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LC-MS Analysis Reveals Hydrolysed Gluten in Beers Crafted to Remove Gluten. Michelle L Colgrave, Keren Byrne, and Crispin A. Howitt J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03742 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 22, 2017

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Journal of Agricultural and Food Chemistry

LC-MS Analysis Reveals Hydrolysed Gluten in Beers Crafted to Remove Gluten. Michelle L. Colgrave1*, Keren Byrne1 and Crispin A. Howitt2 1. CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia QLD 4067, Australia; 2. CSIRO Agriculture and Food, GPO Box 1700, Canberra ACT 2601, Australia *Corresponding author: Michelle L. Colgrave; phone: +61 (0)7 3214 2697; fax: +61 (0)7 3214 2900; email: [email protected]

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Abstract

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During brewing gluten proteins may be solubilised, modified, complexed, hydrolysed and/or

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precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with

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coeliac disease (CD) or gluten-intolerance. Barley-based beers crafted to remove gluten using

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proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade

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the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented

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products remains controversial. The industry standard, a competitive ELISA, may indicate gluten

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values 30 kDa in size. Barley gluten

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(hordeins) were detected in all beers analysed with peptides representing all hordein classes detected

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in conventional beers, but also alarmingly in many gluten-reduced beers. It is evident that PEP

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digestion was incomplete in several commercial beers and peptides comprising missed cleavages were

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identified warranting further optimisation of PEP application in an industrial setting.

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Keywords: gluten; prolyl endopeptidase (PEP); beer; liquid chromatography mass spectrometry (LC-

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MS)

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Journal of Agricultural and Food Chemistry

Introduction

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Coeliac disease (CD) is an inflammatory disorder of the small intestine affecting 1% of

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people in Western populations.1 After exposure to gluten via ingestion, an inappropriate immune

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response results in destruction of the microvilli within the intestine. This leads to conditions

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commonly involving malabsorption of nutrients (anaemia, osteoporosis), gastrointestinal complaints

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(diarrhoea, bloating) and skin conditions (dermatitis), through to endocrine, neurological and

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reproductive disorders.2 The only treatment for people with CD is a strict gluten-free (GF) diet.

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Brewing is considered the oldest biotechnological process known to mankind. Beer represents

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the third most popular beverage after water and tea. The sugars released from malted barley serve as

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the primary nutrient source for yeast during fermentation when they are converted into alcohol.

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Proteins, predominantly from barley and to a lesser extent yeast, that persist in beer have important

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contributions towards end product quality, including haze formation, foam retention, foam stability

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and flavour. The dominant proteins identified include the serpins, lipid transfer proteins (LTPs), α-

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amylase/trypsin inhibitors and storage proteins including gluten.

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the name for the storage proteins found in barley (hordein), wheat (gliadin/glutenin) and rye (secalin).

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Strict gluten avoidance in CD precludes the consumption of beers made from barley, wheat and/or

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rye. There are a number of beers made from non-gluten-containing cereals (corn, rice, sorghum,

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millet) or pseudocereals (buckwheat), however, these products often lack the distinctive flavour and

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aroma imparted by malted barley.

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3-7

In the context of beer, gluten is

It is well established that proteins undergo a number of modifications and hydrolysis during 8-9

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the brewing process, especially during malting and mashing.

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content is removed from wort during boiling and during wort cooling.

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by >30% during malting (up to 65% for the C-hordeins)

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controlled study, gluten content was shown to decrease by 46-79% from first wort to beer.

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modification of gluten during brewing has been comprehensively reviewed by Kerpes et al.

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Extensive hydrolysis, however, does not abolish the epitopes that are known to trigger CD and several

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studies have reported on Coeliac responses to commercial beers. 5, 16-18

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A large proportion of the protein 10-11

The hordeins are reduced

and further during brewing.

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In a The 15

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A range of brewing aids that are used to stabilise beers, through disrupting the polyphenol-

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protein interactions that lead to haze formation, have also seen application in gluten removal. These

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include the use of polyvinylpolypyrrolidone (PVPP) and silica gel or condensed tannins

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(proanthocyanidins) and their use has led to a reduction in the gluten content of treated beers.

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The enzyme transglutaminase (TG) has been employed as a means of detoxifying food and beverages.

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20-21

10-11, 19

For example, microbial TG creates cross-links between gluten proteins/peptides that ultimately

results in the precipitation of these proteins allowing their removal by filtration. 22 Researchers have applied endogenous peptidases from germinated wheat, rye and barley

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23-24

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demonstrating cleavage of coeliac-active epitopes.

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practice to generate gluten-reduced or gluten-free barley-based beers through the addition of enzymes

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during the brewing process, commonly added at the start of fermentation. A commercial preparation

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of a prolyl endopeptidase (PEP, also known as prolyl oligoprotease) from Aspergillus niger (referred

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to as AN-PEP) was first used to debitter protein hydrolysates 25 and subsequently to decrease haze by

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hydrolysing haze-sensitive proteins. PEPs including AN-PEP cleave proteins at proline (Pro, P)

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residues

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gluten proteins.

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al. 28

63

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In recent years, it has become common

and are able to degrade gluten owing to the high frequency of Pro (10-30%) found in 26-27

The enzymatic detoxification of gluten has been recently reviewed by Wieser et

In a series of studies employing the R5 competitive ELISA,

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a range of commercial

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beers was analysed, including those produced from non-gluten-containing grains and from barley with

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and without PEP treatment. Of these, the beers employing PEP treatment were shown to yield a gluten

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content below the CODEX threshold of 20 mg/kg. A further study examining the action of AN-PEP

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on the degradation of gluten peptides

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follow the fate of the gluten peptides qualitatively. Analysis of untreated and AN-PEP-treated beers

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revealed that immunotoxic epitopes were present in the untreated beers, but not in the AN-PEP-

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treated beers. Another study examined the effectiveness of AN-PEP by both ELISA and LC-MS in a

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sorghum beer incurred with wheat gluten.

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content was demonstrated over the first three days of fermentation, whereas AN-PEP-treated beers

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employed ELISA for gluten quantitation and LC-MS to

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In control beers, a gradual reduction (4-fold) in gluten

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showed a marked decrease (>15-fold) in gluten content from 3-14 days. Using Western blotting, the

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HMW-glutenins were shown to be less susceptible to AN-PEP than the LMW-glutenins. From these

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studies and similar applications in wheat, bran and foodstuffs,

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degrade gluten, however it is unclear if all potential immunopathogenic sequences are completely

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eliminated. Moreover, the safety of gluten-reduced beers is still contentious in part owing to questions

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regarding the accuracy of testing fermented and hydrolyzed foods and the ability to equate hydrolyzed

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gluten content to an equivalent amount of intact gluten. This latter issue is addressed by the USA

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FDA proposed rule (FDA-2014-N-1021).

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reduced beers found that serum from active-CD patients bound to residual gluten peptides in

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conventional beers and that a subset of the patient sera also reacted to gluten-removed beers. 18

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33-34

it is apparent that PEP is able to

A recent study on the antibody response to gluten-

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In the current study LC-MS analysis was applied to a selection of gluten-reduced and gluten-

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free commercial barley-based beers to determine the effect of gluten reduction treatments on the

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protein and peptide profiles.

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Materials and Methods

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Reagents and test samples. Chemicals, including formic acid (FA), ammonium bicarbonate,

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dithiothreitol, iodoacetamide, were purchased from Sigma-Aldrich (Sydney, NSW, Australia).

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Acetonitrile were purchased from ChemSupply (Gillman, SA, Australia). Enzymes used for digestion

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(trypsin and chymotrypsin) were purchased from Promega (Sydney, NSW, Australia). A selection of

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beers were purchased internationally from commercial liquor stores based on their ingredients and

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gluten status according to their packaging and/or company website. All beers selected were barley-

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malt based products rather than gluten-free beers based on non-gluten containing grains such as rice,

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sorghum, millet or tef. A number of regular beers that had previously 6 been shown to contain gluten

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were selected as positive controls, C1-C4. The gluten-reduced (or low gluten, LG) beers, LG1-LG7

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and LG9-LG11 are PEP-treated. LG8 is manufactured by an undisclosed proprietary process. LG12 is

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brewed with a novel ultra-low gluten barley. 36

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Digestion of whole beers. Whole beers (n=4 technical replicates) were subjected to enzymatic digest

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using either trypsin or chymotrypsin. Aliquots of degassed beer (50 µL) were diluted 1:1 in 50 mM

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ammonium bicarbonate containing 1 mM CaCl2, pH 8.5 (50 µL). To these solutions, 10 µL of 50 mM

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dithiothreitol was added and the samples were incubated at 60°C for 30 min. Subsequently, the

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samples were cooled and 10 µL of 100 mM iodoacetamide was added and the samples were incubated

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at RT for 20 min in the dark. To these solutions, 10 µL of either trypsin or chymotrypsin (1 µg/µL)

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was added with incubation at 37°C for 16 hours. To quench the digestion, 50 µL of 1% formic acid

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was added and the samples were stored at -20°C until analysis. A 95% confidence in the suite of

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beers tested. As depicted in Figure 2, the control beers show the highest number of unique gluten-

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derived peptides (range: 54-86 for trypsin; 59-121 for chymotrypsin) with the low gluten beers LG11

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and LG12 revealing the least. The spectral count (total number of gluten peptide spectra acquired)

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followed the same trend as the number of unique peptides, but also reflected the abundance of these

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peptides since the more abundant a peptide is in a sample, the greater the spectral redundancy.

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Qualitatively, from the low gluten beers tested, LG7 and LG8 were noted to contain the greatest

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diversity and abundance of gluten-derived peptides, both with approximately twice the number and

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spectral count compared to the average of all low gluten beers. LG3-LG6 also revealed values above

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the average.

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The undigested filtrates (90% of the

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gluten peptides detected in C1-C4 contained P-X sites within their sequences. Examining LG1-LG7

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and LG9-LG11, the majority of peptide fragments resulted from cleavage at P-X (55-73%) and to a

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lesser extent X-P (2-9%) indicating the use of PEP in the production of these beers. Reviewing the

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identified sequences within the PEP treated beer revealed that 60-94% of the gluten peptides

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contained additional PEP cleavage sites. In fact, only LG11 contained more completely digested

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peptides than partially digested (42.1% missed cleavages). The other notable fact was that untreated 10 ACS Paragon Plus Environment

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beers contained a higher number of P-X sites within an individual peptide (up to 5 missed cleavages)

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compared to PEP treated beers where peptides typically only contained 1-2 missed cleavage sites. The

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exception

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QQAELIIP↓QQP↓QQP↓FP↓LQP↓HQP. Notably, this peptide also contained the QQPFP epitope

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recognised by the Mendez R5 antibody. For LG8, PEP activity was not apparent with only 7% of

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gluten-derived fragments cleaved at P-X or X-P which is within the same range as the control beers.

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LG8 also yielded a high proportion of gluten peptide identifications containing P-X sites (82.8%). As

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with all the beers in this study, LG8 was brewed using barley, but the gluten is claimed to be removed

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by a proprietary process. Alongside a handful of B- and γ-hordeins, D-hordein was identified

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confidently in LG8 by 11 peptides that all clustered in the C-terminal region of the protein suggesting

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that a C-terminal fragment persists after brewing. In LG12, which is a gluten-free beer brewed using a

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novel gluten-free barley,

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resulting from non-specific cleavage (hydrolysis during brewing). This was expected since only γ3-

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hordein is detectable in grain of this novel barley

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identified in the trypsin (8 peptides) and chymotrypsin (1 peptide) digests of whole beer (Table 1).

was

LG7

which

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also

contained

a

peptide

with

5

missed

cleavages:

only γ3-hordein (I6TEV2) was detected by seven peptide fragments

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and only peptides derived from I6TEV2 were

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We also examined the fragments identified in the undigested filtrate (