Streamlining Enzyme Analysis In Brewing - Megazyme

Enzymes and BeermakingStreamlining Enzyme Analysis in Brewingwww.megazyme.com

CONTENTSResearch is at the core of Megazyme’s product development. Continual innovation has allowedus to introduce advances and improvements to accepted, industry-standard methods of analysis.Megazyme products offer: reduced reaction timesunrivalled enzyme purityimproved enzyme stability (resulting in a longer ‘shelf-life’)novel assays with ‘best-in-class’ selectivity for the analyte in questionextended cofactor stability (offered in a stable tablet form or as improved liquid formulationswith extended expiry dates)Megazyme test kits - researched and manufactured in-house - have attracted worldwide acclaimfor their novel methodologies and for the exceptional purity of their enzymes.Streamlining Enzyme Analysis in Brewing The traditional approach to malt and beer analysis Enzymes and beer Measuring enzyme activity Starch hydrolases: a-Amylase, b-Amylase and Limit-Dextrinase Cell wall hydrolases: Malt b-Glucanase, Xylanase and CellulaseAssay Kits for the Measurement of Enzyme Activity in Brewing Starch hydrolases: a-Amylase, b-Amylase, Malt Amylase and Limit-Dextrinase/Pullulanase Cell wall hydrolases: b-Glucanase, Xylanase and CellulaseAnalytical Solutions from Megazyme Cultivating excellence in cereal chemistry Tailor-made substrates Megazyme assays explained3911

STREAMLINING ENZYMEANALYSIS IN BREWINGDR. CLAUDIO CORNAGGIAIN THEIR EFFORTS TO INNOVATE IN A DYNAMIC MARKET, BREWERS AIMTO ACHIEVE THE EFFICIENT PRODUCTION OF BETTER BEERS WITH MORECONSISTENT SENSORY PROPERTIES AT A REDUCED COST.Technical advancements have been appliedto all stages of beer production, from cerealharvesting to filtration to bottling and packaging.All advancements have moved towards the samesolution: automation.Brewers are typically comfortable with the methodsthey trained to use at the start of their careers, and areunderstandably reluctant to abandon them.However, a number of new analytical methods areworth considering as substitutes for (or additions to) thetraditional practices, offering the user new information onproperties that are important to brewing. Importantly, thesesubstitutes also offer the potential for brewers to automatethe analysis of malts and beers.While other aspects of beermaking have modernised,analytical aspects of brewing science have arguablylagged behind: maltsters and brewers continue to useanalytical methodologies developed in the 19th and early20th centuries.Enzymes and beerThe traditional approach to maltand beer analysisThere are two main biochemical processes central tobrewing which require enzymes:Malts usually come with a detailed passport includinga long list of properties and parameters. Despite thereliance of cereal breeders, maltsters, and brewers onthese descriptions, many of the measurements includedare based on traditional methods that lack specificityand sensitivity. Others require specialised equipmentor are highly labour-intensive, making them ill-suited toautomation.1.conversion of starch (naturally present in barley) intofermentable sugars, and2.fermentation of sugars by yeasts to produce ethanoland carbon dioxide.Starch conversion involves two classes of enzyme cell wall hydrolases and starch hydrolases - which arereleased by the barley grain itself during malting.3Discover more at www.megazyme.com

Like most cereals, barleycontains starch as its primarystorage polysaccharide. Starchmust be released from insidethe barley endosperm, thenbroken down into maltose andglucose before it becomesuseful for elease of enzymes that allowthe growing seed to break downits endosperm cell walls andaccess its starch reserves.Maltsters take advantage ofthis process, germinating thebarley grain under controlledconditions in such a way that theessential endogenous enzymesare produced and released.During malting, the germinatingseed releases two main classesof carbohydrate hydrolase,which play important rolesat different points in thebeermaking process.EndogenousEnzymesCell Wall HydrolasesBreak down cell walls, allowingstarch hydrolases to access starchin the mashing stage and alsoimprove filterability.β GlucanaseStarch HydrolasesConvert long starch chains intosmaller sugars, which seXylanaseMalting is terminated by dryingwith hot air (‘kilning’), which killsthe barley embryo and preventsfurther starch loss via respiration.However, unavoidably, some of the enzymes producedduring malting are deactivated by high temperatures atkilning.LimitDextrinasecomprise the majority of the free amino nutrients (FAN) are also formed at this stage, mainly due to the action ofprotease enzymes. FAN plays an important role in feedingyeast during the fermentation stage of beer production.Next comes the second enzyme-intensiveprocess in beermaking: fermentation. Yeastsrelease enzymes to convert sugars into ethanoland carbon dioxide. However, the fermentabilityand filterability of the final beer is largely predecided, based on the enzymatic activity thatoccurred in malting and mashing.Brewers become involved from this stage onwards. Theycombine the malts with water and (optionally) adjuncts,then begin cooking the mixture in a process calledmashing.Mashing aims to complete the destruction of the cell walland accelerate the breakdown of starch. Heat is appliedaccording to a pre-established programme intended tomaximise the activity of the remaining enzymes.Once the cell wall is broken down, starch hydrolases areable to reach the starch molecules and break them intosmaller fragments - the smallest being the fermentablesugars maltose and glucose.Other important molecules such as amino acids - which4

ExogenousEnzymesEndogenous enzyme levels may not be sufficient to produce thedesired brew. The careful addition of commercially-available enzymepreparations helps to optimise fermentability and filterability byadjusting levels of different enzymes according to need.Cell Wall HydrolasesStarch HydrolasesUsed to improve filterability.Used to improve aseTo add an unnecessarilylarge amount of exogenousenzymemaybecostly,counterproductive or simplyfutile.PullulanaseXylanaseone enzyme carries out thedesired activity, the presence ofothers may result in undesiredhydrolytic activities with thepotential to affect the finalproduct, e.g. addition of protease(such as papain) to beer toaddress haze formation, withthe undesired result of inferiorfoam stability unless dosage iscarefully controlled.Conversely, the addition of toolittle exogenous enzyme mayfail to address delays in the brewing process.Measuring enzyme activityWith enzymes playing such a critical role in the brewingprocess, maltsters and brewers must be able tomeasure enzyme activity in their raw materials. Only byunderstanding the enzymatic potential of their maltscan maltsters and brewers fully exploit the enzymaticprocesses that influence the consistency and quality oftheir beer.Starch hydrolasesTo be useful for brewing, starch must be broken down intosmaller units, such as maltose and glucose, which canlater be used by yeasts during fermentation. The enzymesresponsible for breaking down the long starch chains intofermentable sugars are called starch hydrolases.Brewers seeking to maximise particular attributes of theirbeer may add exogenous enzymes to promote relevantenzyme activity.Four main enzymes are involved in the transformation, allof which are produced or activated during malting. All arenecessary in order to maximise the fermentability of thebrew:A brewer may use exogenous enzyme supplementationif there is concern that endogenous enzyme levels willnot be sufficient. Barley variety, pre-harvest sprouting andmethods of malting, kilning and mashing may all influenceendogenous enzyme -GlucosidaseEach has its own specific role to play and a unique patternof action, which will be discussed in turn. a-Glucosidaseis present at such low levels that its contribution to thebrewing process is not considered in detail here.Exogenous enzyme mixtures can correct issues likestuck mashes and low extract yields. These enzymesalso enable beermakers to brew efficiently with unmaltedcereal adjuncts (e.g. corn, wheat and rice) to produce lightbeers or gluten-free beers.However, exogenous enzymes should not be viewed asa ‘quick-fix’ solution that can be undertaken lightly: while5Discover more at www.megazyme.com

Starch itself is divided into two structural forms. Amylose iscomposed of relatively linear a-1,4-glucose chains, whileamylopectin is a branched polysaccharide in which a-1,4glucose chains are joined together by a-1,6 branch points.As the most thermostable of the starch hydrolases,a-amylase has a temperature optimum up to 70oC whichallows it to tolerate the temperatures involved in starchgelatinisation. Around 90% of a-amylase persists throughthe mashing phase. Therefore, the amount of a-amylasethat persists after mashing is generally not the limitingfactor in obtaining the maximal hydrolysis of starch.b-AmylaseamyloseamylopectinLike a-amylase, b-amylase is able to cut a-1,4 linkagesin starch chains. However, b-amylase is an exo-actingenzyme, which means that instead of randomly cuttinginternal linkages in starch, it cuts from the end of the chainonly.An important property of starch is its gelatinisationtemperature, that is, the minimum temperature that mustbe reached during mashing. On reaching the gelatinisationtemperature in the presence of water, the tight crystallinestructure of the starch granule is destroyed, making thepolysaccharide readily accessible to starch hydrolases.The gelatinisation temperature is a crucial concept inmashing as it determines the persistence and activity ofall hydrolases, some of which are more thermostable thanothers. This temperature depends on a number of factors(including amylose:amylopectin ratio, the cereal used, andthe use of adjuncts) but is typically 60oC.amylosea-Amylaseb-Amylase activity (example)a-Amylase is an endo-acting enzyme that randomly cutsinternal a-1,4 linkages in the starch molecules. This has twomain effects: firstly, the viscosity of the mash drops rapidly,and secondly, maltodextrins are produced.amylosea-Amylase activity (example)amylopectinfermentable sugarb-Amylase liberates fermentable maltose molecules,which account for 65% of the fermentable sugar in wort.a-Amylase and b-amylase are able to work synergisticallyduring mashing, as b-amylase generally acts on themaltodextrin fragments liberated by the initial hydrolyticaction of a-amylase.b-Amylase is the most abundant starch hydrolase duringmalting, however it is significantly less thermostable thana-amylase. A recent study found that just 40% of the initialb-amylase persisted to continue liberating maltose in themash. Brewers therefore need to quantify the true activityof b-amylase: the presence of too little b-amylase has thepotential to limit the fermentability of the resulting wort.This is a particular concern when brewing with adjuncts.amylopectinmaltodextrin6