chrysanthemum coronarium

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Animal Feed Science and Technology 161 (2010) 2837

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Animal Feed Science and Technology

jo ur na l ho me pa ge : www.elsevier.com/locate/anifeedsci

Chrysanthemum coronarium as a modulator of fatty acidbiohydrogenation in the rumen

T.A. Wood, E. Ramos-Morales, N. McKain, X. Shen 1, C. Atasoglu 2, R.J. Wallace

Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, Aberdeen AB21 9SB, United Kingdom

a r t i c l e i n f o

Article history:

Received 15 March 2010

Received in revised form 5 July 2010

Accepted 29 July 2010

Keywords:

Biohydrogenation

Conjugated linoleic acid

Rumen

Vaccenic acid

a b s t r a c t

Inclusion of a daisy plant, Chrysanthemum coronarium, in a dairy sheep diet has been

reported to result in increased concentrations of health-promoting rumenic acid (RA; cis-

9,trans-11CLA) andvaccenicacid (VA; trans-11-18:1)in milk. Theaims of thepresent study

were to determine if the reported change in milk fatty acid composition was the result of

the effects ofC. coronarium on the biohydrogenation of linoleic acid (LA; cis-9,cis-12-18:2)

by ruminal microorganisms, and to investigate which constituents of C. coronarium may

be responsible for the observed effects. Ruminal digesta from four sheep receiving a mixed

hay-concentrate diet were incubated in vitro with LA in the presence or absence of dried

whole-plant C. coronarium var. Primrose Gem. Rates of LA disappearance and stearic acid

(SA; 18:0) production decreased as a result of C. coronarium addition, and VA accumula-

tion doubled. Chrysanthemum parthenium and Chrysanthemum vulgare had much smaller

effects on biohydrogenation. C. coronarium added to cultures of the only known ruminal

SA-forming bacterium, Butyrivibrio proteoclasticus, also inhibitedLA metabolism by,but not

growth of, this species. Lipid analysis indicated that C. coronarium var. Primrose Gem had ahigh content of-linolenic acid (LNA; cis-9,cis-12,cis-15-18:3; 8.79 mg/g DM) compared to

the other samples (


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T.A. Wood et al. / Animal Feed Science and Technology 161 (2010) 2837 29

1. Introduction

Ruminant milk and meats contain relatively higher amounts of saturated fats than most oils of plant origin. Furthermore,tissue lipids of ruminants have been known for a long time to be more saturated than those of non-ruminant animals. Thesaturated fats are often associated with health disorders in man, including coronary heart disease (Menotti et al., 1999).Although forages grazed by ruminants are rich sources of health-promoting polyunsaturated fatty acids (PUFA), only a smallproportion of the ingested PUFA passes to milk and meat. However, ruminant products also contain conjugated linoleicacids (CLA), which have been shown to be associated with health benefits such as cancer prevention and improved immuneresponse in animal models (Parodi, 1999; Kritchevsky, 2000; Belury, 2002). Beneficial effects of CLA have been shown inhuman intervention studies (Riserus et al., 2001; Gaullier et al., 2005; Song et al., 2005). CLA are produced as intermediateproducts in thebiohydrogenation of linoleic acid (LA;cis-9, cis-12-18:2) to stearic acid (SA;18:0) by certain groups of bacteriain the rumen (Harfoot and Hazlewood, 1997). Maia et al. (2007) recently demonstrated that 11 of 26 predominant speciesof ruminal bacteria metabolised LA substantially, vaccenic acid (VA; trans-11-18:1) being the most common end product,produced by species related to Butyrivibrio fibrisolvens. VA is converted to one of the CLA, rumenic acid (RA; cis-9,trans-11-18:2) in several tissues and can be considered to have equal health-promoting properties to RA ( Palmquist et al., 2005). Itwas also found that Clostridium proteoclasticum, which has been recently renamed as Butyrivibrio proteoclasticus from its 16SrRNA gene sequence (Moon et al., 2008), produced RA together with VA and was the only species to form SA ( Wallace etal., 2006). It seems logical, therefore, that if effective means of selective suppression ofB. proteoclasticus or inhibition of itsbiohydrogenating activity can be found, it may be possible to decrease the extent of saturation of fatty acids in the rumenand to increase the RA content of meat and milk.

Plants and plant extracts are potentially promising alternatives to antibiotics and ionophores for manipulating ruminalfermentation since there has been a major concern over the use of these substances in ruminant nutrition ( Wallace, 2004).The inclusion of a daisy plant, Chrysanthemum coronarium, in dairy sheep diet resulted in higher concentrations of RA andVA in milk (Cabiddu et al., 2006). These observations have been recently confirmed by Lpez (unpublished results).

The aims of the present study were to determine if the reported change in milk fatty acid composition was the result ofthe effects ofC. coronarium altering the activity of ruminal biohydrogenating bacteria, and to investigate which constituentsofC. coronarium could be responsible for the observed effects.

2. Materials and methods

2.1. Animals and diets

Animal experimentation was carried out under conditions governed by a licence issued by the United Kingdom Home

Office. Four mature sheep, each fitted with a ruminal cannula, received 800 g dry matter (DM)/day of ration comprising(g/kg DM) grass hay (300), rolled barley (422.5), soybean meal (167.5), molasses (100) and minerals and vitamins (10) astwo equal meals (2400 g) at 08.00 and 16.00. Samples of ruminal digesta were collected from each animal just beforethe morning feeding. Digesta samples were bubbled with CO2, maintained at 39

C, and strained ruminal fluid (SRF) wasobtained by straining through double-layered muslin gauze.

2.2. Chrysanthemum samples

A sample ofC. coronarium var. Primrose Gem was prepared from fresh plant material grown in Montrose, UK, by freeze-drying and grinding to pass a 1-mm screen. Air-dried samples ofChrysanthemum parthenium and Chrysanthemum vulgarewere obtained from Plantafarm S.A., Len, Spain.

2.3. Incubations with ruminal digesta in vitro

SRF was incubated with LA in the presence and absence of ground, freeze-dried plant material. Additionally, incubationsof SRF with RA and VA (SigmaAldrich Co. Ltd., UK), as substrates for the biohydrogenating bacteria, with or without C.coronarium added were carried out with the aim of studying where in the biohydrogenation sequence the inhibition by C.coronarium occurred. In order to determine the effect of LNA and coronaric acid on the metabolism of LA, SRF was incubatedeither with LA (SigmaAldrich Co. Ltd., UK) or with a combination of LA and LNA (SigmaAldrich Co. Ltd., UK) or LA andcoronaric acid (Larodan Fine Chemicals, Sweden).

One millilitre of SRF was added under CO2 to Pyrex tubes (125 mm16 mm) containing one of the following: 0.2mldistilled water; 5 mg ground plant and 0.2 ml distilled water; 0.1 ml of 2 g LA/l and 0.1 ml distilled water; 0.1 ml of 2 g RA/land 0.1 mldistilled water; 0.1 ml of2 g VA/l and 0.1 ml distilled water; 5 mg ground plant, 0.1 mlof 2 g LA/l and 0.1 mldistilledwater; 5 mg ground plant,0.1 mlof 2 g RA/l and 0.1 mldistilled water; 5 mg ground plant,0.1 mlof 2 g VA/l and 0.1 mldistilledwater;0.1ml of 2 g LA/land 0.1 mlof 2 g LNA/l or 0.1 mlof 2 g LA/land0.1ml of2 g coronaric acid/l. The tubes were incubatedunder CO

2at 39 C. Tubes were removed at 0, 1, 3, 6, 9 and 24 h for fatty acid analysis. Reactions were stopped by heating in

a heating block at 100 C for 10 min and tubes were stored at20 C.


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30 T.A. Wood et al. / Animal Feed Science and Technology 161 (2010) 2837

In order to measure the influence ofC. coronarium var. Primrose Gem on volatile fatty acids (VFA) production, samples ofSRF from the four sheep were pooled, diluted 3-fold in buffer (Menke and Steingass, 1988) and 50 ml of the diluted SRF wasadded to Wheaton bottles containing either 0.4 g of the ration fed to the sheep, as a control, or 0.4 g of the same ration and20mg ofC. coronarium var. Primrose Gem. Five replicate bottles for each treatment were incubated under CO 2 and at 39

Cfor 24 h, then the reaction was stopped by adding 1 ml of saturated mercuric chloride. VFA was analysed by GC (Newbold etal., 1995).

2.4. Incubations with B. proteoclasticus

B. proteoclasticus P-18 is a recently identified SA-producing bacterium isolated from grazing sheep (Wallace et al., 2006;Moon et al., 2008). All transfers and incubations were carried out under O2-free CO2 at 39

C in Hungate-type tubes in theliquid form of medium M2 (Hobson, 1969). Inoculum volumes were 5% (v/v) of a fresh culture into 5 ml of medium. LAwas added to a final concentration of 0.05 g/l. Fatty acids were prepared as a separate solution, sonicated for 4 min in asmall volume of medium and added to the medium before dispensing and autoclaving. Growth of bacteria was measuredin triplicate from the increase in optical density (OD) at 650 nm of the control tubes using a Novaspec II spectrophotometer(Amersham Biosciences, UK). One millilitre was removed periodically for total fatty acid analysis. Thereafter, 0.1 ml of 19:0(0.2g/l in methanol) was added and tubes were stored at70 C and subsequently freeze-dried. When samples derived fromdifferential solvent extraction were tested, each fraction was dissolved in 1.0 ml of DMSO, and 0.1 ml was dispensed to threeculture tubes containing 10 ml of M2 medium. 0.95 ml of fresh culture ofB. proteoclasticus was used as inoculum.

2.5. Differential solvent extraction

Differential solvent extraction of C. coronarium var. Primrose Gem was carried out using Soxhlet apparatus, extractingfrom 0.5 g of freeze-dried material. The solvents used were, in order, petroleum ether (4060), chloroform, ethyl acetate,acetone, methanol and water. Samples were allowed to dry under vacuum in a desiccator, except for the water extract,which was freeze-dried.

2.6. Fatty acid extraction and analysis

Extraction of total fatty acids was based on the method ofFolch et al. (1957), incorporating the modifications ofDevillardet al. (2006). Fatty acid methyl esters and, in case of doubts about peak identity, 4,4-dimethyloxazoline (DMOX) derivativeswere prepared and analysed witha gas chromatographmass spectrometer (GC/MS)consisting of an Agilent Technologies UK(Stockport, Cheshire, UK) GC (6890) coupled to a quadrupole mass selective detector. The GC was fitted with a 100-m fusedsilica capillary column (i.d., 0.25mm) coated with 0.2m film of cyanopropyl polysiloxane (Varian Analytical Instruments,Walton-on-Thames, Surrey, UK) and helium was the carrier gas (Wasowska et al., 2006). Solid-phase extraction (Kaluzny etal., 1985) was used to separate free fatty acids from other lipids following Folch extraction.

Samples of diluted ruminal digesta and cultures of B. proteoclasticus were analysed for VFA content by GC as describedpreviously (Newbold et al., 1995).

2.7. Data analysis

Data were analysed at each time point separately by randomised block analysis of variance, with individual sheep as ablocking term and plant sample, LNA or coronaric acid as a treatment term. Pure-culture data were analysed by ANOVA,again compared at each sampling time.

3. Results

SRF was incubated in vitro with LA (0.2 g/l) in the presence and absence ofC. coronarium var. Primrose Gem. The rate ofmetabolism of LA decreased (P=0.005), as did the rate of production of SA (P0.05), the accumulation of VA overtime was higher when the plant was present (P0.05) (results not shown). Neither C.parthenium (Fig. 2) nor C. vulgare (Fig. 3) had any influence (P>0.05) on biohydrogenation of LA to SA or the accumulation ofintermediate fatty acids.

The concentration of VFA produced after 24h was increased slightly by C. coronarium var. Primrose Gem, but the propor-tional concentrations of individual VFA changed little (Table 1).

When C. coronarium var. Primrose Gem was sterilized by

-irradiation and added to the growth medium of B. proteo-clasticus, growth at 24 h, as indicated by butyrate concentration, was unchanged (not shown). In separate incubations, LA


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Fig. 1. Influence of C. coronarium var. Primrose Gem on metabolism of LA in ruminal fluid from sheep receiving a mixed grass hay/concentrate diet. LA

was added to an initial concentration of 0.2 g/l and C. coronarium to 5 g/l. (a) LA (,), SA (, ). (b) VA (, ). (c) RA (,), trans-9,trans-11-18:2 (, ),trans-10,cis-12-18:2 (,). Closed symbols are from incubations with LA alone; open symbols are from incubations with LA + C. coronarium. Results are

mean SE from four sheep.

metabolism appeared to be decreased, as was accumulation of RA, in the presence of C. coronarium (P


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Fig. 2. Influence of Chrysanthemum parthenium on metabolism of LA in ruminal fluid from sheep receiving a mixed grass hay/concentrate diet. LA was

added to an initial concentration of 0.2 g/l and C. parthenium to 5 g/l. (a) LA (,), SA (, ). (b) VA (, ). Closed symbols are from incubations with LA

alone; open symbols are from incubations with LA + C. parthenium. Results are mean SE from four sheep.

Table 2

Composition of solid-phase (Soxhlet) extracts ofChrysanthemum coronarium var. Primrose Gem, and their effects on LA metabolism by B. proteoclasticus.

Soxhlet fraction DM extracted (mg)a LNA content (mg/g

DM extracted)

Incubation of LA with B. proteoclasticus, 4 h

Mean SE LA loss (mg/l) RA formed (mg/l) trans-9,trans-11-18:2

formed (mg/l)

Mean SE Mean SE Mean SE

Noneb 36.65 1.03 27.84 0.84 3.36 0.33

Ether 24.4 33.8 1.54 33.95 0.12 25.31 0.55 3.30 0.26

Chloroform 25.3 19.9 0.65 21.47** 1.22 15.33** 1.15 2.50 0.06

Ethyl acetate 22.1 0 32.99* 0.76 26.38 0.64 3.76 0.30

Acetone 41.1 0 34.43 1.42 26.57* 0.17 3.78 0.20

Methanol 96.3 0 32.92* 0.39 25.15* 0.22 3.85 0.17

Water 121.2 0 34.99 0.62 24.89 0.29 4.63* 0.23

a From an initial DM of 0.5 g.b Solvent only, DMSO.* P


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Fig. 3. Influence ofChrysanthemum vulgare on metabolism of LA in ruminal fluid from sheep receiving a mixed grass hay/concentrate diet. LA was added

to an initial concentration of 0.2 g/l and C. vulgare to 5 g/l. (a) LA (,), SA (, ). (b) VA (, ). Closed symbols are from incubations with LA alone; open

symbols are from incubations with LA + C. vulgare. Results are mean SE from four sheep.

lation was not inhibited (P=0.06) by the chloroform fraction at 4 h, but was (P


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Fig. 6. Influence of coronaric acid on metabolism of LA in ruminal fluid from sheep receiving a mixed hay-concentrate diet. LA and coronaric acid were

added to an initial concentration of 0.2g/l. (a) LA (,). (b) RA (,). (c) VA (,). (d) SA (,). Closed symbols are from incubations with LA alone; open

symbols are from incubations with LA + coronaric acid. Results are mean SE from four sheep.

of the accumulation of RA in 1 h (P


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be due to an inhibition of the biohydrogenation by the total concentration of unsaturated fatty acids, as LNA and LA wereadded to a final concentration of 0.2g/l each.

Differential solvent extraction supported this impression, whereby only fractions rich in LNA were inhibitory. It is note-worthy that the less polar ether extract contained a higher content of LNA than the chloroform fraction, yet the chloroformfraction was far more inhibitory. Presumably the latter fraction contained the majority of non-esterified LNA. It is thenon-esterified unsaturated fatty acids rather than their esterified form that inhibit biohydrogenation via their toxicity tobiohydrogenating ruminal bacteria (Wasowska et al., 2006). This is exemplified in studies with fish oil and its componentPUFA (AbuGhazaleh and Jenkins, 2004; Lee et al., 2005; Vlaeminck et al., 2008). On the other hand, it is known that C. coro-narium contains an unusual epoxy fatty acid, coronaric acid (14% of the oil; Earle, 1970), which could also contribute tothe inhibitory effects observed. Incubations of coronaric acid with SRF and LA showed an inhibitory effect which did not,however, lead to accumulation of RA or VA. Therefore, it could be concluded that there may be a synergistic inhibition bycombining LNA and coronaric acid in C. coronarium. It is important to note, as well, that other components might be involvedin the reported effect too. Essential oils and essential oil compounds have been shown to be of potential usefulness in con-trolling biohydrogenation (Durmic et al., 2008; Lourenco et al., 2009). C. coronarium contains essential oils, the compositionof which may vary when collected at different localities (Basta et al., 2007; Alvarez Castellanos et al., 2001).

The results presented here highlight the value of a high LNA content of dietary plant materials in inhibiting biohydro-genation and thereby improving the RA content of milk, even if LNA is not converted directly to RA ( Wasowska et al., 2006).However, they do not explain the reason for the results obtained by Cabiddu et al. (2006), because the LNA content of theirChrysanthemum-containing sward was actually lower than the control sward containing the other two plants only. Theconcentration of coronaric acid may be equally important. An issue not addressed by Cabiddu et al. (2006) or here is thatof lipase activity. As the inhibitory effects of PUFA depend on their non-esterified form, the LNA concentration of greatestimportance will be transitory, depending on the rate of release by lipolysis and the subsequent rate of isomerization andbiohydrogenation. Lee et al. (2006) have demonstrated how important lipase is in mediating the effects of dietary lipids.There may also have been an animal effect in the original work. Other factors may be involved too. Cabiddu et al. (2006) sug-gested that9-desaturase might be affected, altering the balance of VA and RA in the mammary gland. Fatty acid oxidationproducts also inhibit biohydrogenation (Lee et al., 2007).

5. Conclusions

LNA in itsnon-esterified form appears to be an importantmediatorof inhibition of fatty acid biohydrogenationby ruminalmicroorganisms. Coronaric acid also has an inhibitory effect on biohydrogenation. Understanding how the LNA content ofdifferent plant species and cultivars varies and the role of microbial lipase in its release will be useful in devising plant-based strategies to increase the RA content of milk. In the specific case ofChrysanthemum, LNA and coronaric acid contentcan explain some, but not all, experimental observations.

Acknowledgements

The Rowett Research Institute receives most of its funding from the Rural and Environment Research and Analysis Direc-torate of the Scottish Government. The results presented here were from the EC Framework 6 project, REPLACE: Plants andtheir extracts and other natural alternatives to antimicrobials in feeds, contract no.: 506487. ERM has a TALENTIA scholar-ship from the Regional Ministry for Innovation, Science and Enterprise, Andalusia, Spain. XS and CA received Fellowshipsfrom the Royal Society, London. XS and CA also received support from the National Natural Science Foundation of China(30710103006) and The Scientific Research and Technological Council of Turkey, respectively. We thank Susan Moir, DavidBrown and Donna Henderson for help with fatty acid analysis. Thanks are due to Graham Horgan for his help with statisticalanalysis of data.

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