Chrysanthemum coronarium as a modulator of fatty acid biohydrogenation in the rumen

Chrysanthemum coronarium as a modulator of fatty acid biohydrogenation in the rumen

Animal Feed Science and Technology 161 (2010) 28–37 Contents lists available at ScienceDirect Animal Feed Science and Technology journal homepage: w...

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Animal Feed Science and Technology 161 (2010) 28–37

Contents lists available at ScienceDirect

Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Chrysanthemum coronarium as a modulator of fatty acid biohydrogenation 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; cis9,trans-11 CLA) and vaccenic acid (VA; trans-11-18:1) in milk. The aims of the present study were to determine if the reported change in milk fatty acid composition was the result of the effects of C. 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 accumulation 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 inhibited LA metabolism by, but not growth of, this species. Lipid analysis indicated that C. coronarium var. Primrose Gem had a high content of ␣-linolenic acid (LNA; cis-9,cis-12,cis-15-18:3; 8.79 mg/g DM) compared to the other samples (<0.50 mg/g DM). In fractions derived from differential (Soxhlet) solvent extraction, only extracts containing LNA affected LA metabolism by B. proteoclasticus. LNA and coronaric acid ((+)-cis-9,10-epoxy,cis-12-18:1) were investigated as the main components present in C. coronarium that could have altered the biohydrogenation of LA in vitro. LNA inhibited biohydrogenation of LA causing a slowdown of RA and VA formation and a subsequent increase of the accumulation of RA and VA over time. Coronaric acid showed an inhibitory effect on the metabolism of LA, although it did not correspond to a higher accumulation of intermediates. It was concluded that the combined effect of LNA and coronaric acid in C. coronarium could be responsible for changes in the biohydrogenating activity of ruminal bacteria causing an increase of VA and a decrease in SA in vitro. This effect would lead to an increased flow of VA from the rumen which in turn would lead to an increase in RA and VA in milk from ruminants receiving C. coronarium. © 2010 Elsevier B.V. All rights reserved.

Abbreviations: CLA, conjugated linoleic acids; DM, dry matter; LA, linoleic acid; LNA, ␣-linolenic acid; PUFA, polyunsaturated fatty acids; OD, optical density; RA, rumenic acid; SA, stearic acid; SRF, strained rumen fluid; VA, vaccenic acid; VFA, volatile fatty acids. ∗ Corresponding author. Tel.: +44 1224 716656; fax: +44 1224 716687. E-mail addresses: [email protected], [email protected] (R.J. Wallace). 1 Present address: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China. 2 Present address: Canakkale Onsekiz Mart Universitesi, 17100 Canakkale, Turkey. 0377-8401/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2010.07.016

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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. The saturated 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 small proportion of the ingested PUFA passes to milk and meat. However, ruminant products also contain conjugated linoleic acids (CLA), which have been shown to be associated with health benefits such as cancer prevention and improved immune response in animal models (Parodi, 1999; Kritchevsky, 2000; Belury, 2002). Beneficial effects of CLA have been shown in human intervention studies (Riserus et al., 2001; Gaullier et al., 2005; Song et al., 2005). CLA are produced as intermediate products in the biohydrogenation of linoleic acid (LA; cis-9, cis-12-18:2) to stearic acid (SA; 18:0) by certain groups of bacteria in the rumen (Harfoot and Hazlewood, 1997). Maia et al. (2007) recently demonstrated that 11 of 26 predominant species of 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-1118:2) in several tissues and can be considered to have equal health-promoting properties to RA (Palmquist et al., 2005). It was also found that Clostridium proteoclasticum, which has been recently renamed as Butyrivibrio proteoclasticus from its 16S rRNA gene sequence (Moon et al., 2008), produced RA together with VA and was the only species to form SA (Wallace et al., 2006). It seems logical, therefore, that if effective means of selective suppression of B. proteoclasticus or inhibition of its biohydrogenating activity can be found, it may be possible to decrease the extent of saturation of fatty acids in the rumen and to increase the RA content of meat and milk. Plants and plant extracts are potentially promising alternatives to antibiotics and ionophores for manipulating ruminal fermentation 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 and VA in milk (Cabiddu et al., 2006). These observations have been recently confirmed by López (unpublished results). The aims of the present study were to determine if the reported change in milk fatty acid composition was the result of the effects of C. coronarium altering the activity of ruminal biohydrogenating bacteria, and to investigate which constituents of C. 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) as two equal meals (2 × 400 g) at 08.00 and 16.00. Samples of ruminal digesta were collected from each animal just before the morning feeding. Digesta samples were bubbled with CO2 , maintained at 39 ◦ C, and strained ruminal fluid (SRF) was obtained by straining through double-layered muslin gauze. 2.2. Chrysanthemum samples A sample of C. coronarium var. Primrose Gem was prepared from fresh plant material grown in Montrose, UK, by freezedrying and grinding to pass a 1-mm screen. Air-dried samples of Chrysanthemum parthenium and Chrysanthemum vulgare were obtained from Plantafarm S.A., León, 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, incubations of SRF with RA and VA (Sigma–Aldrich 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 incubated either with LA (Sigma–Aldrich Co. Ltd., UK) or with a combination of LA and LNA (Sigma–Aldrich Co. Ltd., UK) or LA and coronaric acid (Larodan Fine Chemicals, Sweden). One millilitre of SRF was added under CO2 to Pyrex tubes (125 mm × 16 mm) containing one of the following: 0.2 ml distilled 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/l and 0.1 ml distilled water; 0.1 ml of 2 g VA/l and 0.1 ml distilled water; 5 mg ground plant, 0.1 ml of 2 g LA/l and 0.1 ml distilled water; 5 mg ground plant, 0.1 ml of 2 g RA/l and 0.1 ml distilled water; 5 mg ground plant, 0.1 ml of 2 g VA/l and 0.1 ml distilled water; 0.1 ml of 2 g LA/l and 0.1 ml of 2 g LNA/l or 0.1 ml of 2 g LA/l and 0.1 ml of 2 g coronaric acid/l. The tubes were incubated under CO2 at 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 at −20 ◦ C.

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In order to measure the influence of C. coronarium var. Primrose Gem on volatile fatty acids (VFA) production, samples of SRF from the four sheep were pooled, diluted 3-fold in buffer (Menke and Steingass, 1988) and 50 ml of the diluted SRF was added 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 and 20 mg of C. coronarium var. Primrose Gem. Five replicate bottles for each treatment were incubated under CO2 and at 39 ◦ C for 24 h, then the reaction was stopped by adding 1 ml of saturated mercuric chloride. VFA was analysed by GC (Newbold et al., 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 the liquid form of medium M2 (Hobson, 1969). Inoculum volumes were 5% (v/v) of a fresh culture into 5 ml of medium. LA was added to a final concentration of 0.05 g/l. Fatty acids were prepared as a separate solution, sonicated for 4 min in a small volume of medium and added to the medium before dispensing and autoclaving. Growth of bacteria was measured in 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 (0.2 g/l in methanol) was added and tubes were stored at −70 ◦ C and subsequently freeze-dried. When samples derived from differential solvent extraction were tested, each fraction was dissolved in 1.0 ml of DMSO, and 0.1 ml was dispensed to three culture tubes containing 10 ml of M2 medium. 0.95 ml of fresh culture of B. 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, extracting from 0.5 g of freeze-dried material. The solvents used were, in order, petroleum ether (40–60), 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 of Folch et al. (1957), incorporating the modifications of Devillard et al. (2006). Fatty acid methyl esters and, in case of doubts about peak identity, 4,4-dimethyloxazoline (DMOX) derivatives were prepared and analysed with a gas chromatograph–mass 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 fused silica capillary column (i.d., 0.25 mm) coated with 0.2 ␮m 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 et al., 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 described previously (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 a blocking 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 of C. coronarium var. Primrose Gem. The rate of metabolism of LA decreased (P=0.005), as did the rate of production of SA (P<0.05 up to 12 h) as a result of C. coronarium addition (Fig. 1a). VA accumulated during the incubation, with C. coronarium doubling the VA concentration (P<0.05 at 6 h onwards; Fig. 1b). The accumulation of RA was unaffected (P>0.05) by C. coronarium, but trans-9,trans-11-18:2 tended to accumulate more in the presence of the plant material (P=0.047 at 1 h; Fig. 1c). In vitro incubations of SRF with C. coronarium and RA as substrate showed that although there was no effect on the metabolism of RA (P>0.05), the accumulation of VA over time was higher when the plant was present (P<0.05 at 1 and 3 h) (results not shown). However, when SRF was incubated with VA and C. coronarium, there was little effect on the reduction of VA to SA (P>0.05) (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 of intermediate fatty acids. The concentration of VFA produced after 24 h was increased slightly by C. coronarium var. Primrose Gem, but the proportional 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. proteoclasticus, 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 (䊉,), SA (, ♦). (b) VA (, ). (c) RA (䊉,), 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<0.05; Fig. 4). VA did not accumulate, nor did the concentration of stearic acid increase over the 96 h of the incubation. Differential solvent extraction of 0.5 g of C. coronarium var. Primrose Gem produced fractions extracted in petroleum ether, chloroform, ethyl acetate, acetone, methanol, and water (Table 2). The extracts were then added to cultures of B. proteoclasticus in the presence of LA, at a concentration equivalent to 5 g/l of the original sample, and fatty acid concentrations were measured up to 24 h at 39 ◦ C. The 4-h results are shown in Table 2. The fraction that inhibited the metabolism of LA and accumulation of RA most was the chloroform fraction (Table 2). The geometric isomer of RA, trans-9,trans-11-18:2, accumulated to a lower concentration (0.12% of accumulated CLA) than RA (Table 2). Its accumuTable 1 Effect of Chrysanthemum coronarium var. Primrose Gem on VFA profile in ruminal digesta after 24 h (n = 5). Fatty acid

Total VFA Molar proportion Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate

Control

C. coronarium

Mean

SE

Mean

SE

50.54

0.92

53.97*

0.61

0.636 0.176 0.008 0.149 0.016 0.014

0.011 0.004 0.0002 0.003 0.0002 0.0002

0.648 0.170 0.008 0.144 0.016 0.014

0.009 0.002 0.0001 0.001 0.0002 0.0002

Pooled rumen fluid (50 ml) from 4 sheep was diluted 1:2 with buffer and incubated either with 0.4 g of ration (control) or 0.4 of ration and 20 mg of Chrysanthemum coronarium var. Primrose Gem for 24 h. * P<0.05.

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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 (䊉,), 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 of Chrysanthemum coronarium var. Primrose Gem, and their effects on LA metabolism by B. proteoclasticus. Soxhlet fraction

b

None Ether Chloroform Ethyl acetate Acetone Methanol Water a b * **

DM extracted (mg)a

– 24.4 25.3 22.1 41.1 96.3 121.2

From an initial DM of 0.5 g. Solvent only, DMSO. P<0.05. P<0.001.

LNA content (mg/g DM extracted)

Incubation of LA with B. proteoclasticus, 4 h

Mean

LA loss (mg/l)

RA formed (mg/l)

trans-9,trans-11-18:2 formed (mg/l)

Mean

SE

Mean

SE

Mean

SE

36.65 33.95 21.47** 32.99* 34.43 32.92* 34.99

1.03 0.12 1.22 0.76 1.42 0.39 0.62

27.84 25.31 15.33** 26.38 26.57* 25.15* 24.89

0.84 0.55 1.15 0.64 0.17 0.22 0.29

3.36 3.30 2.50 3.76 3.78 3.85 4.63*

0.33 0.26 0.06 0.30 0.20 0.17 0.23

– 33.8 19.9 0 0 0 0

SE

– 1.54 0.65

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Fig. 3. Influence of Chrysanthemum 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 (䊉,), 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<0.05) thereafter, by an average of 24% (not shown). LNA comprised 8.79 mg/g DM of the total dry matter of C. coronarium var. Primrose Gem (Table 3). LA was present at a lower concentration (3.34 mg/g DM). C. parthenium and C. vulgare had lower total concentrations of both fatty acids. The proportion of LNA and LA in the non-esterified form was lower in C. coronarium var. Primrose Gem than in the other plant

Table 3 Concentrations of LA and LNA in total and non-esterified forms in Chrysanthemum samples. Sample

LA (mg/g DM)

LNA (mg/g DM)

Non-esterified

C. coronarium var. Primrose Gem C. parthenium C. vulgare a

Mean and SE from 3 replicate analyses.

Non-esterified

Total

Meana

SEa

Mean

Total SE

Mean

SE

Mean

SE

0.29 0.44 0.69

0.017 0.016 0.012

3.34 0.79 2.02

0.062 0.005 0.009

0.39 0.17 0.16

0.037 0.015 0.008

8.79 0.42 0.48

0.152 0.005 0.007

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Fig. 4. Influence of C. coronarium on metabolism of LA by B. proteoclasticus. LA (䊉,), RA (, ), VA (,), SA (, ♦). The initial concentration of LA was 0.05 g/l. Closed symbols are from incubations with LA alone; open symbols are from incubations with LA + C. coronarium. Results are mean ± SE from three separate cultures.

samples. Nevertheless, because of the very high total LNA concentration, the concentration of non-esterified LNA was highest in C. coronarium var. Primrose Gem. In vitro incubations of SRF and LA either alone or with LNA were carried out in order to study the influence of LNA on the metabolism of LA (Fig. 5). An inhibition (P<0.01 at 1 and 3 h; Fig. 5a) of the rate of metabolism of LA as well as an inhibition

Fig. 5. Influence of LNA on metabolism of LA in ruminal fluid from sheep receiving a mixed hay-concentrate diet. LA and LNA were added to an initial concentration of 0.2 g/l. (a) LA (,). (b) RA (,). (c) VA (,♦). (d) SA (䊉,). Closed symbols are from incubations with LA alone; open symbols are from incubations with LA + LNA. Results are mean ± SE from four sheep.

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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 0.2 g/l. (a) LA (,). (b) RA (,). (c) VA (,♦). (d) SA (䊉,). 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<0.05) and a slowdown in its metabolism (Fig. 5b) was observed with LNA addition; this effect resulted in higher concentrations of RA from 3 h onwards (P<0.05 at 9 and 24 h). A corresponding slowdown of VA accumulation (P<0.05 at 1–6 h) was also observed. The rate of production of SA decreased (P<0.05 at 24 h) when LNA was present (Fig. 5d). In the same way, samples of SRF were incubated in vitro with LA in the presence or absence of coronaric acid to study its effect on the metabolism of LA (Fig. 6). An inhibition of the accumulation of VA (P<0.05 at 6–24 h) as well as an inhibition of the rate of production of SA (P<0.05) was shown in the presence of coronaric acid (Fig. 6c and d). 4. Discussion The observation upon which this study was based was that of Cabiddu et al. (2006), who reported that incorporating C. coronarium into dairy sheep diets increased the proportion of PUFA, particularly RA, in milk, with no other effects on productivity. This was achieved by sowing a ternary pasture of C. coronarium with Lolium rigidum and Medicago polymorpha, the resulting sward containing 34% C. coronarium. These observations have been recently confirmed by López, who included C. coronarium at 5% and 10% in a sheep diet (unpublished results). Although Cabiddu et al. (2006) interpreted the result as being due to a higher concentration of LA in the mixed sward, the response in milk RA and VA concentrations (both increased by 59%) was higher than the increase in LA intake (38%). We considered it possible that other components of the C. coronarium were perhaps contributory, possibly via an inhibition of fatty acid biohydrogenation in the rumen. If this were an inhibitory effect of a specific phytochemical, the discovery could have wider implications for using specialist plants in dairy cow nutrition to produce milk and other dairy products with a healthier fatty acid composition. Ruminal biohydrogenation of LA in vitro was indeed inhibited by a sample of C. coronarium var. Primrose Gem grown locally. The rate of LA metabolism was decreased, while VA accumulation doubled and SA formation decreased. LA metabolism by the key biohydrogenation ruminal bacterium, B. proteoclasticus, was also inhibited. Thus, an effect on ruminal biohydrogenation was confirmed. However, samples of C. parthenium and C. vulgare were less effective. The exceptional effectiveness of C. coronarium var. Primrose Gem could be associated with its very high concentration of LNA, which was almost 20 times higher than other samples. It has been shown that feeding ruminants with diets rich in LA and LNA increased RA concentration in milk (Dhiman et al., 2000; Lock and Garnsworthy, 2002). Troegeler-Meynadier et al. (2006) suggested that this effect could be explained partly by the amount of VA produced from LNA and desaturated by the mammary gland and also by the saturation of the VA reductase leading to a greater accumulation of VA. On the other hand, it is known that PUFA are toxic to biohydrogenating bacteria, the antimicrobial effect being associated with the degree of unsaturation (reviewed by Lourenc¸o et al., 2010). We believe that the inhibitory effect observed in the present study may

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be due to an inhibition of the biohydrogenation by the total concentration of unsaturated fatty acids, as LNA and LA were added to a final concentration of 0.2 g/l each. Differential solvent extraction supported this impression, whereby only fractions rich in LNA were inhibitory. It is noteworthy that the less polar ether extract contained a higher content of LNA than the chloroform fraction, yet the chloroform fraction was far more inhibitory. Presumably the latter fraction contained the majority of non-esterified LNA. It is the non-esterified unsaturated fatty acids rather than their esterified form that inhibit biohydrogenation via their toxicity to ˛ biohydrogenating ruminal bacteria (Wasowska et al., 2006). This is exemplified in studies with fish oil and its component PUFA (AbuGhazaleh and Jenkins, 2004; Lee et al., 2005; Vlaeminck et al., 2008). On the other hand, it is known that C. coronarium contains an unusual epoxy fatty acid, coronaric acid (14% of the oil; Earle, 1970), which could also contribute to the 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 by combining LNA and coronaric acid in C. coronarium. It is important to note, as well, that other components might be involved in the reported effect too. Essential oils and essential oil compounds have been shown to be of potential usefulness in controlling biohydrogenation (Durmic et al., 2008; Lourenc¸o et al., 2009). C. coronarium contains essential oils, the composition of 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 their Chrysanthemum-containing sward was actually lower than the control sward containing the other two plants only. The concentration of coronaric acid may be equally important. An issue not addressed by Cabiddu et al. (2006) or here is that of lipase activity. As the inhibitory effects of PUFA depend on their non-esterified form, the LNA concentration of greatest importance will be transitory, depending on the rate of release by lipolysis and the subsequent rate of isomerization and biohydrogenation. 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) suggested that 9 -desaturase might be affected, altering the balance of VA and RA in the mammary gland. Fatty acid oxidation products also inhibit biohydrogenation (Lee et al., 2007). 5. Conclusions LNA in its non-esterified form appears to be an important mediator of inhibition of fatty acid biohydrogenation by ruminal microorganisms. Coronaric acid also has an inhibitory effect on biohydrogenation. Understanding how the LNA content of different plant species and cultivars varies and the role of microbial lipase in its release will be useful in devising plantbased strategies to increase the RA content of milk. In the specific case of Chrysanthemum, LNA and coronaric acid content can 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 Directorate of the Scottish Government. The results presented here were from the EC Framework 6 project, ‘REPLACE’: Plants and their extracts and other natural alternatives to antimicrobials in feeds, contract no.: 506487. ERM has a TALENTIA scholarship from the Regional Ministry for Innovation, Science and Enterprise, Andalusia, Spain. XS and CA received Fellowships from 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, David Brown and Donna Henderson for help with fatty acid analysis. Thanks are due to Graham Horgan for his help with statistical analysis of data. References AbuGhazaleh, A.A., Jenkins, T.C., 2004. 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