TX 558 .W5 C6 Copy 1 Flour Strength as Influenced by the Addition of Diastatic Ferments A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA By FERDINAND A. COLLATZ, B, S., M. S. IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY August, 1922 Chicago, III. Flour Strength as Influenced by the Addition of Diastatic Ferments A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA By FERDINAND A. COLLATZ, B. S., M. S. IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY August, io2_* Chicack), 111. r-^' y-\ •o' v.. ^" ^' \) \ \ w v.™^ LIBRARY OF CONGRESS ( RECEIVED i DOCUMENTS DiV.otO. OCT 171922 i Research Fellowship Plan Under Which Fellows of the American Institute of Baking Have Been Enrolled as Graduate Students of the University of Minnesota. The American Institute of Baking in the fall of 1920 detailed L\vo research fellows in the chemistry of baking to work on suitable prob- lems as graduate students of the University of Minnesota. These fellows were regularly registered in the Graduate School of the Uni- versity. They pursued such courses as ordinarily constitute a study program of candidates for the doctorate in philosophy, majoring in the Division of Agricultural Biochemistry. Research problems were selected and outlined in conference with their advisors in this Division, actual work on the problems being pursued, l)y special agreement, chiefly in the laboratories of the American Institute of Baking. Theses based on this research were duly presented in partial fulfillment of the degree of Doctor of Philosophy, and accepted by committees of the Graduate School of the University. These theses are, by agree- ment with the graduate faculty of the Division of Agricultural Bio- chemistry, published by their respective authors as bulletins of the American Institute of Baking. TABLE OF CONTENTS Page I. Introduction: Definition of Hour .stronL;tli 5 A. Historical 6 1. The proteins of wheat flour and their physical rehition to flour strength 6 2. The carbohydrates of wheat flour and their relation to flour strength • • ■ • 9 3. Historical review of the diastatic enzymes 13 a. The Iodine method for the estimation of diastatic activity ... 15 b. Copper reduction method for the determination of reducing sugars formed by the action of diastase 16 c. Influence of temperature on diastatic activity 17 d. Influence of acids, bases and salts on diastatic activity 17 4. Effects of proteolytic enzymes 18 5. Hydrogen ion concentration 19 6. Viscosity 19 II. Experimental 20 A. The problem 20 B. Material 20 1. Description of materials used in these studies 20 a. Analysis of wheat flours 21 b. Analysis of malt flour 21 c. Analysis of malt extract 21 C. The methods 22 1. Determination of diastatic activity 22 2. Determination of proteolytic activity 23 3. Determination of gas producing capacity of flour 24 4. Baking tests 24 D. Influence of varying conditions on diastatic activity 25 1. Determination of the optimum pH for the amylase of malt flour.. 25 2. Influence of time of digestion on the diastatic activity of malt flour ; 26 3. Effect of temperature upon diastatic activity 28 4. Effect of concentration of diastase on hydrolysis of starch in wheat flour 30 5. Effect of increasing amounts of malt flour when digested with a constant quantity of wheat flour 31 6. Production of reducing sugars in the dough during fermentation .32 7. Determination of proteolytic activity as measured by the fall in viscosity of flour-water suspensions when digested with diastatic preparations .41 8. Gas production capacity of wheat flour in relation to strength.. 47 9. Change in hydrogen ion concentration of fermenting dough 48 10. Baking data 4g III. Discussion 54 1. Changes in pH, temperature and concentration and their effects upon the activity of diastases contained in malt flour 54 2. Effect of diastatic enzymes upon starch of different flours 55 3. Production of reducing sugars in the panary fermentation of bread and the effects of diastases added to the dough 56 4. Eifect of proteolytic enzymes contained in malt preparations upon the viscosity of strong and weak flours, following the addi- tion of various amounts of lactic acid 58 5. Gas producing capacities of strong and weak flours and the effect of added malt extract upon them 59 6. Changes in hydrogen ion concentration taking place during the fermentation of the dough .60 7. Effect of malt flour and malt extract upon tlie baking value of flour.60 IV. Summary ^2 V. Literature cited 63 FLOUR STRENGTH AS INFLUENCED BY THE ADDITION OF DIASTATIC FERMENTS By Ferdinand A. Collatz I. INTRODUCTION. The baking strength of flour has received a great deal of attention by scientific workers in the last twenty-five years, due primarily to the economic importance of bread. A number of factors have been thoroughly investigated, in their relation to baking strength, in order to draw some conclusions as to why some flours give a large, light, pal- atable loaf of bread and others an inferior loaf. Certain factors which have been investigated in their relation to baking strength are total nitrogen, ratio of water-soluble nitrogen to total nitrogen, chemical composition of the individual proteins, total gluten, total gliadin, ratio of gliadin to glutenin, ratio of gliadin to total nitrogen, ratio of wet to dry gluten, efifect of electrolytes, hydrogen-ion concentration, total amount of gas evolved during fermentation, and the effects of diastatic and proteolytic enzymes of the flour. Flours which bake out well have been given the arbitrary term of strong flours while the others arc termed weak. Naturally a great number of definitions of strength have found their way into the litera- ture, but the definition that has been most generally accepted is that of Humphries and Biffin (1907), who state that "a strong wheat is one which yields flour capable of making large, well-piled loaves." Flours which do not measure up to this empirical standard are classed as weak. This definition indicates that strength in flour is more desir- able than weakness for the baking of bread. Wood (1907), has called attention to two factors in strength, namely, size and shape of the loaf. This has stimulated a great deal of research by Ford and Guthrie (1908), Baker and Hulton (1908), on the diastatic and proteolytic en- zymes in wheat flour, and also by Upson and Calvin (1915) (1916), Gortner and Doherty (1918), and Sharp and Gortner (1922), on the colloidal properties of wheat gluten as affecting flour strength. To- day we must recognize three groups of factors dealing with strength or weakness in flour, According to Sharp and Gortner (1922). we have "at least three classes of weak flour, i. e., (1) weakness due to an adequate quantity of gluten but of inferior quality, (2) weakness due to an inadequate quantity of a good quality gluten and (3) weak- ness due to factors influencing yeast activity." HISTORICAL. Not until Osborn and \\)urhees (1893) (1894), established the com- position and properties of the wheat proteins was any great advance made in regard to flour strength, and naturally attention was then di- rected to the two main proteins, gliadin and glutenin. Fluerent (1896) asserted that flour strength depended upon the proportions of gliadin to glutenin, the ratio of 75 percent to 25 percent or 3 to 1 being most nearly ideal. Snyder (1899) came to similar conclusions but stated that the ideal ratio was 65 [percent gliadin and 35 percent glutenin. In a later publication Snyder (1901) claims quality rather than quan- tity of protein is of the greater importance. Shutt (1904) (1907) points out that after several years of research "it appears extremely doubtful if the gliadin number (percentage of albuminoids in the form of gliadin) constitutes a factor that can be correlated with bread mak- ing values as obtained l)y baking trials." These conclusions are again verified in a later report. Thatcher's (1907) results show that no single factor or group of chemical tests which he tried give results from wdiich the comparative baking qualities (jf flour can be determined. Blish (1915) states that the gliadin-glutenin ratio is much more con- stant in flours of different baking strength than has heretofore been supposed. Blish found, after careful investigation, that the individual proteins of weak and strong flours are chemically identical. The soluble proteins of flour have also had their share of investiga- tions as to their relation to baking strength. Snyder (1897). Bremer (1907), Wood (1907) and others have found no relation of baking strength to the amounts of water soluble proteins. Quite recently Martin (1920) has attemi)ted to correlate certain I)roperties of flours with baking strength. He finds "for flours having a satisfactory gas producing capacity, bakers' marks, gas retaining ca- pacity, and amended gliadin content are closely related, and it is con- sidered that the estimation of the gas producing capacity will indicate the strength of the flour." Martin's "amended gliadin'' content is the dift'erence in protein (Nx5.7) between the amounts extracted by 50 percent alcohol and that extracted by water acting for three hours at 24-25°. Sharp and Gortner (1922) find that "amended gliadin" values were not correlated with the strength of the flours with which they worked. The Proteins of Wheat Flour and Their Physical Relation to Flour Strength. As far as we know, all proteins belong to that class of colloids known as emulsoids and more recently termed "hydrophylic colloids." As the latter term suggests, this class of colloids has a great affinity for water, which, however, can be moditied to a great extent by the addi- tion of acids, bases and salts to the dispersion medium. Hofmeister was one of the first to investigate the swelling of pro- teins. He found that in solutions of sulphates, tartrates, acetates, alcohol and cane sugar, gelatin-plates take up less water than they do when immersed in distilled water, while in solutions of potassium, so- dium or ammonium chlorides, sodium chlorate, sodium nitrate and so- dium bromide, they take up more water than they do when immersed in distilled water. Hofmeister's work has been enlarged upon by oth- ers, notably Pauli (1899) (1902) (1903) (1905) (1906) and Fischer (1915) (1918). Wood (1907) and Wood and Hardy (1908) have demonstrated wheat gluten to be an emulsoid colloid and as has already been noted the water-holding capacity of hydrophylic colloids can be altered to a marked degree by the addition of electrolytes. Acids and bases cause imbibition up to a certain point, while neutral salts tend to inhibit the imbibition of water. It appears that Wood (1907) was the first to call attention to the physical properties of the proteins in wheat flours rather than the chemical differences in relation to flour strength. He investigated the possible chemical differences between the glutcnin and the gliadin of these two classes of flours. From this he concludes that strength (particularly the shape of the loaf) is closely related to the physical state of the gluten, which in turn is affected by the presence of elec- trolytes. Wood, and Wood and Hardy determined the effects of acids, with and without salts, by suspending bits of gluten from glass rods in the liquid to be investigated. They found that dispersion of the gluten starts immediately when immersed even in the lowest concentration of acid, and dispersion increased with increase in concentration within certain limits. This holds good for sulphuric, phosphoric and oxalic acids, but not for hydrochloric. When the concentration of the latter exceeded N/30 the dispersion began to decrease until at a concentra- tion of N/12 the gluten was more elastic and coherent than in its original condition. The addition of salts decreases dispersion in all cases and such amounts of salts can be added which will prevent the dispersion of the gluten. From this Wood suggests "that the varia- tion in coherence, elasticity and water content, observed in gluten ex- tracted from different flours, is due to varying cancentrations of acids and salts in the natural surroundings of the gluten, rather than to any intrinsic difference in the composition of the glutens themselves." Wood thinks that the direct addition of acids or salts to the flt»ur, in order to strengthen it, is impractical as they would have to be in con- tact for forty-eight hours before baking. lie found that this was the time required for the gluten to come into equilibrium with its sur- roundings. He, therefore, advocates the blending of such fiours which will supply each flour's requirements in this respect. In a later paper Wood and Hardy ( 1908) take up the theoretical discussion of the efl:'ects of acids, alkalis, and salts up(Mi gluten. Upson and Calvin were the next to study the colloidal swelling of gluten. They atacked the problem in a slightly different manner, washing out the gluten with distilled water and pressing it out into thin layers. They next cut out discs of uniform size and weighed them immediately. The discs are then immersed in acid and acid-salt solutions of varying concentrations for a detinite period of time when they are taken out, drained and weighed. They tind that "flours con- taining acids and salts in such combinations as to favor water absorp- tion will behave as weak flours, wdiercas those containing acids and salts in such combinations as inhibit water absorption will behave as strong flours." Their conclusions are very similar to those of Wood. Gortner and Doherty were the next to investigate the colloidal prop- erties of wheat flour gluten. Their method of attack was like that of Upson and Calvin, in that they recorded the increase in imbibition by weighing discs of gluten after immersion in acid and acid-salt solutions of various concentrations. They worked with five different flours, namely a high grade |)atent flour milled from hard spring wheat, a clear flour and three typically soft flours milled from Oregon wheat. Their results show that the gluten from a weak flour has a much low- er rate of imbibition and a much lower hydration capacity than the gluten from a strong flour; also that inorganic salts when added to an acid solution lower the relative imbibition of gluten placed in such solutions. Glutens from the different flours react differently to the addition of inorganic salts. This leads them to believe "that a weak gluten does not owe its 'weakness' nor its imbibition curve its "flat- ness,' to either the acid or the salt content of the flour from which it is derived, 'but rather to the fact that a weak gluten has inherently in- ferior colloidal properties." In 1918 and 1919, a series of articles dealing with the physical prop- erties of wheat flours were published by Henderson and his co-work- ers. Inasmuch as they fail to describe their flours, no conclusions can be drawn as to their effect on fl()ur strength. It is of interest to note, however, that at a hydrogen ion concentration of about pH 5, (the op- timum hydrogen ion concentration in the making of bread as found by Jessen-Hanson) the viscosity of dough, made from four different flours is at a minimum. They further note that salts such as sodium lactate, NaoSO^ MgSC)^, KBrOs tend to decrease the viscosity of the dough, while NaCl, MgClz NH^Cl tend to decrease the viscosity when used in small amounts, but on further addition the viscosity increases. Ostwald (1919) and Liiers (1919) and Liiers and Ostwald (1919) (1920) in a- series of papers show the remarkable parallelism existing between viscosity measurements of flour-water mixtures and grade of flour. They found that flours divide themselves into three distinct groups when measured in this way. The low extraction flours (40%- 60%) constitutes one group, the high extraction flours (60%-94%) a second, and the tailing flours (remains of 60 % - 94 %) constitute the third group. They also find that acids and bases tend to in- crease the viscosity of the flour-water mixtures while salts tend to depress the viscosity. Their results, although obtained by a dif- ferent method, confirm the work of Wood, Wood and Hardy, and Up- son and Calvin. It must be emphasized, however, that their results were obtained on flours of dift'erent milling extraction and the dif- ferences they obtained refer only to the grade or degree of extraction of the flours, and do not necessarily apply directly to the problem of flour strength. Sharp and Gortner have continued the work of Gortner and Do- herty, and confirm the findings of the latter in regard to the action of acids upon gluten. They find a marked difiference in the rate of im- bibition when using the various alkali hydroxides. The action of al- kalis on gluten is markedly different from that of acids, as dispersion takes place at much lower concentrations. They remark in this re- inspect. "Indeed dis]>ersi()n and imbibition arc here almost coincident." During the course of the work Sharp and (iortner washed out the gluten from their strong and weak flours and dried it at low temper- ature in vacuo. After pulverizing, they found that the dried glutens of various flours were much more alike than in the wet state. This observation is in accord with their theory '"that the strong gluten is strong because of its colloidal properties ; inasmuch as it is well known that the alternate wetting and dr}ing of a colloidal gel. breaks down the gel structure." They also find that the optimum hydrogen ion con- centration for imbibition is the same for the different acids used. In their next paper Sharp and Gortner (1921) extend their work on the hydration capacity of glutens. They find that the viscosimeter gives an accurate measure of imbibition in that the curve obtained fol- lowed the previous curves by weighing out the discs of gluten. As might be expected, they found that the strong flours give much greater viscosity measurements than do the weak flours. The Carbohydrates of Wheat Flour and Their Relation to Flour Strength. The carbohydrates in the flour have also been investigated with ref- 9 erence to their rule in flour strength. Wood was perhaps the first to make any definite statement in this respect, although Girard and Fleu- rent (1903) called attention to the great variations in the amounts of sugars in the flours. They found on analysis that glucose and cane sugar were present, the former varying from 0.097o to 0.81%, the latter 0.63% to 1.897o. Bruying in 1906 considered that the sugar present in flour is not glucose and sucrose, but almost entirely maltose. Liebig (1909) supports the views of Girard and Fleurent in that he found wheat flour to contain from 1-1.5% sucrose and 0.1-0.4% dextrose. Wood thinks that strength hinges to a great extent, as far as volume and size of the loaf is concerned, upon the sugar content and the dias- tatic enzymes that the flour contains. He sums up that factor in strength dealing with volume of the loaf as follows : "The factor which primarily determines the size of the loaf which a flour can make is quite distinct. The size of the loaf is shown to depend in the first instance on the amount of sugar contained in the flour together with that formed in vhe dough by diastatic action. Particular attention should be paid lo the rate of gas evolution in the later stages of fer- mentation, as this is shown to be more directly connected with the size of the loaf." Wood's method of measuring the gas-producing capac- ity of a flour consists of mixing 20 grams of flour and 0.5 grams of yeast with 20 cc of water in stoppered flasks and measuring the lib- erated carbon dioxide under brine. Shutt (1907) shortly after Wood's article, determined the sugar ex- tracted by 70 per cent alcohol and by water and could find no evidence "that with increase of sugars there is increase of volume in loaf, but rather the reverse." .Shutt's data is shown in Table I. TABLE I. Sugars in flour extracted by 70 per cent alcohol and water. (Data taken from Shutt [1907] page 20). In aqiK-ous extract In alcoholic extract Directly .\fter Directly After Designation redncint; Inver.sion reducing Inversion of as as Total as as Total Vol. Sample IMaltosc Sucrose Sugars Maltose Sucrose Sugars Bakers % % % % % % Mark No. 1 Hard 1 .Q6 2.14 4.10 .13 .91 1.04 492 No. 1 Northern 2.73 1.9.^ 4.68 .20 .94 1.14 443 No. 2 Northern 1.87 1.79 3.66 .18 .87 1.05 438 No. 3 Northern 3.42 2.10 .S.22 .26 121 1.47 383 No. 4 Com'cial Gi -. 3.62 2.43 6.05 .05 134 1.39 397 No. 5 Com'cial Gi -. 3.63 2.43 6.06 .06 1 42 1.48 366 No. 6 Com'cial Gi r. 4.07 2.43 6.50 .06 1.36 1.42 363 Shortly after the appearance of Wood's paper. Baker and Hulton (1908) reported a paper in which they investigated the action of en- 10 zynies contained in flour with regard to their etifect on flour strength. Unable to demonstrate experimentally the existence of proteolytic en- zymes in flour, they concluded that any which are present have no ef- fect upon the gluten during the time of fermentation. They did show, however, that the proteolytic enzymes contained in yeast play an important part, since one dough containing yeast showed 2.7% sol- uble nitrogen as protein, while a similar dough without yeast had 1.9% soluble nitrogen as protein. Although the fact was known that wheat flour contained amylolytic enzymes, Baker and Hulton demon- strated their presence in dough by an increase in maltose, extracting the latter with water and preparing the maltosazone. They found that contrary to expectatinns, the diastatic activity of flours increased with age. * Baker and Hulton (1908) show that the total volume of gas pro- duced (same method as outlined by Wood) increases roughly with in- crease in baking strength (Baker's Mark) of the flour. They also point out that a weak flour may have a diastatic power as high or even higher than a strong flour. This is explained by the fact that in real- ity the weak flour is deficient in liquifying enzymes and that by the addition of liquifying enzymes a much greater volume of gas is given off, while a strong flour shows no increase in gas production when a diastatic enzyme is added. The liquifying enzymes were added in the form of a malt extract and Table II shows that even such smaller amounts as 0.25 and 1.0 percent caused the gas production to increase enormously in a w^eak flour. They did not state whether the addition of the malt extract did actually increase the baking strength of the flour. From the data submitted, Baker and Hulton concluded that weak flours in some instances give as great a gas production as do strong flours, and that gas production is not a function of the quan- tity of diastases but, as they show, (Table III) it is intimately con- nected with "the additional matter rendered soluble during the process of doughing," i. e. maltose. TABLE II. Effects of added malt extract upon a weak flour with reference to an increase in gas production. (From data of Baker and Hulton [1908] page 372). ,25 Percent 1.0 Per cent Time FI our Alone of Malt of Malt Hours c.c. CO. c.c. CO2 c.c. C0= 0.5 28 30 32 1.0 47 66 69 1.5 55 101 115 14.0 113 245 362 *(It is a well-known fact that flour.s on aging show greater baking strength and this increase in diastatic activity may therefore be the primary cause for increase in baking strength a."^ the flour ages. At any rate it seems to be in accord with Wood's theory). n TABLE III. The relation between gas volume and the additional matter rendered soluble during the process of doughing. (From Baker and Hulton (1908) page 372). I'ercent of Matter sc )luble Differences Volume Percent of in dou; gli Maltose of gas ob- matter sol uble when k( -Pt formed tained from in water at at 40^ C. for durinj.; dough in Bakers our 15.5°C. 3 liour.s doughing 3 hours Mark 1 2.12 3.60 1.48 78 45 X 2.03 4.41 1.38 84 40 w 2.83 5.38 2.53 145 Id 3 2.49 5.53 3.04 155 80 Y 2.69 6.57 3.88 164 95 2 3.19 6.66 3.45 175 78 4 4.19 10.95 6.75 193 90 V 2.83 8.26 5.42 217 90 T 2.84 7.66 4.82 220 80 U 2.65 7.68 5.02 230 91 Z 3.54 11.65 8.11 270 90 It would seem from the above table that low strength flours are de- ficient in liquifying enzymes and the authors conclude that the liqui- fying enzymes are the limiting factors in the production of maltose in the dough stage. Simultaneously with the appearance of Baker and Hulton's article, the work of Ford and Guthrie (1908) was published on the relation of enzymes contained in flour to its baking strength. They conducted experiments of extraction and found that amylases could be greatly stimulated by the use of KCl and also 1)y active and by boiled papain. In testing amylase values from twelve flours, they found differences (using KCl and papain extracts) varyiii!^ from 22.1 to 4().8 expressed in grams maltose per grani of dry flour. They could not correlate diastase activity with flour strength and state "It however indicates that in developing a method of evaluation, the total amylase is one im- l^ortant factor, also that the presence of a proteolytic ferment is an- otlier and possibly more valuable consideration." Ford and Guthrie (1908) were prol^ably the first to demonstrate the action of proteolytic enzymes in flour. They were unable to secure results with nitrogen determinations or with the viscosi- rneter, so they tried 1 percent gelatin. The liquification of the gela- tin gave them positive proof of proteolysis. They also conducted baking tests with a large amount of protease added, and naturally the loaf did not rise during the fermentation period, the resultant bread being a soggy mass. They concluded that proteases decrease gas holding properties of the gluten and point out that this is the chief reason for failure in the use of malt extracts in baking practise. Bailey and Weigley (1922) fotind that flour strength depended 12 upon factors which control the rate of carbon dioxide production and the amount of carbon dioxide lost during fermentation. They found that "the loss of carbon dioxide per unit increase in volume under controlled conditions afifords a useful measure of gas-holding capacity of dough." In some unpublished data Thatcher and Kennedy show that when flour was digested with water, the amounts of reducing sugars in the extract increased regularly with increase in temperature. A centrifuged aliquot of a flour water extract likewise increases in soluble nitrogen with increase in temperature of extraction. They also found that no increase in reducing sugars takes place when a filtered extract (0°-5°C) of flour is allowed to act on soluble starch when incubated at 40°, 50° and 60° C. Under these conditions they assume that absorption of the enzyme or activator has taken place upon the filter paper or uixm the gluten colloids. Historical Review of the Study of Diastatic Enzymes. In taking up the history of the diastases, one is confronted with a voluminous and at times conflicting literature, which extends back over a period of one hundred years or more. Naturally, a great deal has to be discarded, as it would be impossible to review any but the most important papers submitted on this question. Neverthe- less it is my intention to cite a number of papers which are of in- terest from a purely historical viewpoint. Vauquelin in 1811 was the first to record the fact that when starch was heated in water, it gave an opaque solution and had the characteristics of gum arabic. In the same year Kirchofif found that when starch was boiled with dilute H^SQ^j, a crystallized sugar was formed. Two years later he noted that the protein of the embryo of the seed, particularly if the seed had been germinated, acted on starch in much the same manner as did the acid. He real- ly was the first to record diastatic activity but did not realize the importance of his observations. Vogel in 1812 found that when starch was boiled with acid, it gave two products, a sugar and a gummy substance, the latter now known as dextrin. Stromeyer in 1813 found that iodine was a specific reagent for starch and visa versa, while the action of alkaline copper sulphate w^as found by Trommer in 1841 to be a means of distinguishing sugar from starches and gums. The gummy substance found by Vogel was investigated by Biot and Persoz in 1853 and was found to turn the plane of polarized light to the right. For this reason it was given the name of dex- trin. It is of interest to note that the work of Biot and Persoz formed the basis for the development of our present day polaris- 13 cope. In the same year Payen and Persoz conclusively established the fact that an extract of malted grain had a powerful action in liquifying and saccharifying starch. They ascribed this function to some inner substance and named it diastase. It had been the impression of chemists up until this time that glucose was the sugar formed when starch was acted upon by diastase and it was not until 1872-1876 that O'Sullivan showed it to be maltose. O'Sullivan found the optical rotation to be too high and the reducing power too small to correspond with glucose. It might be of interest to call attention to the discovery of maltose at this time. Although DeSassure had accurately described maltose in 1819 the fact had evidently been forgotten until Dubrunfaut called attention to it in 1847 and named it maltose. This rediscovery was again forgotten until it was again described by O'Sullivan in 1872. Marker in 1877 states that at a temperature of 60° C four mole- cules of starch yield three of maltose and one of dextrin, under the influence of diastatic ferments. At 65" the }'iel- in solutions being analyzed. Effects of Proteolytic Enzymes. The action of proteolytic enzymes upou protein material is well understood today and needs very little mention. In regard to the action of proteolytic enzymes in flour, Ford and Guthrie were not able to show by any chemical means that tlicy existed in wdieat flour. However, when 1 per cent gelatin was added to the flour and then 18 allowed to solidify, proteolysis could be followed by the gradual liquification of the gelatin. Baker and Hulton could find no method to establish the presence of proteolytic enzymes in flour and therefore claim that any which may be present would have very little effect ujuju a dough. They did demonstrate, however, that yeast contains a very powerful protein splitting enzyme, as shown by the soluble nitrogen of two identical doughs, one with and the other without yeast. In the dough to which yeast had been added, they report 2.7 ])er cent soluble nitrogen as protein, while in the other dough they found onlv 1.9 per cent soluble nitrogen as protein. Hydrogen Ion Concentration. From the foregoing literature, it has been shown that acids, bases and salts are of the utmost importance in relation to the activity of diastatic ferments. Jessen Hanson has also shown that the optimum conditions for the baking of bread occur when the dough is at a hydrogen ion concentration of about pH 5.0. Bailey and Peterson (1921) find that when acid or alkali is added to buffered water extracts of flour, a characteristic curve is obtained which indicates very accurately the grade and baking qualities of a flour. Bailey and Collatz (1921) have shown that a remarkable parallelism exists between grade of flour and the electrical conduc- tivity of a water extract of flour when digested one hour at 25°C. Viscosity. Although Ford and Guthrie attempted to demonstrate the proteo- lytic action of enzymes in wheat flour, by digesting the flour in water at a set temperature by means of viscosity measurements, they report no success in this method. Ostwald and Liiers in a series of papers show that different mill grades of flours can be distinguished by means of a viscosimeter. From their data, the flours group themselves according to the degree of extraction in milling-. Quite recently Sharp and Gortner have demonstrated the efficiency of the viscosimeter in measuring differences in the imljibitional capac- ity of strong and weak flours when treated with \arious acids, bases and salts. They find that strong flours show a greater viscosity under the conditions of their experiments than do the weak flours. From the literature cited one may judge the amount of work expended upon the question of flour strength. Although the work of the last few years shows progress on this question, we do not have a single test which gives us an absolute criterion of flour strength 19 and it is still necessary tu fall back upon the baking tests to have a final answer to the question. II. EXPERIMENTAL. (a) The Problem. It has been shcnvn in the historical review that flour strength has been studied in a variety <>f ways. Two points of attack are outstand- ing, however, the work of W'ood and Hardy, Upson and Calvin, Gortner and Doherty, and Sharp and Gortner, who have concerned themselves with the physico-chemical properties of the gluten ; and of Baker and llulton. and I-'ord and Guthrie who have attacked the problem from the enzymic standpoint. In this Thesis we are con- cerned with enz}nie relationships. From tiie data presented by Ford and (lUthrie it would appear that the diastatic enzymes were of more importance than the proteolytic enzymes with regard to flour strength. Baker and Hulton indicate in their excellent work that the amy- loclastic enzymes A\ere perhaps the limiting factor in the production of maltose. I have taken up the i)roblem at this ])oint and am con- cerned with the efl:'ect upon the baking strength of wheaten flours when diastatic ferments are added to the dough. As the diastatic preparations a\ailable for the baker are in the form of malt flours and malt extracts which contain ])roteolytic enzymes, the problem is at once broadened to include the later as well as the starch splitting- ferments. (b) Material. The ])resent investigation was conducted with a commercial malt flour, a rei)re^entative malt extract, a commercial sample of wheat starch, and a series of seven wheat flt)urs of ditTerent grades milled from wheats grown in different regions of North America. All of the flour samples and malt preparations were sul)mitted to careful chemical analysis and in most cases were rechecked by other inves- tigators using the same materials. The buffer values of the flour extracts were also determined by the method of Bailey and Peterson in order to have additional data as to the grade of the flour. This data is given in Table VIT. Description of Materials Used in These Studies. The flours used in this inxestigation were flours milled from auth- entic samples of wheat, grown in different regions of North America under different climatic conditions. The A. O. A. C. methods were followed in analyzing the flours and malt preparations, with the results shown in Tables TV, V, and VI. A description of the wheat flours is as follows : 20 Flour 1001 was milled from a sample of hard Kansas wheat from the crop of 1921. The baking tests showed it to be of good strength and the low ash content. Hydrogen ion concentration, in terms of pH, show^ it to be a patent of low extraction. The protein content, which is a trifle low, reflects directly upon its l)aking strength. TABLE IV. Analysis of Wheat Flours on Air Dry Basis. Flour Samples Protein Laboratory Moisture Ash (Nx5.7) Milling pH of Water No. Percent Percent Percent Grade Extract 1001 12.15 .40 11.34 Patent 5.816 1002 12.14 .61 13.00 Clear 6.052 1003 13.06 .46 8.83 Patent 6.002 1007 11.06 .64 14.12 Clear 6.103 1008 11.70 .46 15.32 Patent 6.133 1009 11.61 .42 13.81 Patent 5.981 1011 11.44 .56 10.77 Patent 6.050 TABLE V. Analysis of Malt Flour on Air Dry Basis. Total Reducinf;- Sugars Sugars as Dex- Diastatic Value s Dextrose trose Degrees Percent Percent Lintner Malt Flour Laboratory Moisture Ash Protein No. Percent Percent Percent 24 8.8 1.26 11.25 4.75 10.62 177.05 TABLE VL Analysis of Malt Extract. Reducing Sugars Total Sugars Dias- Calculated Calculated as Pro- tatic Ex- Ash DeX' Dex- tein Value tract Moisture Per- Maltose trose Maltose trose Per- Degrees Specific No. Percent cent Percent Percent Percent Percent cent Lintner Gravity D 25.63 1.35 73.74 42.52 73.80 42.62 6.06 37.1 1.384 Flour 1002 was milled from a sample of hard Kansas wheat. The high ash content and the pH of the w^ater extract indicate a clear flour. The baking tests show it to have a fair degree of strength. Flour 1003 is a patent milled from soft, white winter, Washington wheat. The ash content and the pH of the water extract indicate a patent flour, while the protein content and the baking tests show it to be an exceptionally weak flour. Flour 1007 is a clear flour milled from a sample of selected hard spring wheat grown near Calgary, Canada. The ash content and the ]iH, of the water extract, show it to be a clear flour. Although the ]>rotein content is high the baking tests show it to be of poor baking strength. Flour 1008 is a patent milled from selected hard spring Canadian 21 wheat, and shows up exceptionally strong in the baking tests. This flour, it would seem, is too strong for any ordinary baking purposes and would have to be blended with a weaker flour. Flour 1009 is a composite, commercial patent flour, milled from hard spring wheat for a select trade. The ash content and pH values show it to be a low extraction patent, while the baking tests show it to be a very strong flour. Flmir 1009 does not give the volume of loaf that flour 1008 does, but it produces bread with a better grain and texture. This flour is the only one of the series in which the origin of the wheat is not known. Flour 1011 is a patent flour milled from Ohio winter wheat. The baking tests show it to be of rather poor baking strength. TABLE VII. Hydrogen Ion Concentration after addition of Acid and Alkali to Flour Extracts as an Index of Buffer Value. Malf Flour No. 1001 1002 1003 1007 1008 1009 1011 Flour pH pH pH pH pH pH pH pH cc. N/50 HCl Added 12..S 2.519 2.958 2.654 2.894 2.514 2.510 2.789 3.904 10.0 2.654 3.006 2.874 3.359 2.876 2.705 2.977 4.225 7.5 2.925 3.320 3.144 3.799 3.192 2.992 3.210 4.428 5.0 3.388 3.685 3.761 4.471 3.545 3.496 3.630 4.727 2.5 4.150 4.666 4.623 5.158 4.502 4.272 4.426 5.166 0.0 5.816 6.052 6.002 6.103 6.133 5.981 6.050 5.491 (cc.N/50 NaOH) 2.5 7.371 6.931 7.726 6.<»38 7.499 7.792 7.048 6.071 5.0 9.045 8.021 9.653 7.852 8,906 8.892 8.883 6.390 7.5 9.755 9.146 10.557 8.926 9.609 9.91S 9.540 6.652 10.0 10.253 9.772 10.877 9.535 9.919 10.448 10.177 6.888 12.5 10.617 10202 11.022 10.062 10.249 10.769 10.464 7.136 (c) The Methods. The Munson and Walker method for the determination of reducing sugars was used throughout the investigation for the estimation of sugar resulting from diastatic activity, and all the results are calcu- lated as dextrose. In the cases where proteolytic activity is deter- mined, the amino nitrogen method of Van Slyke, and the viscosity method of Sharp and Gortner were used. The Method of Determining Diastatic Activity. It was evident from the very first that the method of Lintner, for the determination of diastatic activity was out of the question, as were also the other methods which have since been developed. It was also evident that the raw starch of the flours was the natural 22 substrate of the enzymes and consequently should be used to dupli- cate, as far as possible, the changes taking place in the fermentation process. Many difficulties were involved and clarification of the solu- tion was necessary to obtain uniform results. The method finally adopted was one which was developed and perfected in this laboratory. It consisted of adding 3 cc. of l57o Na.jWO^ to the digestion mixture, transferring to a 200 cc. volumetric flask and then adding 20 drops of concentrated HgSO^ and filling up to the mark with water. After careful and thorough shaking the contents were transferred to centri- fuge tubes and whirled 5 minutes. The resulting clear supernatant liquid contained all the soluble sugars and was practically free of soluble protein as demonstrated by repeated Kjeldahl determinations. For further data, see report on diastatic enzymes of wheat flr)ur and their relation to flour strength. (Rumsey, 1922). In determining the diastatic activity of a malt preparation and the amount of soluble sugars produced from the flours by its action, the following method was used : Ten grams of flour were weighed out and transferred to a 400 cc. erlenmeyer flask, the specified amount of flour or malt extract was then added. One hundred cc. of water, previously brought to temperature, was then added and the whole was thoroughly mixed and placed in a water bath, for 1 hour at 27° C. The flasks were agitated every five minutes and at the end of the digestion period the contents of the digestion flasks were transferred to a 200 cc. volumetric flask (any starch particles adhering to the sides of the flask can be removed with a rubber policeman and a stream of water from the wash bottle) and clarified as described above. After clarification 50 cc. aliquots were transferred to 400 cc. beakers and the reducing sugars determined by the Munson and Walker method. In determining the reducing sugars formed in the dough at various stages of fermentation essentially the same methods were followed. At the time specified, ten grams of dough are pinched off from the fermenting mass and rubbed up in a mortar with a little water until a homogeneous suspension is secured, this is then transferred to a 250 cc. volumetric flask and the same procedure followed as outlined above. The Method of Determination of Proteolytic Activity. In the determination of proteolytic activity eighteen grams of flour (calculated on the dry basis) were weighed and transferred to a 500 cc. erlenmeyer flask, malt flour or malt extract was added and 100 cc. of water, previously brought to temperature of digestion, was then added and the whole digested 4 hours at 40° C. The mixture was 23 then cuuled to 25' C, at the close of digestion, and poured into the cup of the MacMichael viscosimeter and the average of three readings taken. Then 5 cc. of N/1 lactic acid was added, the contents thor- oughly mixed, and the average of three successive readings taken. From the calibrated scale reading of the MacMichael Viscosimeter, which is denoted as M, any values such as centipoise or absolute viscosity can be determined by calculation. Method of Determining Buffer Value of Flours. In the determination of the buffer values of the flours, 80 grams of flour are weighed into a 2 liter flask and 400 cc of water added. The whole is well shaken up to get rid of any lumps and digested 1 hour at 25 °C. The digestion mixture is then centrifuged to throw down the suspended matter. Aliquot portions of 25 cc. volume were created with 2.5. 5.0, 7.5, 10.0 and 12.5 cc. respectively of N/50 HCl or NaOH, then brought to a volume of 50 cc. and the hydrogen ion concentraticMT determined by the use of the Bailey electrode and a Leeds and Northrup ty])e K potentiometer in conjunction with an N/10 KCl calomel electrode and a flowing junction of saturated KCl. Method for Determination of Gas Producing Capacity of Flour. In determining tlie gas i)roducing capacity of a flour, twenty grams of flour are weighed out and transferred to a wide mouthed bottle. T(j this is added .5 grams of fresh yeast suspended in 20 cc. of dis- tilled water and the whole is thoroughly stirred and the bottle stop- pered with a cork containing a delivery tube. The bottles are then put in a water bath kept at 3>7°C. and the liberated gas is measured in an inverted cylinder under concentrated brine. Readings are taken every half hour. When malt extract is added it is first incorporated with the yeast and water and added to the flour in this way. Baking Tests. The .baking tests were carried out according to the standard formula adopted by the American Instiutc of Baking with one exception and that consisted of leaving out sucrose in one set of baking experiments. The formula of the dough was as follows: Flour ^•<23 grams Water 173 grams( varit-d ckpLiKling upon absorption of the flour) Yeast 10 grams Sugar 10 grams Salt 5 grams Lard .' 6.5 grams This formula was corrected for the sugar content of the malt flour and malt extract used, the total sugar content being always equivalent to that stated in the formula. The doughs were mixed with a small 24 bench mixer, fermented and baked under as accurately controlled conditions as possible. After the baked loaves were withdrawn from the oven they were placed in a cabinet to cool and were weighed 1 hour after baking. The volumes of the bread were taken the next day and each loaf was then cut and judged for grain, texture, color, flavor and odor. In the case where the development of sugar forma- tion was followed during the course of fermentation, a double portion of dough was mixed and then divided. The samples for anlysis were taken from one portion of the dough while the other was baked, and then judged as were those described above. In these experiments dough was fermented, with and without yeast, to estimate the total production of sugar and that used by the yeast in normal fermenta- tion. The weight and temperature of the dough were taken at stated periods of fermentation. Certain changes in the hydrophyllic properties of the gluten col- loids of this series of doughs, as measured by the viscosity of dough suspensions in water, were followed by Mr. P. F. Sharp, and will be reported by him in a separate paper. (d) Influence of Varying Conditions on Diastatic Activity. Determination of the Optimum pH for the Amylase of Malt Flour. This analysis was made to determine what relation existed between the optimum pH of dough and the optimum pH for the maximum production of maltose by the malt flour used. Sherman and his col- laborators determined the optimum pH for a purified malt amylase and it was thought of interest to know how a commercial preparation behaved in this regard. The process of manipulation varied slightly from that described above for the determination of buffer values, so will be described at this time. Ten grams of malt flour containing both the enzyme and the raw-starch substrate were weighed out into a flask. Enough water was then added so that when the mixture was brought to the desired pH by acid or alkali, the total volume of liquid added would be 50 cc. The mixture was then digested for one hour at 25°C in an accurately regulated water bath. After digestion the whole was centrifuged and 25 cc. (half of sample) was pipetted into a 200 cc. volumetric flask, 2 cc. of 15% Na.,WO, were added and thor- ughly shaken, and 20 drops of concentrated H^SO^ is added and made up to the mark. The preparation was centrifuged again and 50 cc. taken for reducing sugars as described above. The other portion of the unclarified extract was used to determine the pH values. The experimental data showing the influence of diastatic activity by change in pH are given in Table VIII and illustrated graphically in Figure 1. The optimum activity occurred at pH=4.20. TABLE VIII. Relation between hydrogen ion concentration (as pH) and the diastatic activity of malt flour as expressed in grams of dextrose per 1.5 grams of malt flour. Wt. of Wt. of Xormalitv cc. Cu,0 Dextrose Dextrose" of Hcr used pH Grams f^rams Per Cent N710 28.0 1.988 .1075 .0527 3.52 N/10 26.0 2.099 .1115 .0548 3.65 X/10 24.0 2.139 .1147 .0563 3,75 N/10 22.0 2.437 .1111 .0545 3.64 N/25 50.0 2.572 .1070 .0525 3.50 N 25 45.0 2.745 .1123 .0552 3.68 N/25 40.0 2.970 .1239 .0611 4.07 N/25 35 3.156 .1438 .0714 4.76 N/10 13.6 3.224 .1526 .0759 5.03 N/10 12.0 3.420 .1734 .0868 5.78 N/25 30.0 3.499 1828 .0917 6.12 N/10 11.0 3.613 .1937 .0976 6.51 N/25 25.0 3.704 .1935 .0974 6.50 N'25 22 ^ 3.870 .1954 .1000 6.67 N/25 200 4.159 .2238 .1137 7.58 N 25 15.0 4.260 .2305 .1173 7.82 N/25 12.5 4.542 .2224 .1129 7.52 N/25 10.0 4.621 .2190 .1111 7.41 N/25 5.0 5.069 .2081 .1051 7.01 NaOH 0.0 5.548 .1827 .0917 6.11 ■^^" IN/25 5.0 6.069 .1670 .0834 5.56 N/25 10.0 6.4S9 .1548 .0769 5.12 N/25 15.0 6830 .1438 .0713 4.75 N 25 20.0 7.359 .1336 .0661 4.41 N/25 25.0 7.871 .1230 0605 4.03 N 25 30.0 8.419 .1196 .0589 3.93 N/25 35.0 8.920 .1180 .0581 3.87 N 25 40.0 9.306 .1163 .0572 3.81 N/25 -1." 9.649 .1090 .0535 3.57 N 2- : -^ , 9.991 .1028 .0503 3.37 Influence of Time of Digestion on the Diastatic Activity of Malt Flour. The influence of time on the activity of malt flour was investigated to ascertain at what point, or length of time, the activity would decrease. Heyl has noted that at hrst the reaction is logarithmic, but deviates as the products of hydrolysis accumulate. This particular experiment was planned in order to find the optimum length of time for future periods of digestion. It developed that at the end of eight hours the reaction was proceeding at about the same rate of speed as that of one hour so it was decided to make one hour the standard period of all digestions. The efrects of time of digestion up to eight hours is given in Table IX and presented graphically in Figure 2. 26 Figure 1. Effect of pH on the activity of diastase in malt f^our expressed as grams dextrose per 1.5 grams malt flour. TABLE IX. Effects of time of digestion on diastatic activity as expressed in grams of dextrose per 10 grams of malt fiour. \\"t. of Dextrose Time of per 10 erams Digestion Weight of Cu:0 of flour Dextrose Hour? Grams Grams Per Cent 0.00 1112 .3844 3.86 .25 1462 .5128 5.13 .50 1622 .5752 "> "^^ 1.00 1962 .6992 7.00 1.50 2230 .7992 8.00 2.00 2457 .8848 8.85 3.00 2931 1.0696 10.70 4.00 3331 1 2296 12.30 5.00 3650 1.3608 13.61 6.00 3991 1.5024 15.03 7.00 4264 1.6200 16.20 8.00 4560 1.7480 17.48 27 IMO / lym / iKO. / ' imo. ^ ! 3 / 4 ^ lOOOL / 1 / — J 1 / \ MO / 4C00 ^OCC 1 1 //! //Oi Figure 2. Diastatic activity as influenced by time of digestion, expressed in grams of dextrose per 10.000 grams of malt flour. Effect of Temperature Upon the Activity of Diastase. Practically all of the work in this investigation was carried out at 27^C, which is the temperature of fermentation used in the bake shop; however, it was necessary to find the optimum temperature of the diastase in the malt flour, as Sherman notes that 40° is the optimum temperature with a maximum at 55° for pancreatic amylase. Most of the investigators quoted above found 65°C to be the optimum for malt amylase, while Swanson and Calvin found 62.5°C to be the optimum for wheat diastase. It was thought to be of interest to determine at what temperature the diastase in malt flour exerted its 28 ZM /" "^ *>- ■ / IZOOi / zoooc / 1 / / tiax ' i.ooa. > SOX t y v Mi MX i 1 1 lempcroTi/re Figure 3. Effects of temperature upon the activity of diastase in malt flour when 10.00 grams are digested one hour at various temperatures. TABLE X. Effect of temperature upon the activity of diastase in malt flour when 10 grams are digested for 1 hour at various temperatures. Seri es 1 Wt. of Dex- Serie Wt. of Dex- Temp. trose trose of per 10 per 10 Diges- Wt. of Grams Dex- Wt. of Grams Dex- tion Wt. of Dex- Malt trose Wt. of Dex- Malt trose Decrees Cu:0 trose Flour Per CihO trose Flour Per C Grams Grams Grams Cent Grams Grams Grams Cent 27 .1279 .0559 .5590 5.59 .1280 .0559 .5590 5.59 40 .1951 .0867 .8670 8.67 .1940 .0862 .8620 8.62 45 .2305 .1034 1.0340 10.34 .2320 .1041 1.0410 10.41 50 .2922 .1333 1.3330 13.33 .2914 .1329 1.3290 13.29 55 .4144 .1960 1,9600 19.60 .4136 .1956 1.9560 19.56 60x .5704 .2598 2.5980 25.98 .5692 .2592 2.5920 25.92 65x .6030 .2759 2.7590 27.59 .6018 .2754 2.7540 27.54 70x .5760 .2626 2.6260 26.26 .5762 .2627 2.6270 26.27 X Aliquots of one-half the usual quantity were used, and the resulting values multiplied by two. 29 maximum effect. The procedure was as follows: 10 grams of maU flour were weighed out and digested at temperatures of 27°, 40°, 45*. 50°, 55°, 60'. 65 , and 70 for one hour with 100 cc. of water (previ- ously brought to temperature). After clarifying and bringing to a volume of 250 cc. a 25 cc. aliquot was taken for reducing sugars and determined by the Munson and Walker method. These data are given in Table X and illustrated graphically in Figure 3. Effect of Concentration of Diastase on Hydrolysis of Starch in Wheat Flour, As the concentration of diastatic ferments added to the dough is of great importance, the effects of dift'erent concentrations of malt flour up to 50% were tried by mixing definite proportions of wheat and malt flour and digesting it at 27^ C. for one hour. It was necessar>' to first determine the amounts of dextrose formed when different amounts of malt flour were digested separately in order to apply cor- rections for the autolysis of malt flour in the succeeding experiments with the wheat flour. This data is given in Tables XI and XII, and presented graphically in Figure 4. TABLE XI. Autolysis of malt flour at 27 "C. for one hour. Amount of Mah Flour used, grams 0.25 \Vt. Cu,0, grams .0337 \Vt. Dextrose, grams .0142 Dextrose, per cent 5.68 TABLE XII. Relation of concentration of malt flour to the production of reducing Sugars from wheat flour when digested 1 hour at 27 "C. 0.50 0.75 1.0 1.25 .0744 .1080 .1519 .1980 .0320 .0469 .0668 .0852 6.40 6.25 6.68 690 Dextrose weight of corrected Proportion \Vt ofCu.O Dextrose for dex- of wheat flour to per 2.5 formed per 2.5 gms. trose of Malt Dextrose malt flour gms. flour flour malt flour flour formed Grams Grams Grams Percent Percent 10:0 .0944 .0408 .0408 0.0 1.63 9:1 .2167 .0968 .0826 10.0 3.67 8:2 .2583 .1168 .0848 20.0 4.24 7:3 .2918 .1331 .0862 33.3 4.92 6:4 .3030 .1387 .0719 40.0 4.80 5:5 .3279 .1512 .0760 50.0 6.06 30 u 1 J ; : i ■ 1 ; y^ S6 : ^ \ \ \ \ l^^ 1 „ « \ ^ '/^; ' : i ' 1 i y^ ^ 1 ' ' 1 \/\ ' : 1 1 , i -/ i ; ^ i 1 . : . i f, ' /i ' M i i : / 1 ! ' M : ^ . i -li ! 1 ; ■ 1 ^ 1 ^ ! ^ ; f^KCnf fft/T Flour Figure 4. Relation of increasing concentration of malt flour to the production of reducing sugars, when wheat flour is digested one hour at 27 "C. Effects of Increasing Amounts of Malt Flour When Digested With a Constant Quantity of Wheat Flour. Table XII shows the ettects of large quantities of malt flour upon wheat starch, but the amounts are out of all proportioii to that used in practice. In the experiments following, the sugar producing capacity of the malt flour was measured upon a series of seven flours and a com- mercial wheat starch with concentrations varying from 0.2 percent to 5.0 percent. The experimental data showing percent dextrose produced, when 10 grams of flour are digested with 0.02 - 0.50 grams of malt flour, is given in Table XIII and illustrated graphically in Figure 5. 31 TABLE XIII. Percent dextrose produced from 10 grams of flours 1001, 1002, 1003, 1007, 1008, 1009, 1011, and a commercial wheat starch when digested with 0.02 - 0.50 grams of malt flour for 1 hour at 27°C. f: lour San iple Nuniber — — Com- mercial Ma lit Flour U.sed 1001 1002 1003 1007 1008 1009 1011 Wheat Starch Grams % % % % % 7o % % % .0000 0.00 1.85 1.34 .40 .94 2.2S 1.36 .18 .0200 .20 2.35 1.72 .0250 25 2.15 1.50 .74 1.15 .32 .0400 .40 1.80 .0500 .50 2.11 1.64 .88 1.71 2.38 .85 .42 .0600 .60 1.95 .0750 .75 2.47 1.74 1.03 2.04 2.48 .50 .0800 .80 1.98 .1000 .99 2.59 1.85 1.09 2.10 2.54 2.12 1.00 .59 .1200 1.19 2.19 .1250 1.24 2.69 1,94 1.14 2.32 1.06 .62 .1500 1.48 2.77 2.04 1.20 2.50 2.56 2.28 1.14 .69 .1750 1.72 2.58 2.46 1.16 .2000 1.96 2.90 2.11 1.36 2.80 2.62 2.62 1.47 .84 .2250 2.20 2.64 .2500 2.44 3.02 2.21 1.40 2.95 2.63 2.67 1.51 .91 .3000 2.91 3.15 2.31 1.49 3.21 2.72 2.84 1.56 1.07 .4000 3.84 3.34 2.48 1.64 3.35 2.83 3.06 1.72 .5000 4.76 3.47 2.61 1.73 3.7i 3.03 3.24 1.93 1.53 The Production of Reducing Sugars in the Dough During Fermentation. The production of reducing sugar.s in an actively fermenting dough and its subsequent use by the yeast was followed at various stages of fermentation This necessitated running two parallel doughs, one having the required amount of yeast, while the other had no yeast added but identical in every other respect. After mixing the dough, a ten gram sample was pinched off, shaken to a homogeneous suspen- sion, clarified and the reducing sugars determined. At stated periods 10 gram samples were taken and submitted to analysis, as after the mix. Twice the usual quantity of each dough (with and without yeast) was mixed, divided into two equal parts and the samples taken from one pijrtion only in order to have one dough to l^ake in the usual way. 32 JS [y ^ y' "^~ JO ^ ^ /MB £5 00 O 0\ ^ Ov Ov — " CM !< i< r^ f^ CO 00 >n 1 r, "~, u-/ >/--, m "i2 rt^ i; J5 h ■w o be rtP g rt ooo tCM ,-. 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O O ""- r<^ 1^ O O O On O '"O -^ <^) ■— ' 00 tflQ 6 -o •73 CO ^ o C u .2 3 *-> o in •—' s e g l-H Wi ^ ''^ ^ 4> < u bX) Cu tfl "It- c .ti 1?,^ 5 •^— 1 — — _< \0"^ M r-«. ^ t>. On ■^. M rg f^ "^ ■*«^ So m uo O OOnOnvO „ 0\ ■O \0 oorg ' u. cr, (jj o ^. --| — ; f^. O u >> S2 c o X Q c o 0^ . r^. t->. r,! r>! r< t^ t^ lO LD lO to ID ij-no t^ O •— ' U-) O O CO O; fV) (^ •^ fO CNl CL, i/> ^ 3 C" O "; HO u-i vTi O a o. o *-. o o 38 o 3 O D "o w 2 u .2 S X »« '^ *" ^' ^*« ^ aj be •^ (M fO "O f^l t^ t^ Ov f^"^ (TJ ■^ rt uSiT) U^ LT-, to < fV) -J V 3 yi '*^ *j ^ ri I- t/) J- P i: CO ^ ra bX) O 3 en Z bfi V C ts *o "•J 3 3 TJ O «> J3 ^2 -I? O ■* <^) t^ — Tt Tt -"^ rO ^' / '\ 3C /¥/ ■ '\ / ■; / /^\\ / ^' • ^ / A- '* \ // 7 / \ ■•• ♦ k i i ' \ • Figure 7. Production of reducing sugars in the panary fermentation of flour 1003 with different concentrations of malt flour and malt extract. (Full lines indicate doughs to which yeast was added, dotted lines no yeast dough). 40 < .■■■' 1-' .....■-• .1' iJilfci, / ':^' N .-• ,^ <;• ,ji'- J^-.'"^' / > N \ 1 — -"""^T^^l ^ V K, 1 i ! i J M s. -. s^^-' \ \ .^ ...., \' , "~ ^'""■■r ^ i,--- ^^,M fH^- "" "^ ^ \ V X - 1 « 1 — Time V Feimenhtm Figure 8. Productions of reducing sugars in the panary fermentation of flour 1002, with different concentrations of malt flour and malt extract. (Full lines indicate doughs to which yeast was added, dotted Hnes no yeast dough). Determination of Proteolytic Activity as Measured by the Fall in Vis- cosity of Flour-water Suspensions, when Digested with Diastatic Preparations. The action of proteolytic ferments upon gluten is accompanied by a breaking down of the protein material into simpler compounds srch as protesoses, peptones and amino acids. As the latter can be easily measured by the amount of free amino nitrogen, it was thought th^t an increase in amino nitrogen measured by VanSlyke's method, would be a measure of the amount of proteolysis taking place. This, how- ever, was not the case as no appreciable difference in amino nitrogen could be detected when the flour was digested with and without malt preparations. 41 It is well known that dough becunies slack or less viscous during the fermentation period. This has been attributed to the action of the l)roteolytic enzymes of the }e3st and partially to those in n.ialt pr<"p- arations. ford and Guthrie were unable to demonstrate proteolytic enzymes in flour by means of the viscosimeter and concluded that any which were present would have little or no effect upon the gluten dur- ing fermentation. Sharp and Gortner have shown that the hydration capacity and the quality of the gluten can be accurately determined by the use of the viscosimeter. They have shown that the viscosity of a flour-water suspension is tremendously increased and increases to a well defined maximum by the addition of lactic acid in small amounts, while the further addition of lactic acid causes no appreciable change in viscosity value. The suspensions of various flours in water dif- fered slightly in initial viscosity and it was only after the addition of the lactic acid that these differences were increased to such an extent as to make the results of significance. Under the conditions of Sharp and Gortner's experiments the addition of 5 cc N/1 lactic acid was TABLE XXV. The measurements of proteolytic activity in a flour-water suspension showing the effects of different periods of digestion (1-5 hours) at 30°C. with 100 cc of water and concentrations of malt flour (2 and 4 percent) on viscosity (degrees MacMichael) with the addition of various amounts of lactic acid. Viscosity in degrees MacMichael on addition of N/1 lactic acid. N 1 Lactic Acid added cc Malt Flour . . .0.0 0.5 1.0 1.5 2.0 3.0 5.0 Digestion used Degr's Degr's Degr's Degr's Degr's Degr's Degr's Hours Percent M M M M M M M 1 0.0 33 83 119 134 144 152 160 2 0.0 41 107 139 151 156 161 167 3 0.0 36 86 120 133 139 148 156 4 0.0 38 86 117 129 136 144 160 5 0.0 34 83 115 127 133 141 153 1 2.0 35 86 126 135 141 147 151 2 2.0 29 70 101 115 122 130 135 3 2.0 29 67 96 108 116 122 127 4 2.0 30 69 97 106 112 118 122 5 2.0 31 70 95 104 109 115 120 1 4.0 32 71 104 119 127 134 138 2 4.0 25 57 88 101 109 117 121 3 4.0 22 51 77 90 97 105 111 4 4.0 24 48 72 83 88 96 100 5 4.0 22 45 68 77 83 90 96 42 sufficient to bring any flour to the point of highest viscosity ; accord- ingly their method was followed to see whether any differences could be detected when flour was digested alone and when digested with malt preparations and if any differences which existed in the flour- water extracts could l)c increased by the addition of lactic acid. A few preliminary experiments in which flour was digested with and without malt preparations for different periods of time showed that the flour-water suspensions of the different flours varied very little in their initial viscosities, thus showing why Ford and Guthrie were un- successful in measuring proteolysis by means of the viscosimeter. With the addition of lactic acid, however, great differences were notice- able between the different flours and the results justified the following experiments. m M y N, M y s^ \ s. M \ \ ^ \ f» J M \ "^ k 1 ^ \ i i' \ \ i t "^ \ "^ ^ 1 3 N S ^ .^ US \ V lie \ \ ^ ! . 4 J r Time ^ Dioestion m Hour> Figure 9. Effect of the proteolytic enzymes, contained in malt flour, upon wheat flour as measured by the fall in viscosity when digested 1 to 5 hours at 30°C. With lactic acid added. 43 The e::TcCi ■- i time ^'f d!g"e?Lion was nrsi tried to determine the op- tiniuni tin-e .f digestion. The experiment consisted of digesting flour lOC'l ;a strong Kansas patent) for 1 to 5 hours with 100 cc of water, and reoeating with the same liour after adding 2 and 4 percent of malt !l:ur. After digesting fcr the stated period oi tinne the flour-water suspensions were transferred to the cup of the MacMichael viscosi- meter and the average oi three readings taken. X 1 lactic acid was then added in sm.all amounts up to 5 cc. The mixture was thoroughly stirred aJter each addition of acid and the average of three viscosity readings taken. The ex-- erin: ental data of this series when digested at 30' C. is given in Table XX\' and the data in the last column < -i this Table is shown graphically in Figure 9. It a^vcars fr:n". this table that four h'-ur digestion is ample tinie to secure evidence of prcaeolytic action, and this time of digestion was used in all subsequent wc-rk. Also the temperature of digestion was increased from 30" to 40' in 'irder to procure greater activity of the enzyn-.es. The data given in Table XX\T and illustrated graphically in Figure 10 was obtained by digesting a series of flours . 1001-1002-1003-1007 and ICOS.' with varying amounts of malt flj^ur 1.0-1.5-2.0-2.5-3.0 and 4 Dcrcent > with iCO cc of water at -rO'C. and determining the viscosity after adding 5 cc of X 1 lactic acid. Table XX\'II and Figure lOA shrw similar data v/hen malt extract was used. It has ':een sh I'V.m. in the literature cited that even minute traces of salts have a marked affect upon the imbibition capacities Cif gluten. It v.-as. therefore, thought advisable l'j see how the viscosity of the f.:ur reacted v.-hen treated as above but with the salts of the flour and n:alt n:ur washed -ut after the digestion period. The precedure wa^ as foll;v.-s: The equivalent of IS gram.s of water-free flour 1002, was weighed int.^ each of ^hc nasks and digested with 0.0. 1.0. 1.5. 2.0, 2.5. 3.0 and 4.0 percent m.alt flour in lC>Ci cc of v/ater at -K)'C. After for.r hours 910 cc of distilled water v.-ere added, the vrhole well shaken and centrifuge:. The suternatant liquid v."as decanted and the flour residue shaken up with v.-ater and after complete disintegration made uo to ICO cc. olaced in visC'Osim.eter cup. and the readings taken after adomg varym.g am. ^'Unts oi lactic acio. The data sh :'V,-ing the viscosity in the presence of lactic acid of flour lC0i2 v.-ith the salts v.-ashed out is given in Table XX\'III. In order to dem.onstrate still further that the salt content of the added m,alt flour did not vitiate the results obtained in Table XX \ I v.-hen f.i'ur f:r exam.ple i 1C02 v/as digested 4 hours with 4 percent 44 TABLE XXVI. Effect of varying amounts of malt flour upon the viscosity of 18 grams (calculated on the dry basis) of wheat flour when digested for four hours at 40°C. with 100 cc of water (5 cc N 1 lactic added in each instance). Viscosity readings in degrees MacMichael. % Malt Flour Used. . .. 0.0 1.0 1.5 2.0 2.5 3.0 4.0 Degr's Degr's Deer's Degr"s Desr's Degr's Desr Flour Xo. ^f M M M M -M M 1001 145 123 119 114 112 105 93 1002 150 126 118 109 97 99 81 1003 50 44 42.5 40 36.5 37 28 1007 124 106 103 95 97 103 88 1008 151 136 129.5 117.5 112 109 102 nrztr' Ma.'T firfim.-m^'xr ior Figure 10. Changes in viscosity of fiour water suspensions when digested 4 hours at 40 "C. with increasing amounts of malt flour and malt extract. as measured in degrees MacMichael with lactic acid added. (Full line curves flours digested with malt flour, dotted line curves those digested with malt extract). TABLE XXVII. Effect of varying amounts of malt extract upon the viscosity of 18 grams (calculated to the dry basis) of wheat flour when digested 4 hours at 40 C. with 100 cc of water. (5 cc of N/l lactic acid added in each instance). Viscosity readings in degrees MacMichael. % Malt Ext. Used . . . ...0.0 1.0 1.5 2.0 2.S 3.0 4.0 Degr'.s Dear's Decjr's Degr's Degr's Degr"s Degr's F"lour No. M M M M M U M 1002 145 136 131 127.5 120.5 111 106 1003 3/ 51 50 48 43 38 34 1008 ISO 138 134 132 127 122 112 /5 ! 1 1 R 1 i 1 6S 1 1 « ; ' . I J 3e « <" .. «. i : , . 1, "^ 1 ' ^ s i 1 ■ . ■ ' ' 3 1 ' ' i i 1 i *i K. r^- 1 1 i « j\- "i X LV--~ 1 1 ji 1 11 1 ^ 1 , Rrcenf Mair Pnjxtrahon Uied Figure 10-A. Changes in viscosity of water suspensions of flour 1003 when digested 4 hours at 40° with increasing amounts of malt flour and malt extract, as measured in degrees MacMichael. With lactic acid added. (Full line curve digestions with malt flour, dotted line curve digestion with malt extract). malt flour three samples of 1002 were weighed out, two were used as checks and to the third 4 percent malt flour was added. At the end of three and one-half hours 4 percent malt flour was added to ore of the checks and the mixture well shaken. At the end of 4 hours diges- tion, the viscosities of all three preparations were determined as usual. The data showing the viscosities of this experiment are given in Table XXIX, and leave no doubt but that proteolysis has taken place. TABLE XXVIII. The effect on the viscosity of flour 1002 with the salts washed out af- ter digesting with varying amounts of malt flour for 4 hours at 40°C. with 100 cc of water (lactic acid added in each instance.) Lactic Acid C€. 0.5 cc 1.0 cc. 1.5 cc. Viscosity Viscosity Viscosity Percent Degrees Degrees Degrees Malt Flour M MM 388 411 405 1 299 328 340 Z 295 312 315 3 273 295 305 4 245 263 271 TABLE XXIX. Effect of three and one-half hour digestion without, and half an hour digestion with malt flour as compared to a 4 hour digestion with and without 4 percent malt flour upon the viscosity of suspensions in water. Culjic Centimeters N/1 Lactic Acid Added 0.0 5.0 171 XT Degrees Degrees Flour No. |j 1^ 1002 digested vvitiiout mah flour 38 145 1002 digested with 4% malt flour 19 81 1002 digested 3.5 hrs. without and 3 minutes witli 4% malt flour 30 127 Gas Production Capacity of Wheat Flour in Relation to Strength. Although Wood pointed out that the gas production capacity of a flour was an index to one of the factors in flour strength and Baker and Hulton pointed out that weak flours were low in liquifying' en- zymes, they did not sul)mit sufficient data to show that this was ac- tually the case. In the following work the gas producing capacities of a series of flours was determined, according to the method of Wood, and with and without the addition of malt extract. The flours selected consisted of two typically strong patent flours 1008 and 1009, two clear flours of fair baking strength 1002 and 1007, and one typically weak 47 patent flour 1003, milled from Washington wheat. The method fol- lowed was the same as that described in the methods under gas pro- duction. The data giving the cul)ic centimeters of gas produced from flours 1002, 1003, 1007, 1008 and 1009 when fermented with and without the addition of 1 percent malt extract, is given in Table XXX, and illus- trated graj)hicall\' in Figures 11 and 12. TABLE XXX. Effect of added malt extract upon the gas producing capacity of flours 1002, 1003, 1007, 1008 and 1009 when fermented with 2.5 percent yeast for four hours at 35°. No Added Malt Extract One Percent Malt Extract Flour No 1002 1003 1007 1008 1009 | 1002 1003 1007 1008 1009 Time of ■ I Fermentation Hours 0.5 1.0 1.3 2.0 2 =i 3.0 3.S 4.0 4.5 .128 I .. 208 TABLE XXXL Changes in pH during the fermentation of the dough with values for flour extract and the extract of bread crumb. Baking Data. jas Gas Gas Gas Gas Gas Gas Gas Gas Gas cc. cc. cc. cc. cc. 1 cc. cc. cc. cc. cc 13 14 10 10 7 1 14 15 12 14 8 34 M 31 27 2H 35 34 33 36 31 62 57 58 53 60 1 64 59 60 66 61 98 80 92 90 99 88 94 105 101 140 95 132 148 116 141 115 134 153 125 177 104 174 202 148 180 140 178 203 153 214 117 212 250 176 I 218 184 22i 252 181 245 123 243 192 1 254 196 270 196 Flour Ash Flour Mix 1st Pch. pH Valu 2nd Pch. es 3rd Pch. After Pf. Bread No. 7c 1001 .40 5.81 5.33 5.18 5.09 5.02 4.79 4.96 1002 .61 6.05 5.65 5.25 5.25 5.24 5.05 5.2M 1003 .46 6.00 5.24 5.16 5.19 5.37 5.05 5.16 1004 .83 6.17 5.91 5.92 5.87 5.85 5.80 5.52 1005 .43 5.84 5.75 5.40 5.22 5.23 5.17 5.29 1006 .38 5.78 5.70 5.28 5.22 5.17 5.03 5 30 1007 .64 6.10 5.76 5.63 5.58 5.59 5.53 5.. 58 1008 .42 5.98 5.33 5.23 5,17 5.19 5.25 1011 .56 6.15 5.47 5.33 5.30 5.20 4.92 5.28 Change in Hydrogen Ion Concentration of Fermenting Dough. As considerable data has been accumulated upon this series of flours it w\'is thought that the changes in hydrogen ion concentration dur- ing the fermentation period would be of considerable value, inasmuch as the speed of diastatic and proteolytic activity is influenced to such a great degree by changes in hydrogen ion concentration and many ir- 48 \ / f s^ / / K 1/ / o //> / / ,mJ- ^ //// ^ ... / // . Jt ^ f ^ f ^ ^ Time ,•, Hci Figure 11. Gas producing capacity of flours 1002, 1003, 1007, 1008 and 1009, fermented with 2.57c yeast. 3a. 2S0 / / 100 // llfi- S' / /, // 1 /JO // //> ///. /OOf w ■ ' ■SO y A / ^ ^ Time m He. Figure 12. Effect of 1.9' added malt extract upon the gas producing capacity of flours 1002, 1003, 1007, 1008 and 1009, when fermented with 25.7^ yeast. 49 regularities in the data might be accounted for in this manner. The activity of yeast is also very much influenced by changes in the pH of its medium. The doughs were made from the same flours and in the same manner as that reported in the section on reducing sugar? formed during fermentation. The procedure consisted of taking 10 grams of dough and shaking it up with 50 cc of water until a homo- geneous suspension was secured. The whole was then centrifuged and the pH of the supernatant liquid was then determined in the man- ner described above. Samples were taken at the mix, first punch, second punch, third punch and after proof. The pH value of the flour extract is also given as is that of a water extract of the finished bread. The data is shown in Table XXXT. In the past all chemical and physical data accumulated on th.e strength of flour has been accompanied by baking tests which in the final analysis have been the criterion of flour strength. Inasmuch as it was imperative to have accurate knowledge of the flours and the ef- fects of diastatic ferments, a series of baking tests was conducted in ad- dition to the baking tests made in studying formation of reducing sugars and the change in hydrogen ion concentration during the fer- mentation process. All baking tests were conducted as nearly alike as possible to secure comparable data. In the baking experiments conducted to test the elYects of added diastatic ferments upon wheat flours, the two diastatic preparations used throughcHit the entire work were employed, namely a malt flour and a malt extract. These were added in amounts of .5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4 per cent to each of a series of flours and the results are recorded as total lime of fermentation, valume of the loaf, color, grain and texture of the crumb, flavor and odor. The doughs were made in the manner described under the methods of baking tests and where fermentation is spoken of, total fermentation is meant including both the actively fermenting and proofing periods. The data shcnving the efl'ects of varying amounts of malt flour and malt extracts upon flours 1001. 1002, 1003. 1007, and 1008 is given in tables XXXII to XLI. TABLE XXXII. The effects of varying amounts of malt flour upon the baking qualities of flour 1001. Malt Flour % .Standard 5 1.0 \.S 2.0 2.5 3.0 5.0 Fermentation Hrs. 5:00 4:56 4:52 4:48 4:44 4:40 4:36 4:32 Sucrose added gms. 10.00 9.7Q0 0.580 9.370 9.155 8.940 8.730 8.310 \Vt. of dough gm^. 513 515 516 517 517 521 .522 525 \Vt. of loaf gms. 454 452 443 446 450 446 459 457 \ol of loafer. 1870 1890 2020 1955 2030 2060 1910 2010 50 General Remarks : Loaves grade down with respect to grain and texture with each added increment of malt flour. Color of the crumb is markedly influenced by each increase of malt flour. The crumb was full of large gas holes which was probably due to localized yeast ac- tivity. Mean average temperature of fermentation was 81 °F., and the temperature of baking 440° F. Time of baking 26 minutes . TABLE XXXIII. The effects of varying amounts of malt extract upon the baking qual- ities of flour 1001. Malt Extract % Standard .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 5:00 4:52 4:48 4:44 4:38 4:34 4:30 4:26 Sucrose added gms. 10.00 8.94 7.88 6.820 5.76 4.70 3.64 1.52 Wt. of dough gms. 513 514 514 515 516 514 515 517 Wt. of loaf gms. 454 457 450 448 447 450 453 457 Vol. of loaf cc. 1870 1810 1880 1895 1925 1920 1970 1880 General Remarks : The texture and grain was excellent through- out, but the loaf made with 1 percent malt extract seemed to have better grain than any other. Color was very good up to 3 percent of malt extract where increase in the malt extract darkens the color, or shade. The volume of the loaf also increases up to 3 percent malt extract and then drops. A very decided sweet honeyed flavor was im- parted to the bake which grew more pronounced as the percentage of malt extract increased. Mean temperature of fermentation 81°-82°. Time of baking 25 minutes. Temperature of baking 440°-430°C. TABLE XXXIV. The effects of varying amounts of malt flour upon the baking qaulities of flour 1002. Malt Flour % Standard .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 5:18 5:13 5:08 5:03 4:58 4:53 4:48 4:44 Sucrose added gms. 10.00 9.79 9.58 9.37 9.155 8.94 9.73 8.31 Wt. of dough gms. 519 523 525 528 529 530 532 535 Wt. of loaf gms. 454 451 454 451 460 458 470 471 Volof loaf cc. 1740 1890 2040 2010 2050 2010 2050 2030 General Remarks : Color of crumb grades down very quickly with each addition of malt flour. Grain very much alike throughout while texture was even. Color of the crust improves wdth increase in malt flour; very good smell and good taste while malt flavor is not in evi- dence. Mean Temperature of fermentation 82°-83° and baking 470°F. Time of baking 24 minutes. 51 Standarc 1 ,5 1.0 1.5 2.0 2.5 3.0 5.0 5:19 5:12 5 :07 5:02 4:57 4:52 4:47 4:42 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 519 514 513 513 513 514 515 521 454 441 448 445 437 436 440 447 1740 1900 2050 2010 2010 1980 1060 1940 TABLE XXXV. The effect of varying amounts of malt extract upon the baking qual- ities of flour 1002. Malt Extract % Fermentation Hrs. Sucrose added gnis. Wt. of dough gms. Wt. of loaf ;;ms. Vol. of loaf cc. General Remarks : Color of crumb grades off a trifle as percentage of malt extract increases. Texture increases in fineness with increase in malt extract. Grain is decidedly improved with an addition of malt extract up to 2 percent and then falls off. Odor and flavor of malt ex- tract increases as the percentage of malt extract increases. No no- ticeable difference in the color of the crust between the various bakes. Mean temperature of fermentation S2'^-83° and Ixiking 479° F. Time of baking 25 minutes. TABLE XXXVL The effect of varying amounts of malt flour upon the baking qualities of flour 1003. Mah Flour % Fermentation Hrs. Sucrose added gms. Wt. of dough gms. VVt. of loaf gms. Vol. of loaf cc. (Jeneral Remarks: Standard loaf had by far the best color, which grades down very quickly with increase in malt flour. Loaf made with 0.5 percent malt flour possessed the best texture, flavor and odor. That with 1 percent malt flour had the best grain and those loaves with increased quantities grade off to a very coarse grain. Loaves were ^oggy and heavy. Mean temperature of fermentation was 81° and that of baking 470° F. Time of baking 21 minutes. TABLE XXXVIL The effects of varying amounts of malt extract upon the baking quali- ities of flour 1003. Malt Extract % Fermentation Hrs. Sucrose added gms. Wt. of dough gms. Wt. of loaf gms. Vol. of loaf cc. .Standar( 1 .5 1.0 1.5 2.0 2.'? 3.0 5.0 4:42 4:38 4:34 4:28 4:24 4:20 4:16 4:12 10.00 9.79 9.58 9.37 9.155 8.94 8.73 8.31 511 512 515 515 513 514 521 523 468 467 468 464 457 463 469 476 1660 1760 1675 1860 1710 1690 1650 1550 Standat ■d .5 1.0 1.5 2.0 l.S 3.0 5.0 4:42 4:44 4:40 4:36 4:32 4:28 4:25 4:21 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 511 504 507 504 506 508 ' 508 492 468 460 461 454 448 453 460 448 1660 1 730 1775 1810 1770 1700 1750 1680 General Remarks : Color was decidedly the best in the loaf made with 0.5 percent malt extract while the texture and flavor were best in that with 2.5 percent. Best grain was secured when 1.5 percent malt extract was used and seemed to run off as percent malt extract in- creased but nearly as bad as that of the malt flour. The malt flavor was not as pronounced as in the previous bakes when using malt ex- tract. Color of crust good throughout. Mean temperature of fer- mentation 81° and that of baking 525° F. Time of baking 20 minute.s TABLE XXXVIII. The effect of varying amounts malt flour upon the baking qualities of flour 1007. Malt Flour % Standai •d .5 1.0 1.5 2.0 2.5 3.0 5.U Fermentation Hrs. 5:22 5:15 5:10 5:06 5:02 4:58 4:52 4:47 Sucrose added gms. 10.00 9.79 9.50 9.37 9.155 8.94 8.73 8.21 Wt. of dough gms. Wt. of loaf gms. Vol. of loaf cc. 1640 1750 1750 1780 1680 1665 1610 1690 General Remarks: 1.5 percent malt flour gave the largest volume, hnest texture, color and grain. This appeared to be the high point in quality, since all factors decreased as percentage of malt flour in- creased. Mean temperature of fermentation was 84° while that of baking was 470° F. Time of baking 22 minutes. TABLE XXXIX. The effect of varying amounts of malt extract upon the baking qualities of flour 1007. Malt Extract % .Standar ■d .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 5:22 5:17 5:12 5:07 5 :02 4:57 4:52 4:47 Sucrose added gms. 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 Wt. of dough gms. Wt. of loaf gms. Vol. of loaf cc. 1640 1690 1900 1620 1660 18.^0 1890 1760 General Remarks : The tise of 3 percent malt extract gives the best loaf for color, texture and grain and the best general appearing loaf. Malt extract increased the bloom, color of crumb and volume, through- out the bake. Mean temperature of fermentation was 84° while that of baking- was 470° F. The time of baking was 25 minutes. TABLE XL. The effect of varying amounts of malt flour upon the baking qualities of flour 1008. Malt Flour % Fermentation Hrs. Sucrose added gms. Wt. of dough gms. Wt. of loaf gms. Vol. of loaf cc. 53 Standar d .5 1.0 1.5 2.0 2.5 3.0 5.0 4:53 4:57 4:52 4:47 4:42 4:37 4:32 4:27 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 524 524 525 526 630 530 531 532 450 460 455 460 467 466 467 464 2160 2070 2000 2120 2100 1950 1860 1885 General Remarks : The color of the crumb was affected by the addi- tion of malt flour as those preceeding. Best texture and grain was secured by the use of 1.5 percent malt flour. There was a very marked difference between tln»so loaves made with 1.5 and 2.0 percent in texture and grain. A distinct whcaty ^mell was n(,)ticed in the loaves having malt flour. The color and bloom were about alike. Temperature of fermentation was 83° and the temperature of baking 480° F. Time of baking was 24 minutes. TABLE XLI. The effect of varying amounts of malt extract upon the baking quali- ties of flour 1008. Malt Extract % Standai ■d .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 4:53 4:57 4:52 4:47 4:42 4:37 4:32 4:27 Sucrose added gms. 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 Wt. of dough gms. 524 513 513 514 518 521 519 525 Wt. of loaf gms. 450 457 457 454 450 450 453 460 Vol. of loaf cc. 2160 2020 2040 2070 1995 2130 2180 2130 General Remarks : Color was good throughout the bake, the bread made with 3 percent had the best grain and texture. Added malt ex- tract gave the bread a slight sweet odor and taste. Bloom was even throughout the whole bake. Temperature of fermentation 83° and was baked out in 22 minutes at a temperature of 500° F. III. DISCUSSION. Changes in pH, Temperature, Time and Concentration and Their Ef- fects Upon the Activity of the Diastases Contained in a Com- mercial Malt Flour. As already noted in the historical review of the diastase literature, Sherman and his co-workers found that the pH for the optimum ac- tivity of diastase of different origins were not the same and it could hardly be expected that the diastases derived from different sources of barley would have the same activity at the same hydrogen ion concen- tration. A study of Table Villi, and Figure 1 show that the greatest activity of the diastases,- in the malt flour used in this investigation, was at a pH of 4.26, while that found by Sherman for a highly purified malt amylase was very close to a pH of 4.4 which shows relatively close agreement. It will be noted in Table XXXI, where the changes in hydrogen ion concentration of fermenting dough was followed, that in the later stages of fermentation the dough was, with two exceptions, at a pH of about 5.0. Although this is not at the optimum for dias- tatic activity, it will be noted from Figure 1 that the rate of reaction was very high at this point. This is of significance when we consider that the sugars formed in the later stages of fermentation are impor- tant factors in determining the size of the resulting loaf. 54 Table IX and Figure 2 show that the diastatic activity of the malt flour was practically constant over a period of eight hours digestion. A slight decrease in activity was noticeable as time of digestion pro- ceeds, but for all practical purposes, the rate of reaction showed a straight line when the quantity of dextrose formed was plotted against time. Table X and Figure 3 show that increase in temperature up to 65°C. increased the activity of the diastase. From 27° - 45°C. the rate of reaction increased quite regularly with each increment of rise in temperature. Between 45° and 50° the rate was greatly increased, while between 50° and 60° the increase was very rapid, following quite closely the Vant Hofif and LaBelle law. After 60° the increase in activity was not so marked and the diastatic activity was apparently at a maximum at 65 °C., as a decline in activity was noted with further increase in temperature. Table XII shows that when the percentage of malt flour was increased from 0-50 per cent, the percentage of dex- trose formed from malt flour increased from 1.63 to 6.06 percent. This was calculated to show the quantity of raw starch converted to dex- trose. The greatest efifect of added diastase was in the first 10 percent of added malt flour, which gave an increase in dextrose from 1.03 to 3.67 percent. The Effect of Diastatic Enzymes Upon Starch of Different Flours. The addition of diastatic ferments to wheat flours increased the re- ducing sugars, when digested at 27°C. for 1 hour. As the amount of diastatic ferment was increased a corresponding increase in reducing sugars was noted. All of the flours used in the experiment did not react in the same way to the addition of malt flour, as great dififerences were shown not only in the initial amounts of reducing sugars (1 hour digestion without diastase) contained, but with an increase in malt flour more starch was converted in some flours than in others. In general, but for one exception, the weaker flours produce less re- ducing sugars than do the stronger, when digested with the same amounts of malt flour. From the data presented in Table XIII and Figure 5, it will be noted that the commercial wheat starch shows the least amount of initial reducing sugars and responds less to the action of malt flour than any of the wheat flours. Flour 1003, a decidedly weak Pacific Coast flour, is next in the series; it shows a slightly greater amount of initial reducing sugars (digestion 1 hour without added diastases) and responds a trifle more readily to conver- sion by the malt flour, than does the wheat starch. The next flour, 1011, is a patent milled from a soft winter wheat and runs just a trifle higher in initial sugar content and appears to be more easily converted than flour 1003. These two flours, and the wheat starch constitute a 55 special group a> far as sugar content is concerned. W hile the wheat starch has no baking value, the other two, namely 1003 and 1011. showed very poor baking qualities. The patent flours of good baking strength are highest in the list of initial sugar content and with one exception produce under the action of diastatic enzymes, more dextrose than do the weaker flovirs. The exception noted was flour 1008. which showed the largest volume in the baking test, had a greater initial sugar content than any of the other flours, but did nt)t produce as much dextrose as flours 1001 and 1009 when digested with malt flour. Flour 1007. a clear flour of very poor baking qualities, milled from Canadian wheat stood fourth in the series in regard to initial sugar content. Under the action of the dias- tase in malt flour, however, the reducing >ugars increased out of all proportion to its baking strength and on the addition of .5000 grams of malt flour, it contained more reducing sugars than any of the other flours with a like concentration of malt flour. In general the initial >ugar content (digestion 1 hour without added diastases I indicated the baking qualities of the flour quite accurately. This in turn depends to a large extent uj^on the diastatic enzymes con- tained in the flour itself. From the data presented, it would seem that the starch of the strong flours was generally more easily converted than that of weak flours. This was not in\aria1)le. a> flour 1008. a [)ar- ticularly strong patent flour showed only a very slight increase in sol- uble sugars when dige>ted with apjiroximately 5 ])ercent malt flour, whereas flity of a flour- water suspension was noted when a flour was digested, with and without malt flour, for different periods of time, as sliown in Tal)le XXV, and illustrated graphically in Figure 9. Tlu' higher the concentration of malt flour added, the lower was the resulting viscosity reading, and when diges- tion was carried out for varied lengths of lime, a .steady decrease in viscosity occurred as time of digestion progressed. This was very noticeable as the percentage of malt flour was increased. When flours of different baking strengths were digested with in- creasing amounts of malt flour and malt extract, the viscosity of their suspensions in water (])lus lactic acid) decreased (juite decidedly as shown in Tables XXVI and XX\'1I, and graphically in Figures 10 and lOA. The strongest flours groui)ed themselves, and their suspen- .sions in acidified water ha\e a much liigher viscosity tlian those of the medium or weak flours, and when treated w ith 4 percent of malt flour or malt extract the strength of the flours was indicated by its position on the curve. The malt extract used did not decrease the viscosity as much as a like concentration of malt flour, and the conclusion was that it did not contain as large an amount of proteolytic enzymes as did the malt flour. It might be expected that the stronger flours would not show as great a decrease in viscosity as the weak flours. When digested with 4 jier cent malt flour or malt extract over the range given in Tables XX\T and XX\TI, but tlie oi)posite seems to be 58 actually the case. Flour 1008, the strongest flour in the series, showed a decrease in viscosity of 49° MacMichael, when digested with 4 per- cent ftxtract, while the decrease found for fiour 1003 under the same conditions is 22° and 24° M. respectively. Clear flour 1007 is interme- diate in tliis particular and showed a decrease of 36° MacMichael, when digested with 4 percent malt flour. Although it has been demonstrated that salts have a profound in- fluence upon the viscosity of flour- water suspensions, the results in Table XXYIII show that while the viscosity readings were very much higher in a flour-water suspension, from which the salts have been washed out, the same relative values hold, and the results recorded above were not vitiated by the electrolyte content of the flours. This has been demonstrated in another way where a flour was digested alone for four hours, with 4 percent malt flour for four hours, and an- other sample digested alone for three and one-half hours and at the end of this time 4 percent malt flour was added and digested thirty min- utes longer. It was thought that the salts of the added malt flour w(nild be extracted in thirty minutes and would exert their maximum effect in depressing the viscosity. Also, that in this length of time only a small amount of proteolytic activity would take place, thus showing a difference in viscosity between the flour which was digested four hours with 4 percent malt flour and the other which was digested three and one-half hours alone, and thirty minutes with 4 percent malt flour. These expectations were justitied, as demonstrated in Table XXIX, where the flour digested alone gaves a reading of 145°M., and that digested wath 4 i)ercent malt flour for four hours gave a reading of 81 °M., while that digested alone for three and one half hours and then thirty minutes more with malt flour gave a reading of 127°M. These data show that the increase in viscosity was not due entirely to the electrolytes but to the partial disintegration of the protein From the data presented in Tables XXVI and XXVH it has been shown that suspensions of strong flours in water have a higher viscos- ity than weak flours when digested with and without added malt prep- arations. It has also been shown that suspensions of strong flours in water show a greater decrease in viscosity than do similar suspensions of weak flours when digested with malt preparations and that the de- creases in viscosity recorded al)ove were not due entirely to the elec- trolyte content of the flours but to the cleavage of the gluten, thus de- creasing its imbibitional capacity and consecpiently its viscosity. The Gas Producing Capacities of Strong and Weak Flours and the Effect of Added Malt Extract Upon Them. Wood has shown that the gas produced by a flour, especially in the later stages of fermentation, was a factor in strength, while Baker and 59 llulton have shown that in some cases a weak flour produces as much, and in some cases even more, gas than does a strong flour. They be- lieved that weak flours were deficient in liquifying enzymes and that an addition of liquifying enzymes would increase the gas production of a weak flour to a considerable extent, while they would have little or no efifect upon a strcmg flour. The data in Tal)le XXX supports their theorv and shtnvs that flours 1008 and 1009, which showed very g(K)d baking qualities, did not increase to any extent in gas producing capacity when malt extract was added, while flour 1002, a strong clear fl(nir increased only 9 cc. under tlie same conilitions, and 1007, a clear flour of poor baking quality increased 37 cc. under the same treatment. l"he test seems to be conclusi\e l)y the increase shown by flour 1003, a notably weak flour, ^\•hich increased 80 cc. when 1 percent malt ex- tract was added. The Changes in Hydrogen Ion Concentration Taking Place During the Fermentation of the Dough. The clianges in h}-drogen ion concentration taking place during the fermentation of the dough, are recorded in Table XXXI and show that steady increase in hydorgen ion concentration takes ])lace as fermen- tation proceeds. With two excei)tions the doughs wlien ready ("or the oven had a hydrt)gen ion concentration of ap])roximately pll 5. The two flours which had a higher ])I1 were cle;ir flours ot \ cry jxxir baking strength. The Effects of Malt Flour and Malt Extract Upon the Baking Value of Flour. In flour 1001, a strong patent flour, the xolmne was considerably increased by the use of 2.5 i)ercent malt flour. This advantage was materially offset by the decrease in color. A\"ith the use of the malt ex- tract, the volume increased with additions u]) ti> 3 i)ercent with not much decrease in color, while a sweet honey-like flavor is evident which adds to the value of the loaf. The data in 1\Hble XXXIII shows that the baking qualities of the flour were improved when 3 per- cent malt extract was used. Flour 1002, a fairlv strong clear flour, increased in volume with the addition of malt flour. The greatest volume was secured by the use of 2 percent malt flour for the weight of the dough baked out. While the grain and texture were uniform throughout, the decrease in color value ofYset the advantages secured by the increase in volume. In using malt extract the greatest volume was secured by the use of 1.5 and 2.0 percent, and decidedly the best loaves were thus produced, since texture and grain increased in fineness as the amount of malt ex- tract increased. The slight decrease in color value was not a serious 60 objection and the addition of 1.5 to 2.0 percent malt extract had a decided beneficial effect upon the baking qualities of flour 1002 With the use of 1.5 percent malt flour the largest volume was se- cured in baking flour 1003. As the grain was coarse and the color off, however, the advantages gained by the increase in volume were off- set. A decided increase in volume and grain was secured by the use of 1.5 percent malt extract in this weak flour in my opinion, the baking quality of this flour was thus greatly increased. In clear flour 1007, the use of 1.5 percent malt flour increased the volume as well as the texture and grain, and in this flour the addition of malt flour was beneficial. The use of 3 percent malt extract gave a decided improvement in the baking qualities of flmir 1007 as far as volume, grain, texture and color is concerned. In flour 1008, the strongest flour of the series, the use of 1.5 percent malt flour improved the texture and grain but darkened the color con- siderably. The use of malt flour did not increase the baking qualities of this flour, while on the other hand the use of 3 percent malt extract increased the volume slightly, improved the texture and grain thus improving the baking qualities to a marked extent. 61 IV. SUMMARY This paper deals witli the effects of diastatic ferments upon the strength of wheat flours. Tables and graphs have been presented, showing: 1. Optimum tem,perature fur the diastatic actixity of the malt flour used was at temperature oi 65°C. 2. Optimum hydrogen ion concentration for tlie diastatic enzymes in malt tlour was at a pH of 4.2C). 3. Constant diastatic acti\it\' was shown by the malt flour over a perio(l of eight hour> when digeNted at 27^C 4. Concentrations of added diastase exert their greatest effect in tlie first 10 percent of added malt Hour, giving an increase in dextrose from 1.63 to 3.. when dige^ted at 27' C for 1 hour. The strong flours showed a higiier sugar content and greater diastatic activity than did the weaker flours, ddie starch of the strong flours appeared to be more ea>il}' hydroly/ed by diastatic ferments than that of the weaker flours. 6. Addition of (lia>tatic ferments to a dough con\ert tlie starch to reducing sugars and in the earlier stages of fermentation, produce a surj^lus of fermentable sugars in the doughs made from strong flours. This surplu> :^oon di>a|)p(.-ar> as the acti\ity oi the yeast increases, and at the end of the ft-rmentatiou ])eriod the dough is nearly depleted of a\ailal)le sugars. 7. Susi)ension-^ of strong flours in water had a higher \iscosity (on the addition of lactic acid i than simibir >uspen>ions of weak flours when incubated alone or with added on the properties of wiieat proteins. Jour. Amer. Chem. Soc, 28. 1657-1667. Cohn, E. J., and Henderson, L. J. 1918. The physical chemistry of l)rtad making. Science, 48, 501- 505. Cohn, E. J., Wohlback. S. B., Henderson, L. J., and Cathcart, P. H. 1918. On the control of rope in bread. Jour. Gen. Physiol., 1, 221-230. Cohn, E. ]., Cathcart. P. H., and Henderson, L. J. 1918. The measurement of the acidity of bread. Jour. Biol. Chem., 36, 581-586. de Sassure. Theodore. 1819. Sur Taction de I'amidon a la temperature atmospherique par Taction de Tair et de Teau. Ann. chim. et phys., 11, 379-408. Detmer, W. 1882. Ue1)er den Einfluss der Reaction Amylum sowie Diastase enthaltender Elussigkeiten auf den Verlauf der fermenta- tiven Prozcsse. /. Physiol. Chem.. 7. 1-6. Dubrunfaut. 1847. Note >ur Ic gluc^c-. Ann. chim. ct ph\>.. 21 (III), 178-180. Duggan. J. R. 1885-86 (Jn the determination of diastatic activity. Amer. Chem. Jour., 7, 306-312. Effront, J. 1892. Sur les conditions chimique de Taction des diastase. Compt. rend., 115, 1324-1326. Effront, J. 1893. Sur les conditions chimiques de Taction de la diastase. Bull. soc. chim.. 9 (III), 151-156. Effront, J. 1895. Sur Tamylase. Comj). rend., 120, 1281T283. 64 Effront, J. 1904. Action des acides amids ramylase. Bull, soc, chim., 31, (III), 1230-1233. Fernbach, A., and Wolff, J. 1906. Sur la transformation prcsque integrale en maltose en dex- trine provenant de la saccharification de I'amidon. Compt. rend., 142, 1216-1218. , Fernbach, A., Wolff, J. 1907. Etude sur la liquefaction diastasiquc des empois de feculc. Compt. rend., 145, 261-263. Fischer, M. 1915. OEdema and Nephritis. Ed. 2. New York, 1915. Fischer, M. 1918. On the swelling of gelatin in pol\ basic acids and their salts. Jour. Amer. Chem. Soc, 40, 272-292. Fischer, M. 1918. On the swelling of fibrin in polybasic acids and their salts. Jour. Amer. Chem. Soc, 40, 292-303. Fischer, M. 1918. On the liquifaction or "solution" of gelatin in polybasic acids and their salts. Jour. Amer. Chem. Soc, 40, 303-312. Fleurent. 1896. Sur une method chimiquc d'appreciation de la valuer bou- langers des farines de blc. Compt. rend., 123, 755-758. Ford, J. S. 1904. Lintner's soluble starch and the estimation of diastatic power. Jour. Soc. Chem. Indus., 23. 414-422. Ford, J. S., and Guthrie, J. M. 1908. Contributions to the biochemistry of Ixirley 1. Amylases of resting barley. Jour Inst. Brew.. 14, 61-85. Abst. in Jour. Soc Chem. Indus. 27, 239-240. Ford, J. S., and Guthrie. J. M. 1908. The amylolytic and j)roteolytic ferments of wheaten flovirs. and their relation to leaking \alue. Jour. .Soc Chem. In- dus., 27, 389-393. Francis, J. M. 1898. Testing of diastatic digestants. Bull. Pharm., 12, 52-54. Girard and Fleurent. 1903. Le froment et sa mouture. Gauthier-Villars, Paris, 1903. Gortner. R. A., and Doherty, E. H. 1918. Hydration capacity of gluten from "strong" and "weak" flours. Jour. Agr. Res., 13, 389-419. 65 Gortner, R. A., and Sharp, P. F. 1922. Physico-chemical properties of strong and \\eak tlours III. Viscosity as a measure of hydration capacity and the rela- tion of the hydrogen ion concentration to imbibition in the different acids. Reported before Amer. Chem. Soc , New York, Sept., 1921. Abstract in Science N. S. 54, p. 558, 1921. Griefsmeyer, V. 1880. Rundschau auf dem Gel)iete der Bierl)rauerei. Dingler's Polytechn. b)ur.. 235. 379-387; 452-460. Hawkins, Lon A. 1913. The effect (jf certain chh)rides singly and combined in pairs on the activity of malt diastase. Hot. Cdz., 55, 265-285. Henderson, L. J., Fenn. W. O., and Cohn, E. J. 1919. Influence of electrolytes u])on the viscosity of dough. Jour. Gen. Physiol., 1,387-397. Henderson, L .J., Cohn, E. J., Cathcart, P. 11., W'achman, J. D., and Fenn, W. 6. 1919. A stud\- of the action of acid and alkali on gluten. Jour. Gen. Physiol., 1,459-472. Ilofmeister, Franz. 1890. Zur Lehre von der Wirkung der Salze \\ Untersuchungen iiber den Quellungsvorgang. Arch, fur Expt. Path. u. Pharmakol., 27, 305-413. (Cited from Gortner and Do- he rty). Humphries, A. E., and Biffen. R. H. 1907. The improvement of luigli^h Wheat. Jour. Agr. Sci.. 1, 1-16. Humphries, A. E.. and Biffen. R. H. 1910. Quality in wheaten flours. Jour. Board Agr., 17, No. 3, Sup- plement No. 4, 39-45. Jessen-Hansen, H. 1911. Etudes sur la farine de froment. 1. Influence de la concen- tration en ions hydrogen sur la 1)oulangere de la farine. Compt. rend. trav. lab. Carlsberg. 10. 170-206. Johnson. W. A. 1908. .\ proposed method for the routine valuation of diastase ])re|)arations. Jour. Amer. Chem. Soc, 30, 798-805. Jungk, J. F. C. 1884. Analysis of malt-extract. Jour. Chem. Soc. (London), 46, 529-531. Kendall, E. C, and Sherman. H. C. 1910. Studies on amylaso. 11. A >tudy of the action of pan- creatic amylase, jour. Amer. Chem. Soc, 32. 1087-1105. 66 Kirchhoff, G. S. 181 L Ueber die neue vun Kirchhoff entdeckte Zuckergewinnung. Schweigger's Jour. f. Chem. u. Phys., 4. 108-110 Kirchhoff, G. S. 1815. Ueber die Zuckerl^ildung beim Malzen des Getreides und beim bebriihen seines Mehls mit kochenden Wasser. Schweigger's Jour. f. Chem. u. Phvs., 14, 389-398. Kjeldahl, J. 1879. Recherches sur le ferment producteur du sucre. Compt. rend. trav. lab. Carlsberg., (not read ; cited by Griefsmayer) Kjeldahl, J. 1880. Mitteilung aus dem Carlsberger Laboratorium. Dingler's Polytechn. Jour., 235, 3/9-387; 452-460. Koch, Geo. 1914. The diastase and invertase content of wheat flour and their relation to baking strength. A thesis submitted to the graduate school of the University of Minn, for the degree of Master of Science. Lintner, C. J. 1885. Zur bcstimmung dor Diastasewirkung. Z. ges. Brauwesen n. s. 8. 281-285. Lintner, C. J. 1886. Studicn iibcr Diastase. Jour. Prakt. Chem., 34, (2) 378-394. Lintner, C. J. 1887. Studien ul)er Diastase II. T<>ur. Prakt. Chem.. 36. (2) 481- 498. Liebig, H. J. von. 1909. Uber den Zuckergehalt der feinen Weizenmehle der Weizen- mehle teige und der vergorener Mehlteige sowie iiber die diastatische Kraft der \\'eizenmehle. Land. Jahr., 38, 251- 271. (Abst. from C. A. 3. 2327). Lijers. H., and Ostwald, Wo. 1919. Beitrage zur Kolloidcheniie des Brotcs II ; Zur Viskosim^trie der Mehle. Kolloid Z., 25, 82-136. Liiers, H. 1919. Beitrage zur Kolloidchemie des Brotes III; Kolhndchem- ische Studien am Riggen und Weizen gliadin mit beson- derer Berucksichtigung des Kleber und Bachfahigkeits problems. Kolloid Z., 25, 177-240. Liiers, H., and Ostwald, Wo. 1920. Beitrage zur Kolloidchemie des Brotes, I\'; Zur kenntnis von Mehlen von schlechter Backfahigkeit. Kolloid Z., 26, 66-67. 67 Liicrs, H., and Ostwalcl, Wo. 1920. Beitrage zur Kolloidcheniie des Brutes V. Die KcjUoid Quel lung des Weizenklebers. Kolloid Z.. 27. 34-37. Manquenne, L., and Roux, E. 1906. Influence de la reaction du milieu >ur Taotivite de I'aniNlase et la composition des empois saccharifies. Compt. rend., 142, 124-129. Macpicnne, L., and Roux, E. 1906. Nouvelles recherches sur la saccharification diastatique. Compt. rend.. 142. 1059-1055. Marker. 1877. Standpunct unsere Kenntnisse dcr diastatischen \'organge. Landw. X'ersuchs Sta.. 23. 69-70; Ber. dcr Xaturfor., Miin- chen 1877. Martin. F. J. 1920. Properties effecting strength in wheatcn flcnirs. Jour. Soc. Chem. Indus. 39. 246T-251T. Mohr, O. 1903. Einfluss der Kohlensaure auf die Diastasewirkung. Ber.. 35. 1024-1029. Mu>culu>. M. F. 1860. Remar(|ues sur la transformation de la matiere amylases en glucose et dextrine. Ann. chini. et i)hys., 60, 203-207. Musculus, M. F. 18()2. Xouvelle note >ur la tran>-formati(jn de I'amidon en dextrin et glucose. Comi)t. rend.. 54, 194-197. -Mu>culus. M. F., and (Jruber, D. 1S78. Sur I'amidon. lUill. Soc. Chim. Soc, 30, 54-70. Norton, F. A. lOOr.. Crude gluten. Jour. .\m. Chem. Soc, 28. 8-25. Oshorn, T. B., and X'oorhees. C. C. 1893. The proteins of wheat kernels. Amer. Chem. lour., 15, 392- 471. ()sl)orn. T. B.. and \'oorhees. C. G. 1894. The proteins of wheat kernels. .\mer. C hem. Jour.. U), 524- 535. Osborn, T. B. 1907. The proteins of the wheat kernel. Carnegie Inst., Washing- ton Bui. 84. Ostwald, Wo. 1*^19. Beitrage zur Kolloidchemie des Brotes 1. Kolloid Z., 25. 26-45. OH O'SulIivan, C. 1872. On the transformation products of starch. Jour. Chem. Soc. (London), 25, 579-588. ' . O'SulIivan, C. 1870. On the action of malt-extract on starch. Jour. Chem. Soc. (London), 30, 125-144. Payenc and Persoz. 1833. Memoire sur ha diastase, les principaux produits de ses reac- tions, et leurs applications aux arts industriels. Ann. chim. et phys. 53. 73-92. Pauli, Wo. 1899. Die physikaHschen Zustandsanderun^-en der l-jweisskorper. Arch. ges. Physiol., 7S, 315-345. Pauli, Wo. 1902. Untersuchungen iiber physikalische Zustandsanderungen der K)olloide. \>rhalten der Eiweisskorper gegen elektrolyte. Beitr. z. Chem. Physiol, u. Path.. 3. 225-24<). Pauli, Wo. 1903. Untersuchungen iihcr physikalische Zustandesanderungen der Kolloide. Irreversible Eiweissfallung durch elektro- lyte. Beitr. z. Chem. Physiol, u. Path.. 5, 27-55. Pauli, Wo. 1905. L-ntersucluingen iiher ])hysikali.^che Zustandsanderungen der Kolloide. Eiwcissfallung durch Schwermettalle. Beitr. z. Chem. Physiol, u. Path.. 6. 233-259. Pauli, Wo. 1906. Untersuchungen iiber physikalische Zustandsanderungen der Kolloide. Die Eletrische Sadung \on Eiweiss. Beitr. z. Chem. Physiol, u. Path.. 7, 531-547. Petit, P. 1904. Action de la chaleur et de I'acidite sur I'amylase. Compt. rend., 138, 1231-1233. Reychler, M. A. 1889. La saccharitication diastasiciue. Bull. Soc. Chim., 1. (HI), 286-295. Roberts, W. 1881. On the estimation of amylolytic and proteolytic activity of pancreatic extracts. Proc. Roy. Soc, 32, 145-161. Rockwood, E. W. 1917. Some nitrogeneous auxoamylases. Jour. Amer. Chem. Soc, 39, 2745-2752. Rumsey, L. A. 1922. The diastatic enzymes of wheat flour and their relation to flour strength. Am. Inst. Baking. Bui. 8. 69 Schmidt, C. L. A., and Hoagland, D. R. 1919. Table of pH, H- and OH" values corresponding to electromo- tive forces determined in hydrogen electrode measure- ments, with bibliography. Pub. Univ. of Cal., 5, No. 4. 23-69. Schutt, F. T. 1904. A chemical study of the grain and flour uf the grades of wheat. Centr. Expt. Farm (Ottawa. Can.), Bui. 50, pt. 2. Schutt F. T. 1907. The relationship of composition to bread-making value. Centr. Expt. Farm (Ottawa, Can.), Bui. 57. pt. 2. Schutt F. T. 1908. A chemical study of the grain and flour of the grades of wheat— 1907. Centr. Expt. Farm (Ottawa, Can.). Bui. 60. pt. 2. Sharp, P. F., and Gortner, R. A. 1922. Physico-chemical studies of strong and weak flours 11. The imbibitional properties of the glutens from strong and weak flours. J. Phys. Chem., 26, 101-136. Sherman, H. C, Kendall, E. C, and Clark. E. D. 1910. Study on amylases 1. An examination of methods for the determniation of diastatic power. jour. Amer. Chem. Soc, 32, 1073-1087. Sherman, E. C, and Schlesinger. M. D. 1913. Study on amylases V. Experiments upon the purification of the amylases of malt. Jour. Amer. Chem. Soc, 35, 1617- 1624. Sherman, H. C, and Schlesinger, M. D. 1913. Study on amylases \'l. A comparison of amyloclastic and saccharogenic powers. Jour. Amer. Chem. Soc, 35, 1784- 1790. Sherman, 11. C, and Thomas, A. \V. 1915. Study on amylases VIII. The influence of certain acids and salts upon the activity of malt amylase. Jour. .\mcr. Chem. Soc, 37, 623-642. Sherman, H. C, Thomas, A. W., and Baldwin. M. E. 1919. Influence of hydrogen-ion concentration upon enzymic ac- tivity of three typical amylases. Jour. .\mer. Chem. Soc, 41, 231-235. Sherman, H. C, and Neun, Dora E. 1919. The proteolytic activity of pancreatic amylase prejiarations. Jour. Amer. Chem. Soc, 41. 1855-18(.2. 70 Sherman, H. C, and Neun, Dora E. 1921. The influence of certain amino acids upon the enzymic hy drolysis of starch. Jour. Amer. Chem. Soc, 43, 2461-2469. Sherman, E. C, and Caldwell, M. L. 1921. A study of the influence of arginine, histidine, tryptophane and cystine upon the hydrolysis of starch by purified pan- creatic amylase. Jour. Amer. Chem. Soc. 43, 2469-2476. Snyder, Harry. 1899. Proteids of wheat flour. Minn. Exp. Sta.. Bull. 63. 519-533. Snyder, Harr}-. 1901. Studies of hread and liread making-. U. S. Dept. Agr. Bull. 101. Snyder, Harry. 1903. Wheat flour and bread. U. S. De]jt. Agr., Year Book, 347- 362. Snyder, Harry. 1904. The determination of gliadin in wheat flour by means of the polariscope. Jour. Amer. Chem. Soc, 26, 263-266. Snyder, Harry. 1905. Milling tests of wheat. Minn. Agr. Ex]). Sta., Bull. 90 Schneidewind, W., Meyer. D.. an^igcn Zucker aus Starkuiehl und fiber Umwandlung susser Materien in gahrungsfa- higen Zucker. Schweigger's Jour. Chemie u. Physik, 5. 80-93. Wohlgemuth, J. 1908. Uber eine methode zur quantitativcn Bestimmung der diasta- tischen Fermentc. Biochem. Z., 9, 1-9. Wood, T. B. 1907 The chemistry of strength of wheat flour. Jour. Agr. Sci.. 2, 139-160; 267-277. Wood, T. B., and Hardy, W. B. 1908. Electrolytes and colloids. The jdiysical state of gluten. Proc. Roy. Soc. (London). B.. 81, 38-43. 72 BIOGRAPHICAL. Ferdinand Albert Collatz was born in Duluth, Minnesota. He graduated from the Duluth Central Hig-h School in June, 1914, and entered the University of Minnesota the same fall, where he received the degree of Bachelor of Science in June, 1918. Shortly after this he entered the Army and was assigned to the Piiysiological Labora- tory at the Lakeside Hospital, Cleveland, Ohio, under the direction of Major Roy G. Pierce. During 1919-1920, he held the position of Assistant in Agricultural Biochemistry. University of Minnesota, and in June. 1920, received the degree of Master of Science from this department. During 1920-21 he held the American Institute of Bak- ing Research Fellowship, where the experimental work in this Thesis was done, at the same time continuing his graduate work in the de- partment of Agricultural Biochemistry, University of Minnesota. Here he studied for the degree of Doctor of Philosophy. Major subject, Biochemistry. Minor subject. Botany. Member of Sigma Xi, Phi Lambda Upsilon, Gamma Sigma Delta. Gamma Alpha; Mcmbt-r of llie American Chemical .Society. 73 ACKNOWLEDGMENT. This imotigaliim was carried out under \hv direclioii of Dr. Ro.ss .■\iken (lortner. 'Jdic author lakes ilii> opportuuitx- to ex])ress his ai)i)reciation and gratitude for the help and encourajj;"enu-nt wliich wa-- so i^ladly g'iven durin<;' the lime this work was in progress. F. .\. COLL.ATZ. 74 LIBRARY OF CONGRESS 014 338 852 9 •