Improving extract yield and wort viscosity:
Automated measurement of hydrolytic enzymes in the production of fermentable sugars
D. Mangan, Claudio Cornaggia, R. Ivory, A. Liadova, E. Rooney, N. McCormack, B. Culleton, V. McKie & Barry V. McCleary Megazyme International
Megazyme International has developed a range of advanced enzymatic test kits and reagents specifically aimed at the malting and brewing industries and to cereal breeding programmes and research laboratories in brewing sciences.
These new and advanced products now make enzymatic bio-analysis commercially viable for brewhouses and laboratories of any size.
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] INTRODUCTION
AIM OF RESEARCH
CELL WALL HYDROLASES Filterability
STARCH HYDROLASES Fermentability
CONCLUSIONS Introduction – Malting barley & hydrolases
Germinated barley kernel during malting
Hydrolases
endo-(1,3:1,4)-b-glucanase MALTING BARLEY a-amylase, b-amylase MALTOSE endo-xylanase Production of hydrolases & limit-dextrinase (and other (plus exo-acting hydrolases) (plus a-glucosidase) › b-glucanase fermentable
› xylanase sugars) › a-amylase Hydrolysis of barley cell Hydrolysis and › b-amylase walls to allow dextrinizing debranching of starch is available › limit-dextrinase enzymes to access starch into shorter dextrins for alcoholic › proteases granules and into maltose fermentation FILTERABILITY FERMENTABILITY
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Introduction – Malting barley & hydrolases
Hydrolases: endogenous vs exogenous MALTSTERS Endogenous hydrolases : b-glucanase, endo-xylanase, a-amylase, b-amylase & limit-dextrinase
• Produced during malting. NO endogenous hydrolases produced after kilning.
BREWERS
• Is the wort viscosity optimal? Will there be problems on lautering?
• Is the fermentability optimised? (Diastatic power) • Will there be haze problems? (Due to HMW b-glucan, protein, dextrins)
• How will the beer mouthfeel be affected?
Exogenous hydrolases (mostly of bacterial / fungal source) are sometimes added during the brewing process to optimise the wort filterability and the extract yield (fermentability) Exogenous hydrolases • b-Glucanase • Amyloglucosidase • Xylanase • Limit-dextrinase / Pullulanase • a-Amylase (debranching enzyme)
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] INTRODUCTION
AIM OF RESEARCH
CELL WALL HYDROLASES Filterability
STARCH HYDROLASES Fermentability
CONCLUSIONS Aim of research
Hydrolases targeted for development of specific substrates and automatable assays
1) endo-(1,3:1,4)-β-Glucanase (EC 3.2.1.73) 2) endo-1,4-β-Xylanase (EC 3.2.1.8) 3) α-Amylase (EC 3.2.1.1) Suitable substrate and assay format 4) β-Amylase (EC 3.2.1.2) 5) Limit-dextrinase (EC 3.2.1.142)
Main challenges
• Complex matrix – polysaccharides, oligosaccharides, enzymes, amino acids, inhibitors • Selectivity – presence of a range of exo-acting enzymes competing for the same substrate (e.g. a-glucosidase, a-amylase and b-amylase act on starch) • Sensitivity – low enzyme activities in some cases (e.g. limit-dextrinase)
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Aim of research: existing methods
• b-Glucan analysis in barley, malt, wort and beer (fluorimetric) EBC Methods 3.10.2/4.16.1/8.13.2/9.31.2
1) Size (MW) of b-glucan not accounted for: effect on viscosity and haze. 2) Viscosity assay: more practical indicator of how malt will affect brewhouse performance.
• Diastatic power of malt (iodometric - titration) EBC Method 4.12
1) It generally gives good correlations only with b-amylase activity (not a-amylase or limit-dextrinase) 2) Not always a good predictor of fermentability, it can vary between malt varieties.
• a-Amylase content in malt (iodometric - visual) EBC Method 4.13
1) Visual estimation using a colour standard, does not consider limit-dextrinase activity
• Fermentability (AAL Apparent attenuation limit – specific gravity) EBC Method 4.11.1
1) Long and complicated assay 2) Use of congress mashing which is not always representative of commercial brewing techniques LIMITATIONS?? • None of the above methods can be automated • General reproducibility / practicality / reliability issues EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Hydrolases measurement : existing methods
Megazyme existing products for hydrolase measurement
Dyed polysaccharides - insoluble
LIMITATIONS??
Ill-suited for automated assays due to filtration step
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Aim of research – Novel substrates
Blocked colorimetric oligosaccharides
ADVANTAGES Practical and rapid assay format
Ability to measure endogenous enzymes in barley malt / all cereals
Ability to measure exogenous enzymes in commercial enzymatic preparations
Sensitive and quantitative assay
Excellent repeatability and reproducibility
Automatable assay format for auto-analyser
Replace / complement existing methods
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] INTRODUCTION
AIM OF RESEARCH
CELL WALL HYDROLASES Filterability
STARCH HYDROLASES Fermentability
CONCLUSIONS endo-(1,3:1,4)-β-Glucanase – Overview
• (1,3:1,4)-β-Glucan: 75% (w/w) of cell wall in starchy endosperm
• Composed of b-1,4-linked glucose units (DP4:DP3) separated by β-1,3 linkages
• b-Glucan hydrolysis allows starch hydrolases to degrade starch leading to maltose formation
DP3 DP4 DP3
β-1,3 linkage
• endo-(1,3:1,4)-β-Glucanase hydrolyses β-1,4 linkages on the non-reducing side of the β-1,3 linkages
• Extent of b-glucan hydrolysis: key for wort viscosity, filterability, haze formation & starch mobilisation
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] endo-(1,3:1,4)-β-Glucanase – Substrate & assay format
K-MBG4 Assay: Rationale
DP4 (1,3:1,4)-b-gluco-oligosaccharide β-1,3 • optimal recognition by endo-(1,3:1,4)-b-glucanase
Benzylidene acetal blocking group • prevents hydrolysis by exo-acting glycosyl hydrolases present in barley malt extracts (e.g. b-glucosidase)
2-Chloro-4-nitrophenol chromophore (pKa = 5.4) • provides for substrate sensitivity
Directly proportional to endo- (1,3:1,4)-b-glucanase activity
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] endo-(1,3:1,4)-β-Glucanase – Substrate & assay format
K-MBG4 Manual Assay Conditions
Mill barley malt sample to pass 0.5 mm screen. EnzymeExtraction
0.5 g Milled malt sample + 8 mL extraction buffer
(pH 4.5, 0.1M NaOAc). Extract over 15 min.
Centrifuge at 1000 x g for 10 min. Standard curve relating the activity of malt Decant the supernatant. endo-(1,3:1,4)-β-glucanase on barley b-glucan to absorbance increase at 400 nm on hydrolysis of MBG4 under the assay conditions
0.5 mL Enzyme extract + 0.1 mL MBG4 reagent Assay Activity
(10 mM, 50% DMSO/H2O). Barley malt endo-(1,3:1,4)-β-Glucanase o Incubate for 20 min at 30 C. International Units on b-glucan = 3.98 x MBG4 Units
MBG4 substrate suitable also for the
Terminate assay with 0.9 mL Tris buffer (pH 10). Read absorbance value at 400 nm. assay of lichenases of bacterial source
Mangan et al. 2016, Carbohydr. Res. submitted
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] endo-Xylanase – Overview
• Arabinoxylan (pentosan): approximately 20% (w/w) of cell wall in starchy endosperm
• Heterogeneous polysaccharide: backbone of β-1,4-linked xylose monomers (arabinofuranosyl residues, feruloyl esters and glucuronic acid residues also present)
• endo-1,4-β-Xylanase hydrolyses internal β-1,4 linkages between xylose residues in the backbone
• Extent of arabinoxylan hydrolysis is key for wort viscosity, filterability & starch mobilisation
• Soluble arabinoxylan levels are much higher in wort and beer than those of b-glucan (up to 10 times)
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] endo-Xylanase – Overview
XylX6 Substrate + K-XylX6 Assay: Rationale ancillary b-xylosidase = XylX6 Reagent
XylX6 assay suitable also for the measurement of endo-xylanase in:
• Barley malt • Commercial enzyme preparations • Pure enzyme (fungal/ bacterial)
Directly proportional to endo-xylanase activity
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] endo-Xylanase – Substrate & assay format
Graph showing the activity of Standard curve relating the activity of endo-β-xylanase endo-β-xylanase extracted (A. niger) on beechwood xylan (left) or wheat arabinoxylan (right) to from malt flour on XylX6 absorbance increase at 400 nm on hydrolysis of XylX6
A. niger endo-(1,4)-β-Xylanase International Units on xylan beechwood = 1.13 x XylX6 Units A. niger endo-(1,4)-β-Xylanase International Units on wheat arabinoxylan = 1.05 x XylX6 Units
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] INTRODUCTION
AIM OF RESEARCH
CELL WALL HYDROLASES Filterability
STARCH HYDROLASES Fermentability
CONCLUSIONS Amylases and limit-dextrinase – Overview
• Starch: approx. 60-65% (w/w) of barley grain weight.
• Amylose: long linear α-1,4 glucose chains & Amylopectin: contains α-1,6 branch points
Limit-dextrinase hydrolyses internal α-1,6 linkages
α-1,6
α-1,4 α-Amylase hydrolyses internal b-Amylase hydrolyses dextrins from α-1,4 linkages non-reducing end to release MALTOSE
• Starch degrading enzymes balance is key for extract yield, final carbohydrate profile (mouthfeel) and haze due to limit dextrins • Thermal stability of enzymes, key for production of fermentable sugars: a-amylase more thermostable, b-amylase & limit-dextrinase less thermostable
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] a-Amylase – Substrate & assay format
DP7 Malto-oligosaccharide • optimal recognition by a-amylase
Benzylidene acetal blocking group • prevents hydrolysis by exo-hydrolases (e.g. b-amylase, a-glucosidase)
4-Nitrophenol chromophore • substrate sensitivity & stability
Ceralpha Substrate + ancillary a-glucosidase = Ceralpha Reagent
Ceralpha Assay can be used to measure cereal, bacterial & fungal a-amylase. Directly related to a-amylase activity:
McCleary et al. 1987 J. Cereal Sci., 6, 237. McCleary et al. 2002 J. AOAC Int., 85, 1096. EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] a-Amylase – Substrate & assay format
K-CERA Manual Assay Conditions
Mill barley malt sample to pass 0.5 mm screen. EnzymeExtraction
0.5 g Milled malt sample + 100 mL extraction solution. Extract over 15-20 min at room temperature.
Comparison between the Ceralpha and ASBC methods for the
measurement of a-amylase in malt flours
Centrifuge for 10 min. Barley malt a-amylase Decant the supernatant and dilute 1:20. International Units on Starch = 4.10 x Ceralpha Units
Fungal a-amylase (A. niger) 0.2 mL Enzyme extract + 0.2 mL Ceralpha reagent. Assay Activity International Units on Starch = 0.94 x Ceralpha Units
Incubate for 10 min at 40oC. Bacterial a-amylase (B. subtilis) International Units on Starch = 4.60 x Ceralpha Units Validated method Terminate assay with 3.0 mL phosphate buffer (pH 11). Read absorbance value at 400 nm.
McCleary et al. 1987 J. Cereal Sci., 6, 237 – McCleary et al. 2002 J. AOAC Int., 85, 1096. EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] b-Amylase – Substrate & assay format
Betamyl-3 Assay: Rationale
DP3 Malto-oligosaccharide: • recognition by b-amylase • untouched by cereal a-amylase • hydrolysed by fungal a-amylase and amyloglucosidase
NO blocking group: • substrate hydrolysed by a-glucosidase (negligible in barley malt)
4-Nitrophenol chromophore: • substrate sensitivity & stability
Betamyl-3 Substrate + ancillary b-glucosidase = Directly related to Betamyl-3 Reagent b-amylase activity
McCleary et al. 1989 J. Cereal Sci., 9, 17.
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] b-Amylase – Substrate & assay format
K-BETA3 Manual Assay Conditions
Mill barley malt sample to pass 0.5 mm screen. Enzyme ExtractionEnzyme
0.5 g Milled malt sample + 5 mL extraction buffer (Tris/HCl pH 8).
Extract over 1 h at room temperature.
Centrifuge for 10 min. Decant the supernatant and dilute 1:20.
Activity Assay Activity 0.2 mL Enzyme extract + 0.2 mL Betamyl-3 reagent. Barley malt b-amylase Incubate for 10 min at 40oC. International Units on Starch = 39.7 x Betamyl-3 Units
Validated method Terminate assay with 3.0 mL Tris solution (pH 10). Read absorbance value at 400 nm.
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Limit-Dextrinase – Substrate & assay format
DP6 mixed-linkage (a-1,4:1:6) K-PullG6 Assay: Rationale malto-oligosaccharide • ensures optimal recognition by limit- dextrinase
Benzylidene acetal blocking group • prevents hydrolysis by exo-hydrolases (e.g. b-amylase). 4-nitrophenol chromophore: • provides for substrate sensitivity and stability
PullG6 Substrate + ancillary a-glucosidase + ancillary b-glucosidase = PullG6 Reagent
McCleary et al., 2014, Carbohydr. Res., 393, 60. Directly related to Mangan et al., 2015, J. Cereal Sci., 62, 50. limit-dextrinase/ pullulanase activity
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Limit-Dextrinase – Substrate & assay format
K-PullG6 Manual Assay Conditions
Mill barley malt sample to pass 0.5 mm screen. EnzymeExtraction
0.5 g Milled malt sample + 8 mL extraction buffer (pH 5.5, 0.1M sodium maleate). Extract over 5 h at 40oC (vortex for 10 sec every hour). Standard curve relating the activity of limit-dextrinase extracted from rice (left) and bacterial pullulanase
(right) on borohydride-reduced pullulan to absorbance increase at
Centrifuge for 10 min. 400 nm on hydrolysis of PullG6 Decant the supernatant. Rice limit-dextrinase (LD)
International U on BH4 pullulan = 5.31 x PullG6 Units 0.1 mL Enzyme extract + 0.1 mL PullG6 reagent Assay Activity (4.3 mM). Incubate for 30 min at 40oC. PullG6 substrate suitable also for the assay of pullulanases
Terminate assay with 1.5 mL Tris buffer (pH 9). Read absorbance value at 400 nm.
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Hydrolase assays – Automation
Automated formats created for the following hydrolase assays: • endo-(1,3:1,4)-β-Glucanase • endo-β-1,4-Xylanase • endo-β-1,4-Cellulase • a-Amylase • Limit-dextrinase • Pullulanase • Lichenase Assay programmes pre-installed and enzyme activity calculations are automated
Auto-analyser involved in all of the assay steps post-enzyme extraction including blank samples
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Hydrolase assays – Superior predictors of malt fermentability?
• Diastatic power (DP): not a reliable parameter for prediction of malt fermentability (b-amylase bias) • Malt variety does not guarantee reproducible fermentability (high variations in DP and DP enzymes)
Measurement of DP enyzmes – a-amylase, b-amylase and limit-dextrinase – individually provide a superior prediction of fermentability
Commercial cases : Data taken from D. Evans et al. Proc. Congr. Eur. Brew. Conv. 31, 2007 Total limit- Malt Brewers Comment (mashing, DP Total β-amylase α-amylase dextrinase blend fermentation) (WK) (U/g dwt) (U/g dwt) (U/Kg dwt) 1 short stand, high fermentability 215 532 170 220 2 short stand, high fermentability 208 543 171 248
3 long stand, low fermentability 212 504 129 221 4 long stand, low fermentability 212 528 134 231
5 long stand, low fermentability 205 394 139 151 6 long stand, low fermentability 194 402 116 141 b-Amylase measured using Betamyl-3; a-amylase measured using Ceralpha and limit-dextrinase measured using Limit-Dextrizyme tablets (T-LDZ)
Unpredictable fermentability despite similar DP values can be explained with DP enzymes analysis
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Hydrolase assays – Superior predictors of malt fermentability?
Multi-linear regression (MLR):
AAL = 69.9 + 0.17 × A + 9.60 × B + 0.195 × C + 0.007 × D + 0.538 × E – 0.008 × D × E (r2 = 0.91)
A = α-amylase activity | B = total LD activity | C = KI (%) | D = total β-amylase activity | E = β-amylase thermostability (%)
Ceralpha PullG6 Routinely Betamyl-3 Intrinsic varietal characteristics
measured (D. Evans et al., 1998, J. Cereal Sci.,28,301)
MLR taken from D. Evans et al., 2005, J. Am. Soc. Brew. Chem., 63, 185 (and later papers by the same author)
ADVANTAGES:
• Predicts approx. 90% of variation in malt attenuation compared to < 50% by DP alone • One extraction can be used for the three enzymes combined MALTSTERS • Blend malt batches precisely within or between varieties • Satisfy brewers fermentability expectations with malt whose qualities are better described
BREWERS • Unexpected mashing / fermentability behaviour can be predicted in advance
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] INTRODUCTION
AIM OF RESEARCH
CELL WALL HYDROLASES Filterability
STARCH HYDROLASES Fermentability
CONCLUSIONS Megazyme’s enzyme coupled colorimetric substrate platform for endo-hydrolases
Substrates for the prediction of wort filterability endo-1,3:1,4-β-Glucanase / Lichenase – MBG4
endo-1,4-β-Xylanase – XylX6
Substrates for the prediction of malt fermentability
α-Amylase – Ceralpha
b-Amylase – Betamyl-3
Limit-dextrinase/Pullulanase – PullG6
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Conclusions
• Novel substrates / assay formats developed for important endo-hydrolases o Chemically defined completely specific colorimetric substrates. o Suitable for the analysis of crude malt extracts and commercial enzyme preparations o Complete with standardised controls of the relevant enzyme
• Method advantages o Rapid, sensitive and reproducible. NO batch-to-batch variability o Automatable for reduced hands-on time by analysts o Inexpensive instrumentation o Suitable for all cereals
• Suitable for o Maltsters & brewers o Enzyme manufacturers o Barley breeding programmes & enzyme reasearch o Analytical laboratories
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] Thank you
EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected] EBC Symposium 2016 – Wroclaw, Poland 18-20th September Dr. Claudio Cornaggia – Senior Scientist – [email protected]