Lebensm.-Wiss. u.-Technol., 32, 535}539 (1999)

Organic and Phenolic Acids in Beer

Luigi Montanari, Giuseppe Perretti, Fausta Natella, Alessia Guidi and, Paolo Fantozzis

Luigi Montanari: Dept. BDBAF, University of Basilicata, Potenza (Italy)Giuseppe Perretti, Paolo Fantozzi: Institute of Agri-Food Industries, University of Perugia (Italy)

Fausta Natella, Alessia Guidi: National Institute of Nutrition, Rome (Italy)(Received March 11, 1999; accepted August 12, 1999)

In a previous study we evaluated the ewects of brewing on compounds potentially involved in the antioxidant defence system. Theobjectives of this paper were to determine organic acids and to characterize the quantity and quality of phenolic acids in four Italianlager beers. The data obtained on organic acids are in line with the values reported in the literature for malic, pyruvic, lactic, acetic,ketoglutaric, citric, succinic, citramalic and fumaric acid. Phenolic acid composition was determined using two diwerent high-performance liquid chromatography (HPLC) methodologies: HPLC-ECD (amperometric electrochemical detection) and HPLC-DAD(photodiode array detection). The most common phenolic acid was found to be m-coumaric followed by ferulic, o-coumaric, p-coumaricand 3-OH-benzoic. Vanillic, chlorogenic, homovanillic, p-OH-benzoic, 2,6-dihydroxybenzoic, syringic, gallic, protocatechuic, caweicand xnally, 3,5-dihydroxybenzoic acids were present in small quantities. The amount of phenolic acids is quite low in absolute terms andin relationship to data available in the literature. Statistically signixcant diwerences were not found between the two diwerent analyticalmethods utilized.

( 1999 Academic Press

Keywords: beer; phenolic acids; organic acids; beer analysis

Introduction

In a previous study we evaluated the e!ects of di!erenttechnological phases of brewing on some selected com-pounds potentially involved in the antioxidant defencesystem. During the production of beer the phenolic con-tent and antioxidant activity decreased from wort to beer(1). A new study was therefore undertaken to investigateorganic and phenolic acids.Organic acids (in addition to CO

2) a!ect acidity/pH and

the taste of beer (sourness, tartness, acidity) and havepositive physiological e!ects (diuretic, reduction in uricacid) (2}5). Phenolic compounds are present in allvegetable foods. Beer contains many polyphenols, themajority of which come from the malt, the remaindercoming from the hop (6). Due to their speci"c properties,they are very interesting technologically as well asphysiologically (7}14). They are involved in foam main-tenance, physical and chemical stability and shelf life(7,8). Phenolic compounds may also act as antioxidantsin the human body, for example, as protective agents

sTo whom correspondence should be addressed. Istituto di IndustrieAgrarie, Universita degli Studi di Perugia, Via S. Costanzo, Perugia,Italy, I-06126. Fax: #39 075 585 3939, e-mail: paolofan@unipg.it

The research was supported by a grant from ASSOBIRRA (ItalianBrewers' Association), Rome.

0023-6438/99/080539#05 $30.00/0( 1999 Academic Press All art

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against the oxidation of ascorbic acid and unsaturatedfatty acids (9). Both phenolic acids and polyphenols arepresent in beer (1). The number of reports on the com-position of and the analytical procedures for determiningorganic and phenolic acids in beer is limited (15}17).The "rst objective of this paper was to determine all theorganic acids by their action in several metabolic path-ways, and their technological in#uence on the taste andstability of beer (3}5). The second objective was to char-acterize the quantity and quality of phenolic acids andtheir possible interaction with other substances presentin beer.In this paper, the analytical}technological method wasdeveloped in close connection with the nutritional studyby Ghiselli et al. (18). They found that the intake of beersigni"cantly increased plasma antioxidant capacity. Theirdata on phenolic absorption suggest that beer is able totransfer its phenolic compounds to body #uids (18).

Materials and Methods

Analytical evaluations were carried out on the organicand phenolic acids in four representative Italian brandsof lager type beer following the scheme reported in Fig. 1.The same beers were dealcoholized through lyophili-zation and then restored by adding water. The acids werethen determined.

Article No. fstl.1999.0593icles available online at http://www.idealibrary.com on

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Fig. 1 Experimental protocol

lwt/vol. 32 (1999) No. 8

Four di!erent producers brewed the lager beers understudy. These beers were brewed with 30% corn grits andusing high gravity. Their parameters were pH 4.18}4.57,alcohol 0.45}0.48 mL/L, original extract 1.13}1.15 mL/L.

Organic acid determination by high-performance liquidchromatography

Reagents. Propionic acid, sodium hydroxide (NaOH),phosphoric acid, hydrochloric acid (HCl), ethyl acetate(EtOAc), methanol (MeOH), triethanolamine-hydro-chloride, sodium bicarbonate (NaHCO

3), ethylene-

diaminetetraacetic acid (EDTA) disodium salt anddihydrate, nicotinamide adenine dinucleotide reducedform (NADH

2), nicotinamide adenine dinucleotide

(NAD), distilled water (H2O) and activated carbon were

of analytical reagent grade.Lactic dehydrogenase (LDH) from porcine muscle(Sigma, article no. L.0762), glutamic-oxalacetic trans-aminase (GOT), glycylglycine, L-glutamic acid, L-malatedehydrogenase and L-malic acid were obtained fromBoeringer}Mannheim (article no. 139.068).

Instruments. High-performance liquid chromatography(HPLC) was performed on a system consisting of twoVarian 2020 pumps coupled to a 2010 gradient program-mer with an RI-3 Varian di!erential Refractive Indexdetector with a sensitivity of 10]10~6 *R

*. The data

were recorded with a Varian Vista DS-654 integrator.Chromatographic separation was achieved usinga Hamilton C

18precolumn and an Alltima C

18Alltech

(4.6 mm ID]250 mm) column. The mobile phase consis-ted of H

2O/H

3PO

4/MeOH (94:50:1 by vol) pumped at

a #ow rate of 0.8 mL/min at room temperature. Thesample injection volume was 100 kL with a loop injectionsystem using a Rheodyne 7025 valve (19).

Analysis. Lager beers were degassed in a rotary evapo-rator (rotovapor) at a temperature below 303C for30 min. Samples were decolorized with activated carbon(2 g/100 mL of beer) and percolated through "lter paper(Whatman 1; 11 km). Propionic acid (5 mL) was added tothe "ltered solution (internal standard solution 5 mg/mLH

2O) and then neutralized with 1 N NaOH to pH values

of about 7.50/8.00.

Anionic resin adsorption. A chromatographic column(1 cm ID]25 cm) "lled with Amberlite Resin IRA 400(anionic) or Dower 1X2 (ca. 10 mL) was wet with 20 mLof 0.1 N NaOH and washed with distilled water to pH

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7.0. After neutralization, the sample was slowly "lteredthrough this column in order to exchange all the organicacids. Afterwards, the sugar fractions were removed byrinsing with distilled water (ca. 25 mL) and the organicacids were released from the anionic resin using ca.25 mL of 0.1 N HCl and washed with distilled water untilthey reached a volume of 50 mL. The treated sample was"ltered at 0.4 km and was then ready for HPLC analysis.

Pyruvic acid determinationReagents used included a bu!er (pH 7.6). Triethanola-mine hydrochloride (9.32 g) was dissolved in distilledwater (80 mL), then the disodium salt and dehydratedEDTA (0.188 mg) were added. The pH was "xed at 7.6 byadding ca. 8 mL of 0.2 mol/L NaOH and by addingdistilled water until a volume of 100 mL was obtained.For the NADH

2solution, 10 mg of NADH

2disodium

salt were dissolved in 2 mL of NaHCO3/H

2O (5/95 v/v).

A new solution was prepared every week.For the LDH solution, 50 kL of LDH Sigma from por-cine muscle (450 U in 50 kL) were diluted at 2 mL withH

2O (225 U/mL). A new solution was prepared every

week (20).For the determination, 0.3 mL of beer sample, 2.5 mL ofbu!er (pH 7.6) and 0.2 mL of NADH

2solution were

placed in a 4 mL cuvette (l"1 cm light path). The ab-sorbance was measured at j"340 nm, then 0.1 mL ofLDH solution was added and the absorbance was mea-sured again, at the same wavelength, after 5 min. Thedi!erence in absorbance was due to NADH

2oxidation

and was proportional to the amount of pyruvic acid (20).

Malic acid determinationA UV method was adopted for the determination ofL-malic acid in foodstu!s and other materials (21).Solution 1 comprised glycylglycine, L-glutamic acid andstabilizers. Solution 2 comprised freeze-dried NAD, andsolution 3 was a suspension of GOT. Solution 4 com-prised L-malate dehydrogenase and solution 5 wasa standard solution of L-malic acid (21).The following were placed in a 4 mL cuvette: 1.00 mL ofsolution 1; 0.20 mL of solution 2; 0.90 mL of doubledistilled water; 0.01 mL of solution 3; 0.10 mL of sampleof beer (or H

2O for the control). The absorbance was

measured at j"340 nm (A1) then 0.01 mL of solution

4 was added and absorbance was measured again (A2), at

the same wavelength, after 5}10 min. For each sampleand for the control the di!erence in absorbance(A

2!A

1) was calculated and, using the equation

6

lwt/vol. 32 (1999) No. 8

*A"*A4!.1-%

!*A#0/530-

the quantity of L-malic acid(g/L) in the sample was determined using the equation:

C"2.977/e]*A (Eqn 1)

Phenolic acid determinationPhenolic acid determination was conducted on the samebeer samples following two di!erent methods to comparethe usefulness of two di!erent HPLC detectors.The "rst method was based on HPLC with amperomet-ric electrochemical detection (HPLC-ECD) (22), modi-"ed as follows. Reagents used were sodium dodecylsulphate (SDS), nitriloacetic acid and sodium phos-phate of analytical reagent grade.

Preparation of samples. Lager beer samples were dilutedwith the mobile phase (1:10) and were passed througha 0.22 km "lter (SRI nc). A 10 kL volume of "ltratewas used for analysis by HPLC with coulometric arraydetection.

Instruments and conditions. Samples were analysed witha Coulochem Electode Array System (ESA, model 5600,8E) consisting of an ESA 580 pump and an ESA SVSstatic gradient mixer. Chromatographic separation wasachieved using an Inertsil ODS-3 (GL-Science Inc.,Japan) column (4.6 mm ID]250 mm, particle diameter,5 km). For the binary gradient elution, mobile phaseA consisted of MeOH/H

2O (50% by vol) containing

34.7 km SDS, 0.1 M phosphate bu!er (pH 3.35), 50 nmnitriloacetic acid. Mobile phase B was the same composi-tion as A with the addition of 173.0 km SDS. The gradi-ent cycle consisted of an initial 10 min isocratic segment(6% B), a 20 min linear gradient (#1.2% B/min),a 10 min linear gradient (#7% B/min) and a 5 minisocratic segment (100% B) before returning to initialconditions.The detector 8-array was set from 0 to 960 mV in in-crements of 120 mV vs. palladium reference electrode atroom temperature.

Table 1 Organic acid content in four beers (mg/L$Sx)

Beer Malic* Pyruvic* Lactic Aceti

A Alcoholic 57$1aA 48$1aA 99$5aA 28$5Dealcoholized 61$1ba 47$1aa 81$9ba 11$1

B Alcoholic 61$2aA 48$1aA 66$6aB 159$9Dealcoholized 53$2bb 47$1aa 77$6ba 60$

C Alcoholic 40$1aB 33$2aB 186$7aC 153$1Dealcoholized 37$1bc 32$1ab 110$14bb 21$3

D Alcoholic 93$10aC 48$1aA 159$18aD 93$5Dealcoholized 76$3bd 47$3aa 166$8ac 19$1

Literatures 14}105 10}104 44}292 37}17

Values are means$Sx

for six determinationsLowercase Latin letters, internal comparison between alcoholic adi!erent dealcoholized beers; capital letters, comparison betweenstatistically di!erent at P(0.01*Found through enzymatic determinations (22)

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The second method was a modi"cation of the methodused by Montedoro et al. for olive oil using HPLC withphotodiode array detection (HPLC-DAD) (23). Notably,the concentration of phenolic acids by solid phase extrac-tion is followed by ethyl acetate/methanol extraction andby HPLC-DAD determination.

Statistical analysis. When the comparison was made be-tween the means of the two samples, and the number ofrepetitions was equal (organic acid comparison betweenalcoholic and dealcoholized beers), the analysis of vari-ance used was the paired t-test. When the number ofrepetitions was di!erent, however, the t-test was used(phenolic acid comparison between alcoholized and de-alcoholized beers). When the comparison was made be-tween the means of four samples, the one-way analysis ofthe variance was used with the Tukey test (organic acidcomparison among four beer samples).

Results and Discussion

The data reported in Table 1 are in line with the valuesreported in the literature for malic, pyruvic, lactic, acetic,ketoglutaric, citric, succinic, citramalic and fumaric acid(15). Only citric acid and, partly, malic acid are of pri-mary origin; the others are principally secondary prod-ucts of alcoholic fermentation (24,25).The total content of organic acids was between 451 and712 mg/L for the beers examined. The most commonorganic acid found was lactic (average 128 mg/L), fol-lowed by citric (116 mg/L), acetic (108 mg/L), succinic(68 mg/L), malic (63 mg/L), pyruvic and fumaric(44 mg/L). Small amounts of ketoglutaric (25 mg/L) andcitramalic acids (9 mg/L) were also found.Pyruvic and citric acid were not in#uenced by dealco-holization, while acetic acid was reduced by lyophiliz-ation in all beers because of its volatility. Malic, lactic,ketoglutaric, succinic, citramalic and fumaric acids wereslightly in#uenced.

c Ketoglutaric Citric Succinic Citramalic Fumaric

aA 20$3aA 108$10aAC 49$3aAD 2$0aA 42$3aA

ba 18$3aa 108$9aa 44$3aa 1$0ba 43$5aab

aB 8$1aB 131$16aA 62$3aA 21$2aB 41$4aA

2bb 11$3ab 148$5ab 42$9ba 23$3ab 52$3ba

8aB 26$2aA 99$6aBC 43$7aBD 10$1aC 63$4aB

ba 11$2bb 105$7aa 47$4aa 10$2ac 30$5bb

3aC 47$5aC 124$14aA116$11aC 4$1aA 29$4aC

3ba 4$3bc 119$8aa 96$8bb 2$1ba 32$8abd

1 0}20 56}230 36}166 6}15 presence

nd dealcoholized beers; Greek letters, comparison between fourfour di!erent beers. Values bearing the same superscript are not

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Table 2 Comparison between di!erent analytic methods for determination of free phenolic acid content in fourbeers (mg/L)

HPLC-ECD HPLC-DAD

Acids Alcoholic beers Dealcoholized beers Alcoholic beers Dealcoholized beers Literature*

Gallic 0.015$0.006aA 0.008$0.064A 0.034$0.027a 0.017$0.020 12}30Protocatechuic 0.007$0.001aA 0.018$0.043A 0.020$0.011b 0.023$0.004 n.r.3,5-dihydroxybenzoic 0.016$0.002aA 0.025$0.025A 0.014$0.025a 0.005$0.003 n.r.p-OH-benzoic 0.068$0.016aA 0.119$0.034B 0.017$0.013b 0.009$0.002 1}11.2Homovanillic 0.083$0.011aA 0.082$0.047A 0.024$0.012b 0.018$0.001 0.13}28.5Vanillic 0.062$0.063aA 0.232$0.189A 0.097$0.005a 0.054$0.005 n.r.3-OH-benzoic 0.115$0.294aA 0.114$0.067A 0.045$0.021a 0.049$0.043 0.3}3.6Ca!eic 0.019$0.002aA 0.038$0.006B 0.006$0.002b 0.006$0.005 1.4}8Syringic 0.037$0.019aA 0.115$0.025B 0.017$0.011a 0.004$0.006 0.5Chlorogenic 0.089$0.343aA 0.110$0.039A 0.028$0.023a 0.034$0.016 1.92}21p-coumaric 0.027$0.065aA 0.026$0.228A 0.129$0.005b 0.069$0.008 n.r.Ferulic 0.116$0.041aA 0.273$0.548A 0.274$0.051b 0.134$0.020 0.52}20.8m-coumaric 0.071$0.277aA 0.021$1.080A 0.330$0.044a 0.055$0.035 n.r.o-coumaric 0.185$0.748aA 0.517$0.267A 0.152$0.056a 0.056$0.010 n.r.2,6-dihydroxybenzoics 0.039$0.037A 0.259$0.259A n.d. n.d. n.r.

Values are means$Sx

for eight determinationsValues bearing the same superscript are not statistically di!erent P(0.01Small letters, comparison between di!erent methods; Capital letters, comparison by HPLC-ECD between alcoholized anddealcoholized beers* (22)sMeasured only by HPLC-ECD; n.r. not reported; n.d. not detected

lwt/vol. 32 (1999) No. 8

The data presented in Table 2, which reports phenolicacid composition determined by the two methodologiesutilized, show that the amount of each phenolic acid isquite low in absolute value and in relationship to dataavailable in the literature (15).Comparing the two di!erent analytic methods, statist-ically signi"cant di!erences were not found for eight of allthe phenolic acids detected. Comparing alcoholic withdealcoholized beers, statistically signi"cant di!erenceswere not found for 12 of the phenolic acids detected. Themost common phenolic acid was m-coumaric followedby ferulic, o-coumaric, p-coumaric and 3-OH-benzoic.Vanillic, chlorogenic, homovanillic, p-OH-benzoic, 2,6-dihydroxybenzoic, syringic, gallic, protocatechuic, ca!eicand "nally, 3,5-dihydroxybenzoic acids were present insmall quantities.Results from a previous study (1) indicated the presence ofabout 283 mg/L of nontannic and non#avonoidic (NTNF)phenolic compounds. This value is considerably higherthan the total concentration of free phenolic acids re-ported in this paper (0.949 and 1.187 mg/L respectively forHPLC-ECD and HPLC-DAD). This appears to be rea-sonable, as NTNF compounds may be linked by di!erentbonds to many organic compounds (i.e. sugar molecules,organic acids, etc.) and, therefore, not detected as singlemolecules. In fact, direct degassed beer injection (HPLC-ECD and HPLC-DAD) led to detection of only free phen-olic acids, while phenolic acids linked to other moleculeswere not detected as even part of NTNF compounds.Therefore, it appears that free phenolic acids representonly a part of the previously detected NTNF compounds.Beer pHs ranged between 4.18 and 4.57. Di!erent samplepreparation (adopting acidi"cation) or plasma assessmentafter beer intake (digestion at pH 1.2}1.3), leads to higheramounts of free phenolic acids (15}18).

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Conclusions

For this experiment, we can conclude that simple phen-olic substances with antioxidant power have been foundin beer with reliable analytical methodologies. An anti-oxidant e!ect may be exhibited in human physiology asreported by Ghiselli et al. (18). The majority of suchsubstances interact and may combine with each other,with di!erent types of bonds. Phenolic acids in particularcan also bond with other substances which are naturallypresent in beer, such as sugars, organic acids, nitrogenoussubstances and some metallic ions. Data are in agree-ment with the hypothesis that these bonds may a!ect theevaluation of the concentration of phenolic acids. Des-pite this, it is possible to predict that some of these bondscould be broken during digestion, thus forming freephenolic acids (18).In accordance with the recent data reported by Gromusand Lustig (6), our data on the presence of phenoliccompounds in beer show that further research is re-quired which is not limited to a single class ofminor components. Nowadays, it is no longer possible toconsider beer quality merely in terms of beer #avour,astringency, colour and colloidal stability, as done byBradley and Hughes (26). It is also necessary to assessimportance of antioxidants both in beer and in humanphysiology.

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