Effects of condensed and hydrolysable tannins on rumen metabolism

Subscriber access provided by Kaohsiung Medical University

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 35

Journal of Agricultural and Food Chemistry

1

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

2

the biohydrogenation of unsaturated fatty acids.

3

4

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

5

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

6 7 8

†

9

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

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

10

477, Lisboa, Portugal

11

‡

12

del Borghetto, 80 – 56124 Pisa, Italy

13

§

14

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

15 16

*Corresponding author:

17

Rui J. B. Bessa

18

Faculdade de Medicina Veterinária, Universidade de Lisboa,

19

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

20

Tel.: +351 213652876

21

Fax: +351 213652889

22

e-mail: [email protected]

23

1 ACS Paragon Plus Environment


24

Abstract

25

The hypothesis that condensed tannins have higher inhibitory effect on ruminal

26

biohydrogenation than hydrolysable tannins was tested. Condensed tannin extract from

27

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

28

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

29

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

30

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

31

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

32

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

33

succinogenes, Ruminococcus flavefaciens, Ruminococcus albus and Butyrivibrio

34

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

35

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

36

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

37

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

38

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

39

chestnut hydrolysable tannins.

40

41

Keywords:

42

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

43

44


Page 2 of 35

Page 3 of 35


45

Introduction

46

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

47

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

48

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

49

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

50

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

51

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

52

classified into hydrolysable and condensed tannins. Hydrolysable tannins (mainly

53

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

54

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

55

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

56

linkages.3

57

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

58

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

59

demonstrated.4 Depolymerisation can convert the oligomeric hydrolysable tannins into

60

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

61

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

62

membrane lipid bilayers of bacteria.5

63

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

64

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

65

disruption of membranes when added at high concentrations.2

66

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

67

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

68

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

69

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


70

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

71

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

72

ruminal bacterial biohydrogenation.10 In fact, biohydrogenation simultaneously reduces

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

rumen bacteria

89 90

Material and methods

91

Animal handling and management

92

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

93

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

94

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

Page 4 of 35

Page 5 of 35


95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113 114

Sample collection and processing

115

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

116

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

117

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

118

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

119

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



120

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

121

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

122

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

123

fractionation and differential centrifugation of rumen contents using a Beckman

124

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

125

procedure reported by Bessa, et al.

126

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

127

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

128

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

129

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

130

became clear.

131

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

132

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

133

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

134

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

135

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

136

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

137

acids (VFA) analysis.

13

, with slight modifications. These modifications

138 139

Fatty acid and dimethyl acetals analysis

140

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

141

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

142

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

143

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

144

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


Page 6 of 35

Page 7 of 35


145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

Separation and identification was confirmed using gas chromatography-mass

154

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

155

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

156 157

Quantification of microbial 16S rRNA marker genes by qPCR

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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



170

Ruminococcus flavefaciens C94, Selenomonas ruminantium subsp. Lactilytica

171

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

172

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

173

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

174

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

175

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

176

other bacteria were generated with plasmid DNA containing DNA amplicons derived

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

R. albus have already been reported.17,

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

19

For plasmid standards, the amplicons were


Page 8 of 35

Page 9 of 35


195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

GATTAGCCAGTT-3´

207

(reverse)

208

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

209

TACGGTTGGGTCACAGA-3´

210

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

211

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

212

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

213

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

214 215

Calculations and statistical analysis

216

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

217

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

218

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

219

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



220

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

221

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

222

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

223

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

224

biomass as follow: =

(0.5 × + 0.5 × )

225

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

226

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

227

rumen DM.

228

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

229

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

230

to the follow model: = + + +

+ () + !

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

253

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

267

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



268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

the biohydrogenation completeness were numerical higher and much less variable with

283

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

284

0.027) with CT than with HT.

285 286

Fatty acid and dimethyl acetals of rumen bacterial fractions

287

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

288

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

289

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

290

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

291

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

292

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


Page 12 of 35

Page 13 of 35


293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

contents of CT and HT.

301

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

302

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

Effects of condensed and hydrolysable tannins on rumen metabolism

Recommend Documents