Integrated process for extraction of polypeptides, mucilage, and fibre from biomass

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to United States provisional application number 62/930,749, filed November 5, 2019 and United States provisional application number 62/939,089, filed November 22, 2019; each of which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates to the field of extraction of biomolecules from biomass, particularly to processes for the extraction of polypeptides, mucilage, and/or fibre-rich material from biomass, including lignan-rich biomass.

BACKGROUND

[0003] Flax ( Linum usitatissimum L.) is an annual herb of the Linaceae family that produces small flat and oval seeds. Flaxseed, also known as linseed, has been cultivated primarily as a source for oil as a commodity product. T o date, its value as a source of protein and dietary fiber for human consumption has not been as highly recognized as oil (Martinez-Flores et al., 2006; Tirgar et al.,

2017). Due to an increased understanding of the nutraceutical and health benefits of its functional components such as omega-3 fatty acids, dietary fiber, protein, peptide, gum, and lignan, demand for flaxseed and its products has increased (Kajla, 2015; Qian, 2014; Rabetafika et al., 2011). A variety of commercial products produced by extraction of flaxseed, flaxseed meal or flaxseed hull have been developed, such as lignan extracts BeneFlax™, LignaMax™, LinumLife™, AlaLife™ Flax Lignans; the gum product by BioGin™; and Sensiline™, a hydrolyzed flaxseed extract that is enriched in polysaccharides and peptidoglycans. Different health benefits from flaxseed products have been claimed including: antioxidant activity, lower cholesterol levels, balanced blood estrogen levels, effect on lipid and bone/amendment, benefit for women breast health, improved men prostate health, anti-aging effect, weight management and blood health, reduced blood pressure and cardiovascular risks, anti-inflammatory effects, anti-bacterial and anti- carcinogenic activities (Udenigwe et al., 2009 a, b; Bouaziz et al., 2016; Shim, 2015; Wang, 2017; Singh, 2011; Tehrani et al., 2014; Nwachukwu et al., 2014; Hwang et al., 2016; Parikh et al.,

2018). Flaxseed proteins have been shown to have beneficial influence on coronary heart disease, kidney disease and cancer (Oomah and Mazza, 2000). Flaxseed mucilage/gum appears to play a role in reducing diabetes and coronary heart disease, prevention of colon and rectal cancer and reducing the incidence of obesity (Thakur et al., 2009; Singer et al., 2011).

[0004] In addition to being one of the richest sources of olinolenic acid oil and lignans, flaxseed is an essential source of high quality protein, dietary fiber, and functional phenolic compounds. The composition of the major components in flaxseed typically range from 40-45% for lipids, 20- 25% for proteins, 20-25% for dietary fibre and 1% lignans (Hall et al., 2006; Anjum et al., 2013; Gutierrez et al., 2010).

[0005] Flaxseed fibre forms the main foodstuffs and includes soluble and insoluble fractions. Insoluble fibre is composed of cellulose, hemicellulose and lignin. Soluble fibre is composed of gum, sugar and pectin which together form mucilage and collectively account for about 7-14% of dry weight (Rubilar et al., 2010; Singer et al., 2011). Flaxseed gum (FG) comprises anionic polysaccharides that are mainly located in the outermost layer of the flaxseed hull. It is comprised of both neutral (75%) and acidic fractions (25%) (Cui & Mazza, 1996; Qian et al., 2012). The neutral fraction is primarily arabinoxylans with b-ϋ-(1 ,4)-xylan backbones. The acidic fraction is primarily made up of pectic-like polysaccharides containing L-rhamnose, D-galactose, and D- galacturonic acid (Cui & Mazza, 1996; Qian et al., 2012).

[0006] Due to their good emulsifying properties, polysaccharide gums have been commercially used in the food industry for stabilization of emulsions, suspension of particulates, control of crystallization, encapsulation, formation of films and thickening (Parawira, 2008). Flaxseed gum has been used as a food ingredient based on its high water-holding capacity, superior swelling and rheological properties in aqueous solution (Chen, 2006). Meat emulsions extended with flaxseed mucilage show reduced cooking loss and reduced firmness (Dev and Quensel, 1988). Flaxseed gum increases the thermal stability of salt-soluble meat protein and the water holding capacity of porcine myofibrillar protein (Sun et al., 2011; Liu et al., 2015; 2016). It has also been suggested that flaxseed gum can be used to provide similar functionality to other non-gelling gums (Chen et al., 2007; Singh, et al., 2011).

[0007] Mostly located on the seed coat, a considerable amount of mucilage interferes with the process of protein extraction from flaxseed. For efficient protein extraction from flaxseed, mucilage is commonly first removed by treating the whole flaxseed with a large amount of water and chemicals through a multiple-step procedure. Wanasundara and Shahidi (1997) extracted mucilage from flaxseed by soaking in water, sodium bicarbonate solution or treatment with commercially available enzymes such as pectinase, Celluclast™, and Viscozymes™. The demucilaged seed is dried, ground, defatted and then goes through high pH extraction and precipitation at isolelectric point (pi) (Wanasundara & Shahidi. , 1997; Marambe et al., 2008; Tehrani et al., 2014). A patented process for flax protein isolate preparation describes protein extraction by alkaline salt solution followed by iso-electric precipitation or isolation of protein micelles formed at low temperature (Green et al., 2007). This process allows the isolation of high purity protein; however, the flaxseed needs to go through the mucilage removal and defatting process before the protein extraction process is carried out. Wan et al (2014) patented a method for producing flaxseed gum and flaxseed kernels whereby the whole flaxseed is physically polished to separate the outer gum layer from the seed. Flaxseed gum is obtained after degreasing the collected gum powder by an organic solvent and drying.

[0008] The established industrial process for flaxseed oil extraction does not pre-treat the flaxseed by mucilage removal. The flaxseed meal resulting from the pressing process contains a mixture of components including high contents of protein, gum/carbohydrate, fiber, and other functional components. In addition, the previously published process for mucilage removal from whole flaxseed is not effective for flaxseed meal once the flaxseed is broken and other components released. To date, flaxseed meal as the major by-product following flax oil extraction is primarily used as low value animal feed, even though it contains valuable protein, mucilage, fiber and lignans. A review of the literature has failed to identify an efficient integrated process to extract both gum and protein from flaxseed meal for commercial products development.

[0009] Flaxseed protein is commonly obtained by aqueous extraction of flaxseed meal involving solubilisation of proteins at high pH, followed by precipitation of proteins at their isoelectric point. However, high viscosity due to mucilage present in flaxseed meal reduces protein extraction efficiency and impedes the sedimentation of protein during solid-liquid separation (Dev & Quensel, 1988; Mazza & Biliaderis, 1989).

[0010] The first report of flaxseed protein extraction by Osborne (1892) showed the presence of an albumin-like protein and a globulin with 17.5% and 18.6% nitrogen content, respectively. However, the Osborne methodology was not commercially practical (Oomah & Mazza, 1993). Different methods for extraction of flaxseed protein have been investigated, such as alkaline extraction followed by isoelectric precipitation (Dev & Quensel, 1988; Karaca et al., 2011; Krause et al., 2002; Martinez-Flores et al., 2006; Mueller et al., 2010; Silva et al., 2013; Teh et al., 2014), using buffer (Li-Chan and Ma, 2002; Kaushik et al., 2016; Oomah et al., 1994), hexametaphosphate-assisted (Wanasundara & Shahidi, 1996), micellisation (Krause et al. , 2002), salt extraction (Oomah et al., 1994; Karaca et al., 2011), and acid extraction followed by pi precipitation (Teh et al., 2014).

[0011] To achieve high protein content, previous studies typically de-mucilage the whole flaxseed, de-hull, and defat the flaxseed meal before extraction. Using such pretreated flaxseed meal, Wanasundara & Shahidi (1996) optimized the hexametaphosphate-assisted extraction of flaxseed proteins using Response Surface Methodology (RSM). Sodium hexametaphosphate (SHMP, 2.75%, w/v) at pH 8.9 and a meal-to-solvent ratio of 1 :33 (w/v) extracted up to 77% of total nitrogen content of low-mucilage flaxseed meal. The highest amount of protein (57%) was recovered at pH 9.0 with 2.89% SHMP solution and a meal-to-solvent ratio of 1:33. Li-Chan and Ma (2002) reported flaxseed protein extraction conditions as follows: 0.1 M NaCI in 0.1 M Tris buffer, pH 8.6, seed:buffer ratio (w/v) 1 :16, 16 hours at 4 °C. After raw flaxseed demucilaging at 60 °C, defatting, and physical hull separation, Kaushik et al. (2016) isolated flaxseed proteins by alkaline extraction at pH 8.6 followed by isoelectric precipitation at pH 4.2 leading to a protein isolate of 90.60% purity and 20% recovery.

[0012] Without a demucilaging step, Martinez-Flores et al. (2006) obtained flaxseed protein concentrate from defatted and de-hulled flaxseed meal by optimized conditions of alkaline solubilisation at pH 11 and acid precipitation at pH 4.8 leading to a protein concentrate with 66% protein content and 25% carbohydrates.

[0013] In another report, starting with defatted flaxseed meal, flaxseed proteins were isolated by first processing the highly viscous suspension with cellulase to hydrolyze the fibre. The resulting less viscous suspension was then alkaline extracted and collected by pi precipitation. The product yield and quality were not reported (Udenigwe et al., 2009a). Cellulase-assisted extraction of flaxseed protein concentrate from defatted meal led to an increase in protein purity from 51% to 65% and a decrease in carbohydrate impurity from 40% to 26%. An extra step of ethanol extraction further improved the protein purity to 87% and reduced the carbohydrate impurity to 7% (Tirgar et al., 2017).

[0014] Using defatted flaxseed meal, optimum conditions for salt extraction and recovery of protein have been reported. Up to 82% of flaxseed protein can be recovered at neutral pH (pH 6.8), ionic strength of solvent of 1.28 M NaCI, and a solvent-to-meal ratio of 16. Under the reported conditions, solvent pH was not a significant factor affecting protein solubility and recovery but solvent-to-meal ratio and ionic strength were highly significant (Oomah et al. , 1994).

[0015] A US patent application (Rozenszain et al., 2012) described an aqueous process for preparing protein isolate and hydrolyzed protein concentrate from an oilseed in which oil seed meal is mixed with an aqueous solution to form a slurry. After solidiliquid separation followed by liquid phase separation to form an oil phase and an aqueous protein phase, the latter is subjected to ultrafiltration and diafiltration to obtain a retentate suitable to be spray dried to form the protein isolate with high concentrations of soluble protein (>90%) containing less than 2% (w/w) of oil. The solid phase insoluble protein is subjected to enzymatic hydrolysis and membrane filtration, then spray dried to obtain a hydrolyzed protein concentrate comprising amino acids and peptides, and including about 79-95% protein on a dry weight basis.

[0016] Wang et al. (2014) described the preparation of flax protein powder using defatted flaxseed meal wherein a water suspended mixture is directly hydrolyzed by protease to oligopeptides of <2 KDa (80-83%), extracted by alkali and precipitated by acid. The protein extraction rate is 55.7%, with a protein content of 94%.

[0017] Flax mucilage/gum has been extracted from whole flaxseed with hot water, followed by precipitation with ethanol and freeze drying or separation by filtration (Mazza and Oomah, 1995; Thakur et al., 2009; Singer et al., 2011; Ziolkovska, 2012; Bouaziz et al., 2016). Cui et al. (1994) reported optimized gum extraction from flaxseed using RSM exploring the impacts of extraction temperature, pH, and waterseed ratio with respect to yield, apparent viscosity and protein content of the final gum extracts. Temperature and pH were found to have significant influences on both the yield and quality of the extracted crude gum while the waterseed ratio had only minor effects. Optimum reported conditions for the extraction are 85-90 °C, pH 6.5-7.0, and a waterseed ratio of 13. In addition to the impact of extraction conditions, gum yield, composition and physical properties varied significantly with cultivar type, climate and crop age (Cui & Mazza, 1996; Kaewmanee et al., 2014).

[0018] Using whole flaxseed, Barbary et al. (2009) found that flax gum extracted at room temperature is lighter in color with lower protein impurity but also lower mucilage yield compared to boiling water extraction. Also with whole flaxseed, Kaushik et al. (2017) studied the effect of four different extraction temperatures on composition, structure and functional properties of flaxseed gum. As the extraction temperature increased from 30 °C to 90 °C, the gum extraction yield increased four times and the protein content also increased more than three times, the ratio of neutral to acidic monosaccharides decreased, while emulsifying activity and emulsion stability of the gum decreased significantly. Therefore, different extraction conditions may produce flax gum for different applications with different functionalities. In another recent report by Elboutachfaiti et al. (2017), the water-soluble flaxseed gum was extracted from flaxseeds by stirring in distilled water (400 rpm, 1 :25 w/v) for 60 min at 80 °C. After filtration and centrifugation, the supernatant was purified by tangential ultrafiltration on a 100 kDa normal-molecular-weight cutoff polyethersulfone membrane against distilled water, and finally the retentate solution was freeze-dried to obtain the flaxseed mucilage.

[0019] Recently, mechanically separated and defatted flaxseed hull has been used to extract soluble gum by boiling water extraction followed by ethanol precipitation and the product was further purified for analytical and activity analyses (Bouaziz et al., 2016).

[0020] Flaxseed mucilage mixed with protein has been extracted from crushed flaxseed meal at alkaline condition (pH 9-12) and precipitated near the pi of the protein, and protein concentrates containing different amounts of mucilage were prepared (Dev and Quensel, 1988). Starting from cold pressed flaxseed meal, after solvent defatting and solvent detoxification, Singer et al. (2011) reported preparation of mucilage/protein products from flaxseed with four different procedures: 1) co-precipitation method, 2) a modified co-precipitation method, 3) a boiling water method, and 4) an enzymatic method. Method 1) extracted soluble fractions by high pH water suspension at 1 :40 (w/v) ratio, followed by pi precipitation leading to mucilage/protein products with 63-65% protein and 15-16% mucilage/soluble dietary fiber (SDF). Method 2) modified from 1) by precipitation using ethanol instead of pi, led to products of ~9% protein and 74-86% SDF. Method 3) extracted soluble fraction with boiling water followed by ethanol precipitation leading to products with 19- 20% protein and 57-61% SDF. In method 4) water suspended samples were sequentially treated with protease and amylase leading to products with 7-8% protein and 5-7% SDF. It was concluded that the optimum conditions for preparing a high mucilage / low protein product is by method 2): 1 :40 (w/v) meal:water ratio, 1 :50 (v/v) mucilage : ethanol ratio, and 5 °C for precipitation and centrifuge.

[0021] US patent 5925401 (Kankaanpaa-antilla, 1999) suggested the use of an acid and an alkanol to precipitate proteins and mucilage from the alkaline extract. US patent 4915960 (Holmgren, 1990) describes an enzymatic hydrolysis method including phytase, protease and amylase for the preparation of a mucilage product with low protein content, high swelling and water retention functionality.

[0022] Fabre et al. (2015) compared three methods of aqueous extraction of flaxseed mucilage from whole flaxseeds (magnetic stirring, microwaves and ultrasounds) at a concentration of 5% (w/v) in water and a temperature of 50 °C. While microwaves are less efficient than a magnetic stirring, ultrasound-assisted extraction showed the highest mass transfer coefficient and a higher order kinetic. 7% of the seed mass was extracted after only 30 min of treatment. Ultrasound assisted extraction decreases the intrinsic viscosity of the mucilage from 12.5 dL/g (for magnetic stirring) to 6.2 dL/g, and the weight-average molecular weight of the largest polysaccharides from 1.5*10 ⁶ Da to 0.5x10 ^s Da, whilst having a limited impact on protein content and monosaccharide composition.

[0023] To date, there are only a few reports of the preparation of flaxseed peptide and they are mostly carried out for the academic study of biological activities. For such purpose, flaxseed peptides were prepared by enzymatic hydrolysis of purified flaxseed protein isolates and in some studies followed by complicated membrane separation and HPLC purification processes. Different proteases have been tested such as Alcalase® (Omoni & Aluko 2006; Silva et al., 2017), Flavourzyme® (Marambe & Shand, 2008), trypsin, papain, thermolysin, and pancreatin (Udenigwe et al., 2009a; Karamac et al., 2014). In a report by Udenigwe et al., a protein isolate was produced from cellulase-treated defatted flaxseed meal followed by hydrolysis with seven proteases and the hydrolysates were evaluated for antioxidant and anti-inflammatory properties. Low molecular weight and cationic peptide fractions isolated by ultrafiltration and ion-exchange chromatography showed antioxidant and other biological properties that were dependent on the specificity of proteases and size of the resulting peptides. The results suggested that flaxseed protein hydrolysates may act as anti-inflammatory agents and serve as potential ingredients for the formulation of therapeutic products (Udenigwe et al., 2009b).

[0024] When flaxseed protein isolate is hydrolyzed to different degrees of hydrolysis (DH) by pancreatin, the antiradical activity increases along with an increasing DH. The maximum activity was reached by the hydrolysate with DH 20% that was predominated by polypeptides and peptides with MW of 0.238-1.046 kDa (Karamac et al., 2014).

[0025] Ultrafiltration membrane-separated peptide fractions from thermoase-digested flaxseed protein hydrolysate demonstrated that 3% thermoase digestion product exhibited the highest biological activities. Membrane ultrafiltration resulted in significant activity increases in peptides <3 kDa, indicating the potential of flaxseed protein-derived bioactive peptides as ingredients for the formulation of antihypertensive functional foods and nutraceuticals (Nwachukwu et al., 2014).

[0026] Hwang et al. (2016) studied the antioxidant and antibacterial activities of flaxseed protein hydrolysate made from crude protease and further separated into five fractions by ultrafiltration membranes with molecular weight cut-offs of 10, 5, 3 and 1 kDa. Different fractions showed different biological activities, suggesting that it is feasible to derive distinct functional ingredients from flaxseed protein by controlled hydrolysis. Perreault et al. (2017) studied the effect of high hydrostatic pressure (HHP) pretreatment of flaxseed protein isolate on peptide release by trypsin only and trypsin-pronase treatment and confirmed that HHP enhanced the generation of antioxidant peptides.

[0027] Even though many beneficial activities of flaxseed peptide have been confirmed, no process or technology developed for large scale industrial flaxseed peptide production has been found in the literature reviewed so far.

[0028] Using defatted flaxseed meal, different methods have been reported to prepare flax lignan concentrate (FLC). Zanwar et al. (2011) prepared lignan extract from flaxseed meal by trying several methods reported by different authors (Charlet et al., 2002; Eliasson et al., 2003; Muir & Westcott, 2000; Meagher et al., 1999; Fritsche et al., 2002). After analysis of the major lignan, namely secoisolariciresinol diglucoside (SDG), content in the different extracts, the method of Eliasson et al. (2003) led to the highest SDG content. Briefly, defatted flaxseed meal was hydrolysed with 1 M aqueous sodium hydroxide for 1 h at room temperature with intermittent shaking, followed by extraction with 50% ethanol. The filtrate was then acidified to pH 3 and dried on a tray dryer at 50 °C. The yield of FLC was 14.81% w/w (Zanwar et al., 2011).

[0029] A patented process (US 5705618, Westcott and Muir, 1998) describes the extraction of lignans from defatted flaxseed meal with an aliphatic alcohol solvent, e.g. a mixture of methanol or ethanol with water, to obtain a lignan concentrate that was subjected to a base-catalyzed hydrolysis to liberate lignans into a non-complexed form. The hydrolyzed concentrate was subjected to either a liquid/liquid partition, e.g. by an ethyl acetate/water system, or anion exchange to further enrich the lignans followed by chromatographic separation to isolate lignans at a purity of greater than 90 percent. The major lignan (SDG) was found in amounts of up to 20 mg per gram of defatted flaxseed. [0030] Zhang et al. (2007) reported optimization of ethanol-water extraction of lignans from flaxseed. The optimal extraction conditions were: ethanol 70%, extraction time of 28 h at 40 °C. Under this condition, extraction yield of lignans reaches 8.975% (w/w; lignans/defatted flaxseed powder), which is close to the predicted value of 9.316%.

[0031] Lehraiki et al. (2010) optimized a lignan extraction procedure from defatted flaxseed meal as follows: a direct hydrolysis in hydrochloric acid (1 M) at 100 °C for 1 hour followed by an extraction with a mixture of ethyl acetate/hexane (90:10 v/v), resulting in the isolation of secoisolariciresinol and anhydrosecoisolariciresinol with a purity of 97% and 98%, respectively, as determined by high-performance liquid chromatography.

[0032] A microwave-assisted extraction (MAE) method for lignan extraction that used a direct hydrolysis approach was developed by Nemes and Orsat (2010, 2011). The MAE method was used forSDG extraction from defatted flaxseed meal and achieved a 6% increase in the extraction yield as opposed to the conventional direct hydrolysis method developed by Eliasson et al. (2003), with additional benefits such as: reduction in extraction time by 95%, reduction in the NaOH concentration by half and an internal standard (o-coumaric acid) recovery of 97%. MAE methods published by others also showed improvements in the extraction yields of SDG from flax hull (Zhang and Xu 2007) and flaxseed cake (Beejmohun et al. 2007) as opposed to conventional methods such as stirring extraction and Soxhlet extraction.

[0033] A process for isolating and purifying SDG extractions, mainly using supercritical C0 ₂ and chromatographic separation has been patented (Pihlava et al., 2004). Comin et al (2011) reported study of supercritical CO2 extraction of flax Lignans and concluded that SC-CO2 extraction resulted in much lower quantities of SDG compared to traditional extraction. On the contrary, a different report evaluated supercritical antisolvent fractionation (SAF) of lignans from the ethanol extract of defatted flaxseed meal and concluded that SAF is an appropriate technique for the isolation of flaxseed lignans (Perretti et al., 2013). The procedure was optimized using a full factorial design with three factors and two levels and the optimal conditions were calculated through RSM. The optimized SAF process led to significant increases of lignan content from 1.66 g/L to 3.42 to 12.96 g/L. Following such a process, a pilot plant scheme was proposed by the authors.

[0034] Fuentealba et al. (2015) reported an RSM optimized process for flaxseed lignan (SDG) extraction and the isolation by a simple HPLC-UV method. The optimal conditions established for the extractions are: 47 °C, 58 mmo!/L sodium methoxide, and 24 h. This methodology was applied and showed that SDG content in different varieties of flaxseed ranged from 10.8 to 17.9 mg/g in defatted flaxseed flour and from 6.0 to 10.9 mg/g in whole flaxseed.

[0035] Lignan-rich products have also been extracted from defatted flaxseed hulls with aqueous ethanol and commercialized in US and Europe, e.g. Lignans for Life® and LinumLife® EXTRA (Bisson et a!., 2014).

[0036] Renouard et al. (2010) reported cellulase-assisted release of lignan from extracts of flax hulls and whole flaxseeds. Cellulase treatment led to improved lignan extraction with a better yield as compared to b-glucosidase treatment. An optimized extraction process follows the following successive steps: 16 h of 70% hydromethanolic extraction, 6 h of 0.1 M NaOH hydrolysis followed by a 6 h incubation with 1 unit ml ^-1 of cellulase R10 in 0.1 M citrate-phosphate buffer pH 2.8 at 40 °C. Under these conditions, all forms of the main flax lignan were recovered as the aglycone form, i.e. secoisolariciresinol. Highest yields in SDG equivalent reached 7.72% of flaxseed hull dry weight and 2.88% of whole seed weight, thus allowing a significant improvement in comparison with previously published methods.

[0037] Ribeiro et al. (2013) reported an optimized enzyme-enhanced extraction of phenolic compounds and proteins from flaxseed meal using a solvent with 10% ethanol. A solution containing 6.6 g/kg meal of phenolics was obtained after treatment with enzyme mixtures of carbohydrase and protease; corresponding to a 10-fold increase of phenolics extraction yield compared to a conventional nonenzymatic extraction.

[0038] Corbin et al. (2015) developed an efficient ultrasound assisted extraction (UAE) of phenolic compounds from flaxseed hull. The optimal conditions for UAE of phenolic compounds from flaxseeds are: water as solvent supplemented with 0.2 N of NaOH for alkaline hydrolysis of the SDG-HMG complex, 60 min, 25 °C and an ultrasound frequency of 30 kHz. Under these conditions, highest yields of lignan components were extracted in comparison to other published methods. The procedure was suggested as a valuable method for efficient extraction and quantification of the main flaxseed phenolics. Moreover, the UAE method was suggested as of particular interest within the context of green chemistry in terms of reducing energy consumption and valuation of flaxseed meal by-products.

[0039] Extraction of lignans, proteins and carbohydrates from cold pressed flaxseed meal with pressurized low polarity water has been reported (Ho et al., 2007). All three components were successfully extracted. Optimal conditions for extraction of lignans were: pH 9 buffered water at 170 °C and 5.2 MPa, and 100 mL/g solvent to solid ratio. Protein optimal yield was obtained at pH 9, 160 °C and 210mL/g S/S. For carbohydrates, a temperature of 150 °C, 210ml_/g S/S and pH 4 or 6.5 was recommended. However, both protein and carbohydrate may be degraded under such conditions and a commercially viable technology based on this discovery has not been reported so far.

[0040] Gutierrez et al. (2010) reported an integrated process for the fractionations of polysaccharides, polyphenols and proteins from defatted flaxseed meal whereby the meal is first alkaline extracted, with the supernatant containing both protein and soluble polysaccharide, and the insoluble residue used for ethanol extraction of the polyphenol products. The protein isolate was first prepared by pi precipitation and polysaccharide was obtained by ethanol precipitation of the remaining supernatant. Yields of protein and polysaccharide were 53% and 11% (w/w), respectively, of the defatted flaxseed meal. Due to differentiated precipitations, both protein and polysaccharide were quite pure, with low impurities. However, the polyphenol extraction was very inefficient compared to the method reported by Ho et al. (2007). Even though the yield and quality of each product may not have been optimized, this is the only report so far identified assessing the simultaneous extractions of three products from flaxseed meal.

[0041] Mueller et al. (2010) reported a simplified linseed meal fractionation procedure for the extraction of protein and fibre for pilot scale processing using defatted flaxseed meal. Optimal parameters screened by RSM are: 50 °C, pH 8.5, solidiliquid ratio 1 :15, and no salt addition, leading to a soluble protein/fiber fraction with 70% protein yield and an insoluble fiber fraction with 30% protein yield. Even though this process has been scaled up to pilot scale, it only produced one soluble product with all soluble proteins and mucilage as a mixture.

[0042] Ribeiro et al. (2013) reported an optimized enzyme-enhanced extraction of phenolic compounds and proteins from flaxseed meal using a solvent with 10% ethanol. A solution containing 6.6 and 152 g/kg meal of phenolics and proteins was obtained after treatment with enzyme mixtures of carbohydrase and protease, corresponding to a 10- and 14-fold increase of phenolics and proteins extraction yield compared to a conventional nonenzymatic extraction.

[0043] A Chinese patent application (2015, CN104478954A) described a method for directly extracting lignans and removing cyanogenetic glycoside from flaxseed meal with low- concentration ethanol alkali solution by virtue of a one-step extraction. The resulting detoxified, high-protein flaxseed meal can be directly used as feedstuff. A low-energy consumption and low- production cost process allows the comprehensive utilization of the flaxseed meal.

[0044] While many processes have been described for the extraction of protein, mucilage/gum, or lignan from a biomass, such as flaxseed, there remains a desire for a practical and effective integrated process for the extraction of multiple products from a biomass.

SUMMARY

[0045] A first aspect of the disclosure is a process for isolating polypeptides and mucilage from a biomass, the process comprising: i. combining a biomass and a first aqueous solution to form a first mixture having a pH of about 6 or higher; ii. incubating the first mixture at a temperature of about 60 °C or less for a period of time sufficient to solubilize at least a fraction of polypeptides present within the biomass; iii. separating the first mixture into a first solid fraction and a first liquid fraction; iv. combining the first solid fraction and a second aqueous solution to form a second mixture, the second mixture having a pH of about 7 or lower; v. incubating the second mixture at a temperature of about 80 °C or higher for a period of time sufficient to solubilize at least a fraction of mucilage present within the second mixture; and vi. separating the second mixture into a second solid fraction and a second liquid fraction, wherein the first liquid fraction comprises the isolated polypeptide mixture and the second liquid fraction comprises the isolated mucilage.

[0046] In an embodiment, the process further comprises a step of subjecting the isolated polypeptide mixture to treatment with at least one protease to produce hydrolyzed isolated polypeptide mixture.

[0047] In an embodiment, the process further comprises: vii. combining the first liquid fraction with the second solid fraction to form a third mixture; viii. subjecting the third mixture to the protease treatment; and ix. separating the third mixture into a third solid fraction and a third liquid fraction, wherein the third solid fraction is a fibre-rich product and the third liquid fraction comprises the hydrolyzed isolated polypeptide mixture.

[0048] In embodiments, step (i) of the process comprises combining the biomass and the first aqueous solution at a liquid to solid ratio of about 10:1 to about 50: 1 , about 15:1 to about 40: 1 , or about 35:1. In embodiments of the process, the first mixture has a pH of about 6 to about 11, about 9 to about 11 , or about 10.75. In an embodiment of the process, the first aqueous solution has a pH that is lower than the pH of the first mixture and the pH of the first mixture is established by addition of a base or basic solution after the first aqueous solution is combined with the biomass. In an embodiment of the process, the first aqueous solution has a pH of about 6 to about 8. In an embodiment of the process, the first aqueous solution is water.

[0049] In an embodiment of the process, the second aqueous solution has a pH that is higher than the pH of the second mixture and the pH or the second mixture is established by addition of an acid or acidic solution after the second aqueous solution is combined with the biomass. In an embodiment of the process, the second aqueous solution has a pH of about 6 to about 8. In an embodiment of the process, the second aqueous solution is water.

[0050] In embodiments of the process, in step (ii) the first mixture is incubated at a temperature of about 4 °Cto about 60 °C, about 10 °C to about 50 °C, or at room temperature. In embodiments of the process, step (ii) is carried out for a duration of about 10 minutes to about 120 minutes, about 15 minutes to about 60 minutes, or about 30 minutes.

[0051] In an embodiment, the process further comprises washing the first solid fraction with an aqueous wash solution one or more times prior to combining the first solid fraction with the second aqueous solution in step (iv).

[0052] In an embodiment of the process, washing the first solid fraction comprises: a. combining the aqueous wash solution with the first solid fraction to form a wash mixture; b. incubating the wash mixture for a period of time; and c. separating the wash mixture into a washed first solid fraction and a wash liquid fraction, wherein the pH of the aqueous wash solution is selected so that the wash mixture has a pH of about 6 to about 11.

[0053] In embodiments of the process, in step (a) the aqueous wash solution is combined with the first solid fraction at a liquid to solid ratio of between about 10:1 to about 40:1 , between about 15:1 to about 30:1, or about 17:1. In embodiments of the process, step (b) comprises incubating the wash mixture at a temperature of about 4 °C to about 60 °C, about 10 °C to about 50 °C, or at room temperature. In embodiments of the process, step (b) is carried out for a duration of about 10 minutes to about 90 minutes, about 15 minutes to about 60 minutes, or about 15 minutes.

[0054] In embodiments of the process, the pH of the aqueous wash solution is selected so that the wash mixture has a pH of about 7 to about 10 or a pH of about 10. In an embodiment of the process, the aqueous wash solution is water. In an embodiment of the process, step (b) is carried out with mixing.

[0055] In an embodiment of the process, step (vii) comprises combining the second solid fraction with the first liquid fraction and the wash liquid fraction to form the third mixture.

[0056] In embodiments of the process, step (iv) comprises combining the first solid fraction with the second aqueous solution at a liquid to solid ratio of about 10:1 to about 50:1 , about 12:1 to about 30:1 , or about 17:1. In embodiments of the process, the pH of the second mixture is about 1 to about 7, about 2 to about 6, or about 4. In embodiments of the process, in step (v) the second mixture is incubated at a temperature of about 80 °C to about 100 °C or about 100 °C. In embodiments of the process, step (v) is carried out for a duration of about 30 minutes to about 240 minutes, about 60 minutes to about 150 minutes, or greater than or equal to about 110 minutes.

[0057] In embodiments of the process, step (viii) comprises adding at least one protease to the third mixture in an amount of about 0.5% to about 10%, about 2% to about 8%, about 3% to about 7%, or about 3.3% (v/w) relative to the weight of the starting biomass. In an embodiment of the process, the at least one protease comprises an endoprotease. In an embodiment, the protease comprises Subtilisin. In embodiments of the process, the protease treatment is carried out at a temperature of about 30 °C to about 90 °C, about 40 °C to about 80 °C, about 50 °C to about 75 °C, or about 55 °C. In embodiments of the process, the duration of the protease treatment in step (viii) is about 20 minutes to about 360 minutes, about 30 minutes to about 240 minutes, about 40 minutes to about 90 minutes, or about 48 minutes. In embodiments of the process, the protease treatment is carried out at a pH of about 4 to about 11 , about 5 to about 10, about 7 to about 10, or about 10. In an embodiment of the process, the protease treatment is carried out with mixing. In an embodiment, the mixing comprises stirring at a rate of about 30 RPM to about 200 RPM.

[0058] In an embodiment, the process further comprises subjecting the isolated polypeptides to treatment with a second protease. In embodiments, the treatment with the second protease comprises adding the second protease in an amount of about 0.5% to about 10%, about 2% to about 8%, about 3% to about 7% (v/w), or about 3.3% (v/w) relative to the weight of the starting biomass. In an embodiment, the treatment with the second protease is performed concurrently with the protease treatment. In another embodiment, treatment with the second protease is performed subsequent to the protease treatment. In embodiments, the treatment with the second protease is carried out at a pH of about 4 to about 11, about 5 to about 10, about 6 to about 9, or about 7. In embodiments, the treatment with the second protease is carried out at a temperature of about 30 °C to about 70 °C or about 50 °C to about 60 °C. In embodiments, the treatment with the second protease is carried out for a duration of about 20 minutes to about 360 minutes, about 30 minutes to about 240 minutes, about 40 minutes to about 90 minutes, or about 48 minutes. In an embodiment, the treatment with the second protease is carried out with mixing. In an embodiment, the mixing during the treatment with the second protease comprises stirring at a rate of about 30 RPM to about 200 RPM. In an embodiment, the second protease comprises an exoprotease. In an embodiment, the second protease comprises a protease preparation from Aspergillus oryzae. In another embodiment, the second protease comprises at least one endopeptidase and at least one exopeptidase.

[0059] In embodiments of the process, the biomass is a biomass that comprises greater than or equal to about 2% protein by weight and greater than or equal to about 0.5% mucilage by weight; greater than or equal to about 8% protein by weight and greater than or equal to about 4% mucilage by weight; or greater than or equal to about 20% protein by weight and greater than or equal to about 10% mucilage by weight.

[0060] In an embodiment of the process, the biomass comprises an aquatic biomass, an aquatic biomass meal, a plant seed, or a plant seed meal. In an embodiment, the plant seed is a grain crop seed. In an embodiment, the biomass is a biomass that comprises at least 1 mg of lignan per gram of biomass. In other embodiments, the biomass comprises at least 5 mg of lignan per gram of biomass, at least 10 mg of lignan per gram of biomass, at least 15 mg of lignan per gram of biomass, at least 20 mg of lignan per gram of biomass, or at least 25 mg of lignan per gram of biomass.

[0061] In an embodiment of the process, the biomass comprises a seaweed, a microalga, locust bean, carob tree seed, chia seed, flaxseed, or Cyamopsis tetragonolobus seed; or a meal of one or more thereof. In an embodiment, the biomass comprises flaxseed meal.

[0062] A further aspect of the disclosure is an isolated polypeptide mixture, mucilage-rich product, or fibre-rich product isolated by a process described herein. In an embodiment, the polypeptide mixture comprises at least 15 mg of lignin per gram of the isolated polypeptide mixture. In an embodiment, the mucilage-rich product comprises at least 6 mg of lignin per gram of the mucilage- rich product. Yet another aspect of the disclosure is a food, feed, beverage, or nutraceutical product comprising the isolated polypeptide mixture, the mucilage-rich product, the fibre-rich product, or any mixture of two or more thereof.

BRIEF DESCRIPTION OF DRAWINGS

[0063] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0064] Figure 1A is a flow chart illustrating the steps carried out in an embodiment of the purification process.

[0065] Figure 1 B is a flow chart illustrating the steps carried out in an embodiment of the purification process, in which the isolated polypeptides are subjected to protease treatment.

[0066] Figure 1C is a flow chart illustrating the steps carried out in an embodiment of the purification process in which the solid residue remaining after the gum extraction step (the second solid fraction) is combined with the isolated polypeptides (first liquid fraction) prior to protease treatment.

[0067] Figure 2A is a flow chart illustrating the steps carried out in another embodiment of the purification process, in which the first solid fraction is subjected to one or more wash steps. The dashed arrows indicate that the wash step which may be repeated more than once. [0068] Figure 2B is a flow chart illustrating the steps carried out in another embodiment of the purification process, in which the first solid fraction is subjected to one or more wash steps and the isolate polypeptides are subjected to protease treatment. The dashed arrows indicate that the wash step which may be repeated more than once.

[0069] Figure 2C is a flow chart illustrating the steps carried out in another embodiment of the purification process, in which the first solid fraction is subjected to one or more wash steps and in which the solid residue remaining after the gum extraction step (the second solid fraction) is combined with the isolated polypeptides (first liquid fraction) prior to protease treatment. The dashed arrows indicate that the wash step may be repeated more than once.

[0070] Figure 3 is a graph showing the effect of the pH on protein extraction.

[0071] Figure 4 is a graph showing the effect of the pH on protein/carbohydrate selectivity.

[0072] Figure 5 is a graph showing the effect of pH and extraction time on protein/carbohydrate selectivity during the protein extraction step.

[0073] Figure 6 is a graph showing the effect of the liquid to solid (L:S) ratio on protein extraction yield during the protein extraction step.

[0074] Figure 7 is a graph showing the effect of temperature and pH on soluble protein extraction during the protein extraction step.

[0075] Figure 8 is a graph showing the effect of temperature, pH, and extraction time on the mass of protein recoverable during the protein extraction step.

[0076] Figure 9 is a graph showing a comparison of product solid contents of supernatant from direct pH 10 extraction and supernatant after protein hydrolysis of the mixture of extraction supernatant and gum extraction residue.

[0077] Figure 10 shows SDS-PAGE analysis of the polypeptide rich fraction (the third liquid fraction) after protease treatment with varying duration (in minutes) and amount of enzyme.

[0078] Figure 11 is a graph showing the pH profile change during enzyme hydrolysis of the third mixture. [0079] Figure 12 is a graph showing the effect of the inclusion of salts in the first aqueous solution on protein extraction yield.

[0080] Figure 13 is a graph showing the effect of the L:S ratio and extraction time on gum extraction at 100 °C based on carbohydrate content.

[0081] Figure 14 is a graph showing the effect of incubation temperature on gum extraction at pH 4.

[0082] Figure 15 is a graph showing the effect of the L:S ratio on gum extraction yield a pH 4 and 100 °C.

[0083] Figure 16 is a graph showing the effect of the incubation temperature (°C) on gum solubilisation rate at a L:S ratio of 30:1.

[0084] Figure 17 is a graph showing the effect of temperature, pH, and extraction time on the amount of gum extraction based on carbohydrate content.

[0085] Figure 18 is a graph showing the effect of defatting of flaxseed meal and washing of the first solid fraction on protein recovery. 1 = liquid fraction from first mixture without washing, 2 = single wash, 3 = two washes, 4 = three washes.

[0086] Figure 19 is a graph showing the effect of stirring (control), ultrasound, and blending on protein/carbohydrate selectivity and viscosity during protein extraction.

[0087] Figure 20 is a graph showing the effect of salts addition on protein and solids recovery when protein extraction is carried out at pH 11.

[0088] Figure 21 is a graph showing the effect of centrifugation speed on extracted solids recovery after protein extraction.

[0089] Figure 22 is a graph showing the effect of precipitation strategies on total solid recovery.

[0090] Figure 23 is a graph showing the effect of NaOH addition on the pH of the first mixture, when the first mixture is a 35:1 flaxseed meal solution.

[0091] Figure 24 shows a response surface plot of solids recovery as a function of extraction temperature and time used for protein extraction. [0092] Figure 25 shows a response surface plot of solids recovery as a function of extraction temperature and pH used for protein extraction.

[0093] Figure 26 shows a response surface plot of protein/carbohydrate selectivity as a function of extraction pH and temperature used for protein extraction.

[0094] Figure 27 shows a response surface plot of solids recovery as a function of pH and L:S ratio used for protein extraction.

[0095] Figure 28 shows a response surface plot of protein recovery as a function of pH and L:S ratio used for protein extraction.

[0096] Figure 29 shows a response surface plot of protein/carbohydrate selectivity as a function of protein extraction and wash step L:S ratio.

[0097] Figure 30 shows a response surface plot of protein recovery as a function of protein extraction and wash step L:S ratio.

[0098] Figure 31 shows a response surface plot of protein recovery relative to water consumption as a function of protein extraction and wash step L:S ratio.

[0099] Figure 32 is a graph showing protein recovered relative to NaOH added during protein extraction as a function of pH.

[0100] Figure 33 is a desirability function describing the optimal pH for protein extraction in terms of water and NaOH consumption.

[0101] Figure 34 is a graph showing the effect of the mixing speed on gum solids extraction yield from the gum extraction step.

[0102] Figure 35 is a graph showing the effect of extraction time and L:S ratio on gum yield from the gum extraction step

[0103] Figure 36 is a response surface plot for mucilage yield as a function of L:S ratio and extraction time used for the gum extraction step..

[0104] Figure 37 is a desirability plot of favorable mucilage extraction time and L:S ratio at minimum time and maximum yield. [0105] Figure 38 is a contour plot of favorable mucilage extraction time and L:S ratio at minimum time and maximum yield.

[0106] Figure 39 is a desirability plot of favorable mucilage extraction time and L:S ratio at minimum water consumption, minimum time, and maximum yield.

[0107] Figure 40 is a contour plot of favorable mucilage extraction time and L:S ratio at minimum water consumption, minimum time, and maximum yield.

[0108] Figure 41 is a graph showing a mucilage extraction time course comparing yield with boiling versus pressurized extraction.

[0109] Figure 42 is a graph showing loss and recovery of dissolved solids during hydrolysis of the third mixture.

[0110] Figure 43 is a graph showing the effect of: inclusion or exclusion of the second solid fraction (residual solids after the protein extraction step) in the third mixture, enzyme treatment time, and enzyme dose on solids yield from the third mixture.

[0111] Figure 44 is a response surface plot of dissolved solids concentration as a function of enzyme dose and hydrolysis time.

[0112] Figure 45 is a desirability plot for impact on solids yield with respect to enzyme dose and hydrolysis time.

[0113] Figure 46 is a contour plot for impact on solids yield with respect to enzyme dose and hydrolysis time.

[0114] Figure 47 is a response surface plot of the protein content of the third liquid fraction as a function of enzyme dose and hydrolysis time.

[0115] Figure 48 is a graph showing the effect of defatting and enzyme treatments on protein recovery.

[0116] Figure 49 shows SDS-PAGE analysis of changes in protein molecular size upon sequential, combined, and individual enzyme hydrolysis.

[0117] Figure 50 shows a densitometric profile of lane 1 of the SDS-PAGE gel shown in Fig. 49. [0118] Figure 51 shows a densitometric profile of lane 2 of the SDS-PAGE gel shown in Fig. 49.

[0119] Figure 52 shows a densitometric profile of lane 3 of the SDS-PAGE gel shown in Fig. 49.

[0120] Figure 53 shows a densitometric profile of lane 4 of the SDS-PAGE gel shown in Fig. 49.

[0121] Figure 54 shows a densitometric profile of lane 5 of the SDS-PAGE gel shown in Fig. 49.

[0122] Figure 55 shows a densitometric profile of lane 6 of the SDS-PAGE gel shown in Fig. 49.

[0123] Figure 56 shows a densitometric profile of lane 7 of the SDS-PAGE gel shown in Fig. 49.

[0124] Figure 57 shows a densitometric profile of lane 8 of the SDS-PAGE gel shown in Fig. 49.

[0125] Figure 58 shows a densitometric profile of lane 9 of the SDS-PAGE gel shown in Fig. 49.

[0126] Figure 59 is a graph showing the lignan content of a commercial Flax Hull Lignan; defatted flaxseed meal; polypeptides, mucilage-rich product, gum extraction residue, and fibre-rich product produced by extraction of defatted flaxseed meal according to the processes described herein.

DETAILED DESCRIPTION

[0127] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.

Definitions

[0128] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. [0129] As used herein in the specification and in the appended claims, the term “polypeptide” refers to a molecule comprised of two or more amino acid residues joined by peptide bonds. A polypeptide may also be referred to as a “protein” or a “peptide” and these terms are used interchangeably herein.

[0130] As used herein in the specification and in the appended claims, the term “mucilage” refers to a complex mixture of polysaccharides that are naturally produced by mucilage-producing organisms, such as plants or microbes. A large number of plants produce mucilage. The polysaccharides in mucilage are water-soluble, or at least swell perceptibly in water. Mucilages can occur in high concentrations in different plant organs. In flax seed, mucilage is contained mainly in the hull, or, to be more precise, in mucous. Mucilage may also be referred to as “gum” and these terms are used interchangeably herein. Mucilage and gum may also be referred to as “hydrocolloid”.

[0131] As used herein in the specification and in the appended claims, “fibre rich product” refers to the residual biomass remaining after the protease treatment of the third mixture produced by a process as described herein (e.g. see Figure 1C or 2C).

[0132] As used herein in the specification and in the appended claims, “biomass” refers to organisms, microorganisms, parts of organisms, organismal matter, and materials derived from organismal matter; including but not limited to agricultural and marine plants, plant parts, seeds, oilseeds, seaweeds, algae, and by-products thereof.

[0133] As used herein in the specification and in the appended claims, “aqueous solution” refers to a solution in which water is the solvent. The aqueous solution may comprise one or more dissolved water-soluble solutes, such as salts or other water-soluble compounds. As used herein, the term “aqueous solution” includes water. Water used in the processes described herein may or may not be treated to reduce dissolved salts and other impurities, for example by deionization, distillation, and/or filtration.

[0134] As used herein in the specification and in the appended claims, “protease” refer to an enzyme or mixture of enzymes that is capable of the hydrolytic degradation of proteins or polypeptides into smaller amino acid polymers or individual amino acids. A protease may also be referred to as a proteinase, proteolytic enzyme, protein hydrolase, or peptidase. [0135] As used herein in the specification and in the appended claims, “isolating”, “purifying”, or “purification” refers to the process of removing, completely or partially, one or more components present in a biomass to increase the purity of one or more desired components present in the biomass, such as polypeptides, mucilage, or fibre-rich material. The result of the isolation or purification process is a “purified” or “isolated” component.

[0136] As used herein in the specification and in the appended claims, “about” or “approximately” means within ±10% of a given value or range or optionally within ±5% of a given value or range. “About” may also mean within measurement error, or within the degree of variation that would be expected due to experimental error.

[0137] Unless context clearly dictates otherwise, as used herein in the specification and in the appended claims, singular referents such as “a”, “an”, and “the” include both singular and plural references. For example, a composition that is defined to comprise “a” component may include one or more than one of said component.

[0138] As used herein in the specification and in the appended claims, the term “comprising” is an open term that requires the presence of a given element(s), while also allowing for the presence of additional unrecited elements.

[0139] As used herein in the specification and in the appended claims, “incubating” refers to maintaining a composition (e.g. an item, sample, mixture, or reaction mixture) for a period of time under specific conditions. Incubating at a particular temperature means that the composition is maintained for a period of time at the given temperature. Similarly, carrying out a reaction at a particular temperature means carrying out the reaction at the given temperature. For example, the incubation may be carried out in a temperature controlled environment such as, but not limited to, an incubator, water bath, a temperature controlled chamber or vessel, or a temperature controlled atmosphere, where the temperature controlled environment has the given temperature.

[0140] As used herein in the specification and in the appended claims, “liquid to solid” ratio or “L:S” ratio refers to the ratio between liquid and solid components in a mixture, on a volume per weight (v/w) basis.

[0141] As used herein in the specification and in the appended claims, “room temperature” means a temperature of about 15 °C to about 25 °C, preferably about 18 °C to about 23 °C. [0142] As used herein in the specification and in the appended claims, “separating” a mixture into a solid fraction and a liquid fraction means using a separation method such as, but not limited to, centrifugation, gravitational separation, or filtration to separate a majority of the liquid fraction present within the mixture from a majority of the solid fraction present within the mixture.

[0143] As used herein in the specification and in the appended claims, “mixing” refers to the act of applying physical agitation to a mixture, for example by stirring, blending, shaking, swirling, or ultrasound.

[0144] Throughout the specification and in the appended claims, various mixtures are defined to have a particular pH, which may be selected from a range of pH values. In the course of carrying out the processes described herein, these mixtures are subjected to incubation periods, during which the pH of the mixture may change. When a mixture is defined as having a particular pH, this means that the mixture has the given pH, or a pH within the given range, at the start of the incubation period. It is not required for the given pH to be maintained over the course of incubation, as extraction or hydrolysis may cause the pH of the mixture to change over time. For greater clarity, and by way of example, a mixture having a pH of 10 at the start of incubation, but having a pH of 7 at the end of incubation, is considered to be a mixture having a pH of 10.

[0145] Various parameters are described herein, in the specification and in the appended claims, in terms of ranges of values. When a range is provided, it includes any subranges and individual values within the given range, even though specific subranges and individual values may not be individually recited. For example, a pH range of about 9 to about 12 includes subranges and individual values such as 9 to 11.4, 10.5 to 11.8, 10 to 11, 10, and 11.25.

[0146] As used herein in the specification and in the appended claims, "or" or “and/or” should be understood to be inclusive, i.e., meaning the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of' or "exactly one of" or, when used in the claims, "consisting of' will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0147]As used herein in the specification and in the appended claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

Description

[0148] The present application is directed to a process for isolating polypeptides, mucilage, and optionally a residual fibre-rich product from a biomass. The isolated polypeptides may further be hydrolyzed to produce hydrolyzed isolated polypeptides. The steps carried out in different embodiments of the process are outlined in the flow charts shown in Figures 1A, 1 B, 1C, 2A, 2B, and 2C. Generally, the process includes a first protein (aka polypeptide) extraction step and a second gum (aka mucilage) extraction step.

[0149] Polypeptide Extraction

[0150] To extract polypeptides, a biomass is combined with an aqueous solution to form a first mixture having a pH of about 6 or higher. In an embodiment, the aqueous solution has a lower pH than the first mixture, and the pH of the first mixture is established by addition of a base or basic solution after the biomass is combined with the aqueous solution. Any suitable base or basic solution may be used, as will be recognized by one skilled in the art. Examples of suitable bases include sodium hydroxide, sodium carbonate, calcium hydroxide, and potassium hydroxide. In an embodiment, the pH of the first mixture is about 6 or higher. In other embodiments, the pH of the first mixture is about 6 to about 12, about 6 to about 11, about 6 to about 10, about 7 to about 11, about 8 to about 11 , about 9 to about 11 , about 10 to about 11, about 7 to about 12, about 8 to about 12, about 9 to about 12, or about 10 to about 12. In other embodiments, the pH of the first mixture is about 11 or about 10.75.

[0151] The biomass may be a plant or aquatic biomass. The biomass may be a plant, plant part, seed, seaweed, algae, or a by-product of one or more thereof. In an embodiment, the biomass is a meal resulting from seed oil extraction. The meal may be employed as-is, or defatted prior to use in the process. While the process has been exemplified using flax meal, other biomasses and oil seed meals may be used as starting materials for the extraction process. The process is particularly suitable for biomasses that are high in protein and mucilage. Examples of such biomasses include seaweed, microalgae, locust bean, carob tree seed, chia seed, flaxseed, perilla seed, and Cyamopsis tetragonolobus seed. As examples, protein and total carbohydrates content of chia, flax, and perilla seeds are ~21-25% and ~23-45%, respectively (Sargi et al., 2013). Mucilage constitutes part of the carbohydrates and corresponds to ~ 6% of chia seed and 7-14% of flaxseed weight (Reyes-Caudillo et al., 2008; Oomah et al., 1995; Singer et al., 2011). In an embodiment, the biomass is flax meal.

[0152] In an embodiment, the biomass has a protein content of at least 2% protein by weight and a mucilage content of at least 0.5% by weight. In another embodiment, the biomass has a protein content of at least 8% protein by weight and a mucilage content of at least 4% by weight. In yet another embodiment, the biomass has a protein content of at least 20% protein by weight and a mucilage content of at least 10% by weight.

[0153] The first aqueous solution may be water or a salt solution. For example, the first aqueous solution may comprise NaCI or SHMP. In an embodiment, the first aqueous solution includes up to about 1M NaCI or up to about 2% SHMP. In another embodiment, the first aqueous solution is water.

[0154] In embodiments, the first aqueous solution is combined with the biomass at a liquid to solid (L:S) ratio of about 10:1 or higher, about 15:1 or higher, about 20:1 or higher, about 25:1 or higher, about 30:1 or higher, about 35:1 or higher, about 40:1 or higher, about 45:1 or higher, or about 50:1 or higher. In other embodiments, the first aqueous solution is combined with the biomass at a liquid to solid L:S ratio of about 10:1 to about 50:1 , about 10:1 to about 45:1, about 10:1 to about 40: 1 , about 10:1 to about 35: 1 , about 15:1 to about 50: 1 , about 20: 1 to about 50: 1 , about 25:1 to about 50: 1 , about 30: 1 to about 50: 1 , about 35: 1 to about 50: 1 , about 10:1 to about 45:1 , about 15:1 to about 45:1, about 17:1 , about 20:1 to about 45:1, about 25:1 to about 45:1 , about 30: 1 to about 45: 1 , about 10:1 to about 40: 1 , about 15:1 to about 40: 1 , about 20:1 to about 40: 1 , about 25: 1 to about 40: 1 , about 10:1 to about 35: 1 , about 15:1 to about 35: 1 , about 20: 1 to about 35: 1 , about 10:1 to about 30: 1 , about 15:1 to about 30: 1 , about 10:1 to about 25:1 , about 15:1 to about 25: 1 , about 10:1 to about 20:1 , or about 15:1 to about 20: 1.

[0155] The first mixture is then incubated at a relatively low temperature, preferably 60 °C or less, for a period of time sufficient to solubilize at least a fraction of polypeptides present within the biomass. In embodiments, the temperature at which the first mixture is incubated is between about 2 °C and about 60 °C, about 4 °C and about 60 °C, about 6 °C and about 60 °C, about 8 °C and about 60 °C, about 10 °C and about 60 °C, about 12 °C and about 60 °C, about 14 °C and about 60 °C, about 16 °C and about 60 °C, about 18 °C and about 60 °C, about 20 °C and about 60 °C, about 22 °C and about 60 °C, about 24 °C and about 60 °C, about 26 °C and about 60 °C, about 28 °C and about 60 °C, about 30 °C and about 60 °C, about 32 °C and about 60 °C, about 34 °C and about 60 °C, about 36 °C and about 60 °C, about 38 °C and about 60 °C, about 40 °C and about 60 °C, about 42 °C and about 60 °C, about 44 °C and about 60 °C, about 46 °C and about 60 °C, about 48 °C and about 60 °C, about 50 °C and about 60 °C, about 2 °C and about 58 °C, about 2 °C and about 56 °C, about 2 °C and about 54 °C, about 2 °C and about 52 °C, about 2 °C and about 50 °C, about 2 °C and about 48 °C, about 2 °C and about 46 °C, about 2 °C and about 44 °C, about 2 °C and about 42 °C, about 2 °C and about 40 °C, about 2 °C and about 38 °C, about 2 °C and about 36 °C, about 2 °C and about 34 °C, about 2 °C and about 32 °C, about 2 °C and about 30 °C, about 2 °C and about 28 °C, about 2 °C and about 26 °C, about 2 °C and about 24 °C, about 2 °C and about 22 °C, about 2 °C and about 20 °C, about 2 °C and about 18 °C, about 2 °C and about 16 °C, about 2 °C and about 14 °C, about 2 °C and about 12 °C, about 2 °C and about 10 °C, about 2 °C and about 8 °C, about 2 °C and about 6 °C, about 2 °C and about 4 °C, about 4 °C and about 38 °C, about 6 °C and about 36 °C, about 8 °C and about 34 °C, about 10 °C and about 32 °C, about 12 °C and about 30 °C, about 14 °C and about 28 °C, about 16 °C and about 26 °C, about 18 °C and about 24 °C, or about 20 °C and about 22 °C. In embodiments, the temperature at which the first mixture is incubated is between about 4 °C and about 60 °C, about 10 °C and about 50 °C, about 15 °C and about 25 °C, or about 18 °C and about 23 °C.

[0156] The incubation of the first mixture should be carried out for a duration sufficient to solubilize at least a fraction of the polypeptides present within the biomass. In embodiments, the incubation of the first mixture has a duration of about 10 minutes or longer, about 15 minutes or longer, about 20 minutes or longer, about 25 minutes or longer, about 30 minutes or longer, about 90 minutes or less, about 85 minutes or less, about 80 minutes or less, about 75 minutes or less, about 70 minutes or less, about 65 minutes or less, about 60 minutes or less, about 55 minutes or less, about 50 minutes or less, about 45 minutes or less, about 40 minutes or less, about 35 minutes or less, about 30 minutes or less, about 25 minutes or less, about 20 minutes or less, about 15 minutes or less, or about 10 minutes or less. In other embodiments, the incubation of the first mixture is carried out for about 10 to about 120 minutes, about 15 to about 120 minutes, about 20 to about 120 minutes, about 25 to 120 minutes, about 30 to about 120 minutes, about 10 to about 110 minutes, about 10 to about 100 minutes, about 10 to about 90 minutes, about 10 to about 80 minutes, about 10 to about 70 minutes, about 10 to about 60 minutes, about 10 to about 50 minutes, about 10 to about 40 minutes, about 10 to about 30 minutes, about 10 to about 20 minutes, about 15 minutes, about 15 to about 90 minutes, about 15 to about 75 minutes, about 15 to about 60 minutes, about 15 to about 45 minutes, or about 15 to about 30 minutes.

[0157] The incubation of the first mixture may be carried out with mixing, such as stirring, shaking, or ultrasonication. In an embodiment, incubation of the first mixture is carried out with stirring at about 25 RPM or greater, about 50 RPM or greater, about 75 RPM or greater, about 100 RPM or greater, about 125 RPM or greater, about 150 RPM or greater, about 175 RPM or greater, about 200 RPM or greater, about 225 RPM or greater, about 250 RPM or greater, about 275 RPM or greater, about 300 RPM or greater, about 325 RPM or greater, about 350 RPM or greater, about 350 RPM or greater, about 375 RPM or greater, or about 400 RPM or greater. In other embodiments, incubation of the first mixture is carried out with stirring at about 30 to about 200 RPM.

[0158] After incubation, the first mixture is separated into a first liquid fraction comprising isolated polypeptides and a first solid fraction that is used for subsequent gum extraction. In an embodiment, separation is carried out by centrifugation. In embodiments, centrifugation is carried out at about 500 x g or higher, about 1 ,000 x g or higher, about 2,000 x g or higher, about 3000 x g or higher, about 4000 x g or higher, about 5000 x g or higher, about 6000 x g or higher, about 7000 x g or higher, about 8,000 x g or higher, about 9,000 x g or higher, about 10,000 x g or higher, about 10,000 g or lower, about 9,000 x g or lower, about 8,000 x g or lower, about 7,000 x g or lower, about 6,000 x g or lower, about 5,000 x g or lower, about 4,000 x g or lower, about 3,000 x g or lower, about 2,000 x g or lower, or about 1 ,000 x g or lower. In an embodiment, centrifugation is carried out at about 500 x g to about 10,000 x g, about 500 x g to about 5,000 x g, about 500 x g to about 4,000 x g, about 500 x g to about 3,000 x g, about 500 x g to about 2,000 x g, or about 1 ,000 x g to about 2,000 x g. In another embodiment, centrifugation is carried out at about 1000 x g. Other degrees of centrifugation force may be used, so long as they are sufficient to separate the liquid and solid fractions. The duration of centrifugation should be selected to allow adequate separation of the liquid and solid fractions. For example, centrifugation may be carried out for about 10 minutes, although longer or shorter centrifugation times may be employed. In other embodiments, separation is carried out by other physical means, such as continuous centrifuge, decanter centrifuge, super centrifuge, or filter press, as will be known to one skilled in the art. [0159] In an embodiment, polypeptide extraction further comprises one or more wash steps. After separation of the liquid and solid fraction from the first mixture, the first solid fraction may be washed by combining the first solid fraction with an aqueous solution. The first solid fraction may be washed once, twice, three times, four times, five times, or more. In an embodiment, the first solid fraction is washed 1 to 3 times. In other embodiments, the wash step is omitted.

[0160] In an embodiment, for each wash, the wash solution is combined with the first solid fraction at a L:S ratio of about 5:1 or higher, about 6:1 or higher, about 7:1 or higher, about 8:1 or higher, about 9:1 or higher, or about 10:1 or higher. In an embodiment, the wash solution is combined with the first solid fraction at a L:S ratio of about 5:1 to about 50:1 , about 5:1 to about 45:1 , about 5: 1 to about 40: 1 , about 5: 1 to about 35: 1 , about 5: 1 to about 30: 1 , about 5: 1 to about 25: 1 , about 5:1 to about 20:1 , about 5:1 to about 15:1 , about 10:1 to about 50:1 , about 10:1 to about 45:1, about 10:1 to about 40: 1 , about 10:1 to about 35: 1 , about 10:1 to about 30: 1 , about 10:1 to about 25:1 , about 10:1 to about 20:1, about 17:1 , about 15:1 to about 50:1, about 15:1 to about 45:1 , about 15:1 to about 40: 1 , about 15:1 to about 35: 1 , about 15:1 to about 30: 1 , about 15:1 to about 25: 1 , or about 15:1 to about 20: 1.

[0161] In an embodiment, for each wash, the wash step is carried out at a temperature of about 4 °C to about 60 °C, about 10 °C to about 50 °C, or at room temperature. The temperature may be about 4 °C to about 55 °C, about 4 °C to about 50 °C, about 4 °C to about 45 °C, about 4 °C to about 40 °C, about 4 °C to about 35 °C, about 4 °C to about 30 °C, about 4 °C to about 25 °C, about 4 °C to about 20 °C, about 10 °C to about 60 °C, about 10 °C to about 55 °C, about 10 °C to about 50 °C, about 10 °C to about 45 °C, about 10 °C to about 40 °C, about 10 °C to about 35 °C, about 10 °C to about 30 °C, about 10 °C to about 25 °C, about 10 °C to about 20 °C, about 15 °C to about 60 °C, about 15 °C to about 55 °C, about 15 °C to about 50 °C, about 15 °C to about 45 °C, about 15 °C to about 40 °C, about 15 °C to about 35 °C, about 15 °C to about 30 °C, about 15 °C to about 25 °C, about 20 °C to about 60 °C, about 20 °C to about 55 °C, about 20 °C to about 50 °C, about 20 °C to about 45 °C, about 20 °C to about 40 °C, about 20 °C to about 35 °C, about 20 °C to about 30 °C, or about 20 °C to about 25°C.

[0162] In an embodiment, for each wash, the wash step is carried out for a duration of about 10 minutes to about 90 minutes, about 15 minutes to about 60 minutes, or about 15 minutes. The duration may be about 10 minutes to about 90 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 15 minutes to about 90 minutes, about 15 minutes to about 80 minutes, about 15 minutes to about 70 minutes, about 15 minutes to about 60 minutes, about 15 minutes to about 50 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 30 minutes, or about 15 minutes to about 25 minutes.

[0163] In an embodiment, for each wash, the pH of the aqueous wash solution is selected so that the wash mixture has a pH of about 7 to about 10, or about 10. The pH may be about 7 or higher, about 8 or higher, about 9 or higher, about 10 or higher, about 7 to about 11 , about 7.5 to about 11 , about 8 to about 11 , about 8.5 to about 11 , about 9 to about 11 , about 9.5 to about 11 , about 10 to about 11 , about 7 to about 10.5, about 7 to about 10, about 7 to about 9.5, about 7 to about

9, about 7 to about 8.5, about 7 to about 8, about 8 to about 11 , about 8 to about 10.5, about 8 to about 10, about 8 to about 9.5, about 8.5 to about 11 , about 8.5 to about 10.5, about 8.5 to about

10, about 8.5 to about 9.5, about 9 to about 10.5, or about 9 to about 10. In an embodiment, the wash solution is water.

[0164] Gum (Mucilage) Extraction

[0165] To extract gum (aka mucilage), the first solid fraction is combined with a second aqueous solution to form a second mixture having a pH of about 7 or lower. In an embodiment, the pH of the second mixture is about 4. In other embodiments, the pH of the second mixture is about 7 or lower, about 6 or lower, about 5 or lower, about 4 or lower, about 3 or lower, or about 2 or lower. In embodiments, the pH of the second mixture is between about 1 and about 7, about 2 and about 7, about 3 and about 7, about 4 and about 7, about 5 and about 7, about 6 and about 7, about 1 and about 6, about 1 and about 5, about 1 and about 4, about 1 and about 3, about 1 and about 2, about 2 and about 6, about 2 and about 5, about 2 and about 4, about 2 and about 3, about 3 and about 6, about 3 and about 5, about 3 and about 4, about 4 and about 6, about 4 and about 5, about 5 and about 6.

[0166] The second aqueous solution may be water or a salt solution. The salt may be a food- grade salt. For example, the first aqueous solution may comprise NaCI or SHMP. In an embodiment, the first aqueous solution includes up to about 1M NaCI or up to about 2% SHMP. In another embodiment, the first aqueous solution is water. The pH of the second aqueous solution may be adjusted as needed by the addition of base or acid.

[0167] The second mixture is then incubated at a relatively high temperature, preferably about 80 °C or higher, for a period of time sufficient to solubilize at least a fraction of carbohydrates present in the mixture. In an embodiment, the temperature at which the second mixture is incubated is about 80 °C or higher, about 85 °C or higher, about 90 °C or higher, about 95 °C or higher, or about 100 °C or higher. In embodiments, the temperature at which the second mixture is incubated is between about 80 °C and about 120 °C, about 85 °C and about 120 °C, about 90 °C and about 120 °C, about 95 °C and about 120 °C, about 100 °C and about 120 °C, about 80 °C and about 110 °C, about 85 °C and about 110 °C, about 90 °C and about 110 °C, about 95 °C and about 110 °C, about 100 °C and about 110 °C, about 80 °C and about 105 °C, about 85 °C and about 105 °C, about 90 °C and about 105 °C, about 95 °C and about 105 °C, about 100 °C and about 105 °C, about 80 °C and about 100 °C, about 85 °C and about 100 °C, about 90 °C and about 100 °C, or about 95 °C and about 100 °C. In an embodiment, the temperature at which the second mixture is incubated is about 100 °C or about 118 °C. The incubation of the second mixture may be carried out at an elevated pressure, i.e. at a pressure above atmospheric pressure, to obtain an incubation temperature above 100 °C.

[0168] The incubation of the second mixture should be carried out for a duration sufficient to solubilize at least a fraction of the carbohydrates present within the biomass. In an embodiment, the incubation of the second mixture has a duration of at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 70 minutes, at least about 75 minutes, at least about 80 minutes, at least about 85 minutes, at least about 90 minutes, at least about 95 minutes, at least about 100 minutes, at least about 105 minutes, at least about 110 minutes, at least about 115 minutes, or at least about 120 minutes. I n embodiments, the incubation of the second mixture has a duration of about 20 to about 240 minutes, about 30 to about 240 minutes, about 40 to about 240 minutes, about 50 to about 240 minutes, about 60 to about 240 minutes, about 70 to about 240 minutes, about 80 to about 240 minutes, about 90 to about 240 minutes, 20 to about 200 minutes, about 30 to about 200 minutes, about 40 to about 200 minutes, about 50 to about

200 minutes, about 60 to about 200 minutes, about 70 to about 200 minutes, about 80 to about

200 minutes, about 90 to about 200 minutes, about 20 to about 150 minutes, about 30 to about

150 minutes, about 40 to about 150 minutes, about 50 to about 150 minutes, about 60 to about

150 minutes, about 70 to about 150 minutes, about 80 to about 150 minutes, or about 90 to about 150 minutes.

[0169] After incubation, the second mixture is separated into a second liquid fraction comprising isolated mucilage and a second solid fraction (gum extraction residue). In an embodiment, separation is carried out by centrifugation. In embodiments, centrifugation is carried out at about 500 x g or higher, about 1 ,000 x g or higher, about 2,000 x g or higher, about 3000 x g or higher, about 4000 x g or higher, about 5000 x g or higher, about 6000 x g or higher, about 7000 x g or higher, about 8,000 x g or higher, about 9,000 x g or higher, about 10,000 x g or higher, about 10,000 g or lower, about 9,000 x g or lower, about 8,000 x g or lower, about 7,000 x g or lower, about 6,000 x g or lower, about 5,000 x g or lower, about 4,000 x g or lower, about 3,000 x g or lower, about 2,000 x g or lower, or about 1,000 x g or lower. In an embodiment, centrifugation is carried out at about 500 x g to about 10,000 x g, about 500 x g to about 5,000 x g, about 500 x g to about 4,000 x g, about 500 x g to about 3,000 x g, about 500 x g to about 2,000 x g, or about 1 ,000 x g to about 2,000 x g. In another embodiment, centrifugation is carried out at about 1000 x g. Other degrees of centrifugation force may be used, so long as they are sufficient to separate the liquid and solid fractions. Further, the duration of centrifugation should be selected to allow adequate separation of the liquid and solid fractions. For example, centrifugation may be carried out for about 10 minutes, although longer or shorter centrifugation times may be employed. In other embodiments, separation is carried out by other physical means, such as continuous centrifuge, decanter centrifuge, super centrifuge, or filter press, as will be known to one skilled in the art.

[0170] Polypeptide Hydrolysis

[0171] The isolated polypeptides resulting from polypeptide extraction as described herein may be subjected to a protease treatment to produce hydrolyzed isolated polypeptides. Further, to increase the yield of hydrolyzed isolated polypeptides, the gum extraction residue may be combined with the first liquid fraction and any wash fraction(s) comprising the isolated polypeptides, prior to protease treatment.

[0172] Hydrolysis allows the release of short peptides or amino acids with enhanced solubility and potentially higher nutritional value. Hydrolysis may also increase total product recovery.

[0173] Any suitable protease or mixture of proteases may be used, as will be known to one skilled in the art. Examples of suitable proteases include Subtilisin, trypsin, papain, thermolysin, and pancreatin.

[0174] In an embodiment the protease is a non-specific protease or mixture of proteases such as an exopeptidase or mixture of exopeptidases, for example carboxypeptidases and aminopeptidases. Specific proteases cut specific sequence sites within a protein, whereas non- specific proteases do not recognize specific residues, or they recognize a wide variety of residues and digest proteins randomly into small peptide or amino acids. In an embodiment, the protease comprises one or more endoproteases. Endoproteases are proteases that cleave peptide bonds of non-terminal amino acids. In an embodiment, the protease comprises Subtilisin. In an embodiment, the protease is Alcalase®. The isolated polypeptides may be subjected to treatment with a second protease or a second mixture of proteases either concurrently with, or subsequent to, the protease treatment. In an embodiment, the second protease comprises an exoprotease. Exoproteases are proteases that cleave peptide bonds of terminal amino acids. In an embodiment the second protease is a peptidase or a mixture of peptidases such as carboxypeptidases and aminopeptidases. In an embodiment, the protease comprises a peptidase preparation from Aspergillus oryzae. In an embodiment, the protease is Flavourzyme®.

[0175] Conditions for hydrolysis by the protease and/or second protease should be selected to allow effective cleavage by the protease(s). As will be recognized by one skilled in the art, different proteases or mixtures of proteases may require different conditions to promote enzyme activity. As such, the pH, temperature, and duration of protease treatment should be selected based on the protease(s) being used. Any conditions suitable for polypeptide hydrolysis by the protease and/or second protease may be employed.

[0176] In an embodiment, protease treatment by the protease and/or second protease is carried out at a pH of about 4 to about 11 , about 5 to about 11 , about 6 to about 11 , about 7 to about 11 , about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, or about 10.

[0177] In an embodiment, protease treatment by the protease and/or second protease is carried out at a temperature of about 30 °C to about 90 °C, 30 °C to about 70 °C, about 40 °C to about 80 °C, about 50 °C to about 75 °C, about 50 °C to about 60 °C, or about 55 °C.

[0178] After hydrolysis, the third mixture is separated into a third solid fraction and a third liquid fraction. Separation may be carried out by centrifugation or other physical means, such as continuous centrifuge, decanter centrifuge, super centrifuge, or filter press, as will be known to one skilled in the art. The third liquid fraction comprises the hydrolyzed isolated polypeptides. The third solid fraction is a fibre rich product comprising polysaccharides such as cellulose, hemicellulose, lignin, and other leftover fractions such as protein and lipid.

[0179] After isolation, the isolated polypeptides or hydrolyzed isolated polypeptides may be concentrated by reverse osmosis (RO) filtration, vacuum evaporation system, or any other concentrating method such as precipitation by pH adjustment or salt additions. A powdered product can be made by spray drying, freeze drying, oven drying or any other drying method.

[0180] Isolated polypeptides, hydrolyzed isolated polypeptides, isolated mucilage, and/or a fibre- rich product produced as described herein may have a variety of applications, for example as a nutritional product, as a protein/peptide powder, in beverages, in food or feedstuffs, or in cosmetics products. In an embodiment, one or a combination of two or more of the products isolated as described herein may be included in a food, feed, cosmetics, or beverage product. For example, a peptide product may be used in an athletic drink or to replace animal proteins in food and feed; mucilage may be used in cosmetics as a texturing agent, as a food additive, or as a thickening and emulsifying agent; and fiber product may be used in baked goods such as bread and cookies or in other foods or products where additional fiber is desired.

[0181] The following non-limiting examples illustrate various aspects of the invention.

Example 1: Extraction Process

[0182] Flax meal, flax hull, flax kernel, and dried defatted flax kernel were used as starting biomasses. The composition of the various starting materials is shown in Table 1.

Table 1: Composition of biomass starting materials

[0183] Step 1 , protein extraction: The flax meal was mixed with water at a liquid to solid ratio (L:S ratio) of 35:1 to form a first mixture and then the pH was raised to 10.75 by addition of NaOH. The first mixture was then incubated at room temperature with stirring for 30 minutes. After this incubation, the first mixture was centrifuged to separate solid and liquid fractions (the first solid fraction and the first liquid fraction). The supernatant (first liquid fraction) was saved for use in step 3. [0184] Step 2, gum extraction: The first solid fraction from step 1 was mixed with water at a L:S ratio of 17:1 and the pH was adjusted to 4.0 by addition of HCI to form a second mixture. The second mixture was then incubated at 100 °C for over 110 minutes with rapid mixing. After this incubation, the second mixture was centrifuged to separate solid and liquid fractions (the second solid fraction and the second liquid fraction). The supernatant (second liquid fraction) collected in this step is enriched in mucilage. Mucilage content was analyzed based on proximate composition analyses and the total carbohydrate of the mucilage content was determined by difference.

[0185] Step 3, hydrolysis: The second solid fraction from step 3 was mixed with the supernatant (first liquid fraction) from step 1 and Alcalase® was added to 3.3% (v/w) relative to the starting mass of the flax meal. The pH was adjusted to 10 by addition of NaOH. The resulting third mixture was incubated for 48 minutes with stirring at 55 °C. After this incubation, the third mixture was centrifuged to separate solid and liquid fractions (third solid fraction and third liquid fraction). The supernatant (third liquid fraction) collected in this step is enriched in polypeptides and the solid fraction (third solid fraction) is a fibre-rich product. In some experiments, Flavourzyme® was added together with the Alcalase®. In these experiments, Flavourzyme® was added to 3.3% (v/w) relative to the starting mass of the flax meal. Protein content was determined by Kjeldahl total nitrogen analysis and converted to protein content by the generic factor 6.25; fiber content refers to carbohydrates which was determined by difference based on composition analysis.

Table 2: Performance of initial extraction process for flax meal Table 3: Product compositions, compared to starting materials

Example 2: Testing the effect of various parameters on protein extraction [0186] pH, temperature, and time

[0187] The purification process was carried out as described in Example 1 , with the exception that protein extraction in step 1 was carried out at a pH of 4.0, 6.5, 10.75, or 12. As shown in Figure 3, protein recovery increased with increasing pH. However, at pH >10 the solution became unstable and discoloured. The selectivity of extraction for protein or carbohydrate was also analyzed and, as shown in Figure 4, increasing pH was shown to favour selective protein extraction. Selectivity is a dimensionless ratio describing the extent of extraction of one component of the biomass feedstock relative to another; in this investigation, the preferential extraction of protein relative to carbohydrate from biomass was used to measure the effect of extraction conditions in order to optimize extraction of the target solute.

[0188] The purification process was carried out as described in Example 1 , with the exception that protein extraction was carried out at a temperature of 25 °C; with a L:S ratio of 40:1 ; at a pH of either neutral or 10; for a duration of 15, 30, 60, or 90 minutes. As shown in Figure 5, the selectivity of extraction for protein or carbohydrate was higher at pH 10 than at neutral pH. Further, it was observed that protein/carbohydrate selectivity decreased with increased protein extraction time. [0189] The purification process was carried out as described in Example 1 , altering the temperature, pH, and time of the protein extraction in step 1. Extraction was carried out at a temperature of 25 °C and a pH of 4.0, a temperature of 25 °C and a pH of 10.0, a temperature of 100 °C and a pH of 4.0, or a temperature of 100 °C and a pH of 10.0; for a duration of 15, 30, 60, or 120 minutes. As shown in Figure 7, the amount of soluble protein extracted was significantly higher at pH 10.0 than at pH 4.0. The temperature of extraction had relatively little effect on the amount of soluble protein extracted and extending the extraction time beyond 15-30 minutes did not appreciably increase the amount of soluble protein extracted, especially at pH 10.0. The amount of recoverable protein mass produced under the various conditions is shown in Figure 8. Protein recovery was superior at 25 °C and a pH of 10.0. Treatment at elevated temperature and high pH appears to cause biomass swelling and poor solvent recovery.

[0190] L:S ratio

[0191] The purification process was carried out as described in Example 1 , with the exception that the L:S ratio used for protein extraction in step 1 was either 20:1 , 30:1 , or 40:1 and the duration of the protein extraction was 30, 60, or 90 minutes. As shown in Figure 6, protein yield increased over time and with an increased L:S ratio.

[0192] Salts

[0193] The purification process was carried out as described in Example 1 , with the exception that for protein extraction in step 1, the flax meal was mixed with water, 1M NaCI, or 2% SHMP at a L:S ratio of 40:1. As shown in Figure 12, the addition of food-grade salts improved protein extraction. However, the presence of salts imposes complications for downstream purification, as a subsequent desalting treatment may be required.

Example 3: Testing enzyme hydrolysis

[0194] Enzyme hydrolysis was carried out on the supernatant collected after protein extraction (first liquid fraction) or on the supernatant collected after protein extraction (first liquid fraction) combined with the residual biomass following mucilage/gum extraction (second solid fraction). The enzyme treatment was carried out at pH 10, 0.2% Alcalase®, 55 °C, for 60 min. As shown in Figure 9, inclusion of the residual biomass improved total solids extraction yield by approximately 30% relative to the yield from the supernatant alone. [0195] A time course was carried out for varying enzyme dosage and the resulting polypeptide mixtures were analyzed by SDS-PAGE, as shown in Figure 10. Different % of Alcalase® were added to reaction mixtures of individual tubes and reactions were carried out at pH 10, 55 °C, for different times and stopped by boiling for 15 min. Samples were analyzed by 12% SDS-PAGE.

[0196] In one experiment, the pH of the third mixture was adjusted to pH 10 and allowed to decrease to neutral during hydrolysis, taking pH measurements at 0, 10, 15, 30, and 60 minutes. As shown in Figure 11, this indicates that initial Alcalase® 2.4L enzyme activity is rapid.

Example 4: Testing the effect of various parameters on gum extraction

[0197] L:S ratio and time

[0198] The purification process was carried out as described in Example 1 , with the exception that the first solid fraction was mixed with water at a L:S ratio of 10:1 , 20:1 , or 30:1 and gum extraction was carried out for a duration of 30, 60, or 90 minutes. As shown in Figure 13, the carbohydrate yield increased with increased extraction time; increasing the L:S ratio led to reduced carbohydrate concentration in the extract; however, combining the increased volume and concentration, total carbohydrate yield was improved with diminishing returns beyond 20:1 as shown in Figure 15. The total solids extraction yield after 120 min at the different L:S ratios was a representative of total carbohydrate recovery.

[0199] Temperature and time

[0200] The purification process was carried out as described in Example 1 , with the exception that gum extraction was carried out at 60 °C, 80 °C, or 100 °C at 20:1 L:S ratio. As shown in Figure 14, gum recovery was affected strongly by temperature, with higher temperatures resulting in higher carbohydrate recovery.

[0201] Another gum extraction was carried out using a L:S ratio of 30:1 ; an extraction temperature of 60 °C, 80 °C, or 100 °C; and an extraction time of 30, 60, 90, or 120 minutes. As shown in Figure 16, gum extraction was highest at 100°C and 120 minutes.

[0202] pH, temperature, and time

[0203] The effect of temperature, pH, and extraction time on the amount of recoverable carbohydrate after gum extraction was studied by carrying out gum extraction at 25°C and pH 4.0; 25 °C and pH 10.0; 100 °C and pH 4.0; and 100 °C and pH 10.0, for a duration of 15, 30, 60, or 120 minutes. As shown in Figure 17, the amount of recoverable carbohydrate was highest using an extraction temperature of 100 °C, an extraction pH of 4.0, and an extraction time of 60- 120 minutes.

Example 5: Testing the effect of defatting on protein extraction

[0204] The maximum potential recovery of protein from non-defatted (as-received) and hexane- defatted flax meal was evaluated by sequential extraction at pH 11 with a L:S ratio of 35:1, followed by three washes at 30:1, shown in Figure 18. Recovery of nearly 90% of the protein from flax meal was feasible using large L:S ratios and repeated extraction steps, with 87% and 85% recovery obtained for non-defatted and defatted flax meal, respectively. The extent of protein recovery was near 100% based on the aqueous protein concentration, however there was some loss in protein recovery due to water retained by the swollen residual biomass, which holds a significant volume of water under the alkaline extraction conditions. The extent of biomass swelling was observed to increase at higher extraction temperatures and pH, favouring protein extraction at lower temperature at the lowest pH still providing good protein recovery.

[0205] The difference in protein recovery between defatted and as-received flax meal in the first extraction step arises from the greater supernatant volume recovered using non-defatted flax meal. This could be caused by more sites being available to bind water following defatting, causing an increased degree of swelling in the saturated biomass.

[0206] For an industrial process, water consumption should be minimized where feasible, requiring a compromise to balance protein recovery against water consumption. These results demonstrate that using as-received pressed flax meal containing approximately 10% fat resulted in slightly greater protein recovery, particularly under the constraint of limited water consumption. This suggests that a defatting pre-treatment step may be beneficial to avoid the co-extraction of fat with protein, but it is not required for effective protein extraction.

Example 6: Testing the effect of physical enhancements on extraction

[0207] Physical enhancements for extraction, using ultrasound or high-speed blending for 1 and 3 minutes each, were compared to baseline extractions performed with conventional mixing for 30 minutes at equivalent L:S ratios. The effect on extracted solids, protein/carbohydrate selectivity, and solution viscosity is shown in Figure 19. Because the recovery of flaxseed meal proteins is already very good at alkaline pH as demonstrated in Figure 18, it was found that the physical enhancements non-selectively increased the extraction of both protein and carbohydrate components, thereby reducing protein product purity and also decreasing yield in the subsequent gum extraction step.

[0208] Ultrasound treatment gave a lower protein extraction selectivity but had slightly reduced viscosity relative to the untreated controls, while yield was lower than the controls extracted for 30 minutes by stirring. Viscosity increased at the 40:1 L:S ratio relative to 20:1 , presumably due to a greater concentration of extracted solids in the aqueous phase. This was despite the lower initial loading concentration of feedstock, suggesting that ultrasound penetration through the slurry was inefficient at 20:1 , while at 40:1 it had the effect of improving solute extraction.

[0209] High-shear blending at 20:1 L:S quickly produced a solution with viscosity that was more than three-fold greater than controls at equal L:S ratio. This was apparently due to enhanced extraction of carbohydrate polymers as demonstrated by the greater total solids extraction yield with lower protein/carbohydrate selectivity for the blending treatment. The aggressive mixing likely caused strong association and entanglement of protein chains with carbohydrate polymers, greatly increasing solution viscosity. Blending at 40:1 increased extracted solids concentration relative to 20: 1 at the expense of reduced protein selectivity, while the viscosity was similar to that obtained by ultrasound treatment.

[0210] Taken together, these results suggest that aggressive physical enhancement methods, while effective at improving the rate and extent of mass transfer, fail to improve extraction selectivity, and also contribute to increased viscosity which hampers subsequent solid-liquid separation.

Example 7: Testing the effect of salts addition on protein extraction yields

[0211] The food-grade salts, sodium chloride and sodium hexametaphosphate (SHMP) were evaluated for their effect on protein extraction. At alkaline pH where protein solubility is greatest, the addition of up to 1.5% (w/v) SHMP had only a minor effect on protein recovery alone or in combination with NaCI, while NaCI negatively affected recovery, as shown in Figure 20.

[0212] The amount of added salt was similar to the total amount of extracted solids and was far greater than the amount of additional protein extracted. Furthermore, pH adjustment was resisted by the buffering capacity of SHMP, requiring much greater NaOH addition to reach an equal pH value, which further diluted the purity of extracted protein. These aspects indicate that salt addition negatively affects product quality and would also require extensive downstream treatment for desalting.

Example 8: Testing the effect of centrifugation intensity on solids yield

[0213] The challenge posed by the high water absorbance and swelling of the residual biomass pellet causes low volume recovery of supernatant and consequently extracted solids. In an attempt to increase pellet density and supernatant recovery volume, increased centrifugation intensity was evaluated for its potential to improve supernatant recovery volume by producing more compact pellets. Centrifugation was also evaluated for its potential to selectively enrich protein by potentially separating co-extracted non-protein components based on density.

[0214] Centrifugation intensity was compared at 1 ,000 x g, 2,000 x g, and 10,000 x g for 10 minutes, and the primary differences seen were in recoverable supernatant volume and solids content, shown in Figure 21.

[0215] Solids content in the supernatant was reduced by approximately 10% at 2,000 and 30% 10,000 x g due to nonselective sedimentation of extracted solutes, while protein content was not significantly enriched at 10,000 x g.

[0216] Recovered supernatant volume at 1,000 x g was similar to that at 2,000 x g but was reduced by approximately 10% at 10,000 x g due to the larger volume of the pellets produced at greater centrifugation intensity, caused by greater sedimentation of extracted solids. Based on these results, a centrifugation speed of 1,000 x g for 10 minutes was chosen for all separation steps to achieve solids removal while maintaining extraction yield. Furthermore, increasing centrifugation speed did not improve the loose consistency of pellets after protein extraction.

[0217] Following protein extraction and centrifugation, care is required when decanting the supernatant from the loose pellets of residual biomass to maximize recovered supernatant volume and minimize contamination by pellet solids. The loose pellet consistency is likely a result of the presence of swollen polymeric carbohydrate gum interacting with protein remaining in the pellets, which are targeted for recovery by the subsequent gum extraction and protein hydrolysis steps.

[0218] Centrifugation speed for recovery of extracted gum was maintained at the minimum evaluated speed of 1 ,000 x g because this was sufficient to produce compact pellets with minimal potential loss of suspended gum due to sedimentation. Example 9: Testing the effect of precipitation strategies on product separation

[0219] Precipitation strategies including pH adjustment to the isoelectric point, ethanol (anti solvent), and centrifugation were compared for their ability to selectively precipitate either protein or carbohydrate components for product purification. The loss of solids upon precipitation as a percentage of initial solids is shown in Figure 22.

[0220] The data indicate that intensive centrifugation for 25 minutes at 10,000 x g and precipitation by adding 3 volumes of ethanol each removed less than 10% of total extracted solids, with poor selectivity in both cases from co-precipitation of carbohydrates with proteins. Isoelectric precipitation was more effective, removing over 30% of extracted solids; however, the co precipitation of 40% of extracted carbohydrates also made this approach untenable for selective separation.

Example 10: Testing the effect of extraction conditions on protein yield

[0221] Because protein solubility is improved at higher pH values, it is preferable to achieve sufficiently high pH using a minimal amount of NaOH to avoid unnecessary product contamination. The effect of NaOH addition on solution pH was investigated to characterize the change in pH as a function of added salt, shown in Figure 23.

[0222] From this plot, approximately 1% NaOH relative to added flax meal mass achieves the greatest increase in pH, achieving a pH of approximately 10.75 before the point of diminishing returns at the shoulder of the curve, at which point the buffering capacity of the protein in solution resists further pH increase.

[0223] It has been observed that adding flaxseed meal directly to alkaline solution results in aggregation and poor dispersion, thus it is preferable to increase pH after first dispersing the flaxseed meal in water.

Example 11: Process development using Response Surface Methodology

[0224] Following screening of significant process variables as described in the previous Examples, Response Surface Methodology (RSM) was used to characterize the behaviour of each extraction step. Response surfaces were generated by least-squares linear regression modelling of process responses (outputs) to perturbations in operating parameters (inputs / factors). The effect of up to two factors on one response can be plotted visually, producing the three-dimensional graphical plots. Maximum responses within the analyzed range of two factors can be obtained directly from response surface maxima for an individual process.

[0225] To assess multiple responses in the context of practical or logistical constraints, a desirability function can be defined that places weighted emphasis or boundary constraints on certain parameters or responses to predict performance. Optimization of the desirability function aims to maximize desirability by varying the process parameters within the defined constraints, and can define a region of operating conditions that meets these defined criteria.

[0226] Development of protein extraction process

[0227] The effect of extraction temperature and time on total solids yield at pH 11 is shown in Figure 24. The response surface indicates that extraction occurs rapidly, and that overall solids recovery is insensitive to temperature.

[0228] The effect of extraction temperature and pH on total solids yield is shown in Figure 25. Again, this shows that solids recovery is not sensitive to temperature, while pH has a positive effect on total solids recovery due to the improved extraction of protein.

[0229] The effect of extraction temperature and pH on protein extraction selectivity relative to carbohydrate is shown in Figure 26. The response surface indicates that extraction at lower temperature and high pH promotes greater extraction selectivity for protein relative to carbohydrate, likely due to the fraction of gum having a low mass transfer rate at low temperatures due to its very high molecular weight. Extraction at 5 °C may be economical with the availability of a chilled water supply. However, pH has the greatest effect on protein selectivity and recovery.

[0230] The effect of extraction pH and L:S ratio on total solids recovery is shown in Figure 27 and the effect of pH and L:S ratio on protein recovery from a single protein extraction step, without washing, is shown in Figure 28. These response surfaces indicate that protein extraction is most effective at pH 11 with a high L:S ratio, however for practical purposes maximum protein recovery should be balanced against excessive water consumption.

[0231] The effect of extraction and washing L:S ratios on protein/carbohydrate selectivity is shown in Figure 29. This plot demonstrates that higher L:S ratios improve protein extraction selectivity, which follows a similar trend to the effect of L:S ratio on protein recovery. [0232] The effect of L:S ratio used in the extraction and wash steps on protein recovery is shown in Figure 30.

[0233] The effect of extraction and washing L:S ratios on protein recovery as a function of water consumption at pH 11 (% w/v) is shown in Figure 31. This shows maximum protein extraction using an extraction L:S ratio of 35:1 and a wash L:S ratio of 10:1.

[0234] Assessment of protein extraction at pH 11 with the desirability function defined to simultaneously minimize water and NaOH consumption, while maximizing protein recovery and selectivity, shows that an extraction L:S ratio of 33:1 and a wash L:S ratio of 17:1, results in a recovery of 48% of biomass feedstock dry matter, corresponding to a protein recovery of 88% from the starting material. Greater protein recovery approaching complete extraction is achievable at the expense of greater water consumption, which may be acceptable with effective water recycling during product drying.

[0235] The amount of protein recovered relative to the amount of NaOH added for pH control has an effect on the purity of the extracted protein product, making it preferable to use a minimum amount of NaOH that achieves sufficiently high pH for selective protein extraction. The amount of protein recovered relative to the amount of NaOH required across an effective range of pH values for extraction is shown in Figure 32.

[0236] A linear decrease in the amount of protein recovered relative to added NaOH occurs toward higher pH values. Although greater specific protein recovery relative to NaOH consumption is achievable at lower pH, the increase in selectivity and yield at high pH, shown in Figures 29 and 30, favours extraction near pH 11.

[0237] Maximizing of the desirability function for protein extraction under the criteria of minimal NaOH consumption with maximum protein selectivity and recovery gives a pH of 10.75, corresponding to 1.0% w/w NaOH relative to added flax meal. A plot of the generated desirability function is shown in Figure 33.

[0238] Development of gum extraction process

[0239] Because gum extraction demonstrated a kinetic limitation in mass transfer rate that was exploited for selective protein extraction, the physical parameters of mixing speed and temperature were expected to play a significant role in gum extraction. The effect of mixing speed on gum solids yield is shown in Figure 34. [0240] Mixing speed had a slight positive effect on gum yield, in agreement with preliminary findings of improved carbohydrate extraction yield by high-speed blending shown in Figure 19, indicating that maximum available mixing speed should be used during gum extraction, although bench-scale mixing evaluated in this example may not be representative of large-scale equipment.

[0241] The effect of L:S ratio and extraction time are plotted in Figure 35, and the response surface generated from the same yield data is illustrated in Figure 36. The gum extraction response surface illustrates that similar yields are achieved at all L:S ratios at 180 min extraction time, but greater L:S ratios achieve equivalent gum yields at shorter extraction times. Optimization of gum extraction conditions therefore requires a compromise to balance water consumption against processing time. The contour plots in Figures 37 and 38 illustrate the desirability function with constraints set to maximize yield with minimal extraction time (Figure 37), and the yield at varying L:S ratios and extraction times (Figure 38), resulting in a selected extraction time of 60 minutes at 30:1 L:S ratio with a predicted crude gum yield of 12.8% of starting biomass.

[0242] When minimal water consumption is also included in the desirability function criteria at an equal weighting in importance with yield and extraction time, the selected extraction conditions are changed to approximately 110 minutes at 17: 1 L:S ratio, giving a slightly lower yield of 11.4%, shown in Figures 39 and 40.

[0243] Because the resistance to gum extraction appears to be a kinetic limitation, the effect of extraction pressure and temperature was evaluated. Figure 41 shows solids yield during pressurized and boiling gum extraction. Figure 41 demonstrates that gum extraction at elevated pressure of 12.5 psig, corresponding to a temperature of 118 °C, provides a slight improvement of 6% in gum extraction rate and yield at 120 minutes, with a favorable extraction time near 180 minutes, however protein content in the gum is increased from approximately 12% to 16%, presumably due to enhanced non-selective extraction.

[0244] Diminishing returns are reached at longer extraction times, and the yield improvement contributed by pressurization is reduced to 3%.

[0245] Gum solids concentration decreases with increasing L:S ratio, and lower solids contents have been associated with greater yield and lower viscosity after spray-drying (Oomah and Mazza 2001), suggesting that extraction at high pressure and high L:S ratio is favourable for rapid gum extraction if slightly lower gum purity is tolerable and increased drying requirements are acceptable to remove the additional water.

[0246] Development of enzymatic hydrolysis conditions

[0247] Protein extracted following the selected conditions described in Example 11 was subjected to enzyme hydrolysis using a commercial protease (Alcalase® 2.4 FG) to determine optimal reaction conditions and enzyme dosage. The objective was to maintain or increase protein content while reducing the peptide average molecular size.

[0248] Briefly, pooled supernatants from the protein extraction and the wash steps were mixed with the residual biomass pellet following gum extraction, and pH was adjusted to 10.0 by addition of NaOH. Enzyme(s) were added by volume relative to initial biomass feedstock mass (% expressed as mL/g), and release of soluble solids was monitored over the time course of hydrolysis. Solids yield was determined by drying the supernatant following hydrolysis and centrifugation.

[0249] In all hydrolysis experiments, dissolved solids were lost from the initial extraction supernatant at early time points following addition of the residual biomass pellet, possibly due to re-adsorption of dissolved solids by the residual biomass. However, the inclusion of the residual pellet during hydrolysis provides additional material for extraction by enzymatic activity, which offsets the early loss of dissolved solids at later time points, resulting in up to 10% greater dissolved solids within 60 minutes than could be obtained from the protein extraction step alone. The loss of dissolved solids following addition of the pellet is shown in Figure 42.

[0250] No statistically significant effect of incubation temperature was detected between 55 °C and 65 °C, which is the optimal range for Alcalase® 2.4 FG.

[0251] The effect of including the residual pellet on dissolved solids yield during the hydrolysis step at varying enzyme dosages and time points is shown in Figure 43. Figure 43 shows the short-term effects of returning the biomass pellet remaining after gum extraction to the protein extraction supernatant for the hydrolysis step. Increasing the enzyme dose positively affects solids recovery by counteracting the loss of solids occurring due to adsorption onto the pellet.

[0252] A response surface showing the effect of enzyme dosage and hydrolysis time on released solids is shown in Figure 44. At high enzyme dosages there is a plateau in dissolved solids yield above an enzyme dose of approximately 4% (mL/g), especially at long hydrolysis times, potentially resulting from the release of insoluble peptides. These factors led to selection of a treatment time near 48 minutes and an enzyme dosage near 3.3% (v/w), as shown by the desirability and yield contour plots in Figures 45 and 46.

[0253] The effects of enzyme dosage and hydrolysis time on protein content is shown in Figure 47. This figure demonstrates that the protein content of the peptide product is reduced at high enzyme loadings, which likely has the effect of releasing additional non-protein material bound to cleaved proteins, supporting extraction using a relatively low enzyme dosage. This effect is parallel to the plateau in solids content at high enzyme dosages, such that both protein content and solids yield achieve maximum responses at relatively low enzyme dosages.

Example 12: Effect of combined and sequential enzyme hydrolysis

[0254] Following initial protein extraction, the effect of enzyme hydrolysis on protein recovery using Alcalase® alone, or in combination with a second enzyme, Flavourzyme®, was investigated, with protein recovery shown in Figure 48.

[0255] Flavourzyme® possesses exo-peptidase activity that degrades protein chain ends producing very small peptides and amino acids, while Alcalase® possesses endo-peptidase activity which cleaves protein chains internally, producing larger peptides that may be hydrolyzed further.

[0256] Equivalent protein recovery was achieved following Alcalase® treatment of Flaxseed meal biomass that was used as-received or was defatted. Hydrolysis using Alcalase® alone produced similar ultimate protein recoveries, but protein recovery was improved by approximately 10% following Alcalase® hydrolysis of defatted biomass relative to extraction. Sequential treatment using Alcalase® followed by Flavourzyme® increased protein recovery relative to Alcalase® treatment alone, reaching equivalent extents of recovery for non-defatted (as-received) and defatted biomass.

[0257] The effect of individual enzymes and their use in combination on protein molecular size is illustrated in Figure 49. The SDS-PAGE gel shown in Figure 49 shows that Alcalase® produces a drastic reduction in average molecular size within 15 minutes (Figure 49, Lane 3), and subsequent addition of Flavourzyme® for an additional 60 minutes does not further shift the molecular size distribution (Figure 49, Lane 5), while Flavourzyme® in combination with Alcalase® produced a similar molecular weight profile (Figure 49, Lanes 6 and 7). Flavourzyme® alone had very little effect on molecular size (Figure 49, Lanes 8 and 9), in agreement with its exo-peptidase activity. It was found that addition of Flavourzyme® generally had a positive effect on solids yield, but a negative effect on protein content. In some cases the net effect from each contribution slightly increased protein recovery in the range of 10% relative to Alcalase® alone.

[0258] Densitometric lane profiles were generated for lanes 1 to 9 of the SDS-PAGE gel shown in Figure 49 to semi-quantitatively illustrate the relative intensity at each molecular size. As shown in Figure 50, the molecular size profile of extracted proteins in the presence of the residual pellet before hydrolysis (lane 1 in Figure 49) shows a broad distribution with major protein populations near 245, 40, 29, 21 , and 11 kDa. As shown in Figure 51 , the molecular size profile of extracted proteins in the absence of the residual pellet (lane 2 in Figure 49) gives a very similar distribution to that seen in Figure 50. As shown in Figure 52, the molecular size profile following hydrolysis by Alacalase™ for 15 minutes (lane 3 in Figure 49) gives a large reduction in high-molecular-size proteins, with a corresponding shift toward lower values and a narrowing of the size distributions. As shown in Figure 53, following 60 minutes of Alcalase® hydrolysis (lane 4 in Figure 49), the shifted distributions began to decrease in relative abundance, with a further shift toward lower molecular sizes around 11 kDa. As shown in Figure 54, the addition of Flavourzyme® for 60 min following Alcalase® hydrolysis for 60 min (lane 5 in Figure 49) brings a further reduction in relative size and a lower abundance of high-molecular-size peptides. As shown in Figure 55, the addition of Flavourzyme® simultaneously with Alcalase® for 60 minutes (lane 6 in Figure 49) to perform combined hydrolysis gives a very similar profile to the sequential treatment shown in Figure 54. As shown in Figure 56, the combined enzyme hydrolysis for 120 minutes (lane 7 in Figure 49) did not produce a significant change in the molecular size distribution relative to treatment for 60 minutes, indicating low residual enzyme activity at long hydrolysis times. As shown in Figure 57, hydrolysis using Flavourzyme® alone for 60 minutes (lane 8 in Figure 49) had the effect of virtually eliminating the high molecular size fractions and increasing the abundance of the lowest molecular size fraction. As shown in Figure 58, increasing hydrolysis time using Flavourzyme® to 120 minutes (lane 9 in Figure 49) produced a nearly identical profile to that of the 60 minute hydrolysis treatment with Flavourzyme® alone, again suggesting that the majority of enzyme activity occurs within the first hour of hydrolysis.

Example 13: Lignan content analysis

[0259] Extraction of defatted flax seed meal was carried out as described in Example 11. [0260] Analysis of lignan content was performed using the Folin-Denis method by reading optical absorbance at 760 nm. A standard curve was established using a commercial tannic acid standard. The lignan contents of flaxseed meal, peptide (hydrolyzed isolated polypeptide mixture), gum (mucilage-rich product), and residue (fibre-rich product) before and after enzyme hydrolysis from a 1 L extraction process were analyzed; with a commercial product, Concentrated Flax Hull Lignans (Aviva Natural Health Solutions, Winnipeg, MB) also analyzed for comparison.

[0261] Each sample was processed to extract lignan as follows: Direct alkaline hydrolysis was carried out with 100 mg samples of commercial product, flax seed meal (FSM),a polypeptide mixture, a gum product, or the residue as extracted from FSM as described in Example 11, followed by the addition of 1.0 ml methanol and vortexing. The hydrolysate was acidified to pH 3 using 9 M sulfuric acid and centrifuged at 1700 x g, for 10 min at room temperature. The supernatant was re-centrifuged in 1.5 ml. micro centrifuge tubes (11 , 000 x g, 5 min) to get a clear liquid phase witch was mixed with 95% ethanol to precipitate at room temperature for at least 10 min. After centrifugation at 11 ,000 x g for 5 min, the sample was filtrated through 0.45 pm syringe filter and the supernatant was used for lignan content analysis with Folin-Denis method.

[0262] Subsequently, lignan content was analyzed as follows: Each sample (0.1 -0.3 ml.) was added to a 15 mL Falcon tube containing 5.7-6.0 ml. deionized water. After addition of 0.5 mL Folin-Denis reagent for 1 minute and before 8 minutes, 1 mL saturated sodium carbonate solution was added. Total reaction volume was made up to 10 mL with 2.4mL deionized water and absorbance at 760 nm was measured after 30 minutes. Lignan contents in different samples were obtained based on analysis with standard curve and OD760 readings. Results are shown in Figure 59.

[0263] Additional protein content and lignan content analysis was carried out on the polypeptide (hydrolyzed isolated polypeptide mixture), gum (mucilage-rich product), and residual (fibre-rich product) fractions from 20L, 100L, 300L, and 1000L scale extractions. The results of this analysis are provided in Table 4. Table 4: Composition and lignan content analysis of fractions and products of the extraction process

[0264] The lignan content of the isolated polypeptide mixture is notably higher than that of the commercial product Flax Hull Lignan. Thus, the extraction method allows for high recovery of lignan from lignan-rich biomass, such as defatted flax meal.

[0265] While the present application has been described with reference to what are presently considered to be preferred examples, it is to be understood that the application is not limited to the disclosed examples. On the contrary, the present application is intended to cover various modifications and equivalent arrangements encompassed by the appended claims in view of the teachings of the disclosure as a whole.

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