Recipient Organization
GUILD ASSOCIATES, INC.
5750 SHIER-RINGS ROAD
DUBLIN,OH 43016
Performing Department
(N/A)
Non Technical Summary
Challenges in the production of hydrolysate and other milk-derived products center on the efficient generation of a high-yield product. Enzymes enable reaction processes to take place under less harsh conditions than chemical-only methods, increase product specificity, and reduce the difficulty of reaction processing and waste cleanup. The use of enzymes is not without its own challenges. The primary use limitation is the cost of the enzyme, as enzymes are refined and purified protein products. Lower-cost enzyme options are available, but typically sacrifice purity and have broader batch-to-batch variability as a tradeoff to cost. The other significant limitation is that enzymes in their purified form are limited to a single batch reaction. Recovery of enzyme from the reaction mixture for potential reuse is difficult since the substrates, products, and the enzymes all possess similar physical properties. Other cost-drivers are tied to the need to inactivate the enzyme to stop a reaction, and removal of the enzyme from the product if it may cause an allergic or toxic response.To enable the reuse of soluble enzymes, studies have investigated ultrafiltration membranes scaled to retain the larger-size enzymes and simultaneously act as the first purification step and binding enzymes to amembrane to combine the reaction and separation processes. These membrane-based solutions demonstrated efficiency gains, but suffered from increased enzyme deactivation through shear and thermal stress, enzyme leakage into the product, and membrane fouling. As an alternative, enzymes were immobilized on solid supports such as glass, agarose, and silica. Immobilizing enzymes to these supports permitted improved regulation of the reactions, increased enzyme stability, reduced pressure drop issues in the reactors, improved separation of product from enzyme, and reduced contamination. However, the value gained by these improvements was offset by the high cost of the support material.The technology developed on this SBIR similarly seeks to reduce enzyme-related operational costs through immobilization of enzymes. This effort will focus on bead-like pellets made from low-cost and recycled bio-waste materials as the enzyme support. Using these robust and low-cost support materials can enable the use of higher-quality enzyme preparations and leads to more uniform products and simplified post-reaction processes. Similarly, the pellets retain the enzyme so stopping the reaction or separating them from the reactor involves simply filtering them from the reaction solution. Existing plants can be adapted through using abolt-on collection system. The Phase I goal is tovalidate the capability of our patented ImmobiZyme™ technology as a solution for the two primary enzyme-based reactions used by whey processors: protein hydrolysis and lactose removal. Data from this effort will demonstrate that this technology is easily applied to whey processing and will beneficially support the needed enzyme reactions.
Animal Health Component
75%
Research Effort Categories
Basic
0%
Applied
75%
Developmental
25%
Goals / Objectives
The focus of the project is to demonstrate the capabilities of ImmobiZyme™ materials for the processing of whey and other dairy products in comparison to their soluble enzyme counterparts. This will be accomplished by applying GBS's immobilization technology to commercial protease blends and to beta-galactosidase converting these one-time use catalysts to reusable ImmobiZyme™materials that can easily fit into existing production systems and provide an advantageous and cost-effective replacement for currently used enzymes.Success of the conversionwill be determined by meeting the following goals and objectives.Goal 1:Demonstratethe ImmobiZyme™ forms of two commercial protease blendsand of beta-galactosidase perform similarly to their soluble counterparts.Objective 1.1:ImmobiZyme™ materials will achieve a benchmark enzymatic activity of at least 50% of the originally supplied enzyme's activity on an activity unit per gram (U/g) enzyme basis.Objective 1.2:The enzymatic activity profile for each ImmobiZyme™ material at reaction temperatures of 30-60ºC will be similar to the corresponding soluble enzyme.Objective 1.3:ImmobiZyme™ samples stored for up to 24 hours in buffer will produce no detectible enzyme leachingGoal 2:Evaluatethe susceptibly of ImmobiZyme™ materials to microbial contamination and the compatibility of ImmobiZyme™ materials with common clean in place (CIP) processes.Objective 2.1: Susceptibility of ImmobiZyme™ materials to microbial growth under dry and wet storage conditions will be quantified by measurements of ATP activity after 4 weeks.Objective 2.2: Compatibility with individual steps in standard CIP procedures will be quantified by measurements of enzymatic activity retained following exposure to a CIP step.Goal 3:Demonstratethe performance of ImmobiZyme™ materials for extended reaction timesObjective 3.1:ImmobiZyme™ materials will convert substrates to products for up to 360 hours over a series of 15 batch reactions.Objective 3.2:Over 95% of ImmobiZyme™ materials will be recovered between each batch reaction for up to 15 reactions.Objective 3.3:ImmobiZyme™ materials will retain >90% enzyme activity following each batch reaction for up to 15 reactions.
Project Methods
Successful ImmobiZyme™ materials will be defined as those that meet all of the milestones through the testing described below. Together these outcomes will validate the proposed approach for the application of reusable enzyme materials for the production of whey hydrolysate products.The initial effort will develop and evaluate candidate modified enzyme formulations. Formulation variations will be chosen toallow researchers to clearly define how each change leads to material performance variations in terms of both physical andreactive properties. Data on the manufacturing process will be collected to identify any potential areas of process improvement.The assays that will be used to characterize and evaluate each enzyme candidate are based on commonly used protocols andthose from commercial enzyme providers. Protease activity will be measured by its ability to hydrolyzeproteins in both whey protein concentrate (WPC) and whey protein isolate (WPI) substrates. The degree of hydrolysis will be determined by the standard pH-stat method. Beta-galactosidase will be tested for the ability to hydrolyze lactose by monitoring the formation of glucose formed in whey-stocks over time using commercial glucose kits.The measure of success for these candidate materials will be enzyme formulations that demonstratethe capability to react with substrate at a rate greater than 50% of the unmodified enzyme on a per mass enzyme basis.Milestone 1:Candidate ImmobiZyme™ formulations for each enzyme that can react with substrates at greater than 50% the rate of soluble enzyme on a per gram enzyme basis will be identified.Successful candidate materials will be tested for various performance related properties including:temperature response, leaching of the enzyme from the immobilized pellet, antimicrobial performance, and compatibility with clean in place (CIP) methods.Temperature responses will be determined by pre-heating test solutions to temperatures between 30 °C and 60 °Cprior to the addition of enzyme. The reaction temperatures will be held through the assay and returned to room temperature using a water bath prior to the removal of the enzyme. The profile of conversion of substrate to product with respect to temperature will be evaluated for optimum performance conditions.Milestone 2:The enzymatic activity profile for each ImmobiZyme™ material at reaction temperatures of 30-60ºC will be determined and compared to the corresponding soluble enzyme.Leaching of the enzyme from the pellet will be investigated by mixing known amounts of immobilized materials in buffer solutions for up to24 hours. Samples will be regularly taken from the buffer solutions and tested for the presence of protein using the standard bicinchoninic acid assay.Milestone 3:ImmobiZyme™ samples stored for up to 24 hours in buffer will produce no detectible enzyme leachingAntimicrobial properties will be evaluated through dividing candidates into3 test conditions: dry, wet, and wet with a starting bacterial load. For each condition 0.5 g of ImmobiZyme™ pellets will be placed in 50 mL conical tubes. Dry test samples will be capped; wet samples will be mixed with 15 mL of phosphate buffered saline (PBS), agitated by hand, and capped; and controlsamples with a starting bacterial load will be mixed with 15 mL PBS containing 106CFU/mLEnterococcus faecium(E. faecium, ATCC 8459)a non-pathogenic surrogate, and capped. A specific times over a test period of 4weeks, samples will be brought to 50 mL using PBS and placed in an ultrasonic bath for 5 minutes to dislodge any bacteria from biofilms. The processed samples will be passed through a sterile 150-mesh filter to separate bacteria from the solids. The filtrate will be tested for ATP activity and if positive, bacterial numbers will be determined by dilution plate counts from the retained material.Milestone 4:ImmobiZyme™ pellets susceptibility to microbial growth under dry and wet storage conditions will be defined.Compatibility of theImmobiZyme™ candidates will be measured before and after exposure to individual CIP chemicals at both ambient and elevated temperatures for time periods representative of industry-standard processing steps (1 to 15 min at temperatures of25 °C to 60°C).Following exposure candidates will be rinsed with DI water for 5 minutes and their activities will be determined. The post-exposure activity of the immobilized enzymes will be investigated for any relationships between exposure conditions and resulting activity and compared using the recorded activity of unexposed materials as controls. If the ImmobiZyme™ candidates retain more than >80% of their pre-exposure activity to a particular substance, the same exposure process will be repeated up to 5 times to determine the effects of repeat exposure. ImmobiZyme™ materials that demonstrate the highest retained activities to individual CIPsteps will be processed through the full CIP process and evaluated for retained activity.Milestone 5:ImmobiZyme™ compatibility with standard CIP procedures will be quantified and ranked in order of retained enzymatic activity.The final effort in this project will be to demonstrate the reactive capacity of the ImmobiZyme™ candidates.Batch reactions will take place in 500 mL stirred reactors containing up to 20% (w/v) dry WPC, WPI, or lactose powder in buffer solution at pH 8 or pH 5 for protease and lactose tests respectfully. The enzyme and substrate loadings will be selected such that a reaction will reach completion at 24 hours. Samples of the product will be taken at regular intervals from the reactor to measure enzymatic conversion of the substrate over time and to calculate the relative activity of the immobilized enzyme with respect to time in the reactor. At the end of each batch reaction test, the immobilized enzyme will be collected, washed in DI water, weighed, and prepared for the next test. A sample of the immobilized enzyme will be retained and dried. This material will be used to calculate potential loss or degradation of the pellets over time. The dried enzyme material will also be evaluated for activity.The batch reaction process will be continued up to 15 times for up to 360 hours of reaction.Milestone 6:The performance of each ImmobiZyme (MT) material will be characterized over 360 hours of testing with respect to degree of reaction completion, loss of ImmobiZyme™ material, and loss of enzymatic activityThe recovery of immobilized enzymes will be evaluated by comparing the initial mass of dry enzyme supplied for each test to the dry mass of the immobilized enzyme pellets recovered following each reaction. Recovered pellets will be washed 3 times in DI water and spread out and air dried overnight at room temperature. Recovered pellet mass will be measured two times at least 3 hours apart to verify the material has dried to completion. The enzymatic activity of the recovered and dried immobilized enzyme pellets will also be evaluated.Peptide profiles following protease reactions will be performed by HPLC with a 300 Å column at 30 °C, detecting absorbance at 214 nm. If necessary a secondary measurement of the degree of hydrolysis will be tested using o-phthaldialdehyde. Additional compositional analysis of the hydrolysates (total solids, total fat, and minerals/ash content) will be performed through extraction, thermogravimetric analysis, and other commercial kit-based tests.Milestone 7:Over 95% of ImmobiZyme™ materials will be recovered between each batch reaction and ImmobiZyme™ materials will retain >90% enzyme activity following each batch reaction.