Progress 08/01/05 to 07/31/08
Outputs
OUTPUTS: The primary activities of this project included conducting experiments and mentoring graduate students. One PhD student and five MS students worked on various aspects of this project. Results were disseminated via technical conferences and peer-reviewed publications. PARTICIPANTS: Carrie Burbrink, Shannan Sombers-Guck, David Schroeder, Clint Stevenson, Kelsey Ryan and Lindsay Wisener all worked on the project as they were pursuing their MS program in Food Science at Purdue University. Devon Durkee worked on the project as a PhD seeking graduate student. Laura Pillsbury worked on the project as an undergraduate researcher. TARGET AUDIENCES: The target audiences for this project include: State and Federal Dairy Cooperatives, e.g. Dairy Management Incorporated, California Dairies Incorporated, etc. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
New knowledge is the major outcome for this project. Results demonstrate that plasminogen activation and resulting plasmin mediated beta casein hydrolysis during storage of raw and pasteurized milks significantly affect the functional properties of Skim Milk Powder produced from stored milks. Additionally these results demonstrate the need to further investigate the relationship between raw milk quality attributes, for example raw milk somatic cells, and the functionality of Skim Milk Powders. These results are particularly important to U.S. Skim Milk Powder producers as they compete for a larger portion of the growing international Skim Milk Powder market.
Publications
- D. L. Schroeder, S. S Nielsen, and Hayes, K. D. 2008. The effect of raw milk storage temperature on plasmin activity and plasminogen activation in pasteurized milk. International Dairy Journal, 18(2): 114-119.
- Durkee, D and Hayes, K.D. 2008. Plasmin activity, plasminogen activation, and β-casein hydrolysis in reconstituted non-fat dry milk. Dairy Science and Technology, 88:81-94
- Stevenson, C. D., Pillsbury, L., Hayes, K.D. 2007. The effect of cysteine on the thermal inactivation kinetics of plasminogen and plasmin in a model system as well as their activity in heated milk. Presented at the Institute of Food Technologists Annual Meeting, Chicago, IL. Poster
- Durkee, D. L., Hayes, K.D. 2007. Effect of somatic cell count and post-pasteurization storage on the plasmin enzyme system in non-fat dry milk. Presented at the Institute of Food Technologists Annual Meeting, Chicago, IL. Poster
- Ryan, K. N, Hayes, K.D. 2007. Effect of cysteine and temperature on plasminogen activation in a model system. Presented at the Institute of Food Technologists Annual Meeting, Chicago, IL. Received first place in the John Bruhn Poster Presentation Competition sponsored by the IFT Dairy Foods Division.
- Wisener, L.N. and Hayes, K.D. 2008. Effect of heat on plasminogen activation in somatic cells. Presented at the Institute of Food Technologists Annual Meeting, New Orleans, LA. Received first place in the John Bruhn Poster Presentation Competition sponsored by the IFT Dairy Foods Division.
- Ryan, K.N. and Hayes, K.D. 2008 Effect of cystein and heat on the disulfide bond patter of bovine plasminogen. Presented at the Institute of Food Technologists Annual Meeting, New Orleans, LA. Poster
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Progress 08/01/06 to 08/01/07
Outputs
Objective 1- The impact pre-storage thermal treatments, storage time, and concentration of plasminogen activator have on plasminogen activation in fluid milk and cheese curd. We have found that plasminogen (PG) activation and resulting beta casein breakdown occur rapidly once milk had been heated to temperatures between 65 and 75 degrees Celsius, prior to 48 hr cold storage. Additionally, we found that bovine PG denatures at temperatures between 50.1 and 61.6 degrees Celsius. Kinetic analysis of heated PG, in buffer, was conducted and we found that the overall catalytic efficiency increased for PG heated to 60, 80 and 100 degrees Celsius. Therefore, we concluded that the increased activation of heated bovine PG was probably due to the thermal denaturation of PG. Further work was done to study the effect of heat on PG activation in milk where our results confirmed that heating milk to temperatures around 75 degrees Celsius significantly increased plasmin (PL) activity
and therefore promote proteolysis, mainly due to inactivation of inhibitors and enhanced activation of unfolded PG. Objective 2- The effects ionic calcium, beta casein, and temperature have on the kinetic parameters of plasminogen activation in simulated milk buffer. We have studied the effect of refrigerated conditions, serum beta casein and ionic Ca on PG activation in pre-pasteurized milk and in buffer. Higher PL activity was observed along with a significant decrease in PG-derived activity in samples stored at 4.4 degrees Celsius. Inversely, PG and serum Ca concentration were higher in samples stored at 2.2 degrees Celsius. In buffer system, Ca significantly affected the kinetics parameters of PG activation where the Km significantly increased in the presence of Ca chloride (0.2%) at both 2.2 and 4.4 degrees Celsius, which lead to a subsequent decrease in catalytic efficiency (kcat/Km). Thus, we concluded that the higher serum Ca observed in the milk samples stored at 2.2 degrees
Celsius inhibited PG activation. Results implied that lower storage temperatures yield milk with lower PL activity by reducing PG activation. Objective 3- The effects that elevated plasmin activity during cold storage have on the functional properties of non-fat dry milk powders produced from stored milks. We conducted an experiment to study PL activity, PG activation, and beta casein hydrolysis in reconstituted non-fat dry milk (NFDM) samples as affected by various conditions, including heat treatment (low, medium, or high), storage (4 and 21 degrees Celsius), and PA addition (with and without). Low heat and instantized NFDM had significant amount of PL and PG. beta casein proteolysis in reconstituted NFDM significantly increased in instantized NFDM with added PA. Plasminogen present in NFDM can undergo activation by exogenous PA after storage at both refrigerated and room temperature conditions. We concluded that different heat treatment and storage can affect PL activity in NFDM
which will affect its functional properties. Studying change in functional properties of NFDM due to various pre-heat treatments, storage, elevated PL levels, is currently under way.
Impacts
Proteolysis induced by plasmin (PL) is characterized with a complex nature and versatile effects on quality. In fresh milk, plasminogen (PG) is the predominant form, where its concentration is 2-30 times that of plasmin. Therefore, any potential activation of PG by PG-activators (PA) could contribute significantly to PL activity in milk. Thus this project is implemented to investigate closely factors that affect PG activation in milk, cheese and NFDM. So far our work on this project has revealed possible mechanisms and factors that affect PG activation. Mechanisms involved in PG activation, so far, were found to be: PG denaturation, interactions with whey proteins, inactivation of inhibitors, and inhibition of PA by Ca ion. Also, the work on this project, so far, has shown variable effects of different heat treatments and cold storage conditions on PG activation. Furthermore, the work is addressing the consequences of PG activation on functional properties of NFDM.
Results of this work, which is centered on understanding mechanisms and factors involved in PG activation, will give insights to better regulate PL activity in milk depending on the final intended use. This creates the potential for improving the quality and reducing the production costs to make our domestic producers of dairy products more competitive.
Publications
- Burbrink, C.N., & Hayes, K.D. (2006). Effect of thermal treatment on the activation of bovine plasminogen. International Dairy Journal, 16, 580-585.
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Progress 08/01/05 to 08/01/06
Outputs
During the initial eight weeks of this project we (myself and a new M.S. student) began conducting experiments on the impact pre-storage thermal treatments, storage time, and concentration of plasminogen activator have on plasminogen activation in fluid milk and cheese curd. We have found that plasminogen activation and resulting beta casein breakdown occur rapidly once milk had been heated to temperatures between 65 and 75 degrees Celsius regardless of plasminogen activator concentration when kept 48 hours under refrigerated storage conditions. These data suggest that the combination of heating and storage time promote the activation of plasminogen to result in increased protein breakdown in pasteurized milk, while heating milk to temperatures greater than traditional pasteurization temperatures results in less plasminogen activation and subsequently less protein breakdown.
Impacts
These findings suggest that temperatures greater than pasteurization result in slowed plasminogen activation to result in decreased beta casein breakdown; therefore heating milk to higher than traditional pasteurization temperatures results in milk with less protein breakdown and subsequently improved quality.
Publications
- No publications reported this period
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