Free amino nitrogen improvement in sorghum grain brewing By Chisala Charles Ng’andwe Submitted in partial fulfillment of the requirements for the degree MSc (Agric) Food Science and Technology in the Department of Food Science Faculty of Natural and Agricultural Science University of Pretoria Pretoria March 2008 ©© UUnniivveerrssiittyy ooff PPrreettoorriiaa DECLARATION I declare that the dissertation herewith submitted for the degree MSc (Agric) Food Science and Technology at the University of Pretoria, has not previously been submitted by me for a degree at any other university or institution of higher education. i To my Creator, my caring parents Max & Magdalene Ng’andwe, and my dearly beloved late brother Sansa Emmanuel Ng’andwe ii ABSTRACT Free amino nitrogen improvement in sorghum grain brewing By Chisala Charles Ng’andwe Supervisor: Prof. J. R. N. Taylor Free amino nitrogen (FAN) levels in sorghum grain brewing are characteristically low as a direct consequence of the very low levels of FAN in unmalted sorghum grain. During fermentation, FAN provides nutritional support to the yeast, enabling optimal yeast growth and efficient fermentation. Exogenous proteolytic enzymes are required to hydrolyse sorghum grain protein into FAN. Eight commercial proteolytic enzymes were studied in terms of their efficacy to produce FAN levels during mashing considered adequate (150 mg/L) to support optimal yeast functionality and their effect on extract levels. FAN as determined by the ninhydrin assay, ranged between 25-72 mg/100 g sorghum grist (representing 0%-185% increase) when mashing was carried out at 55°C over 45 mins with the 8 different enzymes. The proteolytic enzymes varied greatly in terms of their efficacy, possibly as a result of having different optimal operating conditions. It is also suggested that the proteolytic enzymes differed in terms of their classification and exopeptidase/endopeptidase ratio. In an attempt to further increase FAN levels, the effects of the reducing agent potassium metabisulphite (KMS) and different mashing temperatures (50°C, 55°C and 60°C) were also studied. Three enzymes (Flavourzyme, Neutrase, Papain) were selected for this part of the study. The addition of KMS caused a significant increase in the FAN in mashing systems that utilized Flavourzyme (22% when mashing at 55°C) and Neutrase (14% when mashing at 55°C), but not with Papain. It is suggested that KMS reduced the stabilizing disulphide bonds present in the sorghum kafirin, thus making the kafirin more iii digestible. The KMS could have reduced the effectiveness of Papain because it is a sulphydryl protease which possesses stabilizing disulphide bonds which were reduced by the KMS. Flavourzyme and Papain were more active at 55°C, while Neutrase displayed maximum activity at 50°C. The effect of mashing at a low temperature (40°C) over an extended time period (7 hr) on FAN was studied using Flavourzyme. Samples treated with Flavourzyme and KMS showed a five-fold increase in FAN, while those treated with Flavourzyme only showed a four-fold increase. It is proposed that the low temperature was able to suppress the rate at which disulphide bonds were formed in kafirin, therefore improving its digestibility. The lower temperature also retarded the enzyme denaturation rate, while the long time period allowed the enzyme more contact time with the substrate. Microstructure analysis of sorghum protein bodies using transmission electron microscopy and of sorghum endosperm using confocal laser scanning microscopy revealed that the relative indigestibility of kafirin makes a significant contribution to the relatively low FAN levels. Although the action of the proteolytic enzyme and KMS on the sorghum endosperm protein matrix showed notable degradation, large fragments of undigested protein bodies were observed. This once again suggested that disulphide bond cross-linking played a major role in reducing the digestibility of sorghum protein. The addition of Flavourzyme to a complete mashing cycle was also found to result in a significant increase (9%) in extract. The increase in extract is attributed to the increased availability of starch to amylase hydrolysis as a result of the degradation of the protein matrix which envelopes the starch granules. The addition of KMS to the sorghum grain mashing system and the utilisation of a low temperature protein rest are effective methods of improving FAN in sorghum grain brewing. iv ACKNOWLEDGEMENTS First and foremost to the genius of a man I was privileged to work under, Prof. JRN. Taylor. Your support and guidance throughout the duration of this project has been tremendous and is gratefully acknowledged. I also acknowledge the financial supporters of this project: Novozymes SA (Pty) Ltd; National Research Foundation through the Technology and Human Resources for Industry program; and the International Sorghum and Millet Collaborative Research programme). I would also like to thank all the staff and students of the Department of Food Science at the University of Pretoria for their support and valuable input, in particular Mrs J. Taylor for her technical support and Mr L. Mugode with whom I shared all the joys and frustrations of research work. I also thank Mr A. Hall from the laboratory of microscopy and microanalysis at the University of Pretoria for his kind assistance and valuable contribution to this work. Special thanks to Ms E. Nagel, formerly of student support services, University of Pretoria for always lending a listening ear. My heartfelt thanks go to Ms B. Mashangoane for her tremendous support and encouragement through the duration of this work. I am most thankful to the staff of Novozymes, SA (Pty) Ltd, in particular Mr J. van Aswegen, Mr I. Kennedy and Mr. B. Higgins for presenting us with a problem which has been the basis of this dissertation, and their invaluable technical input. Finally, to my parents, brothers and sisters for their endless encouragement, moral and financial support, I am greatly indebted. v TABLE OF CONTENTS DECLARATION I ABSTRACT III ACKNOWLEDGEMENTS V TABLE OF CONTENTS VI LIST OF TABLES VIII LIST OF FIGURES IX CHAPTER 1: INTRODUCTION 1 1.1 STATEMENT OF THE PROBLEM 1 1.2 LITERATURE REVIEW 5 1.2.1 SORGHUM GRAIN STRUCTURE 5 1.2.2 PROTEINS OF SORGHUM 6 1.2.3 SORGHUM PROTEIN DIGESTIBILITY 12 1.2.4 PROTEOLYTIC ENZYME ACTIVITY IN SORGHUM 15 1.2.5 THE BREWING PROCESS 16 1.2.6 PROTEOLYTIC ENZYMES 24 1.2.7 CONCLUSIONS 29 1.3 OBJECTIVES 30 1.4. HYPOTHESES 31 CHAPTER 2: RESEARCH 32 2.1. EFFECT OF PROTEOLYTIC ENZYMES AND MASHING CONDITIONS ON SORGHUM ENDOSPERM PROTEINS AND WORT PROPERTIES 33 ABSTRACT 33 2.1.1 INTRODUCTION 34 2.1.2 MATERIALS AND METHODS 36 2.1.3 RESULTS AND DISCUSSION 44 2.1.4 CONCLUSIONS 65 2.1.5 REFERENCES 67 vi CHAPTER 3: DISCUSSION 75 3.1 METHODOLOGICAL CONSIDERATIONS 75 3.2. EFFICACY OF PROTEOLYTIC ENZYMES ON SORGHUM PROTEIN DEGRADATION 88 3.2. EFFECT OF MASHING CONDITIONS ON SORGHUM PROTEIN DEGRADATION AND WORT PROPERTIES. 90 3.3 PRACTICALITY OF EMPLOYING OPTIMIZED MASHING CONDITIONS COMMERCIALLY. 94 CHAPTER 4: CONCLUSIONS AND RECOMMENDATIONS 96 5. REFERENCES 98 vii LIST OF TABLES Table I: Amino acid content (mole % of amino acid) of the major Page 8 protein fractions of sorghum Table II: Main steps involved in the brewing process Page 17 Table III: Sorghum mashing procedures used by different workers Page 20 Table IV: Free amino nitrogen levels of worts derived from mashes Page 23 containing increasing levels of Bioprotease Table V: Classification of exopeptidases Page 28 Table VI: Description of the commercial enzyme types used in the study Page 37 Table VII: Effect of different exogenous proteolytic enzymes on free Page 44 amino nitrogen (FAN) levels when sorghum grain mashed at 55°C Table VIII: Effect of mashing temperature (50°C, 55°C and 60°C) and Page 49 potassium metabisulphite (KMS) on free amino nitrogen (FAN) levels in sorghum grain mashed with three different enzymes Table IX: Effect of a combination of 3 different enzymes (Neutrase, Page 50 papain and flavourzyme) and reducing agent on free amino nitrogen levels when mashing with sorghum grain Table X: Effect of proteolytic enzyme (Flavourzyme) and reducing Page 54 agent (KMS) on hot water extract, free amino nitrogen levels and starch presence in sorghum grain wort and spent grain Table. XI: Amino acid (g/100 g protein) spectrum of worts separated Page 56 from sorghum mashed with commercial enzymes ceremix and flavourzyme and KMS at 40°C for 7 hr Table XII: Liqour/Grist ratios and mashing systems used by different Page 78 authors Table XIII: Classification of wort amino acids according to Jones and Page 85 Pierce (1964) based on assimilation by yeast viii LIST OF FIGURES Figure 1: Diagrammatic section through a sorghum kernel Page 5 Figure 2: Micrograph of a sorghum protein body incubated with Page 9 preimmune sera Figure 3: Diagramatic representation of total zein (lane 1) and total Page 10 kafirin (lane 2) polypeptides extracted with 70% alcohol and 2- mercaptoethanol and separated by SDS-PAGE Figure 4: Experimental design for the study of free amino nitrogen Page 32 improvement in sorghum grain brewing Figure 5: Mashing regime used in the s tudy Page 39 Figure 6: Effect of a long time low temperature mashing period and Page 53 reducing agent (KMS) on free amino nitrogen levels in sorghum mashed with Flavourzyme Figure 7: Transmission electron microscopy of sorghum protein bodies Page 58 isolated from sorghum grain mashed at 55°C for 7 hours Figure 8: Confocal laser scanning microscopy of sorghum grain mashed Page 61 at 55°C for 7 hours Figure 9: SDS-PAGE of non solubilised protein of sorghum after Page 64 mashing at 40°C over 7 hours Figure 10: Reaction series of the ninhydrin assay Page 80 Figure 11: Model showing protein denaturation with sodium dodecyl Page 85 sulphate and the creation of a highly negative charged molecule ix