Interactions of Insoluble Residue from Enzymatic Hydrolysis of

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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

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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: [email protected]

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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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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 (

Interactions of Insoluble Residue from Enzymatic Hydrolysis of

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