POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF RYE BRAN EXTRUDATES PRODUCED AT VARYING PARAMETERS OF EXTRUSION PROCESS

Dorota Gumul, Jarosław Korus
Department of Carbohydrates Technology, Agricultural University of Cracow, Poland

ABSTRACT

Extrusion, a popular method of cereal processing, increases the amounts of phenolic acids in cereal grain. These compounds protect the human body from oxidation stress and prevent the development of chronic diseases. The present study determined the composition of phenolic acids in rye bran after extrusion and defined the antioxidant activity of the extrudates. The bran was obtained through the laboratory milling of the grain of three rye cultivars, namely Amilo, Rostockie and Agrikolo. Extrusion was performed in a single-screw laboratory extruder Brabender 20 DN equipped with a 3:1 screw and a 3 mm die. Screw speed was maintained at 190 rpm while two temperature profiles, 80-100-120°C and 120-160-180°C, were applied. Prior to extrusion, the moisture of the rye bran to be processed was equilibrated to 14 or 20%.

The main phenolic acid found in rye bran was ferulic acid. Irrespective of rye cultivar and the parameters of processing, the ferulic acid and diferulic acid contents increased two times, and the p-coumaric acid content increased by 70% due to extrusion. The levels of other phenolic acids and apigenin decreased or did not change. The exception was caffeic acid whose amount in extrudates was higher than in bran for the Amilo and Rostockie cultivars and was lower for Agrikolo. Using a 14% moisture of the original material and a temperature of 120 or 180°C as parameters of the extrusion process produced the highest antioxidant activity of rye bran extrudates.

Key words: antioxidant activity, phenolic acids, rye bran extrudates.

INTRODUCTION

Cereal products as a source of both nutrients and non-nutritive components constitute the base of the food pyramid [9, 22]. The latter components include polyphenols, compounds of low molecular weight that show biological activity and, having antioxidant properties, play a role in preventing civilisation-related diseases [1, 2, 11, 26]. There are two groups of polyphenols: one of these comprises phenolic acids, i.e. the derivatives of cinnamic and benzoic acids, and the other includes flavonoids.

Compared to other cereals, rye has a medium level of polyphenols [26]. Some researchers have found five main phenolic acids in rye grain, four of which, ferulic, sinapic, caffeic and p-coumaric, are the derivatives of cinnamic acid, and one, vanillic, is the derivative of benzoic acid [25].

Ferulic, vanillic and p-coumaric acids constitute the main antioxidants in whole cereal grain: they take part in the scavenging of free radicals and are able to form chelate complexes with transition metal ions catalysing oxidation reactions [25, 27]. Hence, these compounds protect the human body from oxidation stress and prevent the development of chronic diseases, i.a. atherosclerosis, as well as neoplastic changes [5, 10, 21].

According to antioxidant activity, rye grain takes third position in the group of five cereals (after buckwheat and barley, and before oat and wheat) [28]. Many authors, among them Baublis et al.[3], claim that cereal bran, the by-product of milling, contains more polyphenol antioxidants than whole grain and flour which is the worst in this respect.

A popular method of cereal processing that increases the amount of phenolic acids in the plant material is extrusion. The effects of extrusion on the amount and activity of antioxidants contained in cereal grain have been studied so far only sporadically. A more thorough study has been carried out by Zieliński et al. [27] and has revealed that the ferulic acid content may increase even three times due to extrusion. As shown by another research [28], the antioxidant activity of rye grain increases after extrusion.

While much effort has been devoted to investigating the polyphenol content of cereal grain before and after extrusion, there is still not enough information on the levels of those compounds in the extrudates from rye bran. Therefore, it seemed interesting to investigate the polyphenol content and antioxidant activity of the extrudates from rye bran (100% rye bran). The present study aims to define the quantitative changes in the polyphenols contained in rye bran subjected to extrusion with differing parameters of the process and to determine the antioxidant activity of the rye bran extrudates obtained.

MATERIAL AND METHODS

The research used rye bran from three rye cultivars, Amilo, Rostockie and Agrikolo. The bran obtained through the laboratory milling of rye grain was subjected to extrusion in a single-screw laboratory extruder Brabender 20 DN equipped with a 3:1 screw and a 3 mm die. Screw speed was maintained at 190 rpm while two temperature profiles, 80-100-120°C and 120-160-180°C, were applied. Prior to extrusion, the moisture of the rye bran to be processed was equilibrated to 14 or 20%.

Determination of phenolic acids and flavonoids

Mixture of enzymes dissolved in citrate buffer (pH 5.5) was added to the samples which were then incubated for 1 h at 40°C. The samples were stored at room temperature for 20 h in order to accomplish enzymatic hydrolysis. After enzymatic hydrolysis methanol was added to the samples, and they were placed in ultrasound bath and afterwards centrifuged. After centrifugation the samples were used for HPLC analysis.

The HPLC analysis of phenolic acids and flavonoids were carried out on a HPLC apparatus consisting of Merck-Hitachi L-7455 diode array detector (DAD) and pump L-7100 equipped with D-7000 HSM Multisolvent Delivery System (Merck – Hitachi, Tokyo, Japan). The separation was performed on a Li ChroCART^® 125-3 Purospher^® RP-18 (5 µm) Merck column. Column oven temperature was set to 30°C. 80% acetonitrile in 4.5% formic acid (reagent A) and 2.5% acetic acid (reagent B) were used as an eluent. The flow rate was 1 ml/min. The concentration of reagent A was stepwise increased to reach 15% after 7min, 20% after 15 min and 100% after 16 min. After 10 min of elution the concentration of reagent A was reduced to 0% to stabilize the column. During analysis the solvent were degassed in Merck degasser. Data logging were monitored at the following wavelenghts: phenolic acids (sinapic, ferulic, diferulic, caffeic and p-coumaric acids) at 320 nm, vanillic acid at 280 nm and apigenin at 340 nm. Retention times and spectra werre compared to those of pure standards within 200-600 nm.

Determination of antioxidant activity

The antioxidant activity of the methanol-acetone extracts of raw rye bran and rye bran extrudates was determined by the method of Re et al. [18] using the ABTS ·⁺ free radical.

One gram of the milled material was dissolved in 0.16 N HCl in 80% methanol and shaken for 2 h at room temperature. The supernatant was decanted and the residue was treated with 70% acetone and shaken for 2 h. The supernatant was decanted and mixed with the one produced earlier. The methanol-acetone extracts obtained were stored in a refrigerator (temp. –20°C).

For measurements, the ABTS ·⁺ solution was diluted in PBS to an absorbance of 0.700 at 734 nm. The extracts were diluted, too. For a photometric assay, 2 cm³ of the ABTS ·⁺ solution and 1 cm³ of the diluted extract were mixed for 45 s and absorbance was measured after 6 min at 734 nm. Antioxidant activity was expressed in terms of TEAC.

One factional ANOVA was used to estimate the significance of differences between the obtained results. Calculations were made with a computer program Start Skierniewice 1998.

RESULTS AND DISCUSSION

The extrusion of rye bran (cv. Amilo, Rostockie and Agrikolo) of 14 vs. 20% moisture at a temperature of 120 vs. 180°C produced a number of extrudates, an example of which is shown in Figure 1.

Table 1 presents the levels of six phenolic acids and apigenin in the non-processed and extruded rye bran. Five of the acids, namely sinapic, ferulic, diferulic, caffeic and p-coumaric, belong to the derivatives of cinnamic acid, and one, vanillic, is the derivative of benzoic acid.

Among all the bran samples constituting the raw material for extrusion, that of the Amilo cultivar had the highest sinapic acid content while the Rostockie rye bran contained the lowest amount of the acid (Table 1). Regardless of the parameters of extrusion, the level of sinapic acid in the extrudates from the Amilo rye bran remained stable and was about 65% lower than in the non-processed material (Table 1). In contrast, the extrudates from the Rostockie rye bran had a 20% greater amount of that acid than the bran itself, with the largest increase being observed for the extrudate obtained at the 14% moisture of the raw material and the 120°C temperature of the extrusion process (Table 1). The sinapic acid content of the Agrikolo rye bran extrudates also decreased, except for the product obtained at 14% initial moisture and 180°C temperature. This decrease compared to bran, was smaller when the moisture of the raw material was lower (14%) and the temperature of extrusion was higher (180°C) (Table 1). In the research conducted by Zieliński et al. [27], the sinapic acid content of cereal material sharply fell due to extrusion already at the lowest temperature of the process (120°C).

The dominant phenolic acid, both in the non-processed material, i.e. rye bran, and in the extrudates obtained from the bran, was ferulic acid, which is consistent with the results of other studies [24, 25, 27]. The acid constituted 60% of the total amount of polyphenols in the raw material and 85% in the final product (Table 1). In general, the level of this phenolic acid increased almost twofold due to extrusion, regardless of rye cultivar (Table 1). The extrudates produced from the Amilo rye bran contained more ferulic acid than the non-processed material, but the effect of the extrusion parameters was unclear: the greatest amount of the acid was found in the extrudates obtained both at 14% initial moisture and 120°C extrusion temperature and at 20% moisture and 180°C temperature (Table 1). A similar pattern was observed for the Rostockie cultivar, with the increase being the greatest (170%) in the extrudate produced at 14% initial moisture and 120°C temperature. In the Agrikolo cultivar the largest increase (100%) was noted at the 14% moisture of bran and the 180°C temperature of extrusion. The increase in the ferulic acid content due to extrusion was independent of the moisture of the raw material for bran extrudates from the Rostockie and Agrikolo rye (Table 1). The results confirm those obtained by Zieliński et al. [27] who observed a two- to threefold rise in the ferulic acid content of cereal material after extrusion at a temperature of 120-200°C (the highest at 160°C). It is an important finding because ferulic acid was show to act as a chemoprotectant which is thought to deter the process of carcinogenesis by inhibiting the formation of N-nitroso-compounds [12].

The extrudates obtained from the Amilo rye bran had a lower diferulic acid content than the bran, in contrast, the bran extrudates from the other two rye cultivars showed an almost twofold increase in the amount of that acid (Table 1). Regardless of rye cultivar, the level of diferulic acid in the extrudates was the lowest when the parameters of extrusion were 20% moisture and 120°C temperature and the highest at 20% moisture and 180°C temperature. For extrudates obtained from the Rostockie and Agrikolo rye cultivars, the increase in the diferulic acid content in relation to the raw material was greater at a higher temperature of extrusion. In addition, the extrudates produced from bran of 14% moisture at 180°C contained a smaller amount of the acid than those obtained at 20% moisture, with the increase over the raw material being larger for the higher initial moisture. At the 120°C temperature of the hydrothermal process, the diferulic acid content of extrudates was independent of the moisture of the raw material for the Rostockie and Agricolo rye, but not for Amilo (Table 1).

Due to extrusion, the caffeic acid content increased as a rule for the Amilo and Rostockie rye bran (Table 1), which is in contrast with the results reported by other authors [27], showing a dramatic decrease in the level of the acid in extruded cereal material. Irrespective of rye cultivar, the extrudates obtained from bran of 20% moisture at 120°C temperature contained the smallest amount of caffeic acid while those produced at 14% moisture and 120 or 180°C temperature had the biggest amount of the acid. The caffeic acid content of rye bran extrudates depended on the moisture of the raw material subjected to extrusion, as indicated by a greater increase, compared to bran, for the Amilo and Rostockie cultivars and a smaller decrease for Agrikolo in the amount of that acid in the extrudates obtained from the material of 14 than 20% moisture (Table 1).

Extrusion resulted in a 70% increase in the level of p-coumaric acid (Table 1), which corresponds with the results of other authors [27]. Either the moisture of bran or the temperature of the extrusion process did not have an unambiguous effect on the p-coumaric acid content of extrudates from the Amilo rye bran. For the Rostockie cultivar, the amount of the acid increased with the increasing temperature of extrusion irrespective of the moisture of bran, while for the Agrikolo cultivar, the increase over the raw material was 80% irrespective of the parameters of extrusion. Unlike ferulic, diferulic, caffeic and p-coumaric acids, whose amounts usually rose due to extrusion, vanillic acid showed a significantly decreased level in the extrudates. For the Rostockie and Agrikolo rye cultivars, the vanillic acid content of bran extrudates was 50-65% lower than that of non-processed bran while for the Amilo cultivar the decrease amounted to ca 30% (Table 1). In general, a lower temperature of extrusion, and in some cases a lower moisture of the raw material, led to a smaller decrease in the vanillic acid content.

The extrudates obtained from the Amilo and Rostockie rye bran at 14% initial moisture and 120°C temperature and at 20% moisture and 180°C temperature contained 15% more apigenin than the raw material while at the other parameters of extrusion the level of this flavonoid decreased or did not change (Table 1). For the Agrikolo cultivar, the extrudates obtained at 14% moisture and 180°C temperature had a slightly lower apigenin content than the bran while the other bran extrudates exhibited the apigenin levels decreased by ca 20% (Table 1).

Earlier studies carried out by Grela et al. [8] suggested that extrusion lowers the levels of natural antioxidants as well as tocopherols and carotenoids in plant material. Such findings were supported by Camire and Dougherty [6] and Viscidi et al. [23] who, as a consequence, started to add natural or synthetic antioxidants to cereal extrudates. On the other hand investigations other authors [3, 4, 27, 28] and the results of the present study provided a proof to the contrary, indicating that extrusion may positively influence the phenolic content of cereals. The increase in the level of phenolic acids (particularly ferulic acid) after extrusion can be attributed to the release of the acid and its derivatives from the cell walls of the plant material.

Table 2 shows the antioxidant activity of methanol-acetone extracts obtained from extrudates from the bran of three rye cultivars under study. The ability of such extracts to eliminate free radicals depends on the quantitative and qualitative changes in the phenolics contained in the analysed material [7, 20]. The antioxidant activity of polyphenols is in the following order: apigenin > sinapic acid > caffeic acid > ferulic acid > p-coumaric acid [17, 19]. In this study, irrespective of rye cultivar, the extrudates produced from bran of 20% moisture at 120°C temperature showed the smallest antioxidant potential as compared to the other extrudates and the raw material (Table 2). This may be attributed to their lowest levels of phenolic compounds, apigenin and caffeic acid (Table 1). In addition, the low antioxidant potential of those extrudates may result from the low temperature of the extrusion process and the high moisture of the raw material, which, according to Nicoli et al. [15], lead to the formation of oxidation-promoting compounds that reduce the antioxidant activity of the products obtained in such a way. Researches conducted by Őzer et al. [16] indicated that increased moisture of raw material leaded to increased antioxidant activity of product. Polyphenol content after extrusion was not changed in comprison to raw material. This significant increase of antioxidant activity at 20% initial moisture raw material, with no changes in polyphenol content could be explained by the presence of other compounds with antioxidative properties [16].

Among all the rye bran and extrudate samples studied, the extrudates produced from bran of 14% moisture at 180°C displayed the greatest antioxidant activity (Table 2). For the Rostockie cultivar, the high antioxidant potential of the extrudates obtained in the above conditions (Table 2) may result from the high levels of caffeic, p-coumaric and sinapic acids, relative to the other extrudates and bran (Table 1). The high antioxidant potential of the Agrikolo rye bran extrudates obtained in the same conditions (Table 2) can be associated with the greatest levels of apigenin and sinapic, ferulic and diferulic acids, and with the high total phenolic compound content compared to the other bran extrudates from this rye cultivar (Table 1). The high antioxidant potential of the extrudates produced at a moisture of 14% and a temperature of 180°C is partly accounted for by the presence of the high-molecular-weight products of Maillard’s reaction which, according to Nicoli et al. [15] are formed at higher temperatures and act as antioxidants.

At a moisture of 14% and a temperature of 120°C the antioxidant potentials of the extrudates and the non-processed bran were the same (Table 2). For the Amilo cultivar, the latter result may be accounted for by the rise in the levels of apigenin (by 6%), ferulic acid (150%), p-coumaric acid (50%) and caffeic acid (11%) in the extrudate vs. bran, which compensates for the loss of the other phenolic acids (Table 1). For the Agrikolo cultivar, the stabilisation of the antioxidant potential of the extrudates on the level of the raw material (Table 2) can probably be attributed to the increase in the ferulic, diferulic and p-coumaric acid contents, compensating for the decrease in the levels of the other phenolic acids (Table 1).

The increased antioxidant activity of the extrudates obtained from the Amilo and Rostockie rye bran of 20% moisture at 180°C temperature (Table 2) results the almost twofold increase in the ferulic and p-coumaric acid contents (Table 1).

Among all the extrudate samples, those obtained from the bran of the Rostockie and Agrikolo rye exhibited the greatest antioxidant activity (Table 2). Basing on performed researches it could be concluded that antioxidant activity of rye bran extrudates depended on extrusion parameters.

As mentioned before, extrusion caused a release of phenolics from the cell walls of plant material, which most probably enhanced, compared to raw bran, the antioxidant activity of extrudates produced at various parameters of the process.

In the studies of Zieliński and Troszyńska [28], the antioxidant activity of rye extrudates was substantially higher than that of raw grain. Similar conclusions were reached by Baublis et al. [3] and Miller et al. [13] who found that extracts from extruded cereal grain or cereal bran exhibited a greater antioxidant activity than those from unprocessed grain or bran.

In summary, the research confirmed that extrusion of rye bran of 14% moisture at a temperature of 180°C markedly increases the level and antioxidant potential of polyphenols in the plant material and may therefore constitute a method for developing new products of a high polyphenol content and an improved antioxidant activity. Such properties make rye bran extrudates a useful tool for protecting the human body against oxidation stress and preventing both chronic diseases, among them atherosclerosis, and neoplastic changes [5, 10, 21]. Rye bran extrudates may also offer an attractive alternative for those who are compelled to take daily doses of non-processed bran. Apart from increasing the content and activity of phenolic acids, as shown by this study, extrusion raises also the amount of the soluble fraction of dietary fibre [14], thus improving the availability of the product and enhancing its sensory properties.

CONCLUSIONS

1. Ferulic acid formed the largest proportion of phenolic acids both in the raw rye bran (60% of the total polyphenol content) and the final product, i.e. rye bran extrudates (85%).

2. Regardless of rye cultivar and the parameters of the extrusion process, the ferulic and diferulic acid contents increased twofold and the p-coumaric acid content increased by 70% due to extrusion while the levels of sinapic acid, vanillic acid and apigenin decreased or did not change. Caffeic acid showed a different pattern: compared to bran, its amount increased in the extrudates from the Amilo and Rostockie rye bran and decreased in those from the Agrikolo rye bran.

3. The rye bran extrudates obtained from the 20% moisture raw material at a temperature of 120°C had a lower (or the same) total polyphenol content than (as) the extrudates produced at other prameters of the process.

4. Regardless of rye cultivar, antioxidant activity was the highest for the rye bran extrudates obtained at 14% initial moisture and 180°C temperature and the lowest for those produced at 20% moisture and 120°C temperature.

5. The extrudates obtained from the Amilo and Agrikolo rye bran of 14% moisture at 120°C temperature had the same antioxidant potential as the raw bran of the two respective rye cuttivars.

ACKNOWLEDGEMENTS

This project was suppored by the Polish Ministry of Science (grant PBZ-KBN-094/P06/2003/29).

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Accepted for print: 15.10.2006

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