Research article

Received: 22 May 2018 Revised: 15 August 2018 Accepted: 19 August 2018 Published online in Wiley Online Library: 20 November 2018

(wileyonlinelibrary.com) DOI 10.1002/jib.538

Low-molecular-weight materials from heavily

roasted barley and malt with strong
foam-stabilising potential†
Makoto Kanauchi,1 Emily Kultgen2 and Charles Bamforth2*
Extracts of roasted barley and black malt made both at room temperature and using a ramped temperature mashing regime
contained relatively low concentrations of protein. However cold water extracts displayed strong foaming power even at very
low measurable protein concentrations. The observation that very little material appeared to be precipitated out by ammonium
sulphate based salt fractionation is consistent with the very low protein levels and it is inferred that the powerful foaming
potential is not due to polypeptides. The foaming material in black malt was investigated in some detail. It was purified under
conditions that indicated that it is of molecular weight <4000 and is relatively anionic. A solitary foam-active fraction was
recovered using preparative HPLC and time of flight mass spectrometry revealed one large peak and two small peaks. The larger
peak is tentatively identified as pyridyl pyrazine, while we believe that the smaller peaks are due to peptides, which we
tentatively identify as a hexa- and a tetrapeptide. The Fourier transform infrared spectrum is consistent with pyridine, pyrazine
and peptide being components of this foaming entity. © 2018 The Institute of Brewing & Distilling

Keywords: foam; FTIR; mass spectrometry; peptide; pyridyl; roasted adjuncts

Introduction Preparation of cold water extract

One of the long-standing adages in the world of brewing is
that the presence of a proportion of specialty malt in the grist
boosts foam potential (1). This assertion was challenged by
Combe et al. (2), who demonstrated that malts of all types
contain some foam-negative species, primarily lipids, including
oxidised lipids (3). However certain specialty malts, notably
black malt and roasted barley, contain vastly more foam-
positive material than foam-negative substances (2). It has not
hitherto been ascertained why this is the case and what the
nature of these head-enhancing materials are. Certainly Bishop
(4) postulated that during the heating of malt there is a cross-
reaction between polypeptides and polysaccharides to produce
complexes with particularly robust foam performance. However,
Ishibashi et al. (5) demonstrated a clear decrease in the level of
foam-active proteins with increased colour, such that a malt
with a colour of 300 EBC units contained only one seventh as
much foaming protein as is present in a very pale malt. Alterna-
tive materials deemed to be foam-positive in beer are
melanoidins, and indeed Lusk et al. showed them to have inde-
pendent foam stability (6). However, the chemistry involved in
the roasting drum for the production of roasted barley and
black malt has less to do with the Maillard reaction than with
pyrolysis (7).
The present paper investigates the nature of the foaming
species in these heavily roasted adjuncts.
Grain (50 g) was ground using a MIAG Braunschweig mill at a
0.2 mm setting and was stirred with deionised water (200 mL)
for 60 min at room temperature. The total mass was adjusted to
450 g with deionised water and filtered through Whatman 2555
½ filter paper together with a secondary filtration using
Fisherbrand® grade G8 glass fibre filter circles in a plastic 60 mL sy-
ringe with filter housing and gasket.
Protein measurement
Protein was quantified using the Bio-Rad Protein Assay based on
the Bradford method (8).
Foam stability measurement
Foam stability was assessed using the Kapp and Bamforth shake
method (9).
* Correspondence to: Charles W. Bamforth, Department of Food Science &
Technology, University of California, Davis, CA 95616-8598, USA. E-mail:
cwbamforth@ucdavis.edu
†
Preliminary reports of this work were made by M. Kanauchi at the ASBC
Annual Meeting in La Quinta, CA June 2015 and by E. Kultgen to the Young
Scientists Symposium at Chico, CA, April, 2016.

Materials and methods 1

Department of Food Management, Miyagi University, 2-2-1 Hatatate,
Taihaku-ku, Sendai, Miyagi, 982-0215, Japan
Source of cereal materials 2
Department of Food Science and Technology, University of California, Davis,
Roasted barley and black malt were from Simpsons Malt (UK). CA, 95616-8598, USA
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 M. Kanauchi et al.

Colour measurement Purification of the foam fraction

The colour of extracts was evaluated by spectrophotometric absor-
bance at 430 nm (10).
Precipitation using ammonium sulphate
Solid finely ground ammonium sulphate was added to cold water
extracts of grain with continuous stirring at 0°C. Salt additions were
calculated according to the table of Dawson et al. (11) and repre-
sented first adjustment to 30% saturation and then subsequent
additions increasing saturation levels by 10%. At each stage the
mixture was stirred for 15 min before centrifugation at 4000 rpm
for 30 min at 4°C using an Eppendorf 5810 R centrifuge, rotor
radius 13 cm. Upon completion of centrifugation, an aliquot of
the supernatant was retained for dialysis and further study, and
the remainder of the supernatant was decanted into a beaker
for subsequent rounds of ammonium sulphate addition. The
pellets, when present, were re-suspended in deionised water
followed by dialysis using 3.5 K MWCO tubing against a100-fold
excess of deionised water.
Anion exchange chromatography The hot water extract of
black malt was adjusted to pH 8.0 using sodium hydroxide solution
and the wort was applied to a column (50 × 300 mm) of ion
exchange resin (Amberlite IRA400J Cl; Organo Co., Tokyo, Japan).
Elution was by a linear gradient of sodium chloride in acetate
buffer (pH 4.0) and fractions (8 mL) were collected.
Gel permeation chromatography The foam-active fraction col-
lected from the ion-exchange column was applied to a column
(height 450 mm × diameter 10 mm) of Sephadex LH-20 (catalogue
no. 17009001, GE Healthcare, Little Chalfont, UK) and elution was
with 0.1% acetic acid at a slow flow rate of 3.5 mL/h, and
fractions (3.5 mL) were collected.
Preparative high-performance liquid chromatography
The foam-active fraction collected from the gel permeation stage
was separated using preparative high-performance liquid chroma-
tography (HPLC) on a column of Atlantis dC18 OBD (19 × 150 mm,
Water, Milford, MA, USA). The flow rate was 5.0 mL/min, and elu-
tion was using 10 mM phosphate buffer (pH 4.0) containing 70%
acetonitrile by linear gradient from 0 to 70% acetonitrile.

Preparation of hot water extracts

Black malt was extracted according to the method of Combe et al.
(2). Finely ground malt (550 g) was mixed with distilled water (2 L)
at 45°C. The mash was stirred at 45°C for 30 min prior to ramping at
1°C/min until 70°C. A further 1 L of distilled water at 70°C was
added and the mash was held at 70°C for 60 min. After cooling
to room temperature, the mash was filtered.
Analysis of the foam-active fraction
The mass spectrum of the purified foam fraction was obtained
using a 800Plus MALDI TOF/TOF Analyser (AB SCIEX Pte. Ltd,
Ontario, Canada) and UltiMate™ 3000 Rapid System Liquid
Chromatography (Thermo Fisher Scientific). The instrumental
analysis conditions are shown in Table 1.

Table 1. Experimental conditions for Time of Flight mass spectrometry

TOF/TOF
Analytical instrument: 4800Plus MALDI TOF/TOF Analyzer (AB SCIEX)
Ionisation method: MALDI
Ionisation mode: Positive
Matrix: α-cyano-4- hydroxycinnamic acid (CHCA) 10 mg/mL in 50% CH₃CN
Apply sample: sample sol.: matrix sol. (1:1)
Orbitrap
Analytical instrument: Ultimate3000 Rapid System Liquid Chromatography system (Thermo Fisher Scientific)
Column: Acquity UPLC HSS C18 (Waters)
(inner diameter × height; 2.1 × 100 mm, particle size, 1.8 μm)
Mobile: A, 0.1%formic acid in water; B, 0.1%formic acid in CH₃CN
Gradient: B, 2% (0–2 min,hold) → 97% (2–25 min, gradient) → 97% (25–30 min, hold)
Flow rate: 0.3 mL/min
Column temperature: 40°C
Injection: 2–12 μL
UV detector: 220–800 nm
MS condition
Analytical instrument: LTQ Orbitrap Velos (Thermo Fisher Scientific)
Ionisation method: ESI
Ionisation mode: positive
Sheath gas flow setting: ‘40’
Auxiliary gas flow setting: ‘5’
Capillary temperature: 230°C
Scan range: 100–1000 m/z
FTMS resolution: 100,000
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Roasted adjuncts and foam
Fourier transform infrared (FTIR) analysis was performed using
FTIR Spectrum One (PerkinElmer, Waltham, MA, USA).
Results and discussion
Studies with cold water extracts
The relationship between foam stability and protein concentration
in various dilutions of cold water extracts of roasted barley and
Figure 1. The foam stability of cold water extracts of roasted barley and black malt.
Figure 2. The foam stability of precipitates derived from cold water extracts of roasted barley and black malt by treatment with ammonium sulphate.
Figure 3. The foam stability of supernatants derived from cold water extracts of roasted barley and black malt after treatment with ammonium sulphate.
J. Inst. Brew. 2019; 125: 39–46 © 2018 The Institute of Brewing & Distilling
black malt is shown in Fig. 1. At the lower concentrations of protein
the head retention was better for the extracts of roasted barley.
This is not consistent with a prime involvement of Maillard reaction
products that would be expected to be present to a substantially
greater extent in black malt wherein there had been a modification
process in malting.
The protein concentrations measured in the cold water extracts
of roasted barley and black malt were low, at 0.54 and 0.55 mg/mL
respectively.
Having demonstrated that both foaming materials and colour
are entirely removed by filtration through charcoal (data not
shown) we investigated the tendency of the foaming material to
be salted out with ammonium sulphate. Surprisingly, there was
no visible precipitate generated until ammonium sulphate satura-
tion reached 80%. There is an approximately inverse relationship
between the size of a polypeptide and the concentration of salt
needed to precipitate it (12). On this basis the extracts of both
roasted barley and black malt are remarkably devoid of sizeable
polypeptides that can be extracted with cold water. This is consis-
tent with the low protein levels measured in the extracts.
The material that is precipitated at the various salt concentra-
tions (pellets) as well as the supernatants from each stage were
compared for their foam stabilising potential at various dilutions
(Figs 2 and 3). It should be noted that the apparent protein con-
centrations are far lower for the supernatants than for the pellets,
for which it is possible to investigate higher concentrations deter-
mined by the amount of liquid used to re-suspend the pellet. The
most striking observation is the very substantial foam stability
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witnessed in these very dilute supernatant fractions. Other than
the presence of a remarkably foam-active protein species present
in very low quantities, it was surmised that the likelihood is that
foam-stabilising species that are non-proteinaceous are present
in these extracts.
In passing it should be noted that all of the fractions contained
substantial amounts of colour but salt fractionation was not capa-
ble of effecting a separation of colour-forming and foam-forming
Figure 4. Ion exchange chromatography of hot water extracts of black malt.
Figure 5. Gel permeation chromatography of hot water extracts of black malt.
Figure 6. Preparative high-performance liquid chromatography of partially purified hot water extracts of black malt. A 10 mM phosphate B 10 mM phosphate 70% acetonitrile;
0 min (A: 100%) → 30 min (A: 0%). [Colour figure can be viewed at wileyonlinelibrary.com]
42
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M. Kanauchi et al.
species. As the melanoidin colour-forming entities are of very high
molecular weight, it was concluded that the source of colour in
these foam-active products is not a melanoidin. One observation
that is worthy of further investigation is that it proved possible
to remove the colour from pre-formed foams from these coloured
extracts. If the foam was allowed to stand in a column and water at
room temperature was gently dripped onto the top surface of the
foam, then the colour drained out, leaving a ‘white’ foam. This may
be an approach to separating strongly foam-active materials from
the colour, presupposing that the re-dissolved surface-active ma-
terials retain their foam-stabilising ability. The observation also
suggests that the coloured materials are distinct from the foam-
stabilising materials and merely co-fractionate with them in salt
fractionation.
Studies with hot water extracts
This part of the investigation employed black malt only. Typical
protein concentrations in these extracts were 0.54 mg/mL, i.e.
similar to those found in cold water extracts. There is a dearth of
literature addressing the total protein extraction from roasted
barley and intensely heated malts, noting however the work of
Ishibashi et al. (5) refered to earlier.
Anion exchange chromatography demonstrated that the
fraction with foam-stabilising capability demanded 1M sodium
chloride to elute it, suggesting that it was very firmly bound to
the column (i.e. strongly negatively charged, Fig. 4). The foam-
active component was also retained to an extent in gel permeation
chromatography on LH-20 (Fig. 5), which allows the separation of
extremely low-molecular-weight compounds, for example globu-
lar proteins of a molecular mass <4000.
Figure 6 illustrates the fractionation of the material recovered
from gel permeation chromatography on a preparative HPLC
column. The material that forms such columns is designed to
allow better retention of relatively hydrophilic molecules on the
J. Inst. Brew. 2019; 125: 39–46
Roasted adjuncts and foam

Figure 7. Time-of-flight mass spectrometric investigation of foam-active fractions derived from black malt. (a) Analysis using MS; (b, d) the use of MS/MS. (a–c) The use of the
MALDI TOF/TOF analyzer; (d, e)the use of LTQ Orbitrap Velos. [Colour figure can be viewed at wileyonlinelibrary.com]
43

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Figure 7. Continued
reverse-phase matrix. One main and three small peaks were
detected of which only the main peak displayed foam stability.
Mass spectrometry, including time-of-flight MS (13,14), was used
to investigate the nature of the foam-active species recovered by
preparative HPLC (Fig. 7a–e). The time of flight technique revealed
one large peak and two small peaks (Fig. 7a–c). This method is
suited to the detection of non-polar entities, whereas the Orbitrap
is applicable to more polar materials. It is suggested that the larger
peak is possibly pyridyl pyrazine. We interpret the two smaller
peaks as representing peptides, which are tentatively identified
as gly–glu–val–ile (or leu)–ile (or leu)–gln and ala–ile (or leu)–ile
(or leu)–gln, respectively. A peptide database was not employed.
We have not been able to locate any previous publications indi-
cating a role in food or beverage foam activity for molecules incor-
porating pyridyl species. Intuitively it is supposed that there will be
some degree of similarity between extracts of roasted coffee and
roasted barley in respect of certain compositional and functional
activities. Nunes et al. (15) did show a correlation between the de-
gree of bean roasting and the foam stability of espresso coffee;
44
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M. Kanauchi et al.
however, they did not characterise the nature of the foam-active
materials, save for a relationship with the amount of
galactomannan and arabinogalactan. No evidence was found in
the present study for these polysaccharides. Indeed Nunes and
Coimbra (16) confirmed that these polysaccharides, complexed
with protein, were of a far higher molecular weight than the
materials reported here.
A range of volatile pyrazine-containing molecules were reported
in roasted barley by Collins (17), Harding et al. (18) and Wang et al.
(19). Yahya et al. (20) also focused on the impact of roasting pro-
cesses on flavour though not on functional properties. Walker
and Westwood (21) describe a range of pyrazines in extracts of
coloured malts and show that they feature in the low-molecular-
weight fraction. Melanoidins are of much higher molecular weight.
However they showed that such fractions were without effect on
foam stability, whereas the high molecular weight fraction in
which the colour was located were foam negative.
Many chemical species, including proteins, are subject to degra-
dation at intense temperatures through hydrolysis or pyrolysis
J. Inst. Brew. 2019; 125: 39–46
Roasted adjuncts and foam
Figure 8. The UV–vis spectrum of foam-active fractions derived from black malt.
Figure 9. The Fourier transform infrared spectrum of foam-active fractions derived from black malt. [Colour figure can be viewed at wileyonlinelibrary.com]
reactions (22,23). It has certainly been shown that free amino acids,
oligopeptides and polypeptides have a role to play in pyrazine for-
mation induced at high temperatures (24).
The UV–vis spectrum of the recovered material is shown in Fig. 8.
By reference, pyridine displays one peak at around 260 nm
whereas pyrazine has two peaks around 260 and 300 nm (http://
webbook.nist.gov/cgi/cbook.cgi? ID = C290379&Mask = FFF#UV–
Vis-Spec).
FTIR analysis was performed on the isolated foam fraction
(Fig. 9). The peak at 3300 cm 1 relates to an amine (C–N–H) group,
and those at 2920 and 1392 cm 1 indicate methyl groups. With
reference to the report of Ushakumari et al. (25), 1147 and
799 cm 1 may represent peaks from pyrazine. And also -NH2 was
shown by the peak at 1583 cm 1. The FTIR spectrum is consistent
with there being pyridine, pyrazine and peptide in this substance.
The foam active material separates in the various systems as a
single entity, indicating the strong association between the
materials detected as separated fragments in the MS studies, viz.
a putative complex between pyridyl pyrazine and a peptide(s).
Substantially more investigation is needed to complete the identi-
fication of this entity.
Acknowledgements
We thank Matthew Amicucci and Elisha Goonatilleke for their
valuable comments on time-of-flight mass spectrometry.
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