A Comparative Analysis of differentPitching Rates on Castle Lager Beer

Flavour Stability: A Case Study ofDelta Lagers manufacturing company

Harare, Zimbabwe

1Perkins Muredzi; 2Chiridza Lynette Tendai; 3Dinga.N.Moyo; 4Clive Winini1-4Department of Food Processing technology; School of Industrial Sciences and

TechnologyHarare Institute of Technology

Ganges Rd, BelvedereBox BE 277, Harare

pmuredzi@hit.ac.zw; lynchiridza@yahoo.com, dmoyo@hit.ac.zw; cwinini@hit.ac.zw

Abstract— Beer stability is a critical qualityparameter and there is vast amount of researchthat has been done in attempt to understandand optimise it. The research study wasincited by the result of low Lag Time(potential to resist staling) and high T150(degree of staling) in one of the main streambrands, Castle Lager, at Delta Beveragesthereby causing the beer to stale much fasterthan normal. The research focused on theflavour stability aspect of beer stability anddifferent methods of analysis were employed toinvestigate the effect of different pitchingrates on products of yeast metabolism in-turnflavour stability. In the study, beerfermented with pitching rates of 0.7kg/hl and0.9kg/hl were compared to that fermented with0.8kg/hl. 0.8kg/hl is the normal rate used atDelta Beverages; the researcher therefore usedit as the control in the experiments forsulphur dioxide determination, bitternessunits, diacetyl, dissolved oxygen, Lag Timeand T150 analysis and sensory evaluation. TheElectronic Spin Resonance (ESR) machine was

used to measure Lag Time and T150 values andthe sensory evaluation was done by a trainedpanel of tasters from Delta Beverages,Zimbabwe. Results revealed that beer fermentedwith a low pitching rate had a high amount ofsulphur dioxide which meant the Lag Time wasalso high due to sulphur dioxide exertinganti-oxidant properties which preventedoxidation of beer for longer periods than beerfermented with a high pitching rate. Theresults also revealed that there is nosignificant difference in these beers regardssensory analysis following analysis using thet-test.

Keywords-;Lag time, flavour stability, T150 (degree ofstaling),ribes, strecker degradation

I. INTRODUCTION The Delta Corporation phenomenon datesback to the days during the exploration ofAfrica. Beer was an essential provisionbecause water stagnated quickly, while

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malt beer remained a palatable andwholesome addition to the poor diet of theseafarers. Beer was the drink of choice inthe parched climate of Africa. Delta Corporation Ltd is a holding companythat has contributed in the responsibilityand management of a vast businessoperating throughout the whole ofZimbabwe. The Delta Beverages SouthertonPlant majors in clear beer brewing ofCastle, Lion, Pilsner, Zambezi, Eagle,Castle Lite, Zambezi Lite, and CarlingBlack Label lagers as well as carbonatedsparkling beverages (Coke, Fanta, Sprite,Sparletta and Stone). The plant is high inproductivity and this requires the qualityof the beer produced to be very good. InZimbabwe Castle Lager has proved to be themost favoured beer, this is seen in theamount of sales of 40% sales as comparedto the other brands. The trade complaintsof Castle Lager brand since 2008 aremostly viable mould, a cloudy product, theproduct not reaching to its standard shelflife, varying taste complaints of badodour, sweet beer, off taste and“cardboard” taste.Recent research has indicated thatmoderate consumption of beer can be asource of beer specific antioxidants whichhelp reduce the risk of cardiovasculardisease and lower the occurrence ofcertain types of cancers [26]. Appearanceand taste are the two main sensoryattributes on which consumers judge theacceptability of the product and they usethem to critically evaluate every glass ofbeer they drink. The appearance, colour,hue, brightness, foam quality, glassfullness and flavour.Lager beer is by far the biggestproportion of beer distributed and soldcommercially. The major thrust in beerquality lies upon the beers’ foamstability, colloidal stability and mostimportantly flavour stability [4] . Beerflavour stability is of paramountimportance since it has the ability toassault and detrimentally affect foam and

colloidal stability. Due to marketcompetition and the rise of consumerexpectations, standards of beer qualityare set and any deviations must be kept incheck. A flavour stable beer is fresh, hasa clean taste, a pleasurable aroma, and acrisp pleasing bitterness that is thirstquenching.In the brew-house, there are many stepsthat facilitate oxidation. These includegrinding, mixing, mashing, and the ambientfactors which are mainly temperature andhumidity. The quality of the malt and theunmalted cereals is also a very importantaspect of the final flavour stability. Themajor problems due to oxidative conditionsin the brewhouse are; the presence oflipoxygenase and the coloration of thewort by melanoidine oxidation. In thefinal product, we can also find a negativeimpact of the oxygen on the taste of thebeer due to an auto-oxidation of thelipids, with a deviation of the ageing andthe flavour stability. Such situations canget accentuated by oxidative catalysts,principally the presence of heavy metals(Fe2+, Cu2+), high pH (> 5.6), and thetemperature during the mashing (Briggs,1998). The practical solutions to reducethe impact of an oxidized wort followed byDelta beverages Harare include; the use ofmodern machinery, humid grinding, very lowagitation during mashing, the use of de-aerated water, an acidic pH duringmashing, and pumping the mash in frombelow the tank. In the same time thequality of the malt is very evaluativeaccording to the season, such aspectsbring technical problems to standardisethe quality of wort, consequently a stablequality of the bottled beer. Flavour changes that occur during beermaturation play a critical role in theproduct quality and brand identity ofbeer. Successful brands that strive formaximum flavour stability are generallywell received and recognized for such byconsumers. While it is preferred thatflavour improves during the maturationprocess, formation of undesirable flavours

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inevitably occurs during beer storage.More problematic is that occurrence ofaged-flavours varies from one beer styleto another, with lager beer seemingespecially sensitive. Of the manychemicals involved in beer flavourmodification, a few key groups have beenidentified: di-ketones, sulphur compounds,aldehydes and volatile fatty acids [3] .In general, beer aging results indecreased bitter taste, increased sweettaste and increased caramel, ribes (blackcurrant), and toffee-like aromas. Carbonylcompounds such as trans-2-nonenal(cardboard aroma) form during beer storagefrom the oxidation of fatty acids and havebeen attributed to aged-beer flavour dueto their very low flavour thresholds.The researchers challenged establisheddogma by employing new techniques thatenable exploration and innovation, whichare aimed at effectively reducing freeradical species (that have the effect ofoxidising beer) that result in beerflavour instability.The innovative solution the researcherbrought to reduce dissolved oxygen in theproduct for more flavour stable beer hasbeen to increase the amount of sulphurdioxide produced naturally by yeast duringfermentation, which acts as a naturalantioxidant.The Quality Assurance department isresponsible for analysing flavour profilesof beers during the brewing stagesparticularly at the brew-house, fermentvessel, storage vessel, bright beer tankand packaged beer. The analyticalparameter for flavour are usually abovespecifications, and this might be due tobrewing practices or/and brewingprocesses. The major thrust of this research was toimprove Castle lager beer flavourstability by solving technical problems,reducing the amount of dissolved oxygenand free radical species in the product,apart from the improvement of beer flavourstability, this would result in the

improvement of other properties of beerthat are affected by free radical species,such as an improvement in shelf life, foamstability and colloidal stability.Consumer confidence in the brand wouldgreatly increase. Beer would be freshleading to more drinkability.Consumers demand beer freshness. Theirexpectations continue to rise and when itcomes to beer, one of the main things thatconsumers use to determine if they willmake a repeat purchase is taste (flavourstability). If the beer freshness ispreserved for a long time this would be ofconsiderable commercial value to DeltaLagers as well as to consumers as theywill be provided with a product that meetstheir quality expectation. The research would improve theresearchers’ research skills and theirpracticality. Moreover it would improvethe researchers’ knowledge of brewing andalso add on the body of knowledge thatalready exists in brewing. This researchwould also improve the researchers’competitive advantage in the competitiveworld we live in.Sensory changes during storage

Currently, the main quality problem ofbeer is the change of its chemicalcomposition during storage, which altersthe sensory properties. A variety offlavours may arise, depending on the beertype and storage conditions. In contrastto some wines, beer aging is usuallyconsidered negative for flavour quality.The main focus on beer aging has been thestudy of the cardboard-flavoured component(E)-2-nonenal and its formation by lipidoxidation. Other stale flavours are lessdescribed, but may be at least asimportant for the overall sensoryimpression of aged beer. Their origin hasbeen increasingly investigated in recentyears.The literature on beer staling revealsonly a few reports dealing with the actualsensory changes during beer storage.

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Dalgliesh (1977) described the changes inthe most detail. However, the Dalglieshplot (Fig 1) is a generalization of thesensory evolution during beer storage andis by no means applicable to every beer.The graph shows the general tasteacceptance by consumers immediately afterit has been packaged. Over time, thebitterness, fruitiness and drinkability ofthe beer decline while at the same timetaste characteristics such as papery,cardboard-like and sweetness all increaseto a less satisfying product. Freshness islinked with drinkability. The fresher thebeer, the more drinkable

Figure 1: Sensory changes during beeraging (Dalgliesh, 1977)

The changes of flavour in beer are aresult of chemical reactions. As for allsuch reactions, their rate depends greatlyon temperature, a 10°C increase intemperature leading to an approximatedoubling of rate. There is evidence thatthe nature of flavour changes occurring atdifferent temperatures varies. Inparticular it was found that there was anincrease in carbonyl compounds when beerwas aged at 45°C, but actually a decreasein overall carbonyls was seen at roomtemperature, together with a lowering ofester levels. Kaneda et al (1991) say thata beer aged at 25°C tends to develop apredominantly caramel character, whereasat 30 or 37°C more cardboard notes aredominant. In contradiction of the findings

of Bright (1999) and Kaneda (1991),Greenhoff (1981) claimed that the natureof flavour changes, in particular the typeof cardboard character, displayed at 60°Cwas similar to that at 18 or 37°C. Interms of bitterness drop, then it is wellunderstood that the iso-alpha-acids decayrelatively quickly in beer, although theproducts formed are not clearly known.

However there are a great many otherchanges in concentrations of compoundsoccurring. Indeed, Lustig (1993)identified 30 substances by multi-dimensional gas chromatography whose leveleither increases or decreases duringstaling. Classes include esters, O-heterocyclics and carbonyls.

Many flavour active compounds presentin uninfected beer are capable of changingin their level during storage in the finalpackage.

Compounds may:(a) Decrease in level leading to flavourdeterioration by loss of a desirablecharacter(b) Increase in level leading to flavourdeterioration by increase in anundesirable characterIn turn category b compounds may arise:(i) Because they are produced de novo in achemical reaction(ii) By the release of pre-formed materialthat is bound up in the beer with a"holding agent" that prevents theirflavour from being expressed(iii) because conditions have changed in abeer which makes the likelihood of eithertype of change [(i) or (ii)] more likely,e.g. a change in redox conditions [17] .Effect of Pitching Rates on Flavour of beer

Pitching rate refers to the amount ofyeast that is added to cooled wort. Pitchrate is generally referred to in cells permilliter. The amount of yeast variesaccording to the brand profile that isbeing brewed, the yeast strain being used,

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gravity of the wort and the temperature offermentation. Pitching rates make adramatic difference in the final flavourand aroma profile of any beer.The amount of cell growth in fermentationis affected directly by the pitch ratewhereby cell growth decreases as pitchrates decreases . Ester production isdirectly related to yeast growth as aremost other flavour and aroma compounds.Increasing the quantity of yeast pitchedis the most effective method of reducingthe ester profile in the finished beer.Lagers have a reduced ester profile andare characterized as clean withdiscernable malt character.A low pitch rate can lead to:

Excess levels of diacetyl Increase in higher/fusel alcohol

formation Increase in ester formation Increase in volatile sulphur

compounds High terminal gravities Stuck fermentations Increased risk of infection

High pitch rates can lead to:

Very low ester production Very fast fermentations Thin or lacking body/mouth feel Autolysis (Yeasty flavours due to

lysing of cells) [4] With some beer styles, where a complexester profile is desirable it can bebeneficial to under pitch. Over pitchingcan often lead to a very clean beerlacking an ester profile (banana).

II. METHODOLOGYExperimental Research Design

Research Population

In this research the populationcomprised:

Population A: Normal beer fermentedwith a yeast pitching rate of0.8kg/hl

Population B: Beer fermented with ayeast pitching rate of0.9kg/hl

Population C: Beer fermented with ayeast pitching rate of0.7kg/hl

Population D: Delta Lagers tastepanel

Sample Size Sampling TechniquesThe sample size consisted of threesamples each from Castle Lager pitched at0.8kg/hl : Sample A, 0.9kg/hl: Sample B,0.7kg: Sample C and the one pitched at0.8kg/hl was the control as this is thepitching rate normally used at DeltaBeverages for the brand Castle Lager.Samples were purposively sampled in onebatch, these beer samples have similarcharacteristics that require analysis forexample alcohol concentration, hencerequired the same sampling technique andsample size. Samples were taken at dayten of fermentation vessel stage, atracking, at day six of storage vesselstage, bright beer tank and at packagingex-filler and ex-pasteuriser for alllaboratory analysis.

Data Collection TechniquesThe Research ToolsLaboratory analysis was carried out toascertain comparative state of thequality of product. The researcherSemployed the use of a trained panel of 10individuals, consisting of professional

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beer tasters Delta beverages lagersmanufacturing employees.

Experimental WorkAnalysis was done on sample A, B and Cto test parameters which include sulphurdioxide, diacetyl, bitterness units, lagtime and T150 values.

Experimental Data Collection Procedures

Yeast Pitching RatesThe pitching rate that is currently beingused for Castle Lager brewing at DeltaBeverages is 0.8kg/hl. In this research,all parameters remained the same with theexception of sample B (0.9kg/hl pitchingrate) and sample C (0.7kg/hl pitchingrate). After wort cooling the respectiveyeast pitching rates were added torespective batches. The yeast pitchingrate simply refers to the amount of yeastthat is added to cooled wort.Fermentation profile was monitored so wasthe flavour stability profile at eachstage ferment vessel (FV), storage vessel(SV), bright beer tank (BBT) and packagedbeer.3.6.2 Determination of the DissolvedOxygen in the beerThis analysis was done thrice during thefilling of the bright beer tank. Adissolved oxygen meter (Orbisphere 3100)was used for this analysis to measure theamount of dissolved oxygen within asample. The instrument was mounted on thesample point of the filled tank at theBBTs, switched on and sample point tapwas opened to let the beer run throughthe machine. The instrument was left tostabilise for a few minutes and thereading was taken and recorded as a wholenumber or to 1 decimal place. It wasrinsed through with carbonated water(MUL) whose reading was also taken. Thisanalysis was done in order to check theamount of oxygen dissolved in beer whichis not expected in beer since it causes

staling and other off flavours due tooxidation. For the packaged product thebeer analyser was used. A pint was pre-attemperated to 20°C, after which the capwas opened and placed on the analyser torun, the reading was then recorded.

Sulphur Dioxide determinationTubes were chilled to 00C in ice waterprior to sampling of the packagingsample. The flasks were rinsed withethanol and dried using current airbefore sampling. A clean dry centrifugetube was inserted into a beakercontaining crushed ice (Sample must betaken cold without the loss of carbondioxide and sulphur dioxide). A samplecoil was connected to the tank samplecock and thoroughly the line was flushedand coiled with sample. (House the coilin a beaker containing crushed ice.).Slowly the centrifuge tube was filled tothe brim with sample and immediatelycapped. The sample was kept in thecrushed ice. Sample was centrifuged at aforce G2000 for 10 minutes. In removingthe sample from the centrifuge cautionwas maintained so as not to disturb thesediment. The centrifuged sample wasreturned to the beaker containing crushedice. Mercury stabilising solution andsulphuric acid were put into two labelledflask (free and total SO2). Preparedsamples were put into each flask andstoppered. For the blank, starchindicator was added as well as iodinesolution drop-wise until a blue colourpersisted. The samples were diluted withpurified water and stoppered. 25ml ofeach sample was pipetted in acorresponding volumetric flask and colourreagent (p-rosaline) was added to eachflask. Water was added to each flask andstoppered and placed in a water bath at25˚C for exactly 30 minutes. Theintensity of colour was determinedspectrophotometrically (UV-1800,Shimadzu) against purified water at a

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wavelength of 550nm and recorded.Detailed method and calculations arefound in the appendices.

Bitterness Units (BU) AnalysisPrepared samples were pipetted into a BUtube and hydrochloric acid and iso-octanewas added to the tube. Glass beads wereput into the tube and the BU tube wasstoppered and placed on the BU shaker for15 minutes. The tubes were centrifugedusing a centrifuge for 3 minutes and theabsorbance of ultra violet (UV) wasmeasured using a spectrophotometer (UV-1800, Shimadzu) at 275nm against iso-octane and recorded. The detailedprocedure is in the appendices as well asthe calculations of BU.

Determination of Vicinal di-ketones i.e. diacetyl bygas chromatographyThe Gas Chromatograph (Hewlett Packard,Agilent Technologies, 1200 InfinitySeries) is used to measure diacetyl andacetaldehyde. 200 ml sample was measured;it was then filtered through a filterpaper with 1g kieselguhr. The conicalflask collecting the sample had 1 or 2drops of antifoam added. From thefiltered sample 10ml were pipetted intoan HPLC (High Performance LiquidChromatograph) vile (Unfortunately theresearcher had to use these for allanalysis with the gas chromatograph forthey were new and readily available thanthe gas chromatograph vile) and sealedwith a cap. The sample was attemperatedto 0°C for 20-30 minute then it was runthrough the gas chromatography, theresults were then recorded.

Determination of ESR valueThe estimation of the hydroxyl radicaland anti-oxidant values was performedusing a Bruker Biospin, an ElectronicSpin Resonance instrument (model ESG0395), equipped with an auto-sampler, aheating block and a Gibson auto-pump

system for continuous sampling. The ELBASoftware version 3.20 with incorporatedLag-Time analysis software, version 1.09was used for data processing.

Preparation of 4-Hydroxy 2,2,6,6 tetramethylpiperidin-1-oxy (Tempo)

Calibration Solutions: 34mg +/- 0.1mg oftempo was weighed out into a clean amberassay vials, 10ml of de-ionised water wasadded and mixed thoroughly tilldissolved. Absorbance measured at 429nm.Dilutions were calculated (with water)required to get 3.0 and 4.0 γM finalconcentration of tempo. 100ml of eachconcentration was made and freeze 8mlaliquots in assay vials. Defrost onesample of 3 and 4 γM tempo each bystanding the vials in room temperaturewater. Before starting the T150 or lagtime assays, the two tempo concentrationswere run. For each assay run in, the 3and 4 γM stock solutions were insertedtogether with the other wort, green beerand beer samples. This allows thecondition of the peristaltic pump tubingto be monitored throughout the lag timeand T150 assays. Lag Time and T150Assays: N-tert-Butyl-α-Phenylnitrone(PBN) Stock Solution-Out of 678mg γ0.1mgof PBN was weighed into acid-washed amberassay vial. Using a Gilson P1000, 750 γof ethanol was added to the PBN andvortexed at very low speed untilcompletely dissolved. This may takes 2-3minutes. If it takes longer the PBN is ofa poor quality. Using a Gilson P1000,750γl deionized water was added and mixedagain at very low speed.Lag Time Assay: Out of 7 g γ 0.01 g ofwort, green beer and beer was weighedinto acid-washed amber assay vial weighedsamples should be run within 30 minutesof preparation. 280 γof PBN was addedwith a Hamilton syringe to the pre-weighed sample, vortexed briefly, andadded to the heating block. The timer was

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started when the first sample was addedto the heating block and activated theELBA software. PBN was added to eachsuccessive vial every 2 minutesthereafter immediately cleaned theHamilton syringe with 50% ethanol toprevent the PBN crystals from blockingthe needle 8 samples were assayed per runwith at least one standard.T150 Assay: 3g γ0.01g of sample wereincubated with 120γl PBN for 150 min at60°C and one reading was taken at thistime. PBN was added to each successivesample and inserted into the heatingblock every 2 minutes. Once the e-scanwas activated, the auto sampler willrinse and then go back to the startingposition. Once start was activated, thesoftware counted down to 150min andstarted on its own once the time hadelapsed.

Sensory Analysis of BeerBeer flavour evaluations and ageing scoreassessments were carried out by a trainedpanel of beer tasters from DeltaBeverages. For aging score assessmentssample beer was tasted at week one, weekfour, week eight and week twelve and thisdegree of staling assessment in aged beerwas evaluated by ten experienced tasters.Flavour evaluation was used to describeflavour profile and sensory defects ofthe beer, the ten tasters were asked tofill in a flavour score card with 45attributes. The Delta beer ageing scorewas used to judge the degree of beerageing. The panel were asked to taste thethree beer samples, and emphasis was madeon the brand flavour which includesalcohol, sweet, bitter, hoppy, fruity,floral and sour. They were asked to filla brand profile score card with the eightattributes.

III. RESULTS AND DISCUSSIONLag Time and T150

Table 1 below shows the Lag Time and T150of the control taken at different brewingstages.

Sample Identity Lag time

(mins)

T150

Ferment Vessel 60 68792

Storage Vessel 59 58269

BBT 59 88672

Packaged Ex-

Filler

52 70322

Packaged Ex-

Pasteuriser

50 66923

Table 1: Control Pitching RateTable 2 below shows the Lag Time and T150of beer pitched with a high pitching ratemeasured at different brewing stages.

Sample Identity Lag time

(mins)

T150

Ferment Vessel 68 60608

Storage Vessel 68 54102

BBT 67 52136

Packaged Ex-

Filler

64 43870

Packaged Ex-

Pasteuriser

61 56787

Table 2: High Pitching Rate

Table 3 below shows the Lag Time andT150 of beer pitched with a low pitch ratemeasured at different brewing stages.

Sample Identity Lag time

(mins)

T150

Ferment Vessel 84 43567

Storage Vessel 76 52178

BBT 74 34970

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Packaged Ex-

Filler

72 43208

Packaged Ex-

Pasteuriser

70 45230

Table 3: Low Pitching Rate

Figure 1: Graph of Lag Time of beer

samplesThe above graph illustrates the

difference of Lag Times measured atdifferent brewing stages for beers pitchedwith different pitching rates.Sensory Evaluation

Table 4 shows taste scores fordifferent ages for beer pitched atdifferent pitch rates.

Sample Scores out of 10 for

respective ages

Sample

Identity

1 week 4 weeks 8 weeks 12

weeks

Control 9.0 8.2 7.1 6.1

High

Pitch

8.9 7.3 6.9 5.8

Low

Pitch

9.0 8.5 7.7 7.1

Table 4: Sample Scores

Figure 2: Graph showing sensory evaluationscores at different agesThe graph above illustrates the aging ofbeer according to 10 trained Deltatasters. The results show that the scoreof beer thus the acceptability decreaseswith time. By 12 weeks the beer samplepitched with low yeast rate was the onewith the highest score and that of highpitching rate had the lowest score.Hypothesis testing using the student’s t-Test for TwoIndependent samples

The t-Test was used to test the hypothesesabout the differences between the samplesof different pitching rates that wereanalysed in the laboratory. Berenson(2002), states that a t-Test is used toacquire differences between sample means,regardless of their direction ofdifference. The t-Test was applied to lagtime, T150 and the sensory evaluation. Thetest statistic was done using the t-testat 5% significance level.H01: There is no significant difference inthe Lag Time (potential to resist staling)of Castle Lager fermented withlower/higher pitching rates and normalCastle Lager.The calculation in Appendix showed that:High pitch rate and control Tcal = 13.2

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Low pitch rate and control Tcal = 11.2Tcrit = 3.13Decision: Reject H01H02: There is no significant difference inthe T150 (degree of staling) of CastleLager beer fermented with lower/higherpitching rates and normal Castle Lager

High pitch rate and control Tcal = 15.4Low pitch rate and control Tcal = 14.7Tcrit = 3.13Decision: Reject H02H03: There is no significant difference insensory evaluation of Castle Lager beerfermented with different pitching ratesand normal Castle LagerHigh pitch rate and control Tcal = 0.045

Low pitch rate and control Tcal = 0.076Tcrit = 2.23Decision: Accept H03

Table 5: Flavour Wheel Key

Figure 3: Flavour Wheel for High Pitch

The flavour wheel above in figure 3 alsoshows a degree of flavour inconsistency.The major flavour defects arechlorophenol, yeasty, fluidic, Sulphitic,Sulphury, sweet, acetic and acidic fromthe flavour wheel key in table 5.

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Figure 4: Flavour Wheel for Low PitchFrom the flavour wheel in figure 4, it isclear that there is no consistency in theoverall flavour. The major off flavoursdefects being found to be diacetyl,leathery, papery, catty and oxidised fromthe flavour wheel key in table 5.

DISCUSSION

The various experiments conducted wereall focused on attaining information onimproving flavour stability of CastleLager. The comparison of different resultsfrom the different pitching rates providedan insight on the capability of pitchingrates towards improving the flavourstability of beer.

Low pitching rates yielded a higherlevel of SO2 than the higher pitching rateand the control. Sulphur dioxide isproduced in small amounts during thebrewing process usually at concentrationsof <10ppm. According to Hardwick (1994),low pitching rates make the amount ofoxygen utilized to make sterols for newcells lower and overall growth is lowered.Insufficient growth will led to slower andperhaps stuck fermentations. Poor yeast

growth therefore led to high SO2concentrations. Yeast growth patterns hadan influence on the flavour of beer bychanging the proportions of volatileflavour compounds. Sulphur dioxide playsan important role in the flavour andstability of lager beers.

Bitterness units in high pitching ratewere lower than the rest of the samplesand this was due to the uptake of bittersubstances through the yeast cell wall andincreased foaming during fermentation.Bitterness of the beer is an importantquality parameter. It is caused by thealpha-acids of hops which are isomerisedduring wort boiling.

Diacetyl, which is a vicinal diketone,can cause a buttery off-flavour above itsthreshold (Wainwright, 1973) and it is oneof the most important by-products inalcoholic fermentation as it decreases thesensory properties of the final product.In the experiments carried out, lowpitching rate had the highest diacetyllevel of 28.7ppb at Day 10 of fermentationand at 15.6ppb at packaging compared to22.7ppb and 11.1ppb for high pitching rateat Day 10 of fermentation and packagingrespectively. There was a significant dropin the diacetyl concentration fromfermentation day 10 to packaging. Thisphenomenon was probably owing to higherproduction of α-acetolactate during day 1to day 9, due to longer fermentation timethe diacetyl concentration dropped as aresult of complete reduction of diacetyl[33] . Lower diacetyl content in the greenbeer will lead to shorter maturation time.

Formation of diacetyl is alsoinfluenced by the spectrum and totalconcentration of amino acids present inthe wort. In particular the concentrationof valine is significant. Diacetyl isformed by an extracellular oxidativedecarboxylation of α-acetolactate andvaline is a strong inhibitor of the enzymeacetohydroxy synthase which catalyses theformation of α-acetolactate [10] . Inhigh pitching rates, the concentration of

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valine is high and this enzyme isinhibited and accordingly diacetylformation is very low. In low pitchingrates the concentration of valine is lowthus there is high formation of the enzymethus the result of high diacetyl levels.Subsequently, yeast cells assimilate andreduce diacetyl to the flavour inactivecompounds acetoin ad 2,3-butanediol. Theability of lager strains to assimilate andreduce diacetyl is influenced byphysiological conditions. Yeast ofanaerobic repressed physiology, as in thecase of cropped pitching yeast, has alower ability to assimilate [17] andreduce exogenous diacetyl and this couldbe the case for the reduction of diacetylfor Day 10 to Packaging. Pitching at highlevels decreased the pH and the formationof diacetyl was reduced.

Lag time indicates the potential toresist staling. A higher lag time impliesthe presence of more anti-oxidants whichallows the beer to resist staling forlonger. Thus the higher the Lag Time thebetter the flavour stability. The lag timein the low pitching rate was found to behigher than that of the high pitching rateand the control. This was because therewere high levels of SO2 produced duringfermentation in beer pitched with a lowerrate and it acted as an antioxidant. SO2has three main roles in beer, firstly itreacts with carbonyl compounds to formalpha-hydroxysulphonates and these are notflavour active and thus increase thethreshold of carbonyl compoundsresponsible for the stale flavour [4] .Also sulphur dioxide inhibits oxidativereactions. The ESR lag time provides anestimate of the ability of a particularbeer, at a particular point in it shelflife, to prevent the reactions that willfurther oxidize the beer and this isprobably why low pitch rate beer has ahigher lag time than other samples. SO2also exerts antimicrobial properties athigh concentrations.

Sulphur dioxide in beer as highlightedbefore acts as an antioxidant, these

antioxidant properties are seen to have apositive relationship with T150 (degree ofstaling) and lag time (potential to resiststaling). The results from figure 8 andfigure 9 illustrate the potential toresist staling (lag time) and the degreeof staling (T150) and respectively. Thecontrol sample showed very high T150results while showing very low lag times.The two results together show that therewas low concentration of antioxidantshence the beer could not resist stalingfor long hence the lower lag times.Comparing the results with that of sulphurdioxide in Figure 3 indicate that arelationship exists between sulphurdioxide and T150 (degree of staling) andlag time (potential to resist staling). Assulphur dioxide increases the T150 (degreeof staling) decreases and the lag time(potential to resist staling) increases.This is a result of the antioxidantproperties found in soluble sulphurdioxide [1] .

According to the different scores givenby the trained tasters, there was adifference in the beer brewed usingdifferent pitching rates. In as much asthe lag time determines how a beer willstay fresh to a particular time, it is thesensory evaluation that really matters asuse of different pitching rates mightbring out some flavours that might helpprevent staling but change the CastleLager profile. In the sensory evaluation,lower pitching rates were found to resultin higher intensities of the overallaroma, hoppy and fruity aroma notes in thefinished beer. And this was because thehigh BU levels that were observed in theexperiment results. With respect to thearoma compound, the concentration ofisobutanol and isoamyl alcohol increasedwith higher pitching rate. In general, theconcentration of higher alcohols ispositively influenced by the pitchingrate.

The flavour wheel in figure 4 andfigure 5 was presented to facilitate thelocation of terms within a system. The

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wheel was meant as an aid to classifyodours and tastes (Vogel et al., 1999).Figure 4 illustrates the flavour wheel ofthe sample with pitching rate of 0.7kg/hlwhich has flavour defects such as paperywhich is due to the oxidation. Accordingto Bamforth (2014) it can be said that allbeers are susceptible to flavourdegradation due to aging. Thus flavouractive compounds such as furfural, trans-2-nonenal, acetaldehyde and otheroxidative products of normal beer flavourconstituents are responsible forcontributing cardboard, papery orstrawberry like off flavours to beer. Theother flavour notes include iso-amylacetate, ethyl-hexanoate and ethylacetate. These are estery flavours thatare desirable because they impart a fruityor floral characteristic that is generatedfrom yeast. As revealed by figure 13 theseflavours failed to reach the correctthreshold that could be detected by thetrained panel.

Beer pitched with a rate of 0.8kg/hlhad flavour defects which include theacidic, yeasty, sulphitic and sulphurynote. There are numerous sulphurcontaining compounds in finished beer[4] , the factors that contribute tosulphur notes are poor kettle boil, lowoxygenation of the wort, yeast strain,slow fermentation, bacterial contaminationand exposure of bottle beer to light . Asillustrated by figure 11, beer fermentedwith a pitching rate of 0.9kg/hl almosthad the same flavour defects as that of infigure 12, the only difference is thatthey had different intensities.

The flavour profile as illustrated infigure 14, is a descriptive sensoryprocedure, it describes and evaluates in acorresponding manner the aroma and tasteof the product. The test uses a highlytrained panel that systematicallycharacterise a beer based upon theamplitude, which is a rating of the degreeof blend and fullness in the aroma andflavour as a whole. Secondly it focuses onthe identification of the individual

components of aroma and flavour. Thirdlyit looks at the strength or intensity atwhich these components appear. Fourthly itlooks at the order in which they appearand lastly it gives a description of theaftertaste one minute after swallowing.The control sample experiencedinconsistencies in fact that they off onthe floral and sour note. Beer brewed witha pitching rate of 0.9kg/hl was a bit souryeasty and the sweet note was a bit low.Beer fermented with a pitching rate of0.7kg/hl suffered on the sweet note for itwas a bit low.

For the hypothesis testing, H01 wasrejected as there was a significantdifference in Lag Time (potential toresist staling) between beers fermentedwith a high or low pitching rate to thatof the control and it was concluded thatpitching rate had an influence on the lagtime of beer.

For the hypothesis testing, H02 wasrejected as there was a significantdifference in T150 (degree of staling)between beers fermented with a high or lowpitching rate to that of the control andit was concluded that pitching rates didhave an effect on T150 values of a beer.

For the hypothesis testing, H03 wasaccepted as there was no significantdifference in sensory evaluation betweenbeers fermented with a high or lowpitching rate to that of the control andit was concluded pitching rates did nothave a significant effect on the taste ofthe beer.

CONCLUSIONDifferent pitching rates do have an effecton the Lag Time and T150 thus flavourstability of beer. Low pitching rates gavea higher Lag Time and a lower T150 but hadflavour defects such as sweet flavoursthat might not be desirable to people. Onthe other hand, higher pitching rates gavea slightly lower Lag Time compared to that

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given by pitching with a low rate and theT150 is quite high.

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