WO1994018852A1

WO1994018852A1 - Method for extracting soybeam peroxidase from soybean hulls and recovering a product useful as a fiber supplement

Info

Publication number: WO1994018852A1
Application number: PCT/US1994/001995
Authority: WO
Inventors: Mark Alan Johnson; Alexander R. Pokora; Joseph B. Henry
Original assignee: Enzymol International Inc
Current assignee: Enzymol International Inc
Priority date: 1993-02-24
Filing date: 1994-02-24
Publication date: 1994-09-01
Anticipated expiration: 1995-08-24

Abstract

A method for supplementing animal feed which comprises the steps of: extracting soybean peroxidase from soybean seed hulls, recovering a solution of soybean peroxidase in water, recovering fiber-enriched soybean hulls, and feeding the fiber-enriched soybean hulls to animals as a fiber supplement.

Description

METHOD FOR EXTRACTING SOYBEAN PEROXIDASE FROM

SOYBEAN HULLS AND RECOVERING A PRODUCT USEFUL

AS A FIBER SUPPLEMENT

Background of the Invention

In the extraction and recovery of soybean peroxidase enzyme from soybean hulls, there can be a substantial amount of protein carryover from the seed. Additionally, various soluble simple and complex carbohydrates, proteins, fats and oils are associated with the hulls. By washing the hulls with water, preferably hard water, soluble simple and complex carbohydrates, proteins, fats and oils will be extracted leaving a fiber rich product which can be used as a source of fiber/roughage in animal feed and various food products as well as a filler in soybean protein to lower the protein content. This product can be formulated wet or easily dried using a fluidized bed dryer to provide a free- flowing powder and utilized in the same manner as pulverized, non-extracted hulls.

Summary of the Invention

The present invention relates to an improved method for extracting peroxidase enzyme from soybean hulls and recovering the spent soybean hulls as a fiber-enriched product which is formulated into animal feed or a human dietary supplement.

The invention resides in a method comprising the steps of: extracting soybean peroxidase from soybean seed hulls, recovering a solution of said peroxidase in water, recovering said hulls, and feeding said hulls to animals, and particularly livestock, as a fiber supplement.

The invention resides more particularly in a method which comprises the steps of: washing the soybean hulls with water to extract peroxidase enzyme, and separating the particles from the extract to obtain a solution of the peroxidase in ater, and recovering the particles as a fiber enriched soybean hull product which is useful as an animal feed or a dietary supplement for humans.

Detailed Description of the Invention

For use in the present invention, the soybean hulls are typically ground or flaked. Flaked hulls are obtained from dried but untoasted soybeans which are immediately cracked in a cracking mill where the hulls are aspirated away by a separation device.

Ground hulls are obtained by transporting flaked hulls by air to a hammer mill where they are ground to prepare ground hulls. Ground hulls typically contain particles which range between about 2000 microns and 50 microns. Ground hulls are the preferred starting material for enzyme extraction because their high surface area and high density minimize transportation costs. If transportation is not an issue, flaked hulls may be preferred because they are generally considered to have about 30% greater peroxidase activity than the ground hulls. Hulls used for enzyme extraction are generally bagged in 50 pound sacks immediately following grinding. Enzyme yield can be maximized by using the hulls as soon as possible after the hulls are removed from the seed. If the hulls must be stored, temperatures below 25°C -and low humidity are preferred. Hulls stored for longer than one month in some cases may not be suitable as an enzyme source.

It is estimated that about 30-95% of the total peroxidase activity can be obtained from ground hulls by washing alone. About 80% of the activity obtained by washing comes from particles less than 500 microns in size. Using flaked hulls, only about 10% of the peroxidase activity can be obtained by washing.

Washing and extraction of the soybean hulls is preferably accomplished using hard water and, preferably, water containing calcium ions. It has also been found that soybean peroxidase is more stable during processing and upon storage when it has been obtained from an aqueous solution containing calcium ions. It is believed that the calcium ions increase the thermal stability of the enzyme and maintain the yield of fiber recovered. The calcium ion can be present in a concentration of up to about 5M, preferably, up to about 400mM.

As a first step in the recovery process, the soybean hulls, preferably ground, are extracted with water to recover the peroxidase activity. The extraction is typically carried out in a 50 gallon tank using a mechanical stirrer at about 200 rpm maximum. The ratio of hulls to water is typically about 50 pounds hulls to 50 gallon water. The enzyme is extracted from the ground hulls at a temperature of up to about 40°C and preferably at about room temperature for a short period of time (15-30 min) to minimize water uptake by the hulls and leaching of carbohydrates which can interfere with later stages of processing without yield advantages. The extracted hulls are separated from the filtrate using conventional means such as a centrifuge or a 50-100 micron filter (screen, bag, etc.) and the filtrate is then recirculated back to the extraction tank where it is used to extract additional enzyme from a fresh batch of hulls. For example, about 50 pounds of new hulls may be added to 10 gallons of fresh well water followed by about 40 gallons filtrate. This procedure can be repeated up to about 15 times; however, as a practical matter, the procedure is repeated up to about 6 times and preferably about 4 to 6 times. Typically, the extracted hulls are subjected to a second extraction from which the spent particles of soybean hulls can easily be dried to provide fiber-enriched animal feed or a dietary supplement.

It has been found that residual particles remaining in the extract can be efficiently separated to obtain a clear solution of the peroxidase having high specific activity while at the same time recovering a fiber-enriched soybean hull product which is useful as animal feed or dietary supplement.

In one embodiment of the invention, the extract is subjected to a freeze-thaw cycle in which the extract is frozen directly at a temperature of about -20 to -5°C. The temperature will vary with concentration, the more concentrated extracts requiring lower temperatures than the less concentrated ones. The extract may be frozen to a hard solid, although experiments have been performed in which the extract is frozen to a slush, i.e., a partially frozen mixture, with less efficiency. In some cases, the extract may be diluted to 10%-80% of its original concentration.

To thaw the frozen extract, the extract may be heated or simply, allowed to warm at room temperature. In heating, however, because peroxidase is a protein, care must be taken not to heat locally to temperatures which denature it. Upon thawing the extract, the particles readily settle. The particles can then be removed by conventional techniques including filtration (paper, bag, etc.), centrifugation, decantation, etc. Following this procedure, the extract may be used as is, lyophilized or the enzyme may be recovered by ultrafiltration, ion exchange, or the like.

In another embodiment of the invention, the pH of the extract is adjusted to alkaline pH to cause separation of the particles from the extract. Under alkaline conditions particles rapidly coalesce. Preferably the extract is adjusted to a pH of 9 to 13 using 50% sodium hydroxide solution. A control extract had a specific activity of 50 units per mg protein, but after adjusting the pH to 11 with 50% NaOH and allowing the particles to settle overnight, the resulting solution had a specific activity of 66 units per mg protein.

In yet another technique useful in the invention, the extract is heated to a temperature in the range of about 30° to 80°C and held at that temperature for at least about 1 minute and up to about 5 hours to facilitate removal of the particles. Significant levels of particulate matter coalesce on heating at 80°C for 1 hour and settle out of soybean peroxidase extracts upon centrifugation. The half- life of the soybean peroxidase enzyme is approximately 10 hours at 70°C, six hours at 80°C, and one hour at 90°C. The stability of the enzyme is improved by addition of calcium chloride to the extract.

In still another embodiment of the invention, the extract is subjected to a salt treatment in which the concentration of a salt in the extract is increased to facilitate and enhance removal of residual particles. This treatment may be used in combination with the pH and/or thermal treatments. It has been found that soybean hull extract clarifies immediately on centrifugation with trishydroxymethyl ammonium chloride (TRIS) buffer at-pH 8 with a salt concentration up to 50mM. Without the salt, longer settling times and higher pH are generally required. Examples of salts useful in the invention include CaCl₂, KC1, NaCl, Na^0₄, and ( H₄)2S0₄. Generally, the salt is used in an amount of about lOmM to 5M depending on its ionic strength. Calcium chloride is preferred because of its stabilizing effect on the enzyme and because of its good flocculating properties, i.e., calcium chloride enhances the combination of fine particles to provide large particles which are easily recovered along with the fiber.

The above treatments can be used separately or they can be used together in any combination to enhance removal of residual particles. Furthermore, the foregoing treatments namely, pH adjustment, thermal treatment or salt addition, may be conducted prior to or after treatments such as centrifugation which remove the plant particles and other impurities. It is generally more desirable to conduct the pH adjustment, thermal treatment or salt addition before centrifugation, but it is also effective to centrifuge, and treat the extract then allow the extract to stand for 1 or 2 hours whereupon further particles and impurities may settle from the solution of peroxidase.

Particle removal can be effected by conventional techniques including filtration (paper, bag, etc.), centrifugation, decantation, etc. Typical centrifugation conditions are 900Xg or 1500Xg for 15 min.

Separation and clarification of the extract can be enhanced by further cooling and optionally freezing the extract and allowing the extract to warm to room temperature. Generally, this treatment is conducted after centrifugation and other treatments as a means to effect additional sedimentation. The extract may be cooled to about 4°C to 0°C and held at that temperature for about 10 to 60 minutes. In cooling the extract, it may be desirable to freeze the extract to a solid or a slush as described above. To freeze the extract, temperatures on the order of -20 to -5°C are generally required. The temperature will vary with concentration, more concentrated extracts require lower temperatures than less concentrated ones. It is generally not necessary to freeze the extract to a solid.

The thermal treatment described above (either heating or cooling) may be employed in a process including the sequence of steps of: 1) extraction, 2) large particle removal (optional), 3) thermal treatment, and 4) centrifugation or other particle removal; or the sequence of steps of: 1) extraction, 2) large particle removal (optional), 3) centrifugation, 4) thermal treatment, and 5) further sedimentation. Still another process may include the sequence of steps of: 1) extraction, 2) large particle removal (optional), 3) thermal treatment, 4) centrifugation, 5) cooling the extract, 6) further sedimentation. Any of the aforesaid processes may be modified to additionally include a salt addition and/or a pH adjustment in combination with the thermal treatment.

The pH adjustment may be embodied in processes which include the sequence of steps of: 1) extraction, 2) large particle removal (optional), 3) pH adjustment, 4) centrifugation or other particle removal step, 5) optional further sedimentation; or the sequence of steps of: 1) extraction, 2) large particle removal, 3) centrifugation or other particle removal step, 4) pH adjustment, and 5) further sedimentation. The thermal treatment could be conducted in combination with the pH adjustment but after centrifugation or other particle removal step to enhance further sedimentation. The salt treatment, like the thermal treatment, may also be conducted at the same time as, or after the pH adjustment or after centrifugation and preparatory to a further sedimentation step.

Various modifications can be made in the process to enhance recovery of the enzyme. Certain agents such polyethyleneimine, polyvinyl alcohol, polyvinyl pyrrolidone, calcium chloride, and ammonium sulfate, which behave as or somewhat like flocculating agents can also be used to enhance separation.

Other techniques for increasing yield include adjusting the pH to 3, with or without the addition of 100 ppm (20 ppm based on polymer weight alone) cationic flocculent such as Betz polymer 1192 and allowing the particles to settle or removing the particles by centrifugation. The clarity of solution, indicating removal of fines, following this treatment varies depending on the method of extraction of the enzyme.

Regardless of which procedure is used to recover the peroxidase solution, it may be desirable to further treat the solution which is recovered in accordance with the invention to further remove impurities by such well known processes as ultrafiltration, microfiltration, ion exchange, or the like. The enzyme itself may be recovered from the solution by solvent precipitation techniques, through the use of protein fixatives like tannic acid and detergents as described in U.S. Application Serial No. 07/599,584, or by lyophilization.

Microfiltration is a useful technique for further clarifying and removing impurities from the extract. Typically, this may be accomplished using various polymeric or ceramic membranes or depth filters with porosities ranging from 1.0 to 0.2 microns. Microfiltration may be done at 20 to 60C, and pH 3-10.

Ultrafiltration is the preferred technique for concentrating peroxidase activity. Typically, a 30,000 nominal molecular weight cut off membrane, either polymeric or ceramic is suitable. Ultrafiltration may be done at 20 to 60°C and pH 4-10. The greater the removal of fines before ultrafiltration, the faster the process. Ultrafiltration is also faster at higher pH and temperature. High pH causes some particles to dissolve which will reform at pH 4.

Shear and heat generated during ultrafiltration may cause new particles to form in the solution. The enzyme may be microfiltered to provide a finished product or treated by the freeze-thaw technique described above. Peroxidase at this point is typically at 100 to 600 units per ml, 20 to 160 units per mg protein and 2-20 units per mg solids. Peroxidase may be concentrated and purified further by solvent precipitation or by spray drying to a powder, or by lyophilization (freeze-drying) .

Various treatments improve the stability and storage life of the enzyme solution. Solvent precipitation, microfiltration and addition of biostats or biocides such as thimerosal, n-propyl-p-hydroxybenzoate or 50% (v/v) glycerol and refrigeration reduce deterioration due to microorganisms.

Isolation of high purity soybean peroxidase from a concentrated crude extract may also be accomplished by gel- filtration chromatography and the product purity verified by capillary electrophoresis. A crude soybean extract can be concentrated by ultrafiltration to a range from 100-1000 units/ml activity and separated on the gel filtration column. The gel-filtration separation is performed under medium pressure using a semi-rigid crosslinked agarose- dextran polymer gel obtained from Pharmacia LKB Biotechnology, with a nominal particle size of 35 micrometer and a molecular weight separation range of 5000-75000 daltons. This material can be packed into glass columns up to 60 cm in length with internal diameters varying from 1 to 22 cm. This column effects a molecular size separation of the components in the matrix, with the larger, higher molecular weight species eluting first. The molecular weight of the soybean peroxidase is approximately 37,000, which is in the center of the separation range. The solvent used for elution of the sample is deionized water containing 5mM calcium chloride. The concentration and type of salt are important to minimize nonspecific interactions between the column packing and the sample, which cause elution by mechanisms other than size-exclusion. The flow rate required is a function of the column diameter and is chosen to provide a maximum linear velocity of 50 cm/hour. This corresponds to a range of 20-300 ml/min. as column diameter changes from 5 to 22 cm. The volume injected may be from 1- 5% of the column volume for highest resolution, and may be increased to 10% if some loss in resolution can be tolerated. Assuming an 8% load, 40,000 units total activity can be injected per run using a 5 cm i.d. column and 800,000 units injected for a 22 cm i.d. column. The capillary electrophoresis is performed using an uncoated silica capillary of 50 micron i.d. and 50 cm length, with a sodium tetraborate buffer at pH 9. A 20 kV potential effects an excellent separation of the components in the crude peroxidase solution in approximately 20 minutes. Excellent resolution of soybean peroxidase can be obtained from other components in the crude enzyme solution. A capillary electrophoresis profile for the isolated gel-filtration fraction shows that the latter consists of a single peroxidase component with all other proteins having been removed.

Soybean peroxidase shows an unexpected stability across a broad pH range, e.g., 1.5 to 12. Soybean peroxidase is also stable at elevated temperature (up to about 80°C). Other peroxidase such as horseradish peroxidases (HRP) lose activity too quickly to measure. Soybean peroxidase increases reaction rates 8 fold upon increasing the temperature 20 to 60°C and this rate is preserved in solvents such as 60% alcohol which are known to denature HRP and other proteins. The unusual stability of soybean peroxidase makes it useful for many applications. U.S. Application Serial No. 599,584 discloses processes for using soybean peroxidase (SBP) and peroxidases from a number of other plant sources in a variety of biocatalytic oxidative processes including the preparation of phenolic resins. In copending U.S. Application Serial No. 760,999 filed September 17, 1991, it is proposed to use soybean peroxidase in bioremediation of waste waters and in U.S. Application Serial No. 760,870 filed September 16, 1991 it is proposed to use soybean peroxidase in immunoassays.

In addition to being useful for recovering peroxidase, the method of the present invention can be used in conjunction with the recovery of other soybean byproducts such as genistein which is useful as a chemotherapeutic agent and DDMP saponin which is useful in the treatment of AIDS (see COMLINE News Service 8/21/92).

As a result of the removal of these various components, the spent soybean hulls can be recovered as a fiber-enriched product which can be dried to a solid content of about 90% or greater and used as an animal dietary supplement. The spent hulls may be mixed with protein meal or added to animal feed. The product is especially useful in feeds for poultry and cattle. One protein meal that is commonly employed contains about 44% protein and 7% fiber. The spent hulls may be mixed with meal having higher protein and lower fiber content (e.g., a meal containing 48% protein and 3.5% fiber) to give the aforesaid protein and fiber contents.

The invention is illustrated in more detail by the following examples: Example 1

Extraction:

Ground soybean hulls (50 lbs, not toasted, were added to 50 gallons well water and mixed for 30 minutes. The slurry was filtered on a 200 mesh Sweco screen. The hulls retained on the screen were washed in 50 gallons tap water and screened again. The first filtrate was combined and mixed with 50 additional pounds of ground hulls and the above procedure repeated three more times. The second filtrate was used as the wash solution for the spent hulls. At the end of the extractions and filtration, about 100 gallons at 40-110 purpurogallin units of peroxidase per ml were obtained. The mixture was heated up to 70°C for between 1 and 20 minutes with addition of calcium chloride at concentrations ranging from 10-400mM. The mixture was allowed to settle overnight at 4°C. The supernatant containing peroxidase was pumped away from the settled particulates, producing 80 gallons of enzyme solution.

Concentration:

The enzyme solution was concentrated using a Bio-

Recovery XF-60 ultrafiltration unit and 30,000 molecular weight cut off polysulfone membranes. The enzyme was concentrated to 10 to 20 gallons. The concentrate was filtered using a 0.8-0.2 micron Membralox (TM) ceramic membrane (U.S. Filter) and a pilot-scale filtration unit from Cuno Separations. The final product contained 150-800 purpurogallin units per ml. Purification:

Soybean peroxidase concentrate was purified by gel permeation chromatography on a Pharmacia Superdex 75 column as described previously. The eluate containing purified soybean peroxidase was concentrated by ultrafiltration on a Millipore Minitan unit with a 30,000 molecular weight cut off polysulfone membrane. The concentrate was lyophilized, producing purified peroxidase (135 units per mg, 80-85% yield) that is 95% peroxidase by weight. Virtually all the protein coincided with the peroxidase based on capillary electrophoresis analysis. A fraction of lower purity peroxidase from the chromatography was also obtained 50 units per mg, 15-20% yield).

Example 2

Sodium Acetate was prepared at 0.1M concentration and pH 4. The buffer was placed in five containers of 500ml each. To each container, Calcium Chloride was added in differing amounts to 0.0, ImM, and lOmM calcium. Lyophilized soybean peroxidase was prepared by dissolving 10 mg of peroxidase, activity 135 units/mg, in ImL of distilled water. To 2ml of each of. the buffer/calcium solutions, 60ul of peroxidase was added and mixed using a vortex unit. The activity of each sample was measured at room temperature using pyrogallol assay solution. Test tubes containing 2ml each of buffer with the differing levels of calcium and 60ul of peroxidase were placed in a heated bath at 80°C for 30 minutes. The activity of each sample was measured, and continuously checked every half hour while the samples were in the heated bath at 80°C.

Table 1 shows that lOmM calcium stabilizes soybean peroxidase against thermal denaturation with an 8-fold improvement over the control in recovery of activity over five hours at 80°C.

TABLE 1 Effect of Calcium on Thermal Stability of Soybean Peroxidase

Units/ml - Calcium Level Percent Control Activity Time H 0 mM 1 mM lOmM 0 M 1 mM 10 mM

This example shows that selection of tap water for extraction of peroxidase from soybean hulls produces an enzyme that retains activity through processing. Selection of deionized water exclusively leads to an enzyme that loses substantial activity through processing. The reason for loss in activity with deionized water is supported by data showing stabilization due to calcium, a major metal ion in tap water.

Example 3

Ground soybean hulls (40-50 lbs) from Central Soya of Marion, Ohio were mixed with 50 gallons of tap water in a Cowles mixer, screened on a 30 mesh Sweco and washed with 10-15 gallons tap water (65 ppm calcium). The filtrates were combined and mixed in the Cowles with a new batch of hulls. Filtrate was recycled up to three times with hulls. The combined filtrate was allowed to settle 2-4 days. The supernatant was filtered through a 120 mesh screen and ultrafiltered using 30,000 molecular weight cut off cellulose membranes and an XF-60 unit from Bio-Recovery of Hoboken, NJ.

In each set of extractions, enzyme was concentrated from 50 to 8 to 20 gallons. From these extractions 34 gallons were accumulated at 85 purpurogallin units per ml and further concentrated to 17 gallons. The enzyme was frozen at -20°C thawed at room temperature and centrifuged at 1,500 X g for 30 minutes to remove particles. The final yield of activity was 72 units per gram of hulls extracted and 95% of the activity prior to ultrafiltration. Temperature during ultrafiltration was maintained between 45 and 55^βC. The pH was 4. The final activity was 155 units per ml in a volume of 63 liters (16 gallons). A total of 185 gallons was ultrafiltered to produce this enzyme at an average rate of 15 gallons per hour.

The extraction procedure described above was repeated with the use of only deionized water (< 0.1 ppm calcium) in all steps. The other procedural steps were performed in the same way. The final product was 488 liters (129 gallons) prior to ultrafiltration at 25 units per ml (11 million units total, yield; 81 units per gram of hulls). Ultrafiltration was done using the same equipment as above, at a temperature between 45 and 55°C, pH 4 and an average rate of 13 gallons per hour. The concentrated enzyme was separated from particles by the same freeze-thaw procedure described above. The concentrated product was 30 liters (7.8 gallons) at 160 units per ml (4.8 million units, 44% yield).

Similar enzyme extractions were done on a similar scale during the same time period which produced 95% and 44% yield from the starting activity. The only major change between the procedures was the exclusive use of deionized water in one case and the use of tap water in the other. Since the example using tap water gave the better result, tap water is preferred for stabilization of soybean peroxidase activity during further processing.

Having described the invention in detail and by reference to preferred embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.

What is claimed is:

Claims

CLAIMS:

1. A method for supplementing animal feed which comprises the steps of: extracting soybean peroxidase from soybean seed hulls, recovering a solution of said soybean peroxidase in water, recovering fiber-enriched hulls, and feeding said fiber-enriched hulls to animals as a fiber supplement.

2. The method of claim 1 wherein said step of extracting said soybean peroxidase includes the steps of washing said soybean hulls with water, and separating the hulls to obtain a solution of said peroxidase in water.

3. The method of claim 2 wherein the pH of said extract is adjusted to an alkaline pH.

4. The method of claim 3 wherein said pH is adjusted to a pH of about 9 to 13.

5. The method of claim 2 wherein said extract is subjected to a thermal treatment wherein said extract is heated to a temperature of about 30 to 80°C for up to about 5 hours.

6. The method of claim 2 wherein said extract is cooled to a temperature of about -20 to 5°C after removing said hulls.

7. The method of claim 2 wherein said extract is subjected to a salt treatment.

8. The method of claim 2 wherein said hulls are separated from said extract by filtration, centrifugation or decantation.

9. The method of claim 2 wherein said water contains calcium ions.

10. The method of claim 9 wherein said calcium ions are present at a concentration of up to about 5M.

11. The method of claim 10 wherein said calcium ions are present at a concentration of up to about 400mM.

12. The method of claim 2 wherein said separated hulls are subjected to at least one additional extraction step.

13. The method of claim 1 wherein said soybean hulls are ground soybean hulls.

14. The method of claim 13 wherein said ground soybean hulls have a particle distribution in the range of about 50 to 2000 microns.

15. The method of claim 1 wherein said fiber-enriched soybean hulls are dried to at least about 90% solids for use as animal feed.

16. The method of claim 2 wherein said solution of said peroxidase in water is recycled to said extraction step for extraction of fresh soybean seed hulls, said method being repeated up to about 6 times.

17. A method for harvesting soybean peroxidase which comprises extracting soybean peroxidase from soybean hulls with water containing calcium ion.

18. A method for recovering an aqueous solution of soybean peroxidase from ground soybean hulls and producing a fiber supplement for animal feed which comprises the steps of: (a) washing said ground soybean hulls with water containing calcium ions in a stirred extraction vessel;

(b) separating said washed soybean hulls from said water, said water containing extracted soybean peroxidase;

(c) recirculating said water containing said soybean peroxidase to said extraction vessel;

(d) repeating steps (a), (b) and (c) using fresh ground soybean hulls to provide a concentrated solution of said soybean peroxidase in said water;

(e) recovering said concentrated solution of said soybean peroxidase; and

(f) recovering said extracted hulls for use as said fiber supplement.

19. The method of claim 18 wherein said step (d) is repeated up to 6 times.

20. The method of claim 18 wherein said separated hulls are subjected to at least one additional extraction step.