Article pubs.acs.org/JAFC
Extrusion of Barley and Oat Improves the Bioaccessibility of Dietary Phenolic Acids in Growing Pigs Anastasia S. Hole,†,§ Nils Petter Kjos,‡ Stine Grimmer,§ Achim Kohler,§ Per Lea,§ Bård Rasmussen,§ Lene R. Lima,§ Judith Narvhus,† and Stefan Sahlstrøm*,§ †
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway ‡ Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway § Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, Osloveien 1, N-1430 Aas, Norway ABSTRACT: To evaluate the bioaccessibility of phenolic acids in extruded and nonextruded cereal grains, an in vivo experiment was carried out using growing pigs as a model system. Four diets were prepared containing either whole grain barley (BU), dehulled oat (OU), or their respective extruded samples (BE, OE) according to the requirements for crude protein, mineral, and vitamin contents in pig diets. The total contents of free phenolic acids in the OE and BE diets were 22 and 10%, respectively, higher compared with the OU and BU diets, whereas the level of bound phenolic acids was 9% higher in OE than in OU and 11% lower in BE compared with BU. The total tract bioaccessibilities of bound phenolic acids were 29 and 14% higher for the extruded BE and OE diets, respectively, compared with the nonextruded diets. The results of this study indicate an improved bioaccessibility of phenolic acids in extruded cereal grains. KEYWORDS: extrusion, barley, oat, phenolic acids, pig, bioavailability
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INTRODUCTION Intake of whole grain barley and oat is associated with a decreased risk for coronary heart disease and certain types of cancer, as well as a cholesterol-lowering effect.1 Thus, barley and oat have been intensively studied as dietary components with health beneficial properties. Processing of cereal grains is important for sensory properties of cereal-based products. However, the physical, chemical, and nutritional status of the cereal constituents can be modified dramatically in processed food. Significant losses of dietary fiber and associated compounds have been demonstrated during milling and dehulling.2,3 Levels of bioactive compounds such as phytate, alkylresorcinols, and tocopherols have been shown to diminish significantly during baking, whereas the levels of folate and easily extractable phenolic compounds have been shown to increase during germination and sourdough baking.4 Interest in the nutritional aspects of extruded cereals has lately increased due to a broad industrial use. Extrusion is a high-temperature−short-time process used widely in the production of foods and feeds. It has been shown that extrusion of cereal grains can lead to enhanced mineral bioavailability5 and protein digestibility.6 Moreover, an increased content of soluble dietary fiber and phenolic acids and a destruction of antinutritional factors such as trypsin inhibitors and phytates in extruded products have been documented.5,7 Phenolic acids are known to exhibit good antioxidant activities and to have beneficial health effects.8 The most abundant dietary phenolic acids in cereal grains include ferulic, caffeic, p-coumaric, and sinapic acids. However, the bioavailability of phenolic acids in cereals is low due to their low © 2013 American Chemical Society
content in free form. The majority of phenolic acids in cereals is found in bound form esterified to the cell walls. Cereal esterbound phenolic acids are not hydrolyzed by human digestive enzymes. On the other hand, they can be released by the action of bacterial enzymes in the colon and be further metabolized by bacterial microflora by various reactions and cleavage of functional groups before absorption.8−10 Subsequently, numerous phenolic metabolites can be detected in the blood after intake of dietary phenolic acids. However, most studies have mainly focused on the analysis of the phenolic acids themselves in biological samples and not their metabolites after consumption of diets enriched with purified phenolic acids or with cereal bran fractions as rich sources of phenolic acids.11−14 Although the release and metabolism of phenolic acids by colonic microflora have been intensively studied in vitro,15−17 only a few studies have attempted to follow the fate of ingested phenolic acids and their colonic metabolites in vivo.9,18 Furthermore, it has not yet been documented whether the increased content of bound phenolic acids in extruded cereal products would lead to their improved bioaccessibility. Thus, the aim of this study was to investigate the effect of extruded whole grain barley and oat groat based diets, containing increased levels of bound and free phenolic acids, on the bioaccessibility of phenolic acids using growing pigs as a model system. Unextruded whole grain barley and oat groat were used in control diets to evaluate the effect of extrusion of cereals. Data about the content of phenolic acids and their Received: Revised: Accepted: Published: 2739
October 25, 2012 February 1, 2013 February 5, 2013 February 5, 2013 dx.doi.org/10.1021/jf3045236 | J. Agric. Food Chem. 2013, 61, 2739−2747
Journal of Agricultural and Food Chemistry
Article
Table 1. UPLC and Mass Spectrometric Characteristics of Standards for Phenolic Acids and Their Colonic Microbial Metabolites no. (Figure1)
trivial name, phenolic acid/phenolic acid metabolite
systematic name, phenolic acid/phenolic acid metabolite
abbrev
[M − H]¯ (m/z)
fragment ions (m/z)
RT, min
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 IS 17 18
3,4-dihydroxybenzoic 3,4-dihydroxyphenylacetic 4-hydroxyphenylacetic dihydrocaffeic phenylacetic dihydro-p-coumaric vanillic homosyringic benzoic caffeic chlorogenic dihydroferulic syringic phenylpropionic p-coumaric m-coumaric o-coumaric (IS) ferulic sinapic
3,4-dihydroxybenzoic (protocatechuic) 3,4-dihydroxyphenylacetic 2-(4-hydroxyphenyl)acetic 3,4-dihydroxyphenylpropionic phenylacetic 3-(4-hydroxyphenyl)propanoic 4-hydroxy-3-methoxybenzoic 3,5-dimethoxy-4-hydroxyphenylacetic benzoic 3-(3,4-dihydroxyphenyl)propenoic 3-(3,4-dihydroxycinnamoyl)quinic 3-(4-hydroxy-3-methoxyphenyl)propanoic 4-hydroxy-3,5-dimethoxybenzoic 3-phenylpropionic 3-(4-hydroxyphenyl)propenoic acid 3-(3-hydroxyphenyl)propenoic 3-(2-hydroxyphenyl)propenoic (IS) 3-(4-hydroxy-3-methoxyphenyl)propenoic 3-(3,5-dimethoxy-4-hydroxyphenyl)propenoic
PCa DOPAC PHPA HCA PAA HpCA Van homoSyr BA CA CGA HFA Syr PPA pCA mCA o-CA FA SA
153 167 151 181 135 165 167 211 121 179 353 195 197 149 163 163 163 193 223
109 123 107 137 91 121 152 181, 196 77 135 191, 179 136 153, 182
1.07 1.24 1.41 1.49 1.49 2.11 2.17 2.17 2.31 2.64 2.76 2.76 3.06 3.64 3.75 3.85 4.17 4.82 5.74
and feed intake were measured for each pig at every week in the experiment. Experimental Procedure. The total experimental period lasted for 21 days; a 7 day adaptation period followed by a 14 day experimental period. The pigs were fed twice daily (at 8 a.m. and 2 p.m.) according to a restricted Norwegian feeding scale,20 and they had free access to drinking water. They were kept in pens designed for individual feeding in a room with an average temperature of 18 °C. Sample Collection. Fecal samples were collected from each of the pigs at the beginning and during the final two days of the experiment. The samples were frozen immediately at −20 °C. The fecal samples were freeze-dried, ground, and pooled for each dietary treatment before being analyzed. Blood samples were taken at 7 a.m. at the start of the experiment, after 1 week (adaptation period), and at the end of the experiment. The blood samples were centrifuged for 20 min at 3000g, and blood plasma was transferred to TT tubes and frozen at −20 °C for analysis of phenolic metabolites. Analytical Methods. Feed Analysis. The four grain samples, the soybean meal and the four diet samples, were analyzed for dry matter (DM; EU Directive 71/393), ash (EU Dir.ective 71/250), crude protein (Kjeldahl-N × 6.25; EU Directive 93/28), crude fat (EU Directive 98/64), crude fiber (EU Directive 92/89), and starch (AOAC 996.11). The four diets were also analyzed for yttrium by inductively coupled plasma mass spectrometry (ICP-AES analysis, Perkin-Elmer Optia 3000DV; Perkin-Elmer, Wellesley, MA, USA) at 371 nm, after mineralization and solubilization in acid of the pooled sample. In addition, the grain samples and soybean meal were analyzed for β-glucan and nonstarch polysaccharides (total, soluble, and insoluble) as described in ref 21 and for free and bound phenolic acids using the same method as described in our previous study.22 Each sample was prepared in parallels, which were analyzed in duplicates. Fecal Analysis. The final fecal samples were analyzed for dry matter and for yttrium as described earlier and for free and bound phenolic acids as described by Hole et al.22 Plasma Analysis. Total content of free and conjugated phenolic acids and products of their metabolic transformation were determined in plasma samples after glucuronidase/sulfatase hydrolysis. To 500 μL of plasma were added 20 μL of 100 μg/mL o-coumaric acid aqueous solution as internal standard (IS), 20 μL of sulfatase type H-1 solution in 1 M acetate buffer, pH 4.9, containing 2.5 × 105 U/L of sulfatase and 7.5 × 106 U/L of β-glucuronidase, and the mixture was incubated
metabolites in plasma as well as information about the levels of bound and free phenolic acids excreted with the feces were used for estimation of bioaccessibility of dietary phenolic acids. Growth performance of animals and plasma antioxidant status during experiment were also analyzed.
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119 119 119 134 149, 208
MATERIALS AND METHODS
The feeding experiment was performed at the Experimental Farm of the Norwegian University of Life Sciences, Ås, Norway. All pigs were cared for according to laws and regulations controlling experiments with live animals in Norway (Animal Protection Act of December 20, 1974, and the Animal Protection Ordinance concerning experiments with animals of January 15, 1996). Dietary Treatments. Four batches of grain were used in the study: (1) whole grain barley, untreated; (2) whole grain barley, extruded; (3) oat groat, untreated; (4) oat groat, extruded. Each of the batches was produced and pelleted by Lantmännen Cerealia, Moss, Norway. Oat groat samples were produced from the Norwegian oat cultivar Belinda, and whole grain barley samples were produced from the Norwegian barley cultivar Olve. After dehulling, the hull fraction constituted approximately 25% of the grain weight for oat. Whole grain barley and dehulled oat (oat groat) were milled to produce barley flour and oat flour with particle size
