Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Food Science
Non Technical Summary
The overall goal of the proposed research is to fight obesity in children suffering from phenylketonuria by the development of glycomacropeptide-polysaccharide conjugates for use in new protein-rich foods. We aim to be the first to create medical foods for children and adults with phenylketonuria (PKU) that contain intact protein and fruit juice. Wisconsin has been a pioneer in the study of phenylketonuria since the days of Harry Waisman, and more recently through work that has resulted in several patents and pending patents. The first glycomacropeptide (GMP) medical food for people with phenylketonuria resulted from research at Wisconsin completed in 2010. The proposed research builds upon that past by using the dry heating method to conjugate GMP to less expensive polysaccharides than the dextran used in past work, and to develop new separation processes to lower the Phe content of GMP. The proposed work will increase the purity of GMP using charged ultrafiltration membranes. Then, purified GMP will be conjugated to maltodextrin for use in new GMP medical foods made from fruit juices. This will widen the food choices for children with PKU, and improve their health. This new food technology will build on our existing patent on GMP medical foods for PKU, and expand the opportunities for Wisconsin business.We will meld the Wisconsin technology for conjugation of polysaccharides to proteins with the Wisconsin technology to purify bovine kappa-caseinoglycomacropeptide (GMP), and generate useful new GMP-polysaccharide conjugates. These conjugates are expected to be more heat stable, more viscous, and more shielded from the polyphenol haze and Maillard reactions than un-conjugated protein. We will test these conjugates in new PKU foods. This will increase the availability of protein-rich foods for children and adults with PKU. Our ultimate aim is to develop glycomacropeptide-polysaccharide conjugates to improve PKU foods, and to develop new protein-rich foods for all children and reduce the incidence of overweight and obesity.GMP is the only known protein found in nature that is missing the amino acid phe. This odd fact of nature has made GMP useful in PKU foods. Individuals with PKU now have new foods available that allow intact protein consumption for the very first time. These patented PKU foods were developed at Wisconsin.GMP is the second most abundant protein in cheese whey, comprising 15-20% of the protein. GMP is produced during cheese making when bovine kappa-casein is cleaved by chymosin into para-kappa-casin (residues 1-105) that remains with the curd, and GMP (residues 106-169) that remains with the whey. GMP has 64 amino acid residues and a molecular weight of 8.6 kDa. GMP is more acidic and smaller than the other proteins in cheese whey. This has facilitated methods for its purification using ion exchange chromatography and more recently using charged ultrafiltration membranes. These methods of purification have been patented by the University of Wisconsin, and used in research to develop foods for children suffering from the inherited metabolic disorder PKU.Because individuals with PKU eat a low protein diet, it is rich in carbohydrates and fats. Muscle protein synthesis is impaired in low-protein diets, and this combines with the carbohydrate-rich and fat-rich foods of the PKU diet to cause a prevalence of overweight and obesity in children with PKU. For example, in the UK, 25% of 11-15 year-old boys having PKU were overweight compared to 15% in the UK population. Adult PKU patients had a higher BMI and more obesity than the general population.Commercially available GMP is contaminated with other whey proteins that contain Phe. This product has about 2.0 mg Phe per g protein equivalent of GMP, which is too high for many children with PKU. Eating 50 g of GMP adds 100 mg Phe to their diet, which is often limited to 200 to 500 mg Phe. Amino acid medical foods are the current standard and contain no Phe. That means that children must consume less "normal food" such as French fries and potato chips, which contain about 36 mg Phe per g protein (18x more than the commercially-available GMP product). GMP medical foods promote healthy children more than amino acid medical foods, but taking away the ability of children to "cheat" and eat French fries or potato chips with their friends is a high price to pay. Our goalis to solve this problem by lowering the Phe content of GMP using our charged ultrafiltration membrane technology.Our success willcreate the first GMP medical foods for PKU that contain intact protein and fruit juice. This will widen the food choices for children with PKU, and improve their health. This new food technology will build on our existing patent on GMP medical foods for PKU, and expand the opportunities for Wisconsin business.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Goals / Objectives
The overall goal of the proposed research is to fight obesity in children suffering from phenylketonuria by the development of glycomacropeptide-polysaccharide conjugates for use in new protein-rich foods.Objectives:1. Use charged ultrafiltration membranes to increase the purity of GMP.Hypothesis - The low isoelectric point of GMP (pI < 3.8) compared to whey proteins (pI ~ 4.5) makes it possible to retain the negatively charged GMP at pH 5.2 using a 100 kDa negatively charged ultrafiltration membrane, while the uncharged whey proteins permeate the membrane.2. Use the dry heating method to make GMP-polysaccharide conjugates from purified maltodextrin that are suitable for use in new PKU medical foods that contain fruit juice.Hypothesis - GMP-maltodextrin conjugates made using the dry heating method will not cause the protein-polyphenol haze that now limits GMP use in fruit juices. This is because the conjugated polysaccharide shields the Pro residues in GMP that otherwise cause protein-polyphenol haze.
Project Methods
Objective 1. Use charged ultrafiltration membranes to increase the purity of GMP.Composite regenerated cellulose cross-flow membrane modules having a 100 kDa molecular weight cutoff will be used for charge modification following the procedure of Arunkumar and Etzel (2015). This procedure attaches the sulfonic acid moiety in the amino acid taurine to the membrane surface. GMP from Arla (Aarhus, Denmark) will be dissolved in 50 mM sodium acetate, pH 5.2 and the sieving coefficients measured under total recycle conditions, where both the permeate and retentate are recycled back to the feed solution container. Sieving coefficients are a fundamental property of the membrane-protein system, and allow us to use mass balance models to calculate membrane system performance instead of using time-consuming Edisonian experimentation. The impurities in GMP will be dia-filtered out using increasing volumes of pH 5.2 buffer. We will make membranes of different negative charge density by adjusting the reaction time, and evaluate the effect of charge density on purification. More charge means more rejection of GMP, but perhaps less rejection of impurities. Membranes tighter and looser than 100 kDa will be evaluated to find the conditions for the highest purity. Lastly, the chemistry to modify polysulfone (PS) ultrafiltration membranes will be developed. PS membranes are commonly used in industry, and are somewhat less expensive, offering an attractive alternative to regenerated cellulose membranes for some manufacturers. We have never tried to modify the charge on PS membranes, but Nakao (1988) used a phosphate sulfonating compound to successfully prepare a negatively charged ultrafiltration membrane. GMP purity will be assessed by amino acid analysis for Phe, and total Kjeldahl nitrogen for protein. In addition, HPLC and SDS-PAGE will be used to quantify GMP and the protein impurities in GMP.Objective 2. Use the dry heating method to make GMP-polysaccharide conjugates from purified maltodextrin that are suitable for use in new PKU medical foods that contain fruit juice.Mixtures of 10% GMP (Arla Foods, Denmark) and 30% maltodextrin (various mol. weights, Sigma), will be freeze dried and reacted using the dry heating method at 70° C, and 80 % relative humidity. Samples will be taken at times of 0, 1, 2, 4, 8, 16, and 36 h for kinetic analysis. The extent of glycation, molecular mass distribution, and protein purity of each sample will be determined using gel electrophoresis and ion exchange chromatography using past methods. To date, only dextran conjugates with GMP have been made from dextran of molecular weight 10 kDa. Maltodextrin is 1000x less expensive that dextran. In the proposed work, maltodextrin will be tried as a replacement for the more expensive dextran. Maltodextrin has been conjugated to whey proteins in past work (Zhu et al., 2008, 2010), but we will be first use GMP. Maltodextrin is available in various molecular weights, generally much smaller than the dextrans. Maltodextrin DE 4-7 has a MW of 2.6 to 4.5 kDa, and will be used as a substitute for 10 kDa dextran. The maltodextrin will be cleared of low molecular weight sugars that cause Maillard browning by using membrane dialysis prior to conjugation with GMP.SDS-PAGE will be used to separate conjugates from unreacted GMP and maltodextrin with the glycoprotein staining kit obtained from Pierce (Zhu et al., 2008, 2010). This staining kit also stains unreacted sugar groups however, only conjugates have a protein group attached and thus only conjugates will move in the electric field applied during electrophoresis, i.e. unreacted maltodextrin will remain on top of the PAGE gel (unresolved). SDS-PAGE with the Sypro Red fluorescent straining procedure for proteins will be performed under non-reducing and reducing conditions to determine the level of protein denaturation or aggregation after processing. Gels will be scanned on a laser densitometer and concentrations quantified using calibration standards.Reaction kinetics and yield will be quantified by mass balance and used to optimize the reactant concentrations and reaction pH, temperature, and time. Our goal in this optimization is to increase productivity and yield of conjugate. Productivity is the mass of conjugate produced per unit time. In principle, higher reactant concentrations, temperatures, and pH values speed the Maillard reaction. However, these principles must be balanced against restricted diffusion at higher concentrations, forcing the reaction beyond the initial step of Schiff base formation into subsequent color development, and protein denaturation and aggregation at high temperature, concentrations, and pH. Yield is the mass of conjugate produced per mass of reactants. Increasing yield lowers cost. Yield increases as a greater fraction of the reactants are converted into conjugates and not byproducts or left unreacted. Often yield must be balanced against productivity because a slow reaction using lower feed solution concentrations and lower pH values may favor yield, but lower productivity.Commercial cranberry juice concentrate (CJC), white grape juice concentrate (GJC) and apple juice concentrate (AJC) will be purchased from a local grocery store. Grapes, cranberries, and apples are all important crops in Wisconsin. Protein solutions will be prepared at a concentration of 33 mg/ml and various pH values ranging from pH 3.0 to pH 4.6 to fall within the FDA guidelines for acid foods, and within the pH range of different popular fruit juices. Protein solutions will be mixed with juice concentrate to produce a mixture concentration of 21 g/L protein. Both GMP and GMP-maltodextrin conjugates will be evaluated for clarity, color, and viscosity.Protein solutions will be heated to 190°F for 2 min to mimic "hot fill" treatments used for fruit juices in industry. After heat treatment, the sample is cooled to 22°C prior to measurement of clarity, color, and viscosity. The industry standard for fruit juice clarity is Nephelos Turbidity Units (NTU). The minimum for fruit juice is 50 NTU, but juice of this turbidity is noticeably hazy. Values below 30 NTU are more acceptable to consumers, and values below 10 NTU are perceived as clear as water. We will use a calibrated portable nephelometer (Orbeco Hellige, Farmingdale, N.Y.) to measure NTU. Color formation will be evaluated from absorbance at 420 nm, the maximum for Maillard reaction products. Viscosity will be measured using a Cannon-Fenske capillary viscometer. Gelation will be observed after samples are stored at 4°C overnight.