Effects of Condensed and Hydrolyzable Tannins on Rumen

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Effects of condensed and hydrolysable tannins on rumen metabolism with emphasis on the biohydrogenation of unsaturated fatty acids. Mónica Costa, Susana Alves, Alice Cappucci, Shaun R Cook, Ana Duarte, Rui Caldeira, Tim A. McAllister, and Rui J.B. Bessa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04770 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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Journal of Agricultural and Food Chemistry

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Effects of condensed and hydrolysable tannins on rumen metabolism with emphasis on

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the biohydrogenation of unsaturated fatty acids.

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Mónica Costa†, Susana P. Alves†, Alice Cappucci‡, Shaun R. Cook§, Ana Duarte†, Rui

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M. Caldeira†, Tim A. McAllister§, Rui J.B. Bessa†*

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†

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Medicina Veterinária, Universidade de Lisboa; Avenida da Universidade Técnica 1300-

CIISA, Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de

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477, Lisboa, Portugal

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‡

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del Borghetto, 80 – 56124 Pisa, Italy

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§

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4B1, Canada

Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali, University of Pisa, Via

Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB T1J

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*Corresponding author:

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Rui J. B. Bessa

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Faculdade de Medicina Veterinária, Universidade de Lisboa,

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Avenida da Universidade Técnica, 1300-477, Lisboa, Portugal

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Tel.: +351 213652876

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Fax: +351 213652889

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e-mail: [email protected]

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1 ACS Paragon Plus Environment


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Abstract

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The hypothesis that condensed tannins have higher inhibitory effect on ruminal

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biohydrogenation than hydrolysable tannins was tested. Condensed tannin extract from

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mimosa (CT) and hydrolysable tannin extract from chestnut (HT) or their mixture

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(MIX) were incorporated (10%) into oil supplemented diets and fed to rumen fistulated

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sheep. Fatty acid and dimethyl acetal composition of rumen contents and bacterial

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biomass were determined. Selected rumen bacteria were analysed by quantitative real

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time PCR. Lower (P < 0.05) rumen volatile fatty acids concentrations were observed

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with CT compared to HT. Moreover, lower concentration (P < 0.05) of Fibrobacter

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succinogenes, Ruminococcus flavefaciens, Ruminococcus albus and Butyrivibrio

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proteoclasticus were observed with CT compared to HT. The extension of

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biohydrogenation of 18:2n-6 and 18:3n-3 did not differ among treatments, but was

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much more variable with CT and MIX than with HT. The trans-/cis-18:1 ratio in

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bacterial biomass was higher (P < 0.05) with HT than CT. Thus, mimosa condensed

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tannins had a higher inhibitory effect on ruminal metabolism and biohydrogenation than

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chestnut hydrolysable tannins.

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Keywords:

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biohydrogenation; fatty acids; rumen; hydrolysable tannins; condensed tannins.

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Introduction

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The inclusion of tannins in ruminant diets has been reported to modulate

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biohydrogenation of unsaturated fatty acids (FA) in the rumen.1 In general, the effects

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of tannins on rumen microbiota have been attributed to the ability of these phenolic

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compounds to form complexes with polymers of protein and carbohydrates and to

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directly interact with bacterial cell membranes.2 However, the effects of tannins are

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dependent on several factors including their molecular structure. Tannins can be broadly

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classified into hydrolysable and condensed tannins. Hydrolysable tannins (mainly

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gallotannins and ellagitannins) have a polyol as a central core, which is esterified with a

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phenolic group, while condensed tannins (proanthocyanidins) are composed of flavan-

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3-ol (epi)catechin and (epi)gallocatechin units that are connected by interflavonoid

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linkages.3

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Hydrolysable tannins are readily susceptible to depolymerisation in the rumen, whereas

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the depolymerisation of condensed tannins by rumen bacteria has not been clearly

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demonstrated.4 Depolymerisation can convert the oligomeric hydrolysable tannins into

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low molecular weight monomeric subunits that exhibit a lower affinity for extracellular

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or intracellular proteins, such as enzymes, or for cellular lipoproteins within the

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membrane lipid bilayers of bacteria.5

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Conversely, condensed tannins inhibit the activity and growth of microorganisms by

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decreasing membrane permeability as a result of binding to lipoproteins, or even

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disruption of membranes when added at high concentrations.2

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However, it is possible that the monomeric subunits derived from hydrolysable tannins

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also exert toxicity towards bacteria causing disturbances in membrane fluidity6, 7 which

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are lessened through bacteria developing adaptative responses. In the presence of toxins

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that affect membrane integrity, some bacteria are able to reduce membrane fluidity, 3 ACS Paragon Plus Environment


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enriching their membrane lipids with trans-FA.8, 9 The generation of trans-FA and their

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incorporation into microbial cells has been suggested to be one the main purposes of

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ruminal bacterial biohydrogenation.10 In fact, biohydrogenation simultaneously reduces

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the concentration of polyunsaturated FA in the rumen, lowering their direct toxicity to

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bacterial membranes.11 In fact, increasing the level of trans-18:1 in the rumen could be

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a mechanism whereby bacteria improve their ability to cope with the toxicity of

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polyunsaturated FA.10 As the toxic effects of condensed tannins and hydrolysable

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tannins on bacteria are probably mediated by distinct mechanisms of action, it is

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possible that their effects on biohydrogenation also differ. In the present study, we

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hypothesized that condensed tannins will exert a stronger inhibitory action on rumen

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microbiota than hydrolysable tannins, leading to a corresponding general inhibition of

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BH with large accumulation of C18 polyunsaturated FA. Conversely, we anticipated

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that hydrolysable tannins would modulate, instead of inhibit biohydrogenation, leading

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to a larger accumulation of trans-18:1 compared to condensed tannins.

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Thus, a condensed tannin extract from mimosa (Acacia mearnsii) and a hydrolysable

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tannin extract from chestnut (Castanea sativa) or their mixture were incorporated in an

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oil-supplemented basal diet and fed to rumen fistulated sheep, to assess their effects on

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FA composition of rumen digesta and bacteria, as well on the abundance of selected

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rumen bacteria

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Material and methods

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Animal handling and management

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Animal handling followed EU Directive 2010/63/EU concerning animal care and rumen

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fistulated animals were used after approval of the ethical committee of the Faculty of

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Veterinary Medicine, University of Lisbon. Four rumen fistulated ≈ 2-year old sheep 4 ACS Paragon Plus Environment

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(52±3.0 kg) were used in a change-over design with 3 treatments and 4 experimental

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periods. The sheep were kept in individual crates and bedded with wood shavings. The

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dietary treatments were defined by the inclusion of tannin extract in common basal feed

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ingredients: 1) Condensed tanins extract (CT) (100 g/kg dry matter (DM) of mimosa

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tannin extract); 2) Hydrolysable tannins extract (HT) (100 g/kg DM of chestnut tannin

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extract); 3) Mixture of CT and HT (MIX) (50 g/kg DM of each mimosa and chestnut

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tannin extracts). Tannin extracts were incorporated in the diets at a high amount to

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exacerbate their disturbing effects on rumen microbial ecosystem and metabolism. The

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tannin extracts were commercial extracts containing 683 to 723 g/kg of condensed

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tannins from Acacia mearnsii (Mimosa extract ME powder®; Mimosa Extract

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Company (Pty) Ltd, Pietermaritzburg, South Africa) and ≥ 650 g/kg of hydrolysable

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ellagitannins from Castanea sativa (Gallo Tanin B®; Lamothe – Abiet, Canejan,

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France). Compound feeds containing the tannin extracts were pelleted and fed ad

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libitum, and were provided twice daily (09:00 and 17:00) along with grass hay at 100

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g/kg of DM intake. Grass hay contained 35 g/kg DM of crude protein and 764 g/kg DM

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of neutral detergent fiber. The ingredients and chemical and fatty acid composition of

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the pelleted supplement are presented in Table 1. Each experimental period consisted of

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2 weeks of adaptation to the diets and 1 week of sample collection.

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Sample collection and processing

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Feed chemical composition was determined using the methods described by Oliveira, et

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al. 12 Rumen sample collection occurred in the last week over 2 days with an interval of

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3 days between them. On the first sampling day, rumen contents were collected directly

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through the rumen cannula, from various regions in the rumen, before the morning meal

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and used for isolation of bacterial biomass and FA analysis. On the second sampling



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day, rumen contents sampling was repeated using the same procedure. On both

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sampling days, rumen contents were also preserved for DNA extraction.

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Planktonic bacteria (PB) and solid associated bacteria (SAB) pellets were obtained by

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fractionation and differential centrifugation of rumen contents using a Beckman

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Coulter, Avanti J-26 XPITM (Indianapolis, IN, USA) centrifuge, according to the

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procedure reported by Bessa, et al.

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included the use of four layers of surgical gauze to filter the rumen contents, as well as

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the re-suspension and homogenization of rumen washed particles in saline solution with

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carboximetilcellulose at 0.1%, which were then incubated at 39ºC for 15 min. At the

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end, PB and SAB pellets were washed at least three times with saline solution until it

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became clear.

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The aliquots of total rumen contents used for FA analysis and DNA extraction, as well

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the bacterial pellets, were immediately stored at -20ºC, freeze-dried (ScanVac CoolSafe,

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LaboGene ApS, Lynge, Denmark) and preserved at -20ºC until analyzed. The aliquots

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of total rumen contents for evaluation of fermentation activity were immediately filtered

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through four layers of cheesecloth and then used either for pH measurement (Digital pH

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meter “pH-2005”; JP Selecta S.A, Barcelona, Spain) or stored at -20ºC for volatile fatty

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acids (VFA) analysis.

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, with slight modifications. These modifications

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Fatty acid and dimethyl acetals analysis

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Volatile FA were analysed by gas chromatography with flame ionization detection (GC-

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FID) using a Shimadzu GC-2010 Plus chromatograph (Shimadzu, Kyoto, Japan)

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equipped with a Nukol capillary silica column (30 m; 0.25 mm i.d.; 0.25 µm film

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thickness, Supelco Inc., Bellefonte, PA, USA), as previously described.12 For long chain

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FA analysis of rumen content and bacterial pellets, 250 mg of each freeze-dried sample


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was weighed and transesterified into FA methyl esters using a combination of basic

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followed by acidic catalysis and with 19:0 (1 mg/mL) as the internal standard.14

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Fatty acid methyl esters were separated by GC-FID using a Shimadzu GC-2010 Plus

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chromatograph equipped with a SP-2560 capillary column (100 m, 0.25 mm i.d., 0.20

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µm film thickness; Supelco Inc., Bellefont, PA, USA). Identification of FA methyl

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esters and dimethyl acetals (DMA) was achieved by comparison of retention times with

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those of authentic standards (FAME mix 37 components from Supelco Inc., Bellefont,

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PA, USA, and a Bacterial FAME mix from Matreya LLC, Pleasant Gap, PA, USA).

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Separation and identification was confirmed using gas chromatography-mass

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spectrometry (GC-MS) using a Shimadzu GC-MS QP 2010 Plus chromatograph

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(Kyoto, Japan) equipped with a SP-2560 column.15

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Quantification of microbial 16S rRNA marker genes by qPCR

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Total DNA was extracted from freeze-dried rumen contents (30 mg) using a Qiagen

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QIAamp DNA Stool Mini Kit (Qiagen Inc., Valencia, CA, USA), according to

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manufacturer’s instructions and with small modifications to enhance lysis of Gram-

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positive bacteria.16 Supernatant (400 µl) was added to 30 µl of proteinase K (Qiagen

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Inc., Valencia, CA, USA), 400 µl of AL buffer (Lysis buffer; Qiagen Inc., Valencia,

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CA, USA) and 400 µl of ethanol. The eluted DNA was quantified using Quant-iTTM

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PicoGreen® dsDNA Assay Kit (Invitrogen Canada Inc., Burlington, ON, Canada) with a

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NanoDrop 3300 fluorometer (ThermoScientific, Wilmington, DE, USA).

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The extracted DNA was subjected to real-time quantitative polymerase chain reaction

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(qPCR), using a StepOnePlus thermocycler PCR (Applied Biosystems, Foster City, CA,

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USA), for quantification of copies of 16S rRNA sequences specific for Fibrobacter

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succinogenes subsp. S85, Prevotella bryantii B14, Ruminococcus albus 7,



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Ruminococcus flavefaciens C94, Selenomonas ruminantium subsp. Lactilytica

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(ATCC19205), Ruminobacter amylophilus (ATCC 29744), Butyrivibrio fibrisolvens

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JW-11 and Butyrivibrio proteoclasticus PI-18. The PCR standards for the Butyrivibrio

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group were created with genomic DNA from reference bacterial strains originated from

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the Rowett Institute of Nutrition and Health, University of Aberdeen (UK) and cultured

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in Abel Salazar Biomedical Science Institute (ICBAS) (Portugal). The standards for the

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other bacteria were generated with plasmid DNA containing DNA amplicons derived

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from strains of the Lethbridge Research Centre culture collection (Agriculture and Agri-

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Food Canada). The genomic DNA of B. fibrisolvens and B. proteoclasticus was

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extracted from 50 mg of freeze-dried bacterial pellets of pure cultures (ICBAS, Culture

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Collection) using a Qiagen DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA,

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USA), according to manufacturer’s instructions. The genomic DNA of other reference

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strains was also extracted from bacterial pellets of pure cultures (Lethbridge Research

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Centre, Culture Collection) followed by the preparation of plasmid standards, as

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described by Wang, et al. 17. Detailed information about primers and cycling conditions

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for F. succinogenes, P. bryantii, R. flavefaciens, S. ruminantium, R. amylophilus18 and

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R. albus have already been reported.17,

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used at concentrations of 2.0×108 to 2.0×106 and 1.0×106 to 1.0×102 copies per reaction

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obtained by serial 10-fold dilutions. For genomic DNA standards, the number of copies

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considered was covered in a 10-fold dilution series from 3.72×107 to 3.72×101 for B.

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fibrisolvens and from 1.90×107 to 1.90×101 for B. proteoclasticus. For Butyrivibrio spp.,

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the following conditions were applied: 95 ºC for 10 min; 40 cycles of 95 ºC for 15 s,

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annealing temperature of 58 ºC for 1 min (B. fibrisolvens) or 55 ºC for 30 s with a final

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extension step at 72 ºC for 30 s (B. proteoclasticus). For these two bacteria, the real-

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time PCR reaction mixtures (15 µl) contained 1 × Taqman universal PCR master mix

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For plasmid standards, the amplicons were


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(Applied Biosystems®, Foster City, CA, USA), 10 µM of forward and reverse primers

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for B. fibrisolvens and B. proteoclasticus at a final concentration of 100 and 300 nM,

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respectively, 250 nM of both Taqman probes and 100 × purified BSA (BioLabs®

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Ipswich, MA, USA). For the other bacteria, each qPCR reaction (25 µl) contained 1 ×

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iQ SYBR Green Supermix (Bio-Rad Laboratories®, Hercules, CA, USA), 10 µM of

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each primer at a final concentration of 300 nM and 100 × purified BSA (BioLabs®,

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Ipswich, MA, USA). For all bacteria, BSA was used to overcome the presence of

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tannins as PCR inhibitors. The primers and probes for Butyrivibrio spp. were designed

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using Primer Express 2.0 software (Applied Biosystems®, Foster City, CA, USA) and

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the homology of their sequences was searched against GenBank to confirm their

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specificity. The sequences of the primers and probes were as follows: 5´-CCGCGTCA

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GATTAGCCAGTT-3´

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(reverse)

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(probe) for B. proteoclasticus; 5´-ACACACCGCCCGTCACAC-3´ (forward), 5´-TCCT

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TACGGTTGGGTCACAGA-3´

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GGAGTTGGGAATGCCCGA-TAMRA-3´ (probe) for B. fibrisolvens. Primers were

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synthesised by NZYTech (Lisbon, Portugal) and probes were synthesised by Stabvida

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(Almada, Portugal). For all qPCR reactions, 2 µl of extracted DNA template was added

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at 20 ng or 10 ng per reaction and amplified in duplicate.

and

(forward)

and

5´-GTAGGAGTTTGGGCCGTGTCT-3´

5´-FAM-CCAAAGCAACGATCTGTAGCCGGACTG-TAMRA-3´

(reverse)

and

5´-JOE-CATG

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Calculations and statistical analysis

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The ruminal biohydrogenation (% of disappearance) of dietary c9-18:1, 18:2 n-6 and

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18:3 n-3 and the biohydrogenation completeness (18:0 concentration relative to

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potential 18:0 concentration assuming a complete hydrogenation of substrates) were

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calculated as previously reported.15 Membranes of rumen bacteria contain phospholipids



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with alk-1-enyl (vinyl) ether chains (plasmalogens). The vinyl ether chains, quantified

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as DMA, can be used as an internal marker of rumen bacteria.14, 20 Thus, the sum of the

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DMA that were present at similar concentration in SAB and PB pellets (i.e. iso-14:0,

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14:0, anteiso-15:0, 15:0 and 16:0 DMA) was used to estimate the rumen bacteria

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biomass as follow: =

(0.5 × + 0.5 × )

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Where, the Mrumen, MSAB and MPB are the concentration (mg/g DM) of the DMA marker

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in the rumen, SAB and PB pellets, and biomass is expressed as mg bacterial DM/g

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rumen DM.

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Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC,

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USA) considering a change-over design with 3 diets, 4 sheep and 4 periods, according

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to the follow model: = + + +

+ () + !

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Where, yijk are the individual observations, u the overall mean, ti is the fixed effect of

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treatment (i = 1, 2 and 3), aj is the random effect of animal (j = 1, 2, 3 and 4), pk the

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random effect of period (k = 1, 2, 3 and 4), subsampling within experimental units (i.e

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animal×period) (l = 1 and 2) and eijk the residual error. The variance homogeneity was

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tested and, when significant, the variance heterogeneity structure was accommodated in

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the model. The effects of treatments were evaluated using 2 orthogonal contrasts: C1)

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contrasting the least square means of CT against HT, and C2) contrasting the average of

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least square means of CT and HT against MIX (CT + HT)/2 vs. MIX). Statistically

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significant differences or trends were set for P < 0.05 and for 0.05 < P 0.10) between CT and HT,

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but the branched chain volatile FA (iso-butyrate and iso-valerate) presented higher (P
0.05) between HT and CT, a

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higher (P = 0.023) concentration of 16:0 was found with HT than with CT. The



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differences between CT and HT were more extensive for DMA. In fact, concentrations

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of eight DMA (anteiso-15:0¸15:0, 16:0, 13:0, iso-14:0, 14:0, c9-18:1, c11-18:1) and the

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sum of DMA (P < 0.022) were higher with HT than with CT. In general, for DMA,

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MIX did not differ from the average of CT and HT, but concentrations of 14:0- and c11-

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18:1-DMA were higher (P < 0.02) with MIX than the average of CT and HT.

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The means of all C18 FA, except the 10-oxo-18:0, did not differ significantly between

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CT and HT (Table 4). However, CT and MIX were consistently associated with greater

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variances for the majority of C18 FA than HT, as supported by the larger standard error

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of the means (Table 4). This higher variance arose as a result of occasional suppression

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of biohydrogenation (i.e. animal 2, period 2, sampling day 1, treatment CT, animal 3,

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period 1, sampling day 1, treatment MIX, animal 4, period 2, sampling day 1, treatment

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MIX). In these cases, dietary unsaturated FA (i.e. c9-18:1, 18:2n-6, and 18:3n-3)

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remained high, comprising more than 40% of total C18 FA in the rumen. Consequently,

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the estimates of disappearance of unsaturated FA (i.e. biohydrogenation, %), as well as

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the biohydrogenation completeness were numerical higher and much less variable with

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HT than with CT or MIX. Despite such variance, the 10-oxo 18:0 was higher (P =

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0.027) with CT than with HT.

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Fatty acid and dimethyl acetals of rumen bacterial fractions

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The total FA and C18 FA did not differ between SAB and PB populations nor among

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treatments (Table 5). The FA and C18 FA content of bacterial pellets averaged 106 and

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85 mg/g DM, respectively. The majority of FA in these fractions corresponded to C18

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FA followed by 16:0, which also did not differ among treatments or bacterial

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populations. Considering the FA composition in more detail, the sum of branched-chain

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FA was higher (P = 0.019) in PB than in SAB, and this was due to the highest


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concentrations of iso-13:0 and anteiso-15:0 in PB. The treatments did not affect the sum

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of branched-chain FA but iso-14:0 and iso-16:0 were lower (P < 0.01) and iso-13:0

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higher (P = 0.009) with CT than with HT.

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Odd- and linear-chain FA were higher (P = 0.022) in PB than in SAB, mostly due to

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15:0 and 17:0. The only odd- and linear-chain FA that differed between CT and HT was

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the 23:0, which was higher (P = 0.003) with CT compared to HT. However, the

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bacterial 15:0 and 17:0 contents were higher (P < 0.05) with MIX than the average of

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contents of CT and HT.

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Rumen bacteria presented a considerable amount of even chain FA with carbon chain

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length ranging from 20 to 28. The concentrations of 22:0 and 24:0 were higher (P

Effects of Condensed and Hydrolyzable Tannins on Rumen

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