New Technologies Applied to the Brewing Industry Lars Lindqvist and Per Nilsson Thermo Fisher Scientific

The world leader in serving science

Gallery OutlinePlus Beermaster – automated efficiency Analytical Instruments for Beer Analysis • Discrete Analyser – Beermaster • Chromatography • Ion Chromatography (IC) - Instruments and Application overview

• High Performance Liquid Chromatography (HPLC) Example application – Isohumulones

• Gas Chromatography (HPLC) Example application diketones and profiling studies

• Resources

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Gallery GalleryPlus PlusBeermaster Beermaster– –automated automatedefficiency efficiency

• Automated, discrete photometric system for beer, wort and malt analysis • Superior platform for beer, wort and malt quality control and testing • Highly specific colorimetric and enzymatic measurements • Automated, effective bitterness measurement • Compact, easy-to-use system • Wide range of system reagents

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Time and cost savings • Automates all necessary analysis steps

• Bitterness and other photometric tests, like beta- glucan, pH, SO2 etc., can be run parallel • Up to 350 photometric tests/h when running one-reagent methods

• Typical capacity 8 bitterness tests/h • First bitterness result in 10 minutes

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Key Benefits of Gallery Plus Beermaster • Easy to use – once loaded all the steps of analysis are performed automatically • Fully automated pretreatment and measurement for bitterness simultaneously with other tests • Contamination free analysis – disposable cuvettes • Cost-effective – low reagent usage • Reduced environmental impact – low water consumption • Easy to install and maintain 5

Thermo ScientificTM System Reagents for Beer Samples • Acids • Acetic acid (ACS) • Acetic acid • Citric acid • D-Lactic acid • L-Lactic acid

• Sugars • • • • • •

D-Fructose D-Glucose D-Glucose + D-Fructose D-Glucose + D-Fructose + Sucrose Sucrose (Total Glucose) Lactose

• L-Malic acid

• Oxalic acid • Total Acids

• Metals • Calcium

• Total Iron • Magnesium

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• Others

• Alcohols • Glycerol • Ethanol

• • • • • • • •

Acetaldehyde Ammonia SO2 Total Alpha-amino nitrogen (NOPA) pH (ECM or Colorimetric) Bitterness Beta-Glucan (High MW) Total protein

• Calibrators • • • • • • • •

Acetaldehyde standard Acid combination standard Glycerol standard NOPA standard Sugar combination standard Beta-Glucan standard Oxalic acid standard Lactose standard

Over 30 optimized system products for brewing analysis

Key benefits of Thermo Scientific system reagents • Optimized system solution •

System applications for beer analysis •

Continuously expanding menu

•

Electronically loadable application data

•

Optimized kit sizes and on-board stability

•

Wide range of standards

• Productivity and efficiency

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•

Ready-to-use liquid reagents eliminate reagent preparation

•

Minimal reagent waste

•

Bar-coded reagent vials provide easy and reliable identification •

lot, exp date, vial size

•

real-time reagent monitoring

Key benefits of Thermo Scientific system reagents • Optimized system solution •

System applications for beer analysis •

Continuously expanding menu

•

Electronically loadable application data

•

Optimized kit sizes and on-board stability

•

Wide range of standards

• Productivity and efficiency

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•

Ready-to-use liquid reagents eliminate reagent preparation

•

Minimal reagent waste

•

Bar-coded reagent vials provide easy and reliable identification •

lot, exp date, vial size

•

real-time reagent monitoring

Various reporting options • Reports available • Through LIS

• As printouts • Electronically as PDF files • Spreadsheet reporting for further calculations

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Gallery Plus Beermaster - Summary

• Fully automated system for beer analysis • Quick and accurate results securing quality and consistency during the brewing process

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Chromatography Instrumentation - High Performance Liquid Chromatography (HPLC) - Ion Chromatography (IC) - Gas Chromatography

The world leader in serving science

Ion Chromatography Portfolio: For every Beer Analysis Challenge Integrion

ICS-5000+

Aquion ICS-4000

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IC Application Summary • Multi Analyte Detection and Quantification in a Single Run • Applications Available for: Carbohydrates, Alcohols, Organic Acids, Inorganic Anions, and Cations

Download Technical Note 126: Determination of Organic Acids in Beer Samples

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IC Application Examples: Carbohydrates • The power to analyze Fermentable Sugars and Malto-Oligosaccharides in Beer

Download Application Note 46: Ion Chromatography: A Versatile Technique for the Analysis of Beer

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HPLC: Versatile and Powerful Technique

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Example Application: Isohumulones • Different contributions to beer properties • Iso-n-humulones provide balanced bitterness • Isocohumulones considered to provide harsher bitterness • Different contributions to beer flavor, odor and aroma Isohumulone

Foam / Beer Ratio

Isocohumulone

1.3

Iso-n-humulone

2.5

Isoadhumulone

3.3

Total Isohumulones

2.2

• Isoadhumulones stabilize beer foam the most, isocohumulones the least

Source: Handbook of Brewing: Processes, Technology, Markets. H.M. Eßlinger, ed. Wiley-VCH, Weinheim. 2009.

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Isohumulones (Iso-α-acids) Chemistry • Derived from hops during wort boiling

• Foam stability, Bitterness and Anti-microbial preservation chemistry

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Example Solution to the market Brewers need to know the exact isohumulone pattern to be innovative and successful!

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Ready, Steady, Go! One-Click Total Solutions Instrument

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Columns, Reagents and Standards

Method

Get the Result in Less than 10 Minutes

Brewers can create new innovative brands and flavors 20

Information Resources • Brochure: Total Solution for Isohumulones in Beer • Webpage: Thermo Scientific Isohumulones in Beer Application Kit

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Gas Chromatography Solutions: Diketone analysis

Head-space analysis

Trace 1300/1310 Gas Chromatograph with automated head-space sampler

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Static head-space

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Calibration curves 2,3 – butanedione and 2,3 - pentanedione

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Qualitative analysis: Olfactometry + chromatography = true

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The selectivity of the nose

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Experimental - Samples • 9 whisky extracts in ethyl acetate analysed 4 times in random order. • Pool sample prepared from 20 µl each whisky. • Run at 60,000 resolution on Q Exactive GC

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Sample ID

Type

Age

Country of Origin

Region

2263

Single

12

Scotland

Lowlands

2264

Single

18

Scotland

Lowlands

2265*

Single

NAS

Scotland

Lowlands

2281

Single

10

Scotland

Campbeltown

2282

Single

15

Scotland

Campbeltown

2283

Single

15

Scotland

Campbeltown

2284

Single

12

Scotland

Highland

2285

Single

18

Scotland

Highland

2295

Bourbon

-

USA

Kentucky

Analytical approach in this proof of concept study • Are there any chemical differences between whisky samples? • Type of whisky ie Bourbon or Scotch Whisky • Geographical origin ie USA or Scotland, Highland or Lowland

• Age of whisky ie 10 or 15 Year aging

• Approach was to run a non-targeted analysis under generic conditions and use software tools to identify: • If there are differences? Using statistical software ie Sieve 2.2 • What the differences are? Using software tools including NIST libraries, deconvolution software and elemental composition and fragment matching software.

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TIC whisky sample

F:\Work\...\8April_60K_Whisky_025

04/10/15 06:10:59

RT: 3.45 - 34.00 SM: 7B 12.45

100

NL: 2.46E10 TIC MS 8April_60K _Whisky_0 25

95 90 85 14.93

80 75 70 65 60 55 50 45 40

15.69

8.53

35 30

18.90

25

12.55

20

12.28 12.19

15

14.62

10 7.42

5

23.90

20.78 18.99

9.38 9.51

17.15

10.54

26.02

22.18 27.94 29.14

24.89

31.98

0 4

29

6

8

10

12

14

16

18 20 Time (min)

22

24

26

28

30

32

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Software Algorithm for Unknowns Analysis

Deconvolve TIC

Create “clean” spectrum

Acquired

Library Proposed Compounds Library Search 30

PCA from Sieve 2.2 Technical replicates in good agreement and clear differences between samples

2295

2265

2281 2263

Pool 2284

2264

31

2285

2282 2283

PCA from Sieve 2.2 Bourbon and 3 wood aged clearly different from other whiskies. Single distillery whiskies also show clear differences. Bourbon 3 wood aged

Single distillery

10 year Pool

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15 year 15 year

Taking a closer look at the data • Initial approach is to investigate the differences in the bourbon and 3 wood aged from other whiskies

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Peak at 13.61 min. m/z 177.1274 Elevated in Bourbon • From the PCA plot and the table of the detected peaks the following peak was identified as being significantly elevated in bourbon. • The peak was investigated by peak extraction and spectrum assessment.

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NIST Library hit for Trans β Ionone

NIST hits filtered based on high resolution filtering

Acquired spectrum

NIST spectrum

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Trans β Ionone

Literature search for beta ionone supports • Beta-ionone is a breakdown product of beta-carotene and expected to be found in whisky. • Bourbon brewed from corn; Scotch Whisky from barley. • Corn is higher in beta-carotene (53 µg/200g) compared to barley (7 µg/200g (Source: Nutritiondata.com) • This would support the higher levels of beta-ionone found in our data.

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Resources Dedicated Beer Analysis Compendium

thermofisher.com/BeverageTesting 37

Many More Resources • Dedicated Website and Learning Centre

thermofisher.com/BeverageTesting

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Thank You For Your Time – Any Questions…………

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