Free amino nitrogen improvement in sorghum malt brewing

100 Pages·2009·0.66 MB·English

Free amino nitrogen improvement in sorghum malt brewing

By

Luke Mugode

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 Sciences
University of Pretoria
PRETORIA

June 2009

©© 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.

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ABSTRACT

Free amino nitrogen improvement in sorghum malt brewing

By
L. Mugode

Supervisors: Prof. J.R.N. Taylor
Prof: L.W. Rooney

Although sorghum malt is relatively rich in free amino nitrogen (FAN), the 150 mg FAN/L
threshold recommended for brewing is difficult to obtain. The vitreous nature of the
sorghum endosperm hinders proteolysis during brewing. Hence, exogenous proteolytic
enzymes are often required to increase hydrolysis of sorghum malt protein to produce
sufficient FAN in order to support rapid yeast growth during fermentation.
Ten exogenous proteases were examined for their production of FAN in sorghum malt
mashing. Mashing was done at 550C for 45 minutes. Levels of FAN, as determined by
the ninhydrin method, showed great variation among the proteolytic enzymes, ranging
from 96 in control to 182 mg/100 g malt with possibly of most effective proteolytic enzyme.
The variation in FAN level was possibly due to different optimal mashing conditions of
exogenous proteases used and perhaps due to low ratios of exopeptidase/endopeptidase
in the enzyme preparations.
Low temperature (400C) and long duration mashing for (7 hours) gave good FAN
production during mashing to a total of 113 and 138 mg/100 g malt in control and the
treatment with exogenous proteolytic enzyme Flavourzyme plus malt, respectively.
The exogenous enzyme (Flavourzyme) plus potassium metabisulphite (PMB) increased
FAN production during mashing in the ratio of 2 to 1 in a treatment where PMB was added
compared to one without. Similarly, hot wort extract (HWE) increased by 8% during
mashing with exogenous enzyme plus PMB compared to one without PMB, respectively.
PMB was involved in destabilizing the disulphide bonds in the sorghum protein
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polypeptide chains allowing proteolytic enzymes better accessibility to proteins. The
increase in HWE was possibly due to the starch being freed from the sorghum protein
matrix.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed some
oligomeric and polymeric kafirins after mashing. With transmission electron microcopy
(TEM), protein bodies of varying sizes with partially degraded peripheral edges and some
holes were seen after mashing. SDS-PAGE and TEM results suggest insufficient
proteolysis.
High protein digestibility sorghum’s potential for brewing was examined with reference to
FAN production. Although during mashing FAN increased by approx. 82 and 115% for
unmalted normal and high digestibility sorghums, respectively, the 150 mg FAN/L
threshold, recommended for brewing was not achieved.
FAN production to levels above 150 mg/L may only be realized if normal sorghum malt or
high protein digestibility sorghum malt is mashed with exogenous enzymes containing
sufficient exopeptidases coupled with appropriate mashing conditions.

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ACKNOWLEDGEMENTS

I would like to express my gratitude to Profs: J. R. N. Taylor, and L. W. Rooney. Studying
under supervision of these noble and experienced scientists is an experience I will live to
treasure for the rest of my professional life

I am also indebted to the International Sorghum and Millet Collaborative Research
Support Program, (INTSORMIL) for the financial support to enable me undertake this
MSc, under the leadership of the two named dignified scientists, the Zambian National
Institute for Scientific Research (NISIR) for granting me a study leave and other
assistance.

I am also grateful to Novozymes SA (Pty) Ltd; in particular Mr J. van Aswegen, Mr I.
Kennedy and Mr B. Higgins for their technical and material contribution to this study.

Incomparable gratefulness go to Bradly Lawrence Cotterell for his enormous input
particularly in research chapter 4.2, Mr. Chisala C. Ng’andwe with whom I shared pleasure
and anguish, particularly in various research protocol standardization.

Mr. Allan Hall from the Microscopy and Microanalysis Laboratory of the University of
Pretoria is thanked for his priceless contribution to this work.

Dr Janet Taylor is appreciated greatly for rendering enormous technical support in
laboratory work and her critical care in times when hope was almost lost, particularly when
I tragically lost my mother in-law. The one time “Easter Treat” away from home is a
moment I will also live to cherish.

To members of staff of the Department of Food Science of the University of Pretoria for
their support and valuable input is highly appreciated.

To my wife, and children, and my surviving Mom Irene, for their continuous moral support,
I am deeply thankful.

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TABLE OF CONTENTS

DECLARATION ii
ABSTRACT iii
ACKNOWLEDGEMENTS v
TABLE OF CONTENTS vi
LIST OF TABLES viii
LIST OF FIGURES ix
1. INTRODUCTION 1
2. LITERATURE REVIEW 3
2.1 Sorghum grain structure and chemical composition with particular reference 3
to proteins
2.2 Principles and practice of malting and brewing 7
2.3 The science of sorghum malting with particular reference to proteolysis 16
2.4 Mashing science with particular reference to proteolysis 16
2.5 Review of improving FAN when mashing with sorghum malt 17
2.6 Use of exogenous proteases with particular reference to sorghum lager beer 17
brewing
2.7 Conclusions 19
3. HYPOTHESES AND OBJECTIVES 20
3.1 Hypotheses 20
3.2 Objectives 21
4. RESEARCH 22
4.1 Effects of different commercial proteases on free amino nitrogen (FAN) 25
production and their impact on hot water extract when mashing with
malted sorghum
4.1.1 Introduction 26
4.1.2 Materials and methods 26
4.1.3 Results and discussion 33
4.1.4 Conclusions 46
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4.1.5 References 47
4.2 Effect of high protein digestibility sorghum cultivars on free amino 54
nitrogen (FAN) production during mashing
4.2.1 Introduction 55
4.2.2 Materials and methods 56
4.2.3 Results and discussion 59
4.2.4 Conclusions 72
4.2.5 References 73
5. GENERAL DISCUSSIONS 76
5.1 Methodological considerations 76
5.2 Effectiveness of proteolytic enzymes in sorghum endosperm protein matrix 81
degradation
5.3 Practicality of employing a long protein rest at low temperature for production 82
of free amino nitrogen
5.4 Prospects of using high protein digestibility sorghums in lager beer brewing 83
6. CONCLUSIONS AND RECOMMENDATIONS 84
7. REFERENCES 85

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LIST OF TABLES
Page
Table: 2.1 Amino acid compositions of protein fractions from the 6
endosperm,germ and pericap of Barnard Red sorghum
Table:4.1.1 Effect of various exogenous proteases supplied by Novozymes on 33
FAN production when mashing with sorghum malt for 45 minutes at
550
Table:4.1.2 Effect of various exogenous proteases supplied by Kerry-Biosciences 34
on FAN production when mashing with sorghum malt for 45 minutes
at 550C
Table:4.1.3 Effects of exogenous protease (Flavourzyme), with (PMB) on 37
mashing with malted sorghum at low temperature for 7 hours on FAN
and HWE
Table:4.1.4 Effects of Flavourzyme and potassium metabisulphite (PMB) on the 40
wort amino acid profile of sorghum malt protein after mashing at 40oC
for 7 hours
Table:4.2.1 Germinative energy (GE) (%) for normal and high protein digestibility 59
sorghum cultivars
Table 4.2.2 Effects of malting on protein digestibilities of normal and high protein 62
digestibility sorghum cultivars
Table:4.2.3 Effects of mashing with unmalted grains of normal and high protein 64
digestibility sorghum cultivars for 45 minutes at 55°C on FAN
production
Table:4.2.4 Effects of mashing with malted normal and high protein digestibility 65
sorghum cultivars for 45 minutes at 55oC on FAN production
Table:4.2.5 Effect of exogenous protease (Flavourzyme) on FAN production when 67
mashing with unmalted normal and high protein digestibility sorghum
cultivars for 45 minutes at 55oC
Table:4.2.6 Effects of exogenous protease (Bioprotease P. Conc.) on FAN 68
production when mashing with unmalted normal and high protein
digestibility sorghum cultivars for 45 minutes at 55oC
Table:4.2.7 Effect of exogenous protease (Flavourzymes) on FAN production 70
when mashing with malted normal and high protein digestibility
sorghum cultivars for 45 minutes at 55oC
Table:4.2.8 Effect of exogenous protease (Bioprotease P.conc) on FAN 71
production when mashing with malted normal and high protein
digestibility sorghum cultivars for 45 minutes at 55oC

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LIST OF FIGURES

Figure:2.1 A section of sorghum kernel 4
Figure:2.2 Simplified flow diagram of the malting process 7
Figure:2.3 Self-emptying conical steep tank with a mixing “geyser” with rotating 10
hollow arms and aeration rings
Figure:2.4 Flat-bed (Nordon type) steeping tank 11
Figure:2.5 Outdoor floor malting of sorghum 12
Figure:2.6 Saladin box compartment type (a general view of germination vessel, 13
fan and conditioning chamber)
Figure:2.7 Simplified diagram of the lager beer brewing process 14
Figure:2.8 A shortened double decoction process lasting for 2 ½ hours 17
Figure:4.1.1 Flow chart of experimental design for research chapter 4.1 23
Figure:4.1.2 Flow chart of experimental design for research chapter 4.2 24
Figure 4.1.3 Mashing profile for sorghum malt 29
Figure:4.1.4 Effects of Flavourzyme and potassium metabisulphite (PMB) on FAN 39
production during mashing at low temperature
Figure:4.1.5 SDS-PAGE of malted sorghum malt proteins after mashing at low 43
temperature (40°C) for 7 hours
Figure:4.1.6 Effects of Flavourzyme and potassium metabisulphite (PMB) on malt 45
protein after low temperature mashing for 7 hours at 40oC (TEM)
Figure:4.2.1 Effect of malting normal and high protein digestibility sorghums on malt 60
fresh weight basis

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1. INTRODUCTION
Sorghum (Sorghum bicolor (L.) Moench) is a cereal that has significant genetic variability
with more than 30,000 selections present in the world collections in India and elsewhere
(Serna-Saldivar and Rooney, 1995). It is believed that sorghum plant is native to equatorial
Africa (Serna-Saldivar and Rooney, 1995). It is now distributed throughout the semi-arid
equatorial regions and other semi tropics of the world. The sorghum plant is uniquely well
adapted to these conditions in that it is drought-resistant and will withstand periods of
flooding. In fact, sorghum can produce a crop in these regions, where other cereals such as
barley and maize cannot be economically cultivated (Doggett, 1988).
Sorghum is the fifth most important crop in the world after wheat, maize, rice and barley
International Crops Research Institute for the Semi-Arid Tropics/ Food and Agriculture
Organization (ICRISAT/FAO, 1996). The leading producers during 1999-2003 periods
include the USA 10.4 million metric tons (MT), India 8.5 million MT, Nigeria 8.1 million MT,
Mexico 6.5 million MT and Sudan 4.4 million MT (ICRISAT/FAO, 1996).
In Africa, the major sorghum growing areas run across West Africa south of the Sahara to
the coast and eastward into Sudan, Ethiopia, and Somalia (House, 1995). Sorghum is a
staple food crop of millions of poor people in semi-arid tropics of Africa and Asia. Moreover,
it has gained increasing importance as a fodder (green/dry) and feed crop in the last decade
(ICRISAT, 1996).
In some countries, the trend of per capita agricultural production index for cereals has
progressively been increasing during the past 25 years (1980-2005) (FAO, 1996). Although
both area and production of sorghum in Eastern and Southern Africa has increased from the
early 1970's to 2005, there has not been an increase in yield (ICRISAT, 1996). However,
the use of sorghum in commercial production of lager and stout beers (Taylor et al., 2006),
may motivate producers of sorghum and boost production and income to smallholder
farmers as the case is in Uganda and elsewhere where sorghum is being utilized for lager
beer (Mackintosh and Higgins, 2004).
The potential of sorghum as an alternative substrate for lager beer brewing was recognized
over five decades ago (Owuama, 1997). Success in replacing barley malt using sorghum
malt in lager beer brewing has been cited in several parts of Africa, particularly in Nigeria
and elsewhere (Agu and Palmer 1998; Owuama, 1997).
In sub-Saharan African countries, Nigeria has been the leading country in using sorghum for
lager beer production. This development came about due to government ban on importation
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