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University Microfilms International 300 North Zeob Road Ann Arbor, Michigan 48106 USA St John's Road, Tyler’s Green High Wycombe, Bucks, England HP10 8HR 77-31,902 KARDOSH, Kamal B., 1947- EFFECT OF SUBSTRATES ON THE FORMATION OF FUSEL ALCOHOLS IN A SIMULATED BEER FERMENTATION. The Ohio State University, Ph.D., 1977 Food Technology

University Microfilms International, Ann Arbor, Michigan 4aioe EFFECT OF SUBSTRATES ON THE FORMATION

OF FUSEL ALCOHOLS IN A SIMULATED BEER FERMENTATION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By Kamal B. Kardosh, B.Sc., M.Sc.

The Ohio State University

1977

Reading Committee: Approved by

W. H. Harper

T. Kristoffersen

E. Mikolajcik rtment of Food and Nutrition J. L. Blaisdell DEDICATION

I wish to dedicate this dissertation to my parents

Boulous and Yavon Kardosh

for all the things they have meant to me.

ii ACKNOWLEDGMENT

Scientific journals and research papers dealing with beer technology and fermentation are markedly scarce throughout the United States' university libraries. At

Ohio State University, the largest campus in the U.S.A., the libraries have no such journals as Technical Quarterly,

Brewing Digest or The Brewing Institute Journal. In con­ trast, two large beer-producing companies with research facilities with which I was in contact, have excellent libraries equipped with many journals and books. I would have found it very difficult to have completed this research without the use of their facilities. In particular, I am grateful to Dr. Morten C. Meilgaard, Director of Research and Development, The Stroh Brewing Co., Detroit, Michigan, for all the help extended to me throughout my graduate work.

I am also grateful to Ross Laboratories, Columbus, Ohio for their help in analyzing the ‘amino acids samples.

I can not imagine, for a moment, that I could have com­ pleted this study without the help, advice, and encourage­ ment of many people.

iii ACKNOWLEDGMENT (continued)

To my faculty adviser, Dr. W. J. Harper, my sincere thanks and gratitude for his guidance and support throughout this endeavor.

To the chairman of the department, Dr. Thorvald

Kristoffersen, I am thankful for providing me with the opportunity to pursue graduate studies. Above all, Dr.

Kristoffersen's help and guidance in preparing this manu- script were invaluable.

To Dr*. Rory Delaney, the Department of Food Science and

Nutrition, my many thanks for his generous cooperation and advice in preparing the experimental design in some of the experiments.

I acknowledge the friendship and help extended to me by

Drs. Emil Mikolajcik and Paul Hansen, the Department of Food

Science and Nutrition. Also, to all the faculty members who made my stay at Ohio State a pleasant experience.

To the graduate .students at this department, thanks for all the memories, the good times, and above all your friend­ ship.

And last but not least, my love aiid thanks to my wife;

Pamela, for her moral support and for her beautiful gift, our new baby, Adrienne Lee, born a few days prior to my final oral examination.

iv VITA

May 19, 1947 Born - Nazareth, Israel

1972 .... B.Sc., University of Wisconsin, La-Crosse, Wisconsin

1973-1975 . Graduate Research Associate, The Food Science and Nutrition Department, The Ohio State Uni­ versity, Columbus, Ohio

1975 .... M.Sc., The Ohio State University, Columbus, Ohio

1975-1977 . Graduate Teaching Associate, The Food Science and Nutrition Department, The Ohio State Uni­ versity, Columbus, Ohio

1977 .... Consultant Food Technologist Booz Allen and Hamilton Florham Park, New Jersey

v TABLE OF CONTENTS

Page

DEDICATION ...... ii

ACKNOWLEDGMENTS...... ill

VITA ...... V

• LIST OF T A B L E S ...... ix

LIST OF FIGURES ....."...... xii

INTRODUCTION ...... 1

LITERATURE REVIEW ...... 3

Brewing Technology ...... ‘ . . 3 Effect of Steeping on Wort Amino Acids ...... 6 Effect of Steeping on Wort Carbohydrates ...... 8 Nitrogen Utilization ...... 8 Carbohydrate Utilization ...... 10 Flavor Compounds in B e e r ...... 16 Factors Affecting the Production of Fusel Alcohols ...... 1 7 Biosynthesis of Fusel Alcohols 22 Nutritional Properties of B e e r ...... 30

SCOPE OF INVESTIGATION ...... *...... 33

EXPERIMENTAL PROCEDURE ...... ; 34

Fermentation Medium and P r o c e s s ...... 34 Determination of Fu b b I Alcohols ...... 36 Determination of Individual Carbohydrates .... 40 Determination of Free Amino Nitrogen ...... 42 Determination of Amino Acids ...... 46 Determination of Ammonium S u l f a t e ...... 47 Specific Gravity Measurements ...... 50 Analysis of Variance ...... 5 1 Yeast Cell Count ...... 52

vi TABLE OF CONTENTS (continued) » Page

RESULTS ...... 53

Fusel Alcohols in Selected Samples of Commercial B e e r ...... 53 Effect of Source and Concentration of Nitrogen on Selected Characteristic of B e e r ...... 54

A) Free Amino Acids ...... 54 Fusel Alcohol Formation ...... 57 Carbohydrate Utilization ...... 59 Free Amino Nitrogen Utilization ...... 66 Amino Acid Utilization...... 66 pH and Specific Gravity ...... 80

B) Ammonium Sulfate ...... 80 Fusel Alcohol Production 84 ' Carbohydrate Utilization ...... 89 Ammonia Nitrogen Utilization ...... 89 pH and Specific Gravity ...... 94

Comparison of Results for the Fermentations with Amino Acids and Ammonium Sulfate as the Source of Nitrogen ...... 94 Effect of Nitrogen Source on Free Amino Nitrogen Utilization ...... 102 Role of Carbohydrates on Selected Characteristics of B e e r ...... 102 Addition of Single Carbohydrate to Wort . . . . .102 Fusel Alcohol Production ...... 104 Other Characteristics ...... 106 Effect of the Ratio of Fermentable Carbohydrates in W o r t ...... 106 Fusel Alcohol Production...... 108 Carbohydrate Utilization ...... 115 Ammonia Nitrogen Utilization ...... 119 pH and Specific Gravity ...... 119

vii TABLE OP CONTENTS (continued)

Page

Periodic Addition of Carbohydrate to the W o r t ...... 122

Fusel Alcohol Production ...... 125 Carbohydrate Utilization...... 125 Ammonia Nitrogen Utilization...... 133 pH and pH Gravity ...... 135 Yeast Cell Growth ...... 135

DISCUSSION ...... 141

SUMMARY AND CONCLUSIONS...... 158

BIBLIOGRAPHY ...... 162

viii LIST OF TABLES

Table Page

1. Degree o£ Steeping at which Highest Levels of Amino Acids are Obtained in Eli M a l t ...... 7

2. Degree of Steeping at which Highest Levels of Sugars are Obtained in Barley M a l t ...... 9

3. Order of Absorption of Amino Acids From Wort by Brewers' Y e a s t ...... 11’

4. Fusel Alcohol Content of Some Commercial Beer ...... 19

' 5. A Defined Medium Simulating Wort 'Composition...... 35

6. Fusel Alcohol Content of Some Commercial Beer ...... 55

7. Amino Acids Composition of the Basic and the Modified Fermentation Media ...... 56

8. Effect of Amino Acid Concentration on the Production of Fusel Alcohols '...... 58

9. Changes of Amino Acids During Fermentation of Wort (FAN = 150 m g / 1 ) ...... 70

10. Changes of Amino Acids During Fermentation of Wort (FAN = 300 m g / 1 ) ...... 71

11. Changes of Amino Acids During Fermentation of Wort (FAN = 450 mg/1) . . 72

12. Molar Ratios of Fusel Alcohols Produced to Amino Acid Utilized ...... 79

ix LIST OP TABLES (continued)

Table Page

13. Relative Contribution of Carbohydrates to Fusel Alcohols in a Fermentation with Amino Acids as Source of Nitrogen ...... 81

14. Effect of Ammonium Sulfate Concentration on the Production of Fusel Alcohols ...... 85

15. Analysis of Variance of Nitrogen Source on the Production of Fusel Alcohols. FAN = 150 mg/1 Versus 150 mg/1 of Ammonia Nitrogen ...... 98

16. Analysis of Variance of Nitrogen Source on the Production of Fusel Alcohols. FAN = 300 mg/1 Versus 300 mg/1 of Ammonia Nitrogen ...... 99

17. Analysis of Variance of Nitrogen Source on the Production of Fusel Alcohols. FAN = 450 mg/1 Versus 450 mg/1 of Ammonia Nitrogen ...... 100

18. Maltose Concentration (gm/1) After 100 Hours of Fermentation as a Function of Nitrogen Source and Concentration...... 101

19. Free Amino Nitrogen as a Function of Nitrogen Source and Concentrations ...... 103

20. Effect of Single Carbohydrate Source on the Production-of Fusel Alcohols in Wort Fermentation ...... 105

21. Effect of Single Carbohydrate Source on Carbohydrate and Nitrogen Utilization, and Final pH and Specific Gravity of the Fermented W o r t ...... 107 # 22. Composition of Fermentable Sugars in the Experimental Wort Media with Varying Carbohydrate R a t i o ...... 109

x LIST OP TABLES (continued)

Table Page

23. Effect of Carbohydrate Ratio on Fusel Alcohol Production After 100 Hours of Fermentation ...... 110

24. Analysis of Variance of Carbohydrates' Composition on the Production of Fusel Alcohols ...... Ill

25. Order of Addition and Composition of Carbohydrates in the Fermentation Medium ...... 124

26. Effect of Periodic Addition of Carbohydrates on the Production of Fusel Alcohol ...... 126

27. Effect of Periodic Addition of Carbohydrates on Specific Gravity . . . •...... 136

28. Summary of Experimental Data Obtained Using Amino Acids and Ammonium Sulfate as the Source of Nitrogen in the Fermentation M e d i u m ...... 147

29. Summary of Experimental Data Obtained from Fermentations with Varying Carbohydrate R a t i o ...... 153

30. Summary of Experimental Data Obtained from Fermentations with Periodic Addition of Carbohydrates ...... 155

xi LIST OF FIGURES

Figure Page

1. Uptake Mechanism for Maltose into Yeast C e l l s ...... 12'

2. The Fate of Glucose in the Yeast C e l l ...... 14

3. Production of Ethyl Alcohol Via G l y c o l y s i s ...... 1 5

4. Ehrlich Pathway for the Production of Fusel A l c o h o l s ...... 24

5. Pathway of Formation of Isobutyl Alcohol from Pyruvic A c i d ...... 25

6. An Idealized Scheme of Higher Alcohol Formation, Amino Acid Mixture as Nitrogen Source ...... 26

7. Interrelationship Between Biosynthesis of Some Amino Acids and Formation of Some Higher Alcohols in Yeast ...... 29

8. Schematic Representation of Ayrapaa's Hypothesis of Higher Alcohols Formation .'...... 31

9. Chromatogram of Fusel Alcohols Obtained from Fermentation with Sucrose as the Only Carbohydrate...... ,. . . 38

10. Mickrokjeldhal Apparatus for Inorganic Nitrogen Determination ...... 48

11. Effect of Amino Acid Concentration on the Production of Fusel Alcohols - FAN ® 150 m g / 1 ...... 60

xii LIST OP FIGURES (continued)

Figure Page

12. Effect of Amino Acid Concentration on the Production of Fusel Alcohols - FAN » 300 mg / 1 ...... 61

13. -Effect of Amino Acid Concentration on the Production of Fusel Alcohols - FAN « 450 mg / 1 ...... 62

14. Effect of Amino Acid Concentration on the Utilization of Wort Sugars - FAN * 150 mg / 1 ...... 63

15. Effect of Amino Acid Concentration on the Utilization of Wort Sugars - FAN = 300 mg / 1 ...... 64

16. Effect of Amino Acid Concentration on the Utilization of Wort Sugars - FAN = 450 mg / 1 ...... 65

17. Effect of Amino Acid Concentration on the Utilization of Free Amino Nitrogen during Simulated Beer Fermentation ...... 67

16. Chromatogram of Amino Acids Analyzed with Automated Amino Acid Analyzer ...... 69

19. Formation of n-propanol and Utilization of "Threonine" during Wort Fermentation . . . .74

20. Formation of Isobutanol and Utilization of Valine during Wort Fermentation...... 75

21. Formation of 3-methyl-l-butanol and Utilization of Leucine during Wort Fermentation ...... 76

22. Formation of 2-methyl-l-butanol and Utilization of Isobutanol during Wort Fermentation ...... 77

xiii LIST OF FIGURES (continued)

Figure Page

23. Effect of Amino Acid Concentration on Wort p H ...... 82

24. Effect of Amino Acid Concentration on the Specific Gravity during Fermentation of W o r t ...... 83

25. Effect of Ammonium Sulfate Concentration on the Production of Fusel Alcohols -Amm. N s* 150 mg/1 ..... 86

26. Effect of Ammonium Sulfate Concentration on the Production of Fusel Alcohols - Amm. N = 300 m g / 1 ...... 87

27. Effect of Ammonium Sulfate Concentration on the Production of Fusel Alcohols - Amm. N = 4 50 mg/1 .... .88

28. Effect of Ammonium Sulfate Concentrations on the Utilization of Individual Wort Sugars - Ammonia Nitrogen = 150 m g / 1 ...... 90

29. Effect of Ammonium Sulfate Concentrations on the Utilization of Individual Wort Sugars - Ammonia Nitrogen = 300 m g / 1 ...... 91

30. Effect of Ammonium Sulfate Concentrations on the Utilization of Individual Wort Sugars - Ammonia Nitrogen = 450 m g / 1 ...... 92

31. Effect of Ammonium Sulfate Concentration on the Utilization of Ammonia Nitrogen during Simulated Beer Fermentation ......

32. Effect of Ammonium Sulfate Concen­ tration on Wort pH during Fermentation...... 95

xiv LIST OP FIGURES (continued)

Figure Page

33. Effect of Ammonium Sulfate Concentration on the Specific Gravity during Fermentation ...... 96

34. Effect of Carbohydrate Ratio on the Rate of Fusel Alcohol Production. (Maltose:Glucose:Sucrose:75:6.8:18.2) Lot A ...... 112

35. Effect of Carbohydrate Ratio on the Rate of Fusel Alcohol Production (Maltose:Glucose:Sucrose:18.2:75:6.8) Lot B ...... 113.

36. Effect of Carbohydrate Ratio on the Rate of Fusel Alcohol Production. (Maltose:Glucose:Sucrose:18.2:75:6.8) Lot C ...... 114

37. Effect of Carbohydrate Ratio in Wort on the Utilization of Individual Carbohydrates. (Maltose:Glucose: Sucrose:75:6.8:18.2) Lot A ...... 116

38. Effect of Carbohydrate Ratio in Wort on the Utilization of Individual Carbohydrates. (Maltose:Glucose: Sucrose:18.2:75:6.8) Lot B ...... 117

39. Effect of Carbohydrate Ratio in Wort on the Utilization of Individual Carbohydrates. (Maltose:Glucose: Sucrose:6.8:18.2:75) Lot C ...... 118

40. Effect of Carbohydrate Ratio on Ammonia Nitrogen Utilization ...... 120

41. Effect of Carbohydrate Ratio on pH of W o r t ...... 121

42. Effect of Carbohydrate Ratio on Specific Gravity of W o r t ...... 123

xv LIST OP FIGURES (continued)

Figure Page

43. Effect of .Periodic Addition of Carbohydrates on the Rate of Fusel Alcohol Production (Lot A) ...... 127

44. Effect of Periodic Addition of Carbohydrates on the Rate of Fusel Alcohol Production (Lot B) ...... *128

45. Effect of Periodic Addition, of Carbohydrates on the Rate of Fusel Alcohol Production (Lot C) ...... 129

46. Effect of Periodic Addition of Carbohydrates on their Utilization (Lot D) ...... 130

47. Effect of Periodic Addition of Carbohydrates on their Utilization (Lot E) ...... 131

48. Effect of Periodic Addition of Carbohydrates on their Utilization (Lot F) ...... 132

49. Effect of Periodic Addition of Carbohydrates on Ammonia Nitrogen ...... 134

50. Effect of Periodic Addition of Carbohydrates on p H ...... 137

51. Yeast Cell Growth in the Experimental Medium with Varying Carbohydrates R a t i o ...... 138

52. Yeast Cell Growth in the Experimental Medium with Periodic Addition of. Carbohydrates during the Course of Fermentation...... 140

xvi INTRODUCTION

The success of the fermentation industry depends to a large extent on the production of finished products of uni­ form quality characteristics from day to day and season to season. A major obstacle in achieving uniform quality is the variation in the composition of the raw ingredients.

The problems are associated with variations in the composi­ tion of the raw ingredients could be overcome to some‘degree by component analysis of the ingredients and supplementation with those components which are found to.be insufficient or lacking. However, to achieve full control over fermentation it is necessary to have a complete understanding of the role of each individual component and the synergistic effects of components on the formation of desirable end-products.

Specifically, with respect to the brewing industry,' fusel alcohols, which are produced during the fermentation of wort, have a great effect upon the flavor quality and acceptance of beer. Fusel alcohols are needed for flavor . character. However, they also contribute to the "hang­ over" experienced from the consumption of' beer.

For this reason, brewers are interested in controlling the fusel alcohol content of beer within relatively narrow limits. Although studies -have identified possible fermen­

tation pathways for fusel alcohol production in beer,

brewers have yet to achieve control over the fermentation process through adjustment of the wort composition. De­

sirable and uniform levels of fusel alcohol in the marketed product, when controlled, are accomplished by blending two or more lots of beer before bottling.

The effects of substrate and end-products feedback on

the activity and productivity of enzymes are key factors in

the control of the fermentation of beer as well as in any other fermentation.

Control of the fermentation process may be possible if

the yeast can be "directed" to assimilate carbohydrates, proteins, lipids, nucleic acids, and other nutrients only by those metabolic pathways which are desirable. However, as stated, in beer fermentation these controlling factors are incompletely and poorly understood at this time.

The purpose of this investigation was to study the role of the carbohydrate's and the source and concentration of nitrogen in wort on the formation of fusel alcohols. It was expected that the results might lead to a clearer under­

standing of the factors and mechanisms of fusel alcohol pro­

duction and point to means of control over wort composition

which might aid the brewing industry in gaining more com-

plete control over beer fermentation. 4 LITERATURE REVIEW

The review deals briefly with brewing technology and

then covers in detail the effect of steeping on the amino

acid and carbohydrate composition of the wort, the utiliza­

tion of these compounds and the formation of flavor com­

pounds in beer, particularly fusel alcohols.

Brewing Technology

The recipe for beer which was. brewed by the ancient

Egyptians in 3000 B.C. has been reported by Zosimos of

Panopolis (1). The grain was macerated with water. The

mixture was exposed to air for one day, re-moistened and

sun-dried. Then, the malt was placed in fermentation tubs.

The yeast was supplied from risen dough.

Today, breiwing is a major industry. In the United

States, over four thousand (4,000) million gallons of beer

are consumed annually. The principal raw materials are

cereal grains, particularly barley, rice, and corn, which

supply the carbohydrates for fermentation by Saccharomyces

yeast into ethyl alcohol and carbon dioxide. A brief de­

scription of the ingredients and a typical beer making pro­

cess is presented below (2).

3 Malt - The most important ingredient in beer making is

barley that has been germinated and dried. Germination

•is needed to activate the enzymes that convert starches

into sugars that are fermentable by the yeast.

Hops - The flowers of the hops plant (Hujulus lupulus) con­

tain about 10-25% resins, 15% proteins and 4% tannin.

The remainder is water, cellulose and lipids.- The

bittering principals in resins are called alpha acids

and are insoluble in lead acetate. Major alpha acids

are humulones (C21H30C>5) , cohumulones (C2oH28°5* and

prehumulones (C22**32^5^ * Cereal adjuncts - These are cereal grains such as corn, * rice and others that are added to provide additional

carbohydrates for conversion to sugar for subsequent

fermentation.

Mashing - Malted barley, cereal adjuncts and water are

added together and mildly cooked. The purpose of mash­

ing is to extract soluble materials and enhance

enzymatic breakdown. Mashing may start at 100°F and

the temperature is increased gradually to 170°F with a

30 minute rest period between gradual temperature in­

creases. This is done to allow the amylases and

proteinases to function before they are heat inacti­

vated. The mash tank is designed to separate the yeast

fermentable sugars from the spent grains. The liquid

fraction is known as wort. Brewing - Generally, the wort and the added hops are brewed

in the kettle by boiling for 2.5 hours. After that,

the wort is drawn from the kettle and cooled. At this

point the pH of the wort is about 5.6.

Fermentation - The wort is fermented to beer by

Saccharomyces yeast at temperatures ranging from 37°F

to 75°f depending on the strain of yeast. The fermen­

tation is completed in about 9 days. The result is

beer with an alcohol content of about 4.6% by volume

and a pH from 4.6 to 4.0. During this period, the

fusel alcohols and some of the aroma compounds are

formed.

Storage - The beer is chilled quickly to 32°F. The yeast

and other suspended solids are removed through filtra­

tion. The beer is pumped into pressure storage tanks

and generally stored at 32°F for several weeks to sev­

eral months. This is called "secondary fermentation"

where the settling of suspended proteins, yeast cells,

and other remaining materials occurs. Esters, and

other flavor compounds develop during this period.

Carbon dioxide is usually added to the preferred level

and to remove residual oxygen. Proteins or tannins

that cause chill haze are precipitated during this

period.

Finishing steps - After completion of the secondary fermen­

tation the beer is refiltered, C02 is added as 6

required, and the beer is filled into bottles, cans or

other containers. The filled product is pasteurized

at 140°P for several minutes to increase shelf-life.

Effect of Steeping on Wort Amino Acids

The composition of wort varies widely depending on the

type of barley, adjuncts and the manufacturing processes

used. Wagner and Piendl (3) reported on the effect of the

degree of steeping, (immersion of barley in water until the

proper moisture content is reached) germination time, ger­

mination temperatures and final heating temperature on the

amino acid composition of malt. Three main barley varie­

ties were studied; Eli, Wisa, and Bido (3). The effect of

the degree of steeping on the amino acid content of malt

from the Eli variety is shown in Table 1. As the moisture

levels increased, malt protein decreased and wort soluble

nitrogen and malt alpha amino nitrogen both increased. The

concentrations of the majority of the amino acids increased * * with the intensity of steeping. Exceptions were methionine

and proline. On average, malt from the Eli variety of bar­

ley has been found to contain 137 mg of alpha amino nitrogen

per 100 grams of dry substance while malts from Wisa and

Bido each contain 133 mg.

Maendl et. al. (23) have also studied the effect of

' steeping on amino acids. Their results were in agreement

with those of Wagner and Piendl (3). 7

TABLE 1

Degree of steeping at which highest levels of amino acids are obtained in Eli malt after Wagner and Piendl (3).

Amino Acid Before After Degree of mg/1 mg/1 Steeping (% Moisture)

Lysine 65 90 48 Aistidine 30 48 48 Arginine 70 107 48 Tryptophan 21 48 48 Aspartic Acid 40 75 48 Threonine 40 72 48 Serine + Amides 90 115 48 Glutamic Acid 20 35 48 Proline 233 220 48 CO Glycine 21 35 » Alanine 55 92 48 Valine 75 120 48 Methionine 22 10 45 Isoleucine 50 80 48 Leucine 95 130 . 45 Tyrosine 60 100 48 Phenylalanine 82 125 48 Effect of Steeping on Wort Carbohydrates

Piendl (24) investigated the effect of the degree of steeping on the level of carbohydrates. Pour barley varie­ ties, Wisa, Volla, Bido,' and Eli, were studied. His results are summarized in Table 2. Piendl reported that the levels of hexoses and maltose in the malts were directly affected by the degree of steeping. In particular, the glucose and fructose levels increased when the degree of steeping was increased from 45 to 48 percent. He found that the content of hexoses was lower in Wisa barley malt than in Volla, Bido or Eli.

The highest maltose .concentration was obtained when bar­ leys were steeped to 45 percent moisture. Steeping to lower or higher moisture produced lower levels of maltose. The varietal differences were: Wisa and Bido malt contained the highest levels of maltose followed by Eli and Volla malt, respectively.

Nitrogen Utilization

Much information is available in the literature on nitrogen utilization. Most ammonium salts and urea can serve as the sole nitrogen source for brewers yeast while nitrites are not utilized (16). L-amino acids (D and L for glutamate, aspartate, and asparagine) can be used as a single nitrogen source by most yeasts. On the other hand, lysine, giycine, cysteine, and proline are rarely utilized TABLE 2

Degree of steeping at which highest levels of sugars are obtained in barley malt. After Piendl (24).

Sugar Eli Volla Wisa Bido (gm/100 Degree . gm dry of wt.) From To From To From To From To Steeping

Hexoses 7.1 10 8.2 9.0 6.8 8.8 7.7 9*0 48

Sucrose 3.8 4.5 2.8 3.1 3.9 4.5 '3.3 4.8 48

Maltose 32.0 34.5 34.5 35.5 37.5 39.0 37.5 39.0 45 10 as a single nitrogen source. When the complete amino acid

spectrum is available (as in wort) four separate groups of amino acids have been characterized for brewer's yeast, based on the time required for 50 percent assimilation .

(17,- 19). These groups are shown in Table 3.

Carbohydrate Utilization

The individual sugars in wort are utilized in an orderly fashion. The exact sequence will vary, depending on the strain of yeast used and the relative concentration of the sugars (25). For most strains of Saccharomyces cerevisiae and S. carlsbergensis, the general sequence of removal is sucrose, glucose, fructose, maltose, and malto- triose (26). Sucrose, after hydrolysis outside the cyto­ plasmic membrane in the region of the cell wall (27), is metabolized in the same manner as its constituent mono­ saccharides. Thus, the sugars are metabolized in order of increasing molecular complexity.

Since maltose is the major carbohydrate present in wort, a full understanding of the factors affecting its uptake by the yeast is important. Two systems are involved in maltose metabolism: (a) Maltose permease or the system that transports maltose across the cell membrane into the cell; and (b) maltase (alpha-glucosidase) which hydrolyzes maltose, once inside the cell, to yield two' glucose units of maltose (Figure 1). 11

TABLE 3

Order of absorption of amino acids from wort by

brewers' yeast. After Jones and Pierce (19).

Group A Group B Group C Group D

Immediately Absorbed Absorbed Only Slowly Absorbed Gradually after a Lag Absorbed during after 60 hrs. Fermentation

Arginine Histidine Alpha-Alanine Proline Asparagine Isoleucine Glycine Aspartate Leucine Phenylalanine Glutamate Methionine Tryptophane Glutamine Valine Tyrosine Lysine Serine Threonine moltase

(glucose"]

hexokinase

G-6-P ] n maltose permease 1. Uptake mechanism for maltose into yeast cells, After Stewart (25). 13

The fate of glucose inside the yeast cell is summarized in Figure 2. Glucose can be utilized to produce poly­ saccharides, lipids, proteins, or ethanol depending on the metabolic conditions. Detailed information on the several metabolic processes is available in the literature (28) and \ is not discussed here. In the case of beer and other alco­ holic beverage fermentations, the conditions of fermentation are geared to favor ethyl alcohol production which occurs via the glycolysis pathway as shown in Figure 3.

As mentioned, the sequence of removal of individual

sugars from wort depends on the yeast strain and the rela­ tive concentrations of sugars that cause enzyme induction

in the yeast cell. Stewart (25) studied factors affecting

the uptake mechanism for maltose in yeast strains having an

inducible maltose metabolism; First, different strains of

S. carlsbergensia were propagated in a medium with glucose

as the only carbon source and then were added to a complex medium containing glucose, fructose, and maltose as the carbon source. The* rate of removal of these sugars was

always highest for glucose followed by fructose, and only

one strain had an inducible maltose system. However, when

the yeast strains were propagated in a medium with maltose

as the only carbon source, all strains studied were capable

of utilizing the three sugars in the complex medium.

The carbohydrate type and the effect on the induction

of enzymes will determine the relative magnitudes of the cell membrane

giuccso^ y> polysaccharides

proteins 1 // > g tocos i f i CO 2 \ * / TC I LA A \4\4 G-6-R f**pyruvate*“ >/ , 31 = J ^ • * cycle ij

\ pentose cycle a

r ^ ethanol

Figure 2. The fate of glucose in the yeast cell

After Stewart (25). Sucroce I Sucrase Glucose iGK or HK G-6-P

phosphoglucoisomerase > y Fructose — >F-6-P Iphospofructokinase F-l-6 Di-P 1aldolase G3P ^ triose-P-isomerase ^ DHAP l glycerate 3 phosphodehydrogenase 1-3-3 di-P-glycerate i phosphoglycerokinase 3-P-glycerate. p - I phosphoglyceromutase 2-P-glycerate I enolase TCA < O j ------Phosphoenol pyruvate

^ pyruvate kinase

pyruvate ipyruvate decarboxylase Acetaldehyde:eti lalcohol dehydrogenase Ethyl alcohol

Figure 3. Production of ethyl alcohol via glycolysis After Lehninger (52). 16

Pasteur and Crabtree Effect (52). The Pasteur Effect is the

"inhibition of fermentation by respiration" (25). In other words, according to the Pasteur Effect, carbohydrates are metabolized via glycolysis in the absence of oxygen and via the Tricarboxylic Acid cycle in the presence of oxygen. The

Crabtree Effect on the other hand is a concentration effect.

Glucose is metabolized yia glycolysis at high concentration and via TCA at low concentration regardless of oxygen level*

The repressive affect of glucose on the oxidative ac­ tivity of yeast (29, 30, 31) as well as bacteria (32, 33), during growth is a well-known phenomenon. Sugars such as . maltose and galactose, to which yeast has to adapt, cause a repression of enzyme activity in the electron-transport or tricarboxylic acid cycle, but the repression is two to five times less than that caused by glucose.

Flavor Compounds in Beer

The influence of variety, environment and malting technology on the nitrogen and carbohydrate content of bar­ ley, malt and wort along with other factors are responsible for the production of a wide variety of beer flavor com­ pounds. Meilgaard (5) has divided the flavor compounds in beer into four groups:

1. Primary flavor constituents: Ethanol, hop bitter compounds and carbon dioxide. Removal of any of these con­ stituents would produce a major change in flavor. 17 2. Secondary flavor constituents: (a) Volatiles, such as banana esters (e.g., isoamyl acetate), apple esters (e.g., ethyl caproate), fusel alcohols (e.g., isoamyl alcohol), caprylic acid, ethyl acetate butyric acid and isovaleric acid; (b) Non-volatiles, such as polyphenols, organic acids, i hulapornes, nucleotides and amines. These compounds con­ stitute the bulk of the flavor compounds and their removal will cause a small change in flavor. The difference between any two beers of the same type is due to variations in com­ pounds within this group.

3. Tertiary flavor constituents: 2-phenyl ethyl acetate, isovaleraldehyde, and acetoin. These compounds add subsidiary flavor notes. Their removal causes no noticeable change in flavor.

4. Background flavor constituents: Decanol, phenyl acetic acid, lauric acid, and a host of others. It is not known yet whether the background constituents are of im­ portance in beer flavor.

Factors Affecting the Production of Fusel Alcohols

The word fusel (German) means inferior liquor. Fusel alcohols of fusel oils are primary alcohols. Some example of fusel alcohols produced in alcoholic fermentation are n-propanol, isobutanol, 2-methy1-1-butanol, 3-methy1-1- butanol , isopentanol, isopropanol, tyrosol, and phenyl ethyl alcohol. The four underlined alcohols are commonly 18 present in beer.

Fusel alcohols are considered to be secondary flavor constituents. Their presence in small amounts increases beer palatability, but an excessive amount is likely to be deleterious. There is a correlation between "hang-over"

(headaches, nausea, muscle weakness, etc.) and the amounts of fusel alcohols consumed (6). The content of fusel alcohols in commercial beer varies widely as shown in

Table 4 (7). For example, British ale contains three times more total fusel alcohols than Canadian ale. Canadian premium contains on the average 130 ppm total fusel alco­ hols while Canadian ale was 83 ppm total fusel alcohols.

Many factors affect the formation of fusel alcohols

(8) as follows:

(a) Wort Composition. This factor is the most impor­ tant one and can be divided into two sub-factors: The con­ centration of the individual amino acids, and the type and concentration of fermentable Bugars. With respect to amino acids, the L-isomers and not the D-isomers’ (exceptions are glutamate, aspartate, and asparagine) have a great

effect on the production of fusel alcohol. This is because

the D-isomers are not utilized by most yeast strains as a

nitrogen source. 'Ehrlich (8) was the first to describe the

catabolic pathway of fusel alcohol formation from these com­

pounds. The mechanism is described later in this review. 19

TABLE 4

Fusel alcohol contents of some commercial beers (7)

Fusel Alcohol (parts/million)

Sample n-Propanol Isobutanol 2-methyl- 3-methyl- Total 1-butanol 1-butanol

Canadian premium beer 1 8.94 16.29 15.65 73.90 114.78

Canadian premium beer 2 8.03 13.58 27.18 76.48 125.27

Canadian premium beer 3 13.81 29.60 19.89 91.49 154.79

American malt liquor 4.75 25.29 33.45 89.75 153.24

British pale ale 12.59 56.61 33.94 122.46 225.60

Canadian ale 1 9.90 15.05 12.27 57.38 94.60

« t Canadian ale 2 8.04 12.79 10.91 50.20 81.94

Canadian ale 3 10.77 12.82 10.18 42.02 75.79 20

The type of carbohydrates and their concentration in wort also affects the production of fusel alcohols (42),

When 75% of industrial wort was replaced by sucrose, the production of n-propanol was decreased from 30 ppm to 5 ppm while the production of 3-methyl-l-butanol was increased from 62 ppm to 168 ppm.

The effect of sugar addition to wort was investigated by many workers. Jenard and Devreux (45) fermented differ­ ent combinations of wort and sucrose solutions, and found an increase in higher alcohols with increasing sucrose concen­ tration. Kamiyama and Nakagawa (46) studied the effect of the addition of sucrose to wort. They found that n-propanol concentration decreased with increasing addition of sucrose, isobutanol concentration remained unchanged while the concen­ tration o f .2- and 3-methyl-l-butanol increased slightly.

Maule (47) found that addition of glucose to wort led to an increase in isobutanol and a reduction in n-propanol concen­ trations. Hough and Stevens (48) found that addition of sucrose to wort caused a reduction in the concentration of higher alcohols. Thus, the results of sugar addition to wort on fusel alcohol formation vary from one researcher to another. It is difficult to provide a satisfactory explana­ tion with any degree of certainty the reason for these variations in results. However, many researchers who have concerned themselves with the effect of fermenting a wort with added glucose or sucrose on the formation of fusel 21 alcohols have used in their studies a wort of undefined car­ bohydrate and nitrogen characteristics. Replacing part of the wort with sucrose or glucose solution has a two-fold effect. Firstly, the ratio of the fermentable sugars in the wort is changed. Secondly, the nitrogen level in the wort is decreased.

(b) Yeast Strain. The amount of fusel alcohols pro­ duced is greatly affected by the yeast strain (43). Wild yeast, such as S. diastaticus and S. postoriances, produce larger quantities of fusel alcohols than other strains, par­ ticularly under aerobic conditions (9).

(c) Type of Fermentation. Usually, ale beer contains more fusel alcohols than lager beer. This may be directly related to the higher temperatures used in ale fermentation and the increased adjunct (carbohydrate) concentration.

(d) Wort Aeration. Aeration promotes the production of fusel alcohols. Gheluwe and co-workers (8) have shown that the introduction of oxygen during fermentation at a

* rate of 5 ml every 2 minutes for 25 liters of wort will in­ crease the level of fusel alcohols from 90 to 143 ppm.

Aeration also causes a reduction in the fermentation rate (10).

Oxygen is needed by the yeast cells to synthesize lipid constituents of the cell, such as ur\saturated fatty acids and ergosterol. Lipids are necessary for cell growth and reproduction. However, yeast cells can utilize lipids 22 supplied in the medium. Under brewery conditions, there are two occasions when oxygen is not required: (a) when the yeast cells have stored sufficient amounts of lipids to per­ mit distribution to new cells produced during fermentation and (b), when the wort contains sufficient amounts of lipids and unsaturated fatty acids to sustain the cells (3).

(e) Wort pH. Gheluwe et. el. (8) have shown that wort which is initially alkaline {pH 8) or acidic (pH 3-4) give3 rise to higher concentrations of fusel alcohols than normal wort (pH 5.2). These authors suggested that Keto acids are known to be intermediates in the formation of fusel alco­ hols, the lactic and citric acids which were used to lower the pH of wort are preferentially used as a carbon source by the yeast giving rise to the increased fusel alcohol pro­ duction. No explanation was offered for the increased fusel alcohol production at pH 8.

(f) Shape of Fermentor. Generally, a deep fermentation produces less fusel alcohols than a shallow one. This is probably related to the effect of oxygen as discussed in (d).

(g) Yeast Pitching Rate. Usually, the higher the yeast pitching rate the higher the concentration of fusel alcohols formed. Normal pitching rate is 10-15xl06 cells per ml.

Biosynthesis of Fusel Alcohols. Basically there are two metabolic pathways governing the formation of fusel alcohols during fermentation of beer. The first mechanism 23 is catabolism of amino acids, commonly known as the Ehrlich mechanism which is presented in Figure 4 (8). In this mech­ anism the first reaction is deamination of amino acid, followed by decarboxylation of the alpha keto acid and, finally, reduction of the aldehyde to the corresponding alcohol.

The second metabolic pathway for the formation of fusel alcohols is by the anabolic process involving carbohydratest the main carbon source in the medium. Pyruvic acid, an in­ termediate product of carbohydrate metabolism may undergo a series of reactions to produce fusel alcohols (11). As an example, the formation of isobutyl alcohol from pyruvic acid is shown in Figure 5.

The fusel alcohols are therefore the products of two metabolic pathways; Ehrlich*s and the synthetic. However, for either pathway, fusel alcohol production is dependent upon the composition of the medium and in particular the level of nitrogen present.

Ayrapaa (18) has propoised a common idealized scheme

(Figure 6) showing the relationship between fusel alcohols and nitrogen level in the substrate with amino acids as the nitrogen source.

Higher alcohols produced via the synthetic pathway show maximum concentration (line AB) at a low nitrogen level and minimum concentration (line CD) at a high nitrogen level.

In contrast, higher alcohols produced via the Ehrlich 24

NH- I Transaminase R ~ C - COOH -> R_L COOH I H

Amino Acid Alpha - Keto Acid

O Decarboxylase II R— C - COOH R -C - H + C02

Alpha - Keto Acid Aldehyde

Alcohol Dehydrogenase

ii NADH» R— C— H R - CH2— OH

NAD Aldehyde Alcohol

Figure 4: Ehrlich pathway for the Production

of Fusel Alcohols. After Chen (51). 25

COOH C=0 I \ c=o Acetyl CoA Ho-C-COOH

i ■ CH- Thiamine phosphate

Pyruvic acid Alpha acetolactic Acid

H H H Reductive H-C-C C-OH Rearrangement i l i H CH, H

isobutyl alcohol T H CH. CH.

CH3-C-CH3+C02<--- CH3-C-H 4 c h 3-c -o h

o=C-H 0=C-CooH H-C-COOH

Isabutyraldelyde alpha- Ketoisovaleric OH acid 2,3,-Dihydroxy Isovaleric. Acid

Figure 5. Pathway of Formation of Isobutyl Alcohol from

Pyruvic Acid. After Gheluwe et. al. (8). 26

Tototcl

Nitro gen ‘ in sub sf rat e

Figure 6. An idealized scheme o£ higher alcohol formation,

amino acid mixture as nitrogen source. Abscissa:

amount higher alcohol formed. Ordinate: initial

nitrogen level of the medium. After Ayrapaa (18) 27 pathway show less dependence on the nitrogen level of the medium.

The controlling factors that determine the position of lines (AB) and (CD) are not yet known. Ayrapaa hinted that the position of line (AB) may be dependent upon the factors which influence the nitrogen requirement of the system, i.e., sugar concentration. The position of line (CD) may be influenced by factors that control the .synthesis of the corresponding amino acids, i.e., initial sugar concentration of the medium.

Lewis (12) has indicated that the higher alcohols are' derived from keto acids present or produced by yeast in ex­ cess of the amounts needed for amino acid biosynthesis.

Thus, iso£unyl alcohol, 2-methyl-butanol, and isobutanol were shown to be derived from the alpha keto acids required for the synthesis of leucine, isoleucine, and valine, respectively. Ayrapaa (13) indicated that the formation of higher alcohols are from (a) amino acids of the medium and (b) alpha keto acids.

Ayrapaa (18) studied the formation of isobutanol and

3-methyl-l-butanol during fermentation by adding uniformly

^C-labelled valine and leucine to the amino acid pool which was used as nitrogen source. Only 3-methyl-l-butanol 14 became radioactive in the presence of ‘ C-leucine, whereas isobutanol and 3-methyl-l-butanol acquired label in the presence of ^C-valine. The specific radioactivity of 28 these two alcohols was always lower than that of the parent amino acid. This indicates that these higher alcohols are also synthesized simultaneously by the yeast. Thusr if the formation of higher alcohols is to be controlled, the in- fluence of the environmental factors on the catabolic and anabolic modes must be controlled. Ayrapaa's results also indicate that the regulating effect of leucine and valine levels of the medium on the synthesis of these amino acids in yeast cells is rather limited.

Ayrapaa's work on the effect of valine, leucine, isoleucine and phenylalanine on the production of fusel alcohols can be summarized as follows. Firstly, the frac­ tion of an amino acid transformed to higher alcohols is in­ versely _ correlated to the total nitrogen level of the sub­ strate. Secondly, all four amino acids are equally effec­ tive nitrogen donors at nitrogen limitation. Thirdly, at high nitrogen levels, leucine is the preferred nitrogen donor followed by phenylalanine, while isoleucine and valine are utilized very little.

Drews et. al. (14) have.presented a scheme for the formation of n-propanol, isobutanol, 2-methyl-l-butanol,

3-methyl-l-butanol, and their corresponding amino acids threonine, valine, leucine and isoleucine (Figure 7).

Pyruvic acid which is the starting material is produced from the main carbon source in the medium, the sugars.

Following a series of reactions including carboxylation

t

i 29

Leucine^— a-ketoisocaproic acid — ^3-methylbutanol-l

a-keto-£ -carboxyisocaproic acid t ^-hydroxy-^-carboxyisocaproic acid ? + active acetate valine* -a-ketoisovaleric acid >isobutanol

a,(3 -dihydroxyisovaleric acid

.a-acetolactic acid 1 pyruvic acid + active acetate

) > 1 -hydroxy-p-carboxybutyric acid

a-hydroxy-^-carboxybutyric acid

a-keto-p-carboxybutyric. I acid threonine <$- a-ketobutyric acid n-propanol I + pyruvic acid a-aceto-a-hydroxybutyric acid

a, (J-dihydorxy-^-methylvaleric acid

a-keto-p-methyl- J isoleucine ^ valeric acid ------^ 2-methylbutanol-l

Figure 7. Interrelationship between biosynthesis of some

amino acids and formation of some higher alcohols

in yeast. After Drews et al. (1964). 30 and reductive rearrangement, the products may undergo reduc­ tion to produce a fusel alcohol or transamination to produce an amino acid. The factors controlling these pathways are, however, little known. The over-all mechanism presented in

Figure 7 is similar to.that proposed by Guyman et. al. (9).

Ayrapaa (15) has indicated that if the medium is poor in amino acids, the yeast must synthesize the amino acids required for protein synthesis rather early during the fer­ mentation. The keto acids of the required carbon skeleton

* are produced from carbohydrate breakdown in excess of the requirements for amino acid synthesis. The excess keto acids undergo decarboxylation and reduction to produce higher alcohols. Ayrapaa's hypothesis is presented in

Figure 8. The keto acid pool is generated by synthesis from pyruvic acid or from deamination of amino acid.. *

Nutritional Properties of Beer

Few people drink beer for its nutritional value. How- i ever, questions have been raised about the nutritional value of beer. The purpose of this brief review is to answer some of these questions.

Orsi (22) compared the composition of different beers^

The compositions ranged from 90-92% water, 1.0-5.5% ethanol,

0.35-0.45% carbon dioxide and 4-5% extract. Carbohydrates included 60-75% dextrins, 20-30% monosaccharides, and 6-8% pentosans. Tannins were 150-300 mg/1. 31

AMINO ACID

AMINO ACID DEAMINATION WITH KETOGLUTARATE

TRANSAMINATION GLUTAMIC ACID

KETO ACID POOL

SYNTHESIS DECARBOXYLATION AND REDUCTION

FUSEL ALCOHOLS

Figure 8. Schematic representation of Ayrapaa*s hypothesis

of higher alcohols formation (15). 32

The caloric value of regular beer is 400-500 KCal per liter. Low-caloric beer contains about one-third less calories. The lower calory content is due to as complete a removal as possible of residual carbohydrates (non- fermentable) by the use of enzymes such as dextrinase or amylogluconase.

With respect to vitamins, thiamine is present in rela­ tively small amounts (1-2 mg/1). The pyridoxine (vitamin

Bg) content fluctuates widely. Half the daily requirements of pantothenic acid can be provided from one liter of beer.

However, Wagner (20) indicated that the vitamin content of beer is of no practical nutritional significance. For example, one would need to drink five liters of beer to ob­ tain the daily riboflavin requirement. Other B-vitamins are also present in very small amounts.

Beer is easily digested. It enhances digestion and in­ creases the secretion of pepsin (21). It has no allergic side-effects and has a sedative action (21). Beer is a good source of several minerals, such as potassium, magnesium, manganese, zinc, and copper. SCOPE OF INVESTIGATION

Using a defined fermentation medium simulating wort composition, investigations were conducted on the following factors as they affect certain characteristics of been

* 1) Concentration and source of nitrogen

a) amino acids as source of nitrogen

b) ammonium sulfate as source of nitrogen

2) Role of carbohydrates

a) effect of single carbohydrates

b) variation in the ratio of the three major

carbohydrates

c) periodic addition of carbohydrates to the

medium during the course of the fermentation

The specific objectives of this study were to evaluate • the effects of the above factors on:

1) The formation of fusel alcohols

2) The rate of utilization of carbohydrates

3) The rate of nitrogen removal

4) pH

5) Specific gravity

33 EXPERIMENTAL PROCEDURE

Fermentation Medium and Process t The basic substrate used in this study was the de£ined medium of Jones et. al. (41). This medium was used by Jones in studies on the incorporation of individual amino acids into the carbon skeletons of the amino acids-of yeast pro­ teins using uniformally■labelled 14C amino acids in the fer­ mentation medium.

The medium contains amino acids and carbohydrates in the concentrations normally found in hopped.worts together with the vitamins, salts and trace elements known to be factors in yeast metabolism. The detailed composition is listed in Table 5. The sugars and salts were sterilized in an autoclave at 121°C for 15 minutes. .The amino acids and the vitamins were sterilized separately by passing through a Nucleopore Membrane (VWR Scientific, Columbus, Ohio), pore size 0.2 microns.

The yeast, Saccharoroyces carlsbergensis, was selected for the fermentation. Fresh yeast was obtained from a local brewery as first generation culture and was added to the fermentation medium at a concentration of 15x10** cells/ml. 35

TABLE 5

A Defined Medium Simulating Wort Composition (41)

L. aspartic acid 78. 20 mg. L. threonine 54. 28 mg L. serine 43. 48 mg L. asparagine hydrate 149. 17 mg L. glutamine 6. 01 mg L. glutamic acid 90. 24 mg L. proline 316. 56 mg Glycine 32. 94 mg L. alanine 102. 53 mg L. valine 108. 56 mg L. methionine 27. 13 mg L. isoleucine 57. 52 mg L. leucine 141. 29 mg L. tyrosine 93. 02 mg L. phenylalanine 101. 88 mg L. tryptophane 49. 05 mg L. lysine-HCL 130. 15 mg L. histidine-HCl 59..03 mg L. arginine-HCl 155. 90 mg Maltose 75. 00 gm Sucrose 6. 80 gm Glucose 18. 20 gm Biotin 2. 0 ug Calcium pantothenate 400. ug Folic acid 2. ug Myo-inositol 2. ug Niacin 400. ug p-aminobenzoic acid 200. ug Pyridoxin HC1 400. ug Thiamin HC1 400. ug Riboflavin 200. ug Boric acid 500. ug Cupric sulfate 40. ug Potassium, iodide 100. ug Ferric chloride 200. ug Manganese chloride 400. ug Sodium roolybdate 200. ug Zinc sulfate 400. ug Manganese sulfate 1.1 gm Sodium chloride 0. 2 gm Potassium hydrogen phosphate 17. 6 g

Make up to 1 liter with 1^0 36

The fermentation was carried out in 1500 ml- batches contained in two liter Virglass fermentation jars equipped with one-way fermentation air locks. The fermenting medium was continuously stirred magnetically. The temperature of the medium was adjusted and maintained at 10+0.5°C in a thermostatically controlled room. Air was not removed from the' jars nor from the solution. Sterile conditions were maintained throughout the process. The fermentation was carried out for 100 hours. Samples for analysis were re­ moved periodically.

Determination of Fusel Alcohols

Fusel alcohols were extracted according to the method of Ayrapaa (18). To a 100-ml sample of beer, 1 ml of in­ ternal standard (0.2% n-pentanol in 95% ethanol, (V/V)) was added. The sample was distilled in a simple still without reflux and the distillate containing the fusel alcohol was stored in a refrigerator until gas chromatography (GC) analysis.

The GC analysis was accomplished on a Hewlett-Packard

402 instrument with dual flame ionization detectors. The glass column was 6 ft. x 2 mm i.d. and packed with 0.2% car- bowax 1500 on 60/80 carpoback C. Sample size was 5 micro- i liters. The injector and detector temperatures were 90°C and 180°C, respectively. The column temperature was initially at 60°C and programmed at 4°C/min to 150°C. The 37 carrier or nitrogen flow rate was 60 ml/min.; hydrogen flow rate was 60 ml/min and air flow rate viras 400 ml/min. Re­ tention times were obtained by injection of standard fusel alcohol solutions. Quantitative measurements were obtained by cutting and weighing peaks pattern from xeroxed record­ ings- of the chromatograms as follows:

mg of fusel alcohol

(Peak wt. of fusel alcohol)(mg. of (Recovery ______Int. std.) factor) (Peak wt. of Int. Std.)

Recovery factor was calculated by comparing the peaks' pattern weight to the known initial weight of the alcohols that were added at 5% (V/V) to an extraction flask contain­ ing water.

A chromatogram showing the actual separation of the fusel alcohols on 6LC is shown in Figure 9 which is for the fusel alcohols that were extracted from the lot with sucrose as source of carbohydrates and ammonium sulfate as nitrogen source. Peak identification and relative retention times were as follows:

Peak Number Retention Time Identification (Min)

1 0.60 methanol

2 2.00 ethanol . i 3 3.20 isopropanol 38

Figure 9 Chromatogram of Fusel Alcohols Obtained from

Fermentation with Sucrose as the only Carbohy­

drate. 1) methanol, 2) ethanol, 3) isopropanol,

4) ethyl acetate, 5) n-propanol, 6) isobutanol,

7) 2-methyl-l-butanol, 8) 3-methyl-l-butanol,

ahd 9) internal standard. ro RECORDER RECORDER PONSE ES R I

RETENTION 40

Peak Number Retention Time Identification (Min)

4 5.40 ethyl acetate

5 6.60 n-propanol

6 9.60 isobutanol

7 13.20 2-methyl-l-butanol

8 14.00 3-methyl-l-butanol

9 15.80 Internal standard

Ethanol, which was. present in large concentrations in all samples, was not quantitated because the peak had to be off scale to permit quantitation of the fusel alcohols. The * ethanol peak was of the same general magnitude in all ex­ perimental samples o f the same age.

Determination of Individual Carbohydrates

Two methods were used for carbohydrates analysis. GLC was used when more than one sugar was present in the fer­ mentation medium. The Technicon Autoanalyzer II was used when only one sugar was present.

GLC determination of carbohydrates followed the method t * described by Marinelli and Whitney (34, 35). The sample for

GLC analysis was prepared by adding 1.0 ml of pyridine

(silylation grade, Pierce Chemical Co., Rockford, 111.) con­ taining 6 mg of phenyl- -? D-glucopyranoside per ml; 0.9 ml of 1,1,1,3,3,3 hexamethyldisilazane; and 0.1 ml of triflouroacetic acid (Aldrich Chemical Co., Milwaukee, 41

Wis.). The vial was shaken for 30 seconds and then allowed to stand for 20 minutes at room temperature with occasional shaking.

Reactions by this method are quantitative (36). If precipitation occurred, the vials and contents were warmed prior to injection.

GC analysis was accomplished on a Hewlett-Packard 402 instrument with dual flame ionization detectors. The glass column was 6 ft x 20 mm i.d. and packed with 3% SE-30 silicone gum rubber on 60/80 mesh Chromasorb W AW. Sample size was 5 microliter. The injector and detector tempera­ tures were 200°C and 300°C, respectively. The column tem­ perature was initially at 150°C for 1 minute, then programmed at 10°C per minute to 275°C. The carrier gas flow or nitro­ gen flow rate was 60 ml/min; hydrogen flow rate was 60 ml/min; and air flow rate was 400 ml/min. Retention times were obtained by injection of standard sugar solutions.

Quantitative measurements were obtained by cutting and weighing peaks pattern from xeroxed recordings of chromatograms as follow:

mg. of sugar _ (wt. of sugar peak)(response factor) in sample “ wt. of int. std. peak

Response factor was calculated by referring the peak pattern weights of known weights of pure sugars to the peak weight of the internal standard. 42

The Technicon Autoanalyzer II was used when only one sugar was present in the fermentation medium. The indus­ trial method No. 302-73A was used. Wort was hydrolyzed with one normal hydrochloric acid to invert the sugar. The in­ vert sugar was then dialyzed into an alkaline stream of potassium ferricyanide. Invert sugar reduces the yellow ferricyanide to colorless ferrocyanide. The decrease in color at 420 nm is directly proportional to the amount of sugar present.

Determination of Free Amino Nitrogen: The recommended ninhydrin method (37) was used for measurement of free amino nitrogen in wort and beer. The method measures amino acids, ammonia, and to some extent, end-group alpha amino nitrogen in peptides and proteins. Proline is not measured to any extent at the wave-length used. The method is described below.

Reagents

a) Ninhydrin color reagent

Dissolve 10.0 g Na2HP04 12 H2), 6.0 g KH2P04,

0.5 g, ninhydrin (Baker N862), and 0.3 g fructose

in distilled water and dilute to 100 ml.. The pH

should be 6.6-6.8. Will keep for 2 weeks if kept

cold in amber bottle. 43

b) Dilution solution

Dissolve 2 g KIO^ in.600 ml distilled water and add

400 ml of 96% ethanol, store at 5°C.

c) Glycine standard stock solution

Dissolve 107.2 mg glycine (Baker M799) in, distilled

water and dilute to 100 ml. Store at 0°C.■

d) Glycine standard solution

Dilute 1 ml reagent c to 100 ml with distilled

water. This standard contains 2 mg amino nitro­

gen/1 .

Apparatus

a) Test tubes, 16 mm x 150 mm.

b) Glass marbles, 20-25 mm diam.

c) Pipets with rubber suction bulbs.

d) Boillng-water bath.

e) Water bath at 20°C (+ 0.1°C).

f) Spectrophotometer covering the visible range. (See

ASBC Proc. 1972, p. 144, for calibration of

spectrophotometer.)

g) pH Meter. See BEER-9 for calibration of pH meter.

Method

a) Calibration standard: Transfer 2 ml of glycine

standard solution (reagent d) to test, tubes in

triplicate. 44

b) Wort: Clarify wort sample and dilute 1 ml to 100

ml with distilled water. Transfer 2 ml of diluted

wort to test tubes in triplicate.

c) Beer: Decarbonate the beer and dilute .1 ml to 50

ml with distilled water. Transfer 2 ml of diluted

beer to test tubes in triplicate.

d) Blank: Transfer 2 ml of distilled water to test

tubes in triplicate. Add 1 ml of ninhydrin color'

reagent (reagent a) to each test tube.

Stopper all tubes with glass marbles to avoid loss by evapo­ ration. Heat tubes- for exactly 16 min in boiling water.

Cool for 20 min. in 20°C water bath. Add 5 ml of the dilu­ tion solution (reagent b) to each test tube. Mix the con­ tents of each test tube thoroughly and read the absorbance at 570 nm against distilled water within 30 min. after the addition of reagent.b.

Calculations

The absorbance readings obtained for the triplicate tubes were averaged for each sample. The blank absorbance was subtracted from the absorbance of the samples and from the glycine standard. Calculate the free amino nitrogen concentration in the samples as follows: Free amino K, mg/1. *=

net absorbance of test solution Y „ v a • i *- • net absorbance of glycine standard dilution

Example

Average absorbance o£ blanks « 0.050.

Average absorbance of glycine standard « 0.4 90.

Average absorbance of beer diluted 1 to 50 = 0.175.

Net absorbance of glycine standard after blank sub-

straction = 0.440.

Net absorbance of beer after blank subtraction = 0.125

0.125 free amino N (FAN), mg/1. = ------X 2 X 50 = 28.4 0.440

Report FAN in mg/1, to nearest whole number, i.e., 28 mg/1.

Notes

1. Since the quantities of amino nitrogen reacting in this method are very small, it is necessary to. avoid introducing traces from outside sources. Glassware must be carefully washed and handled only on external surfaces. Use suction bulbs for pipets and forceps when handling glass marbles.

2. Times and temperatures stated are critical and should be adhered to very closely. A standard and blank sample must be included in each test to com­ pensate for temperature variations in the boiling- water bath.

3. Suggested dilutions given for wort and beer are appropriate for samples containing from 1 to 3 mg amino N/l. after dilution. 46

Determination of Amino Acids: Amino acid analyses were performed by a private laboratory utilizing an automated amino acid analyzer— Durrum-D-500. Amino acids were measured at 570 nm.

Proline is usually measured at 440 nm. However, since proline is not or only minimally absorbed by. yeast during beer fermentation (19), it was decided to forgo a separate analysis for proline. A peak for proline did appear on the

570 nm chromatogram but the proline content was not esti- mated. Thus, to determine proline content would have re­ quired a separate dilution of the wort sample and addi­ tional analytical work by the private laboratory.

Glutamine, asparagine, and serine eluted together and also interfered somewhat with threonine. There was no way to determine the contribution of each of these amino acids to the total area of the peak. Therefore, the results re­ ported for threonine after onset of fermentation are in reality the sum of the contribution of all four of these amino acids to the peak area.

The difficulty encountered in separating the above four amino acids was due in part to the wide differences in con­ centrations. Additional sample dilution might have solved the problem. However, the additional analyses required would have been an imposition on the private laboratory making the analysis. 47

Occasionally, tryptophan and glutamic acid did not

give a sharp enough peak for the computer to integrate;

therefore, results are shown for some sampling periods but

not for others.

Determination of Ammonium Sulfate

Ammonium sulfate .was determined by a modified micro-

- kjeldhal nitrogen method (44). In this procedure, the di­

gestion step is eliminated and only the distillation and

titration steps are used. The apparatus is shown in Figure

10. The procedure is described below.

1. Distillation procedure (see Figure 10).

a. Turn the rheostat on until steam is generated in the glass steam generator (1). (Rheostat is set at 50 to keep hot and turned up to 75 or 80 to generate steam.)

b. Attach the Kjeldahl flask (2) containing 10 ml of the sample to the distillation apparatus. This should be done with particular care. Wipe the ground glass joint (3) with a paper towel to remove any particles that could prevent closure of the joint. Grease the joint lightly with silicone stopcock grease. The flask should be held firmly in place by a wire screen and ring (4). Be sure the distillation tube (5) is submerged in the Kjeldahl flask con­ tents.

c. Place approximately 10 ml of boric acid (con­ taining indicator) in a 50 ml Ehrlenmeyer flask (6) and place under condenser (7). Be sure the delivery tube is submerged in the boric acid solution.

d. Place 15 ml of concentrated sodium hydroxide in the reagent funnel (9). Fill tube approxi­ mately half-full. 48

STEAM OUT (9 )REAGENT FUNNEL'

ADMISSION STEAM ADMISSION TIISC STOPCOCK (11) STEAM IN

I j 40 OJGBOUHD GLASS JOINT

(5lDlSHLLAHOh TUB (tlSTEAM GEN KEATON (2) KJELDAHL FLASK

(4JWIEE SCREEN

E. FLASK Si Figure 10. MicroKjeldhal Apparatus for In­ organic Nitrogen (44). 49

e. Open the reagent funnel stopcock (10) on the funnel at the top of the apparatus and admit 15 ml of concentrated sodium hydroxide.

f. Turn stopcock (11),on the steam admission tube straight down to permit steam to enter flask from steam generator. See figure 10.

g. Collect about 10 ml of distillate in the Ehrlenmeyer receiving flask (6). With normal boiling, five minutes are usually sufficient to collect 10 ml. The first drop of distil­ late-containing ammonia will cause the indi­ cator in the boric acid to change from red to blue-green.

h. To stop distillation do the following (IN THIS. ORDER):

1. Remove delivery tube (8) from Ehrlenmeyer receiving flask (6).

2. Reduce heat in steam generator (1).

3. Turn the steam admission stopcock (11) so that steam is directed out of the distil­ lation apparatus. (Position indicated in figure).

4. Open reagent funnel stopcock (9).

5. Remove Kjeldahl flask (2) by gentle twist­ ing and lowering of ring and screen (4).

6. Empty contents of the Kjeldahl flask into the sink and rinse the flask with dis­ tilled water.

2. Titration procedure

a. Titrate distillate in the Ehrlenmeyer flask with 1/14 N HCl until neutral end point (gray to very pale pink) is reached and record re­ sult.

b . . Calculations

1. General equation for calculating percent nitrogen: I

50 V acid x N acid x eg. wt. of N „ , M 1000 “ V ° in gms

V acid *= ml of acid used in titration N acid - 1/14 eq. wt. of N - 14 Sample weight - assume 1 ml of sample to equal 1 gm

Specific Gravity Measurements

The apparent specific gravity of the fermented wort was determined according to AOAC (38). A 10 ml pycnometer

(Ace Glass Inc., Vineland, N.J.) was used.

The pycnometer is first calibrated as follows: Pill the cleaned pycnometer with distilled water, stopper, and immerse in constant temperature water bath with bath level above graduation mark on pycnometer. After 30 minutes, re­ move stopper and with.capillary tube adjust water level until bottom of meniscus is tangent to graduation mark.

With small roll of filter paper, dry inside neck of pycnometer, stopper, and immerse in water at room tempera­ ture for 15 minutes. Remove pycnometer, dry, let stand for

15 minutes, and weigh. Empty pycnometer, rinse with acetone, and dry thoroughly in air with suction. Let empty flask come to room temperature, stopper,, and weigh. Wt. in air of contained H20 = wt. filled pycnometer minus Wt. empty pycnometer. Repeat the calibration procedure except this time fill the pycnometer with sample. Wt. in air of contained sample - Wt. pycnometer minus W. empty pycnometer.

The specific gravity is then calculated as S/W, where

S = Wt. sample, and W » Wt. H20.

Analysis of Variance

The "student's" t-test with paired observations was used to calculate the significance of differences between results obtained from two different fermentations.

The mathematical arrangements and calculations are de­ scribed in detail (40) and are summarized below:

1. Calculate the differences between two sets of

results and designate as D

2. Compute the mean and the variance of the differ-

ences

D « mean = (total number of samples)

2 SD = variance = n - 1

3. Calculate the standard deviation, S^- as

4. "t" is calculated as 52

"t" = (D - O) SD

5. Compare the value of *t' obtained from 4 to

another "t" value obtained from Appendix 6 (40) ,

"Percentage Points of the t-distribution."

6. If the calculated value of "t" is larger than the

one obtained from Appendix 6, then the results

are significant; otherwise, the reverse is true.

Yeast Cell Count

Samples from the fermenting wort were collected periodically. The samples were analyzed according to

Standard Method (53). Bacto Plate Count Agar (Difco) was u$ed to enumerate standard plate count. The growth curve wes constructed by plotting the number of yeast cells versus fermentation time. RESULTS

The composition of.the. wort .is the most important fac- tor which influences the production of the fusel alcohols during beer fermentation. In this study, attention was given to two aspects of wort composition and their effect on fusel alcohols and other characteristics of beer (a) source and concentration of free amino nitrogen, and (b) type and concentration of fermentable carbohydrate.

Two sources of amino nitrogen were studied, namely free amino acids and ammonium sulfate. To determine the role of carbohydrates, three situations were examined (a) addition of single carbohydrates to the wort, (b) adjustment- of the ratio of the three major sugars in wort— maltose, glucose, and sucrose, and (c) periodic addition of carbohydrate mix­ tures during the course of the fermentation.

Fusel alcohols in selected samples of commercial beer

At the outset, It was considered desirable to establish the fusel alcohol content of beer in order to obtain a qual­ itative and quantitative basis for comparisons in the suc­ ceeding studies with a defined wort. Five samples.of com­ mercial beer, including both popular and premium brandis,

53 were obtained from a local store and analyzed for fusel alcohols. The results are shown in Table 6. Samples 1-4 were domestic beers whereas sample 5 was imported. The range of total fusel alcohol content was from 70 to 230 ppm.

In all beers, 3-methyl-l-butanol was present in the highest concentration and constituted from 51 to 62% of the total

fusel alcohol content. 2-methyl-l-butanol was present in

the second highest concentration in four out of the five

samples, or from 15 to 24% of the total fusel alcohol con­ tent. N-propanol was present in the lowest concentration

in all beers with concentrations ranging from 3 to 14% of the total fusel alcohols. The low priced beer contained the lowest content of the total fusel alcohols and one of the high priced beer contained the highest content. How­ ever, the number of samples was too small to permit specu­ lation with respect to a relationship between gualtiy

(price) and fusel alcohol content of the beer.

Effect of Source and Concentration

of Free Amino Nitrogen (FAN) on

Selected Characteristics of Beer

Free Amino Acids

In this phase of the study, free amino acids served as the source of free amino nitrogen (FAN). The composition of the defined wort medium was adjusted to provide FAN concen­ trations of 150, 300, and 450-mg/l (Table 7). The lower Table 6: Fusel Alcohol Content of Some Commercial

Beer.

Beer Price n-propanol isobutanol 2-methyl- 3-methyl- Total 1-butanol 1-butanol

ppm (%) ppm (%) ppm (%) ppm (%) ppm

1 low 5.49 (7.91) 9.55 (13.77) 11.18 (16.12) 43.10 (62.17) 69.32

2 medium 19.00 (13.94) 21.00 (15.41) 25.85 (18.97) 70.40 (51.60) 136.25

3 medium 8.01 (9.36) 9.24 (10.78) 20.33 (23.73) 48.06 (56.11) 85.64

4 high 3.80 (3.15) 19.90 (16.51) 27.22 (22.58) 69.61 (57.75) 120.53

5 high 12.59 (5.41) 60.26 (25.92) 35.00 (15.04) 125.64 (54.04) 232.49

in in 56

Table 7: Amino Acids Composition of the Basic and the

Modified Fermentation Media.

# A. A. Concentration IX 2X 3X Component (mg/1) (mg/1) (mg/1) L. aspartic acid 78.29 156.58 234.87 L. threonine 54.28 108.56 162.84 L. serine 43.48 86.96 130.44 L. asparagine hydrate 149.17 298.34 447.51 L. glutamine 6.01 12.02 18.03 L. glutamic acid 90.24 180.48 270.72 L. prollne 316.56 633.12 949.68 Glycine 32.94 65.88 98.82 L. alanine 102.53 203.06 307.59 L. valine .108.56 217.12 325.68 L. methionine 27.13 54.26 81.39 L. isoleucine 57.52 , ‘115.04 172.56 L. leucine 141.29 282.58 423.87 L. tyrosine 93.02 186.04 279.06 L. phenylalanine 110.88 221.76 332.64 L. tryptophane 49.05 98.10 147.15 L. lys1ne-HCl 130.15 260.30 390.45 L. h1st1d1ne-HCl 59.03 118.06 177.09 L. arginine-HCl 155.90 311.80 467.70 Total FAN 150.00 300.00 450.00 57 level of FAN represented that present in the defined wort medium. The two higher levels were accomplished by doubling and tripling the concentration of the free amino acids.

Active yeast, Saccharomyces carlsbergensis, was added g to the medium at the rate of 15 x 1.0 yeast cells/ml (5 grams of wet.yeast per liter). Fermentation was carried out for 100 hours at 10+0.5°C with continuous agitation. Sam­ ples of beer were removed periodically and analyzed for:

1 - Fusel alcohols

2 - Carbohydrates

3 - Free amino nitrogen

4 - pH

5 - Specific gravity

Fusel alcohol formation: The effect of amino acid con­ centrations on the production of fusel alcohols is shown in

Table 8. The total fusel alcohols produced at 150, 300, and

450 mg/1 FAN (Table 8) were 119, 123, and 121 ppm, respec­ tively and were not significantly different ia the three worts with different FAN levels. Of the individual fusel alcohols, the production of n-propanol was the least

(8-10%), followed in order by 2-methyl-l-butanol (10-13.6%), isobutanol (14-24%) and 3-methyl-l-butanol (54-61%).

The concentration of isobutanol decreased from 28 to

17 ppm as the concentration of amino acids increased three­ fold. The concentration of n-propanol increased from 9.5 ppm at the single level of amino acids to 15.38 ppm at the 58

Table 8: Effect of Amino Acid Concentration on the

Production of Fusel Alcohols.

FAN n-propanol isobutanol 2-methyl- ' 3-methyl- Total 1-butanol 1-butanol Alco­ hols mg/1 ppm <%) ppm (%) ppm <%) ppm (%) ppm

150 9.53 (8%) 28.52 (24) 15.76 (13) 65.31 (55) 119.12

300 15.38 (12) 19.80 (16) 12.37 (10) 75.85 (62) 123.40

450 12.50 (10) 17.45 (14) 16.66 (13) 75.18 (62) 121.79 double level then decreased to 12.50 ppm at the triple level

of amino acids in the wort. However, the concentration of

3-methyl-l-butanol increased- by 10 ppm when the concentra­

tion of amino acids was doubled and was the same at the

triple level of amino acids. The concentration of 2-me.thyl-

1-butanol was identical at the single and triple and less at

the double level of amino acids in the wort.

The rate of formation of individual fusel alcohols

during fermentation is illustrated in Figures 11-13. Some

effect of FAN level on fusel alcohol formation is apparent

but, generally, the rate of increase decreased after 40-60

hours of fermentation. Aldo, the order of concentration of fusel alcohols remained constant throughout the fermenta­

tion period.

Carbohydrate Utilization; Utilization of carbohydrates during fermentation of the wort as a function of amino acid concentration is shown in Figures 14, 15, and 16. Of the

three major carbohydrates, sucrose and glucose were uti­

lized completely during the first 32 hours of fermentation.

In.contrast, some maltose remained in the medium even after

102 hours. There was ho direct relationship between the concentration of amino acids in the wort and the rate of utilization or the amount of carbohydrates remaining in'the medium. In the wort with 150 mg/1 FAN, 8.15 mg maltose per ml wort remained in the medium, while with 300 and .450 mg/1 FAN, 5.21 and 8.09 mg maltose per ml wort remained, 60

CL.

U

isobutanol

e— a 2-methyW-butanol

•— —•n-propanol

TIME (HRS)

Figure 11.. Effect of Amino Acid Concentration on the

Production of Fusel Alcohols - FAN = 150 mg/1 iue1: feto mn cdCnetaino the on Concentration Acid Amino of Effect 12: Figure FUSEL ALCOHOLS (PPM) 80 60 Production of Fusel Alcohols - FAN FAN - Alcohols Fusel of Production 20 0 4 20 IE (HRS) TIME 60 100 =300 n - propanol - n ethyl-l-butanol -m 2 isobutanol mg/1

iue1s feto mn oaCnetaino the on Concentration Aoia Amino of Effect 13s Figure FUSEL ALCOHOLS (PPM) rdcino ue loos - Alcohols Fusel of Production 80 * 0 6 20 - ' 20 IE (HRS) TIME 60 h a p = 450 mg/1 450 = 2-methyl-l-butano! ■ - isobutanol * 3-methyM-butanol n - - propanol n

Figure 14; Effect of Amino Acid Concentration on the on Concentration Acid Amino of Effect 14; Figure MG CHO/IML Utilization of Wort Sugars - FAN = 150 mg/1. 150 = FAN - Sugars Wort of Utilization 80* 60i 20J IE (HRS) TIME oSUCROSE a —o o— 60 GLUCOSE MALTOSE

63 iue1: feto mn cdCnetaino the on Concentration Acid Amino of Effect 15: Figure MG CHO/I ML WORT 0 4 Utilization of Wort Sugars - FAN » 300 mg/1. 300 » FAN - Sugars Wort of Utilization I ( ) S R (H E TIM GLUCOSE MALTOSE SUCROSE 64 MG CHO/ML WORT Figure 16: Effect of Amino Acid Concentration on the on Concentration Acid Amino of Effect 16: Figure * 0 4 20 60 Utilization of Wort Sugars - FAN * 450 mg/1. 450 * FAN - Sugars Wort of Utilization ; IE HS . (HRS) TIME MALTOSE GLUCOS E GLUCOS SUCROSE

66 respectively.

Free Amino Nitrogen Utilization; Results in Figure.17

show the effect of amino acid concentration on free amino nitrogen utilization. At the 150 mg/1 level (curve A),

free amino nitrogen decreased during the fermentation up

to 54 hours. After that, the free amino nitrogen level re­ mained constant. In contrast, at the 300 and 450 mg/1 FAN

levels (curves B and C) a steady decrease occurred in con­ centration throughout the fermentation. The slope of curve

A shows that the utilization of free amino nitrogen in the wort with 150 mg/1 FAN reached zero order after 54 hours of

incubation. This may indicate a shift in the metabolic pathways involved in fusel alcohol production. This, in

turn, may account for the differences in the proportion'of

individual fusel alcohols of this wort (Table 8) in compari­

son with the worts containing 300 and 450 mg/1 FAN.

Amino Acid Utilization; Fusel alcohols may be formed

from amino acids (8). The metabolic steps involved are in order (a) deamination to alpha keto acids, (b) decarboxyla­

tion to aldehydes, and (c) reduction to fusel alcohols.

The amino acid precursors of the major fusel alcohols found

in beer are:

Threonine n-propanol

Valine isobutanol

Leucine 3-raethyl-l-butanol

Isoleucine 2-methyl-l-butanol iue 7 Efc fAioAi ocnrto n the on Concentration Acid Amino of Effect 17: Figure FAN MG/L 300 500 Utilization of Free Amino Nitrogen during Simulated during Nitrogen Amino Free of Utilization Beer Fermentation. Beer . IE (HRS) TIME f0 u 6 B =300 mg/l =300 B A= mg/l 150 C =450 mg/l =450 C

(T '(

67 68 The rate of disappearance of the amino acids from the

three media with free amino acids as the source of nitrogen was followed during the fermentation.

Figure 18 is a representative chromatogram of the

amino acid analyses showing the patterns after 43 hours of

fermentation in the wort with 150 mg/l FAN. Detailed re­

sults in Tables 9, 10 and 11 show the calculated concentra­ tions of amino acids after 0, 24, 32, 43, 54, 67 and 89

hours of fermentation in the three lots of wort with 150,

300, and 450 mg/l FAN, respectively.

Utilization of amino acids during fermentation was a

function of the specific amino acids present and their ini­ tial concentration. Generally, more amino acid, regardless of species and concentration, was utilized during the first

24 hours of fermentation than during any subsequent time in­ terval. Also, the amount of a given amino acid utilized for the duration of the fermentation was directly related'to the

initial concentration whereas the percentage utilization was inversely related.

With respect to utilization of specific amino acids, of the non-fusel alcohol precursor amino acids, methionine was completely utilized after 32 hours of fermentation in the wort with a single amino acid level (150 mg/l FAN)• In comparison, 9% and 23% of methionine remained after 89

hours of fermentation in the worts with double and triple

amino acid levels, respectively. Alanine was the least x *

_g CL < •3

( 3 < D 5 —3j|3£S ni0- S o» CL * Q> CO LAJ II L1X 20 80

ELUTION TIME (MIN) rigor* It. Chromatogram of Amino Acids .Analyzed with Automated Amino Acid Analyzer. (Sample Obtained from the Wort with 150 mg/l FAN after 43 Hours of reraentatlon) • 70 Table 9. Changes in Amino Acids during Fermentation

of Wort (FAN = 150 mg/l).

TIME (hrs.) A.A mg/1 0 24 32 43 54 67 89 X-UTILIZED

Aspartic 78.29 58.85 47,90 41.36 36.10 29.40 24.15 69.11

-- • m Threonine^■ & 54.28 98.59 8.50 2.00 3.50 96.61

Seri ne * ---- m • m m ------A I 43.48 Glutamic * 90.24 65.63 32.95 30.55 31.70 M I 32.70 32.55 63.92 Prollne ' 316.56 — -- M * --—— Glycine 32.94 20.25 17.76' 17.30 15.80 14.40 13.50 59.01 Alanine 102.53 66.00 57,B0 57.10 53.40 50.07 46.65 54.50 Valine 108.56 60.30 52.60 45.25 42.40 36.30 29.50 72.68

Methionine 27.13 15.00 -- — -- — -- 100.00 Isolcuclne 57.52 40.15 25.95 16.60 13.35 9.85 7.00 87.83 Leucine 141.29 90.80 45.45 15.45 B.10 3.90 1.85 98.69 Tyrosine 93.02 66.15 57.65 53.10 49.40 44.30 39.50 61.43 Phenylalnlne 110.88 68.10 59.55 45.15 39.90 32.60 25.85 76.68 Histidine 59.03 27.10 16.00 6.35 4.95 3.00 1.20 97.96 Lysine 130.15 45.10 25.15 20.05 18.15 15.20 12.80 90.16

Ammonia 19.55 16.21 20.25 10.05 8.65 11.60 —

Tryptophane3^ 49.05 -- 20.45 15.45 18.72 15.40 10.18 79.24 Arginine 155.90 66.60 34.55 27.35 24.50 20.85 17.46 88.80

Glutamine^ 6.01 — —— — -- -- *

Asparagine1^ 149.17 -- — «*• ‘ — FAN mg/1 150.00 100.85 67.42 57.88 45.27 40.70 38.55 —

^Glutamine, asparagine and sertne eluted together and also Interfered somewhat with threonine. The results for threonine after onset of fermentation are presented as the total of all four of these amino acids. 21'The concentration of proline was not calculated since It was measured at 570 and not 440nm. 3^At times did not give sharp peftks for the computer to Integrate. Table 10. Changes in Amino Acids during Fermentation

o£ Wort. (FAN = 300 mg/l).

TIME (hrs.) A.A mg/1 0 24 32 54 67 89 t UTILIZED

Aspartic 156.58 107.48 105.53 87.56 82.52 74.91 52.15 Threonine1* 108.56 — 312.86 26.26 21.79 6.11 98.78 Serine1* 86.96 ------— -- — Glutamic 180.48 27.84 19.33 30.68 83.00 « t —— Pro11n o f 633.12 -- toto -- ton -- — Glycine 65.88 48.83 40.75 41.23 37.53 40.16 39.04 Alanine 203.06 141.17 126.00 138.09 124.79 136.12 32.96 Valine 217.12 145.90 126.77 117.23 112.29 113.63 47.66 Methionine 54.26 19.26 25.57 6.82 7.99 4.68 91.37 Isoleucine 115.04 79.68 71.67 59.12 61.74 54.81 52.35 Leucine 282.58 156.47 164.90 89.10 95.75 72.61 74.30 Tyrosine 186.04 133.15 117.12 115.18 106.38 115.31 38.01 Phenylalanine 221.76 156.81 135.69 123.65 120.47 115.63 47.85 Histidine 118.06 55.20 57.07 23.82 23.16 27.07 77.07 Lysine 260.30 109.27 99.20 74.13 68.60 72.99 71.95 Ammonia -- 19.77 13.78 19.57 16.84 22.67 —

Tryptophane 98.10 60.19 -- 46.62 — 47.06 52.02 Arginine 311.80 133.53 136.25 110.86 100.41 104.27 66.55

Glytamine1* 12.02 12.02 — — ------

Asparagine1* 298.34 29B.34 — ------FAN mg/l 300.00 172.27 163.00 119.76 122.76 125.43 --

*Glutamine, asparagine and serine eluted together and also Interfered some* what with threonine. The results for threonine after onset of fermentation are presented as the total of all four of these amino acids. Z *The concentration of prollne was not calculated since it was measured at 570 and not 440nm. 31 'At times did not give sharp peaks for the computer to Integrate. 72

Table 11. Changes in Amino Acids during Fermentation

of Wort. (FAN » 450 mg/l).

TIME (hrs.) A.A mg/l 0 24 32 43 54 67 89 X UTILIZED

Aspartic 234,87 215.14 195.12 181.25 169.10 170.70 27.32 Threonine1) 249.01 174.00 108.18 77.60 1 \ 162.84 122.90 67.95 91.03 Serine ' 130.44 -- — -- — —— -- Glutamic 270.72 260.92 234.85 244.00 207.90 217.40 218.30 21.24 P r o l l n e * 949.68 — ------— • • Glycine 98.82 87.71 75.71 76.12 70.65 65.05 64.40 34.83 1 Alanine 307.59 303.64 278.07 276.25 243.65 239.40 234.70 23.69 Valine 325.68 277.14 242.57 230.43 220.05 187.95 185.15 43.14 Methionine 81.39 43.71 33.71 28.56 29.90 20.95 18.75 76.96 Isoleuctne 172.56 155.71 141.57 120.68 115.85 99.65 97.10 43.72 Leucine 423.87 315.07 252.92 206.68 206.40 160.45 151.55 64.17 Tyrosine 279.06 268.35 231.35 217.50 196.60 183,85 181.55 34.94 Phenylalanine 332.64 252.28 215.35 200.43 186.45 163.65 160.45 51.76 Histidine 177.09 26.07 80.00 88.18 70.40 72.50 64.00 63.86 Lysine 390.45 197.57 159.42 160.43 161.90 -- 128.05 67.20 Ammonia 29.64 28.57 15.75 14.90 15.45 18.90 -- Tryptophane 147.15 — 98,85 94.81 86.00 — 80.05 45.59 Arginine 467.70 456.07 396.07 379.75 346.60 323.90 320.90 31.38 Glutamlnel) 18.03 —— — —— -- — Asparagine1^ 447.51 —— 1 — — — -- ■ *** FAN mg/1 450.00 356.82 318.62 267.65 286.60 235.30 238.32 ■ *

^Glutamine, asparagine and serine eluted together and also Interfered somewhat with threonine. The results for threonine after onset of fermentation are presented as the total of all four of these amino acids. 2) 'The concentration of prollne was not calculated since It was measured at 570 and not 440nm. . 3V t times did not give sharp peaks for the computer to Integrate. 73 utilized amino acid. The relative percentage of utilization after 89 hours for the worts with single, double, and triple

levels of amino acids, were 54, 33, and 24, respectively. i i The levels of total free amino nitrogen in the worts during fermentation calculated from the amino acids remain­

ing in Tables 9, 10 and 11 were in agreement with values for

FAN obtained by the ninhydrin method (Figure 17). For example, the calculated FAN values at 24 and 54 hours of

fermentation for the wort with single level of amino acid were 108 and 45 mg/l (Table 9) while the ninhydrin measured

FAN at the same fermentation times was 103 and 51 mg/l, respectively.

With respect to utilization of the four fusel alcohol precursor amino acids, threonine (including serine, glutamine and asparagine) disappeared rapidly from the worts and was 91-99% utilized after 89 hours. Valine was

43-73% utilized, leucine 64-98%, and isoleucine 43-88%.

Again percentage utilization was inversely related to the initial concentration of the amino acid in the wort.

The relationship between the percentage utilization of these four amino acids and the formation of the correspond­ ing fusel alcohols is illustrated in Figures 19, 20, 21, and 22. For threonine, only the values for the wort with a single level of amino acids were plotted, and the

"threonine" value at 24 hours was omitted from the plot.

It may be noted (Figure 19) that when threonine was 74

100.

60.

40-

2 0

004 0.08 012 0J6

n-propanol Formed (mM)

Figure 19. Formation of n-propanol and Utilization of

"Threonine*' during Wort Fermentation.

A - 150 mg/l FAN

Values were plotted at 0, 32, 43, 54, 67 and 89

hours of fermentation. XJ

>8 * 2 0

0.b8 0.16 0'24 0.32 0.'40 Isobutanol Formed (mM)

Figure 2d. Formation of Isobutanol and Utilization of

Valine during Wort Fermentation.

A - 150 mg/l FAN

B - 300 mg/l FAN

C - 450 mg/l FAN * •

Values were plotted at 0, 24, 32, 43, 54, 67, and 89

hours. 76

X> 0> N

0> £ 40. o 3 Q> —I 20.

3-methyUJ>iitanol Formed (mM)

Figure 21. Formation of 3-methyl-l-butanol and Utilization

of Leucine during Wort Fermentation.

A - 150 mg/l FAN

B - 300 mg/l FAN

C - 450 mg/l FAN

Values were plotted at 0, 24, 32, 43, 54, 67, and 89

hours. Figure 22. Formation of. 2-methyl-l-butanol and Utilization and of. 2-methyl-l-butanol Formation 22. Figure % Isoleucine Utilized 100. . 0 6 20 . 0 8 40. 10m/ FAN mg/l 150 - A Values were plotted at 0, 24, 32, 43, 54, 67, and 89 and 67, 54, 43, 32, 24, 0, at plotted were Values FAN mg/l 450 - C 30m/ FAN mg/l 300 - B hours. Fermentation. Wort during Isobutanol of - ty Luao Fre (mM) Formed -Lbutanol ethyl 2-m 64 00 01 01 0'0 0.'24 0.'20 0.16 0.12 0.08 4 .6 0

77

78 exhausted (32 hours and up) the production of n-propanol ceased. Values for threonine utilization— n-propanol pro­ duction for the double and triple levels wort were not * plotted because residual threonine could not be distin­ guished from residual serine, glutamine, and asparagine.

For the other relationships, valine-isobutanol (Figure

20), leucine-3-methyl-l-butanol (Figure 21), and isoleucine-

2-methyl-l-butanol (Figure 22), the production of fusel alcohol appeared to be dependent upon amino acid concentra­ tion. Only at the highest initial concentration of amino acids was there a straight line relationship between fusel alcohol production and amino acid utilization.

Results in Table 12 shows the molar ratios of amino acid utilized to fusel alcohol produced at the time the rate of alcohol prodnction diminished. These results indi­ cate that the conversion of leucine to 3-methyl-l-butanol

(2:1) was the most efficient, followed by the conversion of isoleucine to 2-methyl-l-butanol (4:1) and valine to isobutanol (10:1).

Basically, the total production of fusel alcohols dur­ ing fermentation was independent of amino nitrogen level.

However, the amino acid precursor fusel alcohols and the carbohydrates precursor fusel alcohols were dependent on the amino nitrogen level. There was a direct relationship be­ tween amino acid precursor fusel alcohols and the nitrogen level in the medium and an inverse relationship with the 79

Table 12: Molar Ratios of Fusel Alcohols Produced to Amino Acid Utilized

Amino Acid FAN level A. A. Fusel Alco­ Ratios Utilized hol Formed* mg/l mMole mMole o CO Leucine 150 • 0.4 2.0

300 0.9 0.5 1.8

450 2.0 0.9 2.2

Valine 150 0.4 0.05 8.0

300 0.8 0.08 10.0

450 1.2 0.21 5.7

Isoleucine 150 0.25 0.07 3.5

300 0.4 0.1 4.0

450 0.6 0.17 3.52

*Fuse1 alcohols formed are 3-methy1-1-butanolt Isobutanol, and 2-methyl- l-butanol for the corresponding amino acids leucine, valine and Isoleucine. 80 carbohydrate precursor fusel alcohols (Table 13). For ex­ ample, at the 150 mg/l FAN level, total fusel alcohol forma­ tion was 119.12 ppm with 48.13 ppm originating from amino acids and 70.99 ppm originating from carbohydrates. At the

450 mg/l FAN level, the total concentration of fusel alco­ hols formed without n-propanol was 109.29 ppm (Table 8) with 101.06 ppm originating from amino acids and only 8.23 ppm originating from carbohydrates.

pH and Specific Gravity; The changes in the pH of the worts as a function of FAN level during fermentation are shown in Figure 23. There was no differences in pH of the three worts. The pH decreased from 7.85 to 6.3.

The changes in specific gravity of the worts are shown in Figure 24. The initial specific gravity of the worts was 1.020. The final values ranged from 1.0055 to 1.0075.

There was no apparent relationship between the original FAN level in the wort, utilization of carbohydrate and FAN and formation of fusel alcohols, and the final specific gravity.

Ammonium Sulfate

Much information is available in the literature on nitrogen utilization' by yeast. Most ammonium salts and urea can serve as the sole nitrogen source for brewer's yeast.

This phase of study was conducted to determine the effect of using ammonium sulfate as the source nitrogen (ammonia nitro gen) on the production of fusel alcohols in the defined wort e i Table 13: Relative Contribution of Carbohydrates to Fusel Alcohols 1n a Fermentation with Amino Acids as Source of Nitrogen

Nitrogen level (mg/l) 150 300 450 Total n-propanol Formed (ppm) 9.53 15.38 12.50 n-propanol Formed from A.A. (ppmf 3.00 X X n-propanol Formed from CHO's 6.53 XX Total Isobutanol Formed (ppm) 28.52 19.80 17.45 Isobutanol Formed from A.A. (ppm) 3.70 5.93 15.56 Isobutanol Formed from CKO (ppm) 24.82 13.87 1.89 Total 2-methyl-l-butanol Formed (ppm) 15.76 12.37 16.66 2-methyl-l-butanol from A.A. (ppm) 6.17 8.81 14.98 2-methyl-l-butanol from CHO (ppm) 9.59 3.56 1.68 Total,3-methyl-1-butanol Formed (ppm) 65.31 75.85 75.18 3-methyl-1-butanol from A.A. (ppm) 35.26 44.07 70.52 3-methyl-1-butanol from CHO (ppm) 30.05 31.78 4.66 Total Fusel Alcohols Formed (ppm) 119.12 123.40 121.79

aThe concentration of fusel alcohols formed from amino acids were obtained from Table 12.

^Fusel alcohols formed for carbohydrates were obtained by subtracting the values in (a) from the total concentration of the fusel alcohol (Table 8). 82

72.

x 6 -6 - Q. 6.4.

I 20 40 60 80 100 TIME (HRS) figure 23. Effect of Amino Acid Concentration on Wort pH. (PH changea were similar to the three levels of amino acids— 150, 300, and 450 mg/l FAN). Figure 24: Effect of Amino Acid Concentration on Wort on Concentration Acid Amino of Effect 24: Figure SPECIFIC GRAVITY 1.020 1.0050 1.0070. I.0I90J\. 1.0170 1.0150- 1.0130, 1 . Specific Gravity. Specific 0110 . 0 0 0 100 80 60 0 4 20 I (HRS) E TIM = 1 0 gl FAN A= mg/l 150 =0 gi FAN mg/iB=300 =5m/ FAN C=450mg/I

84 The defined wort was modified in that the amino acids were replaced with ammonium sulfate. Three lots of wort were included with varying ammonium sulfate concentration equal to total free amino nitrogen 150, 300, and 450 mg/1.

The experimental conditions were identical to those used in the previous phase of the study.

Fusel Alcohol Production; The concentration of fusel alcohols produced are shown in Table 14. The results show that the highest amount of total fusel alcohols was produced in the wort with 300 mg/1 ammonia nitrogen (Amm. N) (80 ppm) followed by the wort with 450 mg/1 Amm. N (58 ppm) and the one with 150 mg/1 Amm. N (40 ppm).

The order of concentration of the individual alcohols varied for the different lots of wort. However, in all worts, 3-methy1-1-butanol was produced in the highest con­ centrations and 2-methy1-1-butanol in the lowest, whereas the relative concentrations of isobutanol and n-propanol varied for the lots.

The rate of formation of fusel alcohols during fer­ mentation is illustrated in Figures 25-27. Some effect of nitrogen level on fusel alcohol formation is evident. The rate of fusel alcohol formation increased with time of fer­ mentation. Also, the initial order of concentration of fusel alcohols remained constant throughout the fermentation period. Table 14: Effect of Ammonium Sulfate Concentration on

the Production of Fusel Alcohols

" ■ ■" ■ "

Res. N n-propanol isobutanol 2-methy1- 3-methy1- Total 1-butanol 1-butanol mg/1 ppm (%) • ppm (%) ppm (%) ppm (%)

150 8.55 (29) 6.60 (16) 5.16 (13) 16.48 (41) 39.79

300 14.26 (21) 22.77 (28) 13.01 (17) 27.40 (34) 79.44

450 11.24 (24) 14.44 (25) 9.05 (7) 25.46 (43) 63.19

0 0 tn Figure 25: Effect of Ammonium Sulfate Concentration on Concentration Sulfate Ammonium of Effect 25: Figure FUSEL ALCOHOLS (PPM) 1 “ - 0 1 the Production of Fusel Alcohols - Residual Nitrogen Residual - Alcohols Fusel of Production the 150 mg/1 150 2irethy!-l-butanol 3 methyl-1-butanol 3 isobutanol n-propanol O b ; H I

x

86

Figure 26: Effect of Ammonium Sulfate Concentration on Concentration Sulfate Ammonium of Effect 26: Figure FUSEL. ALCOHOLS (PPM) k 30* J =300 the Production bf Fusel Alcohols - Residual Nitrogen Residual - Alcohols Fusel bf Production the 10 ' O mg/1 0 0 100 60 20 IE (HRS) TIME n-propanol methyl-l-butanol 3 isobutanol 2 methyl-I-butanol 2 i

87

FUSEL ALCOHOLS (PPM) Figure 27: Effect of Ammonium Sulfate Concentration on Concentration Sulfate Ammonium of Effect 27: Figure ■ 450 mg/1, 450 ■ the Production of Fusel Alcohols - Residual Nitrogen Residual - Alcohols Fusel of Production the IE HS ' (HRS) TIME 3 methyl -l-butanol methyl 3 methyl-l-butanol 2 isobutanol n-propanol 60 . 100

88

%

89

Carbohydrate Utilization: Utilization of carbohydrate as a function of ammonium sulfate concentration is shown in

Figures 28, 2 9 , and 30. Of the three major carbohydrates, sucrose was completely utilized after 45 hours of fermenta­ tion, and glucose after 50 hours regardless of ammonia nitrogen concentration. In contrast, approximately, 10-15% of the maltose remained after 100 hours. There was no apparent relationship between the rate of carbohydrate uti­ lization and ammonium sulfate concentration of the wort and fusel alcohol production.

Ammonia Nitrogen Utilization; Results in Figure 31 show the effect of ammonium sulfate concentration on ammonia nitrogen utilization during fermentation. In the fermentation at 150 mg/1 Amm. N, the nitrogen decreased sharply through the first 45 hours followed by a decline in the rate of utilization for the remainder of the period. In contrast, the worts with 300 and 450 mg/1 Amm. N showed a steady and uniform decrease in nitrogen throughout the fer­ mentation period. The fact that there was a sharp change in the rate of utilization in nitrogen at the 150 mg/1 Amm*

N level may indicate a possible shift in the metabolic pathways involved in fusel alcohol production. The 150 mg/1 Amm. N in the form of ammonium sulfate may definitely have been a limiting factor in the production of total fusel alcohol (Table 14). Figure 28: Effect of Ammonium Sulfate Concentrations on Concentrations Sulfate Ammonium of Effect 28: Figure MG. CHO/ML WORT 0 8 Ammonia Nitrogen = 150 mg/1. 150 = Nitrogen - Ammonia Sugars Wort Individual of Utilization the 20 IE (HRS) TIME glucose maltose sucrose

100 90 Figure 29: Effect of Ammonium Sulfate Concentrations on Concentrations Sulfate Ammonium of Effect 29: Figure MG CHO/ML WORT 80 20 Ammonia Nitrogen » 300 mg/1. 300 » Nitrogen Ammonia 40 the Utilization of Individual Wort Sugars - Sugars Wort Individual of Utilization the - 20 IE (HRS) TIME 60 maltose sucrose glucose

92

I— c c o maltose 3 . 6 0 glucose sucrose v % o _ _ x 40 o

TIME (HRS) Figure 30: Effect of Ammonium Sulfate Concentrations on

the Utilization of Individual Wort Sugars -

Ammonia Nitrogen = 450 mg/1. Figure 31: Effect of Ammonium Sulfate Concentration on Concentration Sulfate Ammonium of Effect 31: Figure AMM. N MG/L 5Q0 the Utilization of Ammonia Nitrogen during Nitrogen Ammonia of Utilization the Simulated Beer Fermentation. Beer Simulated 0 60 20 IE (HRS) TIME

=300mg/l 0 0 3 B= =i0 g/l m i50 A= C = 4 5 0 mg/l 0 C 5 = 4

100

94 pH and Specific Gravity; The effect of ammonium sul­ fate concentration on the pH of the wort during fermenta­ tion is shown in Figure 32. The initial wort had a pH of

8.0. Minimum pH values were reached after 45 hours of fer­ mentation regardless of ammonium sulfate level. However, the decrease in pH was directly related to the initial ammonium sulfate concentration, with final pH values of

6.2 for 150 mg/l, 6.2 for 300 mg/l, and 6.15 for 450 mg/l ammonia nitrogen.

The changes in specific gravity of wort are shown in

Figure 33. The initial specific gravity was 1.0190, 1.0192, and 1.0194 for wort with 150, 300, and 450 mg/l ammonia nitrogen, respectively. #

Comparison of Results for the Fermentations

with Amino Acids and Ammonium Sulfate

as the Source of Nitrogen

Effect of Nitrogen Source on Fusel Alcohol Production

The "Student*s" t-test with paired observation was used to determine whether the concentrations of fusel alco­ hols produced in the fermentations with amino acids as the source of FAN were significantly different from those pro- duced with.ammonium sulfate as the source of ammonia nitro­ gen. The concentrations of n-propanol, isobutanol,

2-methyl-l-butanol, and 3-methyl-1-butanol produced at each of the three levels of amino acids were compared with those 95

CL

TIME (HRS) Figure 32. Effect of Ammonium. Sulfate Concentration on

Wort pH during Fermentation. Figure 33: Effect of Ammonium Sulfate Concentration Sulfate Ammonium of Effect 33: Figure SPECIFIC GRAVITY on the Specific Gravity during Fermentation. during Gravity Specific the on 10090. 1.021 1.0050 1.0070, 1.0130, 1.019 I.OIoOJ 1.0170. 1 . 0110

0 . ' 40 4 2'0 IE (HRS) TIME C=A50 C=A50 B=300 A=150 mg mg mg / / / l l l A A A mmonia mmonia mmonia

N N N 97 produced at the corresponding levels of ammonium sulfate.

The results (Tables 15-17) show that in all cases, the amount of the individual fusel alcohols produced was related significantly to the source of nitrogen.

With respect to the order of concentration of the in­ dividual. fusel alcohols as a function of nitrogen source and concentration, 3-methyl-l-butanol was produced in the highest concentrations followed by isobutanol regardless of nitrogen source and concentration. 2-methyl-l-butanol and n-propanol were produced in the lowest concentration with, however, the order in term of relative concentration being reversed for amino acid and ammonium sulfate.

Effect of Nitrogen Source on Carbohydrates Utilizationi

Sucrose and glucose were removed completely from the wort after 45 hours regardless of the source or initial level of nitrogen. However, in all cases, maltose remained in the medium, even after 100 hours, but in varying amounts

(Table 18). Maltose was utilized more completely in wort containing amino acids than in those with' ammonium sulfate.

When the level of free amino nitrogen supplied by amino acids was 150, 300, and 450 mg/l, the corresponding values of percentage of maltose utilized in the fermentation.medium were 89, 93, and 89% respectively. But, when the same levels of nitrogen were supplied by ammonium sulfate, the corresponding values for percentage maltose utilization were

80, 80, and 83% respectively. Table 15: Analysis of Variance of Nitrogen Source on the Production of Fusel Alcohols. FAN = 150 ing/1 versus 150 mg/l of Ammonia Nitrogen.

HOURS S0UPXE PPH PPH OF OF PRODUCED PRODUCED KEAN SIG­ FERHENTATION VARIANCE :AH-150sg/1 An.Na15Qng/l DIFFERENCE 0" VARIANCE V t NIFICANCE ID D* A 8 A-B*D (D-D’ )2 I(D-0’ )Z VARIANCE n n n * 45 4.92 3.61 1.31 0rl024 55 8.83 7.94 0.89 0.5476 8.84 8.35 0.49 1.63 1.2996 1.35 .52 3.13 66 propanol S 90 8.00 11.45 3.45 3.3124 t 100 9.53 11.55 2.02 0.1521 45 13.06 2.92 10.14 58.82 14.97 3.86 11.11 44.89 55 Iso­ 23.33 5.10 18.23 17.81 0.17 50.51 3.17 5.61 VS 66 butanol 90 30.76 6.43 24.33 42.51 •

100 31.87 6.60 25.27 55.65 * 45 8.87 1.34 7.53 20.16 13.00 1.39 11.61 0.16 55 2nnethy1- 16.97 2.63 14.34 12.02 5.38 7.08 1.18 10.18 VS 66 1-butanol 50 17.79 4.74 13.05 1.06 ICO 18.76 5.16 13.60 1.58 45 51.75 4.53 47.22 9.61 55 3-aethyl- 53.54 5.62 47.92 5.76 . 18.35 26.34 66 1-butanol 61.33 11.44 49.89 50.32 0.18 1.91 vs 90 72.98 15.20 57.78 55.65 100 65.31 16.48 48.83 2.22 S - SIGNIFICANT VS - VERY SIGNIFICANT *0.05(4) “ 2,77 Table 16: Analysis of Valrance of Nitrogen Source on the Production of Fusel Alcohols. FAN 3 300 mg/l versus 300 mg/l of Ammonia Nitrogen

HOURS SOURCE PPH PPH OF OF PRODUCED PRODUCED HEAN SIG­ FERHENTATION VARIANCE rAN-3O0Dg/1 Am.N*300ng/1 DIFFERENCE D" VARIANCE V t • NIFICANCE

a BA-B-D 10 (D-D“ )2 E(D-D")ZVARIANCE D* n n n

45 9.72 6.00 3.72 1.36 9.94 8.50 1.44 1.23 55 n* 8.28 11.61 3.33 2.55 0.60 1.33 • 0.51 5.0 VS EG propanol 90 12.99 16.37 3.38 0.68 100 15.38 16.26 0.88 2.78 45 10.62 7.50 3.12 1.14 55 Iso- 13.08 13.36 0.28 3.13 GG butanol 15.76 16.93 - .1.17 2.05 0.77 1.27 0.50 4.1 s 90 19.83 17.08 2.75 0.49 100 19.80 22.77 2.97 0.84 ’ » 45 5.33 - 2.70 2.63 0.05 55 2-aethyl- 8.32 4.23 4.09 2.89 66 1 -butanol 10.37 6.97 3.40 2.39 1.02 1.67 0.57 4.19 S 90 12.49 11.27 1.22 1.36 100 12.37 13.01 0.64 3.06 . 45 41.93 7.54 34.39 47.47 55 3-nrthvl - 42.02 9.34 32.68 73.96 66 51.23 17.17 34.06 41.28 52.12 90 4.24 9.73 VS 90 82.92 26.64 56.28 225.00 100 75.85 27.40 48.45 51.40 S ■ SIGNIFICANT VS * VERY SIGNIFICANT t0.05(4) * 2,77• Table 17: Analysis of Variance of Nitrogen Source on the Production of Fusel Alcohols. FAN ■ 450 mg/l versus 450 mg/l of Ammonia Nitrogen

KJURS SOURCE PPM PM OF OF PRODUCED PRODUCED MEAN SIG­ FERfEtTATICK. VARIANCE FA fMSteg/l Ao.H*450ag/l DIFFERENCE 0* VARIANCE V . t NIFICANCE

k 10 E(0-0")Z VARIANCE D" n 8 A - B aD (D-0')2 n 0 n so-

45 . 6.92 7.18. 0.26 0.33 55 13.03 11.68 1.3S 0.26 66 13.03 11.05 1.93 0.84 1.29 0.49 0.31 2.73 s propanol 1 90 • 12.40 12.56 0.15 0.46 1.09 14.70 14.24 0.46 0.14 . ■ 45 10.24 5,61 __ __ 4.53 1.84 • 55 iso­ 12.10 12.57 0.57 7.29 56 butanol 14.76 9.17 5.59 3.27 5.38 3.77 0.86 3.80 s 90 15.25 12.70 2.55 0.51 100 • 17.45 14.44 3.01 0.06 45 } 11.71 3.19 8.52 4.45

*3 ! J j M t h u l - 3.38 5.12 3.26 9.92 £6 1-butanol 14.17 7.52 6.65 6.41 0.05 4.00 0.89 7.20 vs 50 15.25 9.24 6.01 0.16 100 16.65 9.05 7.61 1.44 45 i £6.12 8.62 47.50 26.01 I 72.64 12.76 59.88 52.99 55 _ 1-butanol 75.76 20.34 55.42 52.60 7.95 24.92 2.23 13.00 vs 90 ' 75.26 25.37 50.49 4.45 ■ 100 75.18 25.46 49.72 8.29 • s - SIGNIFICANT VS « VERT SIGNIFICANT

*0.05(4) ’ ZJ1 101

Table 18: Maltose Concentration (gm/1) After 100 Hours

of Fermentation as a Function of Nitrogen Source £ and Concentration.

Amino Acids as Source Ammonium Sulfate as Source of FAN (mg/l) of Nitrogen (mg/l)

150 300 *50 150 300 450

b 8.15 5.21 8.09 14.90 15.02 12.75

c 89 93 89 80 80 83

aInitial maltose concentration = 7 5 gm/1

kgm/1 of maltose remaining

c% maltose utilized during fermentation

i 102

Effect of Nitrogen Source on Free Amino Nitrogen Uti­

lization; The comparisons in Table 19 show that regardless

of whether free amino acids or ammonium sulfate were the

source of nitrogen, more of the available nitrogen was uti­

lized as the FAN level in the wort was increased from 150

mg/l to 450 mg/l. Nitrogen utilization was consistently

greater in the worts containing ammonium sulfate than in

those with amino acids. In the fermentations with amino

acids as the source of nitrogen at FAN levels of 150, 300,

and 450 mg/l, the corresponding values of FAN utilized

were 66, 65, and 50% respectively. But, when ammonium

sulfate was the source of nitrogen at 150, 300, and 450 mg/l ammonia nitrogen, corresponding values of nitrogen

utilized were 94, 75, and 70% respectively.

Role of Carbohydrates on Selected

Characteristics of Beer

Addition of Single Carbohydrate to Wort

The major carbohydrates present in wort at the level of

10% are maltose, glucose, and sucrose. Normally they are

present in the ratio of 11:3:1. Relatively little is known

about the consequences of variations in the fermentable car­

bohydrates on the production of fusel alcohols and other

characteristics of beer. However, before examining the

effect of varying the ratio of these carbohydrates, the

effect of each individual sugar was examined first. 103

Table 19: Free Amino Nitrogen as a Function of

Nitrogen Source and Concentrations.

Amino Acids as Source Ammonium Sulfate as Source of FAN (gm/1) of Nitrogen (mg/l)

150 300 450 150 300 450

99 195 220 141 226 311

66 65 49 94 75 70

amg/l nitrogen utilized during 100 hours of fermentation

^Percent nitrogen utilized during fermentation 104

In this phase of study, single sugars were added to the fermenting wort. Three lots of wort with ammonium sulfate as the source of nitrogen (450 mg/l nitrogen) were fermented under the same experimental conditions as previously stated except, samples were analyzed only at the end of the fermen­ tation period (100 hours).

As was mentioned earlier, the experiments were not con­ ducted in duplicate due to laboratory limitations. However, in order to determine reproducibility of the results, it was decided to carry out only the fermentation with single car­ bohydrates again after one month. The results obtained from the two experiments were within experimental errors +5% and are presented here as the average of the two experiments.

Fusel alcohol production: The effect of single sugar on the formation of fusel alcohols is presented in Table 20.

The lot with sucrose formed the largest amount of total fusel alcohol (27.50 ppm) followed by the lots with maltose and glucose, 21.00 and 15.74 ppm, respectively.

Of the individual fusel alcohols, 3-methyl-l-butanol was produced in the highest concentrations regardless of the sugar used. However, in the glucose lot, 3-methyl-l- butanol was formed at a concentration of only 4.63 ppm which was almost one-third of the amount formed with sucrose and maltose and considerably less than the amounts formed in the'previous experiments with a mixture of carbohydrates.

The formation of.the other three fusel alcohols was in Table 20: Effect of Single Carbohydrate Source on the Production of Fusel

Alcohols in Wort Fermentation.

Fusel Alcohol n-propanol Isobutanol 2-methyl- 3-methyl- . Total 1-butanol 1-butanol Sugar (ppm) (%) (ppm) (35) (ppm) (25) (ppm) (25) (ppm)

• Sucrose 7.69 27.96 4.1 14.90 4.01 14.58 11.70 42.54 27.50

Maltose 4.31 20.53 1.57 7.47 2.05 9.76 13.06 62.22 20.99

Glucose 4.10 26.04 4.29 27.25 2.72 17.28 4.63 29.41 15.74 106 general in the order of n-propanol, isobutanol, and

2-methyl-l-butanol, with sucrose resulting in the. highest levels of each of these fusel alcohols.

Other characteristics: The effect of single carbohy­ drates on carbohydrate and nitrogen utilization as well as

the final pH and specific gravity in the fermented wort is

summarized in Table 21. Utilization of glucose was 80% in comparison with sucrose and maltose which were 85% and

92.5%, respectively. Nitrogen utilization on the other hand was the highest for the lot with glucose at 71% com­ pared to 54% for each of the lots with sucrose or maltose.

The lot with glucose had a final pH of 6.15 compared with

5.9 for the other two lots. The final specific gravity at all three lots was similar.

Effect of the ratio of fermentable carbohydrates in wort on the formation of fusel alcohols; Generally, the wort contains 10% fermentable carbohydrates. The three major sugars are maltose, which comprises 75% of the total

fermentable sugars, glucose 18.2%, and sucrose at 6.8%, or

a ratio of approximately 11:3:1 of maltose:glucose:sucrose.

In industry, the temperature and extent of steeping is used

to exercise control on the carbohydrate’ composition of wort.

.To determine whether the fusel alcohols content of

beer might be affected by the ratio of the three major car­

bohydrates, an experiment was designed involving three lots

of wort with the following approximate carbohydrate ratios. 107

Table 21; Effect of Single Carbohydrate Source on

Carbohydrate and Nitrogen Utilization, and Final pH

and Specific Gravity of the Fermented Wort.

Fermentation Lot A B C Characteristic

Type of Sugar Sucrose Maltose Glucose

Initial CHO Cone, gm/1 100.00 100.00 100.00

% CHO Utilized 85.00 92.50 80.00

Initial Nitrogen Cone. 450.00 450.00 450.00

% Nitrogen Utilized ^54.00 54.00 71.00 « Final pH 5.90 5.90 6.15

Final Specific Gravity 1.0082 1.0082 1.0082 108

The actual amounts of carbohydrates are shown in Table 22.

Lot Maltos'e Glucose Sucrose

A 11 1 3,

B 3 11 1

C 1 ' 3 11

The choice of ammonium sulfate rather than amino acids as the source of nitrogen was based on the fact that am* monium sulfate does not cause inhibition or activation of enzymes that may be responsible for fusel alcohol produc­ tion such as may be the case with amino acids.

The experimental conditions were the same as previously stated. Because of laboratory limitations a control sample containing the carbohydrates in the normal ratio could not be included in this experiment. 'However, to provide some basis for comparisons, the results obtained in the experi­ ment using ammonium sulfate as the nitrogen source, at the

450 mg/1 Amm. N are reported as "Control."

Fusel alcohol production; The effect of the ratio of the major carbohydrates on fusel alcohols produced is shown in Tables 23 and 24 and Figures 34, 35 and 36. As shown in

Table 23, the amounts of the individual and total fusel alcohols produced after 100 hours of fermentation were con­ siderably less in the worts with adjusted carbohydrate ratio than those produced in the "Control" wort with normal ratio.

The least amounts of fusel alcohols were produced with 109

Table 22: Composition of Fermentable Sugars in the Wort

Experimental Wort Media3 with Varying Carbohydrate.

Lot Maltose Glucose Sucrose Total Sugars Amm. N gm/1 gm/1 gm/1 gm/1 gm/1

A 75.0 6.8 18.2 100.0 450 W GO CM B • 75.0 6.8 100.0 450

C 6.8 18.2 75.0 100.0 450 *

aNutrients other than those listed same as for the basic wort (Table 1). U As ammonium sulfate. Table 23: Effect of Carbohydrate Ratio on Fusel Alcohol

Production after 100 Hours of Fermentation.

Carbohydrate Ratio n-propanol isobutanol 2-methyl- 3-methyl- Total 1-butanol 1-butanol

Lot M G S ppm (%) ppm (%) ppm (%) ppm (%) ppm

* A 11 1. 3 8.50 (23) 7.50 (20) 6.22 (17) 15.00 (40) 37.22

B 3 11 1 8.50 (31) 5.14 (19) 3.46 (12) 10.33 (37) 27.43

C 1 3 11 13.39 (34) 6.60 (17) 4.88 (12) 13.93 (36) 38.80

Con­ trol 11 3 1 . 14.24 (22) 14.44 (23) 9.05 (14) 25.46 (40) 63.19

o Table 24: Analysis of Variance of Carbohydrates

Composition on the Production of Fusel Alcohols

Source of CHO's Comp. d.f. mean variance t Significance SD Variance* £(D-D)2 Control Batch 5 - P variance D . n-1 n n SD

n-propanol Control A 4 5.80 2.45 0.70 8.28 V.S. Control B 4 4.62 1.69 0.58 7.96 V.S. Control C 4 . 4.15 11.08 2.77 1.87 N.S. isobutanol Control A 4 6.51 8.67 1.31 4.96 • S. Control B 4 6.34 10.66 1.46 4.34 s. Control C 4 7.35 6.52 1.14 6.44 V.S. 2-methyl- 1-butanol Control A 4 3.01 3.10 0.78 3.85 s. Control B 4 4.51 2.77 0.74 6.09 V.S. Control C 4 3.91 2.28 0.67 5.83 V.S. 3-methyl- 1-butanol Control A 4 9.69 6.26 1.11 8.72 V.S. Control B 4 11.63 27.23 2.33 4.99 V.S. Control C 4 10.03- . 11.51 1.51 6.64 V.S.

S = Significant V.S. = Very Significant t0.04(4) “ 2,77 Figure 34 : Effect of Carbohydrate Ratio on the Rate of Rate the on Ratio Carbohydrate of Effect 34 :Figure FUSEL ALCOHOLS (PPM) ue loo rdcin (Maltose:Glucose:Sucrose: Production. Alcohol Fusel 75:6.8:18.2) Lot A. Lot 75:6.8:18.2) IE (HRS) TIME 2 methyl-I-butanol 2 3 methyl-l-butanol 3 n- propanol isobutanol

11*2 I 113

Q. CL n-propanol V) isobutanol _J O 2 m ethyl-l-butonol ~T~ o 20 3 methyl-l-butanol o

LU COz> 1 0 * Ll.

20 ' 6’0 ' l(5o TIME (HRS)

Figure 35: Effect of Carbohydrate Ratio on the Rate of

Fusel Alcohol Production. (Maltose:Glucose:Sucrose;

18.2:75:6.8) Lot B. Figure 36: Effect of Carbohydrate Ratio on the Rate of Rate the on Ratio Carbohydrate of Effect 36: Figure FUSEL ALCOHOLS (PPM) ue loo rdcin (Maltose:Glucose:Sucrose: Production. Alcohol Fusel 6.8:18.2:75) Lot C. Lot 6.8:18.2:75) 20 - 0 1 - IE (HRS) TIME 60 methyl-l-butano! 3 n-propanol isobutanol methyl-l-butanol 2

i

114 115 maltose, glucose, sucrose ratio of 3:11:1 (lot B) .

The "Student's" t-test with paired observations was used to determine whether the concentrations of fusel al­ cohols produced with different carbohydrate ratios were significantly different from the "Control." The results are shown in Table 24. The concentration of each individual fusel alcohol except n-propanol in lot C was significantly different from the "Control." This is a clear indication that the ratio of the fermentable carbohydrates in wort is a significant factor in controlling the fusel alcohols pro­ duced during fermentation.

With respect to the rate of alcohol production during fermentation, results in Figures 34, 35, and 36 show that the concentration of the individual fusel alcohols increased gradually during, the fermentation as was the case for the

"Control" (Figure 13). The order of formation and concen­ tration of the fusel alcohols was not affected by the ratio of the carbohydrates.

Carbohydrate utilization: Utilization of the carbohy­ drates during fermentation of the wort with varying sugars ratio is shown in Figures 37, 38, and 39. Maltose was still present in all fermented worts after 100 hours of fermenta­ tion regardless of initial concentration. Glucose was pres­ ent after the fermentation was complete when the initial concentration was 18.2 gm/1 or 75 gm/1 but was completely utilized when the initial concentration was 6.8 gm/1. 116

maltose 75, gm/1 sucrose I8 2 g m /I glucose 6 .8 gm/1

O X o

TIME (HRS)

Figure 37: Effect of Carbohydrate Ratio in Wort on the

Utilization of Individual Carbohydrates. (Maltose:

Glucose:Sucrose:75:6.8:18.2). Lot A. iue3: feto abhdaeRtoi oto the on Wort in Ratio Carbohydrate of Effect 38: Figure MG CHO/ML WORT 2Q GlucoseiSucrose:18.2:75:6.8) Lot B. Lot GlucoseiSucrose:18.2:75:6.8) tlzto fIdvda abhdae. (Maltose Carbohydrates. Individual of Utilization IE (HRS) TIME 60 scoe . gm/1 6.8 .sucrose m altose 182 gm/1 182 maltose lcs 7 gm/1 75 glucose

Figure 39: Effect of Carbohydrate Ratio in Wort on the on Wort in Ratio Carbohydrate of Effect 39: Figure MG CHO/ML WORT Glucose:Sucrose:6.8:18.2:75). Lot C. Lot (Maltose Glucose:Sucrose:6.8:18.2:75). Carbohydrates. Individual of Utilization IE (HRS) TIME 0 6 urs 7 gm/1 75 sucrose lcs 18.2 gm/1 glucose maltose 6.8 gm/1 6.8 maltose

118 119

Sucrose completely was utilized in all fermentations after

20-40 hours regardless of the initial concentration. How­ ever, sucrose lasted about twice as long at the 75 gm/1 concentration in comparison with the 18.2 gm/1 and 6.8 gm/1 ' concentration.

» * * Total fermentable carbohydrates utilized in the wort were 80% for the high maltose wort (lot A), 70% for the high glucose wort (lot B), and 86% for the high sucrose wort

(lot C). This compares with 83% utilized carbohydrate for the •'Control” lot. The results did not indicate a relation­ ship between fusel alcohol production and the utilization of individual carbohydrates.

Ammonia nitrogen utilization; Results in figure 40 show the effect of the carbohydrate ratio in wort on nitro­ gen utilization. Ammonia nitrogen concentration decreased in a straight line function from 450 mg/1 to 250 mg/1 in lot A, and to 305 and 300 mg/1 in lots B and C respectively.

In comparison, the ammonia nitrogen in the "Control” de­ creased to 92 mg/1 after 100 hours of fermentation. Again, there appeared to be no relationship between nitrogen uti­ lization and fusel alcohol production.

■pH and specific gravity; The pH of the worts with varying carbohydrate ratios decreased from a high of 7.8 to a low of 6.2. The rate of decrease in pH and the final value were the same regardless of carbohydrate ratio

(Figure 41). The pH changes were similar to changes which Figure 40: E££ect of Carbohydrate Ratio on Ammonia on Ratio Carbohydrate of E££ect 40: Figure AMM. N MG/L 500 300- 100- Nitrogen utilization. Nitrogen - 0 60 20 IE (HRS) TIME

100 7.8

7.4

7.0 x CL 6.6

6.2 20 60 100 TIME (HRS)

Figure 41; Effect of Carbohydrate Ratio on pH of Wort. 122 occurred in the "Control'' lot.-

The changes in the specific gravity of the worts with

different ratios of carbohydrates are shown in Figure 42.

The specific gravity for lot A with the initial high

maltose level changed from 1.0100 to 1.0064, which compared

with the specific gravity changes for the "Control" lot.

Lots B and C with high glucose and sucrose, respectively

had somewhat higher final specific gravity than lot A, i.e.

1.0074 and 1.0080, respectively.

Periodic addition of carbohydrates to the wort

In the previous experiments the entire amounts of car­

bohydrate were added to the worts before the beginning of

the fermentation. Thus, carbohydrates were not a limiting

substrate in fusel alcohol production.

Since carbohydrate limitation may affect fusel alcohol

production, a study was conducted in which carbohydrate (in

sterile solution) was added to the wort periodically during

progress of the fermentation. Three lots of wort desig­

nated as D, E, and F were included and a functional deBign

of carbohydrate addition was developed to incorporate a)

time of addition, b) amount of addition at each time, and

c) varying carbohydrate ratios (Table 25). Total carbohy-

♦ drate addition was 100 gm/1 of wort, 50 grams of which were

added at the outset followed by 25 grams addition after 24

and 48 hours of fermentation. The ratios of maltose,

-glucose, and sucrose were varied for the three lots as 123

>- H

O U- 1.0110J o Ll I w I.0090J

1.0070,

I.0 0 5 0 L 20 40 60 80 100 TIM E (HRS) Figure 42: Effect of Carbohydrate Ratio on Specific

Gravity of Wort. Table 25: Order of Addition and Composition of Carbohydrates in the Fermentation Medium.

Lot D £ F

Time of Addition (hours) CHO Composition gm/1 0 24 48 0 24 48 0 24 48

maltose 37.50 18.75 18.75 9.10 4.55 4.55 -3.40 1.70 1.70 glucose 3.40 1.70 1.70 37.50 18.75 18.75 9.10 4.55 4.55 sucrose 9.10 4.55 4.55 3.40 1.70 1.70 37.50 18.75 18.75

Total 50.00 25.00 25.00 50.00 25.00 25.00 50.00 25.00 25.00 125 shown on Page 107, Table 21. For each of the carbohydrates,

50% of the designated amount was added at the outset followed by 25% after 24 and 48 hours..

Ammonium sulfate at the 450 mg/1 ammonia nitrogen served as the source of nitrogen. Analyses were as out­ lined previously. .

Fusel alcohol production; The effect of periodic ad­ dition of carbohydrate in various ratios on fusel alcohol production is shown in Table 26 and Figures 43, 44, and 45. t Total fusel alcohol production (Table 26) was 26 ppm in lot

A, 45 ppm in lot B, and 36 ppm in lot C. In comparison, the total fusel alcohol production in the "Control" lot was

63 ppm.

The rate of production of individual alcohols as a factor of periodic carbohydrate addition is shown in Figure

43, 44, and 45. The concentration of each of the fusel alcohols increased gradually with time up to 85 hours of fermentation. (This was in contrast to one-time addition of carbohydrates in varying ratio). The order of fusel alco­ hols produced was similar to the "Control." In all cases,

3-methy1-1-butanol was produced in the highest concentration followed in order by 2-methyl-l-butanol, n-propanol, and isobutanol.

Carbohydrates utilization; The rate of utilization of carbohydrates during fermentation with periodic addition of carbohydrates is shown in Figures 46, 47, and 48. The Table 26: Effect of Periodic Addition of Carbohydrates on the Production of Fusel Alcohol

Fusel Alcohols n-propanol Isobutanol 2-methyl - 3-methyl- Total 1-butanol 1-butanol (ppm) (%) (ppm) (%) (ppm) (S) (ppm) (*) (ppm)

A 8.23 31.70 6.09 23.45 1.68 6.47 9.96 38.36 25.96

B 10.21 22.55 12.10 26.73 5.55 12.26 17.40 38.44 45.26

C 6.50 18.03 8.85 24.55 3.28 9.10 17.41 48.30 35.04

Control 14.24 22.53 14.44 22.85 9.05 14.32 25.46 40.29 63.19 Figure 43: Effect' of Periodic Addition of Carbohydrates of Addition Effect' Periodic of 43: Figure FUSEL ALCOHOLS (PPM) n h ae fFslAchlPouto; LtA). ) (Lot A Production; Alcohol Fusel of Rate the on 10 |u'w.|vMiuati —a»yw*jr*yi*Lwxi J|Mu4''wf.m|iv

IE (HRS) TIME * « 60 isobutanol - -o n-propanol 0 ■o methyl-l-butanol 3 methyl-1-butanol 2

/ O' wii

iihobb

i

i h rjTTT—ni “«3 "“ 100

127 Figure Figure FUSEL ALCOHOLS (PPM) n h aeo uq loo rdcin (Loton.D). FubqL Ratethe of Production.Alcohol 44 ; Effect of Periodic Effectof ofCarbohydrates Addition ; 3 isobutanol* n-propanol » methyl-l-butanol 3 2 m e t h y l - I - b u t a n o l IE (HRS) TIME

/ 128 n -propanol 129

1 8 a — * isobutanol c—o 2 methyl-l-butanol 3 methyl-l-butanol

14*

CL CL CO o 10 Xo o -J <

Ul ( f ) 3 Li-

25 ' 65 ' i(5cr TIME (HRS)

Figure 45: Effect of Periodic Addition of Carbohydrates

on the Rate of Fusel Alcohol Production.' (Lot C ) . Figure 46: Effect of Periodic Addition of Carbohydrates of Addition Periodic of Effect 46: Figure MG CHO/ML WORT nterUiiain [o . ] [Lot D Utilization. their on 50 30 IE (HRS) TIME glucose sucrose maltose m Figure 47: Effect of Periodic Addition of Carbohydrates of Addition Periodic of Effect 47: Figure MG CHO/ML WORT on their Utilization. [Lot E] [Lot Utilization. their on 60- 30- IE (HRS) TIME sucrose maltose glucose R5o 131 Figure 48: £££ect of Periodic Addition o£ Carbohydrates o£ Addition Periodic of £££ect 48: Figure MG C HO/ML WORT nterUiiain [LotF] Utilization. their on —glucose a— i IE (HRS) TIME 9 maltose sucrose

132 133 carbohydrates concentration in the wort is reflected by their periodic addition. Their concentration decreased with increase in time of fermentation and when carbohydrates were added, their concentration was increased, i.e. after

24 and 48 hours.

Maltose in lots D, E, and F and glucose in lot E only remained in the medium after 100 hours of fermentation.

This was true regardless of the initial concentration of maltose added. Sucrose was utilized completely in all three lots.

The rate of utilization of carbohydrates in the three lots was varied. In lot D, 83% of the sugars were utilized, while in lots E and F, 74 and 96% of the sugars were uti­ lized, respectively.

Ammonia Nitrogen Utilization; Results in Figure 49 show the effect of periodic addition of carbohydrates on nitrogen utilization. The level of ammonia nitrogen in the three lots decreased sharply in the first 80 hours of fer­ mentation followed by a decline in the fate of utilization for the remainder of the period. The ammonia nitrogen in lot D decreased from 450 to 254 mg/1 and to 290 and 306 mg/1 ammonia nitrogen for lots E and F, respectively. In con­ trast, the ammonia nitrogen in the "Control** decreased to

92 mg/1 after 100 hours of fermentation.

The leveling off in the rate of utilization of nitro­ gen and fusel alcohol production after 80 hours of Figure 49: E£fect of Periodic Addition of Carbohydrates of Addition Periodic of E£fect 49: Figure AMM.N MG/L on Ammonia Nitrogen. Ammonia on 100- IE (HRS) TIME 60 134 135 fermentation, may indicate a possible shift in the metabolic pathway involved in fusel alcohol production.

pH and Specific Gravity: The effect of periodic addi­ tion of carbohydrate on the pH of the wort during fermenta- « tion is shown in Figure 50. The final pH for lot D was 6.1 while the final pH for lots E and F was 6.2. The rate of change in pH for the lots E and F were identical.

The changes in the specific gravity of the three lots of wort are shown in Table 27. Lot D had the lowest spe­ cific gravity of 1.0077 while lot E had the highest specific gravity of 1.0081. The changes in specific gravity during fermentation reflect periodic carbohydrate addition. Ini­ tial values of specific gravity were lower than others, how­ ever, the final values were similar to those obtained from other experiments.

Yeast cell growth

Since individual fusel alcohol formation differed, in media with different levels of nitrogen, the question was raised as to whether these differences were related to vari­ ation in yeast cell growth. Therefore, it was decided, thereafter, to follow growth of yeast cells in the ferment­ ing wort.

The yeast growth in the three experimental worts with varying carbohydrate ratios is shown in Figure 51, while yeast growth in the three experimental worts with periodic addition of carbohydrates during the course of fermentation 136 Table 27: Effect of Periodic Addition of Carbohydrates

on Specific Gravity.

Time of Addition A B C

hours

0 1.0085 1.0084 1.0090

10 1.0072 1.0075 1.0081

24 1.0070 1.0071 1.0078

34 1.0083 1.0084 1.0093

48 1.0097 1.0080 1.0081

59 1.0089 1.0092 1.0103

72 1.0086 1.0085 1.0098

81 1.0085 1.0082 1.0089

93 1.0083 1.0080 1^ 0085

100 1.0077 1.0078 1.0081 137

X CL

A A i g + Q /\

— -TP- 4 0 6 0 8 0 100 TIME (HRS)

Figure 50i Effect of Periodic Addition of Carbohydrates on pH. 4* MJM 1 0 20 3 0 4 0 5 0 Fermentation Time (Hrs)

Figure 51s Yeast Cell Growth in the Experimental Medium

with Varying Carbohydrates Ratio. 139

to the wort is shown in Figure 52. The varius media (Fig­

ures 51 and 52) supported yeast growth essentially to the

same extent. Therefore, the observed differences in the

• production of fusel alcohol in' these media can not be at­

tributed to lack of growth, or to substantial differences

in yeast growth; in different media. ° 10 .

■— r - T“ 10 20 30 4 0 5 0

Fermentation Time (Hrs) Figure 52: Yeast Cell Growth in the Experimental Medium

with Periodic Addition of Carbohydrates during the

Course of Fermentation. DISCUSSION

This work was undertaken to study the effect of the source and concentration of nitrogen and carbohydrates in wort on some characteristics of beer.

A pH 7.9 defined medium that contained a source of nitrogen, fermentable carbohydrates, and other nutrients and minerals 'that are known to be factors in yeast growth was used as the fermentation medium. The medium was essen­ tially that of Jones et. al., (41) who had demonstrated the efficacy of using such a wort for beer fermentation studies.

The source of nitrogen was either amino acids, as

found in regular wort, or ammonium sulfate both of which were added in three different concentrations but at equiva­

lent nitrogen level. The fermentable carbohydrates were maltose, glucose, and sucrose which were added in concentra­

tions similar to those of regular wort, either singly, in

ratios as in wort, or in varying ratios at once or peri­ odically during fermentation.

In the present study, the concentrations of fusel

alcohols obtained upon fermenting the defined wort with

amino acids as the source of nitrogen were comparable with

those concentrations found in commercial beer analyzed in

141 142 our laboratory (Table 6) and values reported in the lit­ erature (7). In comparison, formation of fusel alcohols was greatly reduced when ammonium sulfate was the source of nitrogen and when the ratio of carbohydrates was varied.

The reduction in fusel alcohol production with ammonium sulfate and varying carbohydrate ratios raises the question of the ability of the medium to support yeast growth under such conditions. Lack of yeast growth would provide a ready explanation for reduced fusel alcohol production.

However, the two indicators of "normal" yeast activity used in this study, i.e. changes in pH and specific gravity of the wort, indicated that the overall yeast activity was apparently unaffected by variations in nitrogen source and carbohydrate ratio. Thus, the reduction in fusel alcohol production may be assumed to have been a function of • substrate variations, only.

Overall, the changes in pH during fermentation were from 7.9, the initial value, to 6.5-6.2 and the changes in

specific gravity, which is a measure of ethyl alcohol pro­ duction and carbohydrate utilization, were from the initial value of approximately 1.'020 to 1.0083-1.0057. Within each trial, the rates of decrease and the final pH and specific . gravity values were the same or very similar for each of

the three lots of wort.

The factors affecting the formation of fusel alcohols

in beer are numerous. However, the most important factor 143 appears to be wort composition, specifically with respect to nitrogen and fermentable carbohydrates (8). The,effect of nitrogen and carbohydrates is two-fold. Both the type and the concentration of these nutrients influence fusel' alcohol formation.

Nitrogen is required by yeast during fermentation to synthesize proteins and enzymes for reproduction. The amino acids needed for synthesis can be either assimilated from the fermentation medium or synthesized from the breakdown of carbohydrates and subsequent transamination of the alpha keto acids.

The level of nitrogen in the wort affects the ability of yeast cells to reproduce and synthesize the enzymes and proteins required, for reproduction. In a wort with inade­ quate nitrogen level, (FAN less than 120 mg/1) (4) the synthetic process and, 'thus, the ability of yeast cells to reproduce is impaired. In a wort with an adequate nitrogen level, in the form of amino acids, yeast cells usually assimilate the amino acids required for enzyme synthesis rather than synthesizing such amino acids. Naturally, when ammonium sulfate is the sole source of nitrogen in the fer­ mentation medium, then the carbon skeletons of the required amino acids originate from breakdown of the 'sugars in the medium and, by transaminase action, nitrogen is added to the carbon skeleton to produce the amino acids. 144

The rate of absorption of the Individual amino acids from the wort by the yeast varied. The rate of absorption was a function of amino acid species and initial concentra­ tion. Threonine, serine, glutamic acid, and methionine were absorbed at a greater rate than glycine, alanine, and a , tryosine. The order of absorption of amino acids was in agreement with Jones (19) w.ho had demonstrated that the amino acids of wort are absorbed in a definite order during fermentation.

With respect to utilization of the four fusel alcohol precursor amino acids, threonine, valine, leucine, and isoleucine disappeared rapidly from the wort during fermen­ tation. The mechanism for the formation of fusel alcohols from the above four amino acids was presented by Ehrlich

(8) and was illustrated in Figure 4. Therefore, it seems that there should be a parallel relationship between the order of absorption of amino acids and fusel alcohol forma­ tion. Previous attempts to demonstrate this relationship have been unsuccessful (49, 50).

The relationship between the rate of absorption of amino acids and fusel alcohol formation was illustrated.

At the 150 mg/1 FAN level, there was a direct relationship only at the early stages of fermentation as the amino acids precursor of fusel alcohols were being actively absorbed.

However, this relationship was true throughout the entire

fermentation period when the FAN level was increased to 145 450 mg/1. Also, as the PAN level was increased in the fer­ mentation medium, the concentration of the fusel alcohols originated from their parent amino acid increased, while the concentration of the fusel alcohols that originated from the carbohydrates decreased. However, the total concentration of fusel alcohols formed, i.e. from amino acids and carbohy­ drates, remained constant regardless, of the initial level of nitrogen in the medium.

The concentration of fusel alcohols that originated from carbohydrates was higher in the presence of ammonium sulfate rather than amino acids as the source of nitrogen.

This may be due to one or possibly two things. First, the yeast cells prefer to utilize the carbon skeleton of amino acid as the building block of a newly synthesized amino acid or secondly, amino acids cause a feedback inhibition on the system responsible for fusel alcohol production.

The fermentable carbohydrates in wort are the major energy source for yeast. Yeast can utilize the carbohy­ drates via glycolysis to produce two moles of ATP for every mole of glucose, or. via the tricarboxylic acid cycle to give 32 moles of ATP for every mole of glucose metabolized.

The utilization of carbohydrates in wort via glycolysis or

TCA cycle is determined by the relative magnitude of the

Pasteur and Crabtree Effects. The Pasteur Effect is utili­ zation of sugars by fermentation in the absence of oxygen and by TCA cycle in the presence of oxygen. The Crabtree 146

Effect is a concentration effect. Glucose at high concen­ tration is metabolized via glycolysis and at low concentra­ tion via the TCA cycle, regardless of oxygen availability.

The production of fusel alcohols in beer may therefore be related to the relative magnitude of the Pasteur vs. the

Crabtree Effect. In a fermentation where the Pasteur Effect is dominating, the concentration of fusel alcohols formed may be expected to be relatively low whereas the reverse may be true if the Crabtree Effect is overriding in the system.

For convenience, a. summary of the experimental data obtained using amino acids and ammonium sulfate as the sources of nitrogen in the fermentation medium is presented in Table 28.

With respect to the effect of nitrogen source on fusel alcohol production, it was found in this study, as would be expected, that amino acids resulted in greater concentra­ tions of fusel alcohols than ammonium sulfate. Particu­ larly, 3-methyl-l-butanol was produced in higher concentra­ tion with amino acids than with ammonium sulfate, with a less effect, usually, on the other fusel alcohols. A logical explanation for this phenomena is provided by the molar ratio of amino acid utilized to fusel alcohol pro­ duced. The conversion of leucine to 3-methyl-l-butanol was the most efficient (2:1) while the conversion of isoleucine to 2-methyl-l-butano,l or valine to isobutanol was much Table 28: Summary of Experimental Data Obtained Using Amino Acids and Ammonium Sulfate as the Source of Nitrogen in the Fermentation Medium.

* *

Variables Amino Acids as Source Ammonium Sulfate as Source of FAN mg/1 - of Nitrogen mg/1

Nitrogen level mg/1 150 300 450 150 300 450 n-propanol (ppm) 9.53 15.38 12.50 8.55 14.26 11.24

Isobutanol (ppm) 28.52 19.80 17.45 6.60 22.77 14.44

2-methylr • 1-butanol (ppm) 15.76 12.37 16.66 5.16 13.01 9.05

3-methyl- l-butanol (ppm) 65.31 75.85 75.18 16.48 27.40 25.46

Total fusel alcohols (ppm) 119.12 123.40 121.79 36.79 77.44 60.19

% Nitrogen utilized 66.00 65.00 49.00 94.00 75.00 70.00

% Carbohydrates utilized 92.00 95.00 . 92.00 85.00 85.00 87.00

Final pH 6.30 6.30 6.30 6.50 6.30 ’ 6.25

Final specific gravity 1.0057 1.0071 1.0064 1.0083 1.0066 1.0073 147 i 148 less efficient.

The source of nitrogen did also appear to affect the order of formation of individual fusel alcohol in terms of relative concentration. In the fermentations with amino acids , formation of individual fusel alcohols, in order of decreasing concentration, was 3-methyl-l-butanol, isobutanol,

2-methyl-l-butanol, and n-propanol. However, with ammonium sulfate, the order of fusel alcohol, in terms of final con­ centration, was 3-methyl-l-butanol, isobutanol, n-propanol, and 2-methyl-l-butanol. « The fusel alcohol 3-methyl-l-butanol was always present at a higher concentration than the other alcohols regardless of the nitrogen or carbohydrate sources or concentration.

This was also true in the commercial beers analyzed in our laboratory (Table 6) and in the literature (Table 4). The reason for this may be explained on the basis of the effi­ ciency by which the yeast converts leucine to 3-methyl-l- butanol .

The fact that higher concentrations of fusel alcohols were produced when amino acids were the source of nitrogen may be explained on the basis of keto acid availability for yeast metabolism. As was shown in Figure 8, the keto acid pool may originate from two sources, deamination of amino acids (Ehrlich pathway) and biosynthesis from pyruvic acid

(Synthetic pathway). The keto acid pool may be utilized in two manners, transamination to form amino acids and 149 reduction to form fusel alcohols.

The total fusel alcohol content produced in beer, therefore, would depend on the manner in which the keto acids are utilized. When amino acids are present in excess of that needed for yeast assimilation, most of the keto acids may be converted to fusel alcohols. However, when amino acids needed for yeast synthesis are not available, then the yeast will synthesize amino acids by transamination of keto acids. The result would be less keto acids for fusel alcohol production. In other words, in fermentation medium with amino acids, keto acids may be formed both via the Ehrlich and Synthetic pathways. But, in a medium with ammonium sulfate, only the Synthetic pathway will contribute keto acids.

The literature values reported for the minimum FAN re­ quired for normal yeast growth vary. The average reported values is 128 mg/1 (4). The three lots of wort with amino.

% acids in this study,contained FAN in excess of the minimum i requirement.

At three levels of FAN (150-450 mg/1), we found that the final concentration of total fusel alcohols was inde­ pendent of the initial concentration of amino acids. This finding is in agreement with Ernest et. al, (51) but is in disagreement with Ayrapaa (18), who postulated that total

fusel alcohol formation is dependent on nitrogen concentra­ tion. However, when ammonium sulfate was the source of 150 nitrogen, the final concentration of the fusel alcohol pro­ duced via the synthetic pathway was affected by the nitro­ gen concentration which is in agreement with Ayrapaa's pro­ posal (18).

The reason for the disagreement with Ayrapaa with re­ spect to the effect of nitrogen level on fusel alcohol for­ mation may be due to the nitrogen level at which Ayrapaa carried-out his fermentation. As was mentioned before, the minimum FAN level required for normal yeast growth is 128 mg/1. Ayrapaa fermented the wort at FAN levels ranging from 108-324 mg/1. It is possible that at the low FAN level of 108 mg/1, the yeast growth was retarded and fusel alcohol formation was adversely affected.

With respect to carbohydrates utilization, the curves for glucose, sucrose, and maltose uptake from the medium were very similar to each other regardless of the source or initial nitrogen level in the fermented wort. Sucrose and glucose were utilized at one constant rate while the maltose curve has a two-step utilization rate; a rapid rate from

0-32 hours and a slow utilization rate from 32-100 hours of fermentation.

The fermentable carbohydrates in regular wort, maltose, glucose, and sucrose, are present in the ratio of 1.1:3tl.

The three sugars, however, are utilized at a different rate.

Sucrose and glucose are metabolized at a faster rate than maltose. The effect of single carbohydrate fermentation on 151

the production of fusel alcohol was examined. It was hoped

that the results would aid in the explanation of results of

other studies conducted with varying carbohydrate ratio.

Ammonium sulfate was the source of nitrogen in this experi­

ment* This should permit speculation as to the reason for

the effect of carbohydrate type on fusel alcohol production

since carbohydrate would be the only source of fusel

alcohols.

Fusel alcohols were formed in higher .concentrations

with sucrose or maltose than with glucose as the source of

carbohydrate. The reduction in fusel alcohol in the lot

with glucose could not be attributed to lack of carbohydrate

or nitrogen utilization. As a matter of fact, carbohydrate

and nitrogen utilization as well as final specific gravity

were very comparable with those values obtained in the lots

with sucrose and maltose. Carbohydrate utilization ranged

from 80-92% and nitrogen utilization from 54-71%. Specific

gravity ranged from 1.00B22-1.00825. The final. pH* however,

was higher (pH = 6.15) than for the lots with sucrose or

’ maltose (pH » 5.9).

The reason for the reduction in fusel alcohol concen­

tration in the lot with glucose is not clear, but it may be

due to the type of sugar used in the fermentation. It is

possible that a high glucose concentration inhibits the ac­

tivity of enzymes that are responsible for the formation of

fusel alcohols. 152 The results obtained from fermentations with varying carbohydrate ratios are summarized for convenience in Table

29. In this study, where the ratio of carbohydrates was varied, but the total concentration was kept constant, a marked reduction occurred in fusel alcohol production in comparison with the production in the defined wort with a normal carbohydrate ratio. The reduction occurred irre­ spective of the relative concentration to which the carbohy­ drates was altered.

The effect of glucose concentration on. the production of fusel alcohol is once again evident. The lot with the highest glucose ratio (M:G:S:3:11:1) contained the lowest concentration of fusel alcohol, 27 ppm. In comparison, the lots with M:G:S ratio of 11:1:3 and 1:3:11 contained higher concentrations of fusel alcohols, namely 37 ppm and 39 ppm, respectively.

The variation in carbohydrate ratio of the wort did not affect the order of individual fusel alcohol formation. The order was in- decreasing concentration, 3-methyl-l-butanol, n-propanol, isobutanol, and 2-methyl-l-butanol.

Nitrogen and carbohydrate utilization was similar-in the three lots with varying carbohydrate ratio. Nitrogen utilization ranged from 56-68% while carbohydrate utiliza­ tion ranged from 75-86%. The final pH was 6.2 for all three lots while the specific gravity was 1.0064-1.0080. Table 29: Summary of Experimental Data Obtained iron Fermentations with Varying Carbohydrate Ratio.

* CHO Ratio CHO Ratio CHO Ratio

. a) H G S H G SH GS b) . VARIABLES 11 1 3 3 11 1 1 3 11 c) * 0. 20 8 0.037 0 . 05 3 0.050 0.416 0.019 0.018 0.101 0.219

Total Holes of Carbohydrates 0.298 0.485 0.338 n-Prpoanol (ppm) 8.50 8.50 13.39 Isobutanol (ppm) 7.50 5.14 6.60 2-Hethyl-l-butanol {ppm) 6.22 3.46 • 4 . 88 3-Hethyl-1-butanol (ppm) 15.00 10.33 13.93 Total fusel alcohols (ppm) 37.22 27.43 38.80 X Nitrogen utilized 56.00 68 .0 0 67.00 X Carbohydrates • u t i l i z e d 85 .0 0 75.00 86.00 Final pH 6.20 6 . 20 ' 6.20 Final specific gravity 1.0064 1.0074 1.0080 a) H s Maltose, G * Glucose, S 3 Sucrose

b} Ratios 153 c) Units In Holes 154

Stewart (25) found that the Individual sugars in reg­ ular wort are utilized in an orderly fashion. The exact sequence will vary, depending on the strain of yeast and the relative concentration of the sugars. Since the rela­ tive concentration of the sugars in this study was changed, and due to the effect of'increased glucose concentration on fusel alcohol formation, it was decided to add the carbohy­ drates to the fermenting wort periodically in order to study the effect of initial carbohydrate level in wort on fusel alcohol formation.

The results obtained in this phase of study are sum­ marized for convenience in Table 30.

Usually, when the entire amount of carbohydrates were added to the fermented wort at the beginning of the fermen­ tation, glucose and sucrose were utilized completely after

40-45 hours. However, when the carbohydrates were added periodically during the time course of the fermentation, glucose in the lot with M:G:S ratio of 3:11:1 remained un­ utilized in the wort even after 100 hours of fermentation

(Figure 33).

The production of fusel alcohol in the lot with high glucose ratio decreased upon periodic addition of carbohy­ drates. This may indicate that there is a concentration effect of glucose on fusel alcohol formation. It may be that high glucose concentration in the wort is inhibiting certain enzymes responsible for fusel alcohol formation. Table 30: Sunraary of Experimental Data Obtained from Fermentations with Periodic Addition of Carbohydrates.

CHO Ratio CHO Ratio CHO Ratio a) M £ S H GS M £ S VIDTARIPC VAKlnDLfcw b) 11 1 3 3 11 1 1 3 11 CHO's added at 0 hours c,0.104 0.0185 0.0265 0.0025 0.208 0.0095 0.009 0.0505 0.10 95 CHO's added at 24 hours 0.052 0.0092 0.0132 0.0125 0.104 0.0047 0.0045 0.0252 0.0547 CHO's added at 48 hours 0.052 0.0092 0.0132 0.0125 0.104 0.0047 0.0045 0.0252 0.0547

n-Propanol (ppm) 8.23 10.21 6.50 Isobutanol (ppv) 6.09 12.10 8.85 2-Hethyl-l-butanol (ppm) 1.68 5.55 3.28 3-Methyl-1-butanol (ppm) 9.96 17.40 17.41 Total fusel alcohols (ppm) 25.96 45.26 36.04 X Nitrogen utilized 56.00 64.00 68.00 % Carbohydrates ut il ize d 84 .0 0 77 .0 0 96.00 Final pH 6.10 6.20 6.20 Final specific gravity 1.0077 1.0078 1.0071

a) H • Maltose, £ ■ Glucose, S * Sucrose b) * Ratios c) - Units 1n soles

in 156

Ayrapaa (18) has indicated that the synthetic formation of

fusel alcohols shows a maximum at a low nitrogen level

(Figure 6), but then decreasing rapidly with increasing

nitrogen level approaching a certain value (line CD - Figure

6}, characteristic of each particular high alcohol. Ayrapaa

proposed that the position of line CD is controlled by (a)

a regulatory mechanism controlling the synthesis of amino

acids and (b) by initial sugar concentration of the medium.

Ayrapaa did not publish any data to support his proposed

scheme. Our results support his proposal that the position

of line CD is indeed affected not only by the initial con­

centration of the sugars but also by the type of sugars

present in the wort. Less fusel alcohols were produced in

the lot, with high glucose ratio that was added periodically

than the lots with either high sucrose or maltose ratio.

Periodic addition of carbohydrate during the time

course did not affect the.rate of carbohydrate or nitrogen

utilization. Carbohydrates were utilized from 77-96% while

nitrogen was utilized from 56-68%. The final pH and spe­

cific gravity values were very similar to those values ob­

tained with complete addition of carbohydrates. The final

pH was 6.1-6.2 and final specific gravity was 1.0071-1.0078.

The order of fusel alcohol formation was not affected

either by the periodic addition of carbohydrates. The .

order, in order of decreasing concentration was 3-methyl-l-

butanol, n-propanol, isobutanol, and 2-methyl-l-butanol. 157 This was the same order as was obtained with complete ini- m tial addition of carbohydrates to the wort.

In summary, more fusel alcohols were formed in fermen­ tation with amino acids as the source of nitrogen than am­ monium sulfate or when the ratio of carbohydrates was varied, or with periodic addition of carbohydrate. However, the final pH and specific gravity were similar for each of the lots of wort.

The order of formation of individual fusel alcohol in terms of relative concentration was also affected by the source of nitrogen. In the fermentation with amino acids, formation of individual fusel alcohols, in order of de­ creasing concentration, was 3-methyl-l-butanol, isobutanol,

2-methyl-l-butanol, and n-propanol. However, with ammonium sulfate, the order was 3-methyl-l-butanol, isobutanol, n-propanol, and 2-methyl-l-butanol. SUMMARY AND CONCLUSIONS

A study was made on the effect of source and concen­ tration of nitrogen and carbohydrates in defined media simulating wort on the production of fusel alcohols, nitro-

• gen and carbohydrate utilization, pH and specific gravity of beer. The source of nitrogen was either amino acids, as found in regular wort, or ammonium sulfate. Amino acids and ammonium sulfate were studied at similar nitrogen lev­ els. The fermentable carbohydrates were maltose, glucose, and sucrose which were added in concentrations similar to those of regular wort, either singly and in ratios as in . wort before onset of fermentation or in varying ratios added at once‘ or periodically during fermentation.

The yeast, Sacharomyces carlsbergensis, was selected for the fermentation and was added as first generation at a concentration of 15 x 106 cells/ml.

The fermentation was carried out anaerobically with continuous magnetic stirring in two liter Virglass fermen­ tation jars. The temperature of the medium was maintained at 10 + 0.5°C and the fermentation was carried out for

100 hours.

158 159

It was found that the fusel alcohol concentrations ob­ tained upon fermenting defined wort with amino acids as the source of nitrogen were comparable to those values found in commercial beer analyzed in our laboratory and values re­ ported in the literature. However, formation of fusel alcohols was greatly reduced when (a) ammonium sulfate was the source of nitrogen, (b) the ratio of carbohydrates was varied, and (c) with periodic addition of carbohydrates.

The difference in fusel alcohol formation in media with amino acids and ammonium sulfate as the source of nitrogen in wort was explained on the basis of keto acid avail­ ability.

The rate of absorption of individual amino acids during fermentation varied but overall, the order of absorption was in agreement with the findings of other investigators. There was a parallel relationship between the rate of absorption of the four amino acids leucine, isoleucine, valine, and threonine and formation of the corresponding fusel alcohols i.e. 3-methyl-l-butanol, 2-methyl-l-butanol, isobutanol, and n-propanol.

The fusel alcohol 3-methyl-l-butanol was formed at higher concentrations than the other three alcohols regard­ less of the initial concentration or source of nitrogen.

The molar ratio of amino acid utilized to fusel alcohol pro­ duced was the most efficient in the conversion of leucine to

3-methyl-l-butanol (2:1). The molar ratio for conversion 160 of isoleucine to 2-methyl-l-butanol was 4:1, and for valine to isobutanol was 8:1.

The-source of nitrogen affected the order of formation of individual fusel alcohol in terms of relative concen­ tration. In the fermentation with amino acids, formation of individual fusel alcohols, in order of decreasing concen­ tration, was 3-methyl-l-butanol, isobutanol, 2-methyl-

1-butanol, and n-propanol. With ammonium sulfate, the order was 3-methyl-l-butanol, isobutanol, n-propanol, and

2-methyl-l-butanol. * The fermentable carbohydrates in regular wort, maltose, glucose, and sucrose, are present in the ratio of 11:3:1.

At this ratio, sucrose and glucose were metabolized at a. faster rate than maltose.

In the fermentations with a single carbohydrate, fusel alcohols were formed in higher concentrations with sucrose or maltose than with glucose. This appears to be attrib­ utable to the type of sugar rather than to difference in the rate of fermentation. Total fusel alcohol formation with single carbohydrate addition was markedly less than with normal and varying carbohydrate ratios or with periodic addition of the carbohydrates.

In the fermentation where the ratio of carbohydrates was varied, less fusel alcohols were produced in comparison with fermented wort with normal carbohydrate ratio. In addition, the fermented wort with high glucose ratio 161

contained less fusel alcohols than worts with high sucrose

or maltose ratio. Variation of the carbohydrate ratio did

not affect the nitrogen or carbohydrate utilization.

In the fermentation with periodic addition of carbohy­

drate, some glucose, contrary to*the other fermentations,

remained unutilized in the high glucose ratio lot even after

100 hours of fermentation. Also, the production of fusel

alcohols in this lot was less than in the other two lots

with high sucrose or maltose ratios. Again, the rate of

nitrogen and carbohydrate utilization were not affected by

the periodic addition of carbohydrates.

The overall changes during fermentation in .pH were

from 7.9, the original value to 6.5-6.2 and the changes in

specific gravity were from the initial value of approxi­

mately 1.020 to 1.0083-1.0057.

It may be concluded from this study that the concentra­

tion of the fusel alcohols may be lowered by modifying

either the amino acid content or carbohydrates in the wort.

Relatively low concentration of the fusel alcohol precursor

amino acids leucine, isoleucine, valine, and threonine or

high concentration of glucose in the wort, or periodic addi­

tion of the carbohydrates to the wort will result in a beer

♦ with low fusel alcohol content. . BIBLIOGRAPHY

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