WO2005036981A1

WO2005036981A1 - Method for enriching nervonic acid in expressed milk of ruminants

Info

Publication number: WO2005036981A1
Application number: PCT/CA2004/001856
Authority: WO
Inventors: William J. Bettger; Brian Mcbride; Tom Wright
Original assignee: University Of Guelph
Priority date: 2003-10-21
Filing date: 2004-10-21
Publication date: 2005-04-28

Abstract

The present invention is directed to a new method and feed additive and a method for enriching nervonic acid (NA) in expressed milk of ruminants, for example, dairy cattle. The feed additive comprises a source of NA and an effective amount of an inhibitor of degradation of NA in the rumen of ruminants consisting of feather meal.

Description

Title: METHOD FOR ENRICHING NERVONIC ACID IN EXPRESSED MILK OF RUMINANTS

Field of the Invention: This invention relates to fortification of ruminant milk, in particular it concerns methods for enriching very long chain fatty acids in dairy milk in particular a feed additive and method for enriching nervonic acid in expressed milk of dairy cattle.

Background of the Invention: Nervonic acid (NA) or ^'s-15-tetracosenoic acid (24:ln-9) is a very long chain fatty acid (VLCFA - greater than or equal to 22 carbons). It has the common name nervonic acid because it is abundant in nerve tissue, particularly in the white matter of brain and in myelinated, peripheral nerves. Except for a few rare plants, nervonic acid is not found in plants in quantities higher than 0.01 % of total fatty acids. Nervonic acid is found from 10 to 100 fold higher concentration in animal tissues. In plants, if present, nervonic acid is found in triglyceride form; in animals, nervonic acid is present as a major fatty acid of the phospholipid sphingomyelin. Nervonic acid is a normal component of the diet of omnivorous humans. Concentrated dietary sources of nervonic acid include human milk. Poor sources of nervonic acid are milk from ruminants, meat and plant foods. Nervonic acid is considered to be a conditionally essential nutrient, particularly in premature infants, formula fed babies and in patients with certain degenerative brain diseases like multiple sclerosis and adrenoleukodystrophy (Medical Hypotheses (1994) 42: 237- 242). The main dietary form of nervonic acid is as sphingomyelin. Nervonic acid- containing sphingomyelin may have special physiological functions. Sphingomyelin is being investigated as a nutraceutical for reducing the risk of gastrointestinal cancers and cardiovascular disease (Eur J Cancer Prev (2002) 11: 193-197; J Nutr (1997) 127: 1055-1060). Dietary nervonic acid readily raises the nervonic acid content in the sphingomyelin of some tissues. Bettger et al. (Lipids (1997) 32: 51-55; Nutr Res (1996) 16: 1761-1765) have shown, using a rat bioassay, that dietary nervonic acid significantly elevates the nervonic acid content of sphingomyelin in liver, heart, skeletal muscle and adipose tissue in developing rats. Barre and Holub (Lipids (1992) 27: 315-320) have shown that human adults fed a dietary supplement rich in nervonic acid raises the nervonic acid content of platelet sphingomyelin. Nervonic acid is a major fatty acid in the sphingomyelin fraction of milk from monogastric animals, including humans. However, the content of nervonic acid in the sphingomyelin in the milk of ruminants is roughly 10-fold lower than other VLCFA. It is not clear why the nervonic acid content is so much lower than other VLCFA in the milk of ruminants. The nervonic acid content in the milk of omnivorous, monogastric animals depends, in part, on the dietary intake of nervonic acid. Researchers have shown that feeding nervonic acid rich diets to rats or mice (model monogastric animals) will increase the nervonic acid of the sphingomyelin fraction of the expressed milk (J Nutr Biochem (2003) 14: 160-165; Lipids (1998) 33: 993-1000). There is commercial interest in the use of nervonic acid-rich lipids as dietary supplements. US 5,194,448 (owned by Croda International, in the United Kingdom), teaches the production and use of nervonic as triglyceride isolated from the Lunaria plant, and US 5,994,404 (also in the name of Croda International) teaches various nervonic acid compositions from plants, animals and/or microbial products, including, the seed oils of Cardamine gracea, Heliphila longifola, Thlaspi perfoliatum, Tropaeolum speciosum, Lunaria biennis, Lunaria annua and Malania oleifera; the moulds Neocallismastix^' frontalis, Erysiphe graminis and Sphaerotheca humuli; the bacterium Pseudomonas atlantica; the yeast Saccharomyces cerevisiae and the marine diatom Nitzschia cylindrus.

There have been a number of reports on efforts to enrich bovine milk with various fatty acids. A recent review outlines a number of these (Ashes, J. R. et al. (1997)). For example, methods have been developed to increase the level of omega-3 fatty acids in the flesh of beef cattle (US 5,290,573), sows (US 5,106,639; DE 3808885; Taugbol, O. et al, Zentralbl. Veterinarmed. A., (1993) 40(6): 437-443), poultry (US 5,012,761; JP 04271754; US 5,133,963; US 5,069,903), and eggs (KR 9311396; US 5,069,903). DHA has also been added as a dietary supplement to infant formula as discussed above, and milk. Sources of DHA for supplementing milk or infant formula include fish products, fatty acid containing microbial oils (US 5,374,657; US 5,397,591; US 5,407,957), or fatty acids extracted from a mixture of egg yolk and coconut oil (US 4,670,285). Indeed, researchers have been able to increase DHA content in the expressed milk of humans (Harris, W.S. et al, Am. J. Clin. Nutr. 40(4): 780-785, 1984; Henderson, R.A., Lipids, 27(11): 863-869, 1992; US 5,069,903), sows (Taugbol, O. et al, Zentralbl, Veterinarmed. A. 40(6): 437-443, 1993), and rats (Yonekubo, A., et al. /. Nutr. 123(10): 1703-1708, 1993). However, researchers have had difficulty obtaining significant levels of DHA in cow's milk. (Hebeisen, D.F., et al. Int. J. Vitam. Nutr. Res., 63(3): 229-233, 1993). A method, a feed additive and a feed to increase DHA content in expressed milk of dairy cattle has been disclosed by one of the present inventors in US 5,932,257 and in Wright, T.; McBride, B. ; and Holub, B, World Rev. Nutr. Diet 83: 160-165, 1998. However, there are no reported studies of an attempt to enrich bovine milk or low- fat milk products with nervonic acid. Yet in light of the importance of nervonic acid in the diet it is clear that it is desirable to have a method of enriching milk products with nervonic acid.

SUMMARY OF THE INVENTION Surprisingly, the present inventors have found the use of a feed additive comprising feather meal and a source of NA such as oil extracted from the seeds of the plant Lunaria annua is able to dramatically increase the content of VLCFA in the expressed milk of a ruminant. The NA is in the whey protein fraction of expressed milk of dairy cattle fed a novel feed additive containing NA, and inhibitors of degradation of NA in the rumen of the cattle. The feed additive does not affect the ability of the cattle to digest the feed by normal symbiotic digestion. The feed additive is also palatable to the cattle, and therefore food consumption is not decreased. As a result, the health of the cattle is maintained and their productivity is not reduced. In addition, because NA is present in the whey protein fraction, the expressed milk is suitable for the production of low-fat dairy products and milk protein fractions. The present inventors found that when dairy cattle are fed the feed additive throughout lactation, the levels of NA in the whey protein fraction of expressed milk are between 26% and 34% of the fatty acids in sphingomyelin (see table 3). Broadly stated, the present invention relates to a feed additive for dairy cattle which comprises a source of NA and inhibitors of degradation of NA in the rumen of dairy cattle. The source of NA and the inhibitors of degradation of NA are present in the feed in an amount sufficient to enhance the concentration of NA in the milk of ruminants, preferably dairy cows, fed with feed containing the additive. In an embodiment of the invention, the source of NA is the seed oil of Lunaria annua and Lunaria biennis (the Honesty plant). Various methods of extracting seed oils from oil bearing seeds are well known to those skilled in the art (see "Baileys Industrial Oil and Fat Products" ed. D. Swern, Vol. 2, pages 175 et. seq. 4th Edition, Pub 1982, John Wiley & Sons Inc.). In one embodiment the inhibitors are inhibitors of degradation and comprise feather meal. Preferably the feed additive comprises an amount of feather meal sufficient to increase the concentration of NA in milk from cattle consuming the feed additive. The invention also contemplates a feed containing the feed additive. The invention also relates to a method of producing milk in dairy cattle which is enriched for NA comprising feeding dairy cattle a diet containing a feed additive of the invention for a period of time longer than one day and preferably for at least about 7 days and milking the dairy cattle to obtain milk enriched for N A. The invention further relates to expressed milk from dairy cattle enriched with NA which is produced by feeding cattle a diet containing a feed additive of the invention for a period of at least two days, preferably at least about 7 days, and milking the dairy cattle to obtain milk enriched for N A. The expressed whole milk of the invention preferably contains about 16% of NA in the sphingomyelin fraction (see Table 2). NA is also enriched in other ceramide-based lipids such as ceramides, phosphoceramides and glycoceramides. The invention further relates to a NA-enriched dairy product produced using . the expressed milk of the invention. The NA-enriched dairy product is preferably selected from the group consisting of fluid milk, cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant milk and butter. In another preferred embodiment the dairy product is a low-fat dairy product. In yet another preferred embodiment the dairy product is a milk protein product. The milk protein products is preferably selected from the group consisting of dried whey protein concentrate, dried whey, dried skim milk, dried milk protein, dried buttermilk, casein, acid casein, a protein drink, a protein bar or a protein supplement. In one embodiment, the invention relates to the use of feather meal as an inhibitor of degradation of NA in the rumen of dairy cattle in an amount sufficient to increase the concentration of NA in milk. In another embodiment the feather meal is added to the feed for dairy cattle. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Table 1. Effect of a Nervonic Acid Supplement (Canstar) on Feed Intake and Milk Production of Dairy Cows*

* Values represent the means of n=6 cows. Values within a parameter are not significantly altered by the nervonic acid supplement (Canstar) or by the number of days on the experiment (Student's t-test, P > 0.05). Table 2. Effect of a Nervonic Acid Supplement (Canstar) on the Fatty Acid Composition of Sphingomyelin from Whole Milk of Dairy Cows*

* Values are means (standard deviations) of n = 6 cows. Value for nervonic acid (24:1) on day 7 of the trial (Canstar Day 7) is significantly higher than that of the Olive Day 7 and the Day 1 controls (Student's t-test, P< 0.001). ^a The range of NA (24:1) values for Canstar Day 7 are 11.5 to 25.0%

Table 3. Effect of a Nervonic Acid Supplement (Canstar) on the Composition of Very Long Chain Fatty Acids in Sphingomyelin of Isolated Whey Protein from Milk*

*Values for the nervonic acid (24:1) content of the sphingomyelin in the whey protein fraction of cows milk, day 7 of the experiment. Nervonic acid content is increased over 10-fold by the Canstar supplement.

As hereinbefore mentioned, the present invention relates to a feed additive for dairy cattle which comprises a source of NA, and inhibitors of degradation of NA in the rumen of dairy cattle. Preferably, these components are present in an amount sufficient to increase the concentration of NA in the whey protein fraction of the expressed milk of dairy cows fed a diet containing the additive. "An amount sufficient" as used herein would be understood by a person skilled in the art to be the amount which results in an increased concentration of NA in milk. The NA in the feed additive may be a NA concentrate containing 4 to 98% NA, or it may be a component of an extract from a source known to contain NA; for example, it may be a component of an extract derived from fish, such as fish meal. NA concentrates may be obtained from commercial sources for example, CROSSENTIAL N25. Indeed any source of NA which is palatable to cattle and which does not compromise the taste qualities of milk produced may be used. According to an embodiment of the invention, the feed additive comprises feather meal and a Lunaria oil based product containing NA. The NA may be made from the seeds ( or any other part ) of the plant Lunaria annua, and contains an amount of NA which will provide about 0.1 to 0.8% NA by weight of the feed supplement as described herein. In another embodiment there is 0.2 to 0.6%NA by weight of the feed supplement, and in yet another embodiment the nervonic acid content of the supplement is 0.375%. The feather meal may be made from the feathers of broiler chickens, although the feathers of any foul including turkeys and geese may be used. Feather meal may be selected which has 93.9% protein, and a total fat content of 2.6% (dry measurement basis). It will be appreciated that the feather meal may be obtained from commercial sources for example, Cargill, Shurgain, Masterf eeds, ADM & Land O' Lakes. In a preferred embodiment, the feed additive contains 5 to 60% feather meal, preferably 10 to 20% by weight of the total feed additive; and 5 to 20 % of Canstar oil preferably 8 to 15% by weight of the total feed additive.

In a more preferred embodiment, the feed additive contains 15% feather meal by weight of the total feed additive and 10% of Canstar oil by weight of the total feed additive. The feed additive may contain a carbohydrate fraction such as soft white wheat or corn. Preferably the feed additive contains soft white wheat. The feed additive may be added to a basal feed which may contain a carbohydrate fraction, a protein fraction, a lipid fraction and/ or a vitamin/ mineral fraction. Examples of components in carbohydrate fractions include corn silage, alfalfa hay, Timothy hay, wheat straw, barley grain, canola meal, oat grain, mixed straw, and corn. Typical components in the vitamin/ mineral fraction include magnesium oxide, limestone, potassium chloride, sodium chloride, and a trace mineral supplement, containing zinc, copper, manganese, selenium, vitamins A, D and E. Commercial sources of these components are Cargill, Shurgain, Masterfeeds, ADM & Land O' Lakes. Typical protein feeds include soybean meal, corn gluten meal and distillers dried grains. Typical lipid feeds including vegetable fat sources (Church & Dwight Co.). The feed containing the feed additive may be pelleted for feeding to dairy cattle or the feed additive and basal feed may be fed to the cattle in a total mixed ration or as separate ingredients. In an embodiment of the invention a feed is provided comprising (a) a basal feed containing 2 to 10%, preferably 5-7%, mixed straw; 40 to 55%, preferably 45 to 47% corn silage; 35, to 50%, preferably 42 to 45% high moisture corn, and 2-4% of a vitamin/ mineral fraction, each percentage being a percentage of the total weight of the basal feed; and (b) a feed additive comprising 1 to 7% feather meal, 0.5 to 3.5% Canstar oil, each percentage being a percentage of the total weight of the feed (dry weight basis). In a preferred embodiment of the invention the feed additive comprises about 2.0% feather meal and about 1.5% Canstar oil , each percentage being a percentage of the total weight of the feed (dry weight basis). In a more preferred embodiment, the feed additive comprises about 1.0% feather meal and about 1.0% Canstar oil, each percentage being a percentage of the total weight of the feed (dry weight basis)

As illustrated in the examples herein, the use of a feed additive comprising feather meal and a source of NA such as oil extracted from the seeds of the plant Lunaria annua provides milk with an increased concentration of NA. Accordingly, the invention also relates to the use of feather meal as an inhibitor of microbial degradation of NA in the rumen of dairy cattle. Although it is to be understood that feather meal may also be functioning as an inhibitor of non- microbial-mediated degradation of NA in the rumen of dairy cattle. The use of feather meal is characterized in that the feather meal is added to the feed for dairy cattle. According to a preferred embodiment, the use is characterized in that an amount sufficient to enrich milk from dairy cattle with NA is added to feed additive, which itself may be added to cattle feed. The invention also relates to a method of producing milk in dairy cattle which is enriched for NA comprising feeding the dairy cattle a diet containing the feed additive of the invention for a period of at least 2 days, and preferably about 7 days, and milking the dairy cattle to obtain milk enriched for NA. The cattle may be fed a basal feed containing the feed additive, or the feed additive and basal feed may be fed to the cattle in a total mixed ration or as separate ingredients. The cattle are preferably fed throughout lactation and for at least two days, preferably for at least 7 days, and more preferably 12 days, in order to obtain expressed milk with a NA content which is greater than or equal to 16% of the total fatty acids of sphingomyelin in whole milk. Whey protein isolated from such milk will have greater than 26% of the VLCFA in sphingomyelin as NA. Typically the cattle are fed the feed additive (which may be part of a basal feed) twice daily. The amount of feed additive given to the cattle ranges from 600 grams to 7.5 kg per animal per day. The method of the invention for producing expressed milk enriched for NA may be applied to any breed of dairy cattle, for example, Ayshire, Guernsey, Holstein, Jersey, Brown Swiss, Dutch Belted, Canadienne and Milking Shorthorn. It will be appreciated that the method may also be applied to other ruminant species such as sheep and goats to produce expressed milk enriched for NA. The expressed milk from dairy cattle enriched for NA produced by a method of the invention contains levels of NA typically in the range of 11.5 to 25% (see Table 2) of the fatty acids in sphingomyelin in the milk. These levels are as high as, or higher than NA levels found in human expressed milk which are typically in the range of 15.5 to 20% (Am J Clin Nutr (1984) 40: 1103-1119). The taste of the milk enriched for NA produced by the method of the invention is not altered and it is therefore suitable for human consumption. Further, the method achieves these concentrations of NA in milk using only feather meal as the inhibitor of NA degradation in the rumen. It will be appreciated that other NA-enriched dairy products can be produced by using the method described herein. For example, cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant formula, and butter enriched for NA may be produced using the method of the invention. In addition, low-fat dairy products and milk protein products can also be produced by using the method described herein. For example, low-fat , cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant formula, and butter enriched for NA may be produced using the method of the invention. Examples of milk protein products enriched for NA include whey protein supplements for athletes and whey protein-fortified beverages and high protein bars. The milk dairy products and milk protein products of the present invention enriched for NA are nutritionally superior products to conventional milk products. The milk and dairy products may be of particular benefit with respect to the various factors for preventing degenerative brain diseases like multiple sclerosis and adrenoleukodystrophy. The benefits of the invention also extend beyond the production of NA-enriched food products for human consumption. For example, dairy cattle that are fed with a feed of the invention can be expected to exhibit improved health effects associated with NA, since NA is an essential nutrient for growth, development, and neuronal functioning in animals. The use of feather meal as a feed additive to 'mask' the impact of the ruminant GI tract on dietary fatty acid digestion, metabolism and absorption and/ or to override the program of directed synthesis of certain molecular species of sphingomyelin in milk is just one manifestation of this NA enrichment technology. The feather meal/ N A may also be administered within microcapsules to be ingested orally or in the coating of microcapsules. Suitable materials for the microcapsules include gelatin, gum arabic, food starch, malto dextrin, lactose, dextrin, corn syrup solids, the materials disclosed in U.S. Pat. No. 3,455,838 (which is hereby incorporated by reference), and mixtures thereof. The feather meal/ N A may also be entrapped in an edible solid vehicle composed of a film forming material or a plasticizing agent. Alternatively the feather meal/ N A could be gavaged or freely ingested in a liquefied form. Other vehicles may also be used for the delivery of feather meal/ N A. In addition, the feather meal and the NA may be administered in separate capsules or vehicles.

Another aspect of the current invention includes inhibitors other than feather meal, which can facilitate the enrichment of nervonic acid in the milk of ruminants. It is to be understood that these other inhibitors which facilitate the enrichment of nervonic acid may include compounds that inhibit NA from microbial or non-microbial- mediated degradation or from a combination of microbial and non-microbial mediated degradation in the rumen of ruminants. Examples of suitable methods that would inhibit the degradation of NA include, but are not limited to coating with a protein such as zeain, or combining with pH-sensitive co-polymers such as vinylpyridine and styrene, or enzymatically-degradable agents, such as zein or chitosan. Other suitable methods for inhibiting the degradation of NA include combining the feed additive with a reducing carbohydrate such as xylose, or with a divalent metal salt where the divalent metal is iron, calcium, or magnesium, and the anion is selected from the group consisting of phosphates, stearates, maleates,. succinates, silicates, pyrophosphates and mixed fatty acids. Degradation of NA may also be inhibited by combining the feed additive with diatomaceous earth, bentonite clay or non-ionic surfactants.

These inhibitors or facilitating agents of N A enrichment may also be administered either separately or in combination with NA as described above. The physical form of the inhibitors or facilitating agents for NA can be a solid or a liquid. They can also be a microbe (living organism) or a mechanical device and they can be administered by orally. The following non-limiting examples are illustrative of the present invention:

EXAMPLES

Example 1

NA Enrichment of Milk by Feeding a Lipid and Protein Rich Supplement containing NA and Feather Meal

Experimental Design

l l Eighteen Holstein cows housed at the Elora Research Station, University of Guelph, were used in this .experiment and divided equally into three groups; control, B, and C. Throughout the study, cows were fed and milked twice daily. The diet of the control group was the total mixed ration (TMR) routinely fed at the research facility. On a percent as-fed basis, it consisted of about: 2.9% hay, 16.7% haylage, 57.8% corn silage, 12.1% high-moisture corn, 10.6% commercial supplement pellet, as well as a mineral premix (see appendix H). The basal diet of groups B and C on a percent as- fed basis was: 4% straw, 60.6% corn silage, 33.6% high-moisture corn. A mineral premix and 2kg of non-pellet supplement were top-dressed onto the basal diet of these two groups. During days 1 to 14 of the trial, groups B and C received the same supplement (supplement A). Supplement A contained the following ingredients on a percent as- fed basis: 25% wheat, 15% feather meal, and 60% canola meal. On days 15 to 21, supplements B and C were fed to groups B and C respectively. Supplement B contained (percent as-fed basis): 25% wheat, 15% feather meal, 50% canola meal, and 10% olive oil. Supplement C was identical to supplement B, only the 10% olive oil was replaced with 10% canstar oil. The canstar oil was composed of 85% canola oil and 15% crossential N25, an oil containing 25% nervonic acid purchased from Croda Oleochemicals, UK. During days 22 to 28 of the trial, groups B and C received supplement A. On the final 7 days of the trial (day 29 to 35), all eighteen cows were fed the routine TMR. Milk samples were collected from the morning and evening milkings every Monday, Wednesday, and Friday on days 1 to 14 and 22 to 35, and daily during days 15 to 21. The samples obtained on these days were frozen at B20°C for fatty acid analysis. After consuming the oil-containing supplement for 7 consecutive days (day 22), two randomly selected cows from groups B and C were segregated during the morning milking. Their milk was collected and stored separately for protein fraction analysis. Sphingomyelin Fatty Acid Analysis of Whole Milk For each sample, 0.12 g of freeze-dried whole milk was placed into a pre- rinsed test tube. 4.0 ml of 2:1 chloroform:methanol and 25 μl of 5% BHT in 2:1 chloroform:methanol were added to each test tube. N₂ was added and the samples were vortexed for 1 minute. Test tubes were stored at 4 °C for 2 hours and centrifuged at 2500 RPM for 20 minutes. After pipetting the supernatant from each test tube into other pre-rinsed test tubes, 0.8 ml of 88% KC1 in distilled water were added to the samples. ₂ was added and the samples were vortexed for 30 seconds followed by centrifugation at 2500 RPM for 20 minutes and placed in the freezer overnight. Once the lower phase was transferred from each sample into pre-rinsed test tubes, the upper phase was discarded. Each sample was dried completely under N₂ and 20μl^'of 2:1 chloroform:methanol was added. Test tubes were capped immediately to avoid evaporation. In the case of evaporation, an additional 20μl of 2:1 chloroform:methanol was added. Samples were vortexed for 15 seconds and the thin layer chromatography (TLC) plate was spotted using a lOμl Hamilton syringe. After the last spot dried, the TLC plate was placed in the TLC tank containing chloroform:methanol:acetic acid:H₂0 (50:37.5:3.5:5:2). The plate ran until it was approximately one inch from the top of the plate and was removed from the tank and dried under a fume hood. The plate was then sprayed with 0.1% 8- anilinonapthalene-1-sulphonic acid (ANS) and viewed under UV light to identify the phospholipid bands. The sphingomyelin and phosphatidylcholine bands were marked, and scraped horizontally with a blade into disposable. 3.0 ml of 6% H2SO4 in methanol and 5.0 μl of 17:0 standard were added to these test tubes and they were filled with N₂ and vortexed for 3 seconds. Samples were placed in an oven at 75- 80°C for 16 hours to transmethylate. After removing the samples from the oven, they were cooled to room temperature and 2.5 ml of hexane was added. After filling with N₂ and vortexing for 15 seconds, 1.0 ml of deionized water was added and each test tube was filled with N2 again and vortexed for an additional 5 seconds. The test tubes were then centrifuged at 1000 RPM for 1 minute. The top layer of each sample was transferred removed into minivials, filled with N_2/ and stored in the freezer. Each sample was dried completely under N₂. 3 μl of CS₂ were added before injecting 1 μl into the gas chromatograph (Hewlett-Packard 5890 A). The amount of each fatty acid was identified by the area under each peak.

The results of this experiment are shown in Tables 1 and 2 , below.

* Values represent the means of n=6 cows. Values within a parameter are not significantly altered by the nervonic acid supplement (Canstar) or by the number of days on the experiment (Student's t-test, P > 0.05).

Table 2. Effect of a Nervonic Acid Supplement (Canstar) on the Fatty Acid Composition of Sphingomyelin from Whole Milk of Dairy Cows*

The data clearly indicate that the supplemented cows had higher levels of nervonic acid than standard bovine milk. The control samples in this trial contained levels similar to the levels of nervonic acid found in commercially available, pasteurized, homogenized milk. The milk obtained from the cows fed the nutritional supplement had nervonic acid levels that were more like human milk than standard cow's milk. No ill effects were observed in animals consuming the feed supplement. The NA content (16% of the fatty acids in the milk sphingomyelin) increased in expressed milk within 3 days after the cows first started consuming the feed supplement containing NA. Milk production and feed consumption were similar to production/ consumption observed when the cows were fed conventional feeds. Example 2

NA Enrichment of Whey Protein from Milk by Feeding a Lipid and Protein Rich Supplement containing NA and Feather Meal Experimental Design Same as for Example 1.

Skimming and Pasteurization Milk Aliquots of each milk sample collected on day 22 were skimmed by centrifugation at 13,000 rpm for 30 minutes at 4°C. The remaining milk was skimmed by allowing the cream to rise to the surface of the milk, and removing the cream with a beaker. This step was repeated four times to ensure most of the fat was removed. All samples were pasteurized according to Ontario government standards by heating to 72°C for not less than 16 seconds.

Preparation ofWliey Whey was separated from the skim milk by first warming the sample to 31°C using a water bath, adding ^"a rennet-water solution, and allowing the milk to curd for 50 minutes. Once a curd was formed, it was gently agitated and gradually heated to 39°C. After holding this temperature for 60 minutes, the whey was removed.

Concentrating the Whey Proteins The whey proteins were concentrated via ultrafiltration (UF) at 4°C. Membranes with a pore size suitable to retain molecules with a molecular weight greater than 10,000 were used. The nitrogen pressure required for the procedure was maintained at 40 psi. Aliquots of whey from each sample were injected into the UF. When 3ml of whey remained in the UF, water was injected to further concentrate the whey proteins (diafiltration). The whey remaining in the UF (retentate) is highly concentrated with whey proteins. Samples were placed in the Dura-Dry MP freeze dryer for 42 hours.

Sphingomyelin Fatty Acid Analysis of Isolated Whey Protein Same procedure, as for whole milk (example 1) except started with 0.15g of freeze-dried whey protein instead of whole milk. Results The results of this experiment are shown in Table 3, below.

* Values for the nervonic acid (24:1) content of the sphingomyelin in the whey protein fraction of cows milk, day 7 of the experiment. Nervonic acid content is increased over 10-fold by the Canstar supplement. These results clearly demonstrate that isolated whey protein from cows fed the NA supplement (Canstar) is enriched in nervonyl-sphingomyelin relative to whey obtained from the milk of unsupplemented (Olive) cows.

It is also noteworthy that the detectable erucic acid (22:1) concentration in both the total milkf at and in the sphingomyelin was zero (see Table 2 and 3), despite the fact that the levels of erucic administered in the Canstar supplement were roughly twice that of nervonic acid. This is an important finding as recent studies have demonstrated an association between dietary erucic acid and myocardial lipidosis in a number of species. In addition, studies have also demonstrated an association between dietary erucic acid and heart lesions in rats.

Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims. All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

WE CLAIM:

1. A feed additive for ruminants comprising the components: (a) a source of nervonic acid; and (b) a source of inhibitors of degradation of nervonic acid in the rumen said inhibitors being present in amounts to enhance the production of nervonic acid in the milk of ruminants.

2. A feed additive for ruminants comprising the components: (a) a source of nervonic acid; and (b) a source of inhibitors of degradation of nervonic acid in the rumen comprising feather meal or its constituents, said inhibitors being present in amounts to enhance the production of nervonic acid in the milk of ruminants.

3. The feed additive of claims 1 or 2 wherein said inhibitors are present in amounts to enhance the production of nervonic acid in the milk of ruminants to levels greater than 16% of the very long chain fatty acids in ceramide-based lipids of the milk.

4. A feed additive for ruminants comprising the components: (a) a source of nervonic acid; and (b) a source of inhibitors of degradation of nervonic acid in the rumen comprising feather meal or its constituents, said inhibitors being present in amounts to enhance the production of nervonic acid in the whey protein fraction of the milk of ruminants.

5. The feed additive of claim 4 wherein the inhibitors are present in amounts to enhance the production of nervonic acid in the whey protein fraction of milk of ruminants to levels of 26% to 34% of the very long chain fatty acids in the ceramide-based lipids in the whey protein fraction of the milk.

6. The feed additive of claims 1, 2, 3, 4 or 5, wherein said source of nervonic acid is the Lunaria plant.

7. The feed additive of claim 6, wherein said source of inhibitors of degradation of nervonic acid in the rumen of the ruminants is feather meal

8. A feed containing the feed additive as claimed in any preceding claim 1 to 7.

9. A method of producing milk in ruminants comprising feeding the ruminants a feed containing a feed additive according to any of claims 1 to 7 for a period of at least 2 days and milking the ruminants to obtain milk enriched for nervonic acid.

10. Expressed milk from ruminants which milk is produced using a method as claimed in claim 9.

11. The expressed milk as claim in claim 10, which contains greater than 16% nervonic acid as percent of the very long chain fatty acids in the milk.

12. A nervonic acid-enriched dairy product produced using expressed milk from ruminants enriched for nervonic acid as claimed in claim 11.

13. A nervonic acid-enriched dairy product as claimed in claim 12 which is cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant formula, or butter.

14. The expressed milk as claimed in claim 10, which contains 26% to 34% nervonic acid as a percent of the very long chain fatty acids in ceramide-based lipids in the whey protein fraction of the milk.

15. A nervonic acid-enriched low-fat dairy product produced using expressed milk from ruminants enriched for nervonic acid as claimed in claim 14.

16. A nervonic acid-enriched low-fat dairy product as claimed in claim 15 which is cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant formula, or butter.

17. A nervonic acid-enriched milk protein product produced using expressed milk from ruminants enriched for nervonic acid as claimed in claim 14.

18. A nervonic acid-enriched milk protein product as claimed in claim 17 which is a dried whey protein concentrate, dried whey, dried skim milk, dried milk protein, dried buttermilk, casein, acid casein, a protein drink, a protein bar or a protein supplement.

19. Use of feather meal as an inhibitor of degradation of NA in the rumen of ruminants.

20. Use of feather meal as claimed in claim 19 characterized in that the feather meal is added to the feed for ruminants.

21. Use of feather meal as claimed in claim 19 characterized in that the feather meal is added to feed additive in an amount sufficient to enrich milk from the ruminants with NA.