COMPOSITION AND METHOD FOR IMPROVING THE QUALITY OF MILK

The present application relates to compositions and methods for improving the quality of milk, in particular to nutritional compositions for feeding to ruminants.

During the production of milk, one of the important considerations to the dairy farmer and consumer is the quality of the milk produced, both in terms of taste and nutritional composition. However, there are a number of factors which can affect the quality of the milk, one being the time of year in which the milk is produced.

Several surveys have shown that concentrations of polyunsaturated fatty acids and the predominant conjugated linoleic acid (CLA) isomer in milk fat are higher for summer milk produced from cows grazing fresh pastures than for winter milk when conserved forages are fed. This makes summer milk more beneficial in terms of quality and human health than winter milk.

It would, therefore, be desirable to eliminate this difference between summer milk and winter nulk so that inter milk had the same high quality and fatty a id contciil of suinuier milk.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a composition comprising one or more green odour compounds or one or more derivatives, analogues or homologues thereof.

Preferably, the one or more green odour compounds are fatty acid oxidation products (FAOPs) selected from (i) one or more hydroperoxides, (ii) one or more alcohols, (iii) one or more aldehydes, (iv) one or more ketones, (v) one or more epoxy hydroperoxides, (vi) one or more divinyl ethers, and (vii) one or more jasmonates. Preferably, the one or more hydroperoxides are selected from (9S)- hydroperoxyoctadecatrienoic acid, (9S)-hydroperoxyoctadecadienoic acid, (13S)- hydroperoxyoctadecatrienoic acid and (13S)-hydroperoxyoctadecadienoic acid.

Preferably, the one or more alcohols are selected from ketol, 3-hexenol and 2-hexenol.

Preferably, the one or more aldehydes are selected from malondialdehyde, 3-hexenal and 2-hexenal.

Preferably, the one or more ketones are selected from small vinyl ketones, actone and butanone.

Preferably, the one or more epoxy hydroperoxides are selected from 9,10- epoxyoctadecatrienoic acid and 12,13- epoxyoctadecatrienoic acid.

Preferably, the one or more divinyl ethers are selected from C18:3n-3 and C18:2n-6 generated divinyl ether polyunsaturated fatty acids, such as etherolenic acid.

Preferably, the one or more jasmonates are selected from jasmonic acid and metabolites such as methyl ester or amino acid conjugates.

In particularly preferred embodiments, the green odour compounds comprise one or more of hydroperoxides and jasmonates.

Preferably, the composition is a nutritional composition.

Preferably, the composition is a nutritional supplement.

Preferably, the composition is an animal feed.

Accordingly, in one aspect of the present invention, there is provided an animal feed comprising a supplement of one or more green odour compounds. According to another aspect of the present invention, there is provided a method for improving the quality of milk produced from a ruminant, the method comprising administering to a ruminant a composition as described herein. Preferably, the method is for improving the fatty acid content of milk.

Preferably, the method is for improving the quality of winter milk production.

Preferably, the ruminant is selected from a cow, goat, reindeer, sheep, water buffalo and yak.

Another aspect of the present invention relates to use of a composition as described herein for improving the quality of milk produced from a ruminant.

Example embodiments of the present invention will now be described with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of unweighted pair group method with arithmetic mean dendogram showing the effect of hydroperoxides on the bacterial community in the rumen following PCR-DGGE (A), Hael l l (B), and MSP1 (C)-based T-RFLP. Scale relates to percent similarity;

Figure 2 shows the results of unweighted pair group method with arithmetic mean dendogram showing the effect of salicylic acid and jasmonic acid on the bacterial community in the rumen following PCR-DGGE (A), Hael l l (B), and MSP1 (C)-based T-RFLP. Scale relates to percent similarity; and

Figure 3 shows the results of unweighted pair group method with arithmetic mean dendogram showing the effect of hydroperoxides, salicylic acid and jasmonic acid on the bacterial community in the rumen following PCR-DGGE (A), Hael l l (B), and MSP1 (C based T-RFLP. Scale relates to percent similarity. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and method for improving the quality of milk.

Winter as opposed to summer milk is of a lower quality due to the lower proportion of beneficial fatty acids namely PUFA and CLA as a consequence of the animals consuming conserved as opposed to fresh grass. This invention aims to improve the composition of winter milk by providing animal feed with a supplement which will mimic the action of 'green odour' compounds released during summer grazing and thought to be partially responsible for the higher levels of PUFA and CLA in summer milk.

In one aspect, the present invention provides an animal winter feed supplement made from synthesised fatty acid oxidation products (FAOPs) which when offered will improve the fatty acid composition of the milk, i.e. milk more akin to pasture grazing than winter conserved grass feeding.

The methods used in the invention and detailed examples of the invention are set out below.

Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the invention.

Within this specification, the terms "comprises" and "comprising" are interpreted to mean "includes, among other things". These terms are not intended to be construed as "consists of only".

Within this specification, the term "derivatives" refers to molecules derived from the above-described compounds. Such derivatives may, for example, be digestion products of the above-described compounds or synthetically altered derivatives of the above described compounds. Within this specification, the term "homologues" refers to molecules having substantial structural similarities to the above-described compounds.

Within this specification, the term "analogues" refers to molecules having substantial biological similarities to the above-described compounds regardless of structural similarities.

Oxidative loss of polyunsaturated fatty acids (PUFA) during field wilting represents a major loss to the food chain, with substantial losses of linolenic acid (C18:3n-3) during haymaking and modest losses during wilting prior to ensiling. These losses are associated with the lipoxygenase system- a plant defence mechanism which is initiated in damaged tissues. Plant lipases release non-esterified C18 PUFA, (C18:3n-3 and C18:2n-6) from damaged membranes and these are rapidly converted to hydroperoxy PUFA by the action of lipoxygenases. The hydroperoxy PUFA are further catabolised to yield a range of volatile compounds, such as leaf aldehydes and alcohols which are released during grass cutting and grazing to give the distinctive 'green odour'.

Several surveys have shown that concentrations of the predominant conjugated linoleic acid (CLA) isomer, namely cis-9 trans-1 1 and total CI 8 PUFA in milk fat were higher for summer milk produced from cows grazing fresh pastures than for winter milk when conserved forages were fed, making summer milk more beneficial in terms of quality and human health than winter milk. Furthermore a similar depression in milk fat cis-9 trans-11 CLA could be achieved if the grass was simply cut and fed after a short wilt. The 'green odour' consists mainly of fatty acid oxidation products (FAOP): hydroperoxides, alcohols, aldehydes and ketones.

We have preliminary evidence that a number of these FAOP have an effect on lipid metabolism, with particular emphasis on CI 8 PUFA biohydrogenation and intermediate formation (VA and CLA), during in vitro batch cultures. The most potent FAOP was then used in a titration study to determine if the marked changes in lipid metabolism were related to any changes in the microbial ecology, thus providing an explanation for the changes in milk fatty acid content between grazing pasture and feeding conserved forage such as silage where these FAOP products are lost. The results indicate that these compounds could be used as a dietary supplement during winter feeding to improve the fatty acid profile of milk. The results show that there is scope to synthesise 'green odour' compounds such as long chain hydroperoxides, aldehydes and peroxides like those produced from freshly cut grass and use them as a supplement in winter feed to simulate the effect of the these compounds at grazing and improve the fatty acid profile of winter milk.

EXAMPLE 1 - The effect of green odour compound on CI 8 fatty acid metabolism and the rumen microbial ecosystem in in vitro batch culture.

Site: Gogerddan

Date: February 2010

Protocol responsibility: MRF Lee, SA Huws

Animal management: M Leyland, N Ellis

Sample collection and storage: MRF Lee, SA Huws, MB Scott

Chemical analysis: MRF Lee, SA Huws, MB Scott, JKS Tweed

Statistical consultant: R. Sanderson

Time recording code: 10316 ESDF Green Odour

Home office Licence: PPL 40-3020 (JM Moorby)

Introduction

The effect of a combination of specific green odour compounds released during cell damage at physiological levels on CI 8 fatty acid metabolism and rumen microbial ecosystems was investigated. The ability of these compounds to actively influence the microbial ecosystem and lipid metabolism provides evidence for their usefulness in vivo to help improve the quality of ruminant products such as milk in their fatty acid profiles.

Experimental design

The experiment was a simple batch culture experiment with four treatments each with a basal level of freeze-dried silage and either: control, no green odour compounds (C); hydroperoxides, the initial oxidation products of linoleic and linolenic acids (HP); terminal green odour compounds, the signal compounds released by the damaged plant (T); and a combination of hydroperoxides and terminal signal compounds (HPT). Each treatment had three replicate incubations per each of the four time points (0, 2, 4 and 6 h). These were incubated under rumen like conditions in the presence of rumen inoculum and then destructively harvested for the measurement of lipid metabolism and microbial ecosystem profiles. The fixed effects were treatment (C vs. HP vs. T vs. HPT); time (0 h vs. 2 h vs. 4 h vs. 6 h) and interaction (treatment * time).

Rumen inoculum

Four fistulated dairy cows maintained on permanent pasture each provided 0.5 L of hand squeezed rumen liquor along with 100 g of digesta solids. The samples from each cow were combined and thoroughly mixed to provide a single 2 L sample of rumen liquor and 400 g of rumen solids. These were transferred back to the laboratory in a temperature regulated container and then once in the lab transferred to a water bath at 39°C with continual C0 ₂ purging. The rumen solids were prepared to extract solid associated bacteria by stomaching in Van Soest medium based on the average DM of the rumen contents of a forage fed dairy cow (12% Dewhurst et al. 2003). Rumen solids (24 g ~ 6 g from each cow) were stomached in 200 mL of Van Soest medium. This was then combined with 200 mL of the rumen liquor and the combined solution used as the inoculum (circa 400 mL).

Anaerobic buffer and inoculation

The day before the start of each experiment, anaerobic incubation ^" medium was made up as described by Goering and Van Soest (1970):

Buffer solution (g/L) Ammonium hydrogen carbonate 4

Sodium hydrogen carbonate 35

Macromineral solution (g/L) Di-sodium hydrogen orthophosphate 12-hydrate 9.45

Potassium di-hydrogen orthophosphate (anhydrous) 6.2

Magnesium sulphate 7-hydrate 0.6

Micromineral solution (g/100 mL) Calcium chloride 2-hydrate 13.2

Manganese chloride 4-hydrate 10.0 Cobalt chloride 6-hydrate 1.0

Ferric chloride 6-hydrate 8.0

Resazurin solution (g/ 100 mL) Resazurin (redox indicator) 0.1

Reducing agent (g/100 mL) Cysteine HC1 0.625

Distilled water 95 mL lM NaOH 4 mL

Sodium sulphide 0.625

Make up according to the recipe:

Distilled water 1500 mL

Micromineral solution 0.3 mL

Buffer solution 600 mL

Macromineral solution 600 mL

Resazurin solution 3 mL

Reducing agent is added at 4.7% of the total volume

Setting up incubations

Freeze-dried and ground grass silage was weighed (1 g DM) into 48 individual extraction tubes with the exact weight accurately recorded. Buffer (6.0 mL) was dispensed into each extraction tube by peristaltic pump gassing headspace with C0 ₂ as tubes were sealed. These were then left overnight in an anaerobic cabinet at 39°C.

Green odour compounds

The concentration of green odour compounds was as described in Lee et al. (2007) derived from the typical release rate of fatty acid oxidation products from cut grass (0.3mmol/kg Fresh weight min, Hatanaka, 1993) and the average dry matter of the rumen contents of a forage fed dairy cow (12%, Dewhurst et al., 2003). Therefore, the green odour compounds were to be added at a final concentration of 200μΜ. To achieve this, two stock solutions were made: HP Stock solution: Ethanol 10ml

13 S-Hydroperoxyoctadecatrienoic acid 49.6 mg

13S-Hydroperoxyoctadecadienoic acid 49.6 mg

T Stock solution: Ethanol 10ml

Jasmonic acid 22.4 mg

Hexanal 5.7 mg

Hexenol 5.6 mg

Salicylic acid 14.9 mg

The two hydroperoxides were prepared from linolenic and linoleic acid as described by Drouet et al. (1994) and Gardner and Grove (2001). Briefly: lOOmg linoleic/linolenic acid was dissolved in Octane which was added to a 2mg solution of LOX-1 in Borate buffer pH9.6 at a ratio of 1 :8, octane : buffer, 1.25ml: 10ml. Reaction was catalysed by oxygen added through a diffuser with vigorous stirring for 30mins. Solution pH was lowered to 4.0 with 1M Oxalic acid. Octane extracted and dried carefully under N ₂ , immediately re-dissolved in 1ml diethyl ether and stored at -80°C. Percentage yield of hydroperoxides was checked by converting to methyl trimethylsilyl ethers for running on GC-FID.

Incubation, sampling and harvesting

The day of the experiment the 48 tubes were split into four groups of 12. These groups were then treated with either: ΙΟΟμί of ethanol (C); ΙΟΟμΙ of HP stock solution (HP); ΙΟΟμΙ, of T stock solution (T); or 50μΙ, of HP stock solution and 50μΙ, of T stock solution (HPT). The tubes were then inoculated with 6.0 mL of the rumen inoculum. A sample (20 mL) of inoculum was retained frozen for later chemical analysis. The tubes were then incubated in the dark at 39°C with rotation at 100 rpm. At the allocated time points three tubes were destructively harvested for each treatment by firstly blending and a 200μ1 aliquot taken for RNA extraction prior to freezing with liquid N ₂ and retaining in the freezer at -20°C before freeze-drying. Weight before freezing and post-freeze drying was taken to calculate dry matter content so that quantitative RNA data could be converted to suitable units/ g DM in line with DNA based data. Molecular techniques

RNA was extracted from the aliquoted 200μ1 using the QBiogene FastRNA direct pro soil kit according to manufacturers instruction. RNA was then DNAse treated before reverse transcription to cDNA using Invitrogen reverse transcriptase 11 1 (See J:Nutrition and microbiology/ruminant microbiology/methods/molecular/reverse transcription). DNA was extracted from freeze-dried, milled samples (10 mg DM) using the QBiogene soil extraction kit. Total eubacterial diversity within cDNA and DNA was investigated using DGGE and/or Terminal Restriction Fragment Length Polymorophisms (T-RFLP) as described in J:Nutrition and microbiology/ruminant microbiology/methods/molecular/T-RFLP. The usual 27F fam and 1389r primer pair were used. Total eubacterial 16S rRNA gene concentration within cDNA and DNA was assessed using the Maeda primer pair (Maeda et al., 2003). C. proteoclasticum group, Anaerovibrio lipolytica, Fibrobacter succinogenes, Megasphaera elsdenii, Prevotella spp. Ruminococcus albus, Ruminococcus flavefaciens, Streptococcus bovis and Butyrivibrio hungatei (VA2 group) QPCR was conducted as described in J:Nutrition and microbiology/ruminant microbiology/methods/molecular/QPCR for cDNA and DNA.

Lipid extraction and fractionation

Once freeze-dried the lipid was extracted by adding 3 x 4 mL of chloroform : methanol (2:1 ; v/v) and ΙΟΟμΙ of internal standard (C21 :0 15 mg/mL CHC13). The extract was collected in a clean extraction tube dried down under N ₂ at 50°C resuspended in 1 mL of heptane and then bimethylated using the procedure of Kramer and Zhou, (2001) for total lipid analysis.

Analysis

Freeze-dried grass: FDM, FAME, WSC, NDF, OM, TN

Inoculum: FAME

Incubations: Total lipid (FAME), microbial diversity, total

eubacteria, A. lipolytica, SA bacterial group. Results

Table 1. Temporal proportional (% of total fatty acids) quantities of prevalent fatty acids within batch cultures in the absence of treatment (control), and presence of hydroperoxides (HP), terminal green odour compounds (Terminal) and a combination of hydroperoxides and terminal green odour compounds (HPT).

Fatty acid

Time Treatment 18:3n-3 C18:2n-6 CLA ¹ CLA C s-9, C18:1 C18:1 C18:1 C18:1 C18:0 BOC ( ) trans- 1 cis trans frans-10 frans-11

2 Control 51.59* 13.63" 0.38" 0.13° 3.57 1.02" 3.99 4.17" 3.51" 3.62"

HP 55.90 14.17 0.23° 0.12° 3.43° 0.64° 3.42° 3.58° 2.73° 2.49"

Terminal 50.60 ^a 13.13° 0.37" 0.13° 3.46 ^a " 1.20" 3.68" 4.05" 3.33°" 2.50°

HPT 52.07° 13.40"" 0.32" 0.11 ° 3.60° 1.05" 3.69" 3.93" 3.74" 3.24"

SED 0.82 0.13 0.02 0.00 0.05 0.09 0.07 0.14 0.30 0.20

P value <0.001 <0.001 0.002 0.11 0.015 0.002 <0.001 0.018 0.024 0.001

4 Control 46.70 ^a 13.30 ^a 0.47 0.04" 0.11 ^a " 1.69 0.08 1.14 4.01 4.15

HP 55.87 14.52 0.24a 0.02° 0.12" 0.54" 0.02 ^a 0.34° 2.66° 2.20°

Terminal 51.68" 13.78" 0.32b 0.06 0.10 ^a " 1.00" 0.04" 0.66° 3.49" 3.18"

HPT 52.20 13.88" 0.34b 0.04" 0.09 ^a 1.01" 0.04" 0.62a 3.55" 3.00°

SED 1.14 0.16 0.01 0.00 0.00 0.16 0.00 0.14 0.20 0.25

P value <0.001 <0.001 O.001 0.003 0.084 <0.001 <0.001 0.002 <0.001 <0.001

6 Control 44.90" 13.49° 0.62 0.64" 0.1 1 ^a " 1.73" 0.09 ^c 1.11" 4.76 4.36

HP 53.91 i4.5a u.31 ⁼ 0.38 ^: 0. 2" 0.70° 0.03° 0.43° 2.91 " 2.51°

Terminal 48.02 ^alJ 13.78° 0.53" 0.75" 0.10 ^a " 1.44" 0.07°" 0.95" 4.14" 3.61 "

HPT 48.97" 13.89° 0.50" 0.69" 0.08° 1.24" 0.06°" 0.81" 4.08" 3.18 ^a "

SED 1.48 0.19 0.03 0.06 0.01 0.21 0.01 0.14 0.30 0.30

P value 0.002 0.002 <0.001 0.001 0.131 0.008 0.006 0.007 <0.001 0.002

SED = standard error of the mean; 1 = Sum of all isomers; CLA = conjugated Iinoleic acid;

BOC = Branched and odd chain fatty acids

Means with different superscripts ^a ' ^{b c} within columns and time differ significantly P<0.05. SUMMARY

As described herein, biotic and biotic rumen processes illicit plant stress responses resulting in the production of chemicals often refereed to as 'Green Odour'. In the examples described herein we investigated the effect of 'Green Odour' products, namely hydroperoxides (initial oxidation products of iinoleic and iinolenic acids) (HP), terminal green odour compounds (signal compounds released due to plant damage) (T), and a combination of both (HPT) on ruminal bacterial diversity and CI 8 fatty acid metabolism. Following 6 h of in vitro incubation 15.3 % of 18:3 n-3 and 4.4 % of 18:2 n-6 were biohydrogenated in control incubations, in the absence of any treatment addition, compared with 1.3, 9.4 and 9.3 % of 18:3 n-3 for HP, T and HPT treatments respectively whilst no 18:2 n-6 biohydrogenation was seen in the presence of these chemicals. Both Denaturing Gradient Gel Electrophoresis (DGGE) and Hae III, MSP 1 Terminal-Restriction Fragment Length Polymorphism (T- RFLP)-based dendograms revealed that the presence of HP, T and HPT caused sub-clustering of the bacterial population present compared to control incubations. Thus, chemicals released due to rumen-based plant cell damage are able to cause changes in ruminal bacterial diversity as well as decreasing fatty acid metabolism.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications are covered by the appended claims.

The content of all references cited herein are incorporated herein by reference in their entirety.

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