RELEASE OF FREE FERULIC ACID AND FERULOYLATED ARABINOXYLANS FROM BREWERY’S SPENT GRAIN BY COMMERCIAL ENZYME PREPARATIONS

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

ABSTRACT

Feruloylated arabinoxylans, obtained after enzymatic hydrolysis of arabinoxylans present in cereal by-products of food industry, can become a very attractive group of natural antioxidants that can be supplemented to human diet. In “in vitro” models, feruloylated arabinoxylans are more effective antioxidants towards low density lipoproteins oxidation and DPPH* free radicals “scavengers” than free ferulic acid. One possible way of obtaining bulk amounts of feruloylated arabinoxylans from industrial by-products (brewery’s spent grain, sugar beet pulp, wheat bran) is application of purified enzymes, xylanase and ferulic acid esterase, but this method is expensive. Another way of obtaining feruloylated arabinoxylans in industrial quantities is application of commercial enzyme preparations possessing many enzymatic activities towards non-starch polysaccharydes. In presented study, five commercial enzyme preparations were used in order to release feruloylated arabinoxylans from brewery’s spent grain: Celluclast, Viscozyme, Shearzyme, Cereflo and Ultraflo. All five enzyme preparations effectively released water-soluble esterified ferulic acid and free ferulic acid from brewery’s spent grain. Application of enzyme preparation Celluclast resulted in the highest yield of feruloylated arabinoxylans and in the lowest yield of free ferulic acid among all five preparations used, probably due to the lack of ferulic acid esterase activity in Celluclast. Enzyme preparations Viscozyme and Shearzyme very effectively released feruloylated arabinoxylans as well as free ferulic acid from brewery’s spent grain. The thermostabilities of xylanases in Viscozyme and Shearzyme were evaluated and attempt to partly inactivate ferulic acid esterases was made. It was proved, that heating of preparations Viscozyme and Shearzyme at 50°C prior addition to brewery’s spent grain resulted in decreased levels of free ferulic acid in solutions, whereas only a moderate decrease of feruloylated arabinoxylans concentrations occurred. In conclusion, it can be stated that enzyme preparations possessing except xylanase activity also ferulic acid esterase activity and other non-starch polysaccharides' hydrolases activities can be effectively used in order to obtain feruloylated arabinoxylans from brewery’s spent grain.

Key words: brewery’s spent grain, ferulic acid, xylanase, ferulic acid esterase, enzyme preparations.

INTRODUCTION

Brewery’s spent grain produced during mashing is the most precious by-product obtained during brewing. In the past the intensive search for useful and effective industrial utilisation of brewery’s spent grain has been performed. The result is a number of patents in this field. Important way of brewery's spent grain utilisation is its use as animal feed [19]. Another possible way is production of preparations containing high contents of easy digestible-proteins [10, 11, 12] or biogas production [5]. This by-product contains significant amounts of valuable components, like proteins, dietary fibre and fatty acids. The chemical composition of brewery’s spent grain is as follows: arabinoxylans 20-30%, other non-starch polysaccharides 44%, proteins ca. 24%, cellulose 17%, lipids 6%, lignins (4%), mineral components and traces of starch < 2% [1]. This composition depends among others on mashing programme and malt quality [27].

Arabinoxylans present in brewery’s spent grain in considerable concentrations are in the form of beta-1,4-xylanopiranose chains with xylose residues substituted with arabinose rests in C2 position (ca. 17% of xylose residues), C3 (ca 7% of xylose residues) or in both positions (approx. 16% of xylose residues). Most of the alpha-L-arabinose residues are present in the form of monomeric side chains, whereas a small part takes part in forming short chains of Araf-Araf-Xylp-Araf [26]. Ferulic acid residues form ester bonds with arabinose in 5-O position. The average content of ferulic acid in brewery’s spent grain is 0.32% of dry matter [24]. A number of in vitro and in vivo studies presented the positive impact of free or water-soluble esterified ferulic acid on the increase of antioxidant status of the measuring system. Ohta et al. [17], using enzymatic methods, isolated feruloylated arabinose and short feruloylated arabinoxylan consisting of two xylopiranose residues and one arabinose esterified to ferulic acid (FA-Araf-Xylp-Xylp). Moreover, a number of compounds consisting of ferulic acid residue and the arabinoxylan moiety of higher molecular mass (not estimated) was identified. Feruloylated arabinose (FA-Araf), in comparison to free ferulic acid, showed higher antioxidant activity towards the low density lipoproteins fractions (LDL) in the presence of Cu²⁺ [16, 17]. The higher antioxidant activity of feruloylated arabinoxylans suggests the affinity of the esters to the lipoproteins. Also, the reason of the higher antioxidant activity of feruloylated arabinose in comparison to free ferulic acid could be the presence the hydrophilic region as well as hydrophobic moiety in the ester structure. Antioxidant activities of ferulic acid-xylooligosaccharides esters in the systems containing free fatty acids 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 [16]. Also, sugar esters of ferulic acid were more potent antioxidants than ferulic acid in the system measuring the peroxidation of lipids originating from rat livers in the presence of CCl₄, as well as more potent DPPH* free radicals “scavengers”, which explains the activity of these compounds against lipid oxidation [16]. Esterified ferulic acid is less susceptible to transformation than free ferulic acid during heating in the presence of oxygen or by the action of bacterial contamination. This can lead to decreased formation of 4-vinylguaiacol, a compound that impairs a negative flavor to the beverages, for example beers [28]. Another study proved that feruloylated arabinoxylans isolated from wheat bran were very potent antioxidants in systems where the erythrocyte hemolysis mediated by the presence of free peroxyl radicals occurred [29].

In presented paper, the research focused on the increase of feruloylated arabinoxylans concentrations in brewery’s spent grain media by the maximal promotion of xylanase activity and decreasing the ferulic acid esterase activity by using five commercial enzyme preparations. The reason of undertaking such experiments was as follows. Application of purified endo-1,4-xylanase or ferulic acid esterase in order to gain high ferulic acid yields is not interesting from the economical point of view, because of the very high costs of purified enzymes. This goal is also well worth pursuing due to the lack of purified feruloylated arabinoxylans concentrates commercially available. Good results are obtained when microbiological enzyme preparations possessing many non-starch polysaccharides’ hydrolases activities are used, so the use of commercial enzyme preparations could decrease the costs of brewery’s spent grain utilisation [3].

MATERIALS AND METHODS

Brewery’s spent grain. Brewery’s spent grain was obtained from local brewery and directly stored at minus 20^oC until use. Brewery’s spent grain was gained after mashing using infusion method and no enzyme preparation was used during the process in the brewery.

Enzyme preparations. The following commercial enzyme preparations from Novozymes Denmark were used: Celluclast 1.5L FG, Cereflo 200 L, Shearzyme 500 L, Ultraflo L and Viscozyme L. The main enzyme activities towards non-starch polysaccharides were estimated in enzyme preparations after dialysis. Dialysis tubes (Sigma-Aldrich, 10 kDa cut-off) were filled with preparations (20 mL) and dipped in Tris/HCl buffer (0.05 mol × L^-1, pH 5.0. The buffer was changed every 12 h. After dialysis, the volume of preparations was adjusted to the initial one using Tris/HCl buffer. Samples were stored in a refrigerator and used within 1 week.

Determination of the optimum pH for xylanase activity in enzyme preparations. The determination was performed using method proposed by Biely et al. [4] with modifications, using birchwod xylan (10 g × L^-1). The optimal pH values for the xylanase activities in enzyme preparations were evaluated in the pH range 4.0-7.0 (in half of unit increments) using Tris/HCl buffer (0.01 mol × L^-1). 4 mL of xylan solutions (solutions prepared using magnetic stirring and at 50^oC in all buffers from the range 4.0-7.0) and 1 mL of preparation were incubated for 1 h at 37°C. Simltaneously, two blanks were prepared:

1. 1 mL of enzymatic preparation and 4 mL of Tris/HCl buffer of adequate pH.

2. 5 mL of Tris/HCl buffer containing xylan (10 g × L^-1).

After incubation, concentration of reducing substances was evaluated using standard method with 3.5-dinitrosalicylic acid and spectrophotometrically measured at 550 nm [15]. The mixture of 0.5 mL of the sample and 3 mL of DNS reagent was boiled for 10 min, cooled and 10 mL of distilled water was added.

Determination of thermostability of xylanases in Shearzyme and Viscozyme. Thermostabilities of xylanases in enzyme preparations were evaluated at temperatures 37°C-63°C. Samples of enzyme preparations (10 mL) were heated for 1 h, cooled and used for the determination of remnant xylanase activity as described above.

Determination of beta-glucanase activity in enzyme preparations. Beta-glucanase activity was determined using Tris/HCl buffer, (pH 5.2, 0.01 mol × L^-1). 0.1 mL of the enzyme preparation was transferred into Eppendorf tubes and 0.4 mL of laminarine solution (1%_w/v) in Tris/ HCl buffer (pH 5.2, 0.01 mol × L^-1) was added. The samples were incubated for 1 h at 50°C. After incubation, concentration of reducing compounds was evaluated using standard method with 3,5-dinitrosalicylic acid [15].

Determination of cellulase activity in enzyme preparations. Cellulase activity determination was performed in Tris/HCl buffer, (pH 5.2, 0.01 mol × L^-1). 0.1 mL of the enzyme preparation, 1.3 mL of Tris/HCl buffer and 50 mg of Whatman cellulose paper were transferred intto Eppendorf tubes. Samples were incubated for 1 h at 50°C. After incubation, concentration of reducing substances was evaluated using standard method with 3,5-dinitrosalicylic acid [15].

Determination of ferulic acid esterase activity in enzyme preparations. The method proposed by Sancho et al., [22] was applied, using methyl ferulate as substrate. The solution of the substrate was prepared by solubilisation in 0.5 mL of pure methanol following addition of 9.5 mL of the buffer solution (50 mmol × L^-1 of Tris/HCl, pH 5.0); the concentration of the substrate was 8 mmol × L^-1. In the next step, 0.7 mL of the substrate solution and the enzyme preparation (0.1 mL) were put into an Eppendorf tube. Samples were incubated for 5 h at 30°C. After incubation the enzyme was inactivated in a boiling water for 5 min and the samples were then cooled and centrifuged (30 min, 5000 g). Simultaneously, blank samples containing enzyme preparation with inactivated ferulic acid esterase (5 min, in boiling water) and methyl ferulate were incubated. After incubation, free ferulic acid was separated from the corresponding methyl ester using HPLC with UV detection according to the method of McCrae et al. [13]. HPLC was performed in an isocratic system. The eluent was water:buthanol:acetic acid 93.8:6.0:0.2. The HPLC system consisted of: Rheodyne loop (20 µL), a KNAUER mini piston pump, uv-vis detector LINEAR 200 (San Jose, CA, USA) and a TZ 4620 recorder (ECOM s.r.o. Praha, Czech Republic) Column Symmetry® C₁₈ (Waters, 250 mm, 4.6 mm i.d.,5 µm) was used for separations. The flow was 0.8 mL × min^-1. The detection of ferulic acid was performed at 320 nm. The eluent was prepared in a distilled water and degassed in an ultrasonic bath before use. Concentrations of free phenolic acid were determined by using the heights of the chromatogram peaks. The series of methanol solutions of phenolic acids were prepared and the calibration curves were constructed.

Determination of the acetic acid esterase activity in enzyme preparations. Enzymatic activity was determined by using p-nitrophenyloacetate as a substrate according to method of McCrae et al. [13] with modifications. P-nitrophenyloacetate solution (0.01 g × 100 mL^-1) was prepared using 96% water solution of ethanol. Enzyme activity was determined after 1 h of incubation at 25°C. The composition of the reaction mixture was as follows: 0.1 mL of the enzyme preparation, 10 mL of 0.01 mol/L Tris buffer (pH 7.0), 0.5 mL of p-nitrophenyloacetate solution. Two blank samples were prepared:

1. mL of enzyme preparation, 0.5 mL of ethanol and 10 mL of 0.01 mol/L Tris buffer (pH 7.0);

2. 10.1 mL of 0.01 mol × L^-1 Tris buffer (pH 7.0) and 0.5 mL of p-nitrophenyloacetate in ethanol solution.

Absorbances of incubated samples were read after 30 min at 430 nm. The standard curve was prepared by dissolving of 5 mg of p-nitrophenyloacetate in 5 mL of ethanol. From this solution, aliquots of 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL and 0.5 mL were completed to the volume of 10.6 mL using 0.1 mol × L^-1 NaOH solution [13].

Inactivation of ferulic acid esterase in Shearzyme and Viscozyme. Partial inactivation of ferulic acid esterase in enzyme preparations was performed at 50°C, pH 5.0 for 15-60 min. The samples of preparations (10 mL) were then cooled and used for the incubation with brewery’s spent grain.

Incubation of brewery’s spent grain with enzyme preparations. 100 mL of Tris/HCl buffer (0.1 mol × L^-1) was added to 100 g of brewery’s spent grain; the pH of the buffer depended on optimal pH level of xylanase activity present in commercial enzyme preparations and was as follows (see Fig. 1): pH 5.0 for Celluclast, Shearzyme and Viscozyme, 6.5 for Cereflo and Ultraflo. Brewery’s spent-grain was incubated with enzyme preparation (0.3 g of the enzyme preparation/ 100 g of brewery’s spent grain) at 30°C for 20 h, and aliquots were taken after 4, 8, 12 and 20 h of incubation. In order to avoid the degradation of the substrate during incubation due to microbial contamination, 0.05% of sodium azide was added. After incubation, samples were boiled for 5 min in order to inactivate enzyme activities, cooled, centrifuged (30 min, 5000g) and frozen until free and water-soluble esterified ferulic acid concentrations were determined using HPLC with UV detection.

HPLC analysis of free ferulic acid. HPLC in isocratic conditions, according to the method presented by Zupfer et al., [30] was performed. The mobile phase was 13% _v/v methanol solution in aqueous citric acid buffer (0.01 mol × L^-1 pH 5.4). The HPLC unit consisted of: Rheodyne 20 µL loop, piston pump KNAUER, uv-vis LINEAR 200 detector (San Jose, CA, USA) and TZ 4620 recorder (ECOM s.r.o. Praha, Czech Republic). Symmetry® C₁₈ (Waters, length: 250 mm, i.d. 4.6 mm, 5 µm) RP column was used for separations. The flow of mobile phase was 0.4 mL µ min^-1. Free ferulic acid was detected at 320 nm. HPLC analyses were performed in triplicates and mean values were calculated. Concentrations of free ferulic acid in brewery’s spent grain samples were expressed on the basis of wet matter. In the same samples, water-soluble esterified ferulic acid concentrations were estimated after mild alkaline hydrolysis (as described below) followed by HPLC and subtracting the free ferulic acid content before hydrolysis. Water-soluble esterified ferulic acid concentrations were calculated from the increase of free ferulic acid in samples.

Mild alkaline hydrolysis. Mild hydrolysis with NaOH solution was performed in order to release the free ferulic acid from feruloylated arabinoxylans present in brewery’s spent grain medium incubated with enzymes preparations. Mild alkaline hydrolysis was performed according to Pan et al., [18] using NaOH solution (0.5 mol × L^-1) by mixing with the sample in ratio 1:1_v/v. After 24 h of incubation at room temperature and in darkness, pH of the samples was adjusted to 4.5 using 0.2 mol × L^-1 HCl solution and samples were analysed using HPLC-UV as described above.

Repetability and reproducibility of the HPLC method. The repeatability and reproducibility of the HPLC method were evaluated as described before [25]. In short, the repeatability of the HPLC method was determined within one day using ferulic acid pure standard solutions. The sample was injected into the HPLC system 5 times. The within-laboratory reproducibility of the HPLC method was calculated over 8-week period. Ferulic acid pure standard solutions were used. During this period, ten injections were performed.

RESULTS

Characterization of main enzyme activities in enzyme preparations

The main enzyme activities present in commercial enzyme preparations used in this study and directly or indirectly involved in ferulic acid release from brewer’s spent grain are presented in Table 1.

Ferulic acid esterase was present in four enzyme preparations and Celluclast lacked this enzyme activity. The lack of the ferulic acid esterase activity in Celluclast was important in the context of the aim of this study, because the aim of the study was to release feruloylated arabinoxylans without significant increase of free ferulic acid concentration in solutions. Cereflo, the only enzyme preparation among the five used, lacked beta-glucanase activity. Acetyloesterase activity was detected in enzyme preparations Celluclast and Ultraflo. Although the key enzyme during arabinoxylan degradation is xylanase, other enzyme activities present in enzyme preparations are important as accessory activities that contribute to the maximal degradation of brewery’s spent grain.

Release of ferulic acid from brewery’s spent grain using commercial enzyme preparations

The concentrations of ferulic acid released from brewery’s spent grain in free form or in the form of feruloylated arabinoxylans (after hydrolysis) were evaluated using UV detection after HPLC separations. The repeatability and reproducibility of the HPLC method were evaluated, in order to check the analytical method (Table 2). Coefficients of variations (% CV) were calculated on the basis of HPLC-UV determinations of ferulic acid. Standard deviations of the results were under 10%-the repeatability and reproducibility of the HPLC method was good. All presented results correspond to the repeatability and reproducibility of the HPLC method used in this study.

Five commercial enzyme preparations are commonly used in the brewing industry in order to assist malt endogenous non-starch polysaccharides’ hydrolases. Indeed, the results of this study show, that feruloylated arabinoxylans were very effectively released from brewery’s spent grain by the action of each of the preparation. In samples of brewery’s spent grain incubated at pH 5.0, the highest concentration of feruloylated arabinoxylans (15.75 mg × 100 g^-1 of brewery’s spent grain) was obtained after 8 h of incubation when Celluclast was used (Table 3). Moreover, Celluclast showed the lowest ability to release free ferulic acid in comparison to the sample incubated without enzyme preparation (Table 4). Enzyme preparations Shearzyme and Viscozyme very effectively released feruloylated arabinoxylans (12.824 mg × 100 g^-1 and 12.804 mg × 100 g^-1 of brewery’s spent grain, respectively), after 8 h of incubation, so the continuation of the process for longer period seems to be pointless in the case of three mentioned enzyme preparations. It must be noticed, that in the case of all samples containing enzyme preparations as well as the sample without any enzyme preparation, there was a clear decrease of both forms of ferulic acid concentrations after 12 or 20 h of incubation (Table 3). The reason of this observation could be the transformation of ferulic acid due to the elevated temperature and presence of oxygen. The optimal pH levels for xylanase activities in enzyme preparations Cereflo and Ultraflo was near 6.5. (Fig. 1) so the incubations of brewery’s spent grain with these enzyme preparations were perfomed close to this pH. As it can be seen in Table 5, esterified ferulic acid was very effectively released from brewery’s spent grain using both enzyme preparations, in comparison to the samples without any enzyme preparations added. Cereflo released 12.57 mg of ferulic acid/100 g of brewery’s spent grain in water-soluble esterified from after 8 h of incubation, whereas Ultraflo released 7.39 mg of ferulic acid/100 g of brewery’s spent grain at the same time of incubation (Table 5). It must be noticed, that similarly as in the experiments performed at pH 5.0 (with Celluclast, Shearzyme and Viscozyme), there was clear decrease of water-soluble esterified ferulic acid concentration in samples after 12 or 20 h of incubation. Enzyme preparations Cereflo and Ultraflo released free ferulic acid in concentrations reaching 6.06 mg of ferulic acid/100 g of brewery’s spent grain and 7.18 mg of ferulic acid/100 g of brewery’s spent grain, respectively, after 8 h of incubation (Table 6). Similarly to the experiments performed at pH 5.0, also at pH 6.5 there was clear decrease of free ferulic acid concentrations in mixtures after 12 and 20 h of incubation.

Ferulic acid release from brewery’s spent grain by Shearzyme and Viscozyme which contained partly inactivated ferulic acid esterase

A possible procedure to increase the concentration of feruloylated arabinoxylans with simultaneous decrease of free ferulic acid concentration can be the thermal inactivation of ferulic acid esterase prior the incubation with brewery’s spent grain. Optimal pH value for xylanases activities in Shearzyme and Viscozyme were close to 5.0 (Fig. 2) and both enzyme preparations were heated at 50°C at pH 5.0 for 15, 30, 45 and 60 min in order to inactivate ferulic acid esterase activity with maximal preservation of xylanase activity. Heating of Shearzyme at 50°C and pH 5.0 for 15 min prior the incubation with brewery’s spent grain resulted in significant decrease of free ferulic acid release (341% in non-heated preparation versus to 183 in the heated one; Table 7) whereas the concentration of esterified form of ferulic acid in studied mixture with brewery’s spent grain decreased in much lower extent (187% versus 150%) (Table 7). It proves that the thermostability of xylanase in Shearzyme was higher in comparison to the thermostability of ferulic acid esterase in this preparation. Heating of Shearzyme for 30 min caused increased ferulic inactivation, but extended time of inactivation up to 60 min did not cause further ferulic acid esterase inactivation. Xylanase activity in Shearzyme was moderately decreased after 15 min of heating and prolonged time of the process had no influence on xylanase activity. Presented results prove, that in order to promote maximal activity of xylanase and to inactivate ferulic acid esterase, Shearzyme should be incubated for 15 min at temperature 50°C and at pH 5.0.

In the case of Viscozyme, heating of the enzyme preparation at 50°C and pH 5.0 for 15-60 min caused significant decrease of xylanase activity. Nevertheless, after 60 min of heating, ferulic acid release was 20% higher than in medium that did not contain any enzyme preparation (Table 7). On the other hand, heating of Viscozyme in the range of 15-60 min caused complete inactivation of ferulic acid esterase activity. Free ferulic acid concentrations released from brewery’s spent grain in the presence of Viscozyme were similar to the concentrations in the blank sample that contained brewery’s spent grain and no enzyme preparation (Table 7). Presented results prove, that complete inactivation of ferulic acid esterase and maximal preservation of xylanase activity in Viscozyme can be realised by incubation of the preparation for 15-30 min at 50°C and pH of 5.0.

DISCUSSION

The purpose of the presented work was to release feruloylated arabinoxylans from brewery’s spent grain, using the enzyme preparations generally applied in the brewing industry during mashing. Feruloylated arabinoxylans can be obtained from industrial by-products (brewery’s spent grain, sugar beet pulp, wheat bran) by using the purified xylanase, ferulic acid esterase and a number of other accessory enzyme activities towards non-starch polysaccharides, but this method is expensive and pointless. On the contrary, commercial enzyme preparations can be used for this purpose with positive results. The reason why enzyme preparations are suitable for this purpose is as follows: it is known, that ferulic acid esterase and xylanase show synergism during degradation of arabinoxylans. Humberstone and Briggs [9] stated, that incubation of malt extract exhibiting “ferulic acid esterase” with brewery’s spent grain caused hydrolysis of low amounts of free ferulic acid, but in the presence of xylanase produced by Trichoderma viridae, release of ferulic acid was about 10-fold higher. When the xylanase from Trichoderma viridae was used alone, the release of free ferulic acid did not occure, what could be the proof of cooperation of “ferulic acid esterase” and xylanase. Another experiment with ferulic acid esterase FAE-III, produced by Aspergillus niger showed, that this enzyme released only about 3.3% ferulic acid present in brewery’s spent grain. However, in the presence of endo-1,4-xylanase produced by Trichoderma viride, the degree of ferulic acid release reached approx. 30% of total ferulic acid present in brewery’s spent grain [2]. The experiments concerning ferulic acid release from wheat bran in the presence of endo-1,4-xylanase produced by Trichoderma viride and ferulic acid esterase from Aspergillus niger in laboratory scale showed the profitability of the process [6]. It must be underlined, that endo-1,4-xylanase produced by Trichoderma viride didn’t release free ferulic acid, but low molecular feruloylated arabinoxylans [8]. Enzyme preparation Ultraflo L, containing main β-glucanase activity and many other accessory activities of hemicellulases produced by Humicola insolens exhibited ferulic acid esterase activity towards methyl esters of cinnamic acid derivatives. The preparation was able to release 65% of ferulic acid in monomer form as well as three forms of dehydrodiferulic acid from brewery’s spent grain. Application of enzyme preparation Ultraflo L considerably improved the ability (from 23% to 47%) of purified ferulic acid esterase produced by Aspergillus niger to release ferulic acid from brewery’s spent grain, especially in 8,5’ – benzofuran form. Coopertion of Ultraflo L and ferulic acid esterase didn’t result in complete release of ferulic acid from ester bond. This suggests, that in order to release the dimeric forms of ferulic acid from arabinoxylans present in brewery’s spent grain, the enzymatic complex of many non-starch polysaccharides’ hydrolases is needed [7]. This is another reason why the use of enzyme preparations with many different activities towards non-starch polysaccharides instead of purified enzymes is justified. Other by-products of food industry can also be the source of feruloylated arabinoxylans. Degradation of sugar beet pulp by enzyme preparation containing the main pectinase activity and other hemicelulases’ activities, as well as esterases and acetylesterase activities resulted in the release of free ferulic acid, feruloylated galactobiose, feruloylated arabinobiose, feruloylated galactose, and, in lower concentrations, feruloylated oligomers [14]. The molecular masses of feruloylated oligosaccharides after enzymatic hydrolysis of sugar beet pulp depended on the enzyme preparation applied, because other authors reported on the isolation of feruloylated arabinose, as well as feruloylated di-, tri, hexa- hepta- and octasaccharides and feruloylated galactose disaccharides after application of fungal enzyme preparation Driselase possessing different carbohydrolases activities [21]. Oligosaccharides, namely ferulic acid arabinose ester (Fa-Ara), disaccharide consisting of arabinose and xylose esterified to ferulic acid (Fa-Ara-Xyl) and trisaccharide consisting of arabinose, xylose and galactose esterified to ferulic acid (Fa-Ara-Ksyl-Gal) were also isolated from wheat bran by the means of acid hydrolysis [23]. Isolated structures prove the fact that there are side chains in heteroxylans forming the cell walls of corn, and ferulic acid is partly responsible for insolubility of polysaccharides by forming the diferulic bridges. The citied works undoubtly suggest the possible potential of commercial enzyme preparations in the obtaining of feruloylated arabinoxylans from by-products of food industry. Low-molecular feruloylated arabinoxylans obtained from waste materials of food industry, can be a very attractive group of antioxidants. Increasing the concentrations of this class of compounds in different foods (e.g. beverages) is interesting alternative for the future industry. Free ferulic acid can be used in food industry directly as antioxidant, or can be transformed into caffeic acid or vanillin. Feruloylated arabinoxylans obtained after enzymatic hydrolysis can be used in food industry as antioxidant additives, for example, in the form of “antioxidant concentrate”, particularly in beverages, e.g. beers and fruit juices where ferulic acid is a common antioxidant. The direction of this research work is strongly supported by the theory, that consumers present more and more open attitude towards natural antioxidants originating from natural sources and vigourously react against synthetic food additives, including antioxidants.

ACKNOWLEDGEMENTS

Research work was financed from the budget resources 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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