Evidence of dextrin hydrolyzing enzymes in Cascade hops

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Food and Beverage Chemistry/Biochemistry

Evidence of dextrin hydrolyzing enzymes in Cascade hops (Humulus lupulus) Kirkpatrick Kaylyn, and Thomas H. Shellhammer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03563 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Journal of Agricultural and Food Chemistry

Title Evidence of Dextrin Hydrolyzing Enzymes in Cascade Hops (Humulus lupulus) Authors Kaylyn R. Kirkpatrick, Thomas H. Shellhammer Department of Food Science & Technology, Oregon State University, Corvallis, Oregon 97330, United States.

Corresponding Author: Thomas H. Shellhammer Phone: 541.737.9308 Fax: 541.737.1877 Email: [email protected]

Kaylyn R. Kirkpatrick Phone: 970.373.7742 Fax: NA Email: [email protected]

ACS Paragon Plus Environment


ABSTRACT

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Dry-hopping, the addition of hops to beer during or after fermentation, is a common practice in

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brewing to impart hoppy flavor to beer. Previously assumed to be inert ingredients, recent

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evidence suggests that hops contain biologically active compounds that may also extract into

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beer and complicate the brewing process by altering the final composition of beer. Experiments

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described herein provide evidence of microbial and/or plant-derived enzymes associated with

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hops (Humulus lupulus) which can impact beer quality by influencing the composition of

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fermentable and nonfermentable carbohydrates in dry-hopped beer. Fully-attenuated and

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packaged commercial lager beer was dry-hopped at a rate of 10 g hops/L beer with pelletized

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Cascade hops, dosed with 106 cells/mL of ale yeast, and incubated at 20 °C. Real extract of the

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treated beer declined significantly within several days with a reduction of 1 °P (% w/w) after 5

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days and then slowly to a total reduction of approximately 2 °P after 40 days. When fully

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fermented, this was equivalent to the production of an additional 4.75% (v/v) of CO2 and an

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additional 1.3% (v/v) of alcohol. The refermentation of beer driven by dry-hopping was

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attributed to the low but persistent activities of several starch degrading enzymes present in

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Cascade hops including amyloglucosidase, α-amylase, β-amylase and limit dextrinase. The effect

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of hop-derived enzymes on beer was time, temperature and dose-dependent. Characterizing

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bioactive enzymes in hops will help hop suppliers and brewers to address the unexpected quality

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and safety issues surrounding hopping practices in beer.

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Keywords dry hopping, hops, humulus lupulus, amylase, maltase, over attenuation,

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refermentation


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INTRODUCTION

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Hops (Humulus lupulus) are used in beer production primarily to impart characteristic bitterness

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and aroma and to lend antibacterial resistance to beer. Their use also facilitates precipitation of

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protein during brewing, aids in filtration post-boiling, enhances beer foam properties, and

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promotes foam lacing.1-2 In the case where distinctive hoppy aroma is desired, late-hopping or

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dry-hopping techniques are commonly employed to preserve the heat sensitive volatile aromatic

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compounds in the essential oil fraction of hops. Late-hop additions are performed at the end of

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wort boiling just prior to wort chilling to impart hop aroma with minimal evaporative losses. In

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contrast, dry-hopping is a cold extraction of hops in beer during the lagering process and

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sometimes during active fermentation on yeast3, and it allows a brewer to add a significant

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quantity of hops, and hence hop aroma, to beer without adding substantial bitterness that

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normally occurs during wort boiling. During fermentation, yeast may transform hop compounds

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thereby modifying hoppy beer aroma.4-5 This knowledge combined with an array of new,

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aromatic hop varieties has led to innovative and radical dry-hopping techniques to create unique

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and intensely hoppy aroma in beer. With greater and broader use of dry-hopping technology by

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the brewing industry, there have been many anecdotal accounts of unexpected changes to beer

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fermentation from dry-hopping such as over-attenuation, out of specification alcohol, and

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excessive package pressurization due to production of carbon dioxide beyond theorized yield at

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the time of packaging. Brewers use the term “attenuation” to refer to a beer’s chemical

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composition when an alcoholic fermentation has completed. The term “over-attenuation” refers

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to a beer that has fermented to a higher alcoholic strength than normally expected. This over-

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attenuation effect following dry-hopping has been colloquially referred to as “hop creep” by the

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brewing community and observed by many breweries globally. Hop creep can be defined as the



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refermentation of fully-fermented beer following the addition of hops and is observed as a slow

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reduction in residual extract and steady increase of alcohol and CO2 over time. Alcohol

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concentration that is unexpectedly high is a quality control issue, while the production of

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additional CO2 post-packaging in glass presents the consumer with a serious safety risk due to

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exploding bottles.

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Over-attenuation of dry-hopped British cask ales was first reported by Dr. Horace Brown over

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100 years ago, citing that “dry-hopping induces an earlier and more persistent cask fermentation

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in beer”.6 The small percentage of endogenous sugars in hops was reported to be insufficient for

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the extent of refermentation. Through additional experiments, the authors found that wild yeasts

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were too slow to grow and influence the fermentation dynamics observed. These observations

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led researchers to conclude that hops contain a “diastase” able to break down residual, non-

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fermentable beer dextrins as seen by the production of reducing sugars in dry-hopped beers.7 In

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another publication, researchers reported that wort treated with hops in the “fermenting vessel”

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showed markedly lower attenuation than the conventionally hopped wort suggesting that hops

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impact fermentability and beer quality.8 With the nearly complete loss of late-hopped and dry-

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hopped beer during the mid-20th century, knowledge of the potential for refermentation to occur

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following dry-hopping seems to have been largely lost or forgotten until now.

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Kirkendall and others provided an updated account of hops ability to stimulate overattenuation

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and from a relevant brewing context, proposed a method to monitor hops in the brewery using

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the production of alcohol formed during refermentation.9

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Recently,

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The study presented herein employs a systematic lab-scale dry-hopping approach to assess the

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ability of hops to alter the fermentability of finished beer in the presence of yeast. The activities

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of four starch degrading enzymes, amyloglucosidase, α-amylase, β-amylase and limit dextrinase,

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are measured and reported in Cascade pellet hops. Finally, various dry-hopping conditions such

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as exposure time, temperature and hop dosage are explored to understand the effects of starch

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degrading enzymes on beer carbohydrates.

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MATERIALS AND METHODS

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Chemicals. The following analytical grade standards were purchased from Sigma Aldrich (St.

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Louis, MO, USA): maltose monohydrate 99.0% (CAS 6363-53-7), glucose >99.5% (CAS 50-99-

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7), fructose >99% (CAS 57-48-7), maltotriose >90% (CAS 1109-28-0). For the preparation of

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enzyme and/or dry-hop extraction buffers, sodium azide 99.5% (CAS 26628-22-8), maleic acid >

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99.0% (CAS 110-16-7), and dithiothreitol molecular biology reagent (CAS 3483-12-3) were

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additionally purchased from Sigma Aldrich (St. Louis, MO, USA). Malt amylase assay kit (K-

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MALTA), Pullulanase/Limit-Dextrinase kit (K-PullG6), Amyloglucosidase assay reagent (R-

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AMGR3), and L-cysteine hydrochloride monohydrate were purchased from Megazyme (Bray,

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Ireland).

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Refermentation of commercial beer. Cascade hops from the 2014 harvest in the form of T90

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pellets were donated by John I. Haas, Yakima, WA. Hops were stored at -10°C under nitrogen

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gas in high barrier, foil packaging until needed. A commercial lager beer (Coors Original

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Banquet, Golden, CO) was used as a base beer for dry-hopping experiments because it is widely

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available, has consistent physiochemical parameters, and low concentrations of fermentable

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sugars (Table 1). To assess refermentation of dry-hopped beer, the base beer was dry-hopped in

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sterilized 1 liter glass bottles with 10 g/L hops, 106 cells/mL yeast (California Ale yeast, Wyeast



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1056, Hood River, OR), and held at 20 °C over a period of 40 days. The beer was sampled

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aseptically at various time points and real extract and alcohol were measured with an Anton Paar

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Alcolyzer. Theoretical CO2 production was calculated based on its stoichiometric relationship

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with fermentable sugar concentration.

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Enzyme activity assays. Activities of amyloglucosidase, α-amylase, β-amylase, and limit

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dextrinase were measured in hops using commercially available spectrophotometric-based kits

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with enzyme specific para-nitrophenyl blocked oligosaccharide substrates (Megazyme, Bray,

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Ireland). Crude homogenized hop pellets were extracted per assay guidelines: amylases and

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amyloglucosidase were extracted in Tris/HCl buffer (pH 8.0) with 1 mM disodium EDTA and

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0.02% (w/v) sodium azide, and limit dextrinase in sodium maleate (100 mM, pH 5.5) plus

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sodium azide, 0.02% (w/v). After extraction in pH buffered sodium azide stabilized solution

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(amylases and amyloglucosidase for 1 hour at room temperature, limit dextrinase for 5 hours at

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40°C), the hop material was removed via centrifugation and remaining extract was diluted in

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appropriate buffer solution. The extracted hop amyloglucosidase was diluted 1:10 in 100 mM

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sodium acetate buffer (pH 4.5), α-amylase 1:300 in 1 M sodium malate/sodium chloride buffer

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(pH 5.4, 40 mM CaCl2, 0.02% w/v sodium azide), β-amylase diluted 1:20 in 0.1 M MES buffer

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(pH 6.2, 2 mM EDTA, 1.0 mg/mL of BSA and 0.02% w/v sodium azide), and undiluted limit

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dextrinase extract in 100 mM sodium maleate buffer (pH 5.5 with 0.02% w/v sodium azide).

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Enzyme extracts were combined with the reactive substrate in 1.5 mL Eppendorf tubes at 40°C.

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Due to the low levels of enzyme activity, assays were carried out over 48 hours alongside a blank

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with readings taken at three time points during the assay’s incubation at 40°C. Reactions were

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terminated with alkaline tris base (pH 9.0) and velocity curves were generated from three time

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points so that assay linearity was well established (Table 2). All four enzyme assays displayed a


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linear response over time despite their low activities. For instance, exploratory work with the

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limit dextrinase assay produced a linear response over 1.5 days at 40°C. The para-nitrophenyl

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compound was detected spectrophotometrically at 400 nm and absorbance related directly to

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hydrolysis of each oligosaccharide substrate at pre-determined concentrations over a known

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amount of time (Figure 1). Units of activity were calculated per gram of hop material.

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Dry-hop treatments. 100 grams of dried hops was homogenized in a blender and stored at 4 °C

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prior to dry-hopping and/or enzyme activity assays. Dry-hop treatments were performed using

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the same base beer in the absence of yeast in 1 liter glass bottles as follows. Hops were added at

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5, 10, and 20 g/L to base beer to which sodium azide (0.02% w/v) had been added to inhibit

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microbial growth and held at either 10 or 20 °C. The production of fermentable sugars was

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monitored over time via HPLC as described below.

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Instrumental analysis. Analysis of lower molecular weight carbohydrates was performed using

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cation exchange high performance liquid chromatography following the ASBC Methods of

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Analysis, Sugars and Syrups 18.10 The method was adapted to the following conditions:

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Phenomenex Rezex RAM-Carbohydrate AG+ 8% column (Phenomenex, Torrance, CA)

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operating at 70 °C under isocratic conditions of 0.380 mL/min Milli-Q treated water for mobile

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phase. Fructose, glucose, maltose and maltotriose were quantitated based on available chemical

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standards (analytical grade, Sigma Aldrich). Fructose and maltotriose were not reported here due

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to low levels in all treatments. Physiochemical analysis of beer was performed using an Anton

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Paar Alcolyzer (Ashland, VA) to measure changes in beer real extract (% w/w), and alcohol (%

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v/v) using ASBC Method of Analysis Beer 4G.11

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RESULTS AND DISCUSSION

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Refermentation of dry-hopped beer in the presence of yeast.

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The real extract of beer, which is expressed as % w/w or °Plato (°P), represents the dissolved

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solids remaining in beer post-fermentation and is composed of carbohydrates, protein, lipids and

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minerals. Carbohydrates represent the largest part of the real extract with 75-80% consisting of

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unfermentable branched and linear dextrins, β-glucans, and a small amount of pentose sugars.12

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A “typical” wort is approximately 65-70% fermentable, which means there is a considerable

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portion of unfermentable carbohydrates remaining in beer. Nevertheless, a brewer may modify

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wort production practices, such as temperature and water to grist ratio, to produce highly

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fermentable (>80%) or poorly fermentable (

Evidence of dextrin hydrolyzing enzymes in Cascade hops

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