Duration: 10/01/2004 to 09/30/2009

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Dairy products are an important source of vital nutrients in the human diet. Nevertheless, many health-conscious consumers perceive dairy products to contain excessive amounts of total fat, saturated fat, and cholesterol. Butter and other high-fat dairy products are excluded from diets designed to decrease blood cholesterol and prevent or treat coronary heart disease.

The research of this committee has produced insights into milk fat production and methods to alter milk fat amount and composition by simple dietary means. Based on the work of individual researchers and collaboration among member research groups, knowledge has been developed that should allow milk fat to be 'tailored' in composition to meet market demands for improved fatty acid composition and milk fat levels. It has been demonstrated that nutritional manipulation can increase oleic acid content and decrease saturated fatty acid content of cow milk. Dietary manipulation can be used to decrease milk fat production and thus have a positive impact on early lactation cows in negative energy balance in terms of general health and reproduction. Allowing cows to pasture feed increases the conjugated linoleic acid (CLA) content of milk as does manipulating the fat content of the diet with grains, fish oils and calcium salt supplements. In addition, the mechanisms at a cellular level are being examined to provide clues to additional regulation that may be possible by targeting specific enzymes controlling triglyceride and phospholipid synthesis in the mammary gland. These enzymes modify fatty acid utilization through effects on de novo synthesis, uptake and transport from the blood, and desaturation and esterification of fatty acids into milk fat triglycerides. Increasing the vaccenic acid content produced in the rumen from the diet leads to increased cis-9, trans-11 CLA in the milk due to the action of the delta-9 desaturase enzyme in the mammary gland. Based on evidence from studies indicating that certain conjugated linoleic acids (CLA) and vaccenic acid are protective in animal models for some cancers, the ability to increase these in milk fat holds promise for improvement of human health. In addition, some CLA isomers have been shown to lead to decreased body fat and increased lean muscle mass in animal models. Lipids from milk and other ruminant animal products are the major natural source of this class of fatty acids.

One clear advantage of a multistate project aimed at milk fat modification is the standardization of fatty acid methods. New advances in analysis of fatty acids, including trans isomers, are occurring at a rapid pace. Members of W-181 share information on new fatty acid methods, which is then used to generate a more reliable database of milk fatty acid analysis. Also, members of the committee cooperate on projects to take advantage of lipid analysis methods available in a few, but not all locations.

Another advantage of a multistate effort on milk fat modification is the success of W-181 in bringing together expertise from industry and more than a dozen international research stations. Discussions not only occur about research results aimed at milk fat modification, but also occur about the practical implications of these results to the consumer and industry in the U.S. and around the world. Other formats do not exist to bring together this diverse level of expertise for discussions aimed at improving the nutritional value and marketing of milk.

A third advantage of the multistate project is the opportunity for committee members to provide input on the advantages and disadvantages of recommended changes in milk fat composition that are being considered as US dietary policy. An example was a previous letter from W-181 to FDA outlining the advantages and disadvantages of requiring labeling of trans fatty acids. The W-181 project allowed scientists who were directly engaged in research projects on milk fatty acids the opportunity to discuss as a group the full implications of potential policy changes and to respond to requests for expertise and input from US Regulatory Agencies.

Related, Current and Previous Work

This section will review the extent to which our three objectives have been accomplished during this period of Project W-181 Modifying Milk Fat composition for Improved Manufacturing Qualities and Consumer Acceptability. During this period of review most of the research has focused on the characterization of and development of methods to increase CLA in cow milk fat. This work was most closely aligned with the second objective of the project. This research area was developed during the duration of this project and there has been much scientific interest in the CLA area. There was some work reported relating to Objective 1 to identify regulatory steps in fatty acid synthesis and to a lesser extent work relating to Objective 3, which was to characterize the physical, chemical, manufacturing and sensory properties of modified milk products. The lower level of activity relating to Objective 3 was largely due to less participation on the committee of groups working in these areas. Although there was some interest and this objective was included into some of the studies reported, there seemed to be a general feeling that this objective was of more immediate interest to industry and manufacturing groups that were not widely represented on the committee.

Objective 1. To identify and characterize important regulatory steps in fatty acid synthesis and desaturation and their positional distribution on glycerol in milk fat.

Several studies have indicated that the synthesis of short chain fatty acids is inhibited by diets and diet supplements that increase the amounts of trans monoenes and CLA fatty acids. Studies have been conducted in mice, rats, goats, and cows. Some studies are still in progress but in those that are finished, the de novo fatty acid synthesis and abundance of mRNA for fatty acid synthase (FAS) and acetyl-CoA Carboxylase (ACC) enzymes were decreased under these conditions (Piperova et al., 2000; Baumgard et al., 2002; Peterson, et al., 2003). Coordinated reductions in gene expression for key enzymes in other processes in milk fat synthesis also occur, including uptake of circulating fatty acids, desaturation and formation of milk fat triglycerides. Mechanisms of these changes are not yet known but there is evidence that at the cellular level they involve the SREBP family of transcription factors (Peterson et al., 2003). Continuing studies are examining the regulation of delta-9 desaturase. Additional tissue culture studies are underway to determine CPT-1 mRNA and PPARa mRNA expression in response to trans-10, cis-12-CLA.

There have been several studies looking at the effects of CLA dietary supplementation or diet modifications on the desaturation index of milk fats by comparing the ratios of saturate to monounsaturate carbon number pairs. The results from different research groups have been mixed - some have seen effects while others have not. The basis for this appears to relate to the dose of trans-10, cis-12 CLA that is used; when low doses are used there are no effects on desaturation index whereas at higher doses (>5 g/d abomasally infused) the activity of D9-desaturase is reduced (Baumgard et al., 2001; Peterson et al., 2002b). The variation among cows in content of CLA and CLA-desaturase index is 3-fold but over a 12-week period the index is maintained even when cows are shifted between diets (Peterson et al., 2002a; Kelsey et al., 2003).

The most consistently observed and perhaps the most important observation for human health and nutrition is the observation that the predominant CLA isomer appearing in cows' milk is made in the gland from the trans-11-18:1, vaccenic acid, produced in the rumen (Griinari et al., 2000; Corl et al., 2001; Piperova et al., 2002). This fatty acid transfers readily to the mammary gland and is desaturated to the cis-9, trans-11 CLA that has been shown to be protective for mammary cancer in the rat model (Ip et al., 1999). The trans-7, cis-9-CLA isomer is also formed by endogenous synthesis via delta-9 desaturase with the substrate being trans-11 18:1 produced in the rumen (Corl et al., 2002; Piperova et al., 2002). Vaccenic acid (trans-11 18: 1) has recently been shown to be protective for mammary tumors in a rat model when provided as a triglyceride in a natural food (Corl et al., 2003). This effect of vaccenic acid may be indirect as a consequence of its use in the formation of the cis-9, trans-11 CLA as observed for dairy cows. The apparent transfer of the preformed CLA from the rumen is of a smaller order of magnitude compared to that formed in the gland.

Objective 2. To quantify modification of milk fat composition by manipulating the diet of the cow.

Much of the effort during this period of the W-181 project has been devoted to defining the dietary conditions that lead to the desired production of milk fat of certain composition. This 'desired composition' varied among research groups and was modified in several ways. Some groups were seeking to decrease saturated fatty acids or to increase polyunsaturated fatty acids. Some groups were interested in decreasing total milk fat and others worked to increase CLA or other fatty acids deemed important in human nutrition such as omega-3 isomers.

The diets fed to lactating cows were modified using many different schemes. Studies were done to investigate the effects of breed, parity, and stage of lactation (Kelsey et al., 2003). A number of studies have included the addition of vegetable oils, fish oils, rumen protected salts, or high oil containing grains or combinations of these treatments (Franklin et al., 1999; Chouinard et al., 2001; AbuGhazaleh et al., 2003a; Abughazaleh et al., 2003b; Whitlock et al., 2002, DePeters et al., 2001; Avila et al., 2000; Donovan et al., 2000; Ruppert et al., 2003). Interactions of fat source with copper concentration in the diet were examined for effects on ruminal and tissue lipid metabolism (Engle et al., 2000; Engle et al., 2001). In addition there have been studies with pasture feeding both cows and steers to increase the CLA content of the milk or meat (Kelly et al., 1998a; 1998b; Dhiman et al., 2000; Dhiman et al., 1999). Some studies have included post-rumen infusions to exclude rumen modification of the diet supplements (Mackle et al., 2003).

It has been shown that diets high in concentrate and lower in forage lead to higher levels of trans (tFA) and CLA fatty acids (Drackley et al., 2001; Onetti et al., 2002). These diets also decrease milk fat. Buffering such diets alleviates the milk fat depression. Addition of oils to these diets increases the levels of tFA and CLA. The combination of a high linoleic fat source with fish oil leads to even higher levels of the tFA and CLA isomers (Bauman et al 2003). The form of the forage and method of lipid feeding have also been studied for effects on milk fatty acid composition including CLA concentration (Chouinard et al., 2001). The type of forage has been shown to affect the production of trans-10-18: 1 fatty acid production. In the dairy goat is was observed that the of trans-10-18: 1 was higher in the milk when the goats were fed a corn silage-based diet as opposed to a diet which was alfalfa hay based. Pasture feeding cows leads to high levels of CLA but the addition of feed sources rich in linoleic acid does not increase the CLA levels beyond only pasture feeding.

There has been a large number of studies in which calcium salts of fatty acids are fed in order to rumen protect the fatty acids. Calcium salts of palm fatty acids are used to increase milk fat production and polyunsaturated fatty acids are being investigated to improve fertility. In this case, the salts of both trans fatty acids and CLA have been fed to reduce milk fat (Geisy et al., 2002; Perfield et al., 2002; Bernal-Santos, 2003; Selberg et al., 2003). Studies in late lactation show that effects on reducing milk fat persisted during a 20-week study with no adverse effects on the cow well-being or subsequent pregnancy. Studies in early lactation also show effects on milk fat persist as long as treatment continues and also indicate that treatment may improve some reproductive variables. Other studies examining the effect of supplemental fat types on the reproductive performance of lactating dairy cows have been done using calcium salt products containing fish oils or polyunsaturated vegetable oils (Mattos et al., 2002).

A number of our member groups have been using in vitro rumen fermentation to study modification of feed components. This is a technique that allows more detailed study of the processes involved in rumen modification of feedstuffs. Products and intermediates can be isolated and studied and quantitative comparisons made since there is not a flow into or out of the fermentation vessel. One study based on gas chromatograph-mass spectrometry has shown that incubation of 13C labeled elaidic acid (trans-9-18: 1) for 48 hours resulted in the formation of all trans-18: 1 isomers from delta position 6 through 16 (Mosley et al., 2002). Furthermore 5 to 10% of the elaidic acid was converted to cis-9- and cis-11-18: 1 (Proell et al., 2002). If the starting fatty acid was oleic acid, a range of trans isomers was also seen. It was also determined that oleamide (Loor et al., 2002) and linoleamide (Jenkins and Adams, 2002) was less biohydrogenated than the parent acids.

Objective 3. To characterize the effects of modified milk fats on physical, chemical, manufacturing, and sensory properties of dairy products.

Several research groups from around the country have fed cows diets designed to enhance the levels of polyunsaturated and CLA fatty acids in the milk. They have made products from this milk, mostly butter, yogurt, and cheese, which have been used in taste panels, human nutrition studies or biomedical studies with animal models (Baer et al., 2001; Bobe et al., 2003a; Bauman et al. 2001; Corl et al. 2003; Maynard and Franklin, 2003). Diets fed to lactating cows modified the fatty acid composition of milk fat, but processing or pasteurization of milk or cheese had almost no effect on fatty acid composition. Therefore, modification of the fatty acid composition at the farm level is critical to enhancing the fatty acid composition and subsequent nutritional value for consumers.

The atherogenic index (AI) was calculated for milk fat obtained from cows fed varying diets (Bobe et al., 2003b). Cows with consistently high or low AI have been identified and tested by feeding diets that vary in unsaturated fatty acids from grains or oils. The oils have varied but include soy oil, sunflower oil, and fish oil. The products made from these milks have been tested and in the case of butter the penetration, creep, etc., have been measured (Bobe et al, 2003a).

We have had some interaction with industry and research institutions on the food science aspects of milk fat use but it has been limited. Likewise, additional biomedical studies examining the health effects of modifications in milk fatty acid composition with animal models are important. These are areas where the knowledge gained over the last few years now make it possible to consider both changes in manufacturing properties and functional food properties in the development of new markets for milk fat.


Areas requiring further investigation

The emphasis of the project is on understanding and regulating the biology of the animal to achieve changes in milk fat composition that will ultimately yield dairy products that have a greater nutritional value and health benefits for consumers. The new objectives encourage research efforts at all levels from whole animal studies using lactating cows to basic subcellular studies using modern metabolic and molecular approaches. Objective 3 applies information from Objectives 1 and 2 toward enhancing the fatty acid composition of milk for improved human health and also keeps members aware of emerging information on the beneficial roles of specific fatty acids that might be pertinent to Objectives 1 and 2.

The above revisions were based on the following areas of incomplete work;

Little effort has been directed at studies on positional distribution on glycerol even though it is explicitly stated in the previous objective 1 (To identify and characterize important regulatory steps in fatty acid synthesis and desaturation and their positional distribution on glycerol in milk fat). Much of the interest in positional distribution is on a crucial enzyme (lysophosphatidic acid acyl transferase) responsible for placement of specific fatty acids in the sn-2 position of milk triacylglycerols, which reportedly controls the plasticity of milk fat and its hypercholesterolemic effects. Although work on positional distribution of fatty acids in milk triacylglycerols should continue to be encouraged, it is but only one of many important regulatory steps in mammary fatty acid synthesis that need to be identified and characterized. Regulation of mammary delta-9 desaturase that produces oleic acid and the major CLA isomers in milk is equally important, and has received more attention by members of the committee. Objective 1 as rewritten avoids mention of work in the positional distribution area and encompasses present and future participation in all areas related to characterizing regulatory steps in the synthesis of milk fat.


Objective 2 as currently written (To quantify modification of milk fat composition by manipulating the diet of the cow) emphasizes only diet manipulation as a means to alter the tissue and mammary supply of beneficial fatty acids. Work in this area should be done in conjunction with studies on transformation of dietary lipids by the microbial ecosystem, exploring opportunities for metabolic and molecular regulation of the processes of lipolysis and biohydrogenation. Thus, the original Objective 2 was rewritten, which now emphasizes manipulation of fatty acid delivery to the tissues and mammary gland through modification of dietary lipid source and its transformations by the rumen microbial population.

Objective 3 (To characterize the effects of modified milk fats on physical, chemical, manufacturing, and sensory properties of dairy products) was designed to determine how information from the feeding and metabolism phases of the project could translate into beneficial changes on the quality of manufactured dairy products. Regular attendance and input on the committee from milk chemists and processors was needed to accomplish this objective. On the positive side, one major commercial milk processor provided continuous input to W-181 through representation at annual meetings, participation on committees, and constructive debate on realistic goals for manipulating the quality of milk fat. The Committee also benefited from the participation and input from an internationally-recognized expert in dairy processing from Cornell University. However, progress toward achieving goals of Objective 3 fell short of expectations because of limited participation from a variety of researchers with recognized programs in milk chemistry and processing. Many attempts were made to contact and recruit additional membership having milk chemistry and processing expertise, but input was only occasional. Therefore, W-181 recommends revision of Objective 3 to shift the emphasis from manufacturing properties of altered dairy products to enhancing fatty acid nutraceuticals in milk for improved human health. The Committee will continue to encourage participation from the manufacturing area, but not as a prime objective. Instead, membership with expertise in emerging areas of fatty acid metabolism as it relates to human nutrition, or individuals having joint dairy/human nutrition interests will be a priority for Objective 3. Several current members of W-181 have expertise in both dairy and human nutrition research areas, thus giving the revised Objective 3 a good start.

Objectives

1. Characterize the regulation of milk fat synthesis.
   
2. Enhance absorption of desired fatty acids for milk fat synthesis through manipulation of diet and lipid transformations by gut microorganisms.
   
3. Develop quantitative models for evaluation of preharvest strategies for production of milk with greater nutritional value.
   

Methods

Objective 1. Characterize the regulation of milk fat synthesis. Of major interest is to identify the cellular regulation and signaling mechanisms for gene expression and activity of key enzymes in mammary synthesis of milk fat. Methods relating to objective 1 will include microarray analysis to explore global gene expression patterns in mammary tissue. Regulatory aspects of de novo fatty acid synthesis, uptake and use of preformed fatty acids, desaturation of fatty acids, and incorporation into triacylglycerol will be studied in mammary tissue explants, in isolated cells, in cell culture, or by preparing subcellular components of mammary tissue using standard enzymatic procedures. Studies are planned for intravenous infusion of stable isotopes of saturated fatty acids to determine the kinetics of desaturase activity in the mammary gland. Metabolic limits for incorporation of specific fatty acids (varying in unsaturation or chain length) in vivo will be characterized by infusing these intestinally or intravenously. Some studies will examine the abundances of mRNA for fatty acid synthase (FAS) and acetyl-CoA Carboxylase in mice, rats, goats, and cows. Interactions between body composition and CLA intake on milk fat synthesis will be examined in rat studies. Objective 2. Enhance absorption of desired fatty acids for milk fat synthesis through manipulation of diet and lipid transformations by gut microorganisms. Cow trials will be conducted to determine the ability of dietary treatments to alter milk fat composition. Work will continue on examining pasture systems and fat supplements to enhance milk fat content of unsaturated fatty acids such as CLA, vaccenic acid and omega-3 fatty acids. Unprotected fat supplements will include processed and unprocessed oilseeds including sunflower, linseed, cottonseed, and fish oils. Protected fat supplements will include fatty acid derivatives such as fatty acyl amides and calcium salts of fatty acids, along with encapsulated fat technologies. Additional in vivo studies will examine the effects of management practices on milk fat composition, including method of lipid supplementation and physical form of the forage. Studies on regulation of ruminal biohydrogenation will be an integral component of objective 2. The in vitro methods will include batch and continuous cultures of mixed ruminal microorganisms, and possibly some work with pure cultures of ruminal microorganisms. A variety of methods will be used to monitor lipid impacts on ruminal fermentation including fiber digestibilities, gas production, and production of volatile organic acids. Additional studies will be done on the pathways of biohydrogenation by tracing fatty acyl carbons in ruminal contents using stable isotopes of unsaturated fatty acids and gas chromatography - mass spectroscopy. Other in vitro experiments will examine the effects of environmental conditions and additives on the completeness of biohydrogenation. Objective 3. Develop quantitative models for evaluation of preharvest strategies for production of milk with greater nutritional value. Objective 3 will consist of developing products for the marketplace based upon the knowledge gained in objectives 1 and 2. Products developed include quantitative models that describe the extent that milk fat composition can be altered by preharvest strategies. Models will predict how dietary lipid supplements are transformed by ruminal microorganisms, as well as how the absorbed fatty acids are incorporated into milk fat or have a regulatory influence on the mammary gland to alter milk fat composition. Genetic influences will be considered, along with their interactions with diet and the metabolic factors that regulate milk fat synthesis. Approaches will include a continuation of biomedical studies with animal models to evaluate beneficial effects of milk fatty acids. The formulation of integrated concepts and models, along with other information, will lead to products such as feed supplements or other treatments that can directly alter milk fat composition. Objective 3 is the transfer of knowledge to technology that is adopted by the industry.

Measurement of Progress and Results

Outputs

• published research results on modification of milk fat
• patents for altering milk fat content and fatty acid composition
• invited presentations by members at scientific symposia and industry conferences
• prediction equations/models to guide changes in milk fat composition

Outcomes or Projected Impacts

• commercial dairy products with modified fat composition
• increased endorsement of dairy products as part of a healthy diet

Milestones

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Refereed publications, patents, abstracts at scientific conferences, proceedings of nutrition conferences and meetings, producer field days

Organization/Governance

The technical committee will consist of a Chair, secretary, and regional administrative adviser. The executive committee will consist of these three persons and the previous Chair, and will the official nominating body. The Chair and secretary will be elected by the voting members from within their ranks. The Chair is responsible for planning and conducting the annual meeting, for submission of the project annual report, and for facilitating and ensuring effective communication and cooperation among participants. The secretary is responsible for recording minutes and distributing them prior to the Chair preparing the annual reports. Individual station members are responsible for preparing brief annual reports and distributing them to other participants at the annual meeting. Additional committees composed of voting or non-voting members may be appointed as needed to solve particular technical problems, to assist in communication with the project, or to report project findings to other interested parties. Participation of representatives from non-voting research groups in the U.S. or internationally, and of representatives from industry, is encouraged.

Literature Cited

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