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Interactions of insoluble residue from enzymatic hydrolysis of brewer’s spent grain with intestinal microbiota in mice Johanna Maukonen, Anna-Marja Aura, Piritta Niemi, Gulam Shere Raza, Klaus Niemela, Jaroslaw Walkowiak, Ismo Mattila, Kaisa Poutanen, Johanna Buchert, and Karl-Heinz Herzig J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on April 26, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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Interactions of insoluble residue from enzymatic hydrolysis of brewer’s
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spent grain with intestinal microbiota in mice
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Johanna Maukonen1*, Anna-Marja Aura1, Piritta Niemi1, Gulam Shere Raza2, Klaus
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Niemelä1, Jaroslaw Walkowiak3, Ismo Mattila1,†, Kaisa Poutanen1 Johanna Buchert1,# &
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Karl-Heinz Herzig2
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VTT Technical Research Centre of Finland Ltd, P.O. Box 1000, Tietotie 2, Espoo, Finland.
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Institute of Biomedicine and Biocenter of Oulu, Medical Research Centre Oulu, Oulu
University Hospital, Oulu, Finland
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3
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Medical Sciences, Poznan, Poland
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†
present address: Steno Diabetes Center, Gentofte, Denmark
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#
present address: Natural Resources Institute Finland, Helsinki, Finland
Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan University of
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*Correspondence and reprints:
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Johanna Maukonen
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VTT Technical Research Centre of Finland Ltd.
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P.O. Box 1000 (Tietotie 2)
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FI-02044 VTT
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Finland
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Tel: +358 20 722 7183
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Fax: +358 20 722 7001
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E-mail:
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Abstract
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Brewer’s spent grain (BSG) is the major side-stream from brewing. As BSG is rich in dietary
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fiber and protein, it could be used in more valuable applications, such as nutritional additives
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for foods. Our aim was to elucidate whether an insoluble lignin-rich fraction (INS) from BSG
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is metabolized by mice gut microbiota and how it affects the microbiota. Our results
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indicated that lignin was partially degraded by the gut microbiota, degradation products were
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absorbed, and finally excreted in urine. Therefore, they contribute to the phenolic pool
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circulating in the mammalian body, and may have systemic effects on health. In addition, the
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effects of the test diets on the microbiota were significant. Most interestingly, diversities of
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predominant cecal and fecal bacteria were higher after the intervention diet containing INS
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than after the intervention diet containing cellulose. Since low fecal bacterial diversity has
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been linked with numerous diseases and disorders, the diversity increasing ability opens very
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interesting perspectives for the future.
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Keywords: brewer’s spent grain, dietary fiber, lignin, fecal mouse microbiota, cecal mouse
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microbiota, urinary metabolites
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Journal of Agricultural and Food Chemistry
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Introduction
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Brewer’s spent grain (BSG) is the major side-stream from the brewing of beer. It is composed
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of the husks and outer layers of malted barley grains together with the residual endosperm
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remaining after mashing. As such, it is rich in protein and dietary fiber (DF), including
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arabinoxylan, cellulose, and lignin.1 So far the utilization of BSG has been limited to
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ruminant feed with low commercial value. However, as BSG is rich in DF and protein, it
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could be used in more valuable applications, such as nutritional additive for foods, if
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appropriate processing methods are developed.
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DF has been defined as edible parts of plants or analogous carbohydrates that are resistant to
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digestion and absorption in the human small intestine with complete or partial fermentation in
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the large intestine.2 In the current EU definition, DFs are defined as carbohydrate polymers
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with three or more monomeric units, which are neither digested nor absorbed in the human
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small intestine. Also lignin and other phytochemicals such as waxes, saponins, cutin, and
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phytosterols are considered as part of DF, when associated with carbohydrate polymers.3
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Health benefits of DF are due to the bulking effect increasing colonic motility4 and
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absorption capacity of the DF, which enhances the removal of harmful components from the
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gut. In addition, the health benefits can be associated with fermentability, providing short-
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chain fatty acids and specifically butyric acid for the renewal of the colonic epithelia,5 as well
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as reducing pH and inhibiting the conversion of primary bile acids to carcinogenic secondary
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bile acids.6 DF is therefore particularly important for colonic health.
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Lignin is a polyphenolic macromolecule acting as glue between the cellulose-hemicellulose
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matrices in plant cell walls. Lignin is formed from three monomers: p-coumaryl alcohol,
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coniferyl alcohol, and sinapyl alcohol, which are linked together by radical-induced coupling
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reactions during the synthesis of plant cell wall.7 In lignin the monomers form p-
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hydroxyphenyl, guaiacyl and syringyl units, respectively and the ratio of these units is
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dependent on the plant species and tissue. Lignin is considered to be poorly digested by
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rumen microbiota,8 and thus it most likely remains in the gut lumen, where it could interact
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with other dietary components or affect conversion activities of gut microbiota. Partial
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degradation of lignin has been demonstrated in the rumen of goats
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shown to be a precursor of the mammalian lignans (enterodiol and enterolactone) in rats,10
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suggesting that non-ruminants could also be able to degrade lignin to a limited extent. Aura et
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al.11 and Niemi et al.12 also demonstrated that human gut microbiota was in vitro able to
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release structurally relevant compounds from lignin. However, the bioavailability in the
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mammalian body is not known.
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The aim of our study was to elucidate whether a lignin-rich fraction isolated from BSG is
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metabolized by the mice gut microbiota, and assess its bioavailability to the animals, and the
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effects on the mice gut microbiota. To our knowledge, this is the first study assessing the
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bioavailability and the effects of lignin-rich fractions on mice gut microbiota in vivo.
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and lignin has been
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Materials and Methods
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Preparation of the insoluble lignin-rich fraction (INS): BSG was obtained from
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Sinebrychoff brewery (Kerava, Finland). It was first wet-milled with a Masuko
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Supermasscolloider MKZA10-15J, (Masuko Sangyo Co. Ltd., Kawaguchi-city, Japan), and
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then subjected to sequential enzymatic treatments. The first step was a carbohydrate digestion
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with Depol740 (Biocatalysts Ltd., Cefn Coed, Wales, U.K.) and Celluclast1.5 (Novozymes,
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Bagsvaerd, Denmark) enzyme preparations. The hydrolysis was carried out in tap water at 50
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°C for 5 h, using 9% solids content with continuous mixing. The enzymes were dosed based
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on activity; 3000 nkat (nano katal) of xylanase/g of dry BSG for Depol, and 15 FPU/g of
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BSG for Celluclast. The second step was a proteolytic treatment using Alcalase2.4
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(Novozymes, Bagsvaerd, Denmark) at alkaline conditions (pH 9.5, 60 °C, 4 h). The pH of the
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slurry was adjusted with NaOH and maintained at 9.5 during the whole reaction. The amount
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of Alcalase used was 20 µl/g of BSG. Finally the first carbohydrase treatment was repeated
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using the same conditions as earlier. The pH was adjusted to 5.8 with HCl. After hydrolyses,
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the insoluble residue was separated from the slurry with an Alfa-Laval separator BTPX
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205SGD-34CDP-50 (Alfa Laval, Tumba, Sweden). The obtained solid fraction was washed
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by mixing with fresh water and separated again. The solid fraction obtained after the second
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separation was freeze-dried and designated as insoluble residue (INS).
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Preparation of feeds: The feeds were prepared by Altromin Spezialfutter GmbH & Co. KG
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(Lage, Germany). The feeds were based on 60% of Altromin Spezialfutter basal chow C1013
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and 40% of a) BSG insoluble residue (INS), b) cellulose (Solka floc; James River
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Corporation, Berlin, NH, USA) or their mixture c) INS and cellulose 3:2. Therefore the
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intervention diet composition was as follows: a) 40% INS, b) 40% cellulose, and c) 24% INS
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+ 16% cellulose of the total diet.
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Animal feeding: The National Animal Experiment Board of Finland approved the animal
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experiments and the work was conducted in accordance with the guidelines set by the Finnish
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Act on Animal Experimentation, Statute of Animal Experimentation, the animal protection
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legislation (62/2006, 36/2006 and HE32/2005), European Union Directive 2010/63/EU, and
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European Union Commission recommendations 2007/526/EC.
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Male C57bl6 mice (22-28 g) were obtained from Animal Lab Center, University of
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Oulu, Finland. The mice were housed individually in plexiglass cages containing nylon
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bedding materials to avoid lignin intake from wood beddings. They were maintained at 22 ±
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1 °C with a relative humidity of 45 ± 5% and a 12 h light: dark cycle. All animals had free
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access to water and pelleted food. The mice at a starting age of approximately 10 weeks were 5 ACS Paragon Plus Environment
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acclimatized on a fiber deficient diet (
