Whey Research Sponsored by CDRF
Whey 1996-97
95 VAL-01
Applicability of Atomic Force Microscopy for the Assessment of Ultrastructural Features and Quality of Dairy Foods and Food Processes
Principal Investigator:
Dr. Linda Vanasupa, Cal Poly SLO
EXECUTIVE SUMMARY
Edible whey-based films have the potential for becoming an application for the billions of kilograms of whey per year that is currently wasted. Some of the issues that must be explored, however, are their moisture barrier properties as well as the link between their properties and the processing methods employed to create the films. The atomic force microscopy work is among the first techniques used to characterize these films in their natural state. This work was undertaken as a preliminary study to demonstrate the usefulness of atomic force microscopy in revealing the ultrastructure of these edible films. The technique has proven useful in revealing fine surface structure (nanoscale) as well as surface porosity.
Objective 1. Compare the ultrastructural features of dairy biofilms on ultrafiltration membranes obtained using scanning electron microscopy and atomic force microscopy. Atomic force microscopy (AFM) was utilized to characterize pore size of membranes before and after fouling during skim milk ultrafiltration. The images of clean polysulphone ultrafiltration membranes with a molecular weight cutoff of 10,000 daltons reveal average pore sizes of 12.9 angstroms and a total surface porosity of 18.2 percent. Images of fouled and rinsed membranes reveal average pore sizes of 11.0 angstroms and total surface porosity of 5.3 percent. Fouled membranes exhibited a 7.5 angstrom average pore size with a total surface porosity of 2.1 percent.
Objective 2. Document differences in ultrastructural features of cheddar cheese made with homogenized milk and non-homogenized milk using AFM. To be addressed.
Objective 3. Characterize ultrastructural features of dairy-based edible films using AFM. 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 (topographical variations of less than 0.5 percent over regions of 150 microns by 150 microns). However, extensive porosity is also evident, most likely due to extensive outgassing during casting and drying. X-ray diffraction studies indicated that the films have little polymeric crystallinity.
95 HSY-01
Conversion of Lactose to High-Volume and/or High-Value Polymer Products
Principal Investigator:
Dr. You-Lo Hsieh, UC Davis
EXECUTIVE SUMMARY
The goal of this research is to produce lactose-containing polymers by either incorporating a large quantity of lactose in the polymer structure or producing high value-added polymers. Polymers with varying chemical structure and morphology have been synthesized from lactitol-based and lactamine-based monomers. The monomers include lactitol polyether polyol (LPEP), acrylactamine (ALAM), methacrylactamine (MALAM), D-lactose-0-vinylbenzyl-hydroxime (LVH). Free radical polymerization of ALAM, MALAM, and LVH monomers has produced a wide variety of linear polymers with side chains containing lactose derivatives. These homopolymers are water-soluble. Copolymerization of these monomers with other vinyl monomers has shown to improve the solubility of the lactose-based polymers in polar solvents and the hydrophilicity of the vinyl polymers. These hompolymers and copolymers have promise in applications such as stabilizers, emulsifiers/dispersants, adhesives and coatings. Some copolymers also show enhanced thermal stability (lowered decomposition temperature) and processability (lower glass transition temperature). Lactose derivatives can also be used as additives and crosslinking agents. Hydrogels have been synthesized from LPEP, ALAM and LVH. These hydrogels exhibit a wide range of water absorbent characteristics and excellent thermal sensitivities. The controlled-delivery characteristics of these hydrogels have been studied using acetylsalicylic acid (aspirin) and proteins (lipase) as model compounds.
95 KRJ-01
Application of Edible Films From Milk Protein and Milkfat to Food Systems
Principal Investigator:
Dr. John M. Krochta, UC Davis
EXECUTIVE SUMMARY
The objectives of this project are to optimize milk protein/milkfat edible film formulations and drying conditions, and to assess the properties of optimized films formed as coatings on food products such as nuts, dried foods and fresh fruits.
Objective 1. Optimization of milk protein/milkfat film formulations. Film additives called plasticizers affect film mechanical properties such as strength and flexibility. Results with whey protein isolate (WPI) films indicate that their mechanical properties can be modified, depending on the type of plasticizer added to the film formulation. Film additives called surfactants improve the ability of film formulations to spread on food surfaces to form well-adhering coatings. Greater understanding of the fundamental relationship of plasticizer and surfactant composition and structure to their effect on film properties will allow optimization of film properties and usefulness. Preliminary investigation of the color and gloss stability of WPI coatings compared to currently used commercial edible coatings (shellac, zein, HPMC, tapioca dextrin) indicated that WPI coatings have color, transparency and gloss equal to these competitive materials at dry to intermediate relative humidity (RH) and superior properties at high RH.
Objective 2. Optimization of milk protein/milkfat coating drying conditions. Progress was made on creation of a food-coating pilot plant. A pan coater, which resembles a small horizontally-rotating cement mixer, with spraying or ladeling of coating formulations onto the bed of food pieces along with blow drying, was installed and used for studies of nut coating with WPI formulation. Successful preliminary WPI-coating of peanuts led to sample preparation for the Orlando Institute of Food Technologists meeting in June 1997. A fluidized bed coater (a vertical column with air blown upwards to suspend and tumble food pieces while they are being coated and dried) was purchased and installed. This system was used to prepare WPI-coated freeze-dried chicken dice for the Orlando IFT meeting. Finally, a continuous food dip-coating line was purchased. Preliminary work has been to add a coating drying tunnel to follow the dipping step.
Objective 3. Assessment of the barrier properties of optimized films as coatings. Methods were developed for measurement of oxygen, carbon dioxide and ethylene in coated fruits to allow assessment of performance of WPI coatings on apples. WPI clearly has a positive effect in reducing oxygen and ethylene levels and increasing the CO2 level on coated fruit. This indicates good potential for extending the shelf-life of fresh fruits and vegetables. Control of RH is important to optimize this effect.
94 TOP-06
CDFRC Graduate Research Fellowship in Dairy Food Science (Development of Extrudable Milk-Protein-Based Edible Films)
Principal Investigator:
Dr. John M. Krochta, UC Davis
EXECUTIVE SUMMARY
The goal of this project is to form whey protein edible films by extrusion and then to determine the extruded film mechanical and barrier properties. 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 (Tg) that indicate the temperature-range over which whey proteins begin to flow, to determine how whey protein can be formulated so that it can be extruded under heat and pressure into films. Using the Tg, a preliminary idea of extrudability and film samples for testing can then be obtained using thermal compression molding.
Objective 1. Determination of the existence of glass transition temperatures (Tg). A differential scanning calorimeter (DSC) device was set up and programs established for heating, holding and cooling cycles for whey protein isolate (WPI). Tg values for the WPI components have been observed. Additional work is necessary to confirm these values, and the WPI will next be tested with a variety of plasticizers to determine their effects on the Tg. Plasticizers, which are low-molecular weight additives, normally lower the Tg and could make the WPI more easily extruded.
Objective 2. Production of edible films using thermal compression molding. A thermal compression molding system has been purchased for placement in an Inston pressure device. After additional data on Tg 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.
Objective 3. Characterization of the resulting films as to transparency, strength, flexibility, stretchability, and permeability to oxygen, moisture and lipids. 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).
96 SMG-01
Tailoring the Structure of b-Lactoglobulin For Improved Functionality
Principal Investigator:
Dr. Gary M. Smith, UC Davis
EXECUTIVE SUMMARY
Substantial progress has been made in developing a system for secreting bovine BLG from the yeast Yarrowia lipolytica. The bovine BLG gene was obtained from C. Batt in a form that could be expressed intracellularly in E. coli. Three gene fusions were constructed in which the BLG gene was fused to the promoter and various parts of the prepro-region of the Y. lipolytica XPR2 gene, which codes for an alkaline protease that is secreted at high levels. The fusion constructs were introduced as single copies into Y. lipolytica by two step gene replacement. All three constructs resulted in the appearance in the extracellular medium of a polypeptide of similar mobility to bovine BLG on SDS/PAGE that reacts with commercial rabbit antibodies against BLG. The N-terminal amino acid sequence of the polypeptide secreted by the SP construct corresponded exactly to that of mature BLG. The DP protein band was not as sharp as the SP band on SDS/PAGE, and it was confirmed by N-terminal sequencing that at least two forms of BLG were present—one with one X-Ala/X-Pro dipeptide and one with two of these dipeptides still attached to the N-terminus of mature BLG—indicating that dipeptidyl aminopeptidase processing was incomplete. For KR, three larger bands reacted with the rabbit antibodies against BLG suggesting that Xpr6p processing was also incomplete. This may be explained by computer predictions that this processing site is on the surface for wild type Xpr2p and partially buried for the fusion protein. In conclusion, we now possess a Y. lipolytica strain that from only a single copy of the XPR2/BLG SP fusion secretes 10-20 mg/L of authentic BLG at a relatively low cell density (8-10 g/L dry weight).
Preliminary NMR studies were hindered by a colored compound that remained bound to BLG even after several different purification steps and dominated and obscured parts of the NMR spectra. It was determined that the source of this colored compound was Proteose peptone, the nitrogen source used to induce the XPR2 promoter. The promoter has been changed to one from the translation elongation factor gene TEF1. In S. cerevisiae, the TEF1 gene is transcribed at high levels in several different growth media. The TEF1 gene was subcloned and 4110 nucleotides sequenced.
The subcloned TEF1 gene was found to have a frameshift mutation which resulted in a stop codon 938 base pair (bp) before the authentic translational stop codon. This should result in synthesis of a polypeptide only about one third the length of Tef1p. This mutation was also found in the original lambda clone, so a short DNA fragment of this region was obtained from DNA using PCR and was sequenced to determine the correct sequence. This genomic DNA fragment did not have the frameshift mutation, suggesting that there was selection in E. coli against strains containing an intact copy of the Y.lipolytica TEF1 gene.
Assembly of the TEF1 promoter/XPR2 signal peptide/ BLG mature coding region/XPR2 terminator construct has begun. We have obtained the plasmids needed for integrating multiple copies of a gene into the Y. lipolytica genome from C. Gaillardin (INRA, Thiverval-Grignon, France). We have all the DNA fragments and strains needed to construct a Y. lipolytica strain with multiple copies of the BLG fusion.
Our NMR studies to date have used milk-derived protein. We have performed many 2D NMR experiments under different conditions of pH and temperature. For denaturation experiments, we monitored the resonances of the amide N-H protons of several B strands. We were able to perform deuterium exchange experiments to evaluate the stability of different regions of the protein to heat-induced and foaming-induced unfolding. We were able to determine that denaturation at 75o C proceeds in a sequential manner and we could monitor the order of the first regions of the b-barrel to open. We identified both the most thermally labile regions of the protein and the thermally more stable regions. This information will be incorporated into our plan for genetic alteration to decrease the stability of the protein. It was also evident that the protein became opened during foaming. When the foam collapsed, there was no evidence of aggregation. Although the protein refolded, it did not recover its original conformation precisely, as evidenced by changes in chemical shifts of NH resonances.
Conclusions: The new promoter should provide labeled protein in good yield. New NMR Instrumentation will be used in the structure determination. There are several potential uses. Cattle containing the gene for a modified b-lactoglobulin would become part of a dairy herd. The milk would be processed by current methodology. In fluid milk, the unstable protein would render the protein less immunogenic. In cheese whey, the proteins (WPI or bulk whey) would exhibit better functionality and would potentially be used in more products, probably at a higher-than-current price.
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