Source: IOWA STATE UNIVERSITY submitted to
ENZYME MODIFICATION TO ENHANCE SOY PROTEIN INGREDIENTS IN FOOD AND INDUSTRIAL PRODUCTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0195728
Grant No.
2003-34432-13326
Project No.
IOW06643
Proposal No.
2003-06135
Multistate No.
(N/A)
Program Code
QC
Project Start Date
Sep 1, 2003
Project End Date
Aug 31, 2005
Grant Year
2003
Project Director
Johnson, L. A.
Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
CENTER FOR CROPS UTILIZATION RESEARCH
Non Technical Summary
Soybean producers are faced with low prices for their crops. Developing new uses in food and biobased products will increase demand for soybeans. Technologies used today to process soybeans emit solvent vapors that pose environmental risks and alternative more environmentally friendly technologies are needed. These technologies are expected to be suitable for small processors and to lead to rural economic development opportunities. The purpose of this project is to develop technologies based on new industrial enzymes for new value-added soybean products to be used in food and biobased consumer products. These enzymes catalyze reactions in water-based systems and should be more environmentally friendly than current traditional methods.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011820200050%
5111820200050%
Goals / Objectives
The long-term goal of this project is to develop technologies that will add value to soybeans in food and industrial products by using industrial enzymes. Our specific objectives are to: 1. develop a long-term strategic plan for capitalizing upon the extensive industrial enzyme library of Genencor, to add value to soybean proteins and to capitalize on current market trends; 2. improve the functionality of soy protein in soy-protein-based adhesives by reducing viscosity and enhancing adhesion, bond strength, and bond water resistance; restore water solubility and other functional properties of heat-treated, acid-treated, and alcohol-treated soy protein products; and enhance protein extraction and recovery from soybeans and soybean white flakes in order to improve yields and processing characteristics of soy protein into value-added products.
Project Methods
Objective 1: Annually, update and hone the long-term commercialization strategic plan developed in previous projects to capitalize on the extensive industrial enzyme library of Genencor, to add value to soybean protein products. Objective 2: Optimize enzymatic hydrolysis for adhesive applications using Genencor Protease 899 and determine the potential of using other enzymes in the Genencor library to produce a hydrolysate with improved properties for manufacturing adhesives. Objective 3: Screen as additional new enzyme preparations under development by Genencor that are designed to restore functional properties of soy protein products, develop an understanding enzyme action on emulsification properties and flavor, and test enzyme-modified soy protein in key food applications. Objective 4: Fully characterize one fungal pectinase- and one cellulase-assisted protein extraction, and characterize soybean fine structure by microscopic observation of the changes induced by cellulases, pectinase and proteases during soy protein extraction.

Progress 09/01/03 to 08/31/05

Outputs
Commercialization activities focused on enzyme-hydrolyzed soybean meal in adhesives. Soy protein hydrolysates were produced at lower pH, temperature and pressure by using enzymes. Carbohydrase treatment may be needed as well as protease to achieve ideal viscosity because both protein and fiber are hydrolyzed by alkali. The pH of the hydrolysate produced by alkali is high (pH 11-12), preventing microbial growth. Enzymes produce lower pH (7-8) permitting microbial growth. Shelf life was extended by adding a small amount of formaldehyde. Functionalities of soybean meals modified by Multifect Neutral and Exopeptidase C at 2-4% degrees of hydrolysis were compared with bromelain treatment, a commercially used endoprotease. All three enzymes produced similar modifications to protein structures and functionalities. We scaled up 4% DH soy protein isolates modified by Multifect Neutral, Exopeptidase C and bromelain in the pilot plant using extruded-expelled soy flour. Fishy aromas of the control and Multifect Neutral samples were greater than bromelain and Exopeptidase C hydrolysates. Jet-cooked treatments had more fishy and beany aromas than did the uncooked treatments. There were no flavor differences between jet-cooked and uncooked treatments. Control and bromelain treatments were sweeter than Exopeptidase C and Multifect Neutral treatments. Soy flour treated with Multifect Neutral had less toasted and nutty flavors than did other treatments and more grassy/beany flavored than the control. Multifect Neutral and Exopeptidase C hydrolysates were more bitter and astringent than the control and bromlain treatments. Soy protein isolates were made from defatted soy flakes with 5% Multifect Pectinase, Cellulase A or Indiage Super L to increase yield and protein extraction increased 17, 4 and 4% for three enzymes, respectively. Emulsification capacity and activity decreased with Multifect Pectinase, whereas foaming capacity and stability increased. Glycoprotein staining after SDS-PAGE revealed hydrolysis of beta-conglycinin with Multifect Pectinase due to protease side-activity. Scanning electron microscopy showed differences in the disturbed linear alignment of the cells for cellulase-treated samples with the junctions between cells weaken and more cracks were observed in the cell walls. Cell walls of pectinase-treated samples had more holes. Data collected from the enzyme-screening program were converted into commercial applications literature and enabled Genencor to better market their enzymes. We completed a review on aqueous processing and developed 8 alternative strategies for aqueous extraction, which formed the basis for a subsequent proposal. We benchmarked aqueous extraction by reproducing the most effective approaches in the literature. Our best oil extraction was 57% when no enzymes were used compared to 62% reported in the literature and 78-85% when using enzymes. We evaluated different mechanical treatments to improve oil extraction: once pin-milled dehulled soybeans, twice pin-milled dehulled soybean, and pin-milled flaked dehulled soybeans. The latter treatment increased oil extraction to 70% of which 25% remained in the skim fraction.

Impacts
This project will lead to improved performance properties in food and adhesive applications as well as improved extraction efficiency in processing soybeans into value-added protein ingredients. These advances will lead to new, lower cost soy protein products and allow food processors to use more soy protein, which has been shown to have health benefits. Enzymatic hydrolysis of soy protein enables an improved process for manufacturing soy protein-based adhesives that promise to reduce worker and consumer exposure to cancer-causing formaldehyde of conventional wood adhesive. Aqueous processing of soybeans will offer alternatives to hexane extraction. Hexane is a regulated pollutant and costs of compliance with emission standards are becoming increasingly difficult and expensive. Aqueous processing may also allow for new opportunities to add value to oil and meal products, and may provide a basis for establishing a biorefinery for soybeans. Understanding of oil stability/de-emulsification phenomenon will help not only aqueous soybean processing but all oil processing that are faced with the same problem.

Publications

  • Jung, S., P. Murphy, and L.A. Johnson. 2003. Pectinases and Proteases: Effects on Protein and Functional Properties of Soy Protein Ingredients. 2003 IFT Annual Meeting and Food Exposition, Chicago, IL. July 12-16. Program Book, Abstract 11-5. p. 13.
  • Jung, S., and L.A. Johnson. 2003. Functional Properties of Soy Protein Modified by an Endopeptidase at Low Degrees of Hydrolysis. 2003 Annual Meeting of American Association of Cereal Chemists, Portland, OR. Sept. 28-Oct. 2. Program Book, Abstract 25. p.70.
  • Jung, S., M. De Lamballerie-Anton, P. Murphy, and L.A. Johnson. 2004. Emulsifying Properties of Protease-modified Soy Protein Substrates. 95th AOCS Annual Meeting and Exposition, Cincinnati, OH. May 9-12. Abstracts, p 123.
  • Jung, S., P. Murphy, and L.A. Johnson. 2004. Effects of Limited Hydrolysis on Physico-chemical and Functional Properties of Commercial Soy Protein Substrates. 36th ACS Great Lakes Regional Meeting, Peoria, IL. Oct. 17-20. Program Book. Abstract 43. p. 73.
  • Jung, S., D. Rickert, N. Deak, E. Aldin, J. Recknor, L.A. Johnson, and P.A. Murphy. 2004. Comparison of Kjeldahl and Dumas Methods for Protein Content Determination in Soy Products for ISS and Planetary Outpost Missions, NASA Food Technology Commercial Space Center Symposium, Ames, IA. April 21. Abstract #FP02, p 62-63.
  • Jung, S., P. Murphy, and L.A. Johnson. 2004. Effects of Limited Hydrolysis on Physio-chemical and Functional Properties of Commercial Soy Protein Substrates. 36th ACS Great Lakes Regional Meeting, Peoria, IL. Oct. 17-20. Abstract #9878.
  • Jung, S., P.A. Murphy, and L.A. Johnson. 2005. Physicochemical and Functional Properties of Soy Protein Substrates Modified by Low Levels of Protease Hydrolysis. J. Food Sci. 70(2):C180-187.
  • Jung, S., B. Lamsal, V. Stepien, L. Johnson, and P. Murphy. 2005. Enzyme-assisted Extraction and Functionality of Soy Proteins from Defatted Flakes on Laboratory- and Pilot-scale. 96th AOCS Annual Meeting and Exposition, Salt Lake City, UT. May 1-4. Abstracts, p 97.
  • Lamsal, B., S. Jung, P.A. Murphy, and L.A. Johnson. 2005. Rheological Properties of Soy Protein Hydrolysates. 2005 IFT Annual Meeting and Food Exposition, New Orleans, LA. July 17-21.


Progress 01/01/04 to 12/31/04

Outputs
The commercialization plan was refocused on enzyme-hydrolyzed soy meal for use in adhesives and performance and cost-sensitive parameters are being verified. Soy protein hydrolysates were produced at lower pH, temperature and pressure using the enzymes compared to alkali. We are scaling-up the enzymatic process in the pilot plant to assess feasibility. Our work shows that a carbohydrase may be required in addition to protease to achieve ideal viscosity. Both protein and complex carbohydrates are hydrolyzed by alkali. The final pH of the hydrolysate produced by alkali is high (pH 11-12), preventing growth of microorganisms. The enzymatic method, however, produces lower pH (7-8) permitting microbial growth and thereby limits shelf life. We have shown that shelf life can be extended by adding a small amount of formaldehyde, which is used in the adhesive formula. The functionalities of soybean meals modified by Multifect Neutral and Exopeptidase C at low degrees of hydrolysis (2 and 4%) were compared with functionalities obtained by using bromelain, a commercially used endoprotease. All three enzymes produced similar modifications to protein structures and functionalities. We scaled up our better treatments (4% DH soy protein isolates modified by Multifect Neutral, Exopeptidase C and bromelain) in the pilot plant using extruded-expelled soy flour. Multifect Neutral soy hydrolysates were bitter and astringent compared to an unhydrolyzed control. The jet-cooked hydrolysates were the most astringent and bitter. Data collected from the enzyme-screening program are being converted into commercial applications literature. Soy protein isolates were also prepared by extracting 15 Kg defatted soy flakes with 5% enzyme (Multifect Pectinase, Cellulase A or Indiage Super L) basis dry protein weight. Protein extraction yield increased 17, 4 and 4% for Multifect Pectinase, Cellulase A and Indiage Super L, respectively. Emulsification capacity and activity decreased for Multifect Pectinase treated isolate whereas foaming capacity and stability both increased. Glycoprotein staining after SDS-PAGE showed that the modification of the beta-conglycinin and appearance of new polypeptides following Multifect Pectinase treatment of soy flakes were probably due to a protease side-activity of the enzyme preparation. We completed an exhaustive literature review on aqueous processing and have been invited to submit it for publication, which we plan to do. As a result, we developed eight alternative strategies for aqueous extraction, which formed the basis for a subsequent proposal. We benchmarked aqueous extraction by reproducing the most effective approaches in the literature. Our best oil extraction was 57% when no enzymes were used compared to 62% reported in the literature when no enzymes were employed and 78-85% when using enzymes. We then evaluated three different types of mechanical pretreatments in an attempt to improve oil extraction: once pin-milled dehulled soybeans, twice pin-milled dehulled soybean, and pin-milled flaked dehulled soybeans. Using the latter treatment we were able to increase the oil extraction to 70% of which 25% remained in the skim fraction.

Impacts
We have shown that enzyme hydrolyzed soy flour performs equivalent to alkali hydrolyzed protein in soy-protein-based adhesives. Enzyme hydrolysis does not require noxious chemicals, capital-intensive reactors and high energy demand. Soy-protein-based adhesives can replace formaldehyde-based adhesive resins that pose health risks to workers and possibly consumers of fabricated wood products. Endo-proteases and pectinase improved the functional properties of soy protein, making it more useful as a food ingredient. Both cellulases and pectinase improved the yield of soy protein isolate and thus the economics.

Publications

  • Jung, S., D. Rickert, N. Deak, E. Aldin, J. Recknor, L.A. Johnson, and P.A. Murphy. 2003. Comparison of Kjeldahl and Dumas Methods for Protein Content Determination in Soy Products. J. Am. Oil Chem. Soc. 80(12):1169-1173.
  • Jung, S., C. Rousel-Philippe, J. Briggs, P.A. Murphy and L. Johnson. 2004. Limited Hydrolysis of Soy Proteins with Endo- and Exo-proteases. J. Am. Oil Chem. Soc. 81:(10):953-960.


Progress 01/01/03 to 12/31/03

Outputs
This project continues to focus on using enzymes to enhance performance of soybean protein in food and industrial products. This is being accomplished by improving hydrolysis of protein for use in wood adhesives and functionality of various soy protein ingredients in food applications and by improving extraction yield of protein isolates from soy flour exposed to a range of acid and heat treatments. The project goals, however, were expanded this year to include benchmarking and developing a strategic plan for aqueous extraction processing (AEP) soybeans, which has the potential to replace hexane with water in extracting oil. AEP also has the potential of forming the basis for a soybean biorefinery to produce biobased products as well as food and feed. The feasibility of using enzymes to produce soy protein hydrolysates from soy flour that can be used in soy-protein-based adhesive resins for wood products was demonstrated. The adhesive resin formulated with enzyme-hydrolyzed soy flour had equal viscosity and solids loading, and produced fiberboard with equal strength, improved water resistance, reduced formaldehyde emissions, equal screw extractability, and lighter color compared to fiberboard bonded with adhesives formulated with more expensive alkali hydrolysate. An enzyme-linked immunosorbent assay (ELISA) was developed to characterize the native state of both soy proteins. Limited proteolysis (2 to 4% DH) of hexane-extracted soyflour, extruded-expelled soyflour, soy protein isolate, soy protein concentrate using endo- and exo-peptidases improved solubility and all other functional properties except for emulsification properties of soyflour, which was discovered to be unique to hydrolyzed soyflour (each soy substrate behaved differently in extent of modification at the same DH). The previously observed loss in emulsification properties of hydrolyzed soyflour versus improved emulsification properties for hydrolyzed soy isolate was confirmed by measuring surface hydrophobicity and droplet size. Cellulases improved protein extraction yield by up to 10% without modifying the native state and properties of the protein; proteases gave similar improvements in protein extractability but degraded the protein; pectinase increased extraction rate by 50% and at the same time modified functional properties of the protein through deglycosylation. Aqueous extraction processing and the potential to utilize enzymes to enhance oil extraction in a water-based system to replace hazardous hexane was benchmarked and a strategic plan was developed to guide future research.

Impacts
We have shown that enzyme hydrolyzed soy flour performs equivalent to alkali hydrolyzed protein in soy-protein-based adhesives. Enzyme hydrolysis does not require noxious chemicals, capital-intensive reactors and high energy demand. Soy-protein-based adhesives can replace formaldehyde-based adhesive resins that pose health risks to workers and possibly consumers of fabricated wood products. Endo-proteases and pectinase improved the functional properties of soy protein, making it more useful as a food ingredient. Both cellulases and pectinase improved the yield of soy protein isolate and thus the economics.

Publications

  • No publications reported this period