Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
  • A23J1/005—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from vegetable waste materials
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
  • A23J1/125—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses by treatment involving enzymes or microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
  • A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
  • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/163—Sugars; Polysaccharides
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
  • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
  • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
  • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
  • Y02A40/81—Aquaculture, e.g. of fish
  • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Definitions

• the present disclosure relates to processing of biomass from oilseed and grain crops, and more particularly to the separation of proteins from cellulose-hemicellulose fibers in waste products or byproducts from oilseed and other grains.
• oilseed crops and grain feedstocks into useful food and beverage products typically results in a large amount of fiber-based waste or byproducts. Although these waste products contain valuable protein and fiber components, they are of low concentration and purity and therefore are often disposed of as waste and are often costly for processors to remove. Some of the oilseed byproducts are usually used as low value animal feed.
• okara For example, worldwide production of tofu and soymilk from ground soybeans generates millions of tons of a solid by-product called okara each year.
• Okara is comprised of 75% moisture.
• okara contains about 50% dietary fiber, 25% protein, 10% lipid, and other nutrients.
• it in its wet form, it is fermentable and gets spoiled in a very short time after it is produced due to its high nutritional value and high moisture content.
• Disposal of large quantities of okara poses a significant environmental and economic problem.
• Currently, only a very small fraction of okara is used in the food industry or as animal feed after drying. The majority of okara is dumped in the field as a fertilizer or is burned as waste at a cost to both the producer and the environment.
• DDG distillers' dried grains
• oilseeds such as soybean, cotton, sunflower, canola and flax are used for animal feed as source of protein for most livestock.
• oilseeds such as soybean, cotton, sunflower, canola and flax
• the oilseed meals of all these crops contain high value proteins mixed with dietary fiber and other components.
• the protein feeding value cannot be fully utilized when oilseed meal is directly consumed for different reasons.
• the high temperature process employed during oil extraction decreases the protein solubility of the meal and also reduces nutrition value.
• animals such as fish, chickens and young pigs
• high levels of fiber dilute the protein and energy content of the meal and have little feeding value.
• antinutritional factors contained in the oilseed biomasses such as trypsin inhibitor and phytic acid also have negative impact.
• the existence of trypsin inhibitor activity in animal feed reduces growth rate and protein efficiency ratio (PER) (Wilson and Poe, 1985, Aquaculture, 46: 19-25).
• PER protein efficiency ratio
• oilseed meal or waste when directly used as a feed component, has limited feeding value as a protein source for monogastric animals such as pigs, chickens and fish due to the high fiber, high antinutritional factor, and high phytate content.
• U.S. Patent No. 5,658,714 describes a process for protein extraction from vegetable flour by first adjusting the pH of the extract media to alkaline condition, after concentration, the extracted protein is precipitated by adjusting the pH of the ultrafiltration permeate to 3.5-6.0.
• U.S. Patent No. 4,420,425 describes an aqueous extraction process of defatted soybean using alkaline conditions. After removing the solid by filtration, the solubilized protein extract is concentrated by ultrafiltration with a molecular weight cut-off of >100 kD to generate a protein concentrate.
• U.S. Patent No. 5,989,600 describes a process to improve vegetable protein solubility with enzymes such as phytase and/or proteolytic enzymes.
• U.S. Patent No. 3,966,971 teaches a vegetable protein extraction process by using acid phytase in an aqueous dispersion.
• a process for producing a protein- and/or peptide-enriched fraction and a dietary fiber- enriched fraction from a biomass comprising;
• liquid fraction is enriched in proteins and/or peptides and the solid fraction is enriched in dietary fibers.
• step a) is performed at a temperature of about 90 °C to about 100 °C.
• step a) is performed at a temperature of about 90°C to about 95 °C.
• step a) is performed for a period of about 15 minutes to about 2 hours, about 30 to about 90 minutes or about 1 hour.
• step b) is performed at a pH of about 7 to about 1 1 .
• step b) is performed at a temperature of about 50 °C to about 80 °C.
• step b) is performed at a temperature of about 14.
• step b) is performed for a period of about 15 minutes to about 2 hours, about 30 to about 90 minutes, or about 1 hour.
• subtilisin is from Bacillus licheniformis.
• step c) comprises centrifuging the proteolytic enzyme-treated slurry of b) to obtain the liquid fraction and solid fraction. 19. The process according to any one of items 1 to 18, wherein the process further comprises inactivating the proteolytic enzyme after step b).
• biomass is a grain biomass, a plant biomass, a distillers' dried grains (DDG), a soybean biomass, canola meal or flax seed meal.
• DDG distillers' dried grains
• a protein and/or peptide-rich dry biomass extract having the following features:
• a drink, cosmetic, food, or feed product comprising the protein and/or peptide-rich dry biomass extract of any one of items 37-39 and or the fiber-rich dry biomass extract of item
• a method of preparing a drink, cosmetic, food, or feed product comprising (i) performing the process of any one of items 31 -33 to obtain a protein and/or peptide-rich dry product; and (ii) incorporating said protein and/or peptide-rich dry product to a drink, cosmetic, food, or feed composition.
• a method of preparing a food or feed product comprising (i) performing the process of item 35 or 36 to obtain a fiber-rich dry product; and (ii) incorporating said fiber-rich dry product to a food or feed composition.
• Fig. 1 shows a schematic diagram of a method described herein with okara.
• Fig. 2 depicts a Coomassie blue-stained SDS-PAGE gel showing the effect of protease E1 concentration on hydrolysis of soybean meal.
• Fig. 3 is a graph showing the effect of protease E1 concentration on protein release of soybean meal and okara extractions.
• Fig. 4 is a graph showing the protein contents in supernatants and pellets after different extraction steps.
• Fig. 5 is a graph depicting the impact of pH and temperature on protein extraction.
• Fig. 6 is a graph depicting the impact of pH and temperature on carbohydrate extraction.
• Fig. 7A is a graph showing the impact of different proteases on trypsin inhibitor activities in okara extracts.
• NE No enzyme; enzymes E1 to E10 are described in Table 4 below.
• Fig. 7B is a graph showing the impact of different proteases on trypsin inhibitor activities in soybean meal (SBM).
• SBM soybean meal
• Fig. 8 is a graph showing the impact of different concentrations of CaCI 2 on phytate content of okara peptide extract.
• Fig. 9 is a graph showing a comparison of the solubility of the okara peptide extract prepared by protease E1 and that of the commercial product Arcon® F.
• Fig. 10 depicts a Coomassie blue-stained SDS-PAGE gel of okara and soybean protein extracts.
• Fig. 1 1 depicts Coomassie blue-stained SDS-PAGE gel of hydrolytic supernatants from different biomasses.
• the term “about” has its ordinary meaning.
• the term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% or 5% of the recited values (or range of values).
• the present inventors have developed an enzymatic process to hydrolyze proteins directly from raw biomass after a pretreatment step.
• a high temperature mild alkaline pretreatment ⁇ e.g., -60 min, -90-95 °C, pH -10) was performed, which permitted sterilization of the raw material, thus minimizing the risk that the following enzymatic process be contaminated, and also helped solubilize and increase the enzyme accessibility of the protein and fiber biomass, leading to good protein recovery and purified fiber component.
• the temperature and pH are adjusted to suit the protease function.
• the present invention thus relates to a process for producing a protein- and/or peptide- enriched fraction and a dietary fiber-enriched fraction from a biomass comprising:
• liquid fraction is enriched in proteins and/or peptides and the solid fraction is enriched in dietary fibers.
• Okara is widely available from tofu or soymilk factories in wet form as a waste and also available as a dry product currently used mostly for feed industry and a small fraction for food industry.
• Soybean meal is also widely available from oilseed processing mills after oil extraction. Such oil extraction may be performed either as a thermomechanical extraction, or a solvent extraction.
• the process developed herein may be suitable for the extraction of protein/peptides and/or fibers from other types of biomasses including plant biomass; grain biomass; other biomasses containing a significant amount (-10% or more) of protein mixed with carbohydrates; aquatic plants such as duckweed and seaweed; and microbial biomasses such as algae, yeasts, fungi and bacteria.
• the process was successfully applied to soybean biomasses (SBM, okara) as well as other biomasses including dried distillers' grains (DDG), canola meal, flax seed meal, whole hemp seed, and dehulled hemp seed.
• DDG dried distillers' grains
• canola meal flax seed meal
• flax seed meal flax seed meal
• whole hemp seed whole hemp seed
• dehulled hemp seed dehulled hemp seed.
• the optimal reaction conditions ⁇ e.g., pH, temperature, and time
• the protein component was effectively hydrolyzed in a short time and the protein recovery was significantly increased after treatment with the process.
• the biomass is in wet form. In another embodiment, the biomass is in dry form.
• Conditions for the aqueous extraction process AEP:
• okara and soybean meal as the major starting biomasses, based on the conventional aqueous extraction process, with water suspension under neutral or alkaline conditions, the impact of different factors on the extraction of soluble fraction containing protein as the major component was tested. These factors include: particle size of starting materials, extraction time and temperature; extraction liquid conditions: pH adjustment and buffer system.
• soybean meal particularly for soybean meal, reducing the size of the solid soy particles by grinding may be preferred to increase extraction efficiency.
• the soybean meal is typically ground to a size such that the particles can pass through a No. 100 mesh (U.S. standard) screen.
• the process defined herein further comprises grinding the biomass (dry biomass) prior to step a).
• the process further comprises isolating the ground particles having a size smaller than about 200, 150, 100, 50, 25 or 10 ⁇ , for example by passing the ground dry biomass through a mesh, preferably a 50 to 200 ⁇ mesh, more preferably a 100 ⁇ mesh.
• the process defined herein further comprises a step of defatting ⁇ e.g., removing oil) the biomass prior to step a).
• a step of defatting e.g., removing oil
• Methods for defatting biomass such as soybean biomass (okara) are known in the art.
• Defatting may be achieved by chemical extraction using suitable solvent or by lipase hydrolysis, for example.
• the pre-treatment step is performed for a period of less than about 2 hours, or less than about 90 minutes, or less than about 75 minutes. In other embodiments, the pre-treatment step is performed for a period of at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 or at least 45 minutes. In further embodiments, the pre- treatment step is performed for a period of about 15 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 45 minutes to about 75 minutes, about 50 minutes to about 70 minutes, or about 60 minutes.
• the pre-treatment step is performed at a temperature of at least about 85 °C, at least about 86 °C, at least about 87 °C, at least about 88 °C, at least about 89 °C or at least about 90 °C.
• the pre-treatment step is performed at a temperature of about 85 °C to about 100 °C, about 85 °C to about 95 °C, or about 88 °C to about 92 °C, e.g., about 90 °C.
• acidic pH -3.5 to 4
• variations from this pH range increased solubility
• mild alkaline conditions led to the highest solubility.
• proteins extracted at different pH showed different composition profile as shown by SDS-PAGE. Particularly, pH 2 extraction led to increased total protein but decreased high molecular weight species.
• the pre-treatment step is performed at a mild alkaline pH, for example at a pH about 7 to about 1 1 , about 8 to about 1 1 , about 9 to about 1 1 , or about 10.
• the aqueous solution may, for example, be water, a saline solution, or a buffer ⁇ e.g., Tris buffer).
• the concentration of biomass in the aqueous solution for the pre-treatment step may be any concentration suitable to perform the process, for example a concentration of at least about 0.1 %, at least about 0.5%, at least about 1 %, at least about 2%, at least about 3%, at least about 4%, at least about 5% or at least about 10% (w/v).
• the concentration of biomass in the aqueous solution is about 0.1 % to about 40% or the concentration of biomass in the aqueous solution is about 0.5%, about 1 %, about 2%, about 3%, about 4%, about 5% or about 10% to about 40%, about 30% or about 20%.
• a reaction condition of about pH 8-10 and about 80-90 °C is favorable for good quality, good yield of protein extraction from okara without increased carbohydrate release.
• the pre-treatment is performed at a temperature of about 88 °C to about 92 °C, e.g., about 90 °C and at a pH of about 9 to about 1 1 , e.g., about 10.
• okara was directly extracted with water three times and the extracted proteins were combined. Compared to total protein in the starting material, a very small fraction was released (7-8%).
• okara water suspension was pretreated for 1 hr at 90 °C, pH 10 (preincubation) and then washed three times. This preincubation step increased the soluble protein recovery. However, only 33% of the total protein in okara was extracted in soluble form. It was thus next assessed whether enzyme treatment could further improve protein recovery.
• Enzyme-assisted aqueous extraction process for product extraction from soybean biomasses
• protease E1 alkaline protease, CAS Number: 9014-01 -1 , referred to herein as protease E1
• protease E1 alkaline protease, CAS Number: 9014-01 -1 , referred to herein as protease E1
• soybean meal and okara were suspended in the reaction buffer (pH 8, 0.03 M Tris-CI) and treated with protease E1 (55 °C, 1 hr), even a very low dosage (0.025% v/v) of the enzyme could effectively hydrolyze the proteins to small peptide directly from the raw biomass within a short time.
• protease treatment also increase the extracted protein content significantly compared to non-enzyme control incubated under the same conditions. More detailed analyses were carried out with okara and extracted protein was increased 66% compared to non-enzyme control.
• proteases see enzymes E1 to E10 in Table 4 below
• the activities of 10 different proteases were characterized, and the soy protein hydrolysis profile was analyzed using both soybean meal and okara with standardized enzyme dosage.
• the present inventors have found that a similar hydrolysis pattern occurred using different proteases including proteases from Bacillus licheniformis (serine-type protease, subtilisin, Sigma® Cat. No. P5459 and EMD Millipore Cat. No. 126741 ), Bacillus amyloliquefaciens (serine-type protease, subtilisin, Sigma® Cat. No. P1236), Aspergillus oryzae (endoproteases and exopeptidases, Sigma® Cat.
• the yield and identity of these peptides may not be the same.
• the physical and neutraceutical functions of peptides from different protease hydrolysis may not be the same due to different compositions.
• the protease (or combination thereof) to be used in the process may be selected based on desired criteria, for example better hydrolysis efficiency under certain conditions, desired activity, etc.
• the 10 proteases were tested for their ability to generate products with reduced trypsin inhibition activities. Compared to non-enzyme treated material, treatment with the different enzymes resulted in a decrease in trypsin inhibition to varying degrees, the differences likely resulting from the different compositions of the hydrolyzed products obtained.
• the process described herein may be performed using any proteolytic enzyme (protease) or combinations thereof, including endoproteases, exopeptidases, serine-type proteases ⁇ e.g., subtilisin), cysteine-type proteases ⁇ e.g., papain), threonine-type proteases, aspartic-type proteases, glutamic-type proteases, metal loproteases and asparagine peptide lyases.
• proteases may be isolated from any suitable organisms (bacteria, fungus, plants, animals, etc.), or produced recombinantly using commonly used techniques.
• the process defined herein comprises the use of one or more serine-type proteases, for example subtitlisins. In another embodiment, the process defined herein comprises the use of one or more cysteine-type proteases, for example papains. In another embodiment, the process defined herein comprises the use of one or more of enzymes E1 to E10 described in Table 4. In an embodiment, the starting biomass is okara and the process defined herein comprises the use of one or more of enzymes E1 , E3, E8, and E10 described in Table 4. In an embodiment, the starting biomass is SBM and the process defined herein comprises the use of one or more of enzymes E3, E4 and E9 described in Table 4.
• the conditions for the proteolysis step may be adapted based the protease(s) used.
• the temperature of the proteolysis step is from about 20 °C to about 80 °C, about 30 °C to about 70 °C, about 40 °C to about 60 °C, or about 50 °C to about 60 °C.
• the proteolysis step is carried out at a pH of about 4 to about 12, about 7 to about 1 1 , about 8 to about 1 1 , about 9 to about 1 1 , or about 10.
• the proteolysis step may be performed in the same aqueous solution as the pre-treatment step, or in a different solution.
• the pH and temperature may be readjusted between the pretreatment step and proteolysis step.
• the proteolysis step is performed for a period of less than about 2 hours, or less than about 90 minutes, or less than about 75 minutes. In other embodiments, the proteolysis step is performed for a period of at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 or at least 45 minutes. In further embodiments, the proteolysis step is performed for a period of about 15 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 45 minutes to about 75 minutes, about 50 minutes to about 70 minutes, or about 60 minutes.
• the process defined herein further comprises a step of inactivating the proteolytic enzyme after the proteolysis step.
• Methods to inactivate proteases are well known in the art and include, for example, chemical inactivation ⁇ e.g., using a protease inhibitor), pH inactivation ⁇ e.g., by adding an acid or a base so that the pH of the mixture/slurry is incompatible with proteolytic activity) or heat inactivation.
• the step of inactivating the proteolytic enzyme comprises heat inactivation, for example by heating the slurry to a temperature of at least 70 or 80 °C for at least 5 or 10 minutes.
• the heat inactivation comprises heating the slurry to a temperature of 80 to about 100 °C (e.g., 80, 85, 90 or 95 °C) for a period of about 5 to about 30 minutes, preferably about 10-20 minutes or about 15 minutes.
• phytate is often referred as an antinutrient because of its interference with the absorption of certain nutrients such as minerals (calcium, magnesium, iron, copper, and zinc).
• selected divalent cations (CaCI 2 , MgCI 2 , MnCI 2 , and FeCI 2 ) were tested to reduce (by precipitation) the concentration of the anti-nutritive phytate in the peptide product.
• MnCI 2 seemed most effective at inducing phytate precipitation, particularly at lower concentration.
• the process defined herein further comprises a step of reducing the phytate content.
• Methods for reducing phytate content in compositions are well known in the art, and include, for example, treatment with a phytate-decomposing enzyme ⁇ e.g., a phytase) or divalent cations.
• Phytases may be from any sources/origins, e.g., fungi, bacteria, yeast, or plants.
• the process comprises treating the proteolytic enzyme-treated slurry of step b) with a solution comprising one or more divalent cations, for example a solution comprising one or more of CaCI 2 , MgCI 2 , MnCI 2 , and FeCI 2 .
• the solution comprises CaCI 2 .
• the amount of divalent cation in the slurry is about 0.5x to about 50x equivalents, or about 1 x to about 40x equivalent, or about 1 .5x to about 25x equivalents.
• the optimal parameters identified are: pretreatment at about 90 °C, pH 10 for about 1 hr, followed by treatment with enzymes of selected dosage for about 1 hr and CaCI 2 was added at identified dosage.
• the supernatant (liquid fraction) is then separated from the solid fraction ⁇ e.g., using centrifugation, filtration, or any other suitable method for separating liquid and solid fractions), and may be concentrated and dried to obtain the protein/peptide-rich soluble product.
• the solid fraction enriched in fibers may be dried for use as a fiber product for food or feed.
• the present invention thus relates to a process for producing a protein- and/or peptide- enriched fraction and a dietary fiber-enriched fraction from a biomass comprising:
• a divalent cation solution ⁇ e.g., CaCI 2
• step e) may be carried out after step g), followed by another round of solid/liquid separation to eliminate the precipitated phytate content.
• the liquid fraction may be further concentrated and dried as final product.
• the liquid fraction may be subjected to one or more purification step(s), for example purification based on molecular mass or size differences using membrane filtration systems based on features of the targeted product.
• the process defined herein does not comprise the use of an organic solvent.
• Table 1 shows a composition comparison between Arcon® F (from Archer Daniels Midland, ADM) and two peptide products obtained by the process described herein.
• Amino acid profile analyses of protein/peptide samples extracted from okara showed similar ratios of both essential and non-essential amino acids when compared to a commercial soy protein concentrate (SPC) Arcon® F from ADM, suggesting equal nutritional value may be expected if other features are the same.
• solubility analyses showed that the extracted protein/peptide product obtained by the process described herein exhibited consistently high solubility (>80%) over a wide range of pH (3 to 1 1 ), whereas the commercial SPC Arcon® F showed only -10% solubility between pH 3 to 9 and raised to 25% only at pH 1 1 .
• the okara-derived peptide product obtained by the process described herein contained a lower amount of protein, mainly due to the lower protein content in the starting material (okara, 25% vs soybean meal, usually > 50%) and the extra lipid in the okara and peptide product.
• lower concentrations of antinutritional factors were detected in the okara extract obtained by the process described herein relative to Arcon® F.
• the enzymatic process also led to protein/peptide products mostly smaller than 20 kDa with a >20% degree of hydrolysis.
• the process also resulted in lower trypsin inhibition activity and phytic acid content. Therefore, the extracted protein/peptide products obtained by the process described herein may be used for wider range of applications including food, feed, drink, cosmetics and with good functionality and nutritional value.
• Table 1 Composition comparison of okara products obtained by the process described herein with a commercial soy protein concentrate, Arcon® F
• the present invention relates to a protein and/or peptide-rich dry biomass extract, preferably a soy biomass extract, comprising one of more of the features described herein.
• the protein and/or peptide-rich dry biomass extract comprises at least 2, 3, 4 or 5 of the features described herein.
• the extract comprises at least 1 , 2, 3 or all of the following features:
• SPC soy protein concentrate
• at least 80%, at least 85%, or at least 90% of the proteins and/or peptides in the extract have a molecular weight of about 20kDa.
• the extract has a trypsin inhibition activity that is less than about 3, 2.5, or 2 TUI/mg as measured using the methods described in the Examples below.
• the extract has a phytate content that is less than about 25 or 20 mg/g as measured using the methods described in the Examples below.
• the protein and/or peptide-rich dry biomass extract is obtained by the process described herein.
• the present invention provides a fiber-rich dry biomass extract, preferably a soy biomass extract, comprising one of more of the features described herein.
• the fiber-rich dry biomass extract is obtained by the process described herein.
• the extracts described herein may be incorporated into various foods such as beverages (e.g., soft drinks); milk products; sauces; confectionery such as baked confectionery, nutrient bars, cereals, candies, gums, jellies and the like; tablets; breads; cooked rice; vegetarian foods (hamburgers, sausages, granola products, pates) and the like.
• beverages e.g., soft drinks
• sauces confectionery such as baked confectionery, nutrient bars, cereals, candies, gums, jellies and the like
• tablets breads; cooked rice; vegetarian foods (hamburgers, sausages, granola products, pates) and the like.
• an extract described herein is incorporated into an animal feed (livestock, pets).
• the extracts described herein may be incorporated into cosmetic products/compositions.
• Such cosmetic products/compositions may be for example in the form of a cream, emulsion, foam, gel, lotion, milk, mousse, ointment, paste, powder, spray, or suspension.
• the cosmetic product/composition optionally comprises at least one cosmetically acceptable auxiliary agent.
• Cosmetically acceptable auxiliary agents include, but are not limited to, carriers, excipients, emulsifiers, surfactants, preservatives, fragrances, perfume oils, thickeners, polymers, gel formers, dyes, absorption pigments, photoprotective agents, consistency regulators, antioxidants, antifoams, antistats, resins, solvents, solubility promoters, neutralizing agents, stabilizers, sterilizing agents, propellants, drying agents, opacifiers, cosmetically active ingredients, hair polymers, hair and skin conditioners, graft polymers, water- soluble or dispersible silicone-containing polymers, bleaches, care agents, colorants, tinting agents, tanning agents, humectants, refatting agents, collagen, protein hydrolyzates, lipids, emollients and softeners, tinting agents, tanning agents, bleaches, keratin-hardening substances, antimicrobial active ingredients, photofilter active ingredients, repellant active ingredients, hyperemic substances,
• the present invention also relates to a method of preparing a beverage, cosmetic, food, or feed product comprising (i) performing the process described herein to obtain a protein and/or peptide-rich dry product; and incorporating said protein and/or peptide-rich dry product to a beverage, cosmetic, food, or feed composition.
• the present invention also relates to a method of preparing a beverage, cosmetic, food, or feed product comprising (i) performing the process described herein to obtain a fiber-rich dry product; and incorporating said fiber-rich dry product to a beverage, cosmetic, food, or feed composition.
• Example 1 Dosage analysis of protease E1 on okara and soybean meal hydrolysis
• protease can efficiently hydrolyze soybean protein directly from raw biomass without first purifying the protein
• freeze-dried defatted soybean meal samples were suspended in 0.03M Tris-HCI (pH 8.0) buffer at a 2.8% solid:liquid ratio (W/V) and incubated with the addition of enzyme for 1 hour at 55°C.
• One of the popular commercially available proteases (Sigma P5459; defined herein as protease E1 ) was used to test the dosage effect on soy protein hydrolysis. For blank, reactions were carried out in separate tubes with no enzyme addition. Enzyme dosages tested were 0.0025, 0.005, 0.01 , 0.02, 0.04, 0.08% (v/v).
• Example 2 The impact of protease on the protein extraction yield from okara
• Example 3 Impact of protease on protein contents in soluble form and fiber pellet of okara
• Protein contents for solid biomass, okara, fiber pellet, and extracted soluble fractions were all determined by Kjeldahl method (AOAC Official Methods 2001 .1 1 ; J AOAC Int. 1999, 82:1389-1398) with the Gerhardt Kjeldatherm Digester, Gerhardt VapodestTM 20s Distiller, and titrated with the SI Analytics TitrolineTM 6000.
• freeze dried okara 250 mg was suspended in water with a 2.8% solid:liquid ratio at room temperature. Solid and liquid were separated by centrifugation at 2800g for 15 minutes on an AllegraTM X-12R (Beckman Coulter) centrifuge and the supernatant was saved. The pellet was resuspended and the process was repeated two more times. The supernatants from the three repeats were combined. Both the pooled supernatant and pellet were freeze dried and protein contents were determined by Kjeldahl method.
• okara pre-incubation and wash freeze dried okara was suspended like last step, incubated at pH 8.0 (adjusted with 4N NaOH) and 90 °C for 1 hour. Supernatants of the incubation and three washes were combined. Protein contents of supernatants and pellet were determined as described in last step.
• okara was washed three times, incubated at 90 °C in pH 8.0 for 1 hour. The liquid was separated from solid and the pellet was washed three times as described above. Pellet was then resuspended in water to the original volume, adjusted pH to 8.0 and incubated at 55° for 1 hour without enzyme. Liquid was separated from solid as described above and pellet was washed three times. All the liquid supernatants were combined. Both the pooled liquid and the washed pellet were freeze dried and protein contents were determined by Kjeldahl method. For enzyme assisted extraction, all operations were the same as the enzyme blank except with the addition of 0.005% (V/V) protease E1 at the 55° for 1 hour.
• Freeze dried okara was suspended in water at 2.5% (W/V) ratio, pH was adjusted with 4N HCI (pH 1 .5- 7) or 4N NaOH (pH 7- 12). Okara suspensions at different pHs were incubated at different temperatures for 1 hour. Solid and liquid separation was carried out by centrifugation at 2800g for 10 minutes. The protein contents in the supernatants were analyzed with the Protein DC Kit (BioRad®) following the manufacturer's protocol.
• proteases listed in Table 4 were tested according to the established EAEP process with okara and defatted soybean meal. Relative activities of proteases were tested based on the Universal Protease Activity Assay procedure (Sigma®) with the non-specific substrate casein (Sigma®). The pretreated okara and soybean meal (SBM) was hydrolyzed with a standardized amount of each enzyme.
• Trypsin inhibitor activity assay was performed following published methods (Kakade, et al., 1969, Cereal Chem 46:518- 526; Kakade, et al., 1974, Cereal Chem 51 :376 - 381 ) with minor modifications. Dried samples (0.5 g) were ground to pass through 60 mesh sieve and extracted with 25 ml 0.01 N NaOH for 3 hours while shaken at 150 rpm at room temperature (pH of the suspension ⁇ 9.5 to 9.8). The suspension was diluted so that 40-60% of trypsin inhibition was achieved.
• Trypsin Unit is arbitrarily defined as an increase of 0.01 absorbance units at 410 nm per 10 ml of the reaction mixture under the conditions used. Trypsin Unit Inhibited (TUI) is the difference of Trypsin Units assayed with and without soy samples.
• Phytate content assay was performed following published methods (Dragicevic et al., 201 1 , Acta periodica technologica 42:1 1 -21 ; Gao et al., 2007, Crop Science 47: 1797-1803) with minor modifications.
• Okara biomass was suspended in water at 10% solid:liquid ratio, pH was adjusted to 10 with 4N NaOH and extracted at 90 °C for 1 hr, The temperature was then adjusted to 55 °C and pH was readjusted to 8 using 4N HCI.
• Protease 1 was added at a dosage of 0.005% and hydrolytic samples were taken at different time points. Experiments were repeated and control experiments without enzyme were run in parallel as comparison. The hydrolytic process was monitored by measuring degree of hydrolysis (% DH).
• % DH was performed with o-phthaldialdehyde (OPA) following published procedures (Nielsen et al., 2001 , J Food Sci. 66: 642-646; Vigo et al., 1992, Food Chem. 44:363-365) with minor modifications. Samples were diluted to contain between 1 - 10 mg protein per ml_, dilution factors were recorded. After all reaction reagents were added, the mixture was allowed to stand for exactly 2 minutes before measuring A 340 .
• OPA o-phthaldialdehyde
• %DH h / h tot * 100;
• Reaction volume (L), sample weight (g) and protein concentration were obtained based on reaction conditions.
• % protein of sample is input as a percentage, not a fraction.
• the results presented in table 5 show an increased degradation of hydrolysis within 5 min of reaction and the DH keep increasing towards the last time point. At 60 min, a % DH of 24 to 27% was reached while the control samples stayed constant between 7 and 10%.
• Protein solubility was measured by using the methods of Lee and Morr (Lee et al., 2003, JAOCS 80, 85-90; Morr et al., 1985, J. Food Sci. 50:1715-1718) with minor modifications.
• One batch of the okara peptide product prepared by protease E1 and the commercial product Arcon® F (ADM) were suspended in 0.1 M NaCI solution at 2% (W/V) concentration. After adjusting the pH with 1 N NaOH or 1 N HCI solution, the suspensions were thoroughly mixed with an air shaker at room temperature, 100 rpm, for 30 min. The suspensions were then centrifuged at 20,000g for 15 min. The protein contents of the supernatants and the original solid powders were measured using the Kjeldahl method and the conversion factor of 6.25. Protein solubility was calculated as a percentage of proteins in the supernatant over the total proteins in the original samples.
• Example 10 Effects of enzyme and CaCI 2 treatment on the protein and anti-nutritional factors concentrations in the okara soluble extracts
• the established procedure including a pre-treatment step, followed by an enzyme treatment was performed with the addition of CaCI 2 at 5 equivalents amount during the enzyme step to reduce the concentration of the anti-nutrient phytate.
• the extraction was performed three times at the 1 L scale. Control extractions with no enzyme and/or no CaCI 2 were also performed three times.
• a sample of the final extract was lyophilized and analyzed for yield and anti- nutritional factors (trypsin inhibition activity and phytic acid).
• the extraction procedure with enzyme and CaCI 2 led to a protein recovery of 53% which is slightly less than the extraction without CaCI 2 (60%), suggesting that CaCI 2 may also reduce the solubility or precipitate some soluble proteins when added to the biomass suspension during enzyme hydrolysis.
• Okara proteins were extracted by suspending dried okara in pH 8 Tris-CI buffer or in water adjusted to different pH (pH 6, 7, 8, 9 and 10) as described above. Soymilk was prepared from the same kind of soybean as the okara by first soaking the dry soybean in water for 2 hrs at room temperature, followed by grinding for 10 min and then boiling of the ground suspension for 30 minute. Soymilk was obtained by filtering through 3 layers of cheese cloth. The extracted samples were compared by running SDS-PAGE gel followed by Coomassie blue staining. The peptide profiles of proteins extracted from okara showed very similar patterns including both the storage soybean 1 1 S/7S proteins and other minor components (Fig. 10). This suggests that there is no significant difference in protein/peptide compositions between okara extracted protein and the whole soybean extracted protein.
• Table 7 Amino acid profile analysis of four different batches of okara peptide extracts (R1 P- R4P) and of the commercial soy protein concentrate Arcon® F
• Example 12 Application of the developed process in other biomasses The established process based on okara extraction was tested on other biomasses at the 100 mL scale; the protein recovery and content in the extracted product were determined for each biomass. Protein recovery is determined by comparing the amount of extracted protein with the total protein in the raw biomass. Protein content is determined by comparing the amount of extracted protein to the total weight of the extracted product on dry matter basis.
• DDG distillers' dried grains
• the pre-treatment pH was raised to pH 1 1 .
• the other steps and other samples followed the procedure described above. For all materials tested, the enzymatic process increased the protein recovery significantly as shown in Table 8.
• DDG Compared to non-enzyme control, the greatest increase in protein recovery was seen in DDG (40% up to 74%) followed by canola meal (65% up to 87%). In terms of protein content in the extracted product, DDG and canola meal showed the largest increases, whereas slight increases were obtained for flax and soybean.
• okara the majority of proteins were hydrolyzed to smaller than 15 kDa peptides, for soybean meal, mostly under 25 kDa, for DDG, mostly under 10 kDa, and for canola meal, mostly under 10 kDa.

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