Accepted Manuscript

Comparison of enzymatic and precipitation treatments for gluten-

free craft beers production

Mauro Fanari, Mauro Forteschi, Manuela Sanna, Manuel Zinellu,

Maria Cristina Porcu, Luca Pretti

PII: S1466-8564(18)30405-3
DOI: doi:10.1016/j.ifset.2018.07.017
Reference: INNFOO 2041
To appear in: Innovative Food Science and Emerging Technologies
Received date: 30 March 2018
Revised date: 30 July 2018
Accepted date: 30 July 2018

Please cite this article as: Mauro Fanari, Mauro Forteschi, Manuela Sanna, Manuel
Zinellu, Maria Cristina Porcu, Luca Pretti , Comparison of enzymatic and precipitation
treatments for gluten-free craft beers production. Innfoo (2018), doi:10.1016/
j.ifset.2018.07.017

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 ACCEPTED MANUSCRIPT

Comparison of enzymatic and precipitation treatments for gluten-free craft beers production

Mauro Fanari1, Mauro Forteschi1, Manuela Sanna1, Manuel Zinellu1, Maria Cristina Porcu2, Luca

Pretti1*
1
Porto Conte Ricerche Srl, Tramariglio, Alghero (Sassari), Italy
2
Istituto di Chimica Biomolecolare (ICB), CNR, Traversa La Crucca, 07100 Li Punti, Sassari, Italy

*Corresponding author: Luca Pretti

Porto Conte Ricerche Strada Provinciale 55, Km 8,400, 07041 Tramariglio, Alghero SS

E-mail: pretti@portocontericerche.it

Fax +39-079998567 - Phone +39-079998400

Mauro Fanari: fanari@portocontericerche.it

Mauro Forteschi: forteschi@portocontericerche.it

Manuela Sanna: sanna@ portocontericerche.it

Manuel Zinellu: zinellu@portocontericerche.it

Maria Cristina Porcu: cristina.porcu@ss.icb.cnr.it

1
Highlights

 Gluten-free craft beer can be produced from traditional ingredients.

 Gluten content decreases in brewing stages.

 Beer deglutinization treatments let unaltered the sensory profile of the original recipe.

Abstract

Celiac disease and non-celiac gluten sensitivity are both related to gluten consumption. In sensitive

subjects, gluten triggers an immune-mediated enteropathy for which no therapy is yet available.

Beer must be avoided by gluten sensitive subjects, since it is generally made from gluten containing

grains such as barley and wheat. In this study we compared two different deglutinization treatments

on an American India Pale Ale, brewed on a craft beer pilot plant: an enzymatic process based on

Aspergillus niger prolyl endopeptidase and a precipitation process by silica gel. The gluten amount

in all stages of brewing, and the effects of treatments on standard quality attributes, chemical

composition and sensory profile were evaluated. Both treatments were able to reduce gluten level

below 20 ppm, showing a different behavior. Only slight differences were observed in the chemico-

physical characteristics of treated and untreated beers, whereas sensory analysis did not highlight

any change.

Industrial relevance

Considering the massive increase of microbreweries and the grown of gluten-free products market,

two different deglutinization treatment were compared in pilot plant production of craft beer.

Results show how it is possible to produce a gluten-free beer from traditional ingredients. The slight

differences observed between treatments had no impact on the sensory profile, allowing to brew

high quality gluten-free beers.

Keywords: Celiac disease; gluten-free; prolyl endopeptidase; silica gel; craft beer; deglutinization.

Abbreviations: AIPA American India Pale Ale; AN-PEP Aspergillus niger prolyl endopeptidase;

CD celiac disease; CntB control Beer; ELISA Enzyme-Linked Immuno Sorbent Assay; ETB

Enzyme-Treated Beer; FAN (Free Amino Nitrogen); GF gluten free; HPLC High Performance

Liquid Chromatography; HS-SPME-GC-MS Head Space Solid Phase Microextraction Gas

Chromatography-Mass Spectrometry; IBU International Bitterness Units. NCGS Non-celiac Gluten

Sensitivity; PTB Precipitation-Treated Beer.

1. Introduction

Gluten is a heterogeneous mixture of insoluble proteins rich in proline and glutamine, comprising

monomeric prolamins and polymeric glutenin, located in the endosperm of some cereals (Moore,

2016). Depending on the source of grain, prolamins are named gliadin, hordein, secalin and avenin,

respectively for wheat, barley, rye and oat. The peculiar primary structure of prolamins triggers, in

sensitive subjects, an immune-mediated enteropathy (Selimoğlu & Karabiber, 2010). Celiac disease

(CD) is an autoimmune syndrome of the small intestine caused by the ingestion of gluten proteins

(Shan, 2002) and non-celiac gluten sensitivity (NCGS) is an undefined syndrome with

gastrointestinal and extra-intestinal manifestations triggered by gluten in patients without celiac

disease and wheat allergy (Czaja-Bulsa, 2015). 6 % of the global population are affected by CD or

NCGS (Genetics home reference, 2015). Currently, the only treatment available for CD and NCGS

patients is a strict life-long gluten-free diet. The codex alimentarius established a gluten

concentration below 20 ppm for gluten-free (GF) food (Shan, 2002).

Beer is produced from barley or other cereals malt, so it is a potentially harmful beverage for

sensitive subjects. The gluten level is highly variable in function of beer style and technology.

During the brewing process, most of the proteins precipitate and a few are hydrolyzed into

polypeptides (Panda, Zoerb, Cho, Jackson, & Garber, 2015). Moreover, stabilization treatments,

such as filtration and the use of fining additives, affect the gluten concentration (Hager, Taylor,

Waters, & Arendt, 2014). The range of gluten content in beer may vary from less than 20 ppm in

some industrial lager beer, to more than 4000 ppm in weissbier (Fanari et al., 2017; Hager, Taylor,

Waters, & Arendt, 2014; Van Landschoot, 2011; Guerdrum, & Bamforth, 2011). Since beer is

considered an unsafe beverage for celiac patient, GF beer are gaining more attention in the market:

most are produced by big companies, whereas GF craft beers are extremely rare. This is mainly due

to the poor knowledge of the required technology among craft breweries. Moreover, because of the
lack of filtration and stabilization processes, the average gluten content in craft beer is generally

higher than in industrial beer (Fanari et al., 2017; Guerdrum & Bamforth, 2011).

To date GF beers have been produced with two different technological approaches: with no gluten

containing cereals (Mayer, Ceccaroni, Marconi, Sileoni, Perretti, & Fantozzi, 2016) or pseudo-

cereals (Deželak, Zarnkow, Becker, & Košir, 2014; De Meo, Freeman, Marconi, Booer, Perretti, &

Fantozzi, 2011) and with standard ingredients processed to reduce peptides content (Di Ghionno,

Marconi, Sileoni, De Francesco, & Perretti, 2017; Van Landschoot, 2011; Guerdrum, & Bamforth,

2012; Knorr, Wieser, & Koehler, 2016; Taylor, Jacob, & Arendt, 2015; Benítez, Acquisgrana,

Peruchena, Sosa, & Lozano, 2016).

Recent research describes two different ways to reduce gluten content of beer: enzymatic treatment

and gluten removal by fining agents (Hager, Taylor, Waters, & Arendt, 2014).

Bamforth (2012) and Knorr, Wieser, & Koehler (2016) investigated the impact of the prolyl

endopeptidase derived from Aspergillus niger (AN-PEP) on the prolamin amount in beers produced

from all-barley malts. This enzyme preparation, already used in brewing to prevent chill haze, is

able to clave the prolammine epitopes responsible for CD (Hager, Taylor, Waters, & Arendt, 2014).

Gluten removal can also be obtained by using standard fining agents such as silica gel (Taylor,

Jacob, & Arendt, 2015; Benítez, Acquisgrana, Peruchena, Sosa, & Lozano, 2016; Leiper, Stewart,

Mckeown, 2003). Silica gel is a synthetic silicon dioxide derivative which, containing a network of

pores, has a very large surface area, covered with silanol groups (Si-OH) possessing a high affinity

for proline residues. This causes the formation of large flocks which can be easily removed by

filtration or precipitation treatments. In beer, protein removal or cleavage may cause changing on

yeast fermentation performance, bitterness, foam retention and sensory profile (Boulton, Quain,

2001b). Silica gel and AN-PEP are widely employed in brewing as fining additives, the published

studies on beer deglutinization are mainly focused on laboratory scale or industrial beer, since the

decrease in gluten content is usually achieved as “side-effect” of haze-active proteins removal.

(Hager, Taylor, Waters, & Arendt, 2014).

The aim of this study was to compare the two aforementioned treatments in a pilot scale production

of an American India Pale Ale (AIPA), considered one of the most popular style of craft beers

(Steele, 2012).

The produced wort was fermented and treated with AN-PEP and silica gel; gluten content was

monitored by competitive ELISA from mash-in to bottle conditioned beers. Quality standard

attributes, iso-alpha acid composition, volatile and sensory profile was also assayed, in order to

highlight differences between control and treated beers.

2. Materials and methods

2.1. Wort production

22 kg of Pale Ale Malt (Weyermann, Bamberg, Germany) and 3 kg of Crystal malt (Thomas

Fawcett & sons, Castleford, England) were ground in a two-roll mill spaced 1 mm and added to 75

L of water with 28 g of calcium sulfate (Mr. Malt, Udine, Italy). Mash was conducted at 68 °C for

60 minutes, then heated at 78 °C and kept for 10 minutes for mash out. Wort was transferred to

kettle and spent grain was washed with water at 78 °C to reach 130 L of total wort. At the start of

the boiling step (60 min) 80 g of Magnum hop (Mr. Malt, Udine, Italy) were added for bittering. 10

minutes prior to end of boil 10 g of Wyeast Beer Nutrient Blend (Wyeast, Oregon, USA) were

added. In the whirlpool phase 300 g of Citra hop (Mr. Malt, Udine, Italy) were added for aroma. A

final volume of 120 L of AIPA wort was obtained and divided into 6 fermenters of 20 L each.

2.2. Deglutinization treatment.

The experimental design is shown in Figure 1. Each trial was conducted in duplicate.

o Precipitation treated beer (PTB): 5 mL L-1 of Biersol (Brewferm, Beverlo, Belgium) silica

gel (active compound Si-OH) was added at the beginning of fermentation. Silica gel is
 normally used to improve proteins clarification in beer wort to avoid the so called “hot-

trub”.

o Enzymatic treated beer (ETB): 40 µL L-1 of Brewers Clarex (DSM, Delft, The Netherlands)

a proline specific endo-protease derived from a selected strain of Aspergillus niger, was

added at the beginning of fermentation.

o Control beer (CtrlB): a further trial without any treatment was conducted.

2.3. Fermentation and bottle conditioning.

0.5 g L-1 of Saccharomyes cerevisiae yeast US-05 (Fermentis, Marcq-en-BaroeulCedex, France)

was added to the wort. Fermentation was conducted at 19 °C for 7 days and then wort was chilled at

4 °C for 14 days. Matured beer was added with 6 g L -1 of glucose and 0.05 g L -1 of yeast F2

(Fermentis, Marcq-en-BaroeulCedex, France) and bottle conditioned at 22 °C for 14 days to obtain

conditioned beer. Both the yeasts were used accordingly with the manufacturer suggestions.

As provided by Italian law for Craft Beer (Legge 28 luglio 2016, n. 154, 2016) no stabilizing

treatments such as filtration or pasteurization were performed (Figure 1).

2.4. Analysis

2.4.1. Gluten quantification.

RIDASCREEN Gliadin competitive assay (Art. No. R7021, R-Biopharm, Darmstadt, Germany)

was used according to the manufacturer's instruction. Bottles were shaken for 3 minutes in order

to suspend the pellet and 100 mL of beer were poured on a beaker and degassed. Gliadin

concentrations were converted into gluten concentrations by multiplying the test results by a

factor of 2 as provided by Codex Alimentarius Commission (Commission, C. A., 2008). A

commercially available gluten-free beer (Peroni gluten-free, Peroni, Roma, Italia) was used as

negative control. All samples were analyzed in triplicate.

 2.4.2. Standard quality attributes.

Original extract (% w/w), real extract (% w/w), apparent extract (% w/w), ethanol (% v/v), apparent

attenuation (%) and real attenuation (%), were measured with a PBA-B generation M (Anton Paar,

Graz, Austria). The following analyses were performed according to the official Analytica-EBC

methods (European Brewery Convention., 1998): free amino nitrogen (mg L -1), by EBC method

8.10; color (EBC-U), by EBC method 9.6; pH, by EBC method 9.35; turbidity (EBC-U), by EBC

method 9.29. Chill haze (EBC-U) was expressed as difference between turbidity measured at 4 °C

and at 20 °C. Foam stability was measured with a NIBEM-TPH foam stability tester (Haffmans,

Zeist, The Netherlands) according to the Analytica-EBC method 9.42.1. Iso-alpha acid (iso-

humulone, iso-cohumulone and iso-adhumulone) (mg L-1), were extracted from beer accordingly

with the EBC method 9.47 as follows: degassed beer (100 mL) was acidified with HCl (6 M; 5 mL)

then partitioned with iso-octane (250 mL; Merck, Darmstadt, Germany) with vigorous shaking.

After separation, the organic Iso-octane layer (125 mL) was collected and the solvent removed

under vacuum at 30°C. The residue was resuspended in 5% methanol water solution (1 mL; HPLC-

grade, Merck). The extract was filtered through a 0.22µm disposable filter assembly (Merck-

Millipore, Darmstadt, Germany) prior to HPLC analysis. Minor modifications were set for

chromatographic separation, 5 µL of sample were injected in an Agilent 1200 series HPLC system

(Palo Alto CA USA) equipped with an analytical 75 X 4,6 mm, 2,6 µm Kinetex C18 column

(Phenomenex, Torrance USA) at 30 °C. Mobile phase was A water/phosphoric acid 0.1 % v/v with

0.1 Mol EDTA (Carlo Erba, Cornaredo, Mi, Italy) and B acetonitrile 100 %, isocratic elution was

performed at 52 % B; for 15minutes and 2 ml/min flow rate (De Cooman, Aerts, Overmiere, & De

Kukeleire 2000). Samples were UV detected at λ 270 nm and compared with ICS I4 (Labor Veritas,

Zürich, Switzerland) standard for quantification. All analyses were performed in triplicate and

results are expressed as mean of three independent experiments.

2.4.3. Semi-quantitative determination of volatile compounds.

Conditioned beer were kept in a cool room at 4 °C overnight in order to preserve the volatile

compounds and 10 mL were transferred in 20 mL headspace vials containing 2 g of sodium

chloride and sealed with PTFE–silicone septa. Analysis of volatile compounds was carried out

using the HS/SPME/GC/MS technique by means of

Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB-CAR-PDMS) fiber (Supelco,

Bellefonte, PA, USA) (Riu-Aumatell, Miró, Serra-Cayuela, Buxaderas, & López-Tamames,

2014). For SPME analysis the samples were incubated for 10 minutes at 40 °C, then extraction

was carried out exposing the fiber to the headspace for 30 minutes. Both incubation and

extraction were performed in agitation. Fiber desorption was done in the injector for 7 minutes

at 250 °C with a split flow of 10 mL min -1. The fiber was activated each day following the

manufacturer instruction. The chromatographic analysis was performed using TRACE GC

coupled with an ISQ single quadrupole (Thermo Scientific, Hudson, USA). The analytes were

separated on a VF-5 ms capillary column (60 m x 0.25 mm x 0.25 μm film thickness) (Supelco,

Bellefonte, PA, USA) using helium as carrier gas at 1 mL min -1 constant flow rate. Oven

temperature program started at 35 °C held for 7 min, and then increased at 8 °C min -1 to 200 °C

and held for 7 min (Charry-Parra, DeJesus-Echevarria, & Perez, 2011). Transfer line and ion

source were both set at 260 °C, quadrupole scan range was 30-250 amu and ionization energy

was 70 eV (Aberl, & Coelhan, 2012). Chromatographic data were acquired by means of

Tracefinder (Thermo Scientific), and identification was carried out by comparison between the

mass spectra with those of the data system library (NIST, 2005 software, Mass Spectral Search

Program V.2.0d, Washington, DC, USA version 2.2 Jun 2014) (Ligor, Stankevičius, Wenda-

Piesik, Obelevičius, Ragažinskienė & Stanius, 2014). All Identity Spectrum Match factor above

850 resulting from the NIST Identity Spectrum Search algorithm (NIST MS Search 2.0) were

considered acceptable for positive identification. All samples were analyzed in triplicate.

Each analysis was undertaken in duplicate using different vials.

 2.4.4. Sensory analysis.

Conditioned beers were analyzed by a triangle test (UNI EN ISO 4120/2004). The test was

performed with 25 judges (13 females and 12 males) aged between 30 and 50 years, belonging

to the staff of the Porto Conte Ricerche Srl. The samples were served at 12 °C in glass cups

coded with three-digit numbers. Panellists were asked to rinse their mouth with water between

samples.

2.4.5. Statistical analysis.

Statistical analysis was performed by GraphPad Prism 5.02 for Windows software. Data were

presented as the mean of measured values and analyzed using the one-way Analysis of Variance

(ANOVA) and Bonferroni multiple-comparison post-test.

3. Result and discussion

3.1. Gluten content variation during brewing process

Gluten content trough different steps of production is shown in Figure 2A. As already reported by

Guerdrum, & Bamforth (2012), gluten content decreases during wort production. In our experiment

the higher gluten reduction was observed during the mash phase (Figure 2A), with a reduction of 84

%, probably due to the barley proteases activity (Steiner, Gastl & Becker, 2011). The filter effect

operated by spent grain on the bottom of lauter tun, caused an additional gluten reduction of about

40 % from the mash out (8189 ± 757 ppm) to the first wort (4782 ± 263 ppm). The dilution by

sparging water brought the gluten concentration to 2586 ± 78 ppm. During the boiling step, protein

coagulation phenomena at high temperatures led to the final gluten concentration observed in wort

(1082 ± 27 ppm). Whirlpool had no effect on gluten reduction. Standard quality attributes of the

wort produced are reported in Table 1. Parameters are within the typical range for the style (BJCP

style guidelines, 2015) and gluten content resulted in 1082 ± 27 ppm.

Yeast inoculum and deglutinization treatment were done simultaneously to take advantage of wort

mixing by CO2 bubbles developed from yeast fermentation. As shown in Figure 2B both treatments

were able to significantly reduce gluten concentration during the seven days of fermentation at 20

°C. Gluten amount in CntB was 120 ± 13 ppm, while 21 ± 5 ppm and 17 ± 4 ppm were recorded

respectively in ETB and PTB. Further reduction was observed in matured beer, with a content of 50

± 2 ppm (CntB), 2 ± 1 ppm (ETB) and 7 ± 1 ppm (PTB) with a relative reduction compared to the

green beer of 58 %, 92 % and 63 % respectively for CntB, ETB and PTB. This is likely due to the

effect of cold temperature that promotes yeast flocculation and precipitation of haze-causing

molecules such as proteins and peptides (Boulton & Quain, 2001a). Bottle conditioned beer showed

a slight increase in gluten amount for CntB and PTB, resulting in a final concentration of 62 ± 3

ppm and 10 ± 2 ppm respectively, while ETB remained unchanged.

Both treated conditioned beers showed a gluten level below 20 ppm, enough to be labeled as gluten-

free beer.

3.2. Standard quality parameters of beers

The standard quality parameters of green, cold matured and conditioned beer are listed in Table 2.

In conditioned beers, apparent and real extract in ETB were significantly lower than in CnTB and

PTB. Apparent and real attenuation reflect this pattern, with ETB significantly higher than PTB and

CntB. As a result, also ethanol content of ETB resulted higher. Hydrolytic activity of added

proteases releases a supplementary source of nitrogen as free amino acids and peptides in the wort

(Lei, Zhao, & Zhao, 2013; Lei, Zheng, Wang, Zhao, & Zhao, 2013) which could increase the

fermentation performance (D’Amore, 1992; Lei, Zhao, Yu & Zhao, 2012).

Analysis of pH did not show significant difference among treatment.

Turbidity significantly decreased from green beer to the two subsequent phases. Different behavior

among treatments was only observed in green beer, where PTB turbidity showed was significantly

lower.
Since beer is usually consumed at cold temperature, chill-haze analysis was performed. Interactions

between proline-rich small peptides and polyphenols at cold temperatures result in turbidity

increase (Asano, Shinagawa, & Hashimoto, 1982). In conditioned beers, higher chill-haze values

were found in CntB, while ETB did not result statistically different from PTB. This is not

surprising, since proline rich small peptides are responsible for this phenomenon resulted lower in

treated beers (Steiner, Becker, Gastl, 2010).

No statistically significant differences were found on color between experimental conditions but a

slight increase for all samples in conditioned beers was observed. This phenomenon is likely due to

oxygen uptake during the bottling phase (Bamforth, 2009a).

Foam stability is one of the parameters that could be highly influenced by protein change

(Bamforth, 2009b) but, as reported by Leiper, Stewart, & Mckeown, (2003), proteins involved in

foam formation contain few proline residues and thus foam stability should not be affected by the

enzymatic treatment. On the other hand, Guerdrum, & Bamforth (2012) demonstrated that the

cleavage of foam active proteins by AN-PEP decreases the foam retention and Fanari et al. (2017)

showed a positive correlation between foam retention and gluten content in craft beers.

Furthermore, as reported by Evans, Finn, Robinson, Eglinton, Sheehy, & Stewart, (2011), the

addition of AN-PEP may cause either an increase or a decrease in the foam stability depending on

barley varieties utilized. In this work no significant differences were observed between CntB e PTB

on foam stability but in ETB it resulted significantly lower.

FAN (Free Amino Nitrogen) content of the wort, a measure of the sum of amino acids, ammonium

ions and small peptides, can affect yeast performance and consequently beer flavour (He et al.,

2014). Literature reports an increase of FAN levels by protease addiction in wort (Lei, Zheng,

Wang, Zhao & Zhao 2013) and a reduction of nitrogen content of beer by use of silica gel (Leiper,

Stewart & Mckeown, 2003). In this work, the analysis of FAN showed a high level in wort (439 ±

14), likely as a result of yeast nutrient addition in the boiling step, and no significant differences

were found among experimental conditions in different phases of production -(Table 2).
During fermentation, iso-alpha acids concentration drops down because of pH reduction and

binding to the yeast cell wall (Boulton, Quain, 2001b). Iso-alpha acids analysis was conducted to

assess possible interaction between deglutinization treatments and amount and composition of iso-

alpha acids in conditioned beers. Analysis of International Bitterness Units (IBU) did not show

significant differences between samples (Tab. 2). Total amount of iso-alpha acids did not show

significant differences between samples, while iso-humulone amount in the ETB was significantly

higher (Figure 3).

3.3. Volatile compounds

The relative abundance of the most important volatile aroma-active compounds, in bottle

conditioned beers, were assayed by HS-SPME-GC-MS analysis (Table 3).

The AIPA style focuses mainly on terpene hop flavours which were significantly higher in CnTB.

Linalool and citronellol concentrations, responsible for citric aroma, were similar, while significant

differences were found on methyl geranate and myrcene amount.

Esters from fermentation were lower in PTB samples, particularly, ethyl caprate and ethyl laurate.

This results appear in contrast with Taylor, Jacob & Arendt (2015).

No significant differences were found in the amount of alcohols between the three experimental

conditions.

3.4. Sensory analysis

Given the differences observed on quality standard attributes and volatile profile, the existence of

perceptible sensorial variation between treated and untreated conditioned beers was evaluated by

triangle test. It is a discriminative method employed in sensory analysis to determine whether shifts

in processing or ingredients have significantly changed a product. It is worthwhile to note that

according to the statistical table (as reported in the UNI ISO 4120 norm) for 25 assessors, the
number of correct responses should be at least 13, so that the panel is able to detect a difference

between two samples. In the present experiment only 10 judges have identified the sample

"different" within CntB and PTB as well as ETB and PTB; and only 7 judges were able to identified

the sample “different” between CntB and ETB. The number of correct answers is then insufficient

to support the conclusion that there is a statistically significant difference between the thesis (α:

0.05)

4. Conclusion

In the last decades craft beer market raised significantly both in terms of interest and variety of

products, but GF craft beers are still extremely rare (Hager, Taylor, Waters, & Arendt, 2014).

As our data clearly show, during brewing phases gluten content becomes lower from mash-in to

conditioned beer, but not below 20 ppm. Starting from a standard recipe and without using specific

equipment or expertise, both studied treatments were able to reduce gluten concentration below this

limit. Differences on yeast performance were observed in both treatments, as an increase of

attenuation on ETB and lower amount of esters on PTB. Hydrolytic activity of protease releases a

supplementary source of nitrogen as free amino acids in the wort (Lei, Zhao, & Zhao, 2013; Lei,

Zheng, Wang, Zhao, & Zhao, 2013) and this nitrogen increment could increase fermentation

performance. The opposite effect could be generated by the reduced availability of protein and

amino acid precipitated by silica gel (Leiper, Stewart, Mckeown, 2003) in PTB. Deeper

investigation is needed to better understand how proteins treatments in wort affect fermentation and

shelf life of beers.

Despite some slight differences on chemico-physical analysis, sensory evaluation did not highlight

significant differences between experimental conditions. Therefore, we demonstrate that is possible

to achieve a craft GF beer keeping unaltered the sensory profile of the original recipe.
Acknowledgements

This research did not receive any specific grant from funding agencies in the public, commercial, or

not-for-profit sectors.

Declarations of interest: none

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Table 1

Parameters Wort

Gluten (ppm) 1082 ± 27

Extract (% w/w) 15.22 ± 0.10

pH at 20°C 5.24 ± 0.08

Turbidity (EBC-U) > 250

Colour (EBC-U) 17.8 ± 0.5

Free Amino Nitrogen (mg L-1) 439 ± 14

Standard quality parameters (average ± standard deviation n=3) of the AIPA wort used in the current study
 Table 2. Standard quality parameters (average ± standard deviation n=3) of beers in different phases of

production

Parameters Green beer Cold-matured beer Conditioned beer

CntB ETB PTB CntB ETB PTB CntB ETB PTB

Apparent extract (% w/w) 3.81 ± 0.01c 3.71 ± 0.02bc 3.82 ± 0.04c 3.80 ± 0.01c 3.72 ± 0.02bc 3.79 ± 0.08c 3.63 ± 0.01b 3.51 ± 0.01a 3.67 ± 0.02b

Real extract (% w/w) 5.97 ± 0.04c 5.90 ± 0.01bc 5.99 ± 0.01c 5.89 ± 0.05bc 5.79 ± 0.01a5.97 ± 0.03c 5.89 ± 0.01b 5.79 ± 0.01a5.88 ± 0.01b

Apparent attenuation (%)74.9 ± 0.09a 75.6 ± 0.14ab 74.9 ± 0.28a 75.0 ± 0.05a75.5 ± 0.14ab 75.1 ± 0.56a 76.1 ± 0.09b 76.9 ± 0.05c75.9 ± 0.14b

Real attenuation (%) 60.7 ± 0.23a 61.6 ± 0.05ab 60.6 ± 0.09a 61.2 ±0.33 ab 61.9 ± 0.09c60.7 ± 0.19ab 61.3 ± 0.05b 61.9 ± 0.09c61.3 ± 0.01b

Ethanol (% v/v) 6.01 ± 0.01ab 6.07 ± 0.04ab 5.97 ± 0.01a 6.03 ± 0.03ab 6.09 ± 0.02b 6.06 ± 0.02ab 6.36 ± 0.01d 6.48 ± 0.01e6.28 ± 0.05c

pH at 20°C 4.22 ± 0.03ab 4.17 ± 0.06a 4.20 ± 0.01a 4.35 ± 0.05b 4.26 ± 0.05ab 4.26 ± 0.01ab 4.32 ± 0.01b 4.29 ± 0.02ab 4.34 ± 0.04ab

Turbidity (EBC units) 19.1 ± 0.57b30.9 ± 5.09c 14.5 ± 1.41b4.8 ± 0.99a5.0 ± 0.50a3.0 ± 0.08a4.5 ± 0.07a3.8 ± 0.16a3.9 ± 0.12a

Chill haze (EBC units)18.6 ± 0.64e11.1 ± 4.74e8.5 ± 0.28c12.6 ± 0.57b1.8 ± 0.08a5.0 ± 0.34a8.5 ± 1.68b2.0 ± 0.05a3.6 ± 0.47a

Colour (EBC units) 14.8 ± 0.39a14.5 ± 0.38a 14.6 ± 077a 14.8 ± 0.46a14.9 ± 0.18a14.7 ± 0.21a 17.7 ± 0.42b 18.0 ± 0.35b18.0 ± 0.49b

Foam stability (sec/3 cm) n.d. n.d. n.d. n.d. n.d. n.d. 334 ± 7.78a 266 ± 27.58b337 ± 1.41a

IBU (mg L-1) n.d. n.d. n.d. n.d. n.d. n.d. 62,4 ± 0.40a 66.7 ± 0.18a 64.1 ± 8.74a

FAN (mg L-1) 146 ± 14.85ab 162 ± 9.10b 148 ± 0.88ab 145 ± 8.20ab 153 ± 2.02b 151 ± 4.92b 126 ± 6.64 a 136 ± 3.51ab 116 ± 7.78a

Values in the same line with different letters are statistically different (P <0.05). n.d. = non determined. IBU, international

bitterness unit. FAN, Free Amino Nitrogen; CntB, Control Beer; ETB, Enzyme-Treated Beer; PTB, Precipitation-Treated

Beer; EBC, European Brewery Convention.

Table 3. Headspace SPME GC-MS analysis of the bottle conditioned beers

Chemical

classes Compound CntB ETB PTB

Alcohols Ethanol 14.74 ± 1.44a 13.29 ± 2.17b 13.64 ± 1.58b

2-Phenylethanol 4.46 ± 0.22a 5.15 ± 0.65b 5.35 ± 1.10b

3-Methyl-1-butanol 2.63 ± 0.13a 2.98 ± 0.50b 3.02 ± 0.56b

2-Methyl-1-butanol 0.61 ± 0.05a 0.75 ± 0.13b 0.74 ± 0.15b

Total alcohols 17.98 ± 1.44 a 17.02 ± 2.17a 17.39 ± 1.58a

Esters Ethyl caprylate 37.40 ± 1.34a 38.93 ± 1.56b 38.41 ± 1.58b

a
Ethyl caprate 15.86 ± 1.51 13.11 ± 1.51b 10.95 ± 1.24c

Ethyl caproate 5.55 ± 0.41a 5.95 ± 0.77a 5.95 ± 0.69a

Isoamyl acetate 3.18 ± 0.37a 3.29 ± 0.66a 2.97 ± 0.77a

Ethyl laurate 1.36 ± 0.28a 1.40 ± 0.26a 0.71 ± 0.23b

Ethyl acetate 1.03 ± 0.08a 1.21 ± 0.19b 1.07 ± 0.35a 2-

phenylethyl acetate 0.92 ± 0.13a 1.21 ± 0.08b 1.10 ± 0.14b Ethyl

nonanoate 0.74 ± 0.15a 0.88 ± 0.18b 0.64 ± 0.08c

Total esters 66.03 ± 0.68a 65.98 ± 0.82a 61.80 ± 0.81b

a
Hop compounds Linalool 2.22 ± 0.17 2.33 ± 0.26a 2.38 ± 0.13a

Methyl geranate 1.07 ± 0.09a 0.52 ± 0.09b 0.62 ± 0.14c

Myrcene 0.98 ± 0.20a 0.28 ± 0.10b 0.25 ± 0.09b

Citronellol 0.58 ± 0.05a 0.62 ± 0.62a 0.61 ± 0.07a

Total hop compounds 4.85 ± 0.14a 3.75 ± 0.82b 3.86 ± 0.81b

Values in the same line with different letters are statistically different (P <0.05). Value are expressed as % of the total

head space (average ± standard deviation n=3).

Figure 1. Overview of experimental design.

Figure 2. Gluten content during wort production (A) and after deglutinization treatment (B).

Figure 3. Composition of iso-alpha acid in conditioned beers.

Figure 1
Figure 2
Figure 3