Carlsberg Res. Commun. Vol. 46, p. 53-64, 1981 ANALYSIS OF PROANTHOCYANIDINS IN WILD-TYPE AND MUTANT BARLEY (HORDEUM VULGARE L.) by BARBRO JENDE-STRID and BIRGER LINDBERG MOLLER Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby Keywords: Beer stability, proanthocyanidin-free barley, high performance liquid chromatography, flavonoids The proanthocyanidin composition of wild-type barley was analyzed by high-performance liquid chromato- graphy. The use of authentic standards allowed tentative identification of procyanidin B-3, prodelphinidin B-3, and catechin as the major constituents. The proanthocyanidin content of different barley varieties (wild types) was found to be qualitatively and quantitatively similar. Mutants belonging to different gene loci and deficient in the biosynthesis of proanthocyanidins and other flavonoids were characterized. 1. INTRODUCTION can be made superfluous by the use of barley Permanent haze and chill haze in beer are varieties in which the biosynthesis of these caused by the precipitation of protein with constituents is genetically blocked. Towards this polyphenols derived from barley and hops, the goal 173 induced proanthocyanidin deficient starting materials in the beer production (l 6, barley mutants have been isolated and eleven of 20). Hitherto, haze formation has been prevented these used for experimental malting and brew- by filtration of the beer through polyvinylpoly- ing. It has been shown that stable beer of pyrrolidone or silica gel (l 9). Alternatively the excellent taste can be obtained from such a beer proteins are degraded with proteolytic variety of barley (29, 30). It is not known enzymes or various additives such as antioxi- whether specific or all proanthocyanidins of dants are used to prevent precipitation. As the barley are equally efficient in haze formation. most important phenols in haze formation are Quantitative as well as qualitative analyses of the proanthocyanidins (5), chemical stabilization these constituents in the mutants and their wild- Abbreviations:cat = catechin; gal = gallocatechin;HPLC = high performance liquid chromatography; PC = procyanidin; PD = prodelphinidin. 0105-1938/81/0046/0053/$ 02.40 B. JENDE-STRID& B. L. MOLLER:Proanthocyanidins in barley types are essential to answer this question and material to develop extraction methods and will also provide basic information on the procedures. In addition to Nordal, the two barley biosynthesis of proanthocyanidins. The quantita- varieties Gula and Triumph were analysed for tive and qualitative analysis of polyphenols is proanthocyanidins. The anthocyanin, proantho- difficult because of the large number of different cyanidin and catechin deficient mutants used in components normally present including products this study were obtained from our collection of of various degrees of polymerization which are 230 induced mutants. The mutants ant 2-15 and either natural constituents or are formed during ant 13-13 were obtained from the variety Foma, extraction procedures. Traditional quantitative mutants ant 2-20, ant 17-139 and ant 18-141 analysis of proanthocyanidins are based on were induced in Nordal, the mutants ant 13- colorimetric measurements after their conver- 152, ant 17-148 and ant 17-150 are derived sion to anthocyanidins upon heating in acid from Triumph, while the mutants ant 18-159, solution (9, 18). This conversion is not quantita- ant 18-168 and ant 19-109 originate from tive and does not permit distinction among Gula, Ark-Royal and Alf, respectively. The five different types of proanthocyanidins. genes ant 2, ant 13, ant 17, ant 18 and ant 19 In recent years gas chromatography and high have been identified by diallelic crosses. Among performance liquid chromatography (HPLC) 38 different genetically analysed mutants, eight have been used for separation and determination mutations were found in the gene ant 13, of flavonoids. VANCRAENENBROECK et al. (27) fourteen in the gene ant 17, fifteen in the gene studied the influence of proanthocyanidin and ant 18, and one in the gene ant 19. All mutants catechin content in beer on colloidal stability used except for ant 19-109 and ant 2-15 are with the aid of gas chromatography and a deficient in anthocyanins, catechins and proan- method for simultaneous quantitative determina- thocyanidins. The mutant ant 19-109 has a tion of biflavans and catechins was devised. normal anthocyanin content in its green parts However, proanthocyanidins of a higher molecu- but is proanthocyanidin and catechin free in the lar weight are less volatile and therefore difficult grain, whereas ant 2-15 is anthocyanin free but to analyse. produces a normal amount of proanthocyanidin High performance liquid chromatography has and catechin. proven to be an excellent tool for the separation of complex mixtures of flavonoids from plant material (8, 24, 25, 31). The HPLC method has 2.2. Extraction procedures also been used to analyse the proanthocyanidin Barley grains (10 g) were ground into a fine content of cider (! 7). Of the different types of powder using a Brown Type MX 32 homoge- column packings used (adsorption, gel permea- nizer. The freshly ground four was extracted tion, reversed phase and partition) a system with 50 ml of 75 % acetone (15) under vigorous based on reversed phase chromatography seemed shaking. The slurry was centrifuged for 5 min at most promising. Recently, the separation obtain- 25.000 x g after which the pellet obtained was ed using HPLC on reversed phase columns has re-extracted twice. The combined supernatants been greatly improved by JERtJMANlS (14), who thus obtained were, depending on their future also used this method to analyse proanthocyani- use, processed by either procedure A or B as dins and catechins from malt, hops and beer. In described below. Procedure A: The acetone the present study we have investigated the extract (approx. 150 ml) was delipidated with proanthocyanidin and catechin composition of 3 x 50 ml of petroleum benzine (bp. 40-60~ the grain from barley varieties and single gene The aqueous acetone phase was evaporated at mutants with an altered flavonoid content. 20 ~ on a Rotary evaporator to a final volume of approx. 30 ml. At this stage most of the acetone is removed and the extract becomes 2. MATERIALS AND METHODS cloudy. To prevent precipitation upon standing, 2.1. Plant material 5 ml of 96 % ethanol was immediately added. Grains of the malting barley variety Nordal The sample was clarified by centrifugation (Hordeum vulgare L.) were used as standard (9.000 • g, 15 min) before HPLC analysis. 54 Carlsberg Res. Commun. Vol. 46, p. 53-64, 1981 B. JENDE-STRID& B. L. MOLLER:Proanthocyanidins in barley Procedure B: The acetone extract (approx. Standard mixtures of dimeric and trimeric 150 ml) was delipidated in a separatory funnel proanthocyanidins isolated from barley were by use of 50 ml of benzene. The aqueous bottom provided by cand. polyt. H. OUTTRUP, Depart- phase was collected and evaporated to a small ment of Brewing Chemistry, Carlsberg Research volume to remove remaining acetone and Laboratory. benzene and was then extracted with 4 x 15 ml of ethylacetate (14). The combined ethyl acetate phases were evaporated to dryness (40 ~ in a 3. RESULTS Rotary evaporator and the residue dissolved in Analyses based on the vanillin test had shown 0.5 ml of 96 % ethanol. The extract was clarified that the proanthocyanidin content of the malting by centrifugation as described above. barley variety Nordal is similar to that of other barley varieties. This variety was used as a standard to develop an extraction procedure for 2.3. High performance liquid chromatography the proanthocyanidins of barley. The procedure Analyses were performed using a Waters is based on repeated extraction of these compo- Assoc. (Milford, Mass., US) instrument compo- nents using 75 % acetone followed by extensive sed of a Model 6000 A solvent delivery system, a removal of lipids through repetitive washings Model U6K injector, a Model 450 variable with petroleum benzine, HPLC analysis of the wavelength detector, a Data Module and a composition of the final extracts thus obtained is System Controller. All separations were carried shown in Figure 1. The efficiency of each out on a pBondapak Phenyl column (Waters extraction step was evaluated by HPLC analysis. Assoc.) by use of a gradient system. The initial The first two acetone extracts were found to conditions of the gradient system was 2 % acetic contain 82 and 17% of the total acetone acid and this was changed to a final concentra- extractable proanthocyanidin. No qualitative tion of 10 % acetic acid using program 8 of the differences between the two extracts were System Controller and maintained at l 0 % acetic observed. When the combined acetone extracts acid for sufficient time to elute all proanthocya- were washed with petroleum benzine no loss of nidins. The flow rate was 0.5 or 1.0 ml. min -I proanthocyanidins could be detected. and the elution was monitored spectrophotome- A different method for the extraction of trically at 281 nm. proanthocyanidins from malt, hops, and beer has been used by JERUMANIS (14). This procedure was also tested except that the fractionation step 2.4. Assays involving polyamide chromatography was omit- The major components absorbing at 281 nm ted. The proanthocyanidin pattern thus obtained were collected when eluted from the laBondapak is shown in Figure 2. It is both quantitatively Phenyl column. Each isolate was divided into and qualitatively different from that obtained aliquots, one of which was tested with vanillin with the previous extraction procedure. The total (30) and the other hydrolyzed with 5N HC1/ yield of proanthocyanidins is less than 50 % and ethanol for 20 min at 80 ~ to localize proanthocy- it is apparent that there is a specific loss of the anidins and catechins. The absorption spectra of more polar proanthocyanidins which cannot be the latter fractions were measured to distinguish quantitatively extracted from an aqueous phase between cyanidin (~, max 535 nm) and delphini- into ethyl acetate.