Subscriber access provided by LAURENTIAN UNIV
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 33
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] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 33
1
Abstract
2
During brewing gluten proteins may be solubilised, modified, complexed, hydrolysed and/or
3
precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with
4
coeliac disease (CD) or gluten-intolerance. Barley-based beers crafted to remove gluten using
5
proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade
6
the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented
7
products remains controversial. The industry standard, a competitive ELISA, may indicate gluten
8
values 30 kDa in size. Barley gluten
10
(hordeins) were detected in all beers analysed with peptides representing all hordein classes detected
11
in conventional beers, but also alarmingly in many gluten-reduced beers. It is evident that PEP
12
digestion was incomplete in several commercial beers and peptides comprising missed cleavages were
13
identified warranting further optimisation of PEP application in an industrial setting.
14 15
Keywords: gluten; prolyl endopeptidase (PEP); beer; liquid chromatography mass spectrometry (LC-
16
MS)
17 18
2 ACS Paragon Plus Environment
Page 3 of 33
19
Journal of Agricultural and Food Chemistry
Introduction
20
Coeliac disease (CD) is an inflammatory disorder of the small intestine affecting 1% of
21
people in Western populations.1 After exposure to gluten via ingestion, an inappropriate immune
22
response results in destruction of the microvilli within the intestine. This leads to conditions
23
commonly involving malabsorption of nutrients (anaemia, osteoporosis), gastrointestinal complaints
24
(diarrhoea, bloating) and skin conditions (dermatitis), through to endocrine, neurological and
25
reproductive disorders.2 The only treatment for people with CD is a strict gluten-free (GF) diet.
26
Brewing is considered the oldest biotechnological process known to mankind. Beer represents
27
the third most popular beverage after water and tea. The sugars released from malted barley serve as
28
the primary nutrient source for yeast during fermentation when they are converted into alcohol.
29
Proteins, predominantly from barley and to a lesser extent yeast, that persist in beer have important
30
contributions towards end product quality, including haze formation, foam retention, foam stability
31
and flavour. The dominant proteins identified include the serpins, lipid transfer proteins (LTPs), α-
32
amylase/trypsin inhibitors and storage proteins including gluten.
33
the name for the storage proteins found in barley (hordein), wheat (gliadin/glutenin) and rye (secalin).
34
Strict gluten avoidance in CD precludes the consumption of beers made from barley, wheat and/or
35
rye. There are a number of beers made from non-gluten-containing cereals (corn, rice, sorghum,
36
millet) or pseudocereals (buckwheat), however, these products often lack the distinctive flavour and
37
aroma imparted by malted barley.
38
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
39
the brewing process, especially during malting and mashing.
40
content is removed from wort during boiling and during wort cooling.
41
by >30% during malting (up to 65% for the C-hordeins)
42
controlled study, gluten content was shown to decrease by 46-79% from first wort to beer.
43
modification of gluten during brewing has been comprehensively reviewed by Kerpes et al.
44
Extensive hydrolysis, however, does not abolish the epitopes that are known to trigger CD and several
45
studies have reported on Coeliac responses to commercial beers. 5, 16-18
12
A large proportion of the protein 10-11
The hordeins are reduced
and further during brewing.
13
14
In a The 15
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 33
46
A range of brewing aids that are used to stabilise beers, through disrupting the polyphenol-
47
protein interactions that lead to haze formation, have also seen application in gluten removal. These
48
include the use of polyvinylpolypyrrolidone (PVPP) and silica gel or condensed tannins
49
(proanthocyanidins) and their use has led to a reduction in the gluten content of treated beers.
50
The enzyme transglutaminase (TG) has been employed as a means of detoxifying food and beverages.
51 52
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
53
23-24
54
demonstrating cleavage of coeliac-active epitopes.
55
practice to generate gluten-reduced or gluten-free barley-based beers through the addition of enzymes
56
during the brewing process, commonly added at the start of fermentation. A commercial preparation
57
of a prolyl endopeptidase (PEP, also known as prolyl oligoprotease) from Aspergillus niger (referred
58
to as AN-PEP) was first used to debitter protein hydrolysates 25 and subsequently to decrease haze by
59
hydrolysing haze-sensitive proteins. PEPs including AN-PEP cleave proteins at proline (Pro, P)
60
residues
61
gluten proteins.
62
al. 28
63
25
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,
24, 29-30
a range of commercial
64
beers was analysed, including those produced from non-gluten-containing grains and from barley with
65
and without PEP treatment. Of these, the beers employing PEP treatment were shown to yield a gluten
66
content below the CODEX threshold of 20 mg/kg. A further study examining the action of AN-PEP
67
on the degradation of gluten peptides
68
follow the fate of the gluten peptides qualitatively. Analysis of untreated and AN-PEP-treated beers
69
revealed that immunotoxic epitopes were present in the untreated beers, but not in the AN-PEP-
70
treated beers. Another study examined the effectiveness of AN-PEP by both ELISA and LC-MS in a
71
sorghum beer incurred with wheat gluten.
72
content was demonstrated over the first three days of fermentation, whereas AN-PEP-treated beers
31
employed ELISA for gluten quantitation and LC-MS to
32
In control beers, a gradual reduction (4-fold) in gluten
4 ACS Paragon Plus Environment
Page 5 of 33
Journal of Agricultural and Food Chemistry
73
showed a marked decrease (>15-fold) in gluten content from 3-14 days. Using Western blotting, the
74
HMW-glutenins were shown to be less susceptible to AN-PEP than the LMW-glutenins. From these
75
studies and similar applications in wheat, bran and foodstuffs,
76
degrade gluten, however it is unclear if all potential immunopathogenic sequences are completely
77
eliminated. Moreover, the safety of gluten-reduced beers is still contentious in part owing to questions
78
regarding the accuracy of testing fermented and hydrolyzed foods and the ability to equate hydrolyzed
79
gluten content to an equivalent amount of intact gluten. This latter issue is addressed by the USA
80
FDA proposed rule (FDA-2014-N-1021).
81
reduced beers found that serum from active-CD patients bound to residual gluten peptides in
82
conventional beers and that a subset of the patient sera also reacted to gluten-removed beers. 18
35
33-34
it is apparent that PEP is able to
A recent study on the antibody response to gluten-
83
In the current study LC-MS analysis was applied to a selection of gluten-reduced and gluten-
84
free commercial barley-based beers to determine the effect of gluten reduction treatments on the
85
protein and peptide profiles.
86 87
Materials and Methods
88
Reagents and test samples. Chemicals, including formic acid (FA), ammonium bicarbonate,
89
dithiothreitol, iodoacetamide, were purchased from Sigma-Aldrich (Sydney, NSW, Australia).
90
Acetonitrile were purchased from ChemSupply (Gillman, SA, Australia). Enzymes used for digestion
91
(trypsin and chymotrypsin) were purchased from Promega (Sydney, NSW, Australia). A selection of
92
beers were purchased internationally from commercial liquor stores based on their ingredients and
93
gluten status according to their packaging and/or company website. All beers selected were barley-
94
malt based products rather than gluten-free beers based on non-gluten containing grains such as rice,
95
sorghum, millet or tef. A number of regular beers that had previously 6 been shown to contain gluten
96
were selected as positive controls, C1-C4. The gluten-reduced (or low gluten, LG) beers, LG1-LG7
97
and LG9-LG11 are PEP-treated. LG8 is manufactured by an undisclosed proprietary process. LG12 is
98
brewed with a novel ultra-low gluten barley. 36
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 33
99 100
Digestion of whole beers. Whole beers (n=4 technical replicates) were subjected to enzymatic digest
101
using either trypsin or chymotrypsin. Aliquots of degassed beer (50 µL) were diluted 1:1 in 50 mM
102
ammonium bicarbonate containing 1 mM CaCl2, pH 8.5 (50 µL). To these solutions, 10 µL of 50 mM
103
dithiothreitol was added and the samples were incubated at 60°C for 30 min. Subsequently, the
104
samples were cooled and 10 µL of 100 mM iodoacetamide was added and the samples were incubated
105
at RT for 20 min in the dark. To these solutions, 10 µL of either trypsin or chymotrypsin (1 µg/µL)
106
was added with incubation at 37°C for 16 hours. To quench the digestion, 50 µL of 1% formic acid
107
was added and the samples were stored at -20°C until analysis. A 95% confidence in the suite of
209
beers tested. As depicted in Figure 2, the control beers show the highest number of unique gluten-
210
derived peptides (range: 54-86 for trypsin; 59-121 for chymotrypsin) with the low gluten beers LG11
211
and LG12 revealing the least. The spectral count (total number of gluten peptide spectra acquired)
212
followed the same trend as the number of unique peptides, but also reflected the abundance of these
213
peptides since the more abundant a peptide is in a sample, the greater the spectral redundancy.
214
Qualitatively, from the low gluten beers tested, LG7 and LG8 were noted to contain the greatest
215
diversity and abundance of gluten-derived peptides, both with approximately twice the number and
216
spectral count compared to the average of all low gluten beers. LG3-LG6 also revealed values above
217
the average.
218 219
The undigested filtrates (90% of the
228
gluten peptides detected in C1-C4 contained P-X sites within their sequences. Examining LG1-LG7
229
and LG9-LG11, the majority of peptide fragments resulted from cleavage at P-X (55-73%) and to a
230
lesser extent X-P (2-9%) indicating the use of PEP in the production of these beers. Reviewing the
231
identified sequences within the PEP treated beer revealed that 60-94% of the gluten peptides
232
contained additional PEP cleavage sites. In fact, only LG11 contained more completely digested
233
peptides than partially digested (42.1% missed cleavages). The other notable fact was that untreated 10 ACS Paragon Plus Environment
Page 11 of 33
Journal of Agricultural and Food Chemistry
234
beers contained a higher number of P-X sites within an individual peptide (up to 5 missed cleavages)
235
compared to PEP treated beers where peptides typically only contained 1-2 missed cleavage sites. The
236
exception
237
QQAELIIP↓QQP↓QQP↓FP↓LQP↓HQP. Notably, this peptide also contained the QQPFP epitope
238
recognised by the Mendez R5 antibody. For LG8, PEP activity was not apparent with only 7% of
239
gluten-derived fragments cleaved at P-X or X-P which is within the same range as the control beers.
240
LG8 also yielded a high proportion of gluten peptide identifications containing P-X sites (82.8%). As
241
with all the beers in this study, LG8 was brewed using barley, but the gluten is claimed to be removed
242
by a proprietary process. Alongside a handful of B- and γ-hordeins, D-hordein was identified
243
confidently in LG8 by 11 peptides that all clustered in the C-terminal region of the protein suggesting
244
that a C-terminal fragment persists after brewing. In LG12, which is a gluten-free beer brewed using a
245
novel gluten-free barley,
246
resulting from non-specific cleavage (hydrolysis during brewing). This was expected since only γ3-
247
hordein is detectable in grain of this novel barley
248
identified in the trypsin (8 peptides) and chymotrypsin (1 peptide) digests of whole beer (Table 1).
was
LG7
which
36
also
contained
a
peptide
with
5
missed
cleavages:
only γ3-hordein (I6TEV2) was detected by seven peptide fragments
35
and only peptides derived from I6TEV2 were
249 250
We also examined the fragments identified in the undigested filtrate (
