Progress 08/15/12 to 08/14/15
Outputs
Target Audience:The primary audience targeted by this research encompasses individuals in both industry and academia. These individuals are in positions that have the potential to, 1) influence the direction of further research and development in the meat industry, and 2) pursue additional academic research that builds on the results of this project. Through interaction with these individuals and presentation of research results at both the 2014 American Meat Institute Reciprocal Meat Conference and the 2014 Institute of Food Technologists Annual Meeting, the relevant points of this project were conveyed. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?cGMP Biomanufacturing Operations. July 8-11, 2014. Raleigh, NC. Completed professional training course at North Carolina State University Biomanufacturing Training and Education Center. This "hands-on" course focused on using laboratory methods and equipment for the production and purification of recombinant proteins in an industrial setting. Attention to accomplishing these tasks according to cGMP (current good manufacturing practices) was emphasized throughout the lecture and laboratory portions of the course. Fermentation Engineering. August 12-14, 2014. Raleigh, NC. Completed professional training course at North Carolina State University Biomanufacturing Training and Education Center. The lecture portion of this course endeavored to explain the biophysical and engineering principals that form the basis of optimizing large scale industrial recombinant protein production. The laboratory portion of the course provided an opportunity to apply those principals through completion of a 30 liter fermentation process in an industrial setting. How have the results been disseminated to communities of interest?Grunwald, E.W., Richards, M.P. June 16, 2014. Studies with the heme binding protein aposhp suggest hemoglobin is the primary promoter of lipid oxidation in pork muscle mince. A poster publishing research results at the American Meat Science Association Reciprocal Meat Conference, Madison, WI. Grunwald, E.W., Richards, M.P. June 22, 2014. Studies with the heme binding protein aposhp suggest hemoglobin is a more effective promoter of lipid oxidation than myoglobin in turkey muscle mince. A poster publishing research results at the Institute of Food Technologists Annual Meeting, New Orleans, LA. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Lipid oxidation is a significant problem that results in undesirable sensory attributes in the post mortem muscle of terrestrial animals that is consumed as meat by consumers. Billions of dollars in profits are lost by the meat industry annually as a result of the biochemical process of lipid oxidation. It is therefore of significant economic interest to develop increasingly effective methods to combat the process of lipid oxidation. To accomplish this complex task, it is necessary to understand the biochemical mechanism of the process. Myoglobin (Mb) and hemoglobin (Hb) are proteins that occur in meat, and have been shown to promote lipid oxidation. Understanding which of these proteins contributes a greater function in the lipid oxidation mechanism would reveal points that could be strategically blocked in order to inhibit the process in meat systems of terrestrial animals. An emphasis of this project was to secure evidence that identified Hb as a primary mediator of lipid oxidation in the muscle of terrestrial animal species, using preparations of the recombinant protein apoShp.The results obtained from this project have contributed data demonstrating that targeted inhibition of Hb activity in turkey and pork systems inhibits the lipid oxidation. This raises the relevance of Hb as a primary promoter of lipid oxidation in meat. Residual Hb in post mortem muscle tissue is found in the regions of blood vessels. Focusing energy on controlling the activity of Hb could lead to more effective midigation of lipid oxidation in meat and subsequent decreases in the lost profits of the meat industry. This would make a significant positive contribution to the improvement of the American economy. Goal #1 The scientific goal of this project was to determine the primary reactant that causes lipid oxidation in different muscle foods. Due to their significant economic value in the animal agriculture industry, this goal sought to determine whether apoShp displayed inhibitory activity on lipid oxidation in turkey and pork muscle. The gene coding for the recombinant protein apoShp was incorporated into the E.coli BL21 cell line. Using a 10 liter expression apparatus, apoShp was successfully expressed in the form of insoluble inclusion bodies in the E.coli cell system. The inclusion bodies were extracted from the cells, dissolved, and the resulting soluble apoShp protein was folded into a functional configuration. Purification of apoShp was accomplished using ion exchange chromatography. While this method was successful in producing usable apoShp in moderate quantities, the purification required considerable unexpected development in order to obtain the apoShp. This resulted in a less than expected quantity of apoShp available for use in the project. Throughout the course of the project, four 10 liter expressions were performed, and a total quantity of 293 mg apoShp was produced. Purity of the apoShp protein was confirmed using SDS-PAGE. The functionality of the prepared apoShp in sequestering heme from Hb was spectrophotometrically confirmed at pH 5.9 in solution using turkey and pork Hb. Statistical analysis of research data obtained in completion of this goal has been completed and a manuscript is nearing completion that will publish the results in a peer reviewed scientific journal. The data contribute evidence that residual Hb in postmortem muscle of terrestrial meat animals should be considered a primary inducer of lipid oxidation in muscle food systems. First Objective of Goal #1. Determine ability of apoShp to inhibit discoloration and lipid oxidation in turkey muscle during refrigerated and frozen storage. The turkey breast and thigh muscles used in lipid oxidation studies were determined to contain 20 µmol/kg and 120 µmol/kg total heme, respectively. It was discovered that turkey breast muscle did not appreciably oxidize under experimental conditions relevant to post mortem muscle (pH 5.9, 0.2ºC, 90% moisture), even in the presence of 1.5% sodium chloride (NaCl). In view of a published scientific report indicating a low ratio of hemoglobin to myoglobin in turkey breast muscle compared to turkey thigh muscle, this is an important finding that supports the relevance of Hb as a primary mediator of lipid oxidation in turkey muscle. It was decided that thigh muscle would be the focus for further lipid oxidation studies in turkey muscle. Experiments comparing lipid oxidation progress in minced turkey muscle in glass vials containing a 2.5 fold excess of apoShp versus total heme content to muscle containing no added apoShp were performed. A 4 fold apoShp excess was originally planned but was not possible due to less than expected apoShp protein quantities produced. For identical reasons, lipid oxidation studies were not performed at frozen conditions. Increases in quantities of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHP), and hexenal are markers for lipid oxidation in muscle tissue. ApoShp inhibits Hb reactivity. Measurements made in turkey muscle mince during0.2ºC storage at pH 6.4 compared changes in TBARS (p=0.0002), LHP (p<0.0001), and hexenal (p=0.0129) content in the presence and absence of apoShp. The measurements show that lipid oxidation is almost completely inhibited in apoShp treatments and not in treatments containing active Hb. This demonstrates the relevance of Hb as a primary promoter of lipid oxidation in turkey muscle. Quantification of color stability during the course of the lipid oxidation studies was not possible due to less than expected quantities of apoShp produced. Second Objective of Goal #1. Determine ability of apoShp to inhibit discoloration and lipid oxidation in pork sausage under light display during refrigerated storage. The pork Semitendinosus muscle used in lipid oxidation studies was determined to contain 130 µmol/kg total heme. A preliminary experiment comparing lipid oxidation in pork mince containing 1.5% added NaCl to mince containing 0% added NaCl showed greater lipid oxidation in the presence of 1.5% NaCl. It was thus decided that the light display was not necessary. Additionally, the investigation of lipid oxidation in pork muscle was originally planned to use pork sausage as a model system. This was not possible due to smaller than expected quantites of apoShp, necessitating smaller reaction sizes. A comparison of the measurements ofTBARS (p=0.0005), LHP (p=0.0021), and hexenal (p=0.0203) in the presence and absence of apoShp at0.2ºC and pH 6.0 in pork muscle mince in glass vials, has shown that inhibition of Hb reactivity by apoShp results in inhibition of lipid oxidation. These results mirror those in turkey muscle and provide evidence for the identity of Hb as a primary promoter of lipid oxidation in pork muscle. For the same reason as in turkey muscle, quantification of color stability was not possible. Goal #2 Prepare the NIFA Fellow for a leadership role in agriculture. First Objective of Goal #2. Increased Proficiency In Production of Peer Reviewed Publications. Fellow contributed to two peer reviewed publications published during the reporting period. Second Objective of Goal #2. Attainment of Additional Experience Applying For Research Funding. Fellow did not apply for further research funding. Third objective of Goal #2. Further Development of Oral Communication Skills. Fellow produced and delivered a guest lecture to Mentor's Chemistry of Food Lipids graduate course. Fourth Objective of Goal #2. Development of Personnel Management Skills. Fellow developed a research project for an undergraduate mentee. The project focused on enhancing efficacy of natural antioxidants in inhibiting lipid oxidation. While this effort did not produce a publication, under the Fellow's guidance, the mentee successfully learned background and techniques relevant to the study of lipid oxidation and antioxidants in muscle foods.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Grunwald, E.W., Richards, M.P. June 16, 2014. Studies with the heme binding protein apoShp suggest hemoglobin is the primary promoter of lipid oxidation in pork muscle. Poster Presented At The American Meat Science Association Reciprocal Meat Conference, Madison, WI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Grunwald, E.W., Richards, M.P. June 22, 2014. Studies with the heme binding protein apoShp suggest hemoglobin is a more effective promoter of lipid oxidation than myoglobin in turkey muscle mince. Poster Presented At The Institute of Food Technologists Annual Meeting, New Orleans, LA.
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Progress 08/15/12 to 08/14/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? An undergraduate student has been selected as a mentee by the NIFA Postdoctoral fellow. The mentee is currently being trained by the NIFA Postdoctoral fellow in techniques to be used in an upcoming research project. The NIFA Postdoctoral fellow is currently developing the research project and will take leadership and management roles in the project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? 1) Preparation of purified apoShp expressed in E. coli in quantities sufficient to complete lipid oxidation and color stability experiments in turkey breast and thigh muscle systems, and in a salted pork sausage system will continue. If preparation of apoShp from the initially insoluble inclusion bodies does not provide sufficient quantities of purified apoShp, then preparation of apoShp from the soluble holoShp will be continued. 2) Lipid oxidation and color stability experiments using apoShp in turkey breast and thigh muscle systems at refrigerated and frozen temperatures, and in a salted pork sausage system under light display at refrigerated temperatures will be initiated and completed. 3) Results of storage studies in turkey and pork muscle systems will be presented at the Institute of Food Technologists 2014 Annual Meeting, the 2014 Reciprocal Meat Conference, and the 2014 NIFA Postdoctoral Program Director’s meeting. 4) Manuscripts based on the results of this research project will be produced and submitted to peer reviewed scientific journals for publication. 5) The NIFA Postdoctoral Fellow will give a guest lecture in the Mentor’s Fall 2013 Food Lipids course. The presentation will likely focus on an aspect of the role of heme proteins in the mechanism of lipid oxidation in muscle food systems. 6) The NIFA Postdoctoral fellow will develop a grant proposal for future research, and identify a funding source. 7) The NIFA Postdoctoral fellow will continue developing and implementing a research project with the selected undergraduate mentee. This project will result in a publication in a peer reviewed scientific journal, or a presentation at an undergraduate research seminar.
Impacts
What was accomplished under these goals?
1) Three 10 liter expressions of the Shp protein using an E. coli BL21(DE3) host cell line have been completed. Each expression yields approximately 120 grams of E. coli cell paste containing the Shp protein expressed as a mixture of initially insoluble apoShp inclusion bodies and soluble holoShp. 2) Preparation from E. coli of the fraction of Shp expressed as initially insoluble apoShp inclusion bodies has been repeatedly attempted. Using SDS-PAGE, the presence of Shp has been confirmed in a preparation of protein from solubilized and refolded inclusion bodies. Based on results thus far, it is estimated that each 10 liter expression yields 300-400 mg of purified apoShp. 3) To increase the yield of Shp produced from each expression, a method was developed to prepare from E. coli the fraction of Shp expressed as soluble holoShp. Each 10 liter expression yields 100-150 mg of purified holoShp. Purity of the holoShp has been confirmed using mass spectrometry and SDS-PAGE. 4) In order to produce apoShp from purified holoShp, the heme group must be removed from holoShp. A method to accomplish this removal is currently under development. Using this method, approximately 150 mg of apoShp have been obtained from purified holoShp. 5) Turkey breast muscle has been obtained. A mince of this muscle has been prepared for lipid oxidation and color stability experiments. The total heme content and pH of this muscle have been determined to be 20 µmol/kg mince and 5.94, respectively. The moisture content of the mince has been determined to be 74%.
Publications
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