25 Cane Malt Glucose and 3 . 5 0 0.46 5.40 1.19 Sugar Extract Malt Extract Total G a s , . . . . . . . . . . . . . . . . . . . . . . . . . 1040 cc. 140 cc. 1140 cc. 10.10 1.34 28.64 6.32 Corrected for Blank.. . . . . . . . . . . . . . . . . . . . . .... 1ooocc. 18.14 2.40 49.00 10.82 Gas equivalent to Cane Sug .. ,,. 80 % (Q) (0) Unfermented Soluble Solids ,, ., , 1:450 g. 1.95Og. Unfermented Glucose.. . . . . . . . . . . . . . . . . . . . .... 8.3% Unfermented Reducing Sugars as Dextrose . . . . . . . 2.9 -0.16 -0.03 (3) Fermentation with 100 g. glucose, digested with 250 cc. cold water Reducing Sugars as Dextrose: extract of malt over night a t 50’ C., then supplied with 20 g. yeast and After boiling with dilute HCl. , . 39. io 5.26 fermented as long as fermentation continued. Gas not collected. After fermentation. . . 1.57 0.21 ... Total Solids (corr. for M a l t Extract). . . . . . . . . . . . . . . . . . . . . . 16.4 Per cent (a) This solution evidently contained no dextrin and the reducing sugars UNFERMENTED REDUCING SUGARS:Calculated as Dextrose.. 3 1 present were all dextrose. Calculated as ,Maltose.. . 5 . 5

.

...

... ...

are shown in Table 4. Evidently a considerable q u a n t i t y of unfermented solids remains from t h e glucose after deducting t h e a m o u n t of unfermented solids due t o t h e malt extract a n d t o t h e water extract used in making t h e fermentation. The a m o u n t of unfermented matter in t h e tables shown, and in other experiments, is variable, as is also t h e amount of unfermented reducing sugars. T h e conditions are found t o be much t h e same whether a large q u a n t i t y of glucose ( r o o g.) is used or a smaller quantity (j or I O g.), b u t t h e difficulties of manipulation are much greater where larger quantities of glucose are used. 1.n addition t o this, with smaller quantities it is possible t o measure t h e amount of gas produced, t h u s giving further d a t a . For this reason, b y far t h e larger number of experiments which have been carried out have been with small quantities of glucose. Several experiments with large quantities of glucose were made, b u t owing t o t h e unsatisfactory results, t h e details of only one of these are given here: T e n lbs. of glucose were diluted t o about 2 5 per cent of solids with water a n d digested over night a t between 40 a n d j o o C. with cold water extract of malt. It was t h e n seeded with about 3 oz. of compressed yeast, a n d fermented as long as fermentation continued (about 5 days). T h e liquor was t h e n filtered a n d evaporated t o a syrupy condition, after which i t was precipitated with 95 per cent alcohol, which was added i n excess or until t h e addition of alcohol produced no further precipitate. T h e precipitate was allowed t o settle, was finally separated b y decantation a n d filtration, a n d t h e n well washed. with fresh alcohol. It must be borne in mind, however, t h a t with a large mass of material of this kind complete separation of insoluble from soluble bodies is difficult, a n d it is probable t h a t some of t h e material which would naturally be present in alcohol was carried down with t h e precipitate. T h e alcoholic filtrate was next evaporated, t h e n made u p t o 1000 cc., a n d di.vided into two portions, one of which was f u r t h e r fermented b y t h e addition of yeast, which reduced t h e solids materially, b u t still left a

... ...

filtered, leaving a residue of 600 g. which also was analyzed with t h e results given in comparison with t h a t of those obtained on t h e alcohol-soluble matter. A portion of t h e insoluble in alcohol was heated with 2’/* per cent hydrochloric acid in t h e usual manner, followed in determining starch b y t r e a t m e n t with this reagent t o convert i t into dextrose. T h a t portion of t h e alcohol-soluble material which h a d been fermented was also treated with hydrochloric acid in t h e same manner. Both these solids were subsequently fermented a n d t h e reducing sugars found after this fermentation. I n t h a t portion of t h e fermented glucose precipitated with alcohol this unfermented residue of reducing sugars calculated as dextrose amounted t o only 3.65 per cent of t h e dry matter of this residue, which, calculated t o t h e original glucose, amounts t o only 0 . 2 1 per cent, while t h e unfermented matter of t h e alcoholic filtrate amounted t o 0.32 per cent of t h e original glucose. I t is very probable t h a t this small amount of unfermented residue when subjected t o more favorable conditions for fermentation would also be completely fermented. As i t is, t h e unfermented m a t t e r amounts t o substantially 0.5 per cent of t h e glucose taken. Owing t o t h e introduction of certain products into t h e fermentation b y t h e use of water extracts of malt a n d wishing t o know t h e influence of other diastasic ferments, especially those of t h e body, a n d such as are used in medicine, experiments along t h e same line as those given above have been made on,glucose, using pancreatin (as described a n d specified i n t h e “ U . S. Pharmacopoeia” a n d also Taka-Diastase, which is extensively recommended a n d used for t h e purpose of assisting in t h e digestion of starchy foods. Pancreatin, usually obtained from t h e hog or ox, contains various ferments a n d is rich in diastase, which, according t o the specifications of t h e U. S. P., “should be capable of converting not less t h a n 2 5 times its own weight of starch into substances soluble in water.” As used, it is a cream-colored, amorphous, dry powder. T a k a Diastase is also a d r y powder a n d contains a ferment said t o be isolated from a certain species of mould,

1014

T H E JOcR-VTdL O F I N D C S T R I A L AiYD E S G I N E E R I N G C H E M I S T R Y

Eurotiuin Oryzae, which has been grown on a suitable medium, such as bran, and, according t o t h e manufacturers, is of such a strength t h a t it will in I O minutes digest I j o times its own weight. of starch. Both of these diastases have been used in a very large number of experiments with glucose alone a n d in comparison with various kinds of starches, and the resulting products of digestion have been subjected t o fermentation with compressed yeast under various conditiohs in such manner as t o determine t h e amount of gas produced and in many instances t h e amount of undigested solids and reducing sugars. The outcome of these experiments is fully set forth in Tables 6 t o 9. I n some instances the completeness of t h e conversion of starch is naturally indicated by the thoroughness of t h e fermentation which follows the action of the diastase. As already noted, in t h e case of digestion with malt extract, this fermentation is not always uniform with different kinds of compressed yeast. Some compressed yeasts appear t o ferment more completely both t h e reducing sugars and some of t h e known reducing dextrins t h a n do others. It is t o be remembered t h a t yeast is a fungus in a measure similar t o t h a t from which Taka-Diastase is obtained, and, as has already been pointed o u t , compressed yeast is likely t o consist of different species of t h e yeast plant; probably these yeast plants are associated with different forms of enzyme-secreting bacteria, which explains in general why these differences (see Table 6) are liltely t o occur. TABLE~--RJ~scLTs OF FsnnlExTINc GLUCOSEWITH THREE DIFFEREXT COMPRESSEDYEASTS, AFTER TAKA-DIASTASE

DIGESTING cc.

Total Yeast A

Gas 940 940 940

OVER

NIGHT

Solids Corrected for Blank

5.54%

Glucose, 6 g. \ n 7.50 i C 8.60 Yeast, 10 g, Gas from j e.. k a n e Suzar ~. (Yeast c ) ,, , , . , . , . , . . . . . , , , , Gas from Blank (1 g. Taka-Diastase, each yeast) . . , , , , , . , Reducing Sugars,. . . . . . . . . . . . . . , , , , . , , , . , , , , , . , , . . , , , , , ,

.

I

.

WITH

1 G.

Reducing Sugars as Dextrose 1.41% 0.94 0.66 I 100 cc. 40 cc. Sone

T h e influence of different conditions of working of t h e diastases and different conditions of fermentations is apparent from Table 7 . TABLE7 Results of fermenting glucose after digesting over night a t near 40' C., two lots of 120 g. each made to 500 cc. with water. One with 10 g. TakaDiastase; the other with 10 g. pancreatin: 25 cc. each, equivalent to 6 g. glucose, taken for fermentation. Per cent Solids Per rent Reducing Total Gas Corrected for Blanks Sugars as Dextrosc Cc. Taka-Diastase l o t . . . . . 1040 14 00 2.60 23.51 1.20 Pancreatin lot. . . . . . . . 940 Cane Sugar (5 6.). . . . . 1 I00

I t is shown in Table 8 t h a t when t h e unfermented solids which remain after treatment with both Taka-Diastase and yeast, and pancreatin and yeast, are treated vrith hydrochloric acid, the unfermented carbohydrates are nearly completely con x-erted int,o dextrose and t h a t when t h i s dextrose is again fermented with yeast, after properly neutralizing t h e solution, the entire amount of reducing sugars present is removed b y the fermentation. To make a more thorough s t u d y of t h e nature of the unfermented solids when acting upon glucose by these diastases and by yeast, a parallel series of experiments was conducted as is shown in Table 9.

V O ~8, . NO. I T

TABLE 8 Results of fermenting glucose after digesting over night a t m a r 40° C., two lots of 120 g . each made to 600 cc. with water. One with 10 g. Taka-Diastase and one with 10 g. pancreatin, 30 cc. of each, corresponding t o 6 g. of glucose, were fermented with 10 g. of yeast and 200 cc. of total liquid. Volume made t o 250 cc. after fermentation and clear filtrate taken foi examination. TakaPancrea5 Grams Cane Sugar Diastase tin Used Total Gas Produced.. , . . . , . . , . . . . . . 1100 cc. 900 cc. 880 cc. Solids after fermentation., , . , , , , , , , . . . . . . . . , . . . 11 .27% 15.0% Ash in solution.. . . .. , , , , , , . . . . . . . . . . . . . , , , , , , 1.10 1.54 REDCCIXG S C G A R S AS DEXTROSE: Alter fermentation , , . . 1.70 1.30 After boiling with 2.5y0HC1.. , , . . . . . . , . . , , . , 10.36 9.8 After fermentation of HC1 product (GO g. Glucose) None None 300 cc. of above digested Glucose solutions were fermented with 20 grams yeast each: 13.07 Unfermented Solids.. . . . . . , . , . . . . . . , . . . , , , , 9.22 2.04 1.87 Unfermented Reducing Sugars as Dextrose., ,

.

Four fermentation tests in each of the t w o series were carried along in parallel experiments with closeIy agreeing results as t o t h e amounts of gas produced. The products of each series of t h e four fermentations were then combined, made u p t o 1000cc., and filtered, after which t h e clear filtrates were used for t h e subsequent analyses as recorded. A blank test was also made with pancreatin and with Taka-Diastase, b u t without glucose, so t h a t corrections might be made for solids and other materials introduced from sources outside t h e glucose. I n these experiments, in addition to t h e determinations of t h e amounts of solids and reducing sugars present, determinations were made of t h e protein a n d t h e ash in solution and also t h e solids introduced and produced by t h e fermentation, as well as the polarization of t h e solution after fermentation1 The acidity given is expressed in cc. N / I O acid for t h e volume of liquid, corresponding t o 6 g. of glucose. This acid is in part t h a t introduced b y the yeast itself and in part t h a t which is produced during the fermentation b y the action of the yeast upon t h e sugars. Both t h e pancreatin and t h e Taka-Diastase contain some milk sugar, which is naturally converted into lactic acid. I n some instances not all of the reducing sugar present in these ferments is removed during t h e process of fermentation, and when such has occurred, t h e quantity of reducing sugars found in t h e blank is deducted from t h e quantity of reducing sugars obtained in t h e parallel experiment where glucose or starch was used. Succinic acid is a well-recognized product of the action of yeast upon sugars; also a small amount of acetic acid is formed and some lactic acid. Thc amount of lactic acid is not great, as has been found where the filtered solution has been shaken with ether and titrated after extracting the lactic acid in this way in comparison viith the titration where the total amount of acid produced was present. I n both of these series of experiments, shown in Table 9 , a portion of t h e fermented liquor was boiled with z . j per cent hydrochloric acid, for t h e purpose of converting t h e remaining carbohydrates into dextrose, and it will be seen t h a t for the pancreatin, vvhere 15.3 per cent of unfermented glucose was left in t h e liquid, reducing sugars were obtained. after boiling with hydrochloric acid, amounting t o 13.7 per cent of t h e glucose used and in case of t h e Taka-Diastase where 9 . j pel1 Polarizations were made in a 200 mm. tube of the filtered solution in a Schmidt and FIaensch polariscope; the results are given in degrees o n the sugar scale as the p u r p o s ~is to show comparisons rather than absolute amollnt.

T H E J O U R N A L O F I , V D L - S T R I A L A N D E iVGI iV E E RI N G C H E M I S T 21 E'

Xov., 1916

TABLE9 Results of fermenting 6 g. of glucose in individual fermentation bottles with 0.5 g. of pancreatin, or with 0.75 g. of Taka-Diastase, acting over night a t a temperature a little below 40' C. Glucose Glucose 6 g. 6 g. PanPanTakaTakaCane GLUCOSE creatin creatin Diastase Diastase Sugar Fermentations 0.5 g. 0.5 g. 0.75 g. (in quadruplicate) 0.75 g. 5 g. Total Gas.. . . . . . . . . . . . . . 60cc. 8OOcc. 60cc. 960cc. 1120cc. Unfermented Solids., . . , . 0 . 5 6 0 g . 1.480 g. 0 . 7 8 0 g 1.350 g. 0.25Og. 0.920 g. . . . . . 0 . si0 g. . . . . . Corrected for Blanks. . . . . . Unfermented Glucose. , , , . . . . 1 5 . 3 % . . . . . 9.5% ..... Cnfermented Sugars: As Dextrose.. . . . . . . . . . . . . . 2.75 . . . . . 1.60 ..... As Maltose.. . . . . . . . . . 4.90 . . . . . 2.80 Protein in 250 cc. solution 0:24()'g. 0.120 g. 0.096 g. 0 . 0 3 0 g. 0:0?9g. Ash in 2.50 cc. solution.. . 0 . 0 8 0 g. 0.095 g. 0.070 g. 0.078 g. 0.037 g. Polarization in 200 mm. 2.30 0.0 1.30 0.0 tube . . . . . . . . . . . . . . . . . 0 . 1 " Acidity of solution, cc. &'/lo Alkali required for 42.0 28.0 50.0 24.0 250 cc.. . . . . . . . . . . . . . . 16.0 After boiling the filtered solution 2112 hrs. with Hvdrochloric Acid 21/2%: Reducing &gars as Dextrose. . . . . 13.7% 10.0% 200 cc. of neutralized solution (equivalent to 4.8 'g.' Glucose) ferment'ed. Kone Unfermented Reducing Sugars None Solids.. 7.160 g. 5.110 g. 0.300 g. Ash ......................... 6.480 g. . . . . . 6.580 g. 0.06Og. Oreanic Matter.. . . . . . . . . . . . . 0.680 e. . . . . . 0.530 e. 0.240 e. Protein., 0.240 . . . . . 0.150 2. 0.150;. Nan-protein Organic Solids. . . . 0.440 g. 0.380 g. 0 . 0 9 0 g. I.,"

.....

.....................

................

9.

....

cent of unfermented glucose remained after t h e first fermentation, 10.8 pel- cent of dextrose was found after t r e a t m e n t with hydrochloric acid. When t h e dextroses produced b y t h e use of hydrochloric acid were again subjected t o fermentation, t h e y were completely removed. I n following out t h e products which remained after this last fermentation, it will be seen t h a t there is still present in t h e solution, besides t h e ash a n d protein, a small a m o u n t of unfermented m a t t e r which, in our opinion, may be justly a t t r i b u t e d t o organic acids produced i n one or both of t h e fermentations. After boiling with t h e hydrochloric acid, t h e solution was carefully neutralized with sodium hydroxide before t h e subsequent fermentation. This would naturally form organic salts with t h e small amounts of organic acids present from t h e previous fermentation: these organic acids would appear as part of t h e organic matter during t h e burning of t h e residue t o determine t h e ash present. Then, too, a small additional amount of acids would be added b y the yeast used for t h e second fermentation. It would appear from a s t u d y of t h e results as outlined in this tabulated series of experiments t h a t all t h e products of glucose are accounted for a n d t h a t there does not remain a n y sulbstance which was a constituent of t h e glucose a n d which cannot b y t h e process used be converted into substances fermentable b y yeast. FERMENTATION EXPERIMENTS

WITH

STARCH F O R

THE

P U R P O S E OF D E T E R X I K I N G TO WHAT E X T E N T T H E RESULTS

OBTAINED B Y T H E FERMENTATION O F

GLUCOSE A F T E R ACTION O F T H E DIASTASE O F MALT E X T R A C T ,

PANCREATIN

AND TAKA-

DIASTASE I S P A R A L L E L E D B Y T H E ACT I O N OE T H E S E SAME R E A G E N T S

O N WELL-COOKED

STARCH

Several series of experiments were made, using different starches in parallel wit$ glucose. T h e starches used included Kingsford's corn starch, a good sample of wheat starch, ordinary rice starch, arrowroot starch (manufactured b y Taylor Bros., London), ordinary p o t a t o starch, p o t a t o starch made soluble b y digesting i n t h e cold with hydrochloric acid (according t o t h e

IO1 j

method of Lintner-the sample -sed being a product of Merck's) a n d a sample of Mazam (previously described). I n t h e use of these several starches t h e q u a n t i t y taken for the test (usually j g . ) was p u t in t h e fermenting bottle with I O O cc. of water a n d placed in a water bath, which was subsequently brought t o boiling so t h a t t h e starch i n t h e bottle was maintained a t the boiling temperature for a t least 1 5 minutes after it h a d become thoroughly gelatinized. Great care was taken in gelatinizing the starch t o avoid lump formation. This method of preparation gives t h e starch a more thorough cooking t h a n it ordinarily receives during t h e cooking of cereals for table use, so t h a t it may be assumed t h a t t h e starch was in t h e best possible condition for t h e working of t h e diastase. A parallel series of experiments was made in which starches a n d 'glucoses were boiled with dilute hydrochloric acid, as described in t h e Official Methods of t h e Association of Official Agricultural Chemists (C. S. Bureau of Chemistry, Bull. 107, 53). The amount of hydrochloric acid used i n tests of this kind was 2.j per cent on t h e liquid used. After boiling with hydrochloric acid, t h e solution was cooled, exactly neutralized with a sufficient q u a n t i t y of normal sodium hydroxide, a n d subjected t o the following tests: amount of dextrose present; polarization of t h e solution in t h e 2 0 0 mm. t u b e ; amount of gas produced in fermentation; a n d presence or absence of reducing sugars after fermentation was completed. T h e results of this test are shown i n Table IO. T h e apparent amount of reducing sugars obtained when calculated as actual starch is somewhat less t h a n we should expect from t h e amount of actual starch present as found b y analysis. Whether this is due t o t h e presence of bodies which have not been converted into dextrose or t o t h e destruction of some of t h e dextrose itself by boiling with hydrochloric acid, or t o what extent i t m a y be due t o variations incident t o t h e method of analysis, we have not a t t e m p t e d t o determine. I t is certain, however, t h a t t h e method of analysis itself is subject t o some variations because of t h e necessity of multiplying a n y small error introduced in t h e determination of t h e dextrose b y a large factor, due t o t h e small amount of solution which it is necessary t o use for t h e purpose of reduction: t h u s t h e results of t h e determination are based upon a small fraction of a gram of starch taken. So, too, in t h e fermentation tests it was possible t o use only 2 0 0 cc. of t h e solution, corresponding t o 2 g. of t h e original starch. I n determining t h e a m o u n t of starch from dextrose obtained in this way, it is necessary t o multiply t h e dextrose obtained by 0.9 t o allow for t h e water hydration. I n t h e case of t h e glucose, however, t h e proper factor t o use for this purpose is somewhat uncertain, b u t m a y be taken on t h e average basis of maltose. T h e factor used was o . g j . A comparison was made i n t h e fermentation of a number of t h e starches digested with malt extract, similar t o t h a t made on cereal foods, as shown in Table 11. Here, as i n t h e case of Mazam, there is a q u a n t i t y of gas produced in excess of what we should expect t o find when we consider t h e amount of sugars which

1016

T H E J O U R N A L O F 11VDUiSTRIAL A N D E S G I N E E R I S G C H E M I S T R Y

Vol. 8, NO. 1 1

TABLE IO-DEXTROSEPRODUCED FROM STARCH AND GLWCOSE B Y BOILING5 G. 2'/2 HRS.WITH 2.5 PERCENT HYDROCHLORIC ACID Solution made t o 500 cc. after cooling and neutralizing with a measured quantity of standardized sodium hydroxide, 200 cc. solution fermented PolarizaGas PERCENTAGE COXPOSITION Dextrose Starch Cc. Gas Equivalent. tion on UnferOF STARCHES USED of 5 g. of mented Solution Found Equivalent from 200 cc. MoisProSample in 200 mm. Tube Per cent Dextrose of ture Ash tein Starch of Sample X 0.9 Solution Sugar Scale Cc. Wheat Starch.. .......................... 8.50 0.23 0.32 90.95 97.3 87.57 4.0' 360 900 Rice Starch 9.00 0.69 0.26 90.05 1000 85.59 4.0 95.1 400 Corn Starch.. ........................... 8.60 0.22 0.26 90.92 88.38 98.2 370 4.2 925 Mazam (prepared Corn Starch) . . . . . . . . . . . 6 . 3 0 0.34 0.26 93.24 925 90.45 100.5 370 4.2 Potato Starch ........................... 10.50 0.26 0.16 89.24 85.10 94.6 925 4.0 370 Soluble Starch (Lintner). . . . . . . . . . . . . . . . . .10.40 0.10 0.35 89.15 950 83.50 4.0 92.8 380 Arrow Root Starch.. ..................... 11.00 0.10 0.00 88.90 93.5 950 54.15 380 4.0 Glucose(b) .............................. 18.30 0.35 0.05 81.30(a) 83.1 800 I 8.90 ( b ) 320 3.7 Total gas from 5 g. cane sugar 1080 cc. Reducing sugars after fermentation, none in any of the solutions (a) Total carbohydrates ( b ) Dextrose X 0.95 as maltose equivalent.

.............................

should normally be produced from t h e starches b y hydrolysis. This is t r u e of all t h e starches in this comparison except in t h e case of rice starch where t h e gas is, for some reason, less.

I n t h e case of t h e malt extract in Table 1 1 , we have corrected for the amount of gas produced from t h e malt extract because there is always a certain a m o u n t of sugars present in this material. I n experiments where Taka-Diastase a n d pancreatin have been used as t h e source of t h e diastase, no correction is made. There is always a small amount of gas apparently produced, even when yeast a n d water only are used a n d i n most instances with a small amount of T a k a Diastase a n d pancreatin added as used, I n a large number of experiments both of these ferments h a v e been used i n excess so as t o carry t h e action of t h e diastase as far as possible. I n many cases one gram of either has been used a n d allowed t o exert its action on t h e starch or glucose for a period of several hours, generally over night. A comparison of this kind is shown in Table 1 2 . I n Table 1 3 , another com-

instances a considerable q u a n t i t y of reducing carbohydrates was not removed b y t h e fermentation. T h e amount is somewhat higher for glucose t h a n for t h e starches, b u t , as shown in former experiments (Table 6 ) in other instances t h e unfermented products were considerably less t h a n is shown in this instance. This was apparently due t o differences in t h e samples of Taka-Diastase used, as well as differences i n t h e action of t h e different yeasts, for i t was shown with T a k a Diastase, as is more clearly pointed out with pancreatin below-,t h a t t h e diastatic activity of t h e material became weaker with t h e age of t h e sample. I t would appear from t h e higher proportion of unfermented solids in t h e case of t h e glucose a n d t h e complex combinations of enzymes of this character, which has been pointed out in a n earlier experiment, t h a t there may be in t h e glucose some form of dextrin which is less readily changed b y t h e enzymes present in this special sample of Taka-Diastase t h a n are those produced in t h e regular series of cha,nges from t h e hydrolyzed starch t o dextrose. We draw this conclusion from a comparison between t h e action of t h e Taka-Diastase a n d t h a t of malt extract a n d pancreatin x h e r e the hydrolysis is apparently as complete with t h e glucose as with t h e starch a n d from t h e fact t h a t t h e different yeasts, which apparently secrete enzymes capable of acting upon starch products less completely hydrolyzed t h a n dextrose, act differently upon t h e hydrolyzed products of glucose, as already shown. Attention should be called t o t h e acidity of t h e fer-

TABLE12 Results of experiments in fermenting products of 5 g. of starch and glucose obtained by digesting same with 1 g of Taka-Diastase for each, over night, a t a little below 40' C., and fermenting with 15 g. of yeast in 200 cc. water, later made to 350 cc. and filtered. Unfermented Acidity for Gas Polariza- Unfermen- Reducing 250 cc. Prolion of ted Solids Sugars Equivalent duced Sol. 200 (corr.) as Dextrose for cc. mm. Tube Per cent (% corr.) N/lO Alkali 0.00 .... 28 Cane Sugar., . . 1080 1.50 35 1.0 Wheat Starch.. 1000 1.68 40 Rice Starch.. . . 950 1. o I .60 40 0.9 Corn S t a r c h . . . 980 2.50 40 M a z a m . . . . . . . 1000 I .o 43 2.30 Potato Starch. 950 1 .O 2.30 40 1 .o Soluble Starch. 940 3.00 43 2.0 Glucose . . . . . . . 780 Taka-Diastase (blank) . . . . . . . . . ... ,.. .... 34

TABLE13 Fermentation with different starches well boiled, 5 6. each, 1 g. T a k a Diastase, added a t time of adding yeast. Yeast, 15 g. Time of fermentation, S1/2 hrs. No material increase of gas during night. Corrected for blanks. Reducing Gas Produced Polarization Per cent Sugars Cc. 200 mm. Tube Solids as Dextrose 11.80 3.70 1.2" Glucose . . . . . . . . . . . . . . . 820 3,10 0. I 8.10 M a z a m . . . . . . . . . . . . . . . 1020 3.20 1.2 9.80 Soluble Starch.. . . . . . . . 920 Corn Starch.. . . . . . . . . . 1000 0.8 9.10 3.00 5 G , Cane Sugar.. . . . . . 1060 0.0 ..... .... Same as above, but digested with Taka-Diastase a t about 30' C. over nighL 10.50 3 .00 Glucose . . . . . . . . . . . . . . 860 1.3O 5.20 1.60 0.2 Corn Starch., . . . . . . . . . 980 2.50 990 0 . 8 6 . 0 0 Mazam . . . . . . . . . . . . . . . 8.40 3.12 1.3 Soluble Starch.. . . . . . . . 880

parison is shown between Taka-Diastase acting over night a n d t h e same quantity acting during t h e fermentation by yeast only. I n this l a t t e r instance t h e TaliaDiastase was added with t h e yeast a n d , of course, acted a t t h e same temperature as t h e yeast, 3 j" C. I t will be seen t h a t with t h e large amount of T a k a Diastase used ( I g. t o each j g. of glucose), t h e action was nearly as rapid and thorough as x-hen t h e diastase was a.llowec1 t o act for a longer time. I n all of these

mented solutions as shown i n Table 12. It will be seen t h a t this is nearly t h e same for t h e several starches and t h e glucose, and is somewhat higher than for cane sugar, a n d for t h e blank-using Taka-Diastase a n d yeast alone. Some p a r t of this acidity may be taken as lactic acid. b u t t h e amount is not large. With t h e Taka-Diastase blank, where t h e total titration for z j o cc. of liquid is equivalent t o 3 4 cc. of decinormal acid, t h e solution was thoroughly shaken twice with

TABLE11 Starches fermented with yeast after boiling thoroughly in water, then digesting at 45' C. for 2 hrs. with cold mater extract of malt 5 g. starch or glucose; 50 cc. malt extract; 15 g. yeast. Total Gas Gas, Corrected Gas Equivalent Produced for T h a t from to Cane Sugar Cc. Malt Extract Per cent Malt Extract.. . . . . . . . . . . . . . . . . . 220 ..'" ..... Wheat Starch.. . . . . . . . . . . . . . . . . . 1280 1060 103.9 960 95,l Rice S t a r c h . . . . . . . . . . . . . . . . . . . . . 1180 Corn Starch.. . . . . . . . . . . . . . . . . . . 1270 1050 102.9 1080 105.8 Potato Starch.. . . . . . . . . . . . . . . . . . 1300 . , , , ~., . , . 1060 Glucose (Solids 82.1 9%). 840 82.3 .. Cane Sugar (5 g.). . . . . . . . . . . . . . . 1020 ..... ..;l

N o v . , 1916

T H E J O U R N A L OF I N D U S T R I A L A N D ELVGINEERING C H E M I S T R Y

ether, which would remove a n y lactic acid present, a n d t h e titration after removing t h e lactic acid i n this way was still 31 cc. of N/ro. I n t h e parallel experiment with t h e j g. of glucose a n d t h e Taka-Diastase t h e t o t a l acidity was equivalent t o 41 cc. of N / I O alkali, a n d after extracting t h e ether-soluble acids, i t was still 34 cc. It is apparent from this t h a t no considerable a m o u n t of t h e glucose had been converted i n t o lactic acid. T o determine concerning t h e action of a lesser a m o u n t of Taka-Diastase when acting upon starch or glucose, under t h e conditions of our experiments, t w o parallel series were r u n , using Lintner's soluble starch, well boiled, a n d glucose. T h e results are shown in Table 14. T h e t o t a l a m o u n t of gas produced

__

'TABLE 14 Experiments with different quantities of Taka-Diastase put in solutions of boiled soluble starch and of glucose a t the time of adding the east and allowed t o act a t 35O C. 5 g. glucose or starch and 15 g. yeast. %me of fermentation, 6 hrs. No material change in readings during night.

SERIES

: i

c?

k

0 000 . 0.025 380 6 : i o 0 050 600 4 . 3 0.125 840 2.0 0.250 860 1 . 3 Cane Sugar 5.000 1040 0 . 0 0.000 390 9 . 0 Glucose. 0.025 480 3 . 9 0.050 620 2 . 8 0.125 700 1 . 5 0.250 770 1 . 0

Starch

...

~~

RATESOF GASDEVELOPXBNT (Cc.) Minutes after Setting 40 80 120 150 180 240 360

D

i0%

4

.

,.

2 34 2 91

...

89

...

3 16 2 54

50 120 240 290 350 380 120 300 440 500 540 600 380 640 770 800 840 840 490 740 840 850 870 870 980 1020 1050 1060 1060 1060 240 300 340 380 380 390 240 320 400 420 440 480 290 400 490 530 570 620 420 560 650 680 680 700 520 660 720 750 760 780

is found t o increase steadily with t h e amount of T a k a Diastase used. Polarization of t h e solution steadily decreased a n d there is a perceptible decrease of t h e amount of unfermented reducing sugars. T h e total unfermented solids were not determined in this experiment, b u t are clear1.y indicated b y t h e polarization. It m a y also be seen b y studying t h e more detailed results of t h e gas production, t h a t t h e action of t h e yeast is much quicker upon t h e glucose, even with Taka-Diastase,than upon starch with t h e small amounts of Taka-Diastase used, t h u s illustrating t h e value of sugars in glucose in aidi:ng t h e more rapid assimilability of t h e product as compared with t h a t of starch. T h e table is also of interest as showing t h e general working of this class of experiments as t o gas production. TABLE15 Results of experiments in fermenting products of 5 g. of starch or glucose, obtained by digesting same with 1 g. of pancreatin for each, over night at a little below 40' C. and fermenting with 15 g. yeast, 200 cc. water, and later made t o 250 cc. and filtered. Acidity for UnferUnfer250 cc. mented mented Re- EquivaGas Polarization of Solids ducing Sugars lent for Produced Sol. 200 rnm. (corr.) a s Dextrose N/10 cc. Tube Per cent (% Corrected) Alkali Cane Sugar.. 1080 28 Wheat Starch.. 800 3.30 19.2 5.2 43 Rice Starch.. 790 3.8 22.6 5.8 43 880 2.5 Corn Starch.. 15.4 5.6 43 Mazam.. 800 45 3.5 20.4 6.0 Potato Starch.. 850 .3 5 18.6 6.4 43 880 Soluble Starch.. 15.2 6.3 2.5 44 Glucose 700 3.0 17.0 5.6 44

..... ... ..... ....

........

..... ..........

.

.

I

allowed t o act upon t h e boiled starches a n d t h e glucose over night. T h e pancreatin was t a k e n from t h e same bottle as was used i n experiment recorded i n Table 9, a n d carr?ed on about 4 weeks earlier. It was t h a t of a s t a n d a r d make in t h e original container a n d was in frequent use during t h e interval; t h e bottle was kept corked except when a sample was being weighed out. It will be seen t h a t t h e activity of t h e pancreatin h a d decreased greatly over what i t was a t t h e time of experiments recorded in Table 9 , so t h a t we may t a k e t h e results in this experiment as under conditions arising when we have a weakened diastasic ferment. T h e amount of unfermented solids obtained in this series of experiments is much more t h a n there was with Taka-Diastase under similar conditions. T h e amount of reducing sugars is more a n d t h e polarization of t h e resulting fermented solution is considerably higher. Following this, another experiment was made (Table 16), where t h e same amount of pancreatin was allowed t o act upon starches a n d glucose during TABLF. 16 Experiments with weakened sample pancreatin acting on boiled starches during time of fermentation. Starch 5 g., pancreatin 1 g., yeast 15 g., water 20 cc. Per cent . . . . . . .

Gas Produced cc.

... ... . . . . .... . . . . . . . . . . . .

25 30 70 95 440 170 140 160 170 220

....

...

.

Following out a line of experiments on starch a n d glucose, similar t o those given above, using pancreatin as t h e source of t h e diastase, we find a series of results as is given in Table 15, where t h e pancreatin was

1017

Cane Sugar (5 g Wheat Starch.. Mazam..

...

Soluble S t a r c h . , . . . . . . 780 Arrowroot Starch,. .... 560 Glucose . . . . . . . . . . . . . 700

Unfermented Polarization Reducing of Fermented Sugars Solution as Dextrose 0.00 0.0 5.0 $.I1 4.5 .28 7.4 8.15 7.4 7.53 9.2 8.53 4.5 7.58 7.0 8.71 3.2 7.85

,

Reaction with Iodine Solution

.... ....

.........

Intense Violet

.........

Intense Violet

.........

Intense Violet

t h e time of fermentation. Here we have a still higher polarization of t h e resulting fermented liquor a n d a much larger proportion of unfermented reducing sugars. This, t o our minds, is very significant, as it indicates t h e formation of a large proportion of reducing bodies which partake of t h e nature of dextrin in t h a t t h e y are not fermented by yeast. It should be noted in this connection t h a t t h e liquid after fermentation with pancreatin a n d yeast in this experiment gave a n intense violet reaction with iodine for t h e corn starch, t h e potato starch a n d t h e arrowroot starch, t h u s showing t h a t a large p a r t of t h e starch was still in t h e form of dextrins a n d t h a t t h e smaller a m o u n t of gas obtained was in no way due t o an incomplete gelatinization of t h e starches acted upon. T h e other solutions did not give a n y color with iodine. T h e proportion of reducing sugars in b o t h these series is fully as high for t h e starch as for t h e glucose, showing t h a t these reducing bodies were produced out of t h e starch as a step i n t h e process of converting t h e starches into t h e more readily fermentable sugar, maltose. T h e amount of gas produced shows t h a t a large amount of maltose also was produced in both cases. T o determine more clearly t o what extent this transformation of starches a n d glucose into unfermentable reducing bodies was due t o t h e nature of t h e pancreatin used, three other s t a n d a r d makes of pancreatin were obtained in original containers a n d used in a series of experiments on soluble starch a n d glucose in connection with t h e glucose used i n t h e two preceding experiments, which in this series of experiments is marked

T H E J O C R N A L OF I N D C S T R I A L AhrD E N G I N E E R I X G C H E M I S T R Y

1018

If (see Table 17). T h e unfermented solids were not determined, b u t t h e extent of t h e change from starch of fermented sugars is indicated b y t h e polarization, a n d in t h e case of t h e glucose b y t h e amount of TABLE17 Soluble starch and glucose fermented b y yeast when treated with different pancreatins added with the yeast. Glucose or starch 5 g., yeast 15 g., pancreatin 1 g. Reducing Gas PolariSugars cc. zation as Dextrose Soluble Starch: Pancreatin h... . , , . . 820 2.99 Pancreatin C.. . , . . . , 800 2.9 5% Pancreatin D. 2.8 .... 4.6 .... Pancreatin M Glucose: Pancreatin A , . . . . . . 2.3 1.91 2 3 2.68 1.69 Pancreatin D . , . . . , . . . , . , . , 10 4.82 Pancreatin nl.,. . . . . . . . , , . !30 Glucose only.. . . . , . , , , , . . . . . , , , , . 360 9.7 .... 3 60 9.8 .... Cane Sugar 1100 0.0 . . .“

.

E;;

..

reducing sugars still unfermented. I n the case of Pancreatin hI much more reducing bodies are present and polarization is much higher on both t h e starch fermentation and t h e pancreatin fermentation t h a n is the case with t h e other pancreatins used, all of which points t o t h e same conclusions which we have drawn from t h e preceding experiments, i. e., t h a t with weakened diastasic action t h e conversion of t h e starch into fermentable sugars is not nearly so complete, and the result is t h e leaving on the way of an increased amount of unfermentable reducing bodies. Xnalyses of a large number of malted liquors, including ale, porter, stout, beer, etc., shorn t h a t in all cases there is a quantity of reducing sugars left in the malted liquor after t h e fermentation has been completed. This fermentation often extends over a considerable period of time, generally amounting t o several n-eeks. The origjnal wort used for fermentation presumably contains in t h e neighborhood of 1 2 t o 13 per cent of extracted matter. After fermentation a greater or less quantity of.-unfermented matter still remains, which is grouped together under t h e general name of cxtractives. T h e average amount of unfermented reducing sugars calculated as maltose, approximates close t o 1.j per cent as is shown in Table 18. While TABLB18-UNFERMENTED PRODCCTS I N MALT LIQVORS Averages of many compiled analyses by various analysts (KGnig, Sahrzings und Genussmittel, Vierte Aupage I, Band)

R duc. ine -

S o . of Lia uor Analvses Ale.. . . . . . , . . . , , 44 Porter (Stout). . . 44 Weiss Beer ..... . . 33 One-half

Per cent Extract 5.59 i.97

02.

Reducing Sugars as Maltose 1.07 2.06

5.29 1.56 per q t . = 1.5 per cent

Sugars 70 of Dextrins Total Extract 1.81 17.8 3.08 25.8 ”,.. 29.5

this is not large when taken upon t h e liquor itself it still is sufficient t o amount t o about I.; 02. per quart of malt liquor, a n d when calculated upon t h e total extractive matter it is from 18 t o 30 per cent of t h e amount present. DISCCSSIOS

I t is stated by R a h l and Henius’ t h a t approximately per cent of maltose remains in beer, and about twice this amount, or I per cent, of so-called malt dextrins, defined as substances t h a t may be considered as in a state of transition from the dextrins t o maltose. These authorities apparently base their conclusions, a t least part, upon the work of Prior and others,? where 0 .j

1

“American Handy Book of Brewing and Rlalting,” p . i 4 8 . LOC.cil.. p. 5 3 i .

Yo]. 8 , KO. 1 1

they also show t h a t t h e extent of t h e fermentation of these so-called malto-dextrins in malted liquors varies within certain limits with t h e species of yeast which is used, just as we have indicated above, with reference t o t h e results of our own experiments. I t will be remembered t h a t in beer-making, the yeasts used are of pure culture, whereas in bread-making, distilling, etc., where compressed yeast is used, such as we have used in our experiments, there may be a mixture of: yeast species and an admixture with the yeast of some proportion of different forms of bacteria. I t has been demonstrated b y t h e work of Hanson’ t h a t certain species of yeast are capable of fermenting maltose because of the presence in t h e m of a n enzyme termed maltase, while others are incapable of fermenting maltose because of the absence of this enzyme, ‘but are still capable of fermenting cane sugar after splitting it u p into dextrose and levulose b y means of the invertase which it secretes. On t h e other hand, most of the yeasts secrete both these ferments and are capable of fermenting both sugars. I n a complex mixture like t h a t obtained from coni-erting boiled s t a r c h ‘ b y the use of diastase, whether from malt, Taka-Diastase or pancreatin, and t h e subsequent action of yeast, it would be extremely difficult t o determine just where t h e action of t h e enzymes present in dlastase preparation leave off their work and where the enzymes produced b y t h e yeast begin theirs. I t is immaterial, however, for our present purpose, because when we have t h e combined action of these t\To agents we are able t o produce t h e fermentation and, therefore, have reason t o believe t h a t such enzymes are somewhere produced. Malted liquors are recognized and often highly extolled as valuable sources of food and yet i t has been shown t h a t a considerable part of the extractive matter which they contain consists of unfermented reducing sugars, of which a considerable part has not progressed t o t h e stage of maltose in t h e changes which t a k e place between starch and fermentable sugars. I t is well recognized that t h e processes of digestion are more active and energetic as they take place in t h e body t h a n they can be when they are carried on outside. A considerable number of enzymes have been isolated from t h e alimentary tract. We do not know t o what extent others may be present, b u t there is evidence t o show t h a t all, or nearly all, of t h e carbohydrates present in glucose are assimilated and used b y t h e body. I t is clearly seen from our own experiments, especially those with pancreatin and with malt extract, t h a t t h e amount of unfermented reducing sugars is not greater where these diastases have been acting upon glucose in connection with yeast t h a n they are where these combined agents have been acting upon t h e various starches, and this in spite of t h e fact t h a t there was originally in the glucose a large proportion of apparently unfermentable reducing sugars already present. I t is also seen in several of the experiments t h a t this proportion of apparently unfermentable reducing sugars is largely decreased and in some instances al1 Jorgsen, “Micro8rganisms and Fermentation,” translated b y Miller and Lennsholm, 3rd Ed., 1900, p. 16T.

Nov., 1916

T H E J O U R N A L OF I N D U S T R I A L A N D E N G I S E E R I X G CHEMISTRY

most entirely removed b y t h e combined action of yeast a n d Taka-Diastase, pancreatin a n d t o a less extent with malt extract. I t must be borne in mind, however, in this connection, t h a t while these reducing sugars ordinarily present in t h e glucose are being removed, other apparently unfermentable reducing sugars are simultaneously produced. I t is apparent, therefore, t h a t t h e material which has been classed as gallisin, or a n unfermentable sugar in glucose, yields t o fermentation when i t is acted upon b y proper diastases. I n this respect i t is like similar products produced b y t h e action of diastases, t h e result of a step in t h e conversion of starch t o maltose. If it so happened t h a t a greater number of t h e yeasts which are used for fermentation were of those species which do not secrete maltase, we might conclude t h a t maltose also is a n unfermentable sugar. Since t h e reverse is true, it is not so classed. S U 31 M A R Y A N D C 0 KC L U SI 0 9S

Commercial glucose is a complex body of viscous consistency, running about 42 t o 4j0 Bir. a n d containing from 80 t o 8 j per cent solids a n d 1 5 t o 2 0 per cent mater. It is nearly water-white a n d possesses a mild, sweet taste. T h e solids are composed almost wholly of sugars a n d dextrins, a minor portion consisting of a trace of mineral matter. T h e ash, present t o t h e extent of mere traces, consists of mineral salts, including phosphates, sulfates, chlorides a n d carbonates, chiefly of sodiuni a n d lime. Tests for arsenic and other poisonous metals show these not t o be prese n t . Nitrogenous substances are present as a mere trace, chiefly as protem bodies, amounting t o about 0.06 per cent. I t has been quite well-established in t h e chemical literature t h a t commercial glucose consists of dextrins a n d reducing sugars. Some authorities hold t h a t t h e reducing bodies consist almost entirely of maltose, while others hold t h a t t h e y are a mixture of maltose a n d dextrose, together with some unknown unfermentable substance which has been called “gallisin” b y some a n d “iso-maltose” b y others. I n our work we have (determined t h a t t h e fermentable reducing sugars are a mixture of maltose a n d dextrose b y calculations based upon their q u a n t i t y as determined b y reduction a n d upon t h e a m o u n t of gas developed b y fermentation with yeast. T h e amount of gas t h u s produced is too large t o allow t h e fermentable sugars as determined b y reduction t o be calculated wholly as dextrose a n d too small t o allow their being calculated wholly as maltose. With different lots of glucose t h e relation of t h e maltose t o t h e dextrose a n d t h e amounts of each will vary t o some extent. Determinations on two samples show 11.7 a n d 1 7 . 2 per cent of dextrose a n d 2 2 . 9 a n d 16.4 per cent of maltose, respectively. F u r t h e r researches lead to t h e conclusion t h a t at least three reducing bodies are normally present; namely, maltose, dextrose, a n d a third, which is less readily fermentable b y ordinary bakers’ yeast, b u t which may be made fermentable b y t h e action of certain enzymes, especially those present in pancreatin,

1019

Taka-Diastase a n d malt, as well as by t h e hydrolytic action of dilute hydrochloric acid under t h e influence of heat. These difficultly fermentable reducing bodies a m o u n t t o about 14 per cent of t h e total glucose when calculated as maltose, or 8 per cent when calculated as dextrose. The unfermentable carbohydrate residue remaining after removing t h e maltose a n d dextrose b y fermentation consists of those bodies commonly recognized as dextrins. These, like t h e unfermentable reducing bodies, when subjected t o hydrolytic action b y diastase result in products which will under suitable conditions undergo almost complete alcoholic fermentation. When t h e y are subjected t o t h e hydrolytic action of hot dilute acid, applied either t o t h e unfermented glucose or t o t h e residue left after t h e glucose has undergone alcoholic fermentation, all of these bodies become wholly fermentable. Cold water extract of malt as t h e source of t h e diastase was found less suited for t h e purpose t h a n pancreatin or Taka-Diastase because of t h e larger proportion of unfermentable bodies which i t contained a n d introduced into t h e products of fermentation. I t was found t h a t a good quality of Taka-Diastase converted t h e unfermentable products into fermentable sugars, leaving only a very small amount of unfermented residue, which contained as reducing sugars less t h a n one per cent of t h e glucose taken. I n t h e most favorable instances t h e total unfermented residue amounted t o from 4 t o j per cent a n d included normal products of fermentation, notably succinic acid a n d possibly some glycerine, which always results from t h e fermentation of sugars b y yeast. T h e diastase present in good samples of pancreatin a n d in t h e cold water extract of malt i n like manner converts t h e unfermentable residue of t h e glucose into fermentable sugars, b u t in some instances less completely t h a n does Taka- Diastase. I n these experiments it was f o u n d t h a t t h e kind of yeast used h a d a considerable influence upon t h e completeness of t h e fermentation of t h e products produced b y t h e action of t h e several diastases. Isolated ferments like pancreatin a n d Taka-Diastase lose their activity with age a n d when these weakened ferments act upon glucose t h e result of t h e decreased vitality is a , decreased proportion of immediately fermentable sugars, with a n increased proportion of t h e unconverted dextrins a n d of t h e intermediate unfermentable reducing carbohydrates or reducing dextrins. These conditions are also clearly apparent where weakened pancreatin or weakened T a k a Diastase or a limited amount of diastase acts upon starch of various kinds. T h e apparent results of such action are a n extended row of products, including t h e well-recognized dextrins, unfermentable reducing bodies (apparently$reducing dextrins) maltose a n d dextrose. T h e fact t h a t t h e first bodies in t h i s series are found t o a limited extent only when there is a sufficient a m o u n t of t h e active diastase present, b u t are abundant when there is a limited amount present, indicates t h a t t h e y belong t o a natural series of changes between starch a n d dextrose. T h e results of our examination of glucose a n d of t h e products of t h e combined action of

I020

T H E J O C R S A L OF I S D C S T R I A L A Y D ESGI-VEERISG CHEMISTIiI’

diastase a n d yeast upon glucose, indicate t h a t all t h e bodies of this series are normally present in glucose which is produced b y t h e incomplete hydrolysis of starch b y acids, just as t h e y are present in liquors containing t h e products of t h e incomplete hydrolysis of starch b y diastase, a n d t h a t these bodies in glucose yield t o further treatment with diastase, just as do those produced b y diastase itself. From these facts, it is apparent t h a t t h e claim for t h e presence in glucose of unfermentable reducing bodies as reversion products brought about by t h e action of t h e acids a t a high heat is untenable. A s t u d y of t h e action of hydrolytic agents and yeast on carbohydrates entering into common foodstuffs, such as potatoes, breakfast cereals, bread, and so forth, and upon pure starches, such as are found in these various food products, has been made in comparison with parallel experiments on glucose. I n these experiments it was found t h a t t h e carbohydrates of glucose agree closely in gas production with t h e carbohydrates of t h e more readily digestible foodstuffs, such as white bread, breakfast cereals and potatoes. It was also found t h a t these several carbohydrates when acted upon by isolated ferments and yeast, as in t h e experiments conducted, yield variable b u t appreciable amounts of unfermentable carbohydrate products, just as t h e mashing of cooked starch with malt diastase in t h e making of malt liquors results in a liquor which after fermentation contains appreciable quantities of such unfermented and apparently unfermentable carbohydrate products. T h e fact t h a t commercial glucose, when i t is treated with diastase and t h e n subjected t o yeast fermentation, is almost wholly converted into alcohol and carbon dioxide goes t o prove t h a t it consists of products t h a t are wholly assimilable and, therefore, i t furnishes a food t o t h e body of a sugar nature. I n this respect i t is a more concentrated and a t the same time a more readily assimilable food t h a n are most of the carbohydrates belonging t o t h e ordinary foodstuffs which first h a t e t o undergo cooking and then complete hydrolysis by t h e action of t h e digestive enzymes before t h e y can be utilized by t h e body. I n this respect glucose. pound for pound of dry weight, will furnish a t least as much energy as does cane sugar. RESEARCH DBPARTMENT

THE C O L ~ M B U LABORATORIES S CHICAGO

VITAMINES AND LIPOIDS IN BUTTER AND MARGARINE By J. DE RLITER Neceived March 2 1 , 1916

Since Osborne a n d hIendel found certain substances in milk and butter, apparently of lipoid nature, necessary in h u m a n food t o maintain health and growth, it is interesting t o compare butter and margarine as t o their respective content of lipoid substances. Of t h e material tested t h e butter was taken from one of the cooperative dairying factories in Frisia; “Klappa” and “Planta,” both “vegetable butter,” f r o m special factories in Xmsterdam and Oss, whereas t h e “margarine proper” was taken from a noted margarine factory. .Is Ear as information could be

YoI. 8,S O .I I

obtained, Klappa and Planta are made b y means of skim milk. First of all, an easy method was required L O find t h e lipoid content of fats and oils. T h a t which t h e writer used is based on t h e observation t h a t lecithin is soluble in concentrated acids, but is precipitated again when t h e acid solution is mixed with water. The first observations were made with lactic acid. b u t more rapid results could be obtained with hydrochloric acid of 1.19 sp. gr. T h e lipoid precipitate could be separated easily from t h e diluted acid. Afterm r d s it was collected on a filter, washed with acid water, dried a t 100’C. and weighed. The lipoids thus obtained, after burning, gave an ash which showed a large phosphorus content on testing with molybdic reagent. Sesame oil, thus shaken with an equal volume of hydrochloric acid (1.19 sp. gr.) proved t o have entirely lost its lipoid content. With t h e method described t h e following results were obtained: GRAXSO F Sesame Oil Arachis Oil Olive Oil Cod Liver Oil

0.100

Traces Traces Traces

L I P O l D S IN

100 CC.

Raffinated Coconut Oil Traces Filtered Butter F a t Traces Butter 0,400

Klappa Planta Margarine “Bran Butter”

0.750 0.475 0.975 1.125

From these figures two conclusions may be drawn: ( I ) T h e seat of the lipoids in butter is not the fat itself b u t t h e casein solution mixed with it. ( 2 ) The lipoids in butter represent only a part of t h e total lipoid content in milk ( * o . o 7 j per cent). The remaining part is responsible for the lipoid content in margarine and vegetable butter as far as they are made by means of skim milk only. The high figure for “margarine proper” may be due t o t h e use of egg yolk besides skim milk as a n emulsifying agent. I n order t o enhance the liptid content of “vegctable butter” the albumin-bearing seeds may be turned t o account. Just as egg yolk, they contain lipoids in a chemical combination or a n adsorption with albumins. So little are t h e y inclined t o give u p their lipoids to solvents t h a t a sesame oil which was hcated Lvitli wheat bran a t 100‘ C. afterwards proved even t o have lost a great deal of its lipoids. From this combination or adsorption t h e lipoids may be set free b y treating t h e seed material with liquors t h a t dissolve t h e albumins. The “steep liquor” of t h e corn starch factories, obtained by treating t h e corn n-ith slightly acidulated 75-ater, is an example of such a solution. T o set free the lipoids of wheat bran the b r a n vias treated v i t h diluted lime-water during 24 hrs. The liquor pressed off and separated from t h e subsiding starch was shaken with a molten mixture of raffinated coconut oil and arachis oil. After t h a t a quantity of skim milk was added, amounting t o one-fifth 01 t h e emulsion, and t h e mixture allowed t o ripen. Then the fat was separated and worked t o a butter-like product, which showed the high lipoid figure above (bran butter). The lipoid content may be increased by altering the proportion of bran and fat. Another interesting point in t h e process described is t h a t the liquor separated from t h e “bran butter’’