Whey Research Sponsored by CDRF
Whey 1998
97-FLR-01
Scaling Up and Feasibility Study of a Novel Absorption Process to Separate and Purify Proteins From Whey
Principal Investigator: Rafael Jiménez-Flores, Cal Poly San Luis Obispo
EXECUTIVE SUMMARY:
As the volume of whey increases in the United States, the need for an increase of value in whey and/or whey products reaches greater importance. The ability of native b-lactoglobulin (LG) to bind lipophilic nutrients to its hydrophobic pocket provides the mechanism for fortifying low-fat or non-fat products with vitamin A and D. The b-LG produced by the bioselective process maintains all the properties of native b-LG and also exhibits excellent food functionality. This means that b-LG, if purified in a way that maintains its native structure, can be developed as an important ingredient in processed foods, resulting in an increase in value and demand for whey. The residual lipids in whey have to be removed as a pretreatment step to isolate b -LG. Alternative methods of lipid removal (centrifugation, ceramic membranes, chitosan, chemical pretreatment and microfiltration) were researched to determine optimal removal of residual lipids from whey. An experiment was conducted to simulate the current industrial treatment of whey by using a pilot-size centrifuge to remove lipids.
Results indicated that the pilot-size centrifuge proved ineffective in removing a high percentage of the lipids from whey, leaving a residual 0.15 percent lipid as measured by the Mojonier method. A pretreatment method using Hyperflo Celitetm is being developed to effectively remove residual lipids from whey. Using a pretreatment method of Hyperflo Celitetm and calcium silicate, whey has been produced with 70 to 80 percent of the residual lipids removed. This is an equivalent of having a finished dry whey powder with only 0.3 percent fat. It is important to note that the initial lipid content for this experiments was ~0.15 percent, while most of the literature considers normal lipid content in whey at ~0.5 percent.
The pretreatment method will be scaled-up to the pilot plant level to determine if large volumes of whey can be treated for removal of residual lipids. A pilot-size ultrafiltration system will be used to compare the flux between treated (lipid removal by biosilicate) and untreated whey (normal lipid content of industrial quality). A trial for purification of proteins by use of the bioselective adsorption process was developed at Southeast Dairy Foods Research Center. 500ml of N-retinyl-Celite bioabsorbent was placed in a column and used to fractionate two liters of acid whey. The process yielded 2 grams of b-LG of >90 percent purity and an a-lactalbumin enriched whey.
Trials will be set up and run at Cal Poly to duplicate the method developed at SDFRC. The adsorption and affinity processes will be optimized and the characteristics of the purified whey will be studied. Studies have been performed to develop methods that would allow a significant reduction in the amount of retinal required. The expense of this reagent represents a major factor in the cost of the bioselective adsorption matrix. Only a small amount of the retinal reacts with the matrix in the derivatization reaction; however, a large concentration is necessary to ensure complete derivatization, thus yielding an effective bioselective adsorbent. We have investigated the possibility of reusing the spent retinal solution to prepare additional lots of the adsorbent. Because very little is removed during one reaction, only a small amount must be added to the solution to give a concentration of retinal comparable to that used for the first batch. We have reused the spent retinal solution 5 times (derivatizing 200 mL of matrix each time). The amount of derivatization appeared to be similar each time. Furthermore, when this material was used as an absorbent, it functioned well in the selective binding of b-LG, yielding purities similar to that previously reported.
The final objective of this project is the production of 100-gram quantities of this protein by treating large volumes (100 gallons) of whey. This should yield enough data in the form of material and energy balances to perform an economic study of the project. The initial experiments using centrifugation to remove residual lipids proved ineffective.
We have concluded that the pretreatment method of Hyperflo Celitetm and calcium silicate should be effective in removing residual lipids from whey. The process will be scaled up to the pilot-plant level and then the adsorption and affinity processes can be performed.
97 & 98-ANG-01
Bovine Undifferentiated Embryonic Germ Cells to Allow Site-Directed Changes in the Bovine Genome
Principal Investigator: Gary B. Anderson, UC Davis
EXECUTIVE SUMMARY:
Genetic engineering has the potential to change the face of agriculture, as it has already changed biology and medicine. Agriculture has always relied on genetic manipulation of its animals and plants to provide a safe and efficient supply of food and fiber. Genetic engineering provides new tools to make genetic changes that are not possible through traditional selection and breeding.
Herbicide-resistant crops and engineered plants that produce natural insecticide are examples of new agricultural commodities available as we move into the next millennium. We foresee a time when the dairy cow can be engineered for improved disease resistance and milk composition that has been tailored for consumer preferences and health, and for manufacturing purposes.
Before genetic engineering can be applied practically to animal systems, a great many questions need to be answered. Most successful results in genetic engineering of animals have been obtained in the laboratory mouse, but for success to be achieved in dairy cattle research efforts must focus on cattle themselves. Our overall aim is to apply to dairy cattle genetic engineering techniques that have been developed and used in other species.
The specific objective of our research was to determine if cultured bovine embryonic germ cells could produce normal embryos by nuclear transplantation. This approach capitalizes on the ability to manipulate embryonic cells genetically and then to use these modified cells to clone an adult animal.
We conducted two experiments, one aimed at identifying a useful marker of undifferentiated bovine embryonic cells, and the other to develop nuclear transfer techniques to test cell lines for the capacity to clone embryos. Our evaluation of cultured embryonic cells was based on their morphology and developmental characteristics. The availability of a cellular marker to identify undifferentiated cells, as is available for mouse embryonic cells, would facilitate research with bovine embryos.
We produced bovine embryos by in vitro fertilization (IVF) and evaluated their expression of a marker protein used with mouse embryos. In parallel, we tested the marker on comparable mouse cells. We determined that, unlike in mouse embryos, expression of the marker is not useful with bovine embryonic cells.
These results are important, because other research laboratories working with bovine embryos have used this marker based on its usefulness in mice. We demonstrated that cellular morphology is as reliable an indicator of undifferentiated bovine embryonic cells as is expression of a specific cellular marker. We also conducted an experiment aimed at developing techniques for nuclear transfer with the ultimate objective to clone genetically engineered dairy cattle. Using embryonic cells, we tested several procedures to clone bovine embryos. We were successful at introducing a novel gene into embryonic cells, and then using the transgenic cells to clone transgenic embryos. To this point, we have not transferred our cloned embryos to recipients for development to term; however, we are prepared now to test various cell types (e.g., mammary epithelial cells in milk) for their capacity to produce cloned embryos.
The combination of genetic engineering and cloning of dairy cattle provides preharvest opportunities to change milk composition to meet consumer preferences and nutritional and health needs.
96-DUS-01
Development of Reversed Micellar Extraction Technology for Whey Proteins
Principal Investigator: Stephanie R. Dungan, UC Davis
EXECUTIVE SUMMARY:
Our goal for this project is to determine whether proteins found in whey may be separated using extraction by water-in-oil microemulsions, also known as reversed micelles. These reversed micelles are nanometer-sized droplets of water that form spontaneously within an oil phase in the presence of specific surfactants.
Protein molecules will often transfer from a water phase into these reversed micellar solvent phases, where they sit inside the tiny water droplets. For most proteins, attractive charge interactions between the protein and the surfactants coating the water droplets provide the driving force for the transfer.
In work previously funded by CDRF, we showed that a-lactalbumin and b-lactoglobulin, the two major proteins in cheese whey, are extracted from an aqueous phase by the reversed micellar solutions over a wide range of salt concentrations and pH values, and mixtures of these two proteins could be separated. In the current project we extended the development of this separation technology further, by (1) exploring whether other valuable proteins in whey, namely immunoglobulins, can be separated by this method, and (2) by looking at ways to recover the proteins from the reversed micellar phase.
We have made great progress on the former goal of exploring the extraction of bovine immunoglobulin G (IgG) into reversed micellar solutions. We have looked at the effect of varying the aqueous phase pH on the extraction of this protein by the reversed micelles, and found that the protein transfers effectively only in a narrow pH range near 5.5. This result is different from what we found for a-lactalbumin and b-lactoglobulin, and we believe this is due to the much larger size of immunoglobulin proteins. This different extraction behavior of immunoglobulin from a-lactalbumin and b-lactoglobulin also indicates that the latter two proteins can be readily separated from immunoglobulins. In addition, we found that salt concentration and temperature influence IgG extraction, but have only a weak effect of surfactant concentration. The latter result is of practical importance because it means that large quantities of the relatively expensive surfactant are not needed to improve the extraction efficiency.
Another important finding of our research is that IgG will undergo a slow aggregation and precipitation within the reversed micelles under some pH and ionic strength conditions. This potential limitation of using reversed micelles for IgG separations can be avoided, using results from our research, by choosing the conditions in which this precipitation is minimized.
Our studies have also focused on recovery of b-lactoglobulin and a-lactalbumin from the reversed micellar solution. Results thus far indicate that 95 percent of a-lactalbumin can be recovered in a single reextraction, while attempts to recover b-lactoglobulin using high salt concentrations resulted in significant interfacial precipitation of this protein.
The kinetics of protein recovery were also much more favorable for a-lactalbumin than for b-lactoglobulin, with the former recovered within 30 minutes of a simple bench-scale contacting experiment, whereas transfer of the latter occurred over more than an hour. Other approaches for affecting recovery of b-lactoglobulin from the micellar droplets must be explored.
97-GEB-01
Controlling the Pro- and Antioxidant Actions of Lactoferrin in Foods
Principal Investigator: J. Bruce German, UC Davis
EXECUTIVE SUMMARY:
Bovine lactoferrin from cheese whey (~93 percent pure) inhibited lipid oxidation when added at the rate of 80–1,600 ppm to corn oil emulsions by 7-59 percent, based on hydroperoxide and by 8-82 percent, based on hexanal formation. This antioxidant effect increased with increasing concentrations of lactoferrin in both phosphate-buffered emulsions and tris-buffered emulsions. In lecithin liposomes, lactoferrin inhibited oxidation at 400–1,600 ppm, but promoted oxidation at 80 ppm. Copper had a stronger pro-oxidant effect than iron in the buffered emulsions. In the corn oil emulsions, 80 ppm of lactoferrin (I uM) inhibited the oxidation-promoting effect of 0.03 ppm added iron (0.5 uM), but not at higher levels of iron (0.06-0.56 ppm).
Lactoferrin from cheese whey is an effective antioxidant in a model corn oil emulsion and prevents the catalytic effect of iron at low concentrations. The iron complexing ability of lactoferrin may be useful in improving the oxidative stability of infant formula supplemented with iron. Cheese whey lactoferrin is an effective antioxidant in emulsions and can prevent the pro-oxidant effect of iron at the 2 lactoferrin to 1 iron molar ratio.
Lactoferrin may not only improve the oxidative stability of food emulsions but may also serve as a stable iron carrier for infant and adult foods supplemented with iron for nutritional purposes.
95-HSY-01
Conversion of Lactose to High-Volume and/or High-Value Polymer Products
Principal Investigator: You-Lo Hsieh, UC Davis
EXECUTIVE SUMMARY:
Converting lactose into polymerizable structures offers new opportunities for the utilization of lactose which is a major component of whey permeate, an underutilized by-product from the cheese industry. Lactose-based and lactose derivative-based polymers offer increasing opportunities because of their unique performance as well as environmental and economic advantages. This project aimed to investigate creative chemical pathways to generate lactose derivatives from which polymerizable structures, or monomers, could be synthesized. Polymerization, copolymerization and crosslinking reactions of these monomers have led to the generation of various novel linear and crosslinked polymeric structures with unique chemical functionalities, such as water solubility, adhesion, chemical reactivities and biocompatibility for high-value applications. Thirteen publications are expected from this project.
Among the chemical pathways studied in this project, three monomers generated from lactitol and lactamine have been most successful. They are lactose-O-(p-vinylbenzyl) hydroxime (LVO or LVH) (Ia, Ib); acrylamido-lactamine (LAM) (Ic); and N-Lactosyl-N’-(4-vinylbenzyl)urea (Id). Synthesis of these monomers and their polymerization have led to water soluble linear polymers (Ia, Id). These three monomers can be polymerized in aqueous medium using free radical or redox initiators. Their homopolymers are water soluble and exhibit adhesive behavior. The hydrophillic nature of the lactose moiety in LVH monomer makes it a desirable candidate for copolymerization with other vinyl monomers to yield resins with enhanced solubility and thermal properties (Ib, IIIa,). Acrylonitrile (AN) has a broad range of applications as fibers, both as textiles and as precursors to carbon fibers, membranes and engineering plastics.
The characteristics of the copolymerization reactions, as well as the thermal properties of the LVH-co-AN copolymers, were studied. LVH was also copolymerized with styrene (ST) in dimethylsulfoxide (DMSO)-toluene. The polymerization was rapid and resultant copolymers had molecular weight in the range from 2.11x104 to 6.53x107 daltons with low polydispersities. The solubility of the copolymers with different monomer compositions was enhanced. Incorporation of up to 65 percent of lactose-based monomer onto polystyrene backbone led to a water-soluble polymer. The synthetic approach documents two chemical routes to prepare novel disaccharide-based polymers with well-defined structures and hydrophilic/hydrophobic balances by adjusting feed ratio of the lactose-based monomer to vinyl monomers. Crosslinking of these polymers and copolymers has generated network structures (hydrogels) which have stimuli-sensitive properties, specifically temperature and pH sensitivities. The crosslinking systems we studied included poly (N-isopropyl-acrylamide), polyethylene oxide (PEO or PEG), and polypropylene oxide (PPO).
Among all lactose derivatives, lactitol (reduced from lactose) appears to have more diverse applications because of its higher chemical stability. Propoxylation of lactitol has yielded lactitol polyether polyol (LPEP) with demonstrated good reactivity in the preparation of polyurethane rigid foams. It also has potential to be used as a component of surfactants. While both the lactitol moiety in LPEP and the PEO crosslink offer good biocompatibility, the PEO crosslink also exhibits excellent swelling properties and good thermosensitivity.
The controlled release properties of these hydrogels for drug and other chemicals have been studied. A series of new thermo-sensitive hydrogels has been produced from reactions of acylated poly(ethylene glycol) bis(carboxymethyl) ether (PEGBCOCl) and lactitol-based poly(ether polyol) (LPEP).
We have shown that the swelling behavior of these gels can be controlled. A longer PPO spacer in the LPEP provides a better gel network and a greater free volume for the more hydrophilic PEO chains to bind water and to swell. These hydrogels expel water when the temperature is raised between 25oC and 40oC. This behavior has been utilized for controlled release of aspirin and proteins. Our work in this area has demonstrated the potential of LPEP hydrogels for controlled release of drug, controlled permeation and controlled immobilization.
94-TOP-06
CDFRC Graduate Research Fellowship in Dairy Food Science: Development of Extrudable Milk Protein-Based Edible Films
Principal Investigator: John M. Krochta, UC Davis
EXECUTIVE SUMMARY:
The broad goal of this project is the value-added utilization of whey protein. More specifically, this project focuses on the development of edible films from whey protein isolate by extrusion. This requires obtaining information on the response of whey protein to the heat and pressure used in the extrusion process. It is necessary to measure the glass transition temperatures (Tgs), which indicate the temperature ranges over which whey proteins begin to flow, to determine how whey protein can be formulated in such a way that it can be extruded under heat and pressure into films.
Using the Tgs, a preliminary idea of extrudability and film samples for testing can then be obtained using thermal compression molding. The first step toward this goal is the determination of the thermal properties of whey protein isolate. We measured the thermal properties of whey protein isolate using Differential Scanning Calorimetry (DSC) and found several thermal transitions in the temperature range of -50 degrees C to +5O degrees C, with a large transition occurring around +35 degrees C. We hypothesize that this transition is due to the possible glass transition of b-lactoglobulin (which is the main protein in whey protein isolate). Thermal degradation of the whey protein was found to occur at approximately 185 degrees C. Thus, compression molding and extrusion of whey protein can likely be accomplished in the temperature range 35-185 degrees C.
A thermal compression molding system was developed for placement in an Instron pressure device. After additional data on Tgs have been obtained using the DSC, the thermal compression molding system will be ready to use for formation of films. Successful production of films using thermal compression molding is an indication that such films can be produced by extrusion.
Finally, methods are in place for assessment of film transparency, strength, flexibility, stretchability, and permeability to oxygen, moisture and lipids. In particular, preliminary work has shown that solution-cast WPI films are excellent lipid barriers. This is especially relevant for the development of extruded WPI films, since such extruded films are greatly needed as lipid barriers in co-extruded food products (e.g., cheese-filled pastries). New uses are desired for value-added utilization of whey protein.
Excellent edible films have been made from whey protein isolate by casting from aqueous solution. Such films can be formed as coatings on foods to provide protection from oxygen, aroma and oil migration. In addition to use as coatings, whey protein edible films can be used as stand-alone films (e.g., as co-extruded films to separate cheese fillings from pastries) if they can be produced via extrusion. Therefore, this project focuses on how and under what conditions whey protein isolate can be used for the production of edible, extrudable films.
Whey protein isolate shows considerable promise for the production of extrudable edible films. The results allow us to propose that the "temperature window of opportunity" for the extrusion of WPI ranges from 35oC up to 195oC, which is a large range. The information developed in this project will be used in additional work on thermal compression and extrusion formation of whey protein edible films.
95-KRJ-01
Application of Edible Films From Milk Protein and Milkfat to Food Systems
Principal Investigator: John M. Krochta, UC Davis
EXECUTIVE SUMMARY:
The objectives of this research and progress against objective follow:
Determination of 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
We have found that coating hydrophobic foods such as nuts requires use of certain food-grade additives (surfactants) in the whey protein (WP) coating formulation to ensure spreading and adhesion of the coating on the food surface. Appropriate food-grade surfactants have been identified and tested with WP coating formulations. Results indicated that it will be possible to coat nuts with WP-based coatings.
- Optimization of milk protein/milkfat coating formulation and coating drying conditions to improve coating adhesion to foods and reduce coating cracking/flaking.
Extensive testing of adhesion of WP coatings on model food surfaces, which simulated peanuts and chocolate, indicated that WP coatings adhere to these surfaces as well as the presently-used commercial shellac, corn zein and hydroxypropyl methylcellulose (HPMC) coatings.
- Assessment of properties of whey protein-based films formed as coatings on nuts, cereals, dried fruits and vegetables, and fresh fruits and vegetables.
WPI coatings were found to have color stability and gloss comparable to, or better than, popular commercial edible coatings (shellac, corn zein, HPMC). This indicates good potential for WP replacing these commercial materials for coating of many foods.
Whey protein (WP) films formed as coatings on foods are attracting considerable attention from the food industry. We are developing data to show that WP coatings are superior to existing popular commercial coatings (shellac, corn zein and HPMC). For example, shellac and corn zein are currently used by the confectionery (candy) industry to coat chocolate, nuts and jelly beans to provide gloss, sticking resistance and protection from moisture, oxygen, oil and aroma migration. However, shellac and zein require use of alcohol as a solvent carrier.
The confectionery industry is under pressure from the EPA to eliminate shellac and corn zein coatings because of the volatile air-pollution emissions caused by evaporation of the alcohol in the coating process. The data we are developing for WP film coatings will provide a rationale for the confectionery segment and other segments of the food industry to choose WP for food coating.
Adoption of WP 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 (WP) 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) of foods. Replacement of shellac and corn zein use by the confectionery industry is a clear target for WP coatings.
We will begin coating foods in a pilot coating facility for generating WP-coated food samples for our evaluation (instrumental and sensory) and for WP-coated food samples requested by the food industry.
98-KRJ-01
Whey Protein Coating for Improving Packaging Material Performance
Principal Investigator: 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:
- Development of whey protein coating on paper and paperboard to improve paper material moisture-barrier, oil-barrier, printability, strength, color and/or gloss.
- Development of whey-protein-coated paper as an oxygen barrier to retard lipid oxidation of packaged food products.
- Development of whey protein coating on plastic film to replace plastic oxygen barrier layers in laminated composite films.
Thus far, the moisture-barrier, printability, oil-resistance, optical and mechancial properties of whey-protein-coated paper have been assessed. The measured water vapor permeability (WVP) of whey-protein-coated paper was half that of uncoated paper. The whey protein coating also decreased the initial contact angle of water, increased the rate of the contact angle change with time, and raised the maximum amount of water absorbed by the paper, suggesting improved printability of water-based ink on WPI-coated paper.
The whey protein coating increased the initial contact angle of corn oil and decreased the rate of contact angle change with time, indicating that whey protein coating increases the oil-resistance of paper. At the same time, the color, gloss and strength of the paper were not significantly affected.
Whey protein coating doubled the water vapor barrier property of paper, and was also smoother and more homogeneous, indicating that it will be easier to print with water-based inks. Whey protein coating also significantly increased the oil-resistance of paper. This was accomplished with no change in the optical or mechanical properties of the paper. This suggest possible use of whey-protein-coated paper in packaging of oily foods.
These initial results indicate the value of developing whey protein coating formulations for paper and then determining the properties of the coated paper. Work is proceeding to quantify the oil-barrier properties of whey-protein-coated paper, using standard ASTM methods to allow comparison with commercial grease-resistant papers.
This additional data on whey-protein coated paper will help attract attention from the paper industry. The result could be use of large amounts of whey protein for coating of paper.
Whey-protein-coated paper has improved water vapor resistance, oil resistance and printability compared to uncoated paper. At the same time, whey-protein-coated paper has the same color, gloss and strength as uncoated paper. Additional properties of whey-protein-coated paper must be determined and then compared to properties of uncoated and commercially coated papers.
98-KRJ-02
CDFRC Graduate Research Fellowship Program in Dairy Food Science: Oil-Barrier Properties of Whey Protein Films
Principal Investigator: John M. Krochta, UC Davis
EXECUTIVE SUMMARY:
The broad goal of this project is the value-added utilization of whey protein. More specifically, this Graduate Research Fellowship project focused on the demonstration that whey protein films are excellent barriers to oil. This has important implications for food systems, since the quality and shelf-life of many foods are limited by oil-migration from oil-rich food components to surrounding components, in which the oil changes the properties of the components and reduces food quality. This first step toward demonstrating the oil-barrier properties of whey protein films was developing an appropriate testing method that allowed quantification of the whey protein film oil permeability.
The successful development of a reliable testing method then allowed testing of whey protein films. Results showed that whey protein films are almost perfect barriers against oil migration at all relevant relative humidity (RH) conditions.
At very high (~90 percent) RH, some permeability occurs. This is due either to swelling of the whey protein matrix with subsequent greater ease of oil permeation, or to some micro-pore or crack formation because of the moisture-weakened films. Results also showed that when lipid material is incorporated in the whey protein film to improve the moisture-barrier properties, the oil-barrier property of the film is preserved. This included lipid contents over 50 percent of the total film weight.
The whey protein-lipid films performed best when the lipid was well-distributed in the film, as in a stable emulsion. New concepts are desirable for value-added utilization of whey protein.
Previously, edible films and coatings made from whey protein were demonstrated to be excellent oxygen and aroma barriers. And when lipid materials are added to the films, the result is a good moisture barrier. This project has demonstrated that, in addition to the earlier-demonstrated attributes, whey protein films are excellent oil barriers.
The quantitative data obtained on whey protein oil-barrier properties in this project will be useful to the food industry in assessing possible applications of whey protein coatings in improving existing food product concepts and in new food product developments.
Whey protein films are excellent barriers to oil. High relative humidity conditions and the addition of lipid to improve the moisture-barrier properties reduced the oil-barrier properties only slightly. Thus, whey protein films clearly have potential to provide an oil barrier for foods.
Two possible applications are being explored: 1) whey protein coatings as oil-barriers on peanuts to prevent migration of peanut oil into chocolate, which reduces chocolate quality; and 2) whey protein coating to reduce oil uptake in frying of foods.
98-MUJ-01
The Application of Transgenic Antisense Technology to Alter the Protein Composition of Milk: Specifically the Down-Regulation of the Level of b-lactoglobulin
Principal Investigator: James D. Murray, UC Davis
EXECUTIVE SUMMARY:
The principle objective of this work is to identify the best antisense transgene construct for down-regulating the level of b-lactoglobulin in milk to less than 25 percent of normal levels. From previous work using a reporter gene construct, we believe that antisense gene constructs based on genomic DNA will be the most effective type of construct for down-regulating the level of the target protein in an animal.
The use of transgenes producing antisense RNA (asRNA), which is RNA complementary to a mRNA, has been relatively successful in significantly decreasing, but not usually completely eliminating, the product of a target gene in mice. Therefore, this technique may be useful for altering the amount of specific proteins or lipids produced in the mammary gland and secreted into the milk by reducing the amount of functional mRNA for these proteins or the amount of mRNA available for producing key enzymes in the lipid biosynthetic pathway.
In this work we will test in mice a number of antisense transgene constructs targeting bovine b-lactoglobulin expressed in the mammary gland of the mouse. We have chosen as a target b-lactoglobulin because published data are available to suggest that a decrease in the level of expression of b-lactoglobulin is accompanied by an increase in the level of casein protein in milk. Different breeds of dairy cattle may demonstrate significant differences in milk composition, suggesting that it should be possible to alter the level of specific proteins or lipids produced in the mammary gland and secreted into milk without disrupting the function of the mammary gland. Milk with altered protein composition or changed fat to protein ratios may have considerable value to the dairy industry.
The current project has been designed to: 1) target bovine b-lactoglobulin, 2) directly compare the relative effectiveness of full-length antisense constructs based on the cDNA (no introns) to antisense constructs based on genomic DNA, and 3) compare antisense constructs having either the first or last intron and exon in the sense orientation. A secondary goal of this project is to identify the antisense transgene construct with the highest probability of significantly down-regulating the amount of b-lactoglobulin in the milk of dairy cattle for future use in the production of transgenic cattle; a project outside the scope of the current work.
We established nine lines of mice transgenic with a construct containing all of the known regulatory elements and coding regions for the bovine b-lactoglobulin gene and confirmed expression of bovine b-lactoglobulin in the milk (Gutierrez-Adan et al., 1999). Two lines of mice have been maintained for use in the current project. We have also constructed the four other constructs necessary for this project. All of these constructs are based on using the same promoter and 3' control regions of the bovine b-lactoglobulin gene used to express b-lactoglobulin in the milk of transgenic mice.
To date we have produced lines of transgenic mice with three of these constructs. In order to detect the presence of both a b-lactoglobulin and an antisense transgene in the same mouse a number of PCR primers were redesigned. In addition, probes specific for the mRNA and the antisense RNA messages have been produced for use in analyzing the amount of RNA present in mammary gland cells.
We are breeding female mice that contain both the b-lactoglobulin transgene and one of the experimental or control antisense transgenes. We then collect milk and mammary gland tissue from these females at day 10 to 12 of lactation for later analysis.
Based on our current information, we estimate that between 10 and 15 double hemizygous transgenic females will need to be tested for each antisense construct evaluated. Analysis of experimental samples had just begun, thus no conclusions could be drawn at the time of publication.
95-VAL-01
Applicability of Atomic Force Microscopy for the Assessment of Ultrastructural Features & Quality of Dairy Foods and Food Processes
Principal Investigator: Linda Vanasupa, UC Davis
EXECUTIVE SUMMARY:
This project had three primary objectives. The first was to compare the ultrastructural features of dairy biofilms on ultrafiltration membranes obtained using scanning electron microscopy and atomic force microscopy. Toward this objective we were able to identify structural features on pre-fouled ultrafiltration membranes. Sample mounting methods included cyanoacrylate, double-sided tape and paraffin. Cyanoacrylate bonding resulted in images that appear to show 2.8x109 pores/m2, approximately 3 nanometers in diameter, creating a porosity of 2 percent.
The second objective was to document differences in ultrastructural features of Cheddar cheese made with homogenized milk and non-homogenized milk using AFM. Our work with this system revealed that the structural differences were outside the window of detection—either macroscopic (greater than 150 micrometers) or too small to be detected. The nature of the cheese system required isolating the imaging system from environmental vibrations.
The third objective was to characterize ultrastructural features of dairy-based edible films using AFM. The system of study was whey-based edible protein films. High-resolution images obtained through atomic force microscopy revealed features on whey-based edible protein films that correlate to the size of beta-lactoglobulin (~7 nanometers). Additionally, protein aggregates with approximate diameters of 13.4 to 15.7 microns were found to extend above the surface of the edible films. Analysis revealed that the surface, with the exception of protein aggregates, displays an extreme flatness. However, extensive porosity is also evident, most likely due to extensive outgassing during casting and drying.
The importance of dairy food ultrastructure cannot be underestimated since these features determine the functional and sensory properties. A current limitation of present imaging techniques, such as scanning electron microscopy, is that they require the sample structure to be chemically altered before imaging. We were able to obtain ultrastructural information on the whey protein and polysulfone membrane without chemical alterations. A technique that provides insight to the ultrastructure in its natural condition has the potential of providing the necessary information to dairy processors to modify and/or improve dairy foods, dairy food ingredients and dairy processing equipment. Specifically, additional ultrastructure information on dairy-based films will accelerate the successful improvement of their barrier properties and commercialization of the technology.
Better documentation of biofilm formation on ultrafiltration membranes will provide needed understanding of this phenomena to improve our ability to maintain high sanitary standards as well as efficiency of dairy processing equipment.
Atomic force microscopy can yield important information on the ultratructural features of dairy systems, such as whey protein edible films and ultrafiltration membranes, in their natural state. Sample preparation is minimal compared to imaging techniques that require freezing, dehydration or chemical alterations, and this technique can provide supplementary information to vacuum imaging techniques such as scanning electron microscopy. However, the minimal sample preparation may in some cases, as we found with the Cheddar cheese system, lead to ambiguous results.
The choice of imaging mode ("contact" or "non-contact") proved to be critical in obtaining information on sub-micron-sized features. Identification of biofilms on ultrafiltration membranes relies heavily on the interpretation of the images and accurate information on the ultrastructural features of the membrane prior to fouling.
The fact that AFM does not provide spectroscopic information complicates the interpretation of the images. However, recent advances in imaging techniques, such as the ability to differentiate between materials of differing elasticity, may advance the application of this technique to dairy systems.
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