FERULOYLATED ARABINOXYLANS AS POTENTIAL BEER ANTIOXIDANTS

Dominik Szwajgier, Jacek Pielecki, Zdzisław Targoński
Department of Food Technology and Storage, Lublin Agricultural Academy, Poland

ABSTRACT

The purification of barley as well as malt extracts was performed, in order to evaluate free ferulic acid and water-soluble esterified ferulic acid contencentrations. In particular, the influence of elevated malting temperature (22°C according to “Activated Germination Malting” versus 14°C) and decreased pH level (5.2 versus 7.4) on bound ferulic acid concentrations was examined. In the case of both barley varietes studied, free ferulic acid comprised less than 0.6% of total concentration of this phenolic acid in barley grain. It was stated, that application of elevated malting temperature and decreased pH level of steeping water caused significant modifications of free and water soluble esterified ferulic acid concentrations in comparison to standard malting conditions (temperature 14°C, pH level 7.4). Water-soluble esterified ferulic acid concentrations in worts produced without exogenous enzymes and using commercial enzyme preparations were a couple of times higher than corresponding free ferulic acid concentrations. Ferulic acid present in wort in significant amounts in water-soluble esterified form of sugar esters could be a very attractive beer antioxidant.

Key words: ferulic acid, arabinoxylans, malt, wort, beer.

INTRODUCTION

Beer is a very popular alcohol beverage consumed in the world, rich in phenolic compounds, reponsible for antioxidant potential of this beverage [3, 4, 22, 33]. Brewing process causes the loss of phenolics in beers because of the precipitation with proteins or removal (e.g. using PVPP). Total antioxidant activity of beer, measured for example in the system containing low density lipoproteins (LDL) and human plasma ex vivo, is low in comparison to the antioxidant activities of wine and grape juice, green and black tea [40]. Ferulic acid (4-hydroxy-3metoxy cinnamic acid) is the main phenolic acid in barley, barley malt and beer and this compound is present in barley mainly in bound form as ester with arabinoxylan polymer, and only a minor part of total ferulic acid in barley kernel is present in free form [19, 20]. The esterificaton of ferulic acid to arabinoxylan polymers, as well as the role of ferulic acid in the structure of cell wall is described in detail [1, 14, 15, 32, 41]. Ferulic acid is a very effective antioxidant and anticancerogenic compound in vitro [24, 9, 21, 6, 34, 42].

From the technological point of view, only water-soluble arabinoxylans are interesting for brewers, because these fractions of arabinoxylans can be present in wort during mashing, and, in consequence, in beer in different stages of beer production, including ready-to-consumption beer. Non-soluble fractions of arabinoxylans, similar to other non soluble non-starch polysaccharides, proteins, minerals and other compounds are part of barley spent grain which is the main by-product of mashing. Because ferulic acid is esterified to arabinoxylan backbone via arabinofuranose residue, this potent and, what is important, natural antioxidant originating from barley can contribute to total antioxidant activity of beer. In presented experiment, the aim was studying the influence of elevated temperature during malting (22°C versus 14°C) according to “Activated Germination Malting” [17] and two pH values of steeping water (5.2 versus 7.4) on both forms of ferulic acid content in malt. Another aim was evaluation of influence of three commercial enzyme preparations application on changes of free and bound ferulic acid concentrations in worts.

MATERIALS AND METHODS

Barleys. Barley Krona was gained from Semundo Saatzucht GmbH, Barley Rudzik was purchased from Hodowla Roœlin Szelejewo, Poland.

Enzyme preparations. Three enzyme preparations used in brewing industry and possessing wide range of enzyme activities towards non-starch polysaccharides: Celluclast, Shearzyme, Viscozyme (Novozymes, Denmark), were used. The amounts of enzyme preparations applied to the grist was: 0.5 kg/1000 kg of malt grist (Ultraflo L), 1 kg/1000 kg of grist (Celluclast or Shearzyme) [38].

Malting in the laboratory scale. Lager-type malt was produced as described Nischwitz et al., [25] with slight modifications. 1 kg of barley grain was malted in a glass, vertical vessel (2 L). The total volume of water in the system was 4.5 L. The time-temperature program was as follows: 0–1 h – steeping the grain with simultaneous disinfection using 0.05% hydrogen peroxide solution 0.05%; 1– 24 h steeping the grain in water at pH level of 7.4 or pH 5.2; 24–34 h – air rest of the grain. Process was performed in chamber equiped with air flow and temperature control. The relative humidity was 95-100%; 36–48 h – steeping the grain in water; 48 h – germination. Malting was continued until the proper rootlets lenth was gained [18]. Kilning was performed in 4 successive steps: 10 h at 40°C; 10 h at 55°C; 4 h at 72°C; 4 h at 83°C. Directly after drying the malt, grain rootlets were removed using a strainer. After this, malt was stored before use for 4 weeks at temperature 4°C.

Extraction of free phenolic acids from barley and malt. Free phenolic acids were extracted from barleys and malts using methanol according to method of Maillard et al. [20].

Extraction of arabinoxylans from barleys and malts. Extraction was performed according to method proposed by Izydorczyk et al. [13] with slight modification. Sample of finely ground sample (40 g) was mixed with 100 cm³ of distilled water containing 0.05% sodium azide. The mixture was then shaken for 24 hours in shaker at ambient temperature and solid parts were centrifuged (5000g, 20 min.). alpha-amylase (porcine pancreas, E.C. 3.2.1.1. type 1-A, Sigma; 25 units) and protease (E.C.3.4.24.31, Type XIV Streptomyces griseus, 25 mg) were added to the supernatant, and the extract was incubated for 24 h at 30°C and pH 7.0 (water bath). Then pH was decreased to 5.5 and beta-amylase (Sigma Aldrich, EC 3.2.1.2, sweet potato, 250 units) and pullulanase (E.C. 3.2.1.41; Klebsiella pneumoniae, 10 units) were added and the mixture was incubated for 8 h at 30°C. After incubation, samples were boiled in water bath for 5 min., and centrifuged (5000 g, 30 min.). Xylanase activities or ferulic acid esterase activities (activities not desired) in alpha-amylase, protease, beta-amylase and pullulanase were absent. Before the application of enzymes, the enzymes were dissolved in Tris/HCl buffer (pH 7.0 for alpha-amylase and protease and pH 5.5 for beta-amylase and pullulanase). It was stated that purfied enzymes didn’t exhibit xylanase or ferulic acid esterase activities.

Wort production. All worts were produced using the infusion method, according to Kunze [18]. Malt for lager-type beer was ground in laboratory mill and two fractions were obtained: flour and bran. Wort was produced in Braun fermentor (B.Braun Biotech International, Typ C10-3) of the working volume 15 L, with the controlling unit Braun Biostat C Typ 884492/5. Heating was performed using steam generator. Tap water (pH 7.4) was used for wort production, and pH was decreased and continouosly controlled at pH level of 5.2 using 20% lactic acid solution. Cooling of worts was performed using tap water. During mashing, wort was continuously mixed (150 rpm). The mashing program comprised the following stages: 2.4 kg of ground malt was mixed in fermentor with water (10 L, 40°C), pH of the medium was brought to pH 5.2 then and mixture was warmed at: 52°C (20 min.), 63°C (20 min.), 75°C (20 min.). Sweet wort was then filtered at 75°C using barley spent grain as filtration medium following barley spent grain removal from fermentor, filling fermentor with sweet wort and boiling the wort with hops (2.011 g of hops granulate, alpha-acids content 4.2%) for 90 min. at 101°C.

Wort preparation for preparative chromatography. 100 mL of wort was centrifuged (7000 g, 30 min.). The sample was then concentrated in vacuum (40°C, 0.9 bar). Concentrated samples were injected into Sephadex LH-20 glass column as described below.

Amberlite XAD-2 and Sephadex LH-20 chromatography. 20 mL of malt extract or wort was injected into glass column (Amberlite XAD-2, 1.5 cm i.d., 80 cm length). Elution was performed according to Saulnier et al., [36] using distilled water with sodium azide (0.05%, 200 mL) and water:methanol solution (1:1_v/v; 200 mL) and methanol (200 mL). The eluent flow was 50 mL*h^-1. The water-methanol fraction and methanol fraction were gathered and concentrated in vacuum (0.95 bar) up to 50 mL. The following separations using Sephadex LH-20 were performed according to Ralet et al. [30]. Concentrated sample (6 mL) was injected into Sephadex LH-20 column (1.5 cm i.d., 80 cm). Column equilibration and separations were performed using distilled water with 0.05% sodium azide. The fractions of 7 mL were collected. The applied flow was 5 mL*10 min^-1. In each fraction obtained from Sephadex LH-20 column, free and bound ferulic acid concentrations were determined in HPLC system as described above. Bound ferulic acid was assayed after enzymatic hydrolysis (8 h) using a-amylase (30°C, Aspergillus oryzae, EC 3.2.1., Type X-A) which contained the side activity of ferulic acid esterase. Each time, 70 mg of a-amylase preparation was added to 7 mL of the fraction eluted from Sephadex LH-20.

HPLC analysis. HPLC in isocratic conditions, according to method presented by Zupfer et al., [43] was performed. The mobile phase was 13% methanol solution in citric acid buffer (0.01 mol*L^-1, pH 5.4). The high pressure chromatography unit consisted of: Rheodyne 0.02 mL loop, piston pump KNAUER, uv-vis LINEAR 200 (USA) detector and TZ 4620 recorder (Czech Republic). Symmetry® C₁₈ (Waters, length: 250 mm, i.d. 4.6 mm, 0.005 mm) RP column was used for separations. The flow of mobile phase was 0.4 mL*min^-1. Ferulic acid was detected at 320 nm. The average value of triplicates were done on the basis the wet matter of the grains and standard deviations were calculated.

RESULTS

Ferulic acid concentrations in samples
Table 1 presents the results of determination of total and free ferulic acid concentrations, in barley Krona and Rudzik.

In the case of both barley varietes, free ferulic acid comprised not more than 0.6% of total concentration of this phenolic acid in barley grain. Barley Rudzik had higher total ferulic acid content (59.79 mg*100 g^-1 of the grain) and simultaneously lower free ferulic acid content (0.129 mg*100 g^-1 of the grain) than barley Krona.

Free ferulic acid and esterified ferulic acid concentrations in barley, malt extracts and wort samples after purifications using Sephadex LH-20
Application of Amberlite XAD-2 resin and Sephadex LH-20 gel allowed to purify barley and malt extracts and to determine the concentrations of ferulic acid in free form as well as in the form of water-soluble arabinoxylan esters.

Figure 1 presents the concentrations of free ferulic acid and total water-soluble ferulic acid (the sum of free and esterified form of ferulic acid) in fractions obtained after purification of Krona barley extract. Total water-soluble ferulic acid was in significantly higher concentration than free ferulic acid. The highest free ferulic acid concentration was obtained in 9^th fraction (63 mL of the elution).

The sum of free ferulic acid content in all fractions purified using Sephadex LH–20 was 0.197 mg*100 mL^-1 and was about 2.5-fold lower than the content of total ferulic acid, which reached 0.698 mg*100 mL^-1.

In the case of all 3 Krona malt extracts, the water-soluble esterified ferulic acid concentrations were 7.0-8.7 times higher than corresponding free ferulic acid concentrations (Figures 2-4).

The lowest concentrations of free and total ferulic acid were determined in malt produced at temperature 14°C and using steeping water of pH 7.4. Both: elevated temperature during malting (Figure 3), and decreased pH value during the steeping and germination (Fig. 4) caused significant increase in concentration of both forms of ferulic acid, but the ratio of free form of ferulic acid to total water-soluble form of ferulic acid was constant in the case of all malts produced.

The results of ferulic acid contents determinations after purifications of Rudzik barley and malts extracts using Amberlite XAD-2 resin and Sephadex LH-20 gel are shown in Table 2. Free ferulic acid concentrations in samples of Rudzik malts were 1.5-4.2 fold lower than corresponding water-soluble esterified ferulic acid contents. Extracts prepared using all three Rudzik malts had the free ferulic acid content 2.8-5.5 fold higher than the extract prepared using Rudzik barley and 1.5-6.2 fold higher total ferulic acid contents than extract prepared using barley.

When comparing 3 malts produced using Rudzik barley, the lowest concentration of free ferulic acid was determined in malt produced at temperature 14°C and using steeping water of pH level 7.4. Elevated temperature during malting (22°C) without decreasing pH from 7.4 to 5.2 caused increase of free ferulic acid concentration and decrease of total ferulic acid concentration. Decreased pH (from 7.4 to 5.2) of water during steeping and germination caused, similarly as in the case of Krona malts, significant increase in concentration of both forms of ferulic acid.

Table 3 presents free ferulic acid and total ferulic acid concentrations in fractions obtained after purification of worts using Amberlite XAD-2 and Sephadex LH-20.

Fractions obtained after purification of wort produced with enzyme preparation Celluclast contained the similar content of free ferulic acid as in the case of wort produced without addition of any enzyme preparation. The reason of this result was probably the lack of ferulic acid esterase activity in Celluclast (two other enzyme preparations Shearzyme and Viscozyme possessed ferulic acid esterase activity; data shown elswhere). The concentration of total ferulic acid in purified wort produced using Celluclast was about 2 fold higher than in purified wort produced without any enzyme preparation. Shearzyme and Viscozyme released free ferulic acid to the wort much more effectively than Celluclast, but both enzyme preparations also effectively increased the total ferulic acid concentrations in experimental worts, in comparison to the wort produced without any enzyme preparation. The concentration of free ferulic acid in wort produced without egzogenous enzymes was 5.5 fold lower than the concentration of total ferulic acid in the same sample. In the worts produced using Celluclast, Shearzyme and Viscozyme, the concentrations of total ferulic acid were 10, 6.1 and i 6.3 times higher, respectively, than the concentrations of free ferulic acid in corresponding worts.

DISCUSSION

Ferulic acid, as the most abundant phenolic acid in barley, is a very attractive, natural antioxidant which could gain applause among consumers, presenting negative attitude towards artificial antioxidants added to foods. Free ferulic acid added to beer in low concentration (2-5 ppm) is very stable, whereas in higher doses its concentration rapidly decreases. The antioxidant activity of ferulic acid in beer is similar to the antioxidant activity of (+)-catechin, but (+)-catechin causes the formation of hazes in much lower concentration than ferulic acid [28]. Therefore, the attention is turned to phenolic acids, because they are easily absorbed to blood plasma in comparison to other, more complex compounds, for example anthocyanins [10]. It was proven, that phenolic acids undergo chemical and enzymic reactions in lesser extend and more slowly than flavonoids during absorbtion to blood plasma [5, 16]. Phenolic acids which are relatively simple structures, although undergo the influence of many factors during digestion and absorbtion into plasma, are not altered in huge extent, and could play important role in increasing the total antioxidant activity in vivo [7, 31, 23, 29, 8, 12].

Modifications of malting parameters like process temperature and pH of steeping water can very strongly influence many processes occuring during malting. “Activated Germination Malting” was proposed by some researchers in order to replace the standard temperature 14°C with elevated malting temperature around 22°C [17]. “Activated Germination Malting” was supposed to decrease the malting time without loosing the malt quality. The aim of the modification of the malting technique was induction of main malt enzymes activities appearance in earlier stages of steeping of the grain, by fast activation of embryo. Except the main desired enzyme activities induced during malting, many accesory enzyme activities are present, including non-starch polysaccharides like xylanases and ferulic acid esterase, which play a crucial role in arabinoxylan degradation during malting.

During malting, the increase of ferulic acid esterase activity was observed, the enzyme that plays a key role during degradation of arabinoxylan polymers present in cell walls of barley grain. [2, 35, 19]. Maillard and Berset [20] proved, that during malting, the proportions of main free phenolic acids (ferulic and coumaric acid) didn’t change, but the concentrations of both phenolic acids initially increased about 130% until the kilning step at 80°C, and then decreased about 20% after heating at 90°C. Free trans-ferulic acid and trans-coumaric acid contents were about 100-fold lower than the corresponding bound forms of both phenolic acids [19]. Also, the increase of free and water-soluble bound ferulic acid concentrations after malting could be caused by the degradation of grain tissues, what improved extraction of phenolic acids.

Arabinoxylans present in beers could comprise up to 10% of the total carbohydrate content, and these non-starch polysaccharides can be present in beers in much higher concentrations than beta-glucans. The dietary fibre content in beers is not negligible and ranges from 183 mg to 3534 mg/100mL, depending on the brand of beer. The highest dietary fibre content was detected in wheat beers, namely Doppelbock and Rauchbier [11]. Schwarz and Han [37] evaluated beta-glucan and arabinoxylan content in 15 commercial beers, differing in extract, alcohol and sugar contents. The arabinoxylan content ranged from 51.4 mg*100 mL^-1 in lager type beers to 421.1 mg*100 mL^-1 in the case of wheat beers. It must be emphasized, that beta-glucan contents in each mentioned beers were a couple of times lower than arabinoxylan contents and reached 24.77 mg*100 mL^-1. Similar results obtained Viëtor et al. [39], evaluating arabinoxylan content in worts.

Ferulic acid in the form of the sugar esters with arabinose or short oligoarabinoxylane chains exhibits higher antioxidant activity towards low density lipoproteins (LDL) than free ferulic acid. [26, 27]. Ohta et al. [26] using enzymatic methods, isolated from insoluble corn bran cell walls short oligoarabinoxylan fragments esterified to ferulic acid. The isolated structures were ferulic acid esters with arabinofuranose (FA-Araf) and trisaccharide consisting from two xylopiranose residues and one arabinofuranose esterified to ferulic acid (FA-Araf-Xylp-Xylp). Moreover, a number of compounds consisting of ferulic acid residue and the arabinoxylane moiety of higher molecular masses was identified. Antioxidant activities of isolated ferulic acid-oligoarabinoxylane molecules in the systems containing free fatty acid undergoing autooxidation, were higher than the corresponding activities of free ferulic acid. Similarly, oligoarabinoxylans of higher molecular masses, esterified by ferulic acid, exhibited higher antioxidant activities than FA-Araf and FA-Araf-Xylp-Xylp [27].

CONCLUSIONS

It can be stated, that malting procedure modification can influence the proportion of free ferulic acid and water-soluble bound ferulic acid concentrations in malts. Increasing the concentrations of water soluble bound ferulic acid, predominantly in the forms of esters with oligoarabinoxylans, can be introduced to the industry using commercial enzyme preparations possessing wide range of non-starch polysaccharides hydrolases, including ferulic acid esterase and xylanase. During the experiment, the molecular masses of feruloylated water-soluble arabinoxylans present in extracts from malts and in experimental worts were not evaluated, only the ratio of free ferulic acid concentration to ferulic acid esterified to arabinoxylans concentration could be stated. In the future, detailed characterisation of structures and compositions of ferulic acid arabinoxylan esters is planned.

ACKNOWLEDGEMENTS

Research work financed from the budget recources by State Committee for Scientific Research in the frames of Scientific Project PBZ/KBN/021/P06/99 in the years 2001-2004

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