Аннотация (русский):
Beer DNA authentication is the process of authentication by identification of barley malt Hordeum vulgare or its substitutes, as well as hops and yeast. The method is based on molecular genetic analysis of residual quantities of nucleic acids extracted from the cellular debris of the final product. The aim of the study was to analyse scientific and methodical approaches to extraction of residual quantities of beer raw materials nucleic acids and beer DNA authentication for their later application in determining brewing products authenticity. The technological level discloses the method of DNA extraction from wines, modified for extraction of nucleic acids from beer samples. The method includes the following characteristic peculiarities: stage enzymatic hydrolysis of polysaccharides and polypeptides of dissolved lyophilisate, multiple sedimentation and resursuspension of nucleoproteid complex, RNA removal followed by DNA extraction by organic solvents, and additional DNA purification by magnetic particle adsorption. This review presents the analysis of genetic targets used as molecular markers for gene identification of malting barley varieties and beer DNA authentication. We also provided the interpretation of PCR analysis of Hordeum vulgare varieties and samples of commercial beer. Data on SSR- and SNP-markers of Hordeum vulgare nuclear DNA, used for barley varieties identification and potentially suitable for beer DNA authentication, are also presented. We also analysed genetic targets used in malting barley substitute detection, as well as hops and yeast identification in beer. Data on correlation of amplified DNA targets with beer quality indicators were systematised.

Ключевые слова:
Alcoholic beverages, malting barley, Hordeum vulgare, DNA, authentication, identification, marker, PCR

INTRODUCTION
Wide assortment of brewery products and their
multicomponent composition refers them to the segment
of difficult-to-identify goods. Their authentication is
aimed at protecting consumers and manufacturers’
rights [1].
One of the strategically important tasks achievable
by multidisciplinary science-intensive approaches is the
search for objective identification criteria with a high
degree of authenticity assessment of brewery products [2].
Molecular and genetic research methods can provide
the technological process of DNA authentication of beer
brands [3], thereby expanding the complex scheme of
brewery products identification, traditionally based on
documentary, visual, sensory and physical and chemical
analyses [4].
Beer brands DNA authentication is a technological
process of the authenticity verification by the gene
identification of Hordeum vulgare barley malt, or
its substitutes, as well as its key ingredients – hops
and yeast, by molecular genetic analysis of residual
quantities of nucleic acids extracted from the cellular
debris of the products [3].
The analysis of scientific and methodological
approaches points to the applicability of DNA
technologies for detecting counterfeit and falsified
brewery products.
RESULTS AND DISCUSSION
Extraction of DNA residues of beer raw
materials. The technological level discloses a method
for DNA extraction from wines [5, 6]. It was later
Review Article DOI: http://doi.org/10.21603/2308-4057-2019-2-364-374
Open Access Available online at http:jfrm.ru
DNA authentication of brewery products: basic principles
and methodological approaches
Lev A. Oganesyants1 , Ramil R. Vafin1,* , Aram G. Galstyan2 , Anastasia E. Ryabova1 ,
Sergey A. Khurshudyan1 , Vladislav K. Semipyatniy
1 All-Russian Research Institute of Brewing, Non-Alcoholic and Wine Industry, Moscow, Russia
2 All-Russian Dairy Research Institute, Moscow, Russia
* e-mail: vafin-ramil@mail.ru
Received May 17, 2019; Accepted in revised form August 08, 2019; Published October 21, 2019
Abstract: Beer DNA authentication is the process of authentication by identification of barley malt Hordeum vulgare or its substitutes,
as well as hops and yeast. The method is based on molecular genetic analysis of residual quantities of nucleic acids extracted from the
cellular debris of the final product. The aim of the study was to analyse scientific and methodical approaches to extraction of residual
quantities of beer raw materials nucleic acids and beer DNA authentication for their later application in determining brewing products
authenticity. The technological level discloses the method of DNA extraction from wines, modified for extraction of nucleic acids
from beer samples. The method includes the following characteristic peculiarities: stage enzymatic hydrolysis of polysaccharides
and polypeptides of dissolved lyophilisate, multiple sedimentation and resursuspension of nucleoproteid complex, RNA removal
followed by DNA extraction by organic solvents, and additional DNA purification by magnetic particle adsorption. This review
presents the analysis of genetic targets used as molecular markers for gene identification of malting barley varieties and beer DNA
authentication. We also provided the interpretation of PCR analysis of Hordeum vulgare varieties and samples of commercial beer.
Data on SSR- and SNP-markers of Hordeum vulgare nuclear DNA, used for barley varieties identification and potentially suitable
for beer DNA authentication, are also presented. We also analysed genetic targets used in malting barley substitute detection, as well
as hops and yeast identification in beer. Data on correlation of amplified DNA targets with beer quality indicators were systematised.
Keywords: Alcoholic beverages, malting barley, Hordeum vulgare, DNA, authentication, identification, marker, PCR
Please cite this article in press as: Oganesyants LA, Vafin RR, Galstyan AG, Ryabova AE, Khurshudyan SA, Semipyatniy VK. DNA
authentication of brewery products: basic principles and methodological approaches. Foods and Raw Materials. 2019;7(2):364–374.
DOI: http://doi.org/10.21603/2308-4057-2019-2-364-374.
Copyright © 2019, Oganesyants et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,
transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Foods and Raw Materials, 2019, vol. 7, no. 2
E-ISSN 2310-9599
ISSN 2308-4057
365
Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374
modified for extraction of nucleic acids from beer
samples [3]. The method includes the following
characteristic peculiarities: stage enzymatic hydrolysis
of polysaccharides and polypeptides of dissolved
lyophilisate, multiple sedimentation and resursuspension
of nucleoproteid complex, RNA removal followed by
DNA extraction by organic solvents, and additional
DNA purification by magnetic particle adsorption.
Figure 1 demonstrates stages of DNA extraction
according to the modified method. In particular,
enzymatic hydrolysis of polysaccharides by α-amylase
(Bacillus licheniformis) takes 3 h instead of 1 h, when
DNA is extracted from wines [3, 5]. The time of
enzymatic hydrolysis of polypeptides by proteinase
K (Tritirachium album) is also increased up to 3 h.
The sedimentation time of non-hydrolysed cellular
debris by centrifugation at 8000 g is reduced to 1 min
instead of 15 min when DNA is extracted from wines.
At the stage of DNA extraction from the lyophilised
beer powder, the sedimentation of the nucleoprotein
complex is carried out by mixing the supernatant with
two volumes of cold absolute ethanol instead of two
volumes of cold isopropanol. At the next stage we mixed
a solution of unpurified DNA with an equal volume
of 70% ethanol. The maturing of the mixture at 0°C
takes 3 min instead of 10 min, as with wines. During
the subsequent nucleoprotein complex sedimentation,
along with the stepwise addition of 10 μL of 3M
sodium acetate and two volumes of cold isopropanol
to the pre-transferred transparent supernatant, 3 μL of
Ethachinmate linear polyacrylamide is added. After
RNA removal and deproteinisation, the sedimentation
of purified DNA is carried out without adding 70%
ethanol. (Сf. DNA extraction from wines involves in
the nucleic acids sedimentation in 0.2 M NaCl and two
volumes of cold ethanol, followed by washing with 70%
ethanol). Later, nucleic acids precipitate, resuspended in
the elution buffer, undergoes an additional purification
by adsorption on magnetic particles, which is one of the
key modification elements of the method for extracting
residual DNA of beer raw materials [3].
The ability of magnetic particles to bind DNA
reversibly and easily be deposited from the suspension in
the magnetic field ensures high quality of nucleic acids
purification and their preservation. Magnetic particles,
as a rule, are a paramagnetic core with a highly
developed surface covered with a polymer film with
exposed covalent-bond carboxylic groups. Magnetic
tripods, used in manual and automated modes, are made
of neodymium magnets resistant to demagnetisation.
The additional purification by adsorption on magnetic
particles of the modified method of extraction of nucleic
acids from beer samples actually took the place of
polymer polyvinylpyrrolidone widely used to reduce the
inhibitory effect of polyphenols on PCR [3, 7–10].
Approaches to beer DNA authentication. Genetic
targets, used as molecular markers for malting barley
varieties identification, can also be analysed for
commercial beer DNA authentication (Table 1) [3].
Polygalacturonase is an enzyme that performs
hydrolytic cleavage of α-1,4-glycoside bonds in
pectin. The DNA target was the locus of its gene
(HvPG1) еamplified by a corresponding pair of primers
constructed by Pulido et al. based on the analysis of
expressed sequence tag (EST) deposited in GenBank
(A/N: EF427919) [11]. The generated PCR products a
and b of the HvPG1 gene locus detected in the barley
and beer samples were 89% and 79% identical to the
previously deposited nucleotide sequence mRNA
polygalacturonase Hordeum vulgare. Among the studied
Japanese barley varieties, only the high quality ‘Ryofu’,
recommended for brewing, generated two discrete
fragments (a, b), like most American and Australian
barley varieties, except for Stimling (Table 2). All
the beer samples were marked only by the country of
manufacture. They generated the PCR product b and
more than half of the samples generated the additional
fragment a (Table 2). The analysed DNA target was
included in the group of DNA markers of identification
and differentiation of beer samples, but did not correlate
with the indicators of beer quality [3].
Hordeins are polymorphic proteins of barley grain
coded by 7 HrdA-G loci which are localised in the
short arm of the 5th Hordeum vulgare chromosome
[12, 13]. Due to the established connection of the
hordein-coding loci alleles with brewing qualities
of barley grain, this block of targets is a priority for
molecular and genetic analysis [14, 15]. From the three
analysed loci (HrdA, HrdB and HrdC) only one (HrdC)
was able to identify a single sample of beer out of 22
investigated by the presence of a specific PCR product e
(Table 2) [3]. However, high variability of HrdA locus
(up to 90% identity of nucleotide sequences of compared
barley varieties with corresponding reference sequence
(GenBank A/N: AF474373) indicates a certain potential
of DNA authentication of beer on the analysed target
by sequencing the amplified locus. The block of DNA
targets under study also did not correlate with the
indicators of beer quality [3].
Amylosis content in barley starch influences the
quality of malt barley. Therefore, waxy-barley varieties
may be a preferred option for their malting in brewing
because starch with low amylosis content is more
susceptible to enzymatic hydrolysis [18]. Molecular
mechanism is embedded in Hordeum vulgare waxygenes
located on 7 HS chromosome. They lead to the
elimination of granule-bound starch synthase (GBSS)
[18, 19]. Primers selected for Waxy-locus amplification
had the positive control status due to generation of
specific PCR product in all the samples of barley and
beer [3]. Their sequeneed nucleotide DNA sequences
were identical to each other and showed 98% identity
to the corresponding reference Hordeum vulgare subsp.
Vulgare sequence, previously deposited to GenBank
(A/N: X07931) [20].
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Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374
1. LIOPHILISED BEER POWDER RESUSPENDING
Lyophilisate dissolving in 500 μl of resuspending buffer
(0.1M Tris-HCl (pH 8.0), 0.1 MNaCl)
2. POLYSACCHARIDES ENZYMATIC HYDROLYSIS
Beer suspension processing with 100 μl of thermostable α-amylase (Bacillus licheniformis)
Incubating the produced mixture at 80°C for 3 h
3. POLYPEPTIDES ENZYMATIC HYDROLYSIS
Suspension processing with 100 μL of proteinase K (Tritirachium album) with 0.2% SDS
Incubating the produced mixture at 55°С for 3 h
4. NON-HYDROLYZED CELL DEBRIS SEDIMENTATION
Centrifugation at 8000 g for 1 min at 40°C
Supernatant transfer to a new tube
5. NUCLEOPROTEID COMPLEX SEDIMENTATION
Mixing the supernatant with two volumes of cold absolute ethanol
and holding the mixture at 0°C (on ice) for 15 min
Centrifugation at 8000 g for 15 min at 4°C
6. NUCLEOPROTEID COMPLEX RESUSPENDING
Sediment resuspending in 300 μL of elution buffer (0.1M Tris-HCl (pH 8.0), 0.1M EDTA)
7. NUCLEOPROTEID COMPLEX SEDIMENTATION
Mixing the crude DNA solution with an equal volume of cold 70% ethanol
and holding the mixture at 0°C (on ice) for 3 min
Transfer the clear supernatant to a new tube and stepwise addition of 10 μL of 3M sodium acetate, 3 μL of linear
polyacrylamide Ethachinmate and 2 volumes of cold isopropanol
Centrifugation at 8000 g for 15 min at 4°C
8. NUCLEOPROTEID COMPLEX RESUSPENDING
Sediment resuspending in 300 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA)
9. RNA REMOVAL
Suspension Treatment with RNAse A at 55°C for 30 min
10. DEPROTEINISATION
Extraction with equal volume of neutral phenol
Recovery of the aqueous phase by centrifugation at 8000 g for 15 min (4°C)
Extraction with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1)
Recovery of the aqueous phase by centrifugation at 8000 g for 15 min at 4°C
11. SEDIMENTATION OF PURIFIED DNA
Repeat stage 7 without adding 70% ethanol
12. RESUSPENDING OF SEDIMENTAL DNA
Sediment resuspending in 125 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA)
13. ADDITIONAL STAGE OF DNA PURIFICATION BY ADSORPTION ON MAGNETIC PARTICLES
Figure 1 Stages of DNA extraction from lyophilised beer powder
L
L
1. LIOPHILISED BEER POWDER RESUSPENDING
Lyophilisate dissolving in 500 μl of resuspending buffer
(0.1M Tris-HCl (pH 8.0), 0.1 MNaCl)
2. POLYSACCHARIDES ENZYMATIC HYDROLYSIS
Beer suspension processing with 100 μl of thermostable α-amylase (Bacillus licheniformis)
Incubating the produced mixture at 80°C for 3 h
3. POLYPEPTIDES ENZYMATIC HYDROLYSIS
Suspension processing with 100 μL of proteinase K (Tritirachium album) with 0.2% SDS
Incubating the produced mixture at 55°С for 3 h
4. NON-HYDROLYZED CELL DEBRIS SEDIMENTATION
Centrifugation at 8000 g for 1 min at 40°C
Supernatant transfer to a new tube
5. NUCLEOPROTEID COMPLEX SEDIMENTATION
Mixing the supernatant with two volumes of cold absolute ethanol
and holding the mixture at 0°C (on ice) for 15 min
Centrifugation at 8000 g for 15 min at 4°C
6. NUCLEOPROTEID COMPLEX RESUSPENDING
Sediment resuspending in 300 μL of elution buffer (0.1M Tris-HCl (pH 8.0), 0.1M EDTA)
7. NUCLEOPROTEID COMPLEX SEDIMENTATION
Mixing the crude DNA solution with an equal volume of cold 70% ethanol
and holding the mixture at 0°C (on ice) for 3 min
Transfer the clear supernatant to a new tube and stepwise addition of 10 μL of 3M sodium acetate, 3 μL of linear
polyacrylamide Ethachinmate and 2 volumes of cold isopropanol
Centrifugation at 8000 g for 15 min at 4°C
8. NUCLEOPROTEID COMPLEX RESUSPENDING
Sediment resuspending in 300 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA)
9. RNA REMOVAL
Suspension Treatment with RNAse A at 55°C for 30 min
10. DEPROTEINISATION
Extraction with equal volume of neutral phenol
Recovery of the aqueous phase by centrifugation at 8000 g for 15 min (4°C)
Extraction with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1)
Recovery of the aqueous phase by centrifugation at 8000 g for 15 min at 4°C
11. SEDIMENTATION OF PURIFIED DNA
Repeat stage 7 without adding 70% ethanol
12. RESUSPENDING OF SEDIMENTAL DNA
Sediment resuspending in 125 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA)
13. ADDITIONAL STAGE OF DNA PURIFICATION BY ADSORPTION ON MAGNETIC PARTICLES
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Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374
Hemicelluloses are vegetable homo- and
heteropolysaccharides, which are an integral part of the
endosperm cell walls. The highest content of xylanes
was reported to be among the main components of
hemicellulose [21]. The malt barley softens as a result of
the decomposition of the cell wall. Xylanase is involved
in the degradation of xylanes to xylooligosarachides,
whose gene locus was used as a target for primers
originally designed for DNA analysis of rice samples [3].
It is noteworthy that among the 16 varieties of barley,
only three varieties (Metcalfe, Nishinohoshi and Ryofu)
showed a positive amplification signal (Table 2). At the
same time, due to possible obtaining inconclusive data,
the authors [3] presented neither the results of PCR of beer
samples, nor data on amplification of the HrdB locus.
Barley Z proteins are the main beer protein which
influence beer quality, especially foam stability [22–
24]. In addition, Z4 and Z7 proteins can be used as
positive and negative markers of foam stability [25].
DNA-markers of foam stability developed by Limure
et al. were also used in by Nakamura et al. for barley
varieties identification and beer DNA-authentication
[3, 25]. Identifying and differentiating barley and
beer samples procedure by the gene locus, encoding
proteins Z 4 a nd Z 7 d iffer. I n t he fi rst c ase P CR
analysis is performed by interpreting three discrete
PCR products (h, i-a, i-b), and in the second –by the
presence or absence of a specific fragment j.
Based on the analysis, the authors recommended the
further use of the tested primers for amplification of the
analysed gene loci [3]. In addition, a negative correlation
of the amplified PCR product h gene locus encoding
Z7 protein with beer bitterness, as well as a positive
correlation of PCR product i-a similar locus with foam
stability (Table 1) were revealed.
Many enzymes, incl. α-amylase and β-amylase,
are activated in the malting process [26, 27]. Their
substrates are amylosis and amylopectin or products
Table 1 Genetic targets used as molecular markers for brewing barley varieties identification and beer DNA authentication
Target PCR product Primer sequence Correlation (+/-) Source
Polygalacturonase (HvPG1) a F: 5/-GACAGAATGGCGTTCAAGAACAT-3/
R: 5/-AGCAAGTTGCCTTCCAGCTTGAT-3/
N/A [3, 11]
b N/A
Hordein A
(HrdA)
с F: 5/-AGATAGCGTTTTGAAGGTCAC-3/
R: 5/-TAGACCTGCAATAATTTCCA-3/
N/A [3, 16]
Hordein B
(HrdB)
d-1 F: 5/-TCACACATAAGGTTGTGTGAC-3/
R: 5/-CAAGCTTTCCCACAACAACCA-3/
N/A [3, 17]
d-2 N/A
Hordein C
(HrdC)
e F: 5/-AATTTAAACAACTAGTTTCGGGTGG-3/
R: 5/-CAAGCTTTCCCACAACAACCACCAT-3/
N/A [3, 16]
Barley starch synthase
(waxy)
f F: 5/-CAATTCATCCGATCACTCAATCAT-3/
R: 5/- CAGGCCGACAAGGTGCTG -3/
N/A [3, 16]
Xylanase g F: 5/-GGTACAACGTCGCGTCGG-3/
R: 5/-CGTGTACCAGACGGTCCAGATACAGC-3/
N/A [3, 21]
Protein Z7 h F: 5/-GGTCACATGACGTGTATTAATCTCC-3/
R: 5/-CGTTGGTGGCAGCAGACTCGGGG-3/
–* [3, 24]
i-a +**
i-b N/A
Protein Z4 j F: 5/-GAGACGTGTAGTAATCTTCG-3/
R: 5/-GCGAGCACAAATTGCACCACC-3/
–*** [3, 24]
α-amylase k F: 5/-AAGGTCTCGTGTCGATCCCAAGGAGGC-3/
R: 5/-CTAAGCCTCGTCTTCGTCCCC-3/
N/A [3]
Barley lipoxygenase
(LOX1)
l F: 5/-GCAACGGAGGGAGTAAAACA-3/
R: 5/-CGATGGCTTGGACCAATTAC-3/
+**** [3, 34]
Barley yellow mosaic virus
(rym5)
m F: 5/-GAGTCGTCACAACGTACCTTGC-3/
R: 5/-GTGGCTGTAAATAGGCTAAGGCC-3/
N/A [3, 34]
Barley powdery mildew
(mlo)
n F: 5/-TAGCAATCACGGTCACGTCAAC-3/
R: 5/-CCGCAAGGCTGCTATGAAAAGGG-3/
N/A [3, 34]
o N/A
Barley trypsin inhibitor
(Itr1)
p F: 5/-CAACTAACAGAAAGTCAGAAAGCAC-3/
R: 5/-CACAATACTGAAAATACTCTGATGC-3/
–***** [3, 37]
Barley β-glucanase
(HvCslF6)
s F: 5/-GCCAAGACCAAGTACGAGAAGC-3/
R: 5/-TGTTCTTGGAGAAGAAGATCTCG-3/
N/A [3, 40]
–* a negative correlation of the amplified PCR product h of the gene locus encoding the protein Z7 with beer bitterness
+** a positive correlation of the amplified PCR product i-a of the gene locus encoding the protein Z7 with foam stability
–*** a negative correlation of the amplified PCR product j of the gene locus encoding the protein Z4 with the detectable PCR product h of the gene
locus encoding the protein Z7
+**** a positive correlation of the amplified DNA target with beer taste saturation
–***** a negative correlation of the detected DNA matrix with the saturation of beer taste
N/A not applicable
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Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374
of their hydrolysis. Primers developed on the basis
of nucleotide sequence of the gene locus encoding
α-amylase initiated the amplification of PCR product k
in most of the barley varieties and beer samples (Table 2)
[3, 28]. It is noteworthy that the amino acid sequence of
the target had 69% identity with Mla-locus of resistance
to powdery mildew Hordeum vulgare (GenBank A/N:
AF427791) [29]. The used set of primers was included
in the group of molecular labeling systems of barley
varieties, and therefore has a certain potential of
practical application for beer authentication, although
the authors did not mention it [3].
Lipoxygenase-deficient barley varieties with reduced
or lost activity of LOX genes have a positive impact on
quality indicators such as beer taste and foam stability
[30–33]. The set of primers constructed by Nagamine et al.
resulted in amplification of the specific PCR product l in
a small number of studied barley varieties and in more
than half of beer samples, whose sequenced nucleotide
sequences had 99% identity with the reference sequence
Table 2 Interpreted results of PCR analysis of brewing barley varieties and beer samples
Barley varieties PCR products
a b c d–1 d–1 e f g h i–a i–b j k l m n o p s
Vlamingh + + – + + – + – – – + – + – – – – – –
Hamelin + + – + + + + – + + + – + – – – – + –
Stimling – + – – + + + – – + – – – – – – – – +
Bardin + + – – + + + – – + – + + – – – – + +
Salute + + + – + – + – – – + – + – – – – – +
Schouner + + – + + – + – – + – + – – – – – + –
Maritime + + – – + + + – – – + + + – – + – + +
Flag ship + + – – + – + – – + – – – – – – – + +
Metkafe + + – + + – + + + + + + – + + + – + +
Harushizuku – + + + – – + – – + – + + – – – + – –
Houshun + – + + – – + – – + – – – – + + – – –
Mikamogolden – – + + – – + – – – + + – – + – + – –
Skygolden – + + + – + + – – – + + + – + – + – –
Nishinohoshi – – + + – + + + – + – + – + + + – – +
Nishinochikara – – – + – + + – – + – + + + + – + + +
Ryofu + + + + – + + + – – + + + + + + – + +
Samples of beer PCR products
a b c d–1 d–1 e f g h i–a i–b j k l m n o p s
Czechoslovakia–a + + + n/a n/a – + n/a + + + – + + n/a n/a n/a + +
USA–a – + + n/a n/a – + n/a – + – – + + n/a n/a n/a + +
Belgium–a – + + n/a n/a – + n/a + + + – – + n/a n/a n/a – +
USA–b + + + n/a n/a – + n/a + + + + + + n/a n/a n/a – +
Netherlands–a + + + n/a n/a + + n/a – + + – + + n/a n/a n/a + +
Thailand–a + + + n/a n/a – + n/a – + + – + + n/a n/a n/a – +
Denmark–a + + + n/a n/a – + n/a – + + – + – n/a n/a n/a – +
England–a – + + n/a n/a – + n/a – + + – + + n/a n/a n/a – +
Germany–a – + + n/a n/a – + n/a – + – – + + n/a n/a n/a – +
Australia–a – + + n/a n/a – + n/a – + – – + – n/a n/a n/a + +
Mexico–a – + + n/a n/a – + n/a – + – – + + n/a n/a n/a – +
USA–c + + + n/a n/a – + n/a + + + – + + n/a n/a n/a + +
Germany–b + + + n/a n/a – + n/a + + + – + + n/a n/a n/a – +
England–b – + + n/a n/a – + n/a – + + – + + n/a n/a n/a + +
Peru–a – + + n/a n/a – + n/a – + – – + + n/a n/a n/a – +
England–c + + + n/a n/a – + n/a + + – + – – n/a n/a n/a + +
Germany–c + + + n/a n/a – + n/a + + – + – – n/a n/a n/a + –
Italy–a + + + n/a n/a – + n/a + – – + + – n/a n/a n/a + +
Japan–a + + + n/a n/a – + n/a + – – + + – n/a n/a n/a + –
Japan–b + + + n/a n/a – + n/a + + – – + – n/a n/a n/a + –
Japan–c + + + n/a n/a – + n/a – + – – + + n/a n/a n/a – +
Japan–d + + + n/a n/a – + n/a + – – – + – n/a n/a n/a + +
+ a positive amplification signal
– a negative amplification signal
n/a not applicable
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Table 3 SSR-markers of nuclear DNA Hordeum vulgare used for genetic identification of brewing barley varieties, potentially
suitable for beer DNA authentication
No. SSR marker The sequence of oligonucleotide primers Allel lenghts, bp Number of alleles
1 Bmac 0040 F: 5/-AGCCCGATCAGATTTACG-3/ 196-226 bp
(196/200/208/214/220/226)
6
R: 5/-TTCTCCCTTTGGTCCTTG-3/
2 Bmac 0134 F: 5/-CCAACTGAGTCGATCTCG-3/ 140-174 bp
(140/144/162/168/174)
5
R: 5/-CTTCGTTGCTTCTCTACCTT-3/
3 Bmag 0125 F: 5/-AATTAGCGAGAACAAAATCAC-3/ 128-148 bp
(128/132/138/144/148)
5
R: 5/-AGATAACGATGCACCACC-3/
4 Bmag 0211 F: 5/-ATTCATCGATCTTGTATTAGTCC-3/ 150-170 bp
(150/154/162/170
4
R: 5/-ACATCATGTCGATCAAAGC-3/
5 Bmag 0222 F: 5/-ATGCTACTCTGGAGTGGAGTA-3/ 140-178 bp
(140/144/162/168/170/174/178)
7
R: 5/-GACCTTCAACTTTGCCTTATA-3/
of locus LoxA-gene Hordeum vulgare (GenBank A/N:
L35931) [3, 34, 35]. The tested set of primers was
recommended for further use in the amplification of the
analysed gene locus for barley varieties identification
and beer brands differentiation. It should also be noted
that the authors [3] additionally revealed a positive
correlation between the amplified DNA target and beer
taste saturation (Table 1).
The selection of barley varieties with genetic
resistance to viral, bacterial and fungal diseases is
aimed at high-quality grain production [36]. A number
of DNA markers of resistance of barley to yellow mosaic
virus (rym5-locus) and powdery mildew (mlo-locus) [34]
integrated into breeding programs can also be used in
molecular labelling of brewing barley varieties, which
is clearly demonstrated in the work [3]. The authors
interpreted the PCR analysis data of barley samples
taking into account the presence or absence of specific
PCR products m (rym), n and o (mlo) recorded on the
corresponding electrophoregrams. But the results of the
PCR analysis of beer samples and their correlation with
quality indicators were not provided [3].
Protein inhibitors of proteolytic enzymes play
an important role both in formation of homeostatic
reactions in plants and in the process of seed maturation
and germination. Selected primers to the trypsin
inhibitor (Itr1) gene locus led to the amplification
of the specific PCR product p in half of the tested
barley varieties and beer samples [37]. Thus, the DNA
marker was concluded to be highly informative [3].
Additionally, the DNA sequences of the Itr1-gene locus
of the material had 94% identity with the same locus
of the Hordeum vulgare subsp. vulgare gene (GenBank
A/N: (X65875) [38]. Also, in the study [3] a negative
correlation of the detected DNA matrix with beer taste
saturation was revealed (Table 1).
The content (1–3, 1–4) of β-D-glucan in barley grain,
which determines its hardness, is much higher compared
to other cereals [39]. However, for barley varieties used
in brewing, a lower the content of this polysaccharide in
the grain is desirable in order to achieve a more effective
flow of the malting process [40]. The amplification
procedure of the locus of the HvCslF6 gene with a
selected primer pair led to the production of a specific
PCR product s in a number of American, Australian
and Japanese brewing barley varieties [3, 40]. The most
of the beer samples also gave a positive amplification
signal (Table 2). The obtained amino acid sequence
of the target had 83% identity with Hordeum vulgare
CslF6-gene (GenBank A/N: EU267181) [41]. The used
primer set was also included in the group of systems
of barley varieties molecular labelling and beer DNAauthentication
[3].
Microsatellites are widely used molecular markers
which are suitable for identification of Hordeum vulgare.
A wide variety of SSR-markers are being used [42–44].
Tomka et al. described a high potential of the five SSRmarkers
for brewing barley varieties identification [45].
Table 3 shows the sequence of oligonucleotide
primers of the corresponding Hordeum vulgare SSRmarkers
of nuclear DNA, as well as the range of lengths
of detected alleles and their number. The genetic
identification procedure includes PCR method with
subsequent data interpretation by horizontal or vertical
gel electrophoresis and DNA fragmentary analysis
of capillary gel electrophoresis. The SSR-markers,
potentially suitable for beer DNA authentication, are
advisable to test in the formulation of single PCR, with
a set of primers of a single SSR-marker to achieve a
reproducible result.
Alongside with SSR-markers, SNP-markers, used for
barley varieties identification, including brewing ones,
also have high identification capacity [46–48].
Table 4 shows oligonucleotide primers sequences
of the corresponding SNP markers of Hordeum
vulgare nuclear DNA, as well as the size of amplified
loci of discriminated alleles [46]. The procedure of
gene identification is carried out by the Amplification
Refractory Mutation System (ARSM-PCR), followed by
data interpretation by horizontal gel electrophoresis or
by high resolution melting curves (HRM) analysis on
PCR platforms in real time. It should be mentioned that
we selected five SNP-markers (out of nine described by
Chiapparino et al. [46] as potentially suitable for beer
DNA authentication due to generation of relatively small
allele-specific PCR products, whose size was not more
than 200 bp (Table 4).
370
Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374
Table 5 Genetic targets used in detecting brewing barley substitutes and identifying hops and yeast in beer
Target PCR product Primer sequence Correlation (+/–) Source
GBSS (rice) t F: 5/-GGATGAAGGCCGGAATCCTG
R: 5/-CTTGCCCGGATACTTCTCCT
missing [3, 49]
Β-conglycinin u F: 5/-TTTGGCATTGCTTACTGGGAAAAAGAG
R: 5/-TCTGTAGGAGTCTCTGTCGTCGTTG
missing [3, 50]
Zein v F: 5/-CACATGTGTAAAGGTGAAGCGAT
R: 5/-GCTCGCCGCAAGCGCTTGTTG
missing [3, 49]
Hop-a w F: 5/-GGAACCGTTGCCTAATCCTAAGATT
R: 5/-GTGTTTTCCGTATCTACGCGCTGGG
missing [3]
Hop-b x F: 5/-AATTAGGGCATGCCATGAATATT
R: 5/-TGGCATAGTTAAATTATTTCG
–*
–**
[3]
Hop-c y F: 5/-AAATAAAACTTTACATGTGATA
R: 5/-CTGAATTGTCGGCGT
missing [3]
Yeast-a
(S. cerevisiae)
z-a F: 5/-GTTTTGCGCTCATTAAAACCTAGTGGGAG
R: 5/-GTCATTTTTTTTAGTGGTGCTAATC
+***
–****
[3]
Yeast-b (thioredoxin) z-b F: 5/-ATGGTCACTCAATTAAAATCCGCTTCT
R: 5/-CTATACGTTGGAAGCAATAGCTTGCTTG
missing [3]
–* a negative correlation of the amplified PCR product t of the corresponding locus of the hop gene (Hop-b) with beer bitterness
–** a negative correlation of the amplified PCR product t of the corresponding hop gene locus (Hop-b) with beer astringency
+*** a positive correlation of the amplified PCR product z-a of the corresponding yeast gene locus with beer acidity
–*** a negative correlation of the amplified PCR product z-a of corresponding locus of the S. cerevisiae gene yeast with beer umami
N/A is not applicable
The detection of brewing barley substitutes in
beer, which is often used as a cheap source of starch,
makes it possible to evaluate the products sold for
qualitative, quantitative, information and complex
falsification. Table 5 demonstrates primer sequences
targeting genetic targets used in the detection of
brewing barley substitutes in beer, such as granulebound
starch synthase of rice, β-conglycinin of soya,
and zein of maize [49, 50]. Nevertheless, other PCR
systems developed for the identification of cereals
in food products can also be suitable for beer DNA
authentication [51].
The effect of hops and yeast on beer quality is
well-known. Thus, hop has a bactericidal effect on
beer as well as provides its bitterness, aroma and foam
stability [52]. Yeast is used in beer fermentation and
Table 4 SSR-markers of Hordeum vulgare nuclear DNA used for brewing barley varieties identification, potentially suitable for
beer DNA authentication
No. Locus (position) The sequence of oligonucleotide primers PCR product, bp
1 MWG2062
(325 A-G)
FOP: 5/-GTTGTGTCAAGCATATCGGTTGCTCTT-3/
ROP: 5/-CAGCACGTTCGAAAACAATAGGATCC-3/
198 bp
FIP: 5/-AAGAATTATGCCAATTATTGGCGTGTCA-3/ 101 bp (A allele)
RIP: 5/-CACACTGCATGTCATCAAACAAGCAC-3/ 151 bp (G allele)
2 ABC465
(254 C-T)
FOP: 5/-CAGGTACACCTGGAAGCTCTACTCAGAG-3/
ROP: 5/-CAGCAGCCTGAATTCAACAAAACATAC-3/
236 bp
FIP: 5/-TGGAGATGTTCTACGCTCTCAAGTACAGT-3/ 130 bp (T allele)
RIP: 5/-CTGTTGGTCAGATAACCTACCAGGATG-3/ 162 bp (C allele)
3 MWG2218
(175 G-C)
FOP: 5/-CTCTCCGACATCGACCGCTTCCTCTTCG-3/
ROP: 5/-GCCGCATCATCCCTGGTGTCATCACCT-3/
215 bp
FIP: 5/-GGGGACGTCATCCACGTCTGTCGACC-3/ 127 bp (C allele)
RIP: 5/-GTTCCCGCGGTGGGCTTTGTTTCCTC-3/ 140 bp (G allele)
4 ABC156
(231 T-G)
FOP: 5/-CTTGGTCCATATAGGTCTCTCTTTTC-3/
ROP: 5/-CCTCCTGATATACTTGAGAGACTCAATA
74 bp
FIP: 5/-TCCATATAGGTCTCTCTTTTCTTATTATG-3/ 70 bp (G allele)
RIP: 5/-TGAGAGACTCAATACTCATGAATTTCA-3/ 60 bp (T allele)
5 MWG801
(344 G-A)
FOP: 5/- CAACAACCCCAATACCAGGCCAGCTCCACA-3/
ROP: 5/-AACCCTCGACTGCTCAAGGCAGAGCCGC-3/
256 bp
FIP: 5/-GAAGCATGCTCGCACGACACCCATCC-3/ 175 bp (C allele)
RIP: 5/-CGGCAGCGGAGGGGAAGGGGAGCAGT-3/ 133 bp (A allele)
FOP is a forward outer primer; ROP is a reverse outer primer; FIP is a forward inner primer; and RIP is a reverse inner primer
371
Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374
impacts its character and taste [53]. Table 5 also
presents sets of primers which initiate the amplification
of specific PCR products of the corresponding loci of
hops and yeast genes. They also allow the identifying
or differentiating of commercial beer samples [3].
In addition, a negative correlation of the amplified
PCR product t of the corresponding locus of the hop
gene (Hop-b) with beer bitterness and astringency
was revealed. The amplified PCR product z-a of the
corresponding locus of the yeast gene S. cerevisiae
showed a positive correlation with beer acidity and
a negative correlation with beer umami [3]. Taking
into account the rapid development of genomic and
bioinformation technologies, metagenomic analysis,
which allows determining yeast species diversity in
beer samples without microorganisms allocating and
cultivating, is one of the promising approaches to beer
DNA authentication [54, 55].
CONCLUSION
Analysis of scientific and methodical approaches to
extraction of residual quantities of nucleic acids of beer
raw materials and beer DNA-authentication indicates
the applicability of molecular and genetic analysis in
detecting counterfeit and falsified brewery products.
The use of DNA technologies helps determine the
authenticity and origin of the brewery industry products.
Molecular labelling systems suitable for identification of
Hordeum vulgare barley malt, or its substitutes, as well
as hops and yeast, can ensure traceability of the product
life cycle. Systematic data on correlation of amplified
DNA targets with beer quality indicators can be of
practical importance when choosing raw materials for
brewery production.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
FUNDING
The study was supported by the section of
storage and processing of agricultural products of the
Department of Agricultural Sciences of the Russian
Academy of Sciences and Federal scientific centre ‘Food
Systems’ of the Russian Academy of Sciences.

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