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TITLE PAGE

EFFECT OF ALKALINE STEEP AND AIR-REST CYCLE ON THE DEVELOPMENT OF

SORGHUM PEROXIDASE ACTIVITY DURING MALTING

A PROJECT WORK SUBMITTED TO IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF

SCIENCE (M.Sc) IN BIOCHEMISTRY (INDUSTRIAL BIOCHEMISTRY AND

BIOTECHNOLOGY) OF THE UNIVERSITY OF NIGERIA, NSUKKA.

By

IGWE, EJIKEME PETER

(PG/M.Sc/09/51922)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA, NSUKKA.

AUGUST, 2012.

CHAPTER ONE

INTRODUCTION

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1.0 Sorghum (Sorghum bicolor (L.) Moench)

Sorghum (Sorghum bicolor (L.) Moench) is the grain of choice to produce traditional cloudy

and opaque beers throughout sub-saharan Africa. The key ingredient of these beers is sorghum

malt, which provides hydrolytic enzymes (especially amylases) to ferment sugars into ethanol

and carbon dioxide. Sorghum is used for food, fodder, and the production of alcoholic

beverages. It is both drought and heat tolerant, and is especially important in arid regions.

Sorghum ranks fifth in the world cereal production, and as of 2008 the world annual sorghum

production stood at 65.5 million tones (Akintayo and Sedgo,2001). It is an important food crop

in Africa, Central America, and South Asia (Akintayo and Sedgo,2001).

1.1 Sorghum as brewing material

In Southern Africa, sorghum is used to produce beer, including the local version of Guinness

stout. In recent years, sorghum has been used as a substitute for other grains in gluten-free beer.

Although the African versions are not "gluten-free", as malt extract is also used, gluten-free

beers are now available using such substitutes as sorghum or buckwheat. Sorghum is used in

the same way as barley to produce "malt" that can form the basis of a mash without gliadin or

hordein and therefore suitable for coeliacs (Smagalski, 2006).

African sorghum beer is a brownish-pink beverage with a fruity, sour taste. It has an alcohol

content that can vary between 1% and 8% (Lermusieau et al., 2001). African sorghum beer is

high in protein, which contributes to foam stability, giving it a milk-like head. Because this

beer is not filtered, its appearance is cloudy and yeasty, and may also contain bits of grain

(Lermusieau et al., 2001).

African sorghum beer is a popular drink primarily amongst the black community. Sorghum

beer is known by many different names in various countries across Africa, such as burukutu

(Nigeria), pombe (East Africa), bil-bil (Cameroon), bjala in Northern Soweto. In Nigeria as

well as other African countries where sorghum is malted commercially, the respective

agricultural departments and commercial breeders breed sorghum cultivars with good malting

quality for brewing. The primary quality criterion is their potential to produce malt with high

diastatic power (amylase activity) (Okolo et al., 2010).

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Traditional and commercial sorghum malting process is split into three unit operations:

steeping, germination, and drying ( Taylor et al.,2005). Steeping involves immersing the grain

in water until it has imbibed sufficient water to initiate the metabolic processes of germination.

During germination the moist grain is allowed to grow under controlled cool conditions in the

dark with or without any further addition of water (Briggs et al., 2004).

Drying involves reducing the moisture content of the green (moist) sorghum malt to around

10% to produce a shelf-stable product (Arnold, 2005). Drying is generally carried out in a box

with a perforated floor, similar to the germination box but with deeper floor. Warm dry air is

blown through the green malt. The air temperature should not be more than 50°C, as higher

temperatures significantly reduce the amylase activity of the malt. In some outdoor floor

malting, the malt is sun-dried by spreading the grain out in thin layer and turning it periodically

(Arnold, 2005).

There are many setbacks in brewing with sorghum such as high lipid content, low extract

recovery, high polyphenol content, absence of hull etc, which affect the quality of the beer.

These problems arising from the use of sorghum to brew beer have been subject of intense

research, especially in Africa (Osagie, 1987;Okolo and Ezeogu, 1996;Nwanguma and Eze

1996; Taylor and Dewar, 2001) .

The absence of hull in sorghum was considered a major problem. This is because when

brewing with barley malt, the hulls act as a filter bed in lautering, the technology traditionally

used to separate the wort (unfermented beer) from the spent grain. In the 1990s, this problem

was solved with the development of tangential-flow mash filters with automatic discharge of

spent grains. Since then the commercial use of sorghum for clear beer brewing in Africa has

become firmly established. Commercial African sorghum beer is packaged in a

microbiologically active state. Packaging does not occur in sterile conditions and many

microorganisms may contaminate the beer. The use of wild lactic acid bacteria also increases

the chances of beer spoilage due to the present of microorganisms. However, the

microbiologically active characteristic of the beer also increases the safety of the product by

creating competition between organisms. Although aflatoxins from mould were found on

sorghum grains, they were not found in industrially produced African sorghum beer (Nakamura

et al., 2003).

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1.2 Enhancing the brewing potential of sorghum

The methods used to enhance the brewing potential of sorghum malt include manipulation of

steeping sequence (alkaline steep treatment, air-rest cycle, cold and hot water extract and warm

water final steep), appropriate cultivar selection, manipulation of germination time,

germination temperature, kilning and mashing temperature and addition of exogenous enzymes

(Okolo and Ezeogu, 1996; Dewar et al., 1997; Ogbonna et al., 2003; Owuama and Adeyemo,

2009; Ukwuru, 2010).

Manipulation of steeping sequence was targeted primarily to increase grain germinability,

develop and increase protein and enzyme synthesis, and to reduce polyphenol influence on

protein content of malts ( Okolo and Ezeogu ,1996 ; Nwanguma and Eze,1996;Ogbonna et al.,

2003). Manipulated malts have improved protein quality characteristics, such as percentage

protein, the nitrogen solubility index and the content of the first limiting amino acid, lysine

(Dewar, 1997; Ogbonna et al., 2003). It also reduces polyphenol content of sorghum which is

known to inhibit the development of enzymes and protein reserves (George et al., 2005). Air-

rest cycle when included as part of alkaline steep also helps to increase the enzymic activities

of sorghum. Some of this method (alkaline steep and final warm water steep) had rather

negative effect on germinative potentials and enzyme development (Okolo and Ezeogu, 1996).

The germination temperature of about 25°C to 30°C seems to favour enzyme development,

while Owuama (1997) suggested that kilning grains in cycles of 45°C to 60°C tend to increase

the number of enzymes than at a single temperature treatment. Mashing temperature of 65°C is

generally used in mashing barley malt, but when sorghum malt was mashed at the same

temperature the result was inadequate gelatinization of the starch and sub-optimal release of

sugars even when commercial enzymes were added. However, at a mashing temperature of

85°C and above, sorghum starch was gelatinised effectively and sugars released into the wort

was higher than at 65°C, and even higher when commercial enzymes were included at a very

low rate. Although higher temperatures and added commercial enzyme preparations used in

mashing sorghum malt dramatically increased the sugars released into the wort of sorghum

mash, the ratio of glucose to maltose did not change. An industrial exogenous enzyme such as

amyloglucosidase contributes more to the release of reducing sugars into the wort during

mashing. For more sugar yield in the wort during yeast fermentation industrial

amyloglucosidase was recommended as enzyme source (Owuama and Adeyemo , 2009).

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1.3 General Outline of the Brewing Process

All beers are brewed using a process based on a simple formula. Key to the process is malted

grains mainly barley or sorghum, although other cereals, such as wheat or rice, may be added.

(Arnold, 2005). The brewing process is made up of 8 main steps namely malting, kilning,

mashing, lautering, fermentation, conditioning, filtration and packaging. Malt is made by

allowing a grain to germinate after which it is then dried in a kiln and sometimes roasted. The

germination process promotes the production of a number of enzymes, notably α-amylase and

β-amylase, which convert the starch in the grain into sugar (Owuama, 1996; Kuntz and

Bamforth, 2007; Ukwuru, 2010).

1.3.1 Malting

Malting is a controlled germination aimed at modifying the grain. The process involves the

germination of the grain until the food store (endosperm), which is available to support the

development of germ (embryo) of the grain, has suffered some degradation from enzymes. This

involves the liberation of the granules from endospermal cell matrix by enzymes which become

active during germination and balancing of the proportion of the various reserve materials of

the grain (Ted, 2000; Wolfgang, 2004). Malters are concerned with the degradation of the

endosperm and the mobilization of the enzymes of the grain during germination. Three steps

are involved in malting, namely steeping, germination and kilning

.

1.3.2 Steeping:

Steeping involves immersing the grains in water until they have imbibed a suitable amount of

water at a temperature of about 30-40oC, until they absorb sufficient moisture to support

growth and biochemical changes during germination (Hough et al.,1981). Additives such as

formaldehyde and lime water could be added to improve germination. Steeping lasts 1-3 days

depending on grain condition. During steeping, the moisture of the steeped grain increases

rapidly at first but progressively slows down and in the absence of germination it effectively

ceases. Water uptake by ungerminated grain is a physical process which is independent of the

grain‟s viability but is accelerated if the grains are so badly damaged that their surface layer

and testa are broken (Hough et al., 1981;Goldhammer, 2008).

Steeping also serves two other functions: dirt, chaff and broken kernels are removed from the

grain by washing and floatation. The steeping step is often also used to inactivate the tannins. If

not inactivated, the tannins bind to the malt‟s amylase enzymes, resulting in reduced sugar

production. A process of inactivating the tannins by soaking sorghum grain for a 4-6 hour

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period at the beginning of steeping in a very dilute solution of formaldehyde is also used.

However, the use of formaldehyde has not been viewed favourably in recent years because of

its potential health risks. Alternative methods of inactivating tannins are now being introduced.

Steeping the grain in dilute alkali (sodium hydroxide) seems to be a safer and almost equally

effective method and is now used commercially in Nigeria (Okolo et al., 2010).

1.3.3 Germination

During germination the moist grain obtained after steeping is allowed to grow under controlled

cool conditions in the dark with no further addition of water (Briggs et al., 2004). Traditionally,

the steeped grains are put in a wetted grain bed and the temperature is maintained within the

range of 19 – 30oC. Enzymes that will modify the endospermal reserves and cell wall materials

to useful extracts are thus developed. Germination rate and modification intensity are

controlled by regulating the moisture content and the temperature of the grain (Hough et

al.,1981). Other changes occurring during germination include increase in activity of hydrolytic

enzymes present in grains. This reduces the strength of tissue and dry malt in comparison with

dry grain (Methner et al., 2003).

During germination, some soluble hydrolysis products are lost through respiration, while others

are used as substrate to synthesize other molecules in the embryo. However, the quantities of

low-molecular weight substances (the cold water extract) increases during malting (Hough et

al., 1981; Ezeogu and Okolo,1995). The germination step in sorghum malting is carried out in

two alternate ways: floor malting and pneumatic malting. In floor malting the steeped grain is

spread out on a concrete floor, normally outdoors, in a layer 10-30 cm deep. The germinating

grain may be covered with sacking or shade cloth to reduce moisture loss. The grain is watered

at intervals with a hosepipe (or by the rain). Pneumatic sorghum malting is in operation in

Nigeria, South Africa and Zimbabwe.

1.3.4 Kilning

Kilning is the final stage of the malting process which involves the drying of the green malt in

a kiln at high temperature ranging from 45oC-60

oC for 8-24 hours depending on brewer‟s aim.

This process preserves the malt and adds colour, and flavour to the finished malt.

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Kilning is controlled to prevent inactivation of the enzymes developed during germination. The

finished malt contains enzymes like α-amylase, β-amylase and maltase, which can breakdown

starch to maltose, glucose and other simple fermentable sugars (Bamforth, 2005).

1.3.4 Mashing

Mashing, also referred to as wort production, is a process that involves mixing crushed malt

and hot water together to extract sugars and nutrients from the malt (Brigg et al., 2004). The

primary aim of mashing is to produce optimal substrate for yeast fermentation. Traditionally,

barley malt was the main ingredient, but today other starchy cereals such as maize, sorghum

and rice are also used for beer production, particularly in sub-Saharan African countries (Taylor

et al., 2006).

There are a number of different mashing methods, namely infusion mashing, double decoction

mashing and temperature programmed mashing (Briggs et al., 2004). In all these mashing

methods, the objective is degradation of starch, proteins, lipids, beta-glucans, pentosans and

xylans to produce fermentable wort (Briggs et al., 2004, Kuntz and Bamforth, 2007). The

starch degrading enzymes, α-amylase and β-amylase, developed during malting are responsible

for hydrolysis of starch into fermentable sugars. The optimum temperature for α-amylase

activity during mashing is in the range of 55°C and 60 °C, while β-amylase is temperature

labile, thus effective between 50°C and 55°C (Sivaramakrishnan et al., 2006 ). In case of

protein hydrolysis, proteases are responsible for degradation of storage protein to produce free

amino nitrogen. Free amino nitrogen is essential for yeast growth and fermentation. After

mashing, sweet wort is produced, while spent grain is gathered as by-product. The wort is then

cooled and hops are added and the wort boiled for approximately 60- 90 minutes. The

importance of adding hops is to allow α- and β- acids present in hops, to provide bitterness and

aroma to the final product (Briggs et al., 2004). During mashing the milled grain is mixed with

water which has been treated to remove temporary hardness caused by carbonates and

bicarbonates. Calcium salts, however, are added as this lowers the pit and increases the wort

extract (Ogbonna et al., 2004).

1.3.6 Lautering

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Lautering is the separation of the wort from the grains. This is done either in a mash tune

outfitted with a false bottom, or a mash filter. Most separation processes have two stages; first

wort run-off, during which extract is separated in an undiluted state from the spent grains, and

sparging, in which remains with the grains are rinsed off with hot water (Micheal, 2004).

1.3.7 Fermentation

The cool wort is pumped or allowed to flow by gravity into the fermentation tanks and yeast is

inoculated or pitched in. During fermentation the wort convert the sugars in the wort chiefly to

alcohol and CO2, plus small amounts of glycerol and acetic acid. Proteins and fat derivatives

yield small amount of higher alcohol and acids. As the carbon dioxide is evolved in increasing

amounts, the foaming increases and gradually disappears as fermentation comes to completion.

At a later stage, the bottom yeast “breaks” i.e. flocculate and settles at the bottom

(Goldhammer, 2008). The beer is then transferred to an airtight container, called a conditioning

tank, for a second fermentation or aging period, where the beer becomes naturally carbonated

(Gibson, 2010). Some brewers inject carbon dioxide gas into the beer when aging is completed

to give it a bubbly and effervescent quality. Aging lasts for a few weeks to several months,

depending on the type of beer being produced. Essentially, fermentation could be of two types:

top fermentation or bottom fermentation.

Top fermentation is used in the United Kingdom for the production of stout and ale, using

strains of Saccharomyces cerevisiae. The wort is introduced into the fermenting bin and the

yeast is pitched in at a rate suitable for the desired temperature. The entire fermentation takes

about six days. Yeast floats to the top during this period after which they are scooped off and

used for future pitching (Omafuvbe et al.; 2000; Goldhammer, 2008).

In bottom fermentation, special beer yeast of the strain Saccharomyces uvarum is used for the

pitching of the cooled wort (Okafor, 2007). The wort temperature during the fermentation

varies in different breweries but is usually in the range of 3.3oC-14

oC. The fermentation process

is completed within 8-10 days (Gibson, 2010).

After aging, the beer may appear somewhat cloudy from yeast cells and other particles that

remain suspended in the liquid.

1.3.8 Conditioning

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During conditioning, the beer is cooled to around freezing, which encourages settling of the

yeast, and causes proteins to coagulate and settle out with the yeast. Unpleasant flavours, such

as phenolic compounds, become insoluble in the cold beer, and the beer‟s flavour becomes

smoother. This can take 2-4 weeks and serves to reduce sulfur compounds produced by the

bottom fermentation yeast and to produce cleaner tasting final product with fewer esters

(Merchant du vin, 2009).

1.3.9 Filtration

Filtering the beer stabilizes the flavour, and gives beer its polish shine and brilliance. Not all

beer is filtered. This is done to remove yeast, hops, and grain‟s particle left in the beer

(Wolfgang, 2004). The most common method of removing these impurities is filtration, a

process in which the finished beer is pumped, under pressure, through a sterile filtering system

that traps nearly all of the suspended particles from the liquid, resulting in a clear liquid

(Abiodun, 2002). At the end of filtration, the beer usually contains some yeast. However,

during pasteurization (82° C or 180° F) the remaining yeast are killed. Draught beer, which is

stored in metal kegs, usually is not pasteurized and must be kept refrigerated to prevent it from

spoiling. Some brewers and beer drinkers believe that filtering and pasteurizing beer robs it of

much of its original flavour and character ( Hui and Smith, 2004).

1.3.10 Packaging

Packaging involves putting the beer into the containers in which it will leave the brewery,

typically, this means putting the beer into bottles, aluminum cans or kegs (Merchant du vin,

2009).

1.4 Role of Lipids in Brewing

Lipids are naturally occurring substances which are soluble in organic solvents but not soluble

in water (Ononogbu, 1988). Lipids are important in brewing because they are essential to yeast

growth and metabolism and also contribute to several quality parameters of the finished beer

(Letters, 1992). Lipid materials present in the wort and beer originate from the malt and, to a

less extent, the adjuncts. The lipid classes extracted in the wort include the free fatty acids,

glycerol, sterols (free and esterified ) and phospholipids (Uchida and Ono, 2000). With respect

to yeast metabolism, the free fatty acids are considered the most important of the lipid classes

present in the wort (Chen, 1980). The transition from wort to beer is accompanied by a

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significant change in the fatty acid composition originally present in the wort. Thus, while

palmitic, linoleic, stearic and oleic acids account for 85-90% of the total fatty acids found in the

wort (Chen, 1980) the alteration involves a change from long-chain to medium chain fatty

acids. Although the possibility of a direct conversion of wort fatty acids to those of beer has

been subject to speculation, it is currently believed that the medium chain fatty acids are not

direct degradation products of the fatty acids in wort. Thus, the long-chain fatty acids in wort

are utilized primarily for the growth and maintenance of yeast cells which subsequently,

synthesize and release the medium chain fatty acids found in beer (Chen, 1980). The roles of

lipids in brewing could be both beneficial and adverse.

1.4.1 Beneficial role of lipid in brewing

As wort nutrients, lipids are essential for the growth, metabolism and viability of yeast cells

(Chen, 1980; Bamforth, 1986). This is because yeast cell membranes require the presence of

lipids to be able to absorb nutrients from the wort. The presence of lipids also endows yeast

cells with the very important property of ethanol tolerance (Bamforth, 1986). This property

enables the yeast cells to survive in the presence of high concentrations of ethanol which are

produced during the process of fermentation.

In addition, medium chain fatty acids are believed to be responsible for the typical beer

flavours in finished beer (Clapperton and Brown, 1978). Thus, fatty acids in different

concentrations in different beers have an additive effect on beer flavour. The presence of high

amounts of lipids in the worts can bring about a reduction in the concentration of these esters

(methyl linolenate, vinyl esters) in the finished beer (Clapperton, 2000). Individual lipids differ

in their ability to destroy beer foam. Dipalmitin is reportedly a more potent foam destroyer than

either palmitic acid or monopalmitin. Similarly, the short or medium-chain fatty acids ( C6– C

10) are less harmful to beer foam than the longer chain fatty acids (Clapperton and Brown,

1978).

1.4.2 Non-beneficial role of lipid in brewing

The adverse effects of high concentration of lipids include reduction of head retention and

promotion of flavor deterioration or staling (Bamforth, 1986). Staling involves the formation of

flavour-active aldehydes (Bamforth, 1986) such as trans-2-nonenal that interferes with the

flavour of the beer. There are also other routes of aldehyde formation such as (i) oxidation of

lipids (ii) oxidation of alcohol, (iii) strecker degradation of amino acids and adol condensation.

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1.4.3 Lipid oxidation in brewing

The oxidation of lipids which occurs during brewing involves both enzymatic and non-

enzymatic mechanisms (Chen, 1980). Linoleic acid is susceptible to both enzymatic and non-

enzymatic oxidation that produces carbonyl compounds, such as trans-2-nonenal (Bamforth,

1986). Peroxidation (auto-oxidation) of lipids exposed to oxygen is responsible not only for

deterioration of food (rancidity) but also for damage to tissues in vivo, where it may be a cause

of cancer, inflammatory diseases, atherosclerosis, etc (Manuel et al,2005). The deleterious

effects are initiated by free radicals (ROO, RO, OH) produced during peroxide formation from

fatty acids containing methyl interrupted double bonds, ie those found in the naturally

occurring polyunsaturated fatty acids. Lipid peroxidation is a chain reaction providing a

continuous supply of free radicals that initiate further peroxidation. The whole process can be

depicted as shown in figure 1.1

(1) Initiation

ROOH + Metal(n)+

→ ROO + Metal(n-1)+

+ H+

Xo + RH → R

o + XH

(2) Propagation: Ro + O2 → ROO

o, R.

ROOo + RH → ROOH + R

o, etc

(3) Termination: ROOo + ROO

o → ROOR + O2

ROOo + R

o → ROOR

Ro + R

o → RR.

Figure 1.1: Lipid peroxidation of fatty acids (Letters,1992).

Since the molecular precursor for the initiation process is generally the hydroperoxide product

ROOH which binds to metal ions releasing free radicals and hydrogen ion. The released free

radicals then undergoes chain reaction that produces more of the free radicals. Lipid

peroxidation is a chain reaction with potentially devastating effects in the brewing industry. To

control and reduce lipid peroxidation, antioxidants such as propyl gallate, butylated

hydroryanisole (BHA), and butylated hydroxytoluene (BHT), vitamin C, are used as food

additives. Naturally occurring antioxidants used to control lipid peroxidation include vitamin E.

(tocopherol), urate and vitamin C. Beta-carotene is also an antioxidant at low pO2

(Devasagayam et al.,2003).

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Antioxidant enzymes include peroxidase, catalase and superoxide dismutase which also

contribute towards the control of lipid peroxidation. Peroxidase, which has high thermostablity,

reduces lipid peroxidation and prolongs the shelf life of beer. Malt contains peroxidase,

lipoxygenase, lipase and hydroperoxide isomerase which are synthesized during germination

and are involved in the oxidation of linoleic acid during malting and mashing (Baxter, 1982).

1.4.4 Enzymatic oxidation of lipids in brewing

The enzymatic oxidation of lipids during brewing is catalyzed sequentially by two malt

enzymes, lipoxygenase and hydroperoxide isomerase. Lipoxygenase catalyses the oxidation of

unsaturated fatty acids with a diene moiety such as linoleic and linolenic acids, to produce the

corresponding hydroperoxides. In the presence of hydroperoxide isomerase, the hydroperoxides

are converted to the corresponding ketols. In its absence, however, the hydroperoxides are

converted non-enzymatically to trihydroxy acids (Bamforth, 1986). These intermediates of fatty

acids oxidation serve as direct precursors of the flavor-active unsaturated aldehydes which are

formed by their non-enzymatic decomposition (Croft et al., 1993; Devasagayam et al., 2003)

(figure 1.2).

The first step in the enzymatic oxidation involves the insertion of hydroperoxyl group into the

unsaturated fatty acid by malt lipoxygenase. Two lipoxygenases have been identified in

germinating barley, one which inserts the hydroperoxyl group at C-13 and the other which

attacks at C-9 to give C-13 and C-9 hydroperoxides. The lipoxygenases require oxygen for

their actions, isomerase in the malt can convert hydroperoxide to ketol, then to dihydroxy acids

(Devasagayam et al., 2003).

Unsaturated fatty acids

O2

Hydroperoxide intermediate

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Figure 1.2: Specific oxidation of linoleic acid (Letters, 1992).

1.4.5 Non- enzymatic oxidation of lipid in brewing

Non- enzymatic oxidation of lipid involves two steps, primary autoxidation reaction that leads

to the formation of hydroperoxides and further reactions of hydroperoxides which lead to the

manifestation of autoxidation in foods.

The non- enzymatic oxidation (autoxidation) of lipids is catalyzed during brewing by oxygen,

divalent metal ions, light, heat and a number of other compounds such as polyphenols

(Bamforth et al., 1993). While the enzymatic oxidation can be limited by heat treatments which

are employed at some stages of the brewing process, the non-enzymatic oxidation is enhanced

by similar treatments, and therefore continues during beer storage (Kobayashi et al., 1993;

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Kobayashi et al., 2000). Although it is not easy to determine which of these mechanisms

accounts for a greater part of the aldehydes present in aged beer, the non-enzymatic oxidation

has proved more difficult to control (Kobayashi et al.,1993). Autoxidation occurs in nature as a

chemical reaction between atmospheric oxygen and a number of unsaturated compounds and

configuration of the double bonds (Chen et al., 2004). The monoperoxides can, however,

undergo a free radical chain reaction resulting in the isomerisation of a single isomer to all

other isomer forms of hydroperoxides and therefore constitutes a problem in the understanding

of the autoxidation of linoleate (Chen et al., 2004). The autoxidation of unsaturated lipids

containing more than 2 methylene interrupted double bonds, for example, methyl linolenate,

results not only in the formation of isomeric monohydroperoxides but also in a mixture of

hydroperoxy-epidioxides ( diperoxides) in which two molecules of oxygen are incorporated to

form a cyclic peroxide function as well as a hydroperoxide function (Chen et al., 2004).

The autoxidation of methyl linolenate is somewhat analogous to that of methyl linoleate in

terms of the oxygenation of pentadienyl radicals except that two 1,3-diene systems, the 9, 13

and the 12, 16 systems are involved instead of one. Thus, unlike the methyl linoleate which

yields equal amounts of the 9-OOH and the 13 – OOH isomers, the autoxidation of methyl

linolenate yields far higher amounts of the 9 –OOH and 16- OOH isomers. The difference in

the proportions is because the other isomer, 12 –OOH and the 13-OOH, whose -OOH groups

occur inside the system of double bonds participate in the reaction (cyclisation) but the isomer

whose -OOH groups exist outside do not cyclise (Chen et al., 2004).

The decomposition occurs by a hemolytic mechanism and is catalyzed in food systems by

transition metal ions or metallo-proteins and in biologically active systems by enzymes. Of all

the known secondary products of lipid oxidation, the carbonyl compounds, especially the

aldehydes, have received much attention in both food and physiological systems. This is mostly

due to their potential to cross-link biological macromolecules including proteins, amino acids

and nucleic acids resulting either in quality deterioration in food or various forms of toxicity,

including carcinogenicity and mutagenicity in living systems (Koshio et al.,1994).

1.4.6 Control of lipid oxidation during brewing

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Antioxidants are defined as compounds able to quench peroxides, (like sulfite), Vitamin C and

Vitamin E, some polyphenols etc., or able to inactivate trace amounts of metals that otherwise

may generate hydroxyl or alkoxyl radicals from the peroxides ( Andersen et al. , 2000). After

depletion of these antioxidants, •OH radicals will accumulate and can cause damage to beer

components. The •OH radical displays a high reactivity toward several beer compounds, such

as ethanol, sugars, isohumulones, polyphenols, alcohols, fatty acids, etc. This can initiate a

series of reactions responsible for the production of carbonyl and phenolic radicals and

ultimately lead to staling compounds in beer. This lack of specificity of the •OH radical makes

it almost impossible to quench this radical intermediate in beer by adding antioxidants that

would react specifically with this radical. Hydrogen peroxide and organic hydroperoxides are

the only reactive oxygen species that are stable enough to be trapped efficiently by antioxidants

which normally only are present in micro-molecular concentrations (Andersen et al., 2000).

Because of the high reactivity of ethanol, being the most abundant compound in beer, this

compound will be the primary reactant of •OH, resulting in the EtO

• radical. This radical will

bind oxygen, resulting in acetaldehyde and the hydroperoxyl radical. The latter can be reduced

to hydrogen peroxide, which can react again with metal ions to continue the radical chain

reactions. As it is impossible to know all of the variables necessary to prevent oxidative

deterioration by reactive oxygen species, minimization of radical formation will be the best

strategy. This can be accomplished by keeping the level of O2 as low as possible, by lowering

the temperature, and by introducing chain breakers or quenchers, generally referred to as

antioxidants. Of all the artificial antioxidants used in the food industry, only sulphites and

ascorbic acid which are water soluble are capable of scavenging oxygen- derived free radicals

in aqueous solutions.

Thus, only these two antioxidants have found wide application in the beer industry where they

are added (either singly or in combination) to beer before packaging (Kocherginsky et al.,

2005). These antioxidants have proved quite effective in improving beer flavour, stability and

their introduction to beer is a standard practice in many countries (Uchida and Ono,1996;

2000).

However, the use of these antioxidants in improving beer shelf –life is associated with a

number of limitations and has therefore, not eliminated the problems of flavor deterioration in

beer. Although ascorbic acid interferes effectively with the oxidation pathway by mopping up

oxygen derived radicals and by terminating radicals chain reactions, in the presence of iron and

16

copper its inhibitory role is reversed to that of acceleration of oxidation (Bamforth et al., 1993).

Similarly, sulphur dioxide can mop up oxygen in addition to binding the aldehydes present in

beer.

However, as beer ages, the ability of sulphite to bind aldehydes is overwhelmed by the

increased production of staling aldehydes (Brown, 1989). This is aggravated by the oxidative

loss of sulphite which occurs normally during beer storage. An enzymatic deoxygenation

method which involves the use of such antioxidant enzymes such as catalase and glucose

oxidase to remove residual oxygen from packaged beer is an additional option in some

breweries. However, the wide application of this method is constrained by high costs of

enzymes, while the efficiency is restricted by the low levels of specific substrates for the

relevant enzymes in beer (Wolfgang, 2004).

Whilst an adequate supply of oxygen (aeration) is necessary for the chemical and biological

processes which take place during preparation of the wort and fermentation, high oxygen

content in the beer itself can have adverse effect on the storage properties and quality. The

compounds formed by oxidation of the tannin substances preferentially react with the

proteins to give insoluble complexes, which can lead to permanent cloudiness. Furthermore,

under the influence of oxygen, colour changes (increase in colour), deterioration in flavour as

a result of destruction of aroma substances and finally biological instability also occur.

Although inadequate amounts of these oxygen acceptors are present, the beer contains

reductones and melanoidins from the brewing process and these, as oxygen acceptors, protect

The polyphenols and the proteins from oxidation. Reliable protection from oxidation can

thus be achieved only by addition of a powerful reducing agent. Apart from apparatus

technology measures for reducing the influence of air during bottling, ascorbic acid,

which is identical to the essential vitamin C and is therefore physiologically absolutely

acceptable, has proved best suitable as an oxygen acceptor in the brewing of beer (Kaneda et

al.,1996). It is today used throughout the entire foods industry as a reliable antioxidant

compared with other reducing agents, eg. salts of sulfurous acid. The major disadvantage of

superoxide dismutase (SOD) is its adverse effect on the sensory properties of the beer and is

usually left behind in the beer. This is not so with ascorbic acid which have powerful

reducing property that enables it to bond to oxygen more rapidly (Kaneda et al.,1996;

Methner et al., 2003).

17

The amount of ascorbic acid added essentially depends on the oxygen content, the

technological requirements on bottling and the turn-around time of the beer. It should be taken

into consideration that about 11 mg of ascorbic acid are required for inactivation of 1 mg of

oxygen. For example, if a beer bottled under a particular operating condition contains 1.5 mg of

oxygen per liter, about 1.65 g of ascorbic acid per hectoliter must be added for inactivation.

Ascorbic acid is very readily soluble in beer. In the small amounts required for stabilization

from oxidation, its acid flavour in no way impairs the taste of the beer. Traces of metals,

especially copper and iron, accelerate the oxidation of the dissolved ascorbic acid by

atmospheric oxygen.

Research attention is also being emphasized as to the possible control of the chemical processes

which affect beer flavour stability during the early stages of brewing namely, malting and

mashing (Clarkson et al.,1992; Vanderhaegen et al., 2006). This was necessitated by the

realisation that large amounts of staling aldehydes and their precursors are formed at these

stages and some of them survive in the finished beer thereby contributing to the total

concentration of staling aldehydes present in beer (Kobayashi et al., 1993).

1.4.7 Role of anti-oxidant enzymes in beer stability

As part of physiological protection against various forms of toxicity caused by reductive forms

of oxygen, living cells synthesize and use a number of enzymes referred to collectively, as

oxygen radical scavenging enzymes or simply, antioxidant enzymes. These enzymes include

superoxide dismutase (E.C. 1.15.1.1), catalases (1.11.1.16) and peroxidase (E.C.1.11.1.7)

Superoxide dismutase (SOD) catalyses the dismutation of superoxide radicals to ground state

oxygen and hydrogen peroxide (Scandalios, 1993; Antoyuk et al., 2009, reaction 1).

2O2- + 2H

+ SOD 2H2O + O2 …………… Reaction 1

The superoxide so generated is a reactive specie and is eliminated subsequently by catalase

viz: ( Clarkson et al.,1992, reaction 2).

Catalase

2H2O2 2H2O + O2 ………….………………. Reaction 2

18

Peroxidase catalyses the reductive destruction of hydrogen peroxide in living system (Hiraga

et al., 2001, reaction 3).

POD

H2O2 + RH2 2H2O + R …………………….Reaction 3

The potential stages in brewing where oxygen radicals might be formed include malting,

kilning and mashing (Clarkson et al.,1992). However, the products of their oxidative reactions

with malt lipids survive in the finished beer and contribute to loss of flavour stability during

storage. All the three antioxidant enzymes have been demonstrated in barley (Clarkson et

al.,1992). Their activities in barley increases during malting and they help limit the rate of

oxidation reactions occurring at this and later stage of brewing. Although the role of all these

enzymes is important for long shelf-life of beer, only peroxidase has been shown to survive the

heat treatments of kilning because of its high thermostability.

19

N

Fe(III)

His

HRP resting(brown)

N

N

N

A2H

2 or A + AH

2

AH.

e H

AH.

AH

ROOH

ROH

2e

N

Fe(IV)

His

HRP - II(green)

N

N

N

O .+

N

Fe(IV)

His

HRP - II(pale-red)

N

N

N

OAHAH.

e H

1.4.8 Mechanism of action of peroxidase

Figure1.3 : The general mechanism of horseradish peroxidase (HRP) showing the conversion

of native enzyme to HRP-II and HRP-II (Veitch, 2004).

The general mechanism of HRP involves two reaction steps.

1) A two-electron oxidation of the native ferric enzyme to compound 1 intermediate

(HRP-1), with the peroxidase substrate cleaved at the O-O

2) A two one-electron reduction of HRP-1 by electron donor substrates to the native

enzyme via compound 11 intermediate (HRP-11)

The common substrate for step 1 and 2 are H2O2 and aromatic compounds, respectively (Figure

1.3).

20

Figure 1.4 :Complete peroxidases catalytic cycle (Villalobos and Buchanan, 2002).

During the catalytic cycle of peroxidase, the ground state enzyme undergoes a two electron

oxidation by H2O2 forming an intermediate state called compound I (E). Compound I (E) will

accept an aromatic compound (AH2) in its active site and will carry out its one-electron

oxidation, liberating a free radical (●AH) that is released back into the solution, and converted

to compound II (Ei). A second aromatic compound (AH2) is accepted in the active site of

compound II (Ei) and is oxidized, resulting in the release of a second free radical (●AH) and the

return of the enzyme to its resting state, completing the catalytic cycle (Figure 1.4). The two

free radicals (●AH) released into the solution combine to produce insoluble precipitate that can

easily be removed by sedimentation or filtration. The stoichiometry report is based on one mole

of peroxidase consumed per mole of aromatic compound removed from the solution; this

stoichiometry depends also on the amount of enzyme consumed in side reaction and the

precipitating oligomers. Therefore, various side reactions that take place during the removal

process are responsible for the enzyme inactivation (E) or inhibition (Eii) leading to a limited

life time, but this form is not permanent since compound III (Eii) decomposes back to the

resting state of peroxidase. Some peroxidases, like horseradish peroxidase (HRP) (figure 1.5),

lead to a permanent inactivation state (P-670) when H2O2 is present in excess or when the end-

product polymer adheres to its active site, causing its permanent inactivation by causing

changes in its geometric configuration (Villalobos and Buchanan, 2002). The optimization of

enzymic activity is very important for related applications. Response Surface Methodology, as

succinctly described below, is a useful tool for this purpose (El Agha et al., 2008, 2009).

21

Figure 1.5: The catalytic cycle of horseradish peroxidase (HRP C) with ferulate as reducing

substrate. The rate constants K1 K2 and K3 represent the rate of compound 1 formation. rate of

compound I reduction and rate of compound II reduction, respectively l (El Agha et al., 2008).

1.4.9 Importance of peroxidase in brewing

Lipid peroxidation is undesirable in malting and brewing because the products from the

reaction, namely the hydroperoxides and their decomposition products, the aldehydes affect the

availability of wort nutrients and affect the beer flavour and colloidal stability. Among the three

antioxidant enzymes (superoxide dismutase, catalase and peroxidase) peroxidase is reported to

have survived the kilning process due to its high temperature stability (Nwanguma and

Eze,1995). The survival of peroxidase during the kilning stage demonstrates that its antioxidant

function or role is guaranteed in the wort, thereby, contributing to the enhancement of the shelf

life of beer.

1.4.10 Aim and objectives of research

22

Steeping sorghum in 0.1N NaOH (alkaline steep) and the introduction of air-rest cycles have

been recommended for enhancing or improving the malting parameters of different sorghum

varieties. Before these modifications, standard steeping methods received widespread

application. The need to determine their effect on a number of other processes that take place

during malting becomes necessary.

Thus, the aim of this study was to determine the effect of alkaline steep and air-rest cycle on

the development (activity) of peroxidase during malting of sorghum. The interest in

peroxidase is based on an earlier report that its activity is important in controlling lipid

peroxidation during malting and mashing. The adopted air-rest cycle and alkaline steep regimes

were those already recommended for commercial scale malting because of their desirable effect

on some malting properties of sorghum.

CHAPTER TWO

23

2.1 Materials

The sorghum variety KSV8 used in this study was obtained from the National Seed

Service Centre (NSSC) Zaria, Kaduna State.

2.2 Equipment

Beaker Pyrex

Conical flasks (100 ml, 500 ml) Pyrex

Centrifuge PAC, Pacific

Electronic pH meter Mettle toledo

Filter paper ( Whatman No. 1 and Ashless 9 cm). Whatman

Measuring cylinders (10 ml, 100 ml, 500 ml ) Pyrex

Mortar and Pestle

Oven Gallenkamp England

Pipettes (I ml, 2 ml, 5 ml, 10 ml) Pyrex

Petri dishes Pyrex

Refrigerator Thermocool

Spectrophotometer – SP 6400 Jenway

Spatula

Thermometer Zeal

Test tubes Pyrex

Weighing balance Mettle toledo

Water bath (CB.2 Sq2) Gallenkamp England

2.3 Chemicals/Reagents

All reagents were of analytical quality.

Acetic acid BDH, England

Bovine serum albumin BDH, England

Boric acid Sigma, USA

Copper sulphate Merck, USA

Disodium hydrogen phosphate Sigma, USA

Folin Cocalteau reagent BDH, England

24

Hydrogen peroxide BDH, England

O – dianisidine BDH, England

Sodium dihydrogen phosphate Sigma, USA

Sodium carbonate Sigma, USA

Sodium potassium tartarate BDH, England

Sodium hydroxide May and Baker, England

2.4 Methods

2.5 Malting

2.5.1 Steeping Methods

The alkaline steep and air-rest steep methods used were those of Okolo and Ezeogu (1996)

Steep regime I (alkaline steep) .

(Test ) Grains (500) were steeped in alkaline water (0.1% sodium hydroxide solution) at

pH 13.25 for 8 h at 30°C followed by a cycle of 4 h dry; 6 h wet for 40 h with final

steep temperature maintained at 40°C for 6 h.

(Control ) Similar to regime I but with distilled water at pH 5.97.

Steep regime II

( Test ) Grains (500) were steeped in 40ml of distilled water at 30°C for 24 h; it was

then subjected to a steep cycle of 10 h wet and 1 h air-rest.

(Control ) Similar to regime II except that a continuous steep was applied.

After 24 h, the water was drained off in both test samples and control and were allowed

to germinate in moisten box inside a dark cupboard for 72 h.

2.5.2 Determination of Germinative Energy

Germinative energy (%), a measure of the number of grains which will germinate in a

malting experiment, (i.e. an index of seed viability) was determined as follows:

Lots of one hundred seeds each were spread evenly at the bottom of 9-cm glass petri dishes;

each lined with 2 layers of whatman No 1 filter paper, wetted uniformly with 4ml of distilled

water. The covered petri dishes were place in the dark and germination counts were taken at

25

24-h interval for 72h. This test was repeated using different steeping regimes (Okolo and

Ezeogu, 1996).

2.5.3 Determination of Water Sensitivity

Water sensitivity tests are carried out in the beer industry before batches of grains are

used for brewing. This is because some grains are sensitive to the volume of water used during

germination thereby limiting the number of seeds that will germinate. Water sensitivity was

calculated as the difference in the number of germinated seeds in the 4ml and 8ml petri dishes

at the end of 72 h (Okolo and Ezeogu, 1996).

2.5.4 Determination of Average Root Length

The root lengths of the malted grains were determined as follows: Twenty seedlings

were picked randomly from a lot of 500 malted sorghum grains, and the length of the radical

(root) of each seedling was measured with a ruler. The mean value of the lengths was

determined as the root length (in centimeter) (Ogbonna et al., 2003).

2.5.5 Determination of Malting Loss

Malting loss is a measure of the material lost during malting. To determine the malting

loss, 500 germinated seed were counted out and the roots removed, then weighed and the

values recorded (W2). Next, 500 ungerminated seeds were weighed out and the weight recorded

(W1) (Okolo and Ezeogu, 1996).

The malting loss was calculated as follows: =

Malting loss = 1

100

1

21

W

WW.

W1 = Weight of 500 ungerminated seed

W2 = Weight of 500 malted seeds

2.6 Preparation of Reagents

2.6.1: Preparation of Phosphate Buffer

26

Preparation of 0.1M sodium dihydrogenphosphate of pH 6.0 and 7.0.

1.1996 g of Na2HPO4 and 1.42 g of NaH2PO4 were each dissolved in 100ml of distilled water.

The acid was titrated against the base till a pH of 6.0 and 7.0 were obtained respectively (

Deangelis, 2007).

2.6. 2 Preparation of Substrate

Exactly 0.1 g O-dianisidine was dissolved in 100 ml methanol and then filtered and stored in

dark bottle (Nwanguma and Eze, 1995).

2. 6.3 Preparation of Stock Hydrogen Peroxide Solution ( 3% H2O2 ).

30% - 100ml

3% - 3%×100

30% = 10 ml

Fresh preparation of 3% H2O2 stock was made by adding 10ml of H2O2 to 100ml of water

(Nwanguma and Eze, 1995).

2.6.4 Preparation of Reagent for Protein Determination

Solution A : 2 g of Na2CO3 and 0.4 g of NaOH all in 100 ml of distilled H2O.

Solution B : 0.5 g of Cu2SO4 and 1 g of Sodium-potassium tartarate in 100 ml of distilled

water.

Solution C: Folin Ciocalteau phenol reagent/distilled water (1:1)

Solution D: 50 ml of solution A + 1 ml of solution B (Lowry et al.,1951).

Different concentration of the Bovin serum albumin (BSA) were prepared according to this

ratio ( protein : water) in order:10:0 ,9:1 8:2,7:3,6:4,5:5 4:6,3:7,2:8:,1:9,10:1

To each test tube, 5ml of solution D (alklaline copper reagent solution) was added to the test

tube and left for 5minutes

0.5ml of solution C (folin cioutatue) was added to the test tubes rapidly and thoroughly mixed.

Solution was left for 30minutes to incubate and the absorbance read at 750nm.

The final concentration of the protein was calculated using the equation C1V1=C2V2

27

2.7 Extraction of Sorghum Peroxidase

Fifty (50) grains of KSV8 were collected and weighed. They were then ground in a

mortar containing 50 ml of phosphate buffer, pH 6.0. The crude extract was then centrifuged at

3500 rpm for 15 minutes and the resulting supernatant was used as the crude enzyme extract

(Nwanguma and Eze, 1995).

2.8 Assay of Sorghum Peroxidase

Exactly 0.3ml of o-dianisidine, 0.1ml 3% H2O2, 2.5ml 0.1M sodium-phosphate buffer,

pH 7.0 and 0.1ml of the crude enzyme extract were used.

In a test tube containing 0.1ml 3% H2O2 , 2.5ml 0.1M sodium-phosphate buffer was added.

0.3ml o-dianisidine plus 0.1ml of the crude enzyme was then added into the test tube. The

reaction mixture was shaken vigorously. From the test tube, a total of 3ml of the reaction

mixture was transferred into the curvette. After 10 minutes of oxidation in a spectrophotometer

the absorbance was obtained and recorded for both the test sample and the control at 30

seconds intervals (Nwanguma and Eze, 1995).

2.9 Kilning Method

At the end of Germination, 50 grains of germinated sorghum were collected from each samples

and placed in a petri dishes. It was then transferred into an electric oven and left for 7h.The

temperature of the oven was 60 °C.

2.9.1 Statistical Method

Statistical analysis was done using standard mean deviation.

SN = √1/𝑁 (𝑥𝑖 − 𝑥⎺)𝑁𝑖=1

2

CHAPTER THREE

28

3.0 RESULTS

Table 3.1: Germinative properties of KSV8.

Regimes

Germinative

Energy

(%)𝑎

Germinative

capacity

(%)𝑎

Water

sensitivity

(%)𝑎

Malting

loss (%)𝑎

Malt yield

𝑏

(%)𝑎

Root

length (cm)

(%)𝑎

Regime (test)

0.1N NaOH

92.0 ± 2.87 98.0 ± 0.57 6.0 20.1 ±0.93 79.9 1.3 ± 0.47

Regime I

Control

89.0 ± 0.71 96.0 ± 1.14 4.0 20.0 ±1.28 80.0 1.4 ±0.28

Regime II

Test

95.0 ± 1.41 99.0 ± 0.0 6.0 5.6 ± 1.28 94.37 1.5 ±0.07

Regime II

Control

95.0 ± 0.57 97.0 ± 1.15 5.0 15.84± 0.19 84.16 2.44± 0.54

a Results are means of triplicate trials

b Calculated as 100 % - malting loss( % )

Germinative energy and germinative capacity of the seeds steeped in 0.1N NaOH were

considerably high and are 92.0 ± 2.87 and 98.0 ± 0.57 respectively. Water sensitive test for the

sorghum seed was found to be low as well as yield in malt and root length.

The germinative properties of sorghum variety KSV8 steeped in distilled water in

regime I (control) showed that the germinative energy and germinative capacity of the sorghum

seeds were considerably high and are 89.0 ± 0.71 and 96.0 ± 1.14 . Yield malt was low ( 80.0)

and most of the malts were lost in form of root length or used in root growth.

29

The germinative properties of sorghum variety KSV8 steeped in distilled water for 10 h

and exposed to 1 h air-rest cycle is shown in regime II (test). Results obtained showed that the

germinative energy (95.0 ± 1.41 ) and germinative capacity (99.0 ± 0.00) of the sorghum seeds

were considerably high as well as malt yield. However, malting loss (5.6 ± 1.28) and root

length (1.5 ± 0.07) of the sorghum seed were low.

The sorghum seed (regime II control) had high germinative energy (95.0 ± 0.57) and

germinative capacity (97.0 ± 1.15). Malting loss 15.84 ± 0.19 and root length 2.44 ± 0.54 of the

sorghum seed increased greatly but there was a decrease in the yield malt.

30

Figure 3.1: Peroxidase activity for alkaline and warm steep at 40°c for regimes I. Results are

means of triplicate trials.

Figure 3.1 represents the plot of the effect of alkaline steep on the development and levels of

peroxidase activity at the end of steeping. The level of peroxidase activity in the seeds steeped

in 0.1N NaOH (alkaline steep) demonstrated a much higher level of activity than the seeds

steeped in distilled water (control).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time(min)

Alkaline steep (test)

Distlled water (control)

31

Figure 3.2: Peroxidase activity at the end of 24h germination for regime I. Results are means

of triplicate trials.

Figure 3.2 shows the level of peroxidase activity at the end of 24 h germination ( Regime 1).

From the result, at the end of germination for 24 h, the difference between the levels of

peroxidase activity in the test ( 0.728) and the control seeds (0.607) had narrowed down.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time(min)

Alkaline steep (test)

Distlled water (control)

32

Figure 3.3: Peroxidase activity at the end of 48h germination for regime I

Figure 3.3 shows the effect of alkaline steep on the levels of peroxidase development at the end

of 48 h germination. The results showed that the levels of peroxidase activity in the test (0.662)

and the control seeds (0.620) had also narrowed down. Peroxidase activity in the control seed

was high compared to peroxidase activity in the test seeds.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time(min)

Alkaline steep (test)

Distlled water (control)

33

Figure 3.4: Peroxidase activity at the end of 72h germination for regime I. Results are means

of triplicate trials.

Figure 3.4 represents plot on the effect of alkaline steep on the development of peroxidase at

the end of 72 h germination. From the result, the control seeds demonstrated a higher level of

peroxidase activity ( 1.764) than the test seeds (1.764) .

00.20.40.60.8

11.21.41.61.8

2

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time(min)

Alkaline steep (test)

Distlled water(control)

34

Figure 3.5: Peroxidase activity at the end of kilning for regime I. Results are means of

triplicate trials.

Figure 3.5 shows peroxidase activity at the end of kilning at 60 °c for 7 h . From the results,

there was a variation in the levels of decrease in peroxidase activity in the test seeds and the

control seeds. At the end of kilning, the level of peroxidase activity in the seeds (0.425) was

much lower than the level in the control ( 0.553).

0

0.1

0.2

0.3

0.4

0.5

0.6

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time(min)

Alkaline steep (test)

Distlled water (control)

35

Fig 3.6: Peroxidase activity at the end of 24 h of steep for regime II. Results are means of

triplicate trials.

Figure 3.6 shows the effect of air-rest cycle on the level of peroxidase activity in the sorghum

malt at the end of 24h steep. From the result, peroxidase activity in the test seeds demonstrated

a slightly higher activity when compared to that in the control seeds.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time (min)

Distilled water steep without air-rest(control)

Distilled water steep with air-rest(test)

36

Fig 3.7: Peroxidase activity at the end of 24 h of germination for regime II

Results are means of triplicate trials.

Figure 3.7 shows the effect of air-rest cycle on the level of peroxidase activity at the end of 24h

germination. From the results, peroxidase activity in the test seeds and the control seeds had

increased considerably. However, peroxidase activity in the test seeds were slightly higher than

peroxidase activity in the control seeds.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time (min)

Distilled water steep without air-rest(control)

Distilled water steep with air-rest(test)

37

Fig 3.8: Peroxidase activity at the end of 48h germination for regime II. Results are means of

triplicate trials.

Figure 3.8 represents the plot of the effect of air-rest cycle on the level of peroxidase activity at

the end of 48 h germination. The test seeds and the control seeds had demonstrated appreciable

level of peroxidase activity overtime. There was a slight difference in the level of peroxidase

activity between the test seeds (1.055) and the control seeds (1.139).

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time (min)

Distilled water steep without air-rest (control)

Distilled water steep with air-rest (test)

38

Fig 3.9: Peroxidase activity at the end of 72 h germination for regime II. Results are means of

triplicate trials.

From the results the level of peroxidase activity in the control seeds had increased appreciable

(1.279) with that in the test seeds (1.229).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4 5 6 7 8 9 10

ΔO

D/m

in

Time (min)

Distilled water steep without air-rest (control)

Distilled water steep without air-rest (test)

39

Fig 3.10: Peroxidase activity at the end of kilning for 7 h for regime II. Results are means of

triplicate trials.

Figure 3.10 shows the effect of air-rest cycle on the level of peroxidase activity at the end of

kilning. The results showed that the peroxidase isolated from both the control and the test seeds

survived kilning. However, much lower level of peroxidase activity was observed in the test

seeds.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4 5 6 7 8 9 10

ΔOD/m

in

Time (min)

Distilled water steep without air-rest (control)

Distilled water steep without air-rest (test)

40

CHAPTER FOUR

4.1 DISCUSSION

This present study showed that the cultivar KSV8 had good germinative energy (Table 3.1-3.4)

which are within the acceptable range (> 95%) prescribed for malting grains ( Briggs et al.,

2004). In regime I control germinative energy was (89% ±0.71) and it is below the

recommended value.

In the alkaline steep (regime I), high germinative capacity of ( 98± 0.57 % ) for test and ( 96±

1.14 % ) for the control were recorded. Germinative capacity (GC ) was also high in regime II

(distilled water steep with air rest),the test recorded ( 99 ± 0.00 % ) while the control had (97 ±

1.15 %). Works by different researchers had shown that physiologically, sorghum malt contains

significantly lower levels of salt soluble proteins which influence some of the biochemical

changes that take place during malting (Okolo and Ezeogu, 1996). Therefore the possibility of

enhancing the enzyme activity through improvement in malting methods such as alkaline steep

and air rest cycle is necessary. Irrespective of the steep treatment applied, the chiting ability of

the grains was suppressed by alkaline steeping. This trend was more pronounced in regime 1

control (alkaline steep) which showed more sensitivity to regime change than the other steep

treatment. Moreover, grain germinability was high in alkaline steep and in the distilled water

steep, since the grains are of the same cultivar it is possible that steeping pre-history and air-

rest cycle had influenced seed germinability. 95% germinative energy was due to possible seed

contamination during malting and perhaps, the methods used to store the seeds after harvest.

The grains were not water sensitive (Ws) (Table 3.1-3.4) possibly because of the cultivar type,

season of grain harvest and method of preservation. However, malting loss was higher in

regime I (alkaline steep) in both the test and the control ( 20. ± 0.93 %; 20 ±1.28 %

respectively). A high value of malting loss was also recorded in regime II (distilled water steep)

control (15.84 ± 0.19 % ),while the lower value for the test (5.63 ± 1.28 % ) would be

attributed possibly to air-rest cycle. The 5.63% malting loss observed in the test was low

because an average of 10 -15 % respiration / metabolic loss is expected in a well malted

sorghum with good diastatic power (Owuama , 1997).

The average root lengths in regime I (alkaline steep) were short and this could be due to the

effect of alkaline steep (Okolo and Ezeogu,1996). Alkaline steep liquor is known to suppress

41

root development, (Okolo and Ezeogu, 1996) causing short root lengths as observed in regime I

(alkaline steep). In regime II (distilled water steep) root length was observed to be more

developed (longer) than that for alkaline steep. Data observed in this study (of Table 3.1-3.4)

suggest that the introduction of air-rest cycle during sorghum steeps would be highly beneficial

in the reduction of malting losses of sorghum malts.

At the end of 40 h steep peroxidase activity for the alkaline steep ( test ) was higher

compared to the control ( figure 3.1). Peroxidase activity in regime 1 control was well below

that of regime 1 (test) which is attributed to the degree of germination suppression by final

warm steep (Okolo and Ezeogu 1996); even though, both the text and the control had the same

pre-steeping history.

For regime II (distilled water steep), at the end of 24 h steep, peroxidase activity was found to

be decreased (control : no exposure to air-rest cycle) and was lower than that of the test ( test :

exposed to air-rest cycle of 10 h wet and 1h dry). This is possibly because steeping causes

physical and biochemical changes that hasten grain modification (Owuama, 1997), and the

effect of air-rest cycle on water retention capacity of the grains. This possibly may have lead to

the decreased peroxidase activity observed in the control seed (figure 3.6).

Peroxidase activity decreased at the end of 24 h germination in regime I (test) but maintained

high activity compared to the control (figure 3.2). This is suspected and may be as a result of

the suppressive effect of alkaline steep, whereas regime 1 control showed increase in

peroxidase activity. Peroxidase activity increased steadily for both regime II (control: not

exposed to air-rest cycle ) and for the test (exposed to air-rest cycle) (figure 3.7). This was also

in line with earlier reportes by Owuama (1997) who demonstrated that peroxidase activity

increases by about 14-fold during germination of grains steeped at 30°C for 24 h without air-

rest cycle.

At the end of 48 h; regime 1(alkaline steep) control continued its progressive increase in

peroxidase activity which may be due to the degree of imbibitions that may have facilitated

protein and enzyme synthesis (figure 3.3). Comparable to the test with decreasing peroxidase

activity; ascribed to fast rate of respiration, moisture and ion uptake engineered presumably by

air-rest. This effect was also observed in diastatic activity where the extent of repression for

ICSV400 was 9 % (Okolo and Ezeogu 1996). This repression effect could also be attribute to

the fact that any character that influences protein binding properties of a molecule also

42

influences its activity in other biochemical processes (Okolo ,1989). The possibility exists that

the differences in the capacity of alkaline steep liquor to influence protein binding properties of

polyphenol /tannins is cultivar dependent and might have contributed to decreased peroxidase

activity. KSV8 have high polyphenol compared to other sorghum cultivars (Okolo, 1989).

However, distribution and concentration of protein binding complexes in the grain is also

specie dependent. Polyphenols are known to inactivate a number of grain enzymes

(Daiber,1975) within the pericarp and testa. The possibility exists that these polyphenols might

have penetrated the endosperm with the imbibed water ( peroxidase in endosperm is 44 %,

acrospires and rootlet is 56 % ) and complexes with reserve seed and enzyme proteins to effect

repression of enzyme synthesis and inactivation of already synthesized enzymes; but the effect

and degree of this is not known (Nwanguma and Eze 1995).

At the end of 48 h, regime II (distilled water steep) test and control: peroxidase activity in

increased steadily (figure 3.8). This might be due to increase in protein and enzyme synthesis

that increases as germination time increases.

At the end of 72h germination, peroxidase activity in the control continued to increase and

surpassed that of regime 1 (test ) which demonstrated increased peroxidase activity at the end

of 72 h (figure 3.4 ). This sudden increase at the end of 72 h for regime1 ( test ) is perhaps as a

result of increased concentration of the imbibed ion, the rate of formation of protein complexes

and time dependent effect of ion protein binding. Furthermore, a possible depletion in the

suppression effect of final warm at 40°C for 6h could be attributed to this sudden increase in

peroxidase activity. It is also possible that the effect of germination times on the development

of proteins and enzymes synthesis may have led to the change in peroxidase activity. Increased

peroxidase activity was also observed in regime I control.

In regime II (distilled water steep) peroxidase activity increased in both the test and the control

but was higher in the control relative to the test (figure 3.9). This might be due to the degree of

imbibitions, sufficient moisture uptake, and the level of oxygen uptake that may have led to

increased peroxidase development. These possibilities are also influenced by the degree of air-

rest cycle the grains were exposed to.

At the end of kilning, peroxidase activity in regimes I and II decreased as expected (figure 3.5

and figure 3.10).This is because proteins generally denature when they are exposed to high

43

temperature. It is therefore possible that some of the peroxidase were denatured at this stage. In

regimes I and II , the peroxidase that survived the heat treatment were more in the control

compared to the test. This perhaps could be due to the nature of the steeped liquor and its

physical properties such as vapour density, evaporation temperature, etc. Heat effect on the ions

of the steeped liquor and their likely interaction with peroxidase proteins could also be a

contributing factor. It is also possible that some of the denatured proteins regained their

conformational integrity afterwards. The survival of peroxidase at this stage (kilning) suggest

that peroxidase activity may be guaranteed even at the mashing stage of beer production.

44

4.2 Conclusion

From the present study, introduction of air-rest cycle as malting condition would be beneficial

to brewers during malting development of peroxidase. It reduces malting loss associated with

sorghum beers, increased the germinative energy and guarantees the defensive role of

peroxidase against lipid peroxidation during malting. Conversely, the alkaline steep had an

inhibitory effect on the activity of peroxidase during malting.

45

4.3 Recommendation/ Suggestion for further studies

Since the results had shown that air rest cycle increases peroxidase activity during malting. It is

also important to look at its effect in other antioxidant enzymes such as catalase and superoxide

dismutase; so as to employ it effectively in reducing lipid peroxidation. The inhibitory effect of

alkaline steep should also be investigated in other antioxidant enzymes since the nature and

mechanism of these enzymes vary Owuama, ( 1997).

46

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51

Appendix

All the results in the appendix are means of triplicate trials.

Table 4.0: Peroxidase activity for regime 1 at the end of 40 h steep and final 6h warm steep at

40 °C. Regime I: Alkaline steep (Test: Alkaline steep with air-rest cycle; Control: Alkaline

steep without air-rest cycle)

Time

(min)

KSV8 with alkaline

steep and air-rest

with (ΔOD/min)

(Test)

KSV8 with distilled

water and air-rest

(ΔOD/min)

Control

1 0.312 0.121

2 0.525 0.189

3 0.650 0.220

4 0.700 0.241

5 0.787 0.254

6 0.818 0.257

7 0.840 0.263

8 0.848 0.259

9 0.850 0.264

10 0.857 0.222

52

Table 4.1: Peroxidase activity at the end of 24h germination for regime I.

Regime I: Alkaline steep (Test: Alkaline steep with air-rest cycle; Control: Alkaline steep

without air-rest cycle)

Time (min) KSV8 with alkaline steep and

air-rest

(ΔOD/min)

(Test)

KSV8 with distilled water

and air-rest

(ΔOD/min)

Control

1 0.304 0.231

2 0.507 0.374

3 0.640 0.430

4 0.717 0.551

5 0.778 0.620

6 0.798 0.663

7 0.814 0.66

8 0.789 0.646

9 0.751 0.608

10 0.728 0.607

53

Table 4.2: Peroxidase activity at the end of 48 h germination for regime I.

Regime I: Alkaline steep (Test: Alkaline steep with air-rest cycle; Control: Alkaline steep

without air-rest cycle)

Time (min) KSV8 with alkaline

steep and air-rest

(ΔOD/min)

(Test)

KSV8 with distilled water

and air-rest (ΔOD/min)

Control

1 0.256 0.192

2 0.367 0.294

3 0.451 0.363

4 0.506 0.419

5 0.549 0.514

6 0.583 0.547

7 0.609 0.572

8 0.630 0.590

9 0.647 0.612

10 0.662 0.620

54

Table 4.3: Peroxidase activity at the end of 72h germination for regime I

Regime I: Alkaline steep (Test: Alkaline steep with air-rest cycle; Control: Alkaline steep

without air-rest cycle).

Time

(min)

KSV8 with alkaline

steep and air-rest

(ΔOD/min)

(Test)

KSV8 with distilled

water and air-rest

(ΔOD/min)

Control

1 0.251 0.343

2 0.478 0.624

3 0.660 0.891

4 0.809 1.116

5 0.949 1.287

6 1.028 1.419

7 1.113 1.538

8 1.165 1.613

9 1.225 1.656

10 1.239 1.764

55

Table 4.4 : Peroxidase activity at the end of kilning for regime I

Regime I: Alkaline steep (Test: Alkaline steep with air-rest cycle; Control: Alkaline steep

without air-rest cycle).

Time (min) KSV8 with alkaline

steep and air-rest

(ΔOD/min)

(Test)

KSV8 with distilled

water and air-rest

(ΔOD/min)

Control

1 0.050 0.051

2 0.102 0.190

3 0.155 0.277

4 0.256 0.342

5 0.285 0.368

6 0.336 0.432

7 0.367 0.457

8 0.396 0.500

9 0.412 0.529

10 0.425 0.553

56

Table 4.5: Peroxidase activity at the end of 24 h steep for regime II

Regime II: KSV8 steeped in distilled water for 24h. (Test: Distilled water steep with air-rest

cycle; Control: Distilled water steep without air-rest cycle).

Time

(min)

Dry KSV8

(ΔOD/min)

KSV8 without air-

rest(ΔOD/min) control

KSV8 with air-

rest(ΔOD/min)(Test)

1 0.078 0.085 0.118

2 0.134 0.131 0.166

3 0.179 0.171 0.206

4 0.216 0.197 0.238

5 0.250 0.219 0.268

6 0.281 0.233 0.295

7 0.310 0.249 0.303

8 0.325 0.264 0.311

9 0.343 0.283 0.321

10 0.360 0.294 0.328

57

Table 4.6: Peroxidase activity at the end of 24h germination for regime II.

Regime II: KSV8 steeped in distilled water for 24h. (Test: Distilled water steep with air-rest

cycle; Control: Distilled water steep without air-rest cycle).

Time

(min)

Dry KSV8

(ΔOD/min)

KSV8 without air-rest

(ΔOD/min)

Control

KSV8 with air-

rest(ΔOD/min)(Test)

1 0.078 0.127 0.136

2 0.134 0.216 0.244

3 0.179 0.288 0.338

4 0.216 0.356 0.423

5 0.250 0.415 0.497

6 0.281 0.462 0.542

7 0.310 0.506 0.593

8 0.325 0.538 0.643

9 0.343 0.568 0.688

10 0.360 0.600 0.731

58

Table 4.7: Peroxidase activity at the end of 42h germination for regime II.

Regime II: KSV8 steeped in distilled water for 24h. (Test: Distilled water steep with air-rest

cycle; Control: Distilled water steep without air-rest cycle).

Time

(min)

Dry KSV8

(ΔOD/min)

KSV8 without air-

rest(ΔOD/min)Control

KSV8 with air-rest

(ΔOD/min)(Test)

1 0.078 0.334 0.287

2 0.134 0.551 0.513

3 0.179 0.697 0.682

4 0.216 0.787 0.809

5 0.250 0.828 0.889

6 0.281 0.934 0.971

7 0.310 0.972 1.026

8 0.325 1.007 1.071

9 0.343 1.035 1.108

10 0.360 1.055 1.139

59

Table 4.8: Peroxidase activity at the end of 72h germination for regime II.

Regime II: KSV8 steeped in distilled water for 24h. (Test: Distilled water steep with air-rest

cycle; Control: Distilled water steep without air-rest cycle).

Time

(min)

Dry KSV8

(ΔOD/min)

KSV8 without air-

rest(ΔOD/min)Control

KSV8 with air-rest

(ΔOD/min)(Test)

1 0.078 0.305 0.348

2 0.134 0.566 0.620

3 0.179 0.764 0.809

4 0.216 0.938 0.948

5 0.250 1.035 1.035

6 0.281 1.117 1.100

7 0.310 1.177 1.146

8 0.325 1.222 1.186

9 0.343 1.252 1.208

10 0.360 1.279 1.229

60

Table 4.9: Peroxidase activity at the end of kilning for regime II.

Regime II: KSV8 steeped in distilled water for 24h.(Test: Distilled water steep with air-rest

cycle; Control: Distilled water steep without air-rest cycle).

Time

(min)

Dry KSV8

(ΔOD/min)

KSV8 without air-

rest(ΔOD/min)Control

KSV8 with air-rest

(ΔOD/min)(Test)

1 0.078 0.169 0.169

2 0.134 0.367 0.291

3 0.179 0.546 0.386

4 0.216 0.700 0.453

5 0.250 0.826 0.511

6 0.281 0.933 0.559

7 0.310 1.017 0.599

8 0.325 1.082 0.633

9 0.343 1.130 0.658

10 0.360 1.175 0.683

61

Table 4.10: Comparing Peroxidase activity of both dry KSV8 and kilning KSV8

Time

(min)

Dry KSV8

(ΔOD/min)

Kilned KSV8 (ΔOD/min)

1 0.078 0.095

2 0.134 0.158

3 0.179 0.205

4 0.216 0.241

5 0.250 0.263

6 0.281 0.278

7 0.310 0.290

8 0.325 0.302

9 0.343 0.305

10 0.360 0.311

62

Table 4.11 : Protocol for Protein Standard Curve

VOL OF BSA

( ml)

Concentration

VOL OF

DISTILLED

WATER ml

ABSORBANCE

1

ABSORBANCE

2

MEAN CONC OF

PROTEIN

mg/ml

0.000 1.000 0.000 0.000 0.000 0.000

0.100 0.900 0.110 0.109 0.110 0.500

0.200 0.800 0.153 0.163 0.153 1.000

0.300 0.700 0.273 0.271 0.272 1.500

0.400 0.600 0.343 0.339 0.341 2.000

0.500 0.500 0.410 0.406 0.408 2.500

0.600 0.400 0.454 0.447 0.451 3.000

0.700 0.300 0.497 0.542 0.519 3.500

0.800 0.200 0.573 0.538 0.555 4.000

0.900 0.100 0.637 0.645 0.641 4.500

63

Table 4.12 : Total protein concentration of the crude enzyme in mg/ml

Regime I

End of

40 h

steep

24h

germination

48h

germination

72h

germination

kilning

Dry KSV8 (mg/ml) 1.29 - - - 1.17

Alkaline steeped KSV8

with air-rest & warm

steep (mg/ml)

Test

1.64 1.36 1.20 1.73 0.85

Distilled water steeped

KSV8 with air-rest &

warm steep (mg/ml)

Control

0.63 0.72 0.88 2.08 1.14

Regime II

24h

steep

24 h

germination

48 h

germination

72 h

germination

kilning

Dry KSV8 (mg/ml) 1.29 - - - 1.17

KSV8 with 24h

continuous steep (mg/ml)

Control

0.83 1.12 1.84 3.07 2.48

KSV8 with air-rest

(mg/ml) Test

0.89 1.29 2.71 2.74 1.01

64

Fig 4.1: Graph of standard Protein Curve using BSA

Preparation of Phosphate Buffer

Molecular weight of NaH2PO4 = 119.96g

0.1M = ?

1M = 119.96 g

0.1×119.96 = 11.996g/10 = 1.1996g/100ml

Na2HPO4 = 142g

1M of Na2HPO4 = 142g

0.1M = ?

0.1×142 = 14.2/10 = 1.42 g / 100ml

1

y = 0.148xR² = 0.976

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

abso

rban

ce a

t 75

0nm

concentration mg/ml