Progress 09/01/04 to 08/31/07
Outputs
OUTPUTS: A patent invention disclosure has been filed and accepted by WARF (UW-Madison) for the preparation of a patent application. Title: Production of protein-polysaccharide conjugates Authors: Lucey, J. A., Zhu, D and S. Damodaran Reference # P08190US. This work was presented as a poster entitled "Conjugation Reaction Between Whey Proteins and Dextran" at the 2007 USDA project directors meeting. This work was presented as an oral presentation at the 2007 Institute of Food Technology 2007 Annual Meeting. The presentation was entitled "The formation of WPI-dextran conjugates in aqueous solutions via the Maillard reaction". Presentation #163-05.
PARTICIPANTS: PI: John Lucey, played the key supervisory role in the project, wrote proposal, reports and patent invention disclosure. Collaborator: Srinivasan Damodaran, assisted in jointly supervising the student and post-docs working on this project. Student: Herlambang, Inggrayani. Investigated conditions that influence conjugation of dilute solutions. Post-doc 1: Tao Wang. He was the initial post-doc/research associate at the start of this project. Developed some of the test methods and preliminary data about reactions and conjugation. Post-doc 2: Dan Zhu, main post-doc on this project. Played a major role in the experimental work, summarized findings and has prepared a draft manuscript.
TARGET AUDIENCES: Food ingredient manufacturers and the scientific/academic community
PROJECT MODIFICATIONS: No major change, but we focused on high concentrations of these two biopolymers when we initially thought that we should work with dilute solutions.
Impacts
We developed a novel method to produce protein-polysaccharide conjugates (PPC) via the formation of a Schiff base using a wet-heat treatment process instead of the lyophilized approach used previously. The reaction occurred by heating aqueous solutions of mixtures of high concentrations of proteins (e.g. 10% whey protein; whey protein isolate, WPI) in the presence of very high concentrations of a polysaccharide that had a reducing sugar (e.g. 30% Dextran, Dx). We discovered that when dilute mixtures of proteins and polysaccharides were heated protein denaturation and protein aggregation occurred much sooner than the conjugation reaction. This was undesirable as denatured and aggregated proteins were probably less available for conjugation. Denatured protein, even if it could be conjugated, probably would have impaired functionality. We discovered that when very high dextran concentrations were used in the reaction mixture that this inhibited protein aggregation.
Polysaccharides also could improve the heat stability of the protein allowing higher reaction temperatures to be used. The high polysaccharide concentration in the mixture caused a molecular crowding effect, which accelerated the conjugation reaction. We tested various process conditions but pH 6.5 and temperatures around 60-70C were considered optimum for increasing this conjugation reaction without causing excessive protein denaturation. During the reaction PPC could be detected within an hour and we have held mixtures for up to 24 hours. We were able to confirm that we had achieved Schiff base formation and therefore the covalent attachment of Dx to whey protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using staining of both protein and carbohydrate moieties. A simple and rapid method was established to determine the formation of PPC, i.e. Schiff base, using Difference UV spectroscopy absorbance peak at ~304 nm. The PPC were purified by anion exchange chromatography and
affinity chromatography. After dialysis and freeze-drying, the purified PPC appeared white in color and had much higher thermal stability (denaturation temperature was 88C) which was much higher than the native whey protein (74C). The PPC had excellent emulsifying properties, which were superior to the native whey protein and a commercial emulsifier, gum arabic. Our PPC product could compete for the emulsification applications currently performed by gum arabic as the price of this ingredient is high and supply varies. There is commercial interest in producing a substitute for gum arabic. The functionality of gum arabic is due to the fact that it also contains some PPC material naturally produced by the acacia tree. It is expected that our PPC they will have significantly improved pH stability compared with native whey protein. This process should be possible with other proteins (if they contain available lysine groups) and polysaccharides with reducing sugars. We could easily control
the extent of the Maillard reaction to stop at the initial Schiff base formation (i.e. the formation of the PPC) and we could limit the formation of unwanted intermediate or advanced products with our process.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The Maillard reaction has been widely utilized to prepare the conjugates of protein and polysaccharide with improved functionalities. However, the processing is usually conducted by dry heating over a long period of time. The formation of WPI-dextran conjugates in aqueous solution was investigated under various conditions. The conjugates of WPI-dextran were obtained at the initial stage of Maillard reaction. Highly concentrated solutions (10% WPI-30% dextran) were used to facilitate the macromolecular crowding effect in order to encourage conjugation and inhibit protein denaturation. This approach was successful. The impact of temperature, pH, concentrations of two macromolecules, time and hydrostatic pressure were investigated for conjugates formation. Increasing WPI and dextran concentrations promoted the formation of conjugates. The reaction was also favored by lowering pH from 8.5 to 6.5. Under hydrostatic pressure (7.9 MPa), the formation of the conjugates was
enhanced at pH 7.0 but significantly suppressed at pH 6.5. The optimal conjugation conditions were chosen: 10% WPI-30% dextran, pH 6.5, at 60C for 24 h. The covalent attachment of dextran to WPI proteins was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with both protein staining and carbohydrate staining. A simple, rapid difference UV spectroscopy (DUV) method was developed to identify the conjugates formed during the initial stage of the Maillard reaction. The absorbance peak value at 304 nm of DUV was used to assess the extent of conjugation between WPI-dextran. Dextran not only worked as a reactant taking part in the conjugates formation, but also as a crowding reagent in promoting conjugation as well as a protective reagent in preventing excessive WPI protein denaturation. The emulsifying properties of the protein-dextran conjugates are being studied. Ion exchange chromatography coupled with a salt gradient was used to separate purified conjugate from unreacted
carbohydrate/protein. Fractions were collected and analyzed for protein and carbohydrate content.
Impacts
The long term goal is to develop novel food ingredients that have enhanced solubility and thermal stability. We have demonstrated a novel approach to generate these conjugates and methods to quantify the conjugate formation. These are key initial steps in achieving the goal of new ingredients with enhanced functionality.
Publications
- Dan Zhu, Srinivasan Damodaran and John A. Lucey. 2007. The formation of WPI - dextran conjugates in aqueous solutions via the Maillard reaction. Abstract for 2007 IFT annual meeting.
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Progress 01/01/05 to 12/31/05
Outputs
In the initial part of this research we have focused on trying to make protein-polysaccharide conjugates from whey protein and dextran. Whey protein isolate was used as the source of whey protein. Dextran (from Sigma) with molecular weights ranging from 8,500-11,000 and 400,000-500,000 has been used as the polysaccharide. We are trying to use the Maillard reaction as the crosslinking method and looking for the formation of a Schiff base as an indication that we have been successful in this reaction. The most critical objective in the initial phase of the project was to develop methods to confirm Schiff base or conjugate formation. We developed two methods. The first method involves sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate conjugates and a glycoprotein straining kit (from Pierce) to strain conjugate bands depending on their molecular weight. This straining kit also strains sugar group however only conjugates have a protein group
attached and thus only conjugates will move in the electric field applied during electrophoresis, i.e. unreacted dextran will remain on top of the PAGE gel (unresolved). We also developed a difference ultraviolet absorbance spectroscopy (DUV) method where there is an increase in the DUV absorbance at approximately 305 nm, which is taken as an indication of Schiff base formation. We investigated the impact of various conditions on the formation of the Schiff bases between whey protein and dextran, such as, the concentrations of whey protein and dextran; ratio of whey protein and dextran; temperature, pH, time, heating and pressure treatment. Our initial results indicate that the formation of Schiff base increased with the increase of dextran concentration from 10 to 30% but decreased at higher concentrations. One suitable ratio of whey protein and dextran was 1:3 by weight. Raising the temperature and extending the incubation time assist in Schiff base formation. A decrease in pH value
(range pH 6.8-8.5) promoted the formation of Schiff Base. Elevated pressure (7.9 MPa) and shear also appeared to enhance Schiff base formation compared with the static heating at atmospheric pressure.
Impacts
Protein-polysaccharide conjugate formation was demonstrated in aqueous systems in contrast to previous published work by other groups that were exclusively performed with freeze-dried powder. Methods have been developed that allow the detection of this conjugation reaction; this will help in optimizing processing conditions to increase the conjugate yield.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The project investigates the interactions between food proteins and polysaccharides and the possible formation of complexes between these two components. Initial work was conducted on whey protein isolate and 500k Dalton dextran (1:1) mixtures that were processed in the pressure cell of the rheometer. A response surface design was used for this experiment where temperatures ranged from 58 to 75C, shear rates from 40 to 250 1/s, and pH values from 4.0 to 9.0. All mixtures for analyzed by size-exclusion chromatography using three detectors: UV, refractive index and multiangle laser light scattering. Several process conditions indicated the possible formation of low levels of complexes; this is being further investigated at present. Mixtures (1:1) of WPI and dextran were also processed using a very high pressure microfluidizer unit using a design where temperature ranged from 58 to 75C and pH values from 4.0 to 9.0. Samples from the microfluidizer were used to prepare
model emulsions and the particle size changes over time were examined using a Malvern Mastersizer. A model was developed for the average particle size of emulsions where pH, temperature, the interaction between shear rate and temperature, and the interaction between pH and temperature, were significant terms.
Impacts
Possible development of new types of food ingredients. Improved knowledge of the interactions during processing of food components.
Publications
- No publications reported this period
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