Whey Research Sponsored by CDRF
Whey 1999
96 DUS-01
Development of Reversed Micellar Extraction Technology for Whey Proteins
Stephanie R. Dungan, UC Davis
Executive summary
Whey is a water-based fluid that is produced as a by-product of cheese manufacturing. It contains milk components that are soluble in water, including many proteins, the sugar lactose and minerals. By "refining" whey (i.e., processing the whey in order to isolate individual components) it can be divided into several high-value products. This report summarizes the development of a method for separating the proteins found in whey using reversed micellar (RM) solvents. These solvents are oil-based and thus do not dissolve in water but contain tiny (nanometer-sized) surfactant-stabilized water droplets, which can selectively remove proteins from whey. By altering properties of these reversed micellar droplets, protein molecules will move into the droplets from an aqueous solution. Our research has established the feasibility of using RM solvents to extract, recover and separate three proteins in whey: beta-lactoglobulin, alpha-lactalbumin, immunoglobulin. The proteins are extracted by the reversed micellar droplet phase due to charge-charge interactions, hydrophobic effects and the size of the protein relative to the droplet. The pH and salt concentration in the aqueous feed and aqueous product phases can be used to control these interactions and therefore influence the protein recovery.
Bovine immunoglobulin (IgG) tends to be extracted by the RM solvent better at lower pH and salt concentration, in which electrostatic interactions between the protein and reversed micelles are maximized. At extremes of pH and ionic strength; however, IgG extraction is followed by a slow (~24 h) precipitation of the protein to the middle region, between solvent (organic) and aqueous phases. We believe that the kinetics of this precipitation process are more rapid for IgG than for most proteins, due to its very large molecular weight, but that the metastability of the protein-containing solution is a general feature of these phases at extremes of pH or low ionic strength.
At moderate values of pH and ionic strength, IgG can be extracted from aqueous solution to the RM solvent. Decreasing temperature shifts the pH dependence of the extraction to lower values. Increasing surfactant concentration has only a small effect, indicating that the cost of surfactant needed to effect protein recovery can readily be limited. The difference between the partitioning behavior of IgG and that of two other proteins in whey, alpha-lactalbumin and beta-lactoglobulin, make it possible to separate these three proteins from one another using this approach.
Recovery of alpha-lactalbumin and beta-lactoglobulin from the RM solvent can be easily accomplished by contacting the organic solvent with an aqueous stripping solution at high pH. Aqueous salt solutions at high (2 M) NaCl concentrations but neutral pH can alternatively be used to recover alpha-lactalbumin from the RM solvent phase. Concentrated salt stripping solutions likewise caused beta-lactoglobulin to exit the reversed micellar droplets, but in this case protein removal was followed by precipitation of the protein to a region between the aqueous and organic phases. Such precipitation was not observed with alpha-lactalbumin, or with beta-lactoglobulin at high pH. When lactose was added with whey proteins to the initial aqueous feed stream, the RM solvent removed the protein but little of the lactose, so that the final aqueous stripping solution contained only protein and no sugar. Thus RM solvents can be used not only to separate proteins from each other, but also to eliminate 97 percent of the lactose.
Partitioning studies such as those described here establish guidelines for the design of a liquid/liquid extraction process for obtaining desired individual whey proteins. The yield of the process can be augmented by increasing the volume of the RM solvent relative to the aqueous feed during the extraction step, and by increasing the volume of the aqueous stripping solution relative to the RM solvent during the recovery step. These extraction methods should have significant cost advantages over membrane or chromatographic methods, due to the ease of scale-up, low cost of equipment, flexibility and potential for continuous processing.
97 FLR-01
Scaling-Up and Feasibility Study of a Novel Adsorption Process to Separate and Purify Proteins from Whey
Rafael Jiménez-Flores, Cal Poly
San Luis Obispo
Executive summary
Beta-lactoglobulin is a pure, natural food stabilizer with impressive gelation and water-binding properties that are useful in frozen dairy desserts and many other foods that require stabilization. Therefore, the isolation of beta-lactoglobulin from whey has the potential to become an important aspect of the dairy processing industry.
As volumes of whey increase in the United States, its role in the profitability of cheese production will gain more importance. However, the increase in value of raw whey to the current status of a valuable commodity, is reaching a plateau.
In a effort to improve the value of whey, the Southeast Dairy Foods Research Center (SDFRC) at North Carolina State University, in Raleigh, NC, investigated techniques to pre-bind fat soluble nutrients Vitamin A and Vitamin E to beta-lactoglobulin. The ability of beta-lactoglobulin to bind lipophilic nutrients to its hydrophobic pocket provides the mechanism for fortifying low-fat or non-fat products with vitamins A, D, E and K, and essential fatty acids. This provides processors with the means to combat the nutritional fortification dilemma by adding fat-soluble vitamins to reduced-fat and fat-free products.
The first part of our project focuses on the pretreatment of whey, with delipidation of whey as the main objective. Delipidation of whey helps to prevent fouling and allows for a clean whey stream needed by the bioselective absorption process. Current methods involve the use of chitosan to selectively precipitate and remove lipids from whey. A method was developed for this project using Celite cake filtration and a chemical pretreatment.
The fractionation of proteins from whey provides the dairy industry with a challenge and opportunity to develop markets for increased use of whey products.
Current trends in food processing have justified the need for more functional ingredients, such as purified beta-lactoglobulin. In order to achieve this while maintaining the bioactivity of whey proteins, we have scaled up a laboratory method previously developed by Swaisgood et al. for the rapid purification of beta-lactoglobulin from whey. Specifically, trans-retinal covalently immobilized on a R648 Celite‰ matrix was used to isolate beta-lactoglobulin from acidified whey. The matrix acts as a bioselective absorbent for beta-lactoglobulin at pH 5.1. Elution of the specifically absorbed protein yields beta-lactoglobulin with greater than 95 percent purity as determined by capillary electrophoresis.
For our scaled-up pilot plant size column (6-liter volume, plus 2.3 kilograms of R648 Celite‰), the total loading capacity, flux rate and contact time were determined. The total loading capacity of the column was determined to be 0.01 grams of beta-lactoglobulin per gram of the R648 Celite‰ matrix, which allowed for the isolation of approximately 23 grams of beta-lactoglobulin per elution. A flux rate of 10 to 100 L/min resulted in a linear increase in pressure from 0 to 20 psi. When contact time was 15 minutes, the efficiency was similar to that obtained in the lab. At high flow rates where contact time was minimal efficiency dropped only 50 percent. Consequently, in our scaled-up setting at a high flow rate, the column has the ability to isolate 12 grams of beta-lactoglobulin in five minutes of operation.
A pretreatment method using Hyperflo Celite‘ and calcium silicate was also developed to effectively remove residual lipids from whey. The whey was cooled to 5oC and the pH was adjusted to 5.1 before Hyperflo Celite and calcium silicate were added. Using a pretreatment method of Hyperflo Celite and calcium silicate, whey has been produced with 90 percent of the residual lipids removed. The pretreatment method was scaled up to the pilot plant level and the process proved effective in the removal of residual lipids from large volumes of whey.
97 GEB-01
Controlling the Pro-oxidant and Antioxidant Actions of Lactoferrin in Foods
J. Bruce German, UC Davis
Executive summary
Lactoferrin is a whey by-product from the cheese industry with great potential as a value-added component for foods. The ability of lactoferrin to bind iron improves the oxidative stability of infant formula, a food product fortified with iron. Therefore, lactoferrin is expected to have applications in other iron-supplemented foods such as flour and cereals.
Lactoferrin inhibits iron-catalyzed oxidation at high protein:iron ratios; however, it stimulates oxidation at low protein to oxidation ratios. Our first study showed that lactoferrin does not affect copper-catalyzed oxidation. In this study of lactoferrin:iron interaction in the inhibition and promotion of oxidation, emulsion systems were sensitive not only to the amount of lactoferrin and iron, but to the chemical form of the emulsifier.
Our second study proved bovine lactoferrin to be an effective antioxidant in infant formula fortified with iron. Lactoferrin:iron ratios as low as 1:18 decreased the pro-oxidant effect of iron by one-half. Lactoferrin can be an excellent iron carrier to improve the oxidative stability of foods fortified with iron for nutritional purposes.
The overall goal of this project was to define, quantify and ultimately resolve the effect of lactoferrin on oxidation of foods. We first set out to determine in model systems the effect of lactoferrin on the oxidative stability of oil emulsions and liposomes, and to
- develop methods to measure oxidation in infant formulas,
- test the effect on oxidation of lactoferrin in infant formulas, and
- determine adequate levels of lactoferrin to prevent oxidation in infant formulas
Interest in the use of lactoferrin (an iron-transport protein present in milk) in foods for its antimicrobial activity inspired this study. Natural bovine lactoferrin inhibited oxidation in buffered corn oil emulsions and lecithin liposome systems at pH 6.6 and 50 degrees C.
The antioxidant activity increased with lactoferrin concentration in both phosphate- and Tris-buffered emulsions, but not in both buffered liposome systems. A mixture of 1 µM lactoferrin and 0.5 µM ferrous ions was a more effective antioxidant than 1 µM lactoferrin alone in Tris-buffered emulsions and in phosphate-buffered liposomes.
At a concentration of 1 µM lactoferrin, the protein was a pro-oxidant in phosphate-buffered liposomes, and at concentrations of 15 and 20 µM, it was a pro-oxidant in Tris-buffered liposomes. Copper was a stronger pro-oxidant than iron in both buffered emulsions.
Lactoferrin decreased the pro-oxidant effect of iron, but not of copper, in emulsions. The anti-oxidant or pro-oxidant activity of lactoferrin depended on the lipid system, the buffer, lactoferrin concentration, the presence of metal ions and oxidation time.
Fat in infant formulas must be extracted in order to measure oxidation. Common fat extraction methods that we tested (e.g., Folch, Bligh and Dyer, and Radin) were not suitable for extraction of fat from infant formula.
We developed a liquid extraction method that had 100 percent recovery with a 4 percent variation coefficient for both fresh and oxidized infant formulas. Using this method of extraction, we isolated the fat and measured the hydroperoxide (primary lipid oxidation products) content. We also measured fluorescence in the aqueous fraction to determine changes in proteins due to their interaction with oxidized lipids. We analyzed the volatile compounds produced by decomposition of the hydroperoxides and responsible for rancidity, using headspace gas-chromatography.
We tested the antioxidant effects of lactoferrin in a commercial infant formula fortified with iron, adding lactoferrin to the formula at several concentrations, ranging from its content in human milk (1 mg/ml), to the amount required to bind all the iron in the formula (11 mg/ml). Lactoferrin decreased oxidation in a concentration-dependent manner. A small amount of lactoferrin (1 mg/ml) inhibited the formation of hydroperoxides by 48 percent. The volatile decomposition products of hydroperoxides are responsible for rancidity, and we measured these by headspace gas chromatography. At the highest ratio tested (lactoferrin:iron ratio 1:2), lactoferrin completely inhibited both the formation of hydroperoxides and hexanal during the testing period.
95 KRJ-01
Application of Edible Films from Milk Protein and Milkfat to Food Systems
John M. Krochta, UC Davis
Executive summary
Objective 1: To determine milk protein/milkfat coating formulation surface tension and viscosity effects on wetting and spreading of coating formulations on foods with hydrophobic and/or porous surfaces.
To maximize the potential of whey-protein coatings on foods, it is necessary to achieve good whey-protein coating formulation spreading, coverage, adhesion and durability on the food surfaces. We have determined that addition of food-grade additives called plasticizers is necessary to maintain coating flexibility and durability. For certain foods with hydrophobic (oily) surfaces, such as nuts, it is also necessary to add food-grade additives called surfactants for the whey-protein coating formulation to spread on the food surface. For coating other foods, such as porous freeze-dried foods and breakfast cereals, surfacants are not necessary. Overall, effective food-grade plasticizers and surfactants that provide efficient whey-protein coating formulation coverage, adhesion and durability on foods have been identified.
Objective 2: To optimize milk protein/milkfat coating formulation and drying conditions to improve film properties, improve coating adhesion to foods and reduce coating cracking and flaking.
Besides achieving efficient whey-protein coating formulation spreading on food surfaces, it is also necessary to achieve and maintain good dry coating adhesion to the food surfaces. We found that acrylic and polypropylene surface properties approximate the surface properties of chocolate and roasted peanuts, respectively. Thus, we selected acrylic and polypropylene as model surfaces for investigating improved whey-protein coating adhesion to chocolate and peanuts. This made data collection and analysis much easier. Using this approach, we found adhesion of whey-protein coating to acrylic (model chocolate surface) to be identical to that of commercial shellac coating and better than that of commercial corn zein coating adhesion. A surfactant was necessary to coat polypropylene (model peanut surface) with whey-protein-coating formulation. With the surfactant added, adhesion of whey-protein coating to the polypropylene (model peanut surface) was identical to that of commercial shellac coating and better than that of commercial corn zein coating. Thus, a low level of surfactant can allow good coverage of peanuts and other nuts by whey-protein coatings and achieve good adhesion.
The properties of films made from whey-protein concentrate (WPC) were compared to those of films made from whey-protein isolate (WPI). Good quality edible films can be made using 80 percent protein WPC. Using the proper formulations, 80 percent WPC films can be made with mechanical properties and barrier properties equivalent to WPI films. The main differences between 80 percent WPC and WPI films are that 80 percent WPC films have increased solubility and color development compared to WPI films. In certain situations, both of these differences could be positive aspects of the films. Examples include a dissolvable ingredient pouch or a coating developing a yellow color to accentuate product color.
We compared films made from native WPI to films made from heat-denatured WPI. Although native WPI films were found to be totally water soluble, heat-denatured WPI films were insoluble. Heat-denatured WPI films also had greater strength and stretchiness than native WPI films. These results provide a better understanding of the molecular forces involved in whey-protein film formation and the ability to form films with different solubility or mechanical properties suited to film application.
Films made from hydrolyzed WPI required less plasticizer addition to achieve desired film strength and flexibility. At the same time, the hydrolyzed WPI films had better moisture- and oxygen-barrier properties and were more soluble. Thus, depending on intended application, use of hydrolyzed WPI films may provide advantages.
We identified certain plasticizers that achieve desired film strength and flexibility while optimizing the film oxygen-barrier properties. Use of these plasticizers will optimize the effectiveness of whey protein films and coatings.
We found that a high-melting milkfat fraction (HMMF) gives WPI-HMMF emulsion coatings more flexibility and makes them less likely to crack than coatings from WPI combined with harder plant-based waxes commonly used for food coating. In addition, we found the water vapor permeability of such WPI-HMMF emulsion films is lower than WPI-wax emulsion films. Drying such films at elevated temperatures simulating commercial operations improved film strength and barrier properties.
Objective 3: To assess properties of whey-protein based films formed as coatings on nuts, cereals, dried fruits and vegetables, and fresh fruits and vegetables.
WPI coatings are flexible and transparent. In addition, WPI coatings have excellent oxygen-, aroma- and oil-barrier properties. We have determined that WPI coatings have gloss comparable to shellac and better than other commercial coatings. In addition, WPI coatings have lower color and better color stability than shellac and other commercial coating materials. WPC coatings yellowed at a faster rate than shellac or WPI coatings. Thus, if one wishes low-yellow color, WPI coating is equivalent to HPMC and better than shellac and corn zein. If one wishes some yellow color, WPC is a better choice.
Bench-scale and pilot-scale coating of peanuts with a WPI coating formulation produced coatings with significant reduction in peanut oxidation. Results suggest a significant increase in peanut shelf life or in shelf life of products containing peanuts.
Bench-scale and pilot-scale coating of chocolate-covered raisins and almonds with WPI-based formulations produced coatings with good uniformity, coverage, adhesion and gloss.
During a bench-scale process, we tested freeze-dried diced chicken coated with a WPI coating formulation and found that it had better resistance to mechanical damage. The coating produced up to 70 percent reduction in product damage in simulated product handling tests.
In addition, we coated breakfast cereal flakes with a WPI coating formulation in a bench-scale process. The coating reduced flake fragmentation in simulated product handling tests. In addition, the flakes retained their crispiness in milk for a longer period of time.
We also found WPI coating to have a positive effect on reducing O2 and ethylene levels and increasing CO2 level in coated fruit. This indicates good potential for extending the shelf-life of fresh fruits and vegetables. Control of relative humidity (RH) is important to optimize this effect, since coating permeability is strongly affected by RH.
Whey protein films formed as coatings on foods continue to attract considerable attention from the food industry. The data developed is necessary to show that whey protein coatings are superior to existing popular commercial coatings (shellac, corn zein and HPMC). Food processors will consider replacing existing coating materials and also consider whey protein coatings where the existing commercial coating materials have not performed adequately. Adoption of whey protein as a coating material by the food industry will result in large, value-added utilization of whey protein.
Our research continues to lay the groundwork for utilization of whey protein as films and coatings by the food industry, to protect foods from moisture, oxygen, oil and aroma migration, provide food integrity and improve appearance (e.g., gloss and color) of foods. Replacement of shellac and corn zein use by the confectionery industry is a clear target for WP coatings, with many other foods also possible. We have now begun coating such foods in a pilot coating facility for generating food samples for instrumental and sensory evaluation and for generating whey-protein coated food samples requested by the food industry.
98 KRJ-01
Whey Protein Coating for Improving Packaging Material Performance
John M. Krochta, UC Davis
Executive summary
The overall objective of this project is the large-volume utilization of whey protein as a packaging material. To accomplish this, the following sub-objectives have been defined:
- Develop whey protein coating on paper and paperboard to improve paper material moisture-barrier, oil-barrier, printability, strength, color and/or gloss.
- Develop whey-protein-coated paper as an oxygen barrier to retard lipid oxidation of packaged food products.
- Develop whey protein coating on plastic film to replace plastic oxygen barrier layers in laminated composite films.
The moisture-barrier and printability properties of whey-protein coated pulp paper have been assessed. Whey-protein coating on paperboard was found to reduce the water vapor permeability and to improve the printability.
The grease-barrier property of whey-protein coated paperboard was determined using a standard test method that quantifies grease penetration. Whey-protein coating imparts excellent oil resistance to paperboard which is comparable to current commercial paperboard grease coatings.
Whey-protein coated paperboard improves gloss, a desirable attribute for packaging purposes. By forming a continuous coating-layer, the whey-protein coating creates a smooth surface over the rough paper fibers and imparts a significantly more glossy surface than non-coated paperboard.
Color measurements determined that no noticeable color changes occurred by coating paperboard with whey protein. The whey-protein coating on paperboard maintained the transparency that is important for packaging appearance.
Our research has shown that whey-protein coating on paperboard imparts excellent grease resistance. Gloss studies have shown that gloss values are higher with the whey-protein coating, and color studies have shown that transparency is maintained. Results from ongoing oxygen, crease and storage tests may further enhance the attractiveness of whey protein as a paperboard coating.
Examples of commercial applications include donut boxes and pizza boxes, which require a significant level of grease barrier on their packaging to retard grease penetration and unsightly stains. An example of an application that will benefit from a good oxygen barrier in addition to grease barrier is pet-food containers. These are just a few examples of the range of applications whey-protein coating on paperboard can have. The result can be the large-volume utilization of whey protein for coating paper and paperboard.
Whey-protein coated paper has been shown to be multi-functional. Whey protein improves water vapor resistance and printability on paper. Whey-protein coated paperboard imparts excellent grease resistance. Comparisons with commercially-coated papers show that whey-protein coated paperboard is comparable in resisting oil penetration. The completion of the remaining objectives in studying the oxygen-barrier properties will allow us to explore the full potential of both whey-protein coated paperboard and whey-protein coated plastic as packaging materials.
98 KUM-01
Lactose-based Functional Polymers: Amphiphilic Polymers, Liquid-crystalline Polymers and Drug Delivery Systems
Mark J. Kurth, You-Lo Hsieh and John M. Krochta, UC Davis
Executive summary
Converting lactose into polymeric materials offers new opportunities for the high-value utilization of lactose, the major component of whey permeate. Lactose-based polymers offer opportunities because of their unique performance, as well as potential environmental and economic advantages. This project aimed to investigate creative chemical pathways to generate lactose derivatives (monomers) that can be polymerized to high-value polymers. This research has led to the generation of various novel polymeric structures with unique chemical functionalities, such as water solubility, swelling and biocompatibility for high-value applications.
The project consisted of two parts: 1) lactose-based monomer and polymer synthesis and characterization, and 2) applications of the polymers to controlled-release drug delivery systems. The polymers that were developed have monomer repeating units that contain lactose. Thus, potential new markets for lactose are possible. Because of the biologically-friendly structure of lactose, the polymers may be applied for various biochemical and biophysical utilization such as sequestering, biosensing and drug delivery.
One of the chemical pathways studied in this project led to a styrene main-chain polymer with attached lactose units. This kind of polymer may be useful as a new type of biocompatible material. The methods developed to synthesize this polymer can provide a range of different water-soluble polymers with different structures. These polymers have potential application for a number of uses, including drug-delivery systems and chromatographic supports for the separation of proteins.
Another chemical pathway studied led to synthesis of a lactose-containing acrylic-type polymer. This polymer shows large swelling in water and, thus, qualifies as a hydrogel. Such hydrogels have potential applications as biocompatible materials, such as for controlled release of biologically-active substances in the human body. Other potential uses include temperature- and pH-sensing biosensors and artificial organs.
Finally, another lactose-based polymer hydrogel involving lactitol as an intermediate was synthesized and studied for control-release of biologically-active substances. The swelling behavior and controlled-release characteristics of these hydrogels can be controlled by the extent of polymer crosslinking. Control releases of aspirin and several proteins, including beta-lactoglobulin and bovine serum albumin, were studied as model systems. The effects of temperature and protein molecular weight on the controlled release rates of the model compounds were determined.
Further characterization of the lactose-polymers already synthesized will allow evaluation of their market potential. Additional opportunities for synthesizing lactose-based polymers have also been defined and await exploration.
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