Progress 10/01/04 to 09/30/07
Outputs
OUTPUTS: Egg yolks contain large amounts of chemicals called phospholipids, which are collectively referred to as lecithin. Lecithin is used as an emulsifier in food, cosmetic, and pharmaceutical products. In pharmaceuticals, it is used to formulate nutritional or drug delivery systems for injection and its suitability is determined by certain chemical characteristics. The lecithin is composed of phospholipids that are divided into different classes. In egg yolks, the major classes are phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Lecithin quality is also influenced by phospholipid species within each class. We conducted egg yolk chemical research to establish if egg lecithins from the State of New York have potential for pharmaceutical applications. These applications require that the phospholipids that comprise the lecithin conform to a certain chemical profile. To that end, all phases proposed for the project were successfully completed,
these included the establishment of all instrumental and chromatographic techniques required for the isolation and chemical profiling of lecithin from eggs produced in the state of New York and comparison with pharmaceutical industry grade lecithin. Specifically, an egg lecithin isolation technique was developed, implemented, and optimized for the subsequent qualitative and quantitative analysis of phospholipid species composition by class according to headgroup and acyl chain substitution pattern. Thin layer chromatography (TLC) was used initially to establish phospholipids class and their relative amount in egg lecithin from the state of New York. This preliminary assessment was followed the development and use of sophisticated liquid chromatography (HPLC) for the quantitative analysis of phospholipid classes and Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) analytical techniques to establish phospholipid detailed species composition within each major phospholipid
class in eggs. Commercial egg lecithin samples were subjected to the same analytical protocols for rigorous comparison and a better evaluation of lecithin form the state of New York. Additionally, TLC techniques were developed, and used to analyze phospholipid mixtures and free omega-3 fatty acids in commercial eggs sold under the label of omega-3 eggs. Also, a Cornell student majoring in Food Science, Mr. Matthew Ginsberg (class 2007), who was a Rawlings Cornell Presidential Research Scholar fellow, received a two-year training under this project. Mr. Ginsberg actively participated in all aspects of the project and presented in the spring of 2007 a research poster titled: Chemical Profiling of Eggs from the State of New York and Their Suitability in the Manufacture of Non-food Derivatives in the Presidential Research Scholars Expo that was highlighted in the Cornell community weekly Cornell Chronicle. In general, there is no other detailed analysis of egg yolk in the scientific
literature comparable to the one conducted in this project, therefore its results will yield a high quality publication in an specialized peer-reviewed scientific journal that is currently in preparation.
PARTICIPANTS: Dr. Manuel Aregullin (PI): Actively participated in the planning and execution of all phases and aspects of the project for the duration of the project. Specific activities included: 1. The comprehensive revision of the scientific literature to determine the state of the art of basic and applied egg research. 2. The development and implementation of all research techniques utilized in the project in pursuing the objectives, data collection and data analysis. 3. The active collection and analysis of data. 4. The training and supervision of a senior undergraduate students who actively participated in data collection and analysis. Mr. Matthew Ginsberg (Senior undergraduate, Class 2007, Food Science Major): Actively participated in data collection and analysis. Specific activities included: 1. Egg processing for lecithin isolation and purification. 2. Sample preparation and standardization for analysis. 3. Sample analysis and data processing Mr. Ira Herbst (Senior
Undergraduate, Class 2006, Biology Major): Actively participated in the early stages of the program, and was responsible for the implementation of preliminary Thin Layer Chromatography analytical conditions for omega-3 fatty acids. Mr. Kurt Kreher (Egg producer and collaborator, Kreher's Farm Fresh Eggs, LLC) Mr. Kreher was a key participant in the project. Mr. Kreher provided production eggs and laying hen diet samples that were used in the analysis of lecithin. He was responsible of all the farm activities involved in the selection of samples to establish an average lecithin chemical profile representative of the farm's eggs.
TARGET AUDIENCES: The intended target audiences are first and foremost the stakeholders, and those are the egg producers of the state of New York. This project was conceptually developed to establish the feasibility of creating alternative markets to egg derived products to benefit this important industry. Having reached successfully the final stage of the project, its results will disseminated in various forums such as scientific conferences and specialized peer-reviewed journals, as well as, egg community information vehicles. Equally important audiences are researchers in the area of avian/poultry science. Detailed studies like the one conducted for this project are basically very rare in the literature. Therefore, the results yielded will expand the knowledge regarding egg biochemistry and factors that affect it. A manuscript for publication is in preparation and there are plans for participation in upcoming poultry science events. Other efforts included the scientific training
of undergraduate students majoring in biology and food science through their participation in various stages of the project.
Impacts
We received samples of chicken feeds and eggs from Kurt Kreher (Kreher's Farm Fresh Eggs, LLC). These belonged to two diet groups, labeled "EB" and "Non-EB" that corresponded to proprietary diet formulas. The ingredients of each diet were and remain unknown. Each egg was treated as a separate sample and three eggs were examined from each feeding group. Triplicate samples of 25g were also taken from each of the chicken feeds for analysis after mixing. A second sampling including six eggs for each category (i.e., EB and Non-EB) was made and analyzed . Eggs yolks were treated with organic solvents to extract the phospholipids of interest and the extract was analyzed by mass spectrometry to determine the presence and relative ratios of the phospholipid species. Feeds were also treated in the same fashion with organic solvents for phospholipid isolation. The feeds levels of the three main phospholipid classes were calculated and compared with commercial lecithin values. The
levels of PC were lower in the feeds than the commercial lecithin values and the levels of PE were higher in the feeds than in the commercial lecithin for both feed samples. PI was not reported in the commercial lecithin and was present in the feed samples at levels under 10%. The EB feed had a higher level of PC and a lower level of PE compared with the non-EB samples. The two had similar levels of PI. These data indicate that the non-EB yolks contain the correct proportions of phospholipid classes for pharmaceutical applications. Further analysis was needed to confirm these results by repeating mass spectrometry analysis and carrying out HPLC analysis. The latter provided details on the total amounts of lecithin present and validate results from the spectrometry experiments. Comparison of feeds and eggs phospholipid profiles show that while in the EB group, feed and lecithin proportions were very similar, they were much different in the non-EB group. There is not enough information
to establish the factors that determine these relationships. And, we feel that the results require confirmation, before we can make suggestions in diet changes. In summary, the activities and results accomplished in the project include: Isolation and analytical methodologies for egg lecithin and feeds from two diet groups were analyzed for phospholipid profiles. Phospholipid composition of samples was compared with commercial pharmaceutical samples and specifications. Egg lecithin from the non-EB group closely matches pharmaceutical products for non-parenteral drug formulation. There was not a direct correlation between feed and egg phospholipid composition in the non-EB group, but there is an apparent one in the EB group; their relationship is unclear. Other chemical attributes of the lecithin such as fatty acid composition and levels of cholesterol would be required, however these were outside the scope of the project.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
In the second year of the project substantial progress was made in the analysis of egg lecithin and the assessment of qualitative and quantitative phospholipid species composition in commercially marketed eggs available to the public in local supermarkets. This stage of the proposed research has been completed, and the isolation and purification techniques of lecithin and instrumental procedures developed specifically for the project have been evaluated yielding positive and satisfactory data sets detailing egg lecithin composition. In summary, eggs are mechanically cracked and the yolk is physically separated from the egg white in a bowl. The egg yolk is extracted with a mixture of chloroform:methanol (2:1, v/v) with vigorous mixing using a whisk, the mixture is transferred to a separatory funnel, and the lecithin containing lower organic phase is collected in an Erlenmeyer flask. The organic phase is taken to dryness in a rotary evaporator under vacuum, and the
residue is retaken in a mixture of chloroform:methanol (1:2, v/v) and filtered through a 0.2 ?m syringe filter. The lecithin phospholipid mixture is analyzed by electrospray ionization mass spectrometry (ESI-MS) in the negative and positive modes, and data compiled regarding phospholipid classes, species and relative concentrations within each class present in each egg type. The applicability and usefulness of these techniques was established by analyzing lecithin from eggs available in the local supermarkets P&C and Wegmans. Large AA Grade Eggs were purchased at P&C (P&C doesn't carry Omega-3 eggs), and Large AA Grade Eggs and Omega-3 eggs were purchase at Wegmans. Lecithin was isolated from these egg samples and analyzed for phospholipid composition. The results of the analyses show that in all three egg samples the major phospholipids are: phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC). PE is always present in the highest concentration and PI
in the lowest. The results of the analyses show that in the Large AA P&C eggs there is only one species of PI (38:4); four species of PE (34:1, 36:1, 38:4, and 40:4); and, four species of PC (34:1, 34:2, 36:2, and 38:4). In the Wegmans Large AA eggs there are two species of PI (36:2, and 38:4); three PE species (36:1, 38;4, and 40:6); and, four species of PC (34:1, 34:2, 36:1, and 36:2). In the Wegmans Omega-3 eggs there are two species of PI (38:3, and 40:5); three species of PE (36:1, 38:6, and 40:6); and, three species of PC (34:1, 34:2, and 38:6). In conclusion, the Large AA P&C eggs are fairly different from Wegmans, and the Large AA and Omega-3 eggs from Wegmans are very similar despite the different label. Also, these results suggest dietary characteristics such as in the production of Omega-3 eggs seaweed (kelp) instead of fishmeal is being fed to the chickens. Overall, these results indicate that the isolation and analytical techniques used in lecithin quality assessment are
appropriate in detail to establish diet dependence and applicability in the pharmaceutical industry of lecithin from eggs produced in the State of New York to be completed in the final year of the project.
Impacts
The intended outcome of this project involves determining alternative market opportunities for eggs produced in the State of New York based on the assessment of their phospholipid chemical composition. This profile will allow us to establish egg quality in terms of its potential for yielding high-value derivatives. We anticipate that the phospholipid profile will depend on the diet of the hen and we will make alterations to diet regimes to optimize the profile for the production of specialty eggs (i.e., omega-3 or lecithin production). We expect this project to have an impact on improving the profit margin of egg production in the state, with the possibility of family owned egg farms finding market opportunities outside the traditional food industry which have not previously been utilized. This intended impact is consistent with CSREES Goal 1, for the creation of value added products and increment of competitiveness of the U.S. agro-production in the global economy,
and with Research and Extension Priorities in identifying market channels for value-added products.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Progress was made in three areas of the project as summarized below: 1) New collaborators-As part of an attempt to expand the number of egg producing farms included in the project, a third collaborator has been recruited. Edwards Eggs located in Genoa, NY. 2) Isolation of egg phospholipids for analysis and preliminary HPTLC characterization-Egg phospholipids (lecithin) are suitable for specialty applications, such as, in the pharmaceutical industry if it meets certain performance requirements determined by its composition. Thus, methods in modern analytical instrumentation have been adapted to perform sophisticated qualitative and quantitative compositional analysis of egg phospholipids. Specifically, in this initial phase of the project, methodology to isolate and analyze the lecithin phospholipids has been completed. An acetone liquid-liquid extraction of the egg yellow permits the selective isolation of a phospholipid mixture with main components being
phosphatidylinositol, phosphatidylcholine and the minor being phosphatidylethanolamine and phosphatidylserine.This mixture is concentrated in an flash evaporator under vacuum and taken to dryness under a gentle stream of nitrogen. The residue is retaken in a mixture of ethanol and chloroform (2:1) and filtered through and syringe filter with pore size of 0.2 microns. A preliminary characterization of the phospholipids mixture is accomplished using silica-gel HPTLC using the solvent system: chloroform: ethyl acetate: acetone: isopropanol: ethanol: methanol: water: acetic acid (30:6:6:6:16:28:6:2, v/v/v/v/v/v/v/v), developing the chromatogram with iodine, and reporting the various phospholipids with discrete Rf values and intensities which can be quantified by optical densitometry methods. In similar fashion the free omega-3 fatty acids can be characterized by HPTLC using a solvent mixture: chloroform: methanol (9:1) and developing the chromatogram with 2,7-dichlorofluorescein and
visualizing under short ultraviolet light (254 nm). 3) Chemical Profiling of Egg Lecithin by Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)-Experimental methods for the metabolic profiling of phospholipid mixtures isolated from the egg yellow were developed for the utilization of a Bruker Esquire LC Mass Spectrometer (Hewlett-Packard, Inc.) equipped with a standard orthogonal electrospray source and a three-dimensional ion trap. The phospholipid mixtures were introduced into the mass spectrometer at a rate of infusion of 120 microliters/hr. The capillary voltage was set at plus 3500 or minus 3500 volts for positive or negative modes respectively. PtdInos, PtdSer, and PtdEt are detected as negative species (deprotonated) in the positive mode and PtdCho is detected as sodiated species in the negative mode. Therefore, egg lecithin phospholipid species composition profiling by headgroup and acyl chain can now be conducted by ESI-MS/MS, which owing to its semi-quantitative
nature relative amounts of such chemical species can also be achieved.
Impacts
The intended outcome of this project involves determining alternative market opportunities for eggs produced in the State of New York based on the assessment of their phospholipid chemical composition. This profile will allow us to establish egg quality in terms of its potential for yielding high-value derivatives. We anticipate that the phospholipid profile will depend on the diet of the hen and we will make alterations to diet regimes to optimize the profile for the production of specialty eggs (i.e., omega-3 or lecithin production). We expect this project to have an impact on improving the profit margin of egg production in the state, with the possibility of family owned egg farms finding market opportunities outside the traditional food industry which have not previously been utilized. This intended impact is consistent with CSREES Goal 1, for the creation of value added products and increment of competitiveness of the U.S. agro-production in the global economy,
and with Research and Extension Priorities in identifying market channels for value-added products.
Publications
- No publications reported this period
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