RELATEQBQS. H‘FQ W! ‘i \HABlT A5533 11.35243 QUALITY EN: WENTERX Y$PREN" 35.3332; 391‘ SEE-(23:57:35 Thais {-0.3 £5226 93:?33 éi‘ 23%;. ‘1‘; M iCHIGAN STAY-g. l; é3‘1’3fl3§.u\: 1‘ o a: an ‘3‘: 1 mm vi $51132, an W63 THESIS This is to certify that the thesis entitled RELATIONSHIP OF WINTER HABIT AND MALTING QUALITY IN WINTER X SPRING BARLEY CROSSES presented by David H. Smith, Jr. has been accepted towards fulfillment of the requirements for PheDe degree in Farm CI‘OES 25%» Major profe‘sor Date M6 0-169 LIBRARY Michigan State University ABSTRACT RELATIONSHIP OF WINTER HABIT AND MALIING QUALITY IN.UINTBR X SPRING BARLEY‘CROSSES by David H. Smith, Jr. Twelve crosses of winter x spring barley were made. F3 rows of the progenies of these crosses were classified as spring, winter and intermediate based on heading date. Bulks of each growth habit class were made and malted in micro-malting equipment designed and .built during the course of this research. Quality evaluation of the bulk malts show that malt quality is independent of growth habit. MUcro-malts of the progeny of a cross between Kindred and “Hudson.were subjected to cold water extraction. Lines of high, low and intermediate cold water extract values were tested for cytolytic activity on gums isolated from the parental lines. Cytolytic activities of all lines was lower on Hudson gum than on Kindred gum indicating a difference in gum structure. The correlation of cold water extract ’with cytolytic activity was not statistically significant. Fractional precipitation with (NRA)2304 of water extracts of grist, alcohol refluxed, of the two parents were made which gave further evidence of a structural difference between the two gums. RELATIONSHIP OF “INTER HABIT AND MALTING QUALITY IN WINTER X SPRING BARLEY CROSSES “w 0 J ‘a\ ‘i David H3 Smith, Jr. A THESIS submitted to ‘Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Farm Crops 1963 ACKNOWLEDGEMENTS The author wishes to thank Drs. J. E. Grafius, C. R. Olien, and C. M. Harrison for their help in conducting this research and in writing the manuscript. The financial support of the Melting Barley Improvement Association, Milwaukee, Wisconsin during the course of this investigation is gratefully acknowledged. ”Dr. A. D. Dickson and Noel Standridge of the USDA Barley and Malt Laboratory deserve special thanks for analyzing the malts on which portions of this work are based. The moral support and encouragement of the author's wife and family during the course of this investigation is deeply appreciated. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . v INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Growth Habit Distribution.Within Crosses . . . . . . . . . . . 15 Progeny versus Mid- -parent Comparison . u . . . . . . . . . 15 Cold water Extract as a Measure of Mbdification . . . . . . . . 20 Cytase Activity . . . . . . . . . . . . . . . . . . . . . . . . 20 Barley Gums . . . . . . . . . . . . . . . . . . . . . . . . . . 20 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . 32 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 33 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 iii Table LIST OF TABLES Composition of the principal cereal gum fractions Parentage of winter x spring barley crosses made in 1959 and 1960 at East Lansing Midparental melting trait values for 12 spring x winter barley crosses compared to the spring variety Traill . Number of rows of each category of growth habit of 12 winter x spring barley crosses grown at East Lansing in 1962 . . . . . . . . . . . . . . . . . Malting traits of progeny mean. mid-parent, parents, (P1 to P2) progeny mean percentage, and mid-parent percentage for 11 spring x winter crosses Average correlation of midparent with progeny mean for 6 malting traits . Cytase activity of green malt extracts on Hudson and Kindred gum compared to specific gravity of cold water extracts . . . . . . . . . . . . . iv Page 11 15 16 19 22 Figure LIST OF FIGURES Effect of substrate concentration on the velocity of enzyme reaction Frequency distribution of cold water extract specific gravities for spring, winter, and intermediate growth habits for the progeny of the cross of Hudson x Kindred, number 60 302 . Reciprocal specific viscosity plotted against reaction time of Kindred and Hudson green malt extracts on Kindred and Hudson gums Reciprocal specific viscosity plotted against reaction time of Kindred green malt extract on Kindred gum, Hudson gum and a 50:50 mixture of the two gums . Page 21 24 25 INTRODUCTION The possibility of a winter barley variety which would be accept- able to the Melting and Brewing Industry is of interest both from the academic and the economic viewpoint. Since winter barley, in contrast to spring barley, is adapted to the climatic areas surrounding popula- tion centers such as Detroit, Chicago and New York, a winter type melting barley might prove advantageous to both the farmer and the industry. The reason why such a variety has never been developed may be traced to custom, lack of interest, and to a conservative attitude which maintained that since it had not been done it could not be done. Whether or not it can be done is still a matter of conjecture, but this thesis allows at least one step towards such a goal. It was the intent of this research to investigate the degree of independence of factors associated with melting quality and growth habit. In brief, was there any real bar to the production of a winter barley with a spring type quality pattern? Comparison of the melting characteristics of the three growth habit categories, spring, winter and intermediate, within crosses of winter x spring varieties when vernelized and grown under the same conditions should furnish some answers to the problem. F Melting in very simple terms involves germination of barley under closely controlled conditions which is terminated at the time the acrospire reaches the length of the kernel. During germination a number 1 2 of changes occur within the seed most of which are directed toward the breakdown of the complex storage forms of energy-containing substances into simpler forms. This breakdown is largely enzymatic and involves a host of enzymes of varying specificities. Modification is a term used to describe the overall breakdown of the contents of the endosperm into simpler and more soluble forms. Poorly modified kernels will be less friable and will have a lower quantity of extractable material ~than kernels that are well modified. In general winter barley varieties tend to produce poorly modified malts under commercial melting condi- tions. Dicktoo is one of the poorer in this respect while Hudson is one of the better varieties. It would appear that differences in modifiability are related to the relative amounts of gum and cytolytic enzymes present in the seed. Enzymes known as cytases are those which in effect open the door to other digestive enzymes. The cytases are those which have been recognized and defined as involved with cell wall degradation. Rupture of the‘cell wall must occur in order for further degradation of the cellular contents to be brought about by other digestive enzymes. In order to study the problems inherent in the development of a winter type barley for melting purposes, the differences in the chem» istry between the spring and winter types must be taken into account. Certain physiological differences between the two types should also be considered. LITERATURE Melting quality is a complex of a number of chemical and mor- phological traits. These traits are varietal and therefore heritable, but the inheritance is mainly quantitative. Grafius (12), Luedders (17), and Whitehouse e£_gl. (30) working with barley, oats and wheat, respectively, have shown that the components of the complex trait yield can be predicted in progenies from mid-parental values. Dickson and Grafius (8) have found a high correlation between progeny mean melting characteristics andmidparent values. Any substance which preVents or slows down cytolytic processes should have a profound effect on the overall modification of the endosperm contents from barley sub- stance to melt substance. Materials of this nature either pre- existing or derived from cell wall hemicelluloses have been recognized as occurring in especially large amount in barleys that are difficult to melt while easily modified barleys contain much smaller amounts (19). These non-starchy water soluble polysaccharides are known as gums or mucilages, and the non-starchy water insoluble polysaccharides which dissolve in 4% sodium hydroxide are hemicellulose (27). Hemi- celluloses play an unusual role in cereal endosperms because little or no cellulose and no pectins are present (7) (9, 10, 11). It has been suggested that they function along with protein as the cementing agent between endosperm cells (18) (31). . O'Sullivan (22) isolated two gums from barley flour extracted with water which he called alpha and beta amylan. Alpha amylan is now 3 4 called beta-D-glucan. Methylation studies have shown that beta-D- glucan is a long unbranched chain of D glucopyranose units (27) (2) made up of l-i3 and 1-94 linkages of approximately equal proportions. Whether the beta-D-glucan molecule is cyclic i.e. in the form of a loop (2) or strictly linear (27) has not been resolved. . Aqueous solutions of beta-D-glucan have very high specific viscosities (26) (20), but according to Preece and McKenzie (26) the beta-D-glucan disappears rapidly during melting and is almost absent from finished malt. Thus wort viscosities are not due to the presence of beta-D-glucan. Harris and McWilliam (15) have shown that the amount of water soluble glucose polymers that are insoluble in 80% ethyl alcohol rises during steeping and decreases as germination begins. Since Hall g£_gl. (14) have found little change in starch content during steeping, this rise and fall in amount of such polymers is related to their liberation from insoluble non-starchy material and subsequent hydrolysis to less complex forms. Preece and Aitkin (23) have found a similar increase in beta-D-glucan content measured at intervals in barley grists treated with water followed by a decrease in the amount of gum. Varieties which are poor in melting quality were found to release gum during the entire period of extraction while gum quantity from good melting varieties increased for 2 hours and then decreased (23). Two cytases have been found in barley and green malt which attack beta-D-glucan, endo-beta-glucosanase (24) or endo-beta- glucosidase (4) and exo-beta glucosanase (24) and exo-beta-glucosidase (4). Endo-beta-glucosanase attacks the polymer at points remote from the ends of the chain thus producing a marked decrease in viscosity 5 without increasing reducing power significantly (4, 24). Exo-beta- glucosanase attacks the ends of the polymer splitting off units of cellobiose (4, 24). Bass and Meredith (5) and Bass, Bendelow and Meredith (3) have shown a relationship between green malt cytase activity on beta-D- glucan and melting quality. Cytase activity of most of the lines tested correlated well with malt quality although 3 lines of low cytase activity were of satisfactory quality and 2 lines with high enzyme activities were unsatisfactory in one or more malt properties. It was also reported (3) that cold water extract is the best single criterion of overall malt quality currently available because of the high correlations found between cold water extract and high levels of melting quality traits. Other seed gums have been isolated from water extracts of ground barley. Alpha-D-glucan and an araboxylan, a branched pentosan have been found (27). Smith and Montgomery (27) state that the viscosity of gum preparations of mixtures of pentosan and hexosan is due to beta-D-glucan. Table l is taken from Preece and Hobkirk (25). It shows the composition of gums from different cereals as isolated using different concentrations of (NH4)2804. The velocity of enzyme catalyzed reactions is a function of substrate concentration only when the concentration of substrate is less than that which allows maximum velocity to be obtained. When sufficient substrate is present to allow maximum velocity the reaction curve becomes that of a zero order reaction as shown in Figure 1. 6 TABLE 1.--Composition of the principal cereal gum fractions (25) Precipitation Level (NH4)ZSO4 Z 20 3O 40' 50 60 saturation Mother liquor Sugar Unit Glucose Glucose Xylose Arabinose Glucose Xylose Arabinose Glucose Xylose Arabinose Glucose Xylose Arabinose Glucose Xylose Arabinose Mannose Glucose Xylose Arabinose Galactose Rye 17 45 38 O 61 39 O 55 45- 2 68 3O 25 9 42 22 Wheat 11 64 25 6 61 33 23 47 3O 56 24 20 18 9 47 ' 26 Barley 100 96 2 2 12 56 32 7 65 28 17 61 1.3. 15 10 62 13 Oats 100 61 20 19 15 14 4O 31 Maize aContains approximately 1% Galactose. bContains approximately 2% Galactose. Velocity of Reaction \ I Substrate Concentration Fig. 1.--Effect of substrate concentration on the velocity of an enzyme reaction (16). MATERIALS AND METHODS Twelve crosses were made using spring and winter barleys as parents (Table 2). TABLE 2.--Parentage of winter x spring barley crosses made in 1959 and 1960 at East Lansing Cross Number Winter Parent Spring Parent 59 301 Dicktoo CI 5529 (Moore x Anoidium) x Montcalm 59 302 Dicktoo CI 5529 (Moore x Anoidium) x Montcalm 59 303 Dicktoo CI 5529 Moore x2 Montcalm 59 304 Dicktoo CI 5529 Liberty x Kindred 59 305 Dicktoo CI 5529 Moore x2 Montcalm 59 306 Dicktoo C1 5529 Moore x2 Montcalm 59 307 Dicktoo CI 5529 Moore x (Kindred x Bay) 59 308 Dicktoo CI 5529 Moore x (Kindred x Bay) 60 301 Dicktoo CI 5529 Montcalm CI 7149 60 302 Hudson C1 8067 Kindred CI 6969 60 303 Dicktoo CI 5529 Kindred CI 6969 60 304 Hudson CI 8067 Traill CI 9538 Spring parents were selected for their superior melting qualities as shown by quality evaluations made at the USDA Barley and Melt Laboratory in Madison, Wisconsin. .Winter parents were picked for their agronomic characteristics, particularly winter hardiness. Progenies of these crosses were grown in the greenhouse for 2 generations and then seed from each F2 plant was planted in a single hill in the field. -Fie1d plantings were made in the early spring when the soil was thawed only to a depth of about 1 inch. This early planting gave sufficient cold temperature during the early development of the plants to vernalize the winter types in the population. Each hill was harvested and threshed individually. Such a procedure made 8 9 it possible to obtain seed for melting from all growth types, winter, intermediate and spring, grown in the same environment. This nursery will henceforth be referred to as the vernalized nursery. Since all seed was vernalized in the experimental planting, it was necessary to plant a nursery at a time when vernalization could not occur of the same material to determine the growth habit of the plants in each row. In the spring of 1962 seed samples were taken from each line and approximately 30 seeds of each sample were planted on May 16, 1962 in 3 foot rows in a nursery. Heading notes were taken and the rows in each cross were classified into growth habit categories: spring, intermediate and winter. Spring habit included all uniform rows heading at the same time as or before the spring parent of the cross. Intermediate included all uniform plants in rows heading later than the spring parent and winter all uniform plants in rows failing to head. Rows of segregating plants were not included in the three groups. Plants in this nursery were not harvested, but used only for indication of the types of growth. Each growth type for each cross from the nursery where plants were vernalized was bulked by taking an equal amount of seed from each line within a growth habit type for each cross. In two crosses there was insufficient seed to group into the three categories and these crosses were analyzed as bulks ignoring growth type. All of the seed in each cross was pro- duced in the same year in the same nursery i.e. the spring, inter- mediate and winter types were all grown under conditions as close to identical as is possible in the field. Three bulks, winter, intermediate, spring, and the two parental lines for each cross were malted using a modification of the technique 10 of Whitemore and Sparrow (30) described in the appendix. All but two crosses contained over 100 lines making a total of about 1300 lines for the group. Detailed analysis of this many lines appeared to be an impossibly long task. It was decided that one cross should be selected for such analysis. The selection was made by cal- culating the mid-parental values for a set of melting characteristics for each cross and choosing the cross having a midparent most closely approaching a typical Spring quality pattern. The midparents are shown in Table 3. Cross number 60 302, Hudson x Kindred, appeared to be the best of the group by this method of prediction. The individual lines within 60 302, Hudson, Kindred, and a standardized check were melted in large test tubes according to the technique of Whitemore and Sparrow (30) outlined in the appendix. Prior to kilning, five grams of green malt were taken from each tube in the group and stored at 0° F. in a deep freeze. Individual kilned malts were ground in a Wiley mill using a #40 sieve. Cold water extracts were made on five grams of seed from each line. The ground malt was mashed with 39 ml of deionized water and 1 ml of 0.05 molar mercuric chloride at 180 C. for 120 minutes. The mixture was then filtered and the Specific gravity of the filtrate determined using a 10 m1 specific gravity bottle. The procedure followed was that of Bass, Bendelow and Meredith (3) except that they used 25 grams of malt and proportional amounts of water and mercuric chloride. Using these specific gravities as an index of modification (3, 6) seed from lines of high, low and intermediate extract were chosen for analysis of their cytolytic activity contained in their green malts. .h~m>wuooamou ommfihsm.xuou ommHham A owumu sandman 10.0mwaham a .uosoo uwumummwv .aowouuw: name cu :mwouuwa uuoB mo oaumu cowouuwa Dana .awwouuwn Duos uomnuxm N .daafid N unwwma noon ”muwmuu wcfiuHmzm o.- c.o~ ¢.a o.- ¢.o~ «.0H n.NH 0.0H N.NH n.0H m.NH N.a N.HH Jhwn N.m¢ o.m¢ m.H¢ H.mm o.~q ¢.wN w.¢m ~.m¢ N.o¢ w.m¢ H.n¢ e.mm o.m¢ [Kw mun wnq awn Hoe seq seq com mnq Ham Hus mmn saw «on n me omH omH o¢~ nqa mmH me umfi OOH mmH mNH oma mod m: 1. m.wm m.om o.Hm m.¢m ¢.Hm ¢.Hm a.mm «.mm o.~m m.mm m.mm ¢.wm o.mm Z\3 1 om.H mw.H no.N oa.H mw.H mm.H ow.H om.~ mw.H mo.H mm.H Nw.H oo.~ 22 who. who. mmo. ace. Mme. mam. woo. moo. 0H0 mac cmo. man. mmo. 23 w.NN N.mm m.NN o.mn H.mm N.mn m.mm o.mm a.m~ o.mm ¢.mm «.05 o.mn uxm «.mm ¢.oN m.mm ¢.Hm ~.m~ o.mm H.mN m.~¢ n.¢m o.mm N.wm «.mq o.o¢ ¢o\o N m.~m m.o~ m.m~ H.NN N.©N w.w~ o.o~ H.¢N w.w~ ~.mm o.w~ o.mN w.NN mm ”Hanna won an Non an 00m an mom on «em mm mom mm won an Hon am «on ow mom 00 Non oc Hon co rib“ 7" I il" Ill ill HHHmuB humwnm> weaken use on nonmanu mammouu Amfiumn Hausa? x magnum NH you mmafim> uwmuu wcwuHmE Hmucoummvwxuu.m MAQ owmwomam Hmooumfiummu-.m .wwm mmuscwz a“ oEwH :0wuomom onH ONH oo so on o b P p b b ONH mmmoo. u odon .Eow comes: so uomuuxo onE noonw nomvsm u xx umHoo. u oaofim .Enw common co uomuuxo bass :mouw convsm 1 mm ommoo. u macaw .esw cayenne so uomnoxo uHmE coouw ponbcwx . xx xx mHmoo. u oaofim .Eow Gowns: so uomuuxm uHmE nomuw boupnwm .vomm 3 O H <7 I O \9 U3 KatsoostA orgioads Iaooadroau .owo .maaw 03u mau mo manuxfla omuon m mam 53w GOmbam .Enw nouncwx co uomuuxo uHmE :oouw nonmawx mo mafia nowuomou unawmwm vouuofid huwmoomw> owmwooom Hmoondwuomuu.¢ .wwm mound“: cw oEHH :oHuomom owe own as . ow om o a . ,OBH cameo. u macaw .Esw umuwCMM co uomuuxm Umuvcfix u o mamoo. u macaw .Esw comma: co uomuuxo wouncwx n m ommoo. u maofim .Esw Umuwcfix cam convex mo muouxwa omnon no oomuuxm noanHx scam 25 fogs Town AntsoostA otgroads Iaooxdroau rcmc 26 of the dry residue of hydrolysis products from the fractional precipi- tation obtained using (NH4)2SO4. More pentosan was found in Kindred pellicle than in Hudson. The lower initial viscosity of the Hudson gum, the lack of evidence for the presence of some form of inhibition, the different amount of (NH4)2SO4 to precipitate the pellicle, and the lower cytase activity of all green malt extracts on Hudson gum all indicate that the glucan from Hudson is different in structure from that of Kindred and also that this difference is one of more irregularity of structure in the Hudson polymer. DISCUSSION One of the most interesting results of this study was the dif- ference found between Kindred and Hudson gums. Without the supporting evidence of the analysis of the bulk malts (Appendix) which show that there was no apparent association of malt quality and growth habit, and the cold water extracts that indicate the independence of winter habit and modification, it might be suggested that there is something i.e. the gum structure, inherent in winter barleys that would prevent their being accepted for malting. This was not true for Cross Number 60 302, Hudson x Kindred, which was analyzed in this research. The difference between the gums of Kindred and Hudson barley contributes to the complexity of developing high quality malting winter types from such crosses but does not mitigate against the eventual production of such lines. Since the winter and Spring beta-D-glucans were precipitated by different levels of (NH4)2804, it would be fairly simple to test for the type of gum present in larger samples of populations from winter x spring crosses than was done in this thesis by extracting alcohol re- fluxed grist with water and adding 20% (NH4)2804. If no pellicle formed at 20% then enough additional salt could be added to make the concentration 30% (NH4)2804. This technique would lend itself well to analyzing the genetic control of gum production in populations from crosses involving winter parents. Gums from both winter and Spring types should be purified through 27 28 fractional precipitation and crystallization to yield pure beta-D- glucans. These could be used for enzymatic studies of cytase activity as well as for structural analysis of the glucans by means of methyla— tion and periodate degradation. Comparisons should also be made of the physical properties of the purified glucans using osmotic pressure measurements, freezing point depression tests, ultracentrifugation, and viscosity measurements. The role of pentosans and proteins associated with gums must also be illucidated. Since the beta-D—glucans from spring and winter barleys are different there might also be differences in the pentosans and proteins in each. Since cytase activity is of a zero order reaction (Figure l) and thus not a function of substrate concentration the reduction of cytase activity of Hudson gum (Figure 3) must be due to some sort of inhibitive action. Such action could be due to the nature of the gum as indicated by its lower initial viscosity, and the fact that in all cases the enzyme reaction rate is at maximum velocity. It is also possible that complexing of the Hudson gum with other substances occurred as a result of the addition of 30% (NH4)2 These substances could be other non-starchy polysaccharides such as 804 in its preparation. pentosans as shown in Table l or possibly proteins or protein deriva- tives. A final possibility exists, a specific inhibitor could be present in the Hudson gum. If the lower cytase activity of green malt extracts on Hudson gum were due to a complexing of the Hudson polymer with a pentosan in some way that the pentosan covered or protected sites on the glucan molecule from enzymic rupture, a sharp decrease in reaction rate on 29 the mixture of the gums would be expected. Such an effect would occur at low concentration of pentosan and increased amounts of the pro- tective substance would not give increased lowering of enzymatic activity because there are limited numbers of cites within the polymer which can be attacked. If the lowering of cytase activity on Hudson gum were due to an additive type of interference the reaction rate of the green malt ex- tract on the mixture of the two gums should have fallen at the mean of the cytase activity on the two pure gum substrates. The reaction rates of the Kindred green malt cytase were linear for all three reaction mixtures (Figure 4). If the lower activity on Hudson gum was due to the presence of a substance which had only a small effect at low concentrations and the effect increased exponen- tially with increasing concentration of the inhibitive substance the curve for cytase activity on the mixture of the two gums should have left a zero order type because as the reaction progressed the relative concentration of inhibitor would rise in the reaction mixture. The cytase activity of Kindred green malt extract was higher on the gum mixture than either of the other substrates (Figure 4) in- dicating that no highly specific inhibitor was present in the Hudson gum. The reason for the higher cytase activity on the gum mixture is not clear. The result cannot be explained on the basis of the presence of other enzymes in the green malt extract simultaneously degrading substances other than beta-D-glucan because this same effect would be present when using the individual gums alone. Perhaps it is due to some interaction of the two gums which makes the mixture more easily attacked by the enzyme. Replication of this result is necessary before 30 its real meaning can be determined. The simplest explanation of the lowered activity of green malt extract cytases on Hudson gum is that the Hudson polymer is more irregular in structure than that isolated from Kindred. The irregu- larity of structure of the gum of Hudson barley may not exist in all winter types. It would be interesting to isolate gums from other winter varieties and analyze them for such differences. The lack of association of cold water extract and cytase activity (Table 7) is not in agreement with Bass, Bendelow, and Meredith (3) who found a highly significant relationship between these two phenomena in spring barleys. Perhaps this lack of agreement is due to the fact that the lines tested are from a winter x spring cross in which recom- binations occur that are not common to progenies from spring x winter crosses. Improved lines of hardy winter barley that have malting properties approaching a spring barley quality pattern have been pro- duced at Michigan State University. These lines contain no spring barley germ plasm, but have been produced by recurrent selection and vector technique for parental choice (13). Gum analysis of these lines may provide some fascinating insights into the effects of selection and genetic recombination on the structure and types of gums present. From the standpoint of malting, there is no apparent reason for preventing the use of improved winter barleys. Winter barleys, up to 20%, are being used in Canada in blends for malting. Malting of winter barleys may require some adjustment in technique on the part of the maltster as was the case with the micro-malting of the winter bulks 31 used to standardize the micro-malting equipment and procedures utilized in this study. The micro-malts exhibited very high levels of modification thus indicating that with proper technique winter barleys can be successfully malted. Acceptance of winter barley malts by brewers will depend on the quality of beer which can be made from them. A few micro and pilot scale brewing tests of winter barleys have been made in the past and the results have been inconclusive. Work on improving winter lines for better brewing properties must be intensified to determine if such lines are possible of development. SUMMARY AND CONCLUSIONS 1. Micromalting of bulks within growth habit classes has been shown to be of use for screening early generation progenies of crosses made for malting quality. 2. Winter growth habit has been shown to be independent of any association detrimental to malting quality of eleven spring x winter barley crosses. 3. Modification of progeny lines of a spring x winter cross appears to be well independent of cytase activity on Kindred gum. 4. Polysaccharide gums isolated from Kindred and Hudson barleys have been shown to have structural differences which effect cytase activity of green malt extracts reacted on these gums. 5. The structural difference between the gums is in the direction of more irregularity of the molecule on the part of the gum isolated from the winter variety Hudson as shown by differences in viscosity, fractional precipitation, and lower cytase activity of green malt ex- tracts on Hudson gum. 32 10. ll. 12. 13. 14. LITERATURE CITED American Society of Brewing Chemists. 1949. Methods of Analysis (5th Ed.). Madison, Wisc. Aspinall, G. O. and R. G. Telfer. 1954. Cereal gums. Part I. The methylation of barley glucosans. J. Chem. Soc. IV:3519- 3522. Bass, E. J., Bendelow, V. M. and Meredith, W. O. S. 1957. Enzymes that degrade barley gums. V. Relation between endo-beta- polyglucosidase activity and other barley and malt properties. Cereal Chem. 34:219-221. Bass, E. J. and Meredith, W. O. S. 1955. Enzymes that degrade barley gums. III. Studies of beta-polyglucosidases of green malt. Cereal Chem. 32:374-383. 1956. Enzymes that degrade barley gums. IV. Studies of varietal differences in endo-beta-polyglucosidase activity. Cereal Chem. 33:129-133. - DeClerck, J. 1958. A textbook of brewing. Vol. I. (Transl. by K. Barton-Wright). Chapman and Hall Ltd. London, England. Devereux, A. 1951. Carbohydrates of barley and malt. Rev. Ferment. 6(6):203-207. (Chem. Abst. 45:5219.) Dickson, A. D. and Grafius, J. E. Unpublished data. Fink, H. and Just, F. 1935. Hop pectin and its significance in brewing. Wschr. Brau. 52:401-405. (Chem. Abst. 29:2918). 1936. Hop pectin. III. Significance in brewing. Wschr. Brau. 53:225-226. (Chem. Abst. 30:5718). 1937. Hop pectin. IV. Effect of different pectin prep- aration on finished beer. Wschr. Brau. 54:281-286. (Chem. Abst. 30:6503). Grafius, J. E. 1959. Heterosis in barley. Agron. J. 51:551-554. Grafius, J. E. and Adams, M. W. 1960. Eugenics in crops. Agron. J. 52:519-523. Hall, R. D., Harris, G. and McWilliam, I. C. 1956. Carbohydrates in malting and brewing. V. Further studies on the carbo- hydrates of barley malt and wort. J. Inst. Brew. 62:232-238. 33 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 34 Harris, G. and McWilliam, I. C. 1954. Chemical aSpects of malting. III. Changes in the carbohydrates of barley during malting. J. Inst. Brew. 60:149-152. Lardy, H. A. 1949. Respiratory enzymes (Rev. Ed.). Burgess, Minneapolis, Minnesota. Luedders, V. D. 1960. An analysis of the components of yield in 18 oat crosses. Michigan State University. M.S. Thesis (Unpub.). McLeod, A. M. and McCorquodale, H. 1958. Water soluble carbohy- drates of seeds and the graminae. New Phytologist. 57:168-182. (Abst. J. Inst. Brew. 64:162). Massart, L. and C. Van Sumere. 1955. Cellulases, Hemicellulases and their substrates. Breuwiss. 289. (Abst., Wallerstein Lab. Comm. 4:163). Meredith, W. O. 8., Watts, T. A. and Anderson, J. A. 1953. Com- parison of barley gums isolated by various procedures. Can. J. Chem. 31:653-664. Meredith, W. O. S. and Anderson, J. 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The chemistry of plant gums and mucilages and some related polysaccharides. Reinhold, New York. Snedecor, George. 1946. Statistical Methods. The Iowa State College Press. Ames, Iowa. 35 29. Whitehouse, R. N. H., Thompson, J. B. and DoValle Ribeiro, M. A. M. 1958. Studies on the breeding of self polinating cereals. 2. The use of a diallel cross analysis in yield prediction. Euphytica. 7:147-169. 30. Whitmore, E. T. and Sparrow, D. H. B. 1957. Laboratory micro- malting technique. J. Inst. Brew. 63:397-398. 31. Wolf, M. J., MacMasters, M. M. and Seckinger, H. L. 1958. Com- position of the cementing layer and adjacent tissues as related to germ-endosperm separation in corn. Cereal Chem. 35:127-131. APPENDIX The second major objective of this study was to develop micro— malting equipment capable of handling large numbers of early generation samples. Seed supplies of such material are generally very small and this was also taken into consideration. The equipment designed in this study was built to accommodate malting trays of stainless steel which are capable of handling 20 grams of each of 100 lines of barley at a time. Trays are divided into 100 2" x 2" x 3" compartments and have stainless steel screen covering the bottom of the entire tray. This design allows malt to be produced under conditions similar to commercial pneumatic malting. The 3 pieces of equipment are: steep tank, germinator, and kiln. The steep tank is of heavy #16 galvanized metal and is large enough to hold one tray. The tray is supported on hangers so that it is about 4 inches off the bottom of the tank. Two methods of steeping were used. With spring barleys 20 grams of grain were placed in each compartment of the tray which was covered with a piece of screen to prevent seeds from floating out and becoming mixed. The covered tray was then lowered into the steep tank. With winter barleys, malted in test tubes and cottage cheese dishes, 20 grams of grain were placed in small cheesecloth bags, one bag per cubicle. The bags facilitated the transfer of the steeped grain from the tray to the test tubes and cottage cheese dishes. The steep tank was maintained at 160 C. in the germinator. In the early phases of the work the steep tank temperature was maintained by running 36 37 tap water through the grain. This proved to be unsatisfactory as the temperature of the tap water was not sufficiently uniform. The germinator is a large plywood box with a hinged cover. It is equipped with a double squirrel cage fan which blows air taken into the box from the room over a refrigerator coil. After leaving the refrigeration coil the air flows over a furnace humidifier which saturates the air with water. The saturated air is evenly distributed by means of internal baffles in the box and passes up through the bottom of the tray and out a 6" hole in the back of the cover. The refrigera- tion coil is connected to a compressor through an evaporator pressure regulating valve. The compressor runs continuously and the coil temperature is regulated by setting the evaporator pressure valve. Two heaters are used; a 600 watt cone heater located at the air intake of the fan controlled by a Fenwal thermostat, and an electric stove element located under the water reservoir of the humidifier controlled by a variable transformer. Heating the water in the reservoir produces finer droplet size from the mechanical disperser as well as warming the air. The various controls make this unit highly flexible. The kiln is also a plywood box equipped with a squirrel cage fan that blows air over a group of thermostatically controlled cone heaters and up through the grain which is in a tray sitting on the top of the kiln. Originally these 3 pieces of equipment were standardized using a lot of plump, bright Traill barley furnished by Dr. A. D. Dickson of the USDA Barley and Malt Laboratory. This sample of Traill had been malted and evaluated at the Barley and Malt Laboratory and was of ex- cellent quality. The malting schedules used in this work are shown in Table I. 38 TABLE I.--Comparison of malting schedules used for spring and winter barleys malted in micro malting equipment at Michigan State University. _ __‘ Spring Barley Winter Barley Steeping temperature control running tap water germinator 15 - 20° c. 16° c. time 48 hrs. 40 hrs. final moisture 42 - 44% 42 - 44% Germination temperature 16° C. 16° C. time 2-1/2 days 8 days container compartmented trays test tubes and cottage cheese dishes Kilning time and temperature 24 hrs. at 45° C. 24 hrs. at 45° C. 8 hrs. at 55° C. 8 hrs. at 55° C. Three lots of Traill were malted according to the spring schedule in Table 1. Lot 3 was deliberately oversteeped (60 hrs.). Two samples of each of the three lots were analyzed by the Barley and Malt Labora- tory (Tables II and III). The quality of Lot 3 was lower than Lots 1 and 2 which were satisfactory. The results of malting winter barley lines and growth habit bulks of crosses (Tables IV and V) indicated that changes in technique were needed. A bulk lot containing more than 40 lines of winter barley was germinated according to the two schedules in Table I. Both samples of the bulk were steeped for 40 hours in cheesecloth bags. Part of the grain was placed in the compartments of the malting tray and germinated for 3 days, the remainder being placed in large (5" x 1") test tubes, 30 grams of steeped grain per tube. The tubes were equipped with single hole rubber st0ppers and germination was allowed to procede for 8 days. This follows the method of Whitemore and Sparrow (31). 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