-^ it; BXCHANGE OCT 27 192: Flour Strength as Influenced by the Addition of Diastatic Ferments f 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, 1922 Chicago, III. TABLE OF CONTENTS Page I. Introduction : Definition of flour strength 5 A. Historical 6 1. The proteins of wheat flour and their physical relation to flour strength i • • 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 48 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. Effect 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 the baking value of flour.60 IV. Summary 62 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, eflFect 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 are 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." \ s * *. HISTORICAL. Not until Osborn and Voorhees (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 by 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 which the comparative baking qualities of 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 attempted to correlate certain properties 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 diflference 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 6 water, which, however, can be modified 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 glutenin 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 flour, in order to strengthen it, is impractical as they would have to be in con- 7 tact for forty-eight hours before baking. He 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 flours 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 effects of acids, alkalis, and salts upon 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 definite period of time when they are taken out, drained and weighed. They find that "flours con- taining acids and salts in such combinations as to favor water absorp- tion will behave as weak flours, whereas 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 patent 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 flour 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, NagSO^ MgSO^, KBrO* tend to decrease the viscosity of the 8 dough, while NaCl, MgClg 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%- 607o) 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 diflferent 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 diflference 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 dispersion and imbibition are here almost coincident." During the course of the work Sharp and Gortner 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 drying 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 dififerent 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 role 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 0.09% to 0.81%, the latter 0.63% to 1.89%. 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 the dough by diastatic action. Particular attention should be paid to 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 sugar's 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 alcohql and water. (Data taken from Shutt [1907] page 20). In aqueous extract In alcoholic extract Directly After Directly After Designation reducing Inversion reducing Inversion of as as Total as as Total Vol. Sample Maltose Sucrose Sugars Maltose Sucrose Sugars Bakers % % % % % % Mark No. 1 Hard 1.96 2.14 4.10 .13 .91 1.04 492 No. 1 Northern 2.73 1.95 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 5.22 .26 1.21 1.47 383 No. 4 Com'cial Gr. 3.62 2.43 6.05 .05 1.34 1.39 397 No. 5 Com'cial Gr. 3.63 2.43 6.06 .06 1.42 1.48 366 No. 6 Com'cial Gr. 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 zymes contained in flour with regard to their effect 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 expectations, 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 weak 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). 0.25 Percent 1.0 Per cent Time Flour Alone of Malt of Malt Hours c.c. CO2 c.c. CO2 c.c. CO2 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 flours on aging show greater jjaking strength and this increase in diastatic activity may therefore be the primary cause for increase in baking strength as the flour ages. At any rate it seems to be in accord with Wood's theory). 11 Differences Volume Maltose of gas ob- formed tained from during dough in Bakers doughing 3 hours Mark 1.48 78 45 1.38 84 40 2.53 145 76 3.04 155 80 3.88 164 95 3.45 175 78 6.75 193 90 5.42 217 90 4.S2 220 80 5.02 230 91 8.11 270 90 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). Percent of Matter solubl Percent of in dough matter soluble when kept in water at at 40° C. for Flour 15.5°C. 3 hours 1 2.12 3.60 X 2.03 4.41 W 2.83 5.38 3 2.49 5.53 Y 2.69 6.57 2 3.19 6.66 4 4.19 1095 V 2.83 8.26 T 2.84 7.66 • U 2.65 7.68 Z 3.54 11.65 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 by active and by boiled papain. In testing amylase values from twelve flours, they found differences (using KCl and papain extracts) varying from 22. L to 46.8 expressed in grams maltose per gram 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- portant factor, also that the presence of a proteolytic ferment is an- other and possibly more valuable consideration." Ford and Guthrie (1908) were probably the first to demonstrate the action of proteolytic enzymes in flour. They were unable to secure results with nitrogen determinations or with the viscosi- meter, 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) found 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 w^ater, 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 upon 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 Kirchoff found that when starch was boiled with dilute HgSO^, 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 tbe action of alkaline copper sulphate was 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 yield of maltose is lowered and at still higher temperatures two molecules of starch yield one of dextrin and one of maltose. Marker concludes that there are two diastatic ferments, one producing dextrin and the oth- er maltose. This is our present day conception of the diastases, one being termed the liquifying and the other the saccharifying enzyme. Musculus and Gruber in 1878 regarded starch as a polysaccharide, containing five or six times the group C12H20O10. Under the action of diastase or acids, the carbohydrate undergoes a series of changes of hydration and successive decomposition, resulting in maltose and dextrin of less molecular weight. Brown and Heron in 1879 found that the heating of a diastatic solution diminishes its activity and that an increase in temperature increases its activity up to 66°, beyond which not much activity is shown. They also found that alkalies markedly reduce the activity of a malt extract. It was in 1879 that Kjeldahl stated his law of proportionality in regard to the action of diastases. He determined the reducing power of malt extract and saliva on an excess of starch at 57°-59°. He conisdered that the reduced copper was directly proportional to the amount of amylase present and was a true measure of diastatic power so long as digestion was not carried above 40 per cent of the starch present. Grief smayer a year later, confirmed Kjeldahl's work and the law of proportionality. Up to about this time the polariscope and the cupric reducing method had been used to estimate the diastatic power of malt ex- tract by the amount of maltose formed. The iodine reaction was made use of by Roberts in 1881, however, and he defined the dias- 14 tatic power of pancreatic extracts, saliva, and malt extracts, as the number of cubic centimeters of a standard starch paste which could be converted by one cubic centimeter of the active solution during five minutes at 40° into products giving no color reaction with iodine. Jungk two years later published a method of determining the diastatic activity of a malt extract by the iodine method which was similar to the method of Roberts. He determined the time re- quired for 10 cc of extract to convert 10 grams of starch which he considered should not exceed 10 minutes, for a good malt. His temperature of digestion was 40°. From the literature already cited, two methods of determining the diastatic activity of an amylase preparation had come into use, namely the iodine or the so-called liquifaction method, which mea- sures the power of the amylase to completely convert the starch into products which no longer give the characteristic color with iodine, and the reduction or saccharification method in which the amounts of reducing sugars are estimated by means of alkaline copper sulphate. The development of the iodine method from the time of Jungk will be the first considered. Iodine Method for the Estimation of Diastatic Activity. Francis in 1898 made the next improvement in the iodine method by laying down very exact rules for the determination of the end point in the iodine starch reaction. He also extended the time of digestion from 10 minutes to half an hour. Takamine in the same year developed a different procedure. He first standardized a sample of taka-diastase, which was found to keep. its diastatic power for a considerable length of time. He then determined the relative amounts of standard and sample to be tested, which are required to accomplish the same amount of conversion in the same length of time. In these determinations he used iodine to test for the end point. The next important work was carried out by Wohlgemuth in 1908. He established a new standard which was based on the number of cubic centimeters of 1 percent starch solution which 1 cc of dias- tatic ferment could convert in 30 minutes at 40°C. The method consists of measuring out 5 cc of a 1 percent starch solution into each of a series of test tubes and then adding different amounts of a diastatic solution to each. At the end of a half hour at 40° the test tubes are transferred to an ice bath. After cooling each tube was shaken up with a definite amount of iodine solution and the one which showed no trace of color was taken for the end point. Johnson in 1908 improved the technique of Francis, and Jungk, by 15 preparing a starch paste (potato) of constant value. He added his diastatic preparations to a fixed amount of starch and withdrew portions and tested with iodine, at the end of ten minutes. When close to the end point, he tested more frequently, repeating with smaller increments of sample, until the amount was found which would in ten minutes just convert the starch. Sherman and Schlesinger (1913) and Sherman and Thomas (1915), used the method of Wohlgemuth in determining amyloclastic activity. They stipulate a definite color for the end point of the iodine reaction, using the Milton Bradley color chart as given by Mullikin in his "Identification of Pure Organic Compounds." If the iodine method is to be used it appears probable that the most accurate results can be obtained by following the technic described by Sherman and his various co-workers. Copper Reduction Method for the Determination of Reducing Sugars Formed by the Action of Diastase. Although Kjeldahl showed that the reducing sugars formed by the action of diastatic enzymes upon an excess of starch was a measure of their activity, no great advance was made in the exact valuation of diastase preparations until 1886, when Lintner modified Kjeldahl's method. He first prepared a soluble starch possessing rather definite characteristics. He was then enabled to base his calcu- lations of diastatic power upon the production of a constant quantity of maltose formed by the action of the malt extract upon a definite amount of soluble starch. His method consisted of measuring 10 cc. of a 2 per cent soluble starch solution into a series of ten test tubes, to each tube he added a slightly diflferent amount of malt extract. After digesting exactly one hour at 21 °C., 5 cc. of Fehling solution is added to each tube and the whole series of tubes are placed in boiling water for ten minutes, and then examined to determine the first tube in which all the copper was reduced. Lintner prepared a diastase, of which .12 mg. was able to produce the required amount of maltose, under the above conditions, to completely reduce 5 cc. of Fehling's solution. To this preparation Lintner gave the value of 100 and the power of the samples were calculated as inversely pro- portional to the^ amount of sample required to produce this fixed amount of reducing sugar. Ford (1904) prepared a soluble starch from various sources and after careful purification, similar to the Lintner method, concluded that no diflference was attributable to the source of the starch. He specified that soluble starch should be neutral to rosolic acid in order to give concordant results by the Lintner method. 16 Ford and Guthrie (1908) expressed amylolytic activity as grams of maltose produced by a filtered extract of one gram of mashed barley in an excess of soluble starch for one hour at 40°C. Several other copper reduction methods have found their way into use since Lintner established his method for determining diastatic activity by measuring the reducing sugars formed. The most notable is that of Sherman, Kendall and Clark (1910). This method consists of placing different amounts of enzyme e. g., 0.2, 0.5, 0.8, and 1.0 mgms. into four erlenmeyer flasks, to this is added 100 cc. of a 2 per cent soluble starch and the whole digested 30 minutes at 40°C. At the end of this time, 50 cc. of Fehling solution are added and the flasks are immersed in a boiling water bath for 15 minutes. The reduced CU2O is then quickly filtered and determined gravimetrically. They supply a table which gives diastatic activity of the preparation called the "new scale." Influence of Temperature on Diastatic Activity. In all of the earlier work high temperatures were employed, that is between 40°-60°C. Marker found that at 60° four molecules of starch formed three of maltose and one of dextrin, while at 65° the yield was lowered and only one molecule of maltose and one of dextrin were formed from two of starch. It is quite obvious from our present day knowledge that the optimum temperature for diastatic activity lies between 63°-65°C., which would explain why Marker got a decreased production of maltose at 65 °C. Brown and Heron (1879) found that previous heating of a malt extract decreased its activity to a great extent. They found that it showed very litle activity when heated above 66° C. Vernon (1901-1902) gives the optimum temperature for the activity of diastase as 35°'C. with continued activity up to 65°C. Kendall and Sherman (1910) find the optimum temperature of puri- fied amylases to be 40° in the presence of salts and a trace of alkali. They find that between 20°-40° the activity is about doubled for each 10° increase in temperature with a considerable falling off in rate of increase of activity betwen 40°-50°, where the maximum activity was found. Influence of Acids, Bases and Salts on Diastatic Activity. The effects of acids and bases upon diastatic activity has received a great deal of attention, some investigators claiming that an acid medium was the most favorable, while others advocate a neutral medium, for the optimum activity of the enzyme. Baswitz (1878-1879) and Mohr (1903) found that when carbon dioxide was passed through the reacting medium, a great increase in 17 diastatic activity took place. Detmer (1882) reached the same con- clusion and noted that small amounts of citric, phosphoric and hydro- chloric acids had an activating effect. Reychler (1889) found that KH2PO4 had a stimulating effect, while Effront noted that HCl, HF, H2SO4 and phosphoric acid as well as KHoPO^ had a very favorable action. Petit (1904) found an acid medium to be the best. Kjeldahl (1880) reported that H2SO4 when in a concentration of .005 N. increased diastatic activity, but was decidedly detrimental when .01 N. was used. The same concentrations hold for citric and acetic acids as reported by Schneidewind, Meyer and Miinter (1906). Heyl finds that KHgPO^ has an activating as well as a conserving action, while Hawkins (1913) reports that small additions of phos- phoric, acetic and tartaric acids increase the saccharogenic power of the malt extract but did not effect the amyloclastic activity. Chittenden and Cummins (1884), Duggan (1885), Lintner (1885), Ford (1904), Maquenne and Roux (1906) and Fernbach and Wolff (1907) are all agreed that a neutral medium is the most favorable for diastatic activity. Sherman and Thomas (1915) and Sherman, Thomas and Baldwin (1919) find that the optimum diastatic activity for various diastatic preparations, depending upon their source, is at a pH of 4.2-4.6. They report an optimum hydrogen ion concentration of about pH 4.4 for a diastase prepared from malt extract, a pH of about 4.7 for the amylase of Aspergillus oryzae and a pH of 6.8-7.0 for pancreatic amylase. After reaching the optimum pH the activity falls off very sharply on the alkaline side. Many substances have likewise been reported which activate dia- static preparations such as asparagine, aspartic acid, different amino acids and proteins. Chief among the workers who have reported such results are Effront (1904), Ford (1904), Rockwood (1917), Sher- man and Walker (1921) and Sherman and Caldwell (1921). Very little work is reported on the action of alkalis upon diastatic activity. Brown and Heron report a decided falling off in diastatic activity when Ba(OH)2, KOH or NaOH is added to the medium. In fact, it has been the custom to use Alkalies to stop diastatic activity in solutions being analyzed. Effects of Proteolytic Enzymes. The action of proteolytic enzymes upon 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 they existed in wheat 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 upon 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 per cent soluble nitrogen as protein, while in the other dough they found only 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 imbibitional capac- ity of strong and weak flours when treated with various 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 to fall back upon the baking tests to have a final answer to the question. II. EXPERIMENTAL, (a) The Problem. It has been shown in the historical review that flour strength has been studied in a variety of ways. Two points of attack are outstand- ing, however, the work of Wood 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 Hulton, and Ford and Guthrie who have attacked the problem from the enzymic standpoint. In this Thesis we are con- cerned with enzyme relationships. From the data presented by Ford and Guthrie 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 were perhaps the limiting factor in the production of maltose. I have taken up the problem at this point and am con- cerned with the effect upon the baking strength of wheaten flours when diastatic ferments are added to the dough. As the diastatic preparations available for the baker are in the form of malt flours and malt extracts which contain proteolytic enzymes, the problem is at once broadened to include the later as well as the starch splitting ferments. (b) Material. The present investigation was conducted with a commercial malt •flour, a representative malt extract, a commercial sample of wheat starch, and a series of seven wheat flours of different grades milled from wheats grown in different regions of North America. All of the flour samples and malt preparations were submitted 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 VII. Description of Materials Used in These Studies. The flours used in this investigation 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 IV, 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 baking strength. TABLE IV. Analysis of Wheat Flours on Air Dry Basis. ir Samples Protein )oratory 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. Malt Flour Laboratory Moisture Ash Protein No. Percent Percent Percent Total Reducing Sugars Sugars as Dex- Diastatic Value as Dextrose trose Degrees Percent Percent Lintner 24 8.8 1.26 11.25 4.75 10.62 177.05 TABLE VI. 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 water 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 pH, of the water extract, show it to be a clear flour. Although the protein 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. Flour 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. Malt Flour No. 1001 1002 10O3 1007 1008 1009 1011 Flour cc. N/50 HCl Added pH pH pH pH pH pH pH pH 12.5 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 6.153 5.981 6.050 5.491 (cc.N/50 NaOH) 2.5 7.371 6.931 7.726 6.938 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 10.202 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 15% NagWO^ 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 flour 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 th» 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 cooled 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, w^hich 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 w^eighed 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 treated 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 concentration determined by the use of the Bailey electrode and a Leeds and Northrup type 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 the gas producing capacity of a flour, twenty grams of flour are weighed out and transferred to a wide mouthed bottle. To 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 w^ater bath kept at 37°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 Instiute 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 325 grams Water 173 grams(varied depending 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% NagWO^ were added and thor- ughly shaken, and 20 drops of concentrated HgSO^ 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=4.26. 25 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 Normality cc. CU3O Dextrose Dextrose- of HCl used pH Grams Grams Per Cent N/10 28.0 1.988 .1075 .0527 3.52 N/10 26.0 2.099 .1115 .0548 3.65 N/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 35.0 3.156 .1438 .0714 4.76 N/10 13.0 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 22.5 3.870 .1984 .1000 6.67 N/25 20.0 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 ff!^f|«[|N/25 5.0 6.069 .1670 .0834 5.56 N/25 10.0 6.489 .1548 .0769 5.12 N/25 15.0 6.830 .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 45.0 9.649 .1090 .0535 3.57 N/25 50.0 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 first 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 effects of time of digestion up to eight hours is given in Table IX and presented graphically in Figure 2. 26 tm A. r \ / \ / \ \ / \ \ am \ \, , \ J. / \ \ s. OIK \ / \ 0U» ; f \ /S / ^ esx / N / 1 1 Figure 1. Effect of pH on the activity of diastase in malt flour 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 flour. Wt. of Dextrose Time of per 10 grams Digestion Weight of CusO of flour Dextrose Hours Grams Grams Per Cent 0.00 .1112 .3844 3.86 .25 .1462 .5128 5.13 .50 .1622 .5752 5.55 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 / laoiXi / / iim / / / /looA ) / "^.ooa / / f i 1 0O(H / / / (MO / ] i / .1000 / 1 1 i JCOC » I 3 4- S Time in Mourj 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 UM ^ k,^ zm / N / sm / im A. mIML 1 L ^im ZHOU 1 L / im J / MX / / ^ y ' ^ m Z5 ■30 ^ ■*0 43 JO JS iO iJ 70 Tempertiture 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. Series 1 Wt. of Series 2 Wt.of Dex- 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 Degrees Cu.O trose Flour Per CU2O 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 xAliquots 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 different 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 necessary 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 Malt Flour used, grams 0.25 0.50 0.75 1.0 1.25 Wt. CuaO. grams .0337 .0744 .1080 .1519 .1980 Wt. Dextrose, grams .0142 .0320 .0469 .0668 .0852 Dextrose, per cent 5.68 6.40 6.25 6.68 6.90 TABLE XII. Relation of concentration of malt flour to the production of reducing Sugars from wheat flour when digested 1 hour at 27 °C. weight of Proportion Wt. of Cu^O Dextrose of wheat per 2.S formed per flour to 2.5 gms. malt flour gms. flour flour Grams Grams 10:0 9:1 8:2 7:3 6:4 5:5 .0944 .2167 .25^3 .2918 .3030 .3279 .0408 .0968 .1168 .1331 .1387 .1512 Dextrose corrected for dex- trose of malt flour Grams .0408 .0826 .0848 .0862 .0719 .0760 Malt flour Percent 0.0 10.0 20.0 33.3 40.0 50.0 Dextrose formed Percent 1.63 3.67 4.24 4.92 4.80 6.06 30 it _^. J ^ / sc y x^ ^ ^ .. ^ ^ 1 <^ i / U / j.e C iO 20 30 10 St fhxeiif ffe/r Hour 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 effects 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. ri our ;:3arn pie iNumoer — L.om- mercial Malt Flour 1001 1002 1003 1007 1008 1009 1011 Wheat Used Starch Grams Jc % % % % % % % % .0000 0.00 1.85 1.34 .40 .94 2.28 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 2.32 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 2>.1Z 3.03 3.24 1.93 1.53 The Production of Reducing Sugars in the Dough During Fermentation. The production of reducing sugars 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 portion only in order to have one dough to bake in the usual way. 32 40 dS X ^ ^ «w JO jt ^<^ 1^ ^^ MAS '^ / ^ ^ X ^ li J X A ^ MM <^ / 7 / ^ ■^ ■*. / y '/ ^ 1 /.s ( fy / ^ ^ mi Y ^^^ ^ Hoi-' y ^ ^ ^^ ,^' m /• ><; ^ ^. X^ ^^ ,-^ / K ^^ r^ y'^ Ai / ,^ i^ / y CO Y OS JO li JO 23 JO JS .40 *S M Gramj Malt Flour Figure 5. Percent of dextrose formed from a series of flours when digested one hour at 27 ^'C. with various amounts of malt flour. Tables XIV-XXII give this data on three flours representing a strong type Canadian patent flour (1008), a decidedly weak Pacific Coast patent flour (1003), and a clear flour from Kansas (1002). Each flour was mixed with varying amounts of diastatic preparations as fol- lows. 1.5 percent malt flour, 4.0 percent malt flour and 3.0 percent malt extract. Controls with no diastase were included in each series. No sugars were added to any of the doughs except that amount con- tained in the malt flour and malt extract used, but this amount was deemed negligible in its effect upon frementation. 33 t/3 4; "o U^ LO »-0 *£> 00 O ■" "^ LO LT) VO l-n U-) 10 ^ o or^ On OOro U3 w w« ns 2"o hjr I D en ■*-' s s g ^'i ^ 4J O O ^S « o TJ--*!- 000 rt-CM 0\ 00 »-• 'O r-< 00 to VO fO ^ CsJ «— I 1-H* (\i f^ fo ""t -^ r^ go a\ "^ N© Tj- O »— I T-J »-J »-H l-H .^ P t) ooooopoor^O\ o ^ " rf Tt ^ Ti- 10 vo >^ £ .' 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"I *"" *"! ""I .H 888iQS^ O e« w. w, « 3 to O Oi^fM cort -^ II I b.5 O cS»< ?^% o SQOOO ocoTtcg o»-< eg CO '^to o vt-i*rr O rt - E C/5 u « u ^ a ^ o S»-H^rgHH e o O rt to ^ ^- a g. a o 35 ■s 2 o O CO c! «» I ^ •M HI 6 > fe •J < T3 C cd C ^ CO O CO CO cd CO u to CO bjO 'o 3 'O 5 "^ CC CO 4-* CO to O O o 2 ^ u •s Pi * c to ..^ o X O bo ri i- '^ ^ 3^ u: -,^^ O Q east otal gars Dex- rose ams >H H 3 +j « o ;^ _^ jl^^ on dry- basis ercen « o Q:: P^ xS^ from 10 gm. Dough Grams W o Q^ «« . 1^ 4> P 000008 c • ^•2c oSg 8§8SJ^:^ S CO 4^ *J CVJ fM Tf \0 NO to Oj flj " O 1^ 2 coO be CJ 5 a CO V- •ofj; be (d 3 (d u cd i-i •^ O CO M > T3 O c -o o d u 3 I en p^ ininoo 0000 t>. t^ in Lo 1— I »-H j< K t< K 06 00 invo vo urj to 10 -. 5^ « o o -M ^ rt bcQ en C o a 8 LOO 00 OOOn OQcvitn KKcjo'od LT) 10 10 10 X X - < u Hcd bA 3 CO °? c « .2 o « « a Wi t-i 3 O 4J o en •" r* en u en u VW O " L "•< w -. , . . •5 4-r i> fd o % " > cd c >. 10 1?5 t< r>! vd ^c ^ ^ Lo u-} vn Lo m to en?, t^<\J00PO00ON — '^ 00"*00fO00tn c3 cOTj-co^ S OOtoO 5 OOCvJfO s a _ »^ O 4> 3 o o bC o u- - > rt C u bo'rtQ o rt 'O Oi-HfM ro ii O C SX o o • £ en £ O O b03 ^ c o . 5 «L>'^ fe o o si (Z4 CM \0 "t r:! Ov <5| CM TtOqOgO r>.\ovpcMOvo ootn-^ooopo 1-t CM* CNJ cm' r-i ^ lOOtOlOOO On On VO tN. 00 On t^OOOOt^in Somtotoin Si-iCvjOrh O^CMfO-TfTt X u P- p, P- O 37 I 2S O (U O 2 ^ Kg X a; PQ "^ 3 1-1 a> S ^ S *- O u5 Q S O 4> jj k^ — C^ l-i . 00 O 1-1 cm' rrj ro rrJ rj-' ro»-< 0^^0 OO 00 r\j CO m vo t^ .— I »— t (VJ (M lO lO fvj T? "^ TJ Cvf "» O 4> O CO rH O i^ ^ 5 =* O M « o ^2 .2 3 ti o X ^ 'O X 0) TS W -^ "J CO ^ ^ 2 o ^ o 2 II O TJ (-> c ?^*- o o be ^ iiTj CO o o be 3 c en ^ bo SI o Qo 1J^ >^' c 5 y rt w o K K t< IN." I< 1-1 vO uo fvl tN. f-i bx On Tj; fVJ Cvi <^ PO -^ i/^ «--. be O O c« Q^ go bfl g cu :-• d w "" O rvj jt^ \nmvn\nxr) mvotN.Oi— < LO Cv OcOOv cm' cri "^ CO eg torn 0»J^ O 88^5^ K Oi-iCM POTt C o bo ^ ^^ W O C n) S B O ^ v; Ci g, <=^ o .^g c/) •2§2S •" X ♦J o o 38 o o o w « P vr, vo lO lO » Tt fvi ro VO f*^ t->» r>. OS ^. »'^ lomin O O fMtvjinONO vo Ov O >"* f^ r-< .-H (M rvj fVJ > 6 H-l l-l X O o -^3 .2 ^ C ^ 4) "^ 43 '^ bX) O bA 4> S ^ 73 O .2 g ■4-» o 3 O 3 c 00r^»-H TfON . /-\.;« «-! I.V I->^ t-' -TT W> X C SS u. "^TfTtfoeO Q° tflO O— ' OvOvvO u. ;_; ;i, ;_,;_; , O rt .Scot 8SS^§ JlJ O'-'Mf0'«f a o O (T) ^ 6 5=" S ^ .5^ ^ o Q 39 r. g (jramj Dc.r. Dje g 1 1 ""S ^ ••"^ "^ ^ ■: \ ■ - 1 \ \- ^^ ^ -aV^ \, .. ^ I \ •:5 ^ j\ J ••. X \] / ' •( / V / '• J y ■ . % / /■, / / / " / A \ / / / / » / "\ '■•. A / 1 / y / / ;••-.,, ,,^ , A V / ^ /\ / / \ •, '■■ ""s , •' w y • Z' '■•• '' Figure 6. Productions of reducing sugars in the panary fermentation of flour 1008 with different concentrations of malt flour and malt extract. (Full lines indicate doughs to which yeast was added, dotted lines no yeast dough). ^ G ami Defrost. 1 i '^ - .. ■'N. ^ ^ \ ■■•••. V. '>:.. \ 1 N ^^ ■••■.. \ » 1* •^ V K \ A • \ ■•• \ 1 \l V > \. ^ '■< \ / ^'.. ; \\ '""A ) Tv ? ■// ■■••• '■•;/ / r. / \\ /■: \ ., r / ./■"■■ "*. K ■■••., ■•, // / / "■• ■^., '■••. ■•• t \ 11 ^ > • 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 ...-•' Mi iv*-*"" P-"' -"' ~ -a 424^ / r^ ■ N N-" •-' ..•' . • ^■; ■\ . L ■^ i.1*.'- _j£..^ .,^ ~\ \ J c ... ^ / V v • , y \ N S .^^' ' \ iM \ ^ , ' \ .^ v^-^ ^ N N L. ^ • *o "^ — , t J 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 lines 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 sr.ch 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 becomes slack or less viscous during the fermentation period. This has been attributed to the action of the proteolytic enzymes of the yeast and partially to those in malt prep- 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. Nl Lactic Acid added cc 0.0 0.5 1.0 1.5 2.0 3.0 5.0 Malt Flour Digestion used Degr's Degr's Degr's Degr's Degr's Degr's Degr's Hours Percent M M M M MM 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 n 90 97 105 111 4 4.0 24 48 n 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 be 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. 1 — m Mi c^ \ M — - y ^ \ s \ ^ \ \ AS I N 1 -.I \ 4 Jl At > \ \ ft n \ \ 1,. \ \ \ \ \ s. J J \ < ^ . MS \ M \ s M \ Mt \ — » ^ -^ / 2 ^ Time ef Dioeition in Houry 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 effect of time of digestion was first tried to determine the op- timum time of digestion. The experiment consisted of digesting flour 1001 (a strong Kansas patent) for 1 to 5 hours with 100 cc of water, and repeating with the same flour after adding 2 and 4 percent of malt flour. After digesting for the stated period of time the flour-water suspensions were transferred to the cup of the MacMichael viscosi- meter and the average of three readings taken, N/1 lactic acid was then added in small amounts up to 5 cc. The mixture was thoroughly stirred after each addition of acid and the average of three viscosity readings taken. The experimental data of this series when digested at 30° C. is given in Table XXV and the data in the last column of this Table is shown graphically in Figure 9. It appears from this table that four hour digestion is ample time to secure evidence of proteolytic action, and this time of digestion was used in all subsequent work. Also the temperature of digestion was increased from 30° to 40° in order to procure greater activity of the enzymes. The data given in Table XXVI and illustrated graphically in Figure 10 was obtained by digesting a series of flours (1001-1002-1003-1007 and 1008) with varying amounts of malt flour (1.0-1.5-2.0-2.5-3.0 and 4 percent) with 100 cc of water at 40° C. and determining the viscosity after adding 5 cc of N/1 lactic acid. Table XXVII and Figure lOA show similar data when malt extract was used. It has been shown in the literature cited that even minute traces of salts have a marked affect upon the imbibition capacities of gluten. It was, therefore, thought advisable to see how the viscosity of the flour reacted when treated as above but with the salts of the flour and malt flour washed out after the digestion period. The precedure was as follows : The equivalent of 18 grams of water-free flour 1002, was weighed into each of six flasks and digested with 0.0, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 percent malt flour in 100 cc of water at 40° C. After four hours 900 cc of distilled water were added, the whole well shaken and centrifuged. The supernatant liquid was decanted and the flour residue shaken up with water and after complete disintegration made up to 100 cc, placed in viscosimeter cup, and the readings taken after adding varying amounts of lactic acid. The data showing the viscosity in the presence of lactic acid of flour 1002 with the salts washed out is given in Table XXVIIL In order to demonstrate still further that the salt content of the added malt flour did not vitiate the results obtained in Table XXVI when flour (for example) 1002 was digested 4 hours with 4 percent 44 TABLE XXVI. EflFect 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 I (J Flour Used 0.0 1.0 1.5 2.0 2.5 3.0 Degr's Degr's Degr's Degr's Degr's Degr's Flour No. M M M M M M 1001 1002 1003 1007 1008 145 150 50 124 151 123 126 H 106 136 119 118 42.5 103 129.5 114 109 40 95 117.5 112 97 36.5 97 112 105 99 37 103 109 4.0 Degr's M 93 81 28 88 102 IS) *x \, /* \ fit \\ \ fJi \ \;^ <> }' \ A, N *'"*-.. ^•/r \ \ \-- ^^ 1- \ S ^\ \ \ \, s \ ^ V ^.u-. \ N, 4 ,,. \ s. \ ^ N '\ \ w \ s \^ N \ HX X \ \, \, « ^^ ^ ^ 10 \ \^ \ * \ ^ s « t i J 4 ' Kr^^nt M^lt J^tpwmtftn UitJ Figure 10. Changes in viscosity of flour 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). 45 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/1 lactic acid added in each instance). Viscosity readings in degrees MacMichael. % Malt Ext. Used. Flour No. 1002 1003 1008 ...0.0 1.0 Degr's Degr's M M 145 57 150 136 51 138 1.5 Degr's M 131 50 134 2.0 Degr's M 127.5 48 132 2.5 Degr's M 120.5 43 127 3.0 Degr's M 111 38 122 4.0 Degr's M 106 34 112 too (K 4 L 5 ^- ^ X ^■- "^v m ^\ ^^ ^ \ ^ ""••. jU N — \- -..^ •»». Jl -"V, \ ■ ---, fi N Tin,enf McJT PreporaTion Used 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). 46 411 405 328 340 312 315 295 305 263 271 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 cc. 0.5 cc. 1.0 cc 1.5 cc. Viscosity Viscosity Viscosity Percent Degrees Degrees Degrees Malt Flour M MM 388 1 299 2 295 3 273 4 245 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. Cubic Centimeters N/1 Lactic Acid Added 0.0 5.0 Flour No. Degrtts Degrees 1002 digested without malt flour 38 145 1002 digested with 4% malt flour 19 81 1002 digested 3.5 hrs. without and 3 minutes with 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 submit 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 cubic 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 graphically 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 Add( id Malt Extract One Percent Malt Extract Flour No . 1002 1003 1007 1008 1009 1002 1003 1007 1008 1009 Time of Fermentation Gas Gas Gas Gas Gas Gas Gas Gas Gas Gas Hours cc. cc. cc. cc. cc. cc. cc. cc. cc. cc. 0.5 13 14 10 10 7 14 15 12 14 8 1.0 34 33 31 27 28 35 34 33 36 31 1.5 62 57 58 53 60 64 59 60 66 61 2.0 98 80 92 90 99 88 94 105 101 2.5 140 95 132 148 116 141 115 134 153 125 3.0 177 104 174 202 148 180 140 178 203 153 3.5 214 117 212 250 176 218 184 223 252 181 4.0 245 123 243 192 254 196 270 196 4.5 . 128 208 TABLE XXXL Changes in pH during the fermentation of the dough with values for flour extract and the extract of bread crumb. Baking Data. pH Values Flour Ash Flour Mix 1st Pch. 2nd Pch. 3rd Pch. After Pf. Bread No. % 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 5.29 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 5.2& 5.22 5.17 5.03 5.3U 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 was 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 ■XK tsi f / / / ^ / / ^ 1 //j / i //. y i §9 ^ ... ■ -" -'"" / .•''"' JD .(< f ./( f ^ r1 Time in Hours Figure 11. Gas producing capacity of flours 1002, 1003, 1007, 1008 and 1009, fermented with 2.5% yeast. Joo m I / too i // ^ 8" A / ^ ^ ^ ISO // ' / A ^ i. ^ / JO / ^ A / ^ ^ Time in Hoon Figure 12. Effect of 1.% added malt extract upon the gas producing capacity of flours 1002, 1003, 1007, 1008 and 1009, when fermented with 25.% yeast. 49 regularities in the data might be accounted for in this manner. The activity of yeast is also vfery 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 sugars 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 XXXI. In the past all chemical and physical data accumulated on the 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 effects of added diastatic ferments upon wheat flours, the two diastatic preparations used throughout 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 time 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 showing the effects 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 1.5 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 9.790 9.580 9.370 9.155 8.940 8.730 8.310 Wt. of dough gms. 513 515 516 517 517 521 522 525 Wt. of loaf gms. 454 452 443 446 450 446 459 457 Vol of loaf cc. 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 Vol. of 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 with increase in matt 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 TABLE XXXV. The effect of varying amounts of malt extract upon the baking qual- ities of flour 1002. Malt Extract % Standard .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 5:19 5:12 5:07 5:02 4:57 4:52 4:47 4:42 Sucrose added gms. 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 Wt. of dough gms. 519 514 513 513 513 514 515 521 Wt. of loaf gms. 454 441 448 445 437 436 440 447 Vol. of loaf cc. 1740 1900 2050 2010 2010 1980 1960 1940 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 82°-83° and baking 479°F. Time of baking 25 minutes. TABLE XXXVI. The effect of varying amounts of malt flour upon the baking qualities of flour 1003. Malt Flour % Standard .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 4:42 4:38 4:34 4:28 4:24 4:20 4:16 4:12 Sucrose added gms. 10.00 9.79 9.58 9.37 9.155 8.94 8.73 8.31 Wt. of dough gms. 511 512 515 515 513 514 521 523 Wt. of loaf gms. 468 467 468 464 457 463 469 476 Vol. of loaf cc. 1660 1760 1675 1860 1710 1690 1650 1550 General 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 soggy 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 % Standarc I .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. 4:42 4:44 4:40 4:36 4:32 4:28 4:25 4:21 Sucrose added gms. 10.00 8.94 7.88 6.82 5.76 4.70 3.64 1.52 Wt. of dough gms. 511 504 507 504 506 508 . 508 492 Wt. of loaf gms. 468 460 461 454 448 453 460 448 Vol. of loaf cc. 1660 1730 1775 1810 1770 1700 1750 1680 52 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 minutes TABLE XXXVIII. The effect of varying amounts malt flour upon the baking qualities of flour 1007. Malt Flour % Standard .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, finest 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 % Standard .5 1.0 1.5 2.0 2.5 3.0 5.0 Fermentation Hrs. Sucrose added gms. Wt. of dough gms. Wt. of lt)af gms. Vol. of loaf cc. 5:22 5:17 10.00 8.94 1640 1690 5:12 7.88 1900 5:07 6.82 1620 5:02 5.76 1660 4:57 4.70 1850 4:52 3.64 1890 4:47 1.52 1760 General Remarks : The use 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 % Standard .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. 524 524 525 526 630 530 531 532 Wt. of loaf gms. 450 460 455 460 467 466 467 464 Vol. of loaf cc. 2160 2070 2000 2120 2100 1950 1860 •1885 53 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 those loaves made with 1.5 and 2.0 percent in texture and grain. A distinct wheaty smell was noticed 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 % Standard .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 VIII, 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 Hoflf 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 effect 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 differences 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 ^eries; 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 as far as sugar content is concerned. While 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 flours. 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 not produce as much dextrose as flours 1001 and -i009 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 sugars 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 sugar content (digestion 1 hour without added diastases) indicated the baking qualities of the flour quite accurately. This in turn depends to a large extent upon 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 invariable, as flour 1008, a par- ticularly strong patent flour showed only a very slight increase in sol- uble sugars when digested with approximately 5 percent malt flour, whereas flour 1007, a clear flour of notably poor baking qualities, showed a phenomenal increase under the same experimental condi- tions. The Production of Reducing Sugars in the Panary Fermentation of Bread and the Effects of Diastases Added in the Dough. In this phase of the investigation, flours 1008 a strong patent, 1003 a weak flour, and 1002 a clear flour of good baking strength, were used. The data in Tables XIV to XXI and in Figures 6, 7 and 8 show how these three typical flours behave with regard to producing reducing sugars, when fermented normally and with different amounts of added malt preparations. In order to have a check on the reducing sugars actually produced during the fermentation period, a check series was run at the same time, identical in all respects but having no yeast. In every instance where a diastatic enzyme was added, there was an increase in reducing sugars, over that of the normal dough, throughout the fermentation period. Flour 1008 had by far the greater amounts of reducing sugars, when the same additions of malt flour or malt extract were made, than did 56 the other two flours. Flour 1002 was next and 1003 was at the bot- tom of the list. The curves in Figure 6, representing the production of sugars in Flour 1008, are all of the same general type, that is, the yeast dough curves are alike and the "no yeast" curves are alike. In the yeast doughs the peak of sugar formation or the point where the diastases were producing as much available sugar as the yeast was us- ing up, was at the' first punch after two and one-half hours of fermen- tation. From this time on the yeast seems to be stimulated and uses up the sugar faster than it is produced, steadily diminishing the sur- plus that the diastases have piled up in the first half of the fermenta- tion period. In the doughs which have no yeast, conversion seems to be slightly faster in the later stages of fermentation than in the begin- ning. If this is the case in the yeast dough, the yeast undoubtedly increases in activity as fermentation proceeds. When 3 percent malt extract is used, the sugar content is decidedly higher than in any of the other doughs. This is of decided advantage as the yeast has a large surplus of sugars to draw from. The curves for flour 1002 (Figure 8), are very similar to those for flour 1008 (Figure 6), with the exception that sugar production ap- peared to have reached a maximum at the end of 1 hour of fermenta- tion in the dough to which no diastatic enzymes were added and the one to which 3 percent malt extract was added. The doughs, to which 1.5 and 4.0 percent malt flour were added, reached their maximum of sugar production at the first punch after a fermentation period of 2 hours and fifteen minutes. The amounts of sugars produced by the diastatic enzymes are remarkably constant during the time of fermen- tation, with the greatest conversion produced by the addition of 4 per- cent malt flour. As in the preceding series, the dough to which the malt extract was added showed a larger amount of reducing sugars throughout the entire fermentation period. A difference in the shape of the curves is at once noticed (Figure 7) where the sugars produced, in the fermentation of flour 1003, are fol- lowed. Instead of an initial increase in reducing sugars, as was the case with the preceding strong flours, the yeast utiltizes all available sugars immediately, with a continued decrease in sugar content as fer- mentation proceeded. A slight increase in reducing sugars was se- cured by the use of 4 percent malt flour, which reached the highest point after 2 hours fermentation. The addition of 3 percent malt extract seemed to be able to convert enough starch to hold the avail- able reducing sugars constant for one hour, but after this time the yeast increased in activity and the available sugars dropped oflf rap- idly. 57 From an inspection of Tables XIV-XVI and XXI-XXIV, and Fig- ures 6 and 8, it appeared that an addition of diastatic enzymes to a dough resulted in a surplus of reducing sugars during the earlier stages of fermentation. This surplus was used up in the later stages of fermentation along with the sugars simultaneously produced by the diastatic enzymes. It also appeared that yeast activity was increased to a considerable extent when the carbon dioxide was punched out of the dough ; at least it seems to have been coincident with the punch- ing of the dough in the case of the stronger flours. It was also evi- dent from the amounts of reducing sugars available, that the flours which showed good baking qualities had a greater diastatic activity than did the flour of poor baking strength. The malt preparations when added to weak flours produced less sugars in proportion than when added to strong flours. Whether or not the starch of the former was harder to hydrolyze than that of the latter is problematical but the data presented in section 2, and also in this section, might be taken as indicating such a possibility. Effects of the Proteolytic Enzymes, Contained in Malt Preparations, Upon the Viscosity of Strong and Weak Flours Following the Addition of Various Amounts of N/1 Lactic Acid. A very decided difference in the viscosity of a flour-water suspension was noted when a flour was digested, with and without malt flour, for different periods of time, as shown in Table XXV, and illustrated graphically in Figure 9. The 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 time, 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 (plus lactic acid) decreased quite decidedly as shown in Tables XXVI and XXVII, and graphically in Figures 10 and lOA. The strongest flours grouped themselves, and their suspen- sions in acidified water have a much higher viscosity than those of the medium or weak flours, and when treated with 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 per cent malt flour or malt extract over the range given in Tables XXVI and XXVII, but the opposite 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 extract, while the decrease found for flour 1003 under the same conditions is 22° and 24° M. respectively. Clear flour 1007 is interme- diate in this 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 XXVIII 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 would 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 justified, as demonstrated in Table XXIX, where the flour digested alone gaves a reading of 145°M., and that digested with 4 percent 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 XXVII 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 above 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 consequently 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 Hulton 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 effect upon a strong flour. The data in Table XXX supports their theory and shows that flours 1008 and 1009, which showed very good baking qualities, did not increase to any extent in gas producing capacity when malt extract was added, while flour 1002, a strong clear flour increased only 9 cc. under the same conditions, and 1007, a clear flour of poor baking quality increased 37 cc. under the same treatment. The test seems to be conclusive by the increase shown by flour 1003, a notably weak flour, which 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 changes in hydrogen 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 place as fermen- tation proceeds. With two exceptions the doughs when ready for the oven had a hydrogen ion concentration of approximately pH 5. The two flours which had a higher pH were clear flours of very poor baking strength. The Effects of Malt Flour and Malt Extract Upon the Baking Value of Flour. In flour 1001, a strong patent flour, the volume was considerably increased by the use of 2.5 percent malt flour. This advantage was materially offset by the decrease in color. With the use of the malt ex- tract, the volume increased with additions up to 3 percent 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 Table XXXIII shows that the baking qualities of the flour were improved when 3 per- cent malt extract was used. Flour 1002, a fairly 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 offset 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 flour 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 with the effects of diastatic ferments upon the strength of wheat flours. Tables and graphs have been presented, showing: 1. Optimum temiperature for the diastatic activity of the malt flour used was at temperature of 65 °C. 2. Optimum hydrogen ion concentration for the diastatic enzymes in malt flour was at a pH of 4.26. 3. Constant diastatic activity was shown by the malt flour over a period of eight hours when digested at 27° C. 4. Concentrations of added diastase exert their greatest effect in the first 10 percent of added malt flour, giving an increase in dextrose from 1.63 to 3.67 percent. 5. Diastatic ferments when added to wheat flours increase the reducing sugars, when digested at 27° C for 1 hour. The strong flours showed a higher sugar content and greater diastatic activity than did the weaker flours. The starch of the strong flours appeared to be more easily hydrolyzed by diastatic ferments than that of the weaker flours. 6. Addition of diastatic ferments to a dough convert the starch to reducing sugars and in the earlier stages of fermentation, produce a surplus of fermentable sugars in the doughs made from strong flours. This surplus soon disappears as the activity of the yeast increases, and at the end of the fermentation period the dough is nearly depleted of available sugars. 7. Suspensions of strong flours in water had a higher viscosity (on the addition of lactic acid) than similar suspensions of weak flours when incubated alone or with added diastatic ferments in the form of malt flour and malt extract. The course of proteolytic activity could be accurately followed by the change in viscosity when wheat flour was digested with added malt flour. The presence of naturally occurring salts of the w^heat flours did not vitiate the viscosity readings. 8. Gas producing capacity of weak flours was greatly increased when fermented with added malt extract. This was not the case when strong flours were fermented with added malt extract. 9. Hydrogen ion concentration of the dough steadily increased as fermentation proceeded. With two exceptions, the doughs were at approximately a pH of 5.0 when ready for the oven. 10. Addition of malt flour and malt extract to doughs increased the volume of the resulting bread. In all cases the use of malt extract gave a superior loaf of bread in volume, grain and texture, thus increasing the baking strength of the flours. 62 V. LITERATURE CITED Bailey, C H. 1920. A simple hydrogen electrode. Jour. Amer. Chem. Soc, 42, 45-48. Bailey, C. H., and Collatz, F. A. 1921. Studies of wheat flour grades 1. .Electrical conductivity of water extracts. Jour. Indus. Engin. Chem., 13, 319-321. Bailey, C. H., and Peterson, A. C. Studies of wheat flour grades II. Buffer action of water Extracts. Jour. Indus. Engin. Chem., 13, 916-918. Bailey, C. H., and Weigley, Mildred. 1922. 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