INTRODUCTIONWide assortment of brewery products and theirmulticomponent composition refers them to the segmentof difficult-to-identify goods. Their authentication isaimed at protecting consumers and manufacturers’rights [1].One of the strategically important tasks achievableby multidisciplinary science-intensive approaches is thesearch for objective identification criteria with a highdegree of authenticity assessment of brewery products [2].Molecular and genetic research methods can providethe technological process of DNA authentication of beerbrands [3], thereby expanding the complex scheme ofbrewery products identification, traditionally based ondocumentary, visual, sensory and physical and chemicalanalyses [4].Beer brands DNA authentication is a technologicalprocess of the authenticity verification by the geneidentification of Hordeum vulgare barley malt, orits substitutes, as well as its key ingredients – hopsand yeast, by molecular genetic analysis of residualquantities of nucleic acids extracted from the cellulardebris of the products [3].The analysis of scientific and methodologicalapproaches points to the applicability of DNAtechnologies for detecting counterfeit and falsifiedbrewery products.RESULTS AND DISCUSSIONExtraction of DNA residues of beer rawmaterials. The technological level discloses a methodfor DNA extraction from wines [5, 6]. It was laterReview Article DOI: http://doi.org/10.21603/2308-4057-2019-2-364-374Open Access Available online at http:jfrm.ruDNA authentication of brewery products: basic principlesand methodological approachesLev A. Oganesyants1 , Ramil R. Vafin1,* , Aram G. Galstyan2 , Anastasia E. Ryabova1 ,Sergey A. Khurshudyan1 , Vladislav K. Semipyatniy1 All-Russian Research Institute of Brewing, Non-Alcoholic and Wine Industry, Moscow, Russia2 All-Russian Dairy Research Institute, Moscow, Russia* e-mail: vafin-ramil@mail.ruReceived May 17, 2019; Accepted in revised form August 08, 2019; Published October 21, 2019Abstract: 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 thecellular debris of the final product. The aim of the study was to analyse scientific and methodical approaches to extraction of residualquantities of beer raw materials nucleic acids and beer DNA authentication for their later application in determining brewing productsauthenticity. The technological level discloses the method of DNA extraction from wines, modified for extraction of nucleic acidsfrom beer samples. The method includes the following characteristic peculiarities: stage enzymatic hydrolysis of polysaccharidesand polypeptides of dissolved lyophilisate, multiple sedimentation and resursuspension of nucleoproteid complex, RNA removalfollowed by DNA extraction by organic solvents, and additional DNA purification by magnetic particle adsorption. This reviewpresents the analysis of genetic targets used as molecular markers for gene identification of malting barley varieties and beer DNAauthentication. 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 suitablefor beer DNA authentication, are also presented. We also analysed genetic targets used in malting barley substitute detection, as wellas 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, PCRPlease cite this article in press as: Oganesyants LA, Vafin RR, Galstyan AG, Ryabova AE, Khurshudyan SA, Semipyatniy VK. DNAauthentication 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 InternationalLicense (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. 2E-ISSN 2310-9599ISSN 2308-4057365Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374modified for extraction of nucleic acids from beersamples [3]. The method includes the followingcharacteristic peculiarities: stage enzymatic hydrolysisof polysaccharides and polypeptides of dissolvedlyophilisate, multiple sedimentation and resursuspensionof nucleoproteid complex, RNA removal followed byDNA extraction by organic solvents, and additionalDNA purification by magnetic particle adsorption.Figure 1 demonstrates stages of DNA extractionaccording to the modified method. In particular,enzymatic hydrolysis of polysaccharides by α-amylase(Bacillus licheniformis) takes 3 h instead of 1 h, whenDNA is extracted from wines [3, 5]. The time ofenzymatic hydrolysis of polypeptides by proteinaseK (Tritirachium album) is also increased up to 3 h.The sedimentation time of non-hydrolysed cellulardebris by centrifugation at 8000 g is reduced to 1 mininstead of 15 min when DNA is extracted from wines.At the stage of DNA extraction from the lyophilisedbeer powder, the sedimentation of the nucleoproteincomplex is carried out by mixing the supernatant withtwo volumes of cold absolute ethanol instead of twovolumes of cold isopropanol. At the next stage we mixeda solution of unpurified DNA with an equal volumeof 70% ethanol. The maturing of the mixture at 0°Ctakes 3 min instead of 10 min, as with wines. Duringthe subsequent nucleoprotein complex sedimentation,along with the stepwise addition of 10 μL of 3Msodium acetate and two volumes of cold isopropanolto the pre-transferred transparent supernatant, 3 μL ofEthachinmate linear polyacrylamide is added. AfterRNA removal and deproteinisation, the sedimentationof purified DNA is carried out without adding 70%ethanol. (Сf. DNA extraction from wines involves inthe nucleic acids sedimentation in 0.2 M NaCl and twovolumes of cold ethanol, followed by washing with 70%ethanol). Later, nucleic acids precipitate, resuspended inthe elution buffer, undergoes an additional purificationby adsorption on magnetic particles, which is one of thekey modification elements of the method for extractingresidual DNA of beer raw materials [3].The ability of magnetic particles to bind DNAreversibly and easily be deposited from the suspension inthe magnetic field ensures high quality of nucleic acidspurification and their preservation. Magnetic particles,as a rule, are a paramagnetic core with a highlydeveloped surface covered with a polymer film withexposed covalent-bond carboxylic groups. Magnetictripods, used in manual and automated modes, are madeof neodymium magnets resistant to demagnetisation.The additional purification by adsorption on magneticparticles of the modified method of extraction of nucleicacids from beer samples actually took the place ofpolymer polyvinylpyrrolidone widely used to reduce theinhibitory effect of polyphenols on PCR [3, 7–10].Approaches to beer DNA authentication. Genetictargets, used as molecular markers for malting barleyvarieties identification, can also be analysed forcommercial beer DNA authentication (Table 1) [3].Polygalacturonase is an enzyme that performshydrolytic cleavage of α-1,4-glycoside bonds inpectin. The DNA target was the locus of its gene(HvPG1) еamplified by a corresponding pair of primersconstructed by Pulido et al. based on the analysis ofexpressed sequence tag (EST) deposited in GenBank(A/N: EF427919) [11]. The generated PCR products aand b of the HvPG1 gene locus detected in the barleyand beer samples were 89% and 79% identical to thepreviously deposited nucleotide sequence mRNApolygalacturonase Hordeum vulgare. Among the studiedJapanese barley varieties, only the high quality ‘Ryofu’,recommended for brewing, generated two discretefragments (a, b), like most American and Australianbarley varieties, except for Stimling (Table 2). Allthe beer samples were marked only by the country ofmanufacture. They generated the PCR product b andmore than half of the samples generated the additionalfragment a (Table 2). The analysed DNA target wasincluded in the group of DNA markers of identificationand differentiation of beer samples, but did not correlatewith the indicators of beer quality [3].Hordeins are polymorphic proteins of barley graincoded by 7 HrdA-G loci which are localised in theshort arm of the 5th Hordeum vulgare chromosome[12, 13]. Due to the established connection of thehordein-coding loci alleles with brewing qualitiesof barley grain, this block of targets is a priority formolecular and genetic analysis [14, 15]. From the threeanalysed loci (HrdA, HrdB and HrdC) only one (HrdC)was able to identify a single sample of beer out of 22investigated 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 comparedbarley varieties with corresponding reference sequence(GenBank A/N: AF474373) indicates a certain potentialof DNA authentication of beer on the analysed targetby sequencing the amplified locus. The block of DNAtargets under study also did not correlate with theindicators of beer quality [3].Amylosis content in barley starch influences thequality of malt barley. Therefore, waxy-barley varietiesmay be a preferred option for their malting in brewingbecause starch with low amylosis content is moresusceptible to enzymatic hydrolysis [18]. Molecularmechanism is embedded in Hordeum vulgare waxygeneslocated on 7 HS chromosome. They lead to theelimination of granule-bound starch synthase (GBSS)[18, 19]. Primers selected for Waxy-locus amplificationhad the positive control status due to generation ofspecific PCR product in all the samples of barley andbeer [3]. Their sequeneed nucleotide DNA sequenceswere identical to each other and showed 98% identityto the corresponding reference Hordeum vulgare subsp.Vulgare sequence, previously deposited to GenBank(A/N: X07931) [20].366Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–3741. LIOPHILISED BEER POWDER RESUSPENDINGLyophilisate dissolving in 500 μl of resuspending buffer(0.1M Tris-HCl (pH 8.0), 0.1 MNaCl)2. POLYSACCHARIDES ENZYMATIC HYDROLYSISBeer suspension processing with 100 μl of thermostable α-amylase (Bacillus licheniformis)Incubating the produced mixture at 80°C for 3 h3. POLYPEPTIDES ENZYMATIC HYDROLYSISSuspension processing with 100 μL of proteinase K (Tritirachium album) with 0.2% SDSIncubating the produced mixture at 55°С for 3 h4. NON-HYDROLYZED CELL DEBRIS SEDIMENTATIONCentrifugation at 8000 g for 1 min at 40°CSupernatant transfer to a new tube5. NUCLEOPROTEID COMPLEX SEDIMENTATIONMixing the supernatant with two volumes of cold absolute ethanoland holding the mixture at 0°C (on ice) for 15 minCentrifugation at 8000 g for 15 min at 4°C6. NUCLEOPROTEID COMPLEX RESUSPENDINGSediment resuspending in 300 μL of elution buffer (0.1M Tris-HCl (pH 8.0), 0.1M EDTA)7. NUCLEOPROTEID COMPLEX SEDIMENTATIONMixing the crude DNA solution with an equal volume of cold 70% ethanoland holding the mixture at 0°C (on ice) for 3 minTransfer the clear supernatant to a new tube and stepwise addition of 10 μL of 3M sodium acetate, 3 μL of linearpolyacrylamide Ethachinmate and 2 volumes of cold isopropanolCentrifugation at 8000 g for 15 min at 4°C8. NUCLEOPROTEID COMPLEX RESUSPENDINGSediment resuspending in 300 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA)9. RNA REMOVALSuspension Treatment with RNAse A at 55°C for 30 min10. DEPROTEINISATIONExtraction with equal volume of neutral phenolRecovery 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°C11. SEDIMENTATION OF PURIFIED DNARepeat stage 7 without adding 70% ethanol12. RESUSPENDING OF SEDIMENTAL DNASediment 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 PARTICLESFigure 1 Stages of DNA extraction from lyophilised beer powderLL1. LIOPHILISED BEER POWDER RESUSPENDINGLyophilisate dissolving in 500 μl of resuspending buffer(0.1M Tris-HCl (pH 8.0), 0.1 MNaCl)2. POLYSACCHARIDES ENZYMATIC HYDROLYSISBeer suspension processing with 100 μl of thermostable α-amylase (Bacillus licheniformis)Incubating the produced mixture at 80°C for 3 h3. POLYPEPTIDES ENZYMATIC HYDROLYSISSuspension processing with 100 μL of proteinase K (Tritirachium album) with 0.2% SDSIncubating the produced mixture at 55°С for 3 h4. NON-HYDROLYZED CELL DEBRIS SEDIMENTATIONCentrifugation at 8000 g for 1 min at 40°CSupernatant transfer to a new tube5. NUCLEOPROTEID COMPLEX SEDIMENTATIONMixing the supernatant with two volumes of cold absolute ethanoland holding the mixture at 0°C (on ice) for 15 minCentrifugation at 8000 g for 15 min at 4°C6. NUCLEOPROTEID COMPLEX RESUSPENDINGSediment resuspending in 300 μL of elution buffer (0.1M Tris-HCl (pH 8.0), 0.1M EDTA)7. NUCLEOPROTEID COMPLEX SEDIMENTATIONMixing the crude DNA solution with an equal volume of cold 70% ethanoland holding the mixture at 0°C (on ice) for 3 minTransfer the clear supernatant to a new tube and stepwise addition of 10 μL of 3M sodium acetate, 3 μL of linearpolyacrylamide Ethachinmate and 2 volumes of cold isopropanolCentrifugation at 8000 g for 15 min at 4°C8. NUCLEOPROTEID COMPLEX RESUSPENDINGSediment resuspending in 300 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA)9. RNA REMOVALSuspension Treatment with RNAse A at 55°C for 30 min10. DEPROTEINISATIONExtraction with equal volume of neutral phenolRecovery 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°C11. SEDIMENTATION OF PURIFIED DNARepeat stage 7 without adding 70% ethanol12. RESUSPENDING OF SEDIMENTAL DNASediment 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 PARTICLES367Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374Hemicelluloses are vegetable homo- andheteropolysaccharides, which are an integral part of theendosperm cell walls. The highest content of xylaneswas reported to be among the main components ofhemicellulose [21]. The malt barley softens as a result ofthe decomposition of the cell wall. Xylanase is involvedin the degradation of xylanes to xylooligosarachides,whose gene locus was used as a target for primersoriginally 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 thesame time, due to possible obtaining inconclusive data,the authors [3] presented neither the results of PCR of beersamples, nor data on amplification of the HrdB locus.Barley Z proteins are the main beer protein whichinfluence beer quality, especially foam stability [22–24]. In addition, Z4 and Z7 proteins can be used aspositive and negative markers of foam stability [25].DNA-markers of foam stability developed by Limureet al. were also used in by Nakamura et al. for barleyvarieties identification and beer DNA-authentication[3, 25]. Identifying and differentiating barley andbeer samples procedure by the gene locus, encodingproteins Z 4 a nd Z 7 d iffer. I n t he fi rst c ase P CRanalysis is performed by interpreting three discretePCR products (h, i-a, i-b), and in the second –by thepresence or absence of a specific fragment j.Based on the analysis, the authors recommended thefurther use of the tested primers for amplification of theanalysed gene loci [3]. In addition, a negative correlationof the amplified PCR product h gene locus encodingZ7 protein with beer bitterness, as well as a positivecorrelation of PCR product i-a similar locus with foamstability (Table 1) were revealed.Many enzymes, incl. α-amylase and β-amylase,are activated in the malting process [26, 27]. Theirsubstrates are amylosis and amylopectin or productsTable 1 Genetic targets used as molecular markers for brewing barley varieties identification and beer DNA authenticationTarget PCR product Primer sequence Correlation (+/-) SourcePolygalacturonase (HvPG1) a F: 5/-GACAGAATGGCGTTCAAGAACAT-3/R: 5/-AGCAAGTTGCCTTCCAGCTTGAT-3/N/A [3, 11]b N/AHordein 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/AHordein 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/AProtein 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/ABarley 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 genelocus 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 tasteN/A not applicable368Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374of their hydrolysis. Primers developed on the basisof nucleotide sequence of the gene locus encodingα-amylase initiated the amplification of PCR product kin most of the barley varieties and beer samples (Table 2)[3, 28]. It is noteworthy that the amino acid sequence ofthe target had 69% identity with Mla-locus of resistanceto powdery mildew Hordeum vulgare (GenBank A/N:AF427791) [29]. The used set of primers was includedin the group of molecular labeling systems of barleyvarieties, and therefore has a certain potential ofpractical application for beer authentication, althoughthe authors did not mention it [3].Lipoxygenase-deficient barley varieties with reducedor lost activity of LOX genes have a positive impact onquality 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 ina small number of studied barley varieties and in morethan half of beer samples, whose sequenced nucleotidesequences had 99% identity with the reference sequenceTable 2 Interpreted results of PCR analysis of brewing barley varieties and beer samplesBarley varieties PCR productsa b c d–1 d–1 e f g h i–a i–b j k l m n o p sVlamingh + + – + + – + – – – + – + – – – – – –Hamelin + + – + + + + – + + + – + – – – – + –Stimling – + – – + + + – – + – – – – – – – – +Bardin + + – – + + + – – + – + + – – – – + +Salute + + + – + – + – – – + – + – – – – – +Schouner + + – + + – + – – + – + – – – – – + –Maritime + + – – + + + – – – + + + – – + – + +Flag ship + + – – + – + – – + – – – – – – – + +Metkafe + + – + + – + + + + + + – + + + – + +Harushizuku – + + + – – + – – + – + + – – – + – –Houshun + – + + – – + – – + – – – – + + – – –Mikamogolden – – + + – – + – – – + + – – + – + – –Skygolden – + + + – + + – – – + + + – + – + – –Nishinohoshi – – + + – + + + – + – + – + + + – – +Nishinochikara – – – + – + + – – + – + + + + – + + +Ryofu + + + + – + + + – – + + + + + + – + +Samples of beer PCR productsa b c d–1 d–1 e f g h i–a i–b j k l m n o p sCzechoslovakia–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 signaln/a not applicable369Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374Table 3 SSR-markers of nuclear DNA Hordeum vulgare used for genetic identification of brewing barley varieties, potentiallysuitable for beer DNA authenticationNo. SSR marker The sequence of oligonucleotide primers Allel lenghts, bp Number of alleles1 Bmac 0040 F: 5/-AGCCCGATCAGATTTACG-3/ 196-226 bp(196/200/208/214/220/226)6R: 5/-TTCTCCCTTTGGTCCTTG-3/2 Bmac 0134 F: 5/-CCAACTGAGTCGATCTCG-3/ 140-174 bp(140/144/162/168/174)5R: 5/-CTTCGTTGCTTCTCTACCTT-3/3 Bmag 0125 F: 5/-AATTAGCGAGAACAAAATCAC-3/ 128-148 bp(128/132/138/144/148)5R: 5/-AGATAACGATGCACCACC-3/4 Bmag 0211 F: 5/-ATTCATCGATCTTGTATTAGTCC-3/ 150-170 bp(150/154/162/1704R: 5/-ACATCATGTCGATCAAAGC-3/5 Bmag 0222 F: 5/-ATGCTACTCTGGAGTGGAGTA-3/ 140-178 bp(140/144/162/168/170/174/178)7R: 5/-GACCTTCAACTTTGCCTTATA-3/of locus LoxA-gene Hordeum vulgare (GenBank A/N:L35931) [3, 34, 35]. The tested set of primers wasrecommended for further use in the amplification of theanalysed gene locus for barley varieties identificationand beer brands differentiation. It should also be notedthat the authors [3] additionally revealed a positivecorrelation between the amplified DNA target and beertaste saturation (Table 1).The selection of barley varieties with geneticresistance to viral, bacterial and fungal diseases isaimed at high-quality grain production [36]. A numberof DNA markers of resistance of barley to yellow mosaicvirus (rym5-locus) and powdery mildew (mlo-locus) [34]integrated into breeding programs can also be used inmolecular labelling of brewing barley varieties, whichis clearly demonstrated in the work [3]. The authorsinterpreted the PCR analysis data of barley samplestaking into account the presence or absence of specificPCR products m (rym), n and o (mlo) recorded on thecorresponding electrophoregrams. But the results of thePCR analysis of beer samples and their correlation withquality indicators were not provided [3].Protein inhibitors of proteolytic enzymes playan important role both in formation of homeostaticreactions in plants and in the process of seed maturationand germination. Selected primers to the trypsininhibitor (Itr1) gene locus led to the amplificationof the specific PCR product p in half of the testedbarley varieties and beer samples [37]. Thus, the DNAmarker was concluded to be highly informative [3].Additionally, the DNA sequences of the Itr1-gene locusof the material had 94% identity with the same locusof the Hordeum vulgare subsp. vulgare gene (GenBankA/N: (X65875) [38]. Also, in the study [3] a negativecorrelation of the detected DNA matrix with beer tastesaturation was revealed (Table 1).The content (1–3, 1–4) of β-D-glucan in barley grain,which determines its hardness, is much higher comparedto other cereals [39]. However, for barley varieties usedin brewing, a lower the content of this polysaccharide inthe grain is desirable in order to achieve a more effectiveflow of the malting process [40]. The amplificationprocedure of the locus of the HvCslF6 gene with aselected primer pair led to the production of a specificPCR product s in a number of American, Australianand Japanese brewing barley varieties [3, 40]. The mostof the beer samples also gave a positive amplificationsignal (Table 2). The obtained amino acid sequenceof the target had 83% identity with Hordeum vulgareCslF6-gene (GenBank A/N: EU267181) [41]. The usedprimer set was also included in the group of systemsof barley varieties molecular labelling and beer DNAauthentication[3].Microsatellites are widely used molecular markerswhich 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 SSRmarkersfor brewing barley varieties identification [45].Table 3 shows the sequence of oligonucleotideprimers of the corresponding Hordeum vulgare SSRmarkersof nuclear DNA, as well as the range of lengthsof detected alleles and their number. The geneticidentification procedure includes PCR method withsubsequent data interpretation by horizontal or verticalgel electrophoresis and DNA fragmentary analysisof capillary gel electrophoresis. The SSR-markers,potentially suitable for beer DNA authentication, areadvisable to test in the formulation of single PCR, witha set of primers of a single SSR-marker to achieve areproducible result.Alongside with SSR-markers, SNP-markers, used forbarley varieties identification, including brewing ones,also have high identification capacity [46–48].Table 4 shows oligonucleotide primers sequencesof the corresponding SNP markers of Hordeumvulgare nuclear DNA, as well as the size of amplifiedloci of discriminated alleles [46]. The procedure ofgene identification is carried out by the AmplificationRefractory Mutation System (ARSM-PCR), followed bydata interpretation by horizontal gel electrophoresis orby high resolution melting curves (HRM) analysis onPCR platforms in real time. It should be mentioned thatwe selected five SNP-markers (out of nine described byChiapparino et al. [46] as potentially suitable for beerDNA authentication due to generation of relatively smallallele-specific PCR products, whose size was not morethan 200 bp (Table 4).370Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374Table 5 Genetic targets used in detecting brewing barley substitutes and identifying hops and yeast in beerTarget PCR product Primer sequence Correlation (+/–) SourceGBSS (rice) t F: 5/-GGATGAAGGCCGGAATCCTGR: 5/-CTTGCCCGGATACTTCTCCTmissing [3, 49]Β-conglycinin u F: 5/-TTTGGCATTGCTTACTGGGAAAAAGAGR: 5/-TCTGTAGGAGTCTCTGTCGTCGTTGmissing [3, 50]Zein v F: 5/-CACATGTGTAAAGGTGAAGCGATR: 5/-GCTCGCCGCAAGCGCTTGTTGmissing [3, 49]Hop-a w F: 5/-GGAACCGTTGCCTAATCCTAAGATTR: 5/-GTGTTTTCCGTATCTACGCGCTGGGmissing [3]Hop-b x F: 5/-AATTAGGGCATGCCATGAATATTR: 5/-TGGCATAGTTAAATTATTTCG–*–**[3]Hop-c y F: 5/-AAATAAAACTTTACATGTGATAR: 5/-CTGAATTGTCGGCGTmissing [3]Yeast-a(S. cerevisiae)z-a F: 5/-GTTTTGCGCTCATTAAAACCTAGTGGGAGR: 5/-GTCATTTTTTTTAGTGGTGCTAATC+***–****[3]Yeast-b (thioredoxin) z-b F: 5/-ATGGTCACTCAATTAAAATCCGCTTCTR: 5/-CTATACGTTGGAAGCAATAGCTTGCTTGmissing [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 umamiN/A is not applicableThe detection of brewing barley substitutes inbeer, which is often used as a cheap source of starch,makes it possible to evaluate the products sold forqualitative, quantitative, information and complexfalsification. Table 5 demonstrates primer sequencestargeting genetic targets used in the detection ofbrewing barley substitutes in beer, such as granuleboundstarch synthase of rice, β-conglycinin of soya,and zein of maize [49, 50]. Nevertheless, other PCRsystems developed for the identification of cerealsin food products can also be suitable for beer DNAauthentication [51].The effect of hops and yeast on beer quality iswell-known. Thus, hop has a bactericidal effect onbeer as well as provides its bitterness, aroma and foamstability [52]. Yeast is used in beer fermentation andTable 4 SSR-markers of Hordeum vulgare nuclear DNA used for brewing barley varieties identification, potentially suitable forbeer DNA authenticationNo. Locus (position) The sequence of oligonucleotide primers PCR product, bp1 MWG2062(325 A-G)FOP: 5/-GTTGTGTCAAGCATATCGGTTGCTCTT-3/ROP: 5/-CAGCACGTTCGAAAACAATAGGATCC-3/198 bpFIP: 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 bpFIP: 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 bpFIP: 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/-CCTCCTGATATACTTGAGAGACTCAATA74 bpFIP: 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 bpFIP: 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 primer371Oganesyants L.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 364–374impacts its character and taste [53]. Table 5 alsopresents sets of primers which initiate the amplificationof specific PCR products of the corresponding loci ofhops and yeast genes. They also allow the identifyingor differentiating of commercial beer samples [3].In addition, a negative correlation of the amplifiedPCR product t of the corresponding locus of the hopgene (Hop-b) with beer bitterness and astringencywas revealed. The amplified PCR product z-a of thecorresponding locus of the yeast gene S. cerevisiaeshowed a positive correlation with beer acidity anda negative correlation with beer umami [3]. Takinginto account the rapid development of genomic andbioinformation technologies, metagenomic analysis,which allows determining yeast species diversity inbeer samples without microorganisms allocating andcultivating, is one of the promising approaches to beerDNA authentication [54, 55].CONCLUSIONAnalysis of scientific and methodical approaches toextraction of residual quantities of nucleic acids of beerraw materials and beer DNA-authentication indicatesthe applicability of molecular and genetic analysis indetecting counterfeit and falsified brewery products.The use of DNA technologies helps determine theauthenticity and origin of the brewery industry products.Molecular labelling systems suitable for identification ofHordeum vulgare barley malt, or its substitutes, as wellas hops and yeast, can ensure traceability of the productlife cycle. Systematic data on correlation of amplifiedDNA targets with beer quality indicators can be ofpractical importance when choosing raw materials forbrewery production.CONFLICT OF INTERESTThe authors state that there is no conflict of interest.FUNDINGThe study was supported by the section ofstorage and processing of agricultural products of theDepartment of Agricultural Sciences of the RussianAcademy of Sciences and Federal scientific centre ‘FoodSystems’ of the Russian Academy of Sciences.