EFFECT OF ALKALINE STEEP AND AIR-REST CYCLE ON THE DEVELOPMENT OF SORGHUM PEROXIDASE ACTIVITY DURING MALTING

ABSTRACT

The effect of  alkaline steep and air-rest cycle on the development of  peroxidase activity during malting  was  investigated  in  sorghum  variety,  KSV8.  Preliminary  experiment  showed  that alkaline steep (test) and the distilled water steep (control) had germinative energy of 92± 2.87 % and 89± 0.57 % respectively. In regime II (sorghum grains steeped in distilled water for 24h), both the test experiment and the control had germinative energy of 95± 1.41 %. Germinative capacity was high in both regimes. The two regimes were not water sensitive, however malting loss were high in alkaline (20.1± 0.93 %) and in distilled water steep (20.0± 1.28 %).Malting loss for distilled water in regime II  (sorghum grains steeped in distilled water for 24h) was 5.6±

1.28 % and it is relatively comparable to that of barley. Malting loss was also high in distilled water steep (control) (15.84± 0.19 %). From the results, there was an appreciable increase in peroxidase activity from day 1 through day 3 of germination for distilled water steep in regime I (control) when compared to the test with regression in peroxidase activity. There was a positive gradual increase in peroxidase  activity  influence by  air-rest cycle  from day 1 through day 3 in regime II (distilled water steep for 24h).   At the end of kilning at 60   C for 7 h, peroxidase activity dropped sharply in both regimes. Consequently, the introduction of air-rest cycle as malting condition will be beneficiary to brewers. It reduces malting loss associated with sorghum beers,  increases  the  germinative energy and  the  defensive  role  of peroxidase against  lipid peroxidation during  malting.  Conversely,  the  alkaline  steep  with  final  warm  steep  had  an inhibitory effect on the development of peroxidase during malting.

CHAPTER ONE

INTRODUCTION

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).

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).

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).

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 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-60oC for 8-24 hours depending on brewer’s aim. This process preserves the malt and adds colour, and flavour to the finished malt.

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

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-14oC.  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

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 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.

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 → Ro + XH

(2)       Propagation: Ro + O2 → ROOo, R.

ROOo + RH → ROOH + Ro, etc

(3)       Termination: ROOo + ROOo → ROOR + O2

ROOo + Ro → ROOR Ro + Ro → 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).

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 (Devasagayamet al., 2003).

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