Process for recovering proteins using enzyme extraction

Abstract

Methods of producing concentrated protein products from non-animal organic matter, such as seaweed, are described herein. According to some implementations, the method includes obtaining the organic matter, which can include algae. In some implementations, the method includes creating a suspension containing the organic matter, and treating the suspension with an enzyme mixture. Some implementations include extracting a high-protein concentrate from the suspension after treatment with the enzyme mixture. Other implementations are also described.

Description

PROCESS FOR RECOVERING PROTEINS USING ENZYME EXTRACTIONFIELD OF THE INVENTION

[0001] The present disclosure relates to the field of food grade protein ingredients (such as protein concentrates and hydrolysates) for human, pet or livestock consumption, and in particular to methods for extracting protein ingredients from seaweed and byproducts thereof.BACKGROUND

[0002] It is anticipated that in the upcoming years, a shortage of high-quality nutritional proteins may arise due to the growing global population, projected to potentially exceed 9.8 billion by 2050. Presently, the food system may contribute to over 25% of greenhouse gas emissions, with livestock activities alone potentially accounting for as much as 60% of these emissions. Consequently, exploration of alternative nutritional options is attractive from an economic and humanitarian standpoint.

[0003] In recent years, consumers have witnessed a shift in global dietary patterns. Notably, there has been a reduction in the consumption of animal-derived products driven at least in part by ethical concerns regarding animal welfare and environmental sustainability. Additionally, some concerns may he in the consumption of marine-origin food. Over the past three decades, certain marine resources have been excessively depleted through practices like overfishing, leading to habitat damage and a drastic reduction in fish populations by as much as 50% (or more). In extreme cases, top-tier predators like the Atlantic and oceanic bluefin tuna have suffered up to a 97% decline, potentially rendering them critically endangered. Furthermore, practices such as trawl fishing have possibly caused underwater soil deforestation equivalent to as much as three times the expanse of the Amazon rainforest.

[0004] Possibly as a result of the factors enumerated above, an increment in the consumption of plant-based foods, including protein-rich sources such as legumes and cereals, has recently occurred. This transition has led to as many as 75 million people embracing flexitarian, vegetarian, vegan, or other specialized diets. In response to consumer demand, the food industry has introduced plant-based protein products as alternatives to animal meat to the market. Notably, products have been developed to closely mimic the appearance, taste, and texture of traditional animal products, addressing the sensory preferences of consumers.

[0005] Many of those who venture into alternative protein products lean towards economically viable vegetable protein sources, with soy being a prominent choice. Soy has long been utilized in the food industry, including in the meat and pet food sectors, as a cost-effective substitute for more expensive animal protein. However, the prevalence and extensive cultivation of soy and other plant-based protein sources are anticipated to escalate to a point where the mass monoculture encounters substantial sustainability issues. For example, some projections indicate an increment of up to 80% in the intensive cultivation of soybeans and peas, signifying a looming environmental challenge. Moreover, monoculture-induced deforestation may exacerbate the loss of biodiversity, causing numerous disruptions to ecosystems and global biological diversity.

[0006] Thus, while techniques currently exist that are used to produce alternative protein sources, challenges still exist, including those listed above. Accordingly, it would be an improvement in the art to augment or even replace current techniques for producing alternative protein sources with other techniques for producing such protein sources.SUMMARY

[0007] Methods of producing concentrated protein products from non-animal organic matter — and seaweed and seaweed byproducts in particular — are described herein. According to some implementations, the method includes obtaining the organic matter. In some implementations, the method includes creating a suspension containing the organic matter. Some implementations of the method include treating the suspension with an enzyme mixture. Some implementations include extracting a high-protein concentrate from the suspension.

[0008] In accordance with the above, some implementations of the method include obtaining non-animal organic matter including algae or byproducts from processing of algae. In some cases, the algae includes seaweed, such as red seaweed, brown seaweed, or green seaweed. In some cases, the algae is selected from at least one of Gracilaria, Gelidium, Cystoseira, Laminaria, and Rugulopteryx okamurae. In some cases, the algae is selected from Alaria, Palmaria, Euchema, Chondrus, Gigartina, Furcellaria, Kappaphycus, Porphyra, Halymenia, Undaria, Ulva, Laminaria and Cystoseira. Where the organic matter includes algae byproducts, some implementations include carbohydrate-extraction byproducts (e.g., byproducts produced when carbohydrates, such as agars, alginates, or carragenans, are extracted from the algae). Some implementations include extracting carbohydrates from seaweed to obtain the byproducts.

[0009] In accordance with any of the implementations above, the method can include creating a suspension containing the organic matter. In some embodiments, creating the suspension includes one or more of the following: suspending the organic matter in a homogenization medium; homogenizing the organic matter and the homogenization medium to form an organic mixture (e.g., using a high-speed homogenizer); separating the organic mixture into a supernatant and a precipitate; and hydrating the precipitate by suspending the precipitate in a suspension medium, thereby forming the suspension. In some embodiments, homogenization involves size reduction of the organic matter.

[0010] According to any of the above, some implementations of the homogenization medium include water. In some cases, pure water is used. In some cases, the water contains one or more additives, such as salt. In some cases, the homogenization medium includes a buffer. In some implementations, a concentration of the organic matter in the homogenization medium is between 1% and 50% w / v, or any subrange thereof (e.g., between 5% and 20%, between 8% and 12%, approximately 10% (±1%), or any other subrange).

[0011] According to any of the above, some implementations include removing residual carbohydrates from the organic matter. In some cases, removing residual carbohydrates includes agitating the organic mixture. The agitation can be done at any temperature, but in some cases it is done at a temperature greater than 70°C. Some implementations include agitation at a temperature of 90°C, or greater, or at any set of temperature and pressure conditions that cause the organic mixture to boil. In some cases, the agitation is done for at least 5 minutes (and in some cases, for between 10-60 minutes, with 30 minutes being preferred).

[0012] According to any of the above, some implementations involve separating the organic mixture into a supernatant (containing the removed residual carbohydrates) and a precipitate (containing the desired protein product in non-concentrated form) using at least one of centrifuging, filtering, and decanting the organic mixture.

[0013] According to any of the above, some implementations of the method include hydrating the precipitate. In some cases, this includes suspending the precipitate in a suspension medium. In some cases, this includes allowing the precipitate to soak in the suspension medium, or agitating the precipitate in the suspension medium, until a desired degree of hydration is achieved (e.g., until there are no lumps in the suspension, until the suspension has a constant shear, until a consistent consistency is achieved, or until a satisfactory degree of hydration is otherwise determined). Some implementations of the suspension medium include at least one of water, salt, and a buffer. In some implementations, the precipitate is hydrated in the suspension medium at a concentration of between 1% and 50% w / v, or any subrange thereof (e.g., between 5% and 20%, 8% and 12%, approximately 10% (±1%), or any other concentration or concentration range between 1% and 50%). In some implementations, the hydration is carried out for at least 5 minutes (and in some cases, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes or within any other timeframe required for satisfactory hydration).

[0014] According to any of the above, the enzyme mixture can include one or more additional or alternative enzymes. In some cases, the additional or alternative enzyme or enzymes are selected from any of the following types of enzymes: arabinose, beta glucosidase, endogalactouronase, hemicellulase, protease, papain, aminopeptidase, carboxypeptidase, glutaminase, transpeptidase, endoprotease, casein protease, or other types of carbohydrases or proteases.

[0015] According to any of the above, the concentration of the enzyme mixture in the suspension can be any concentration suitable for treating the suspension. In some cases, the concentration of the enzyme mixture in the suspension is approximately 0.01%-20%, or any suitable subrange thereof (e.g., approximately 1-2%, etc.). In some cases, after the enzyme mixture is added to the suspension, the concentration of each enzyme within the suspension is between approximately 0.01% and 20%, or any subrange thereof (e.g., approximately 1% cellulase, approximately 1% beta glucanase, etc.).

[0016] According to any of the above, some implementations include adjusting the parameters of the suspension to comport with working parameters of the enzyme mixture. In some cases, the suspension is created to have parameters consistent with working parameters of the enzyme mixture. The parameters in question can include any parameter that might affect the function of an enzyme, such as pH, temperature, enzyme concentration, substrate concentration, product concentration, the presence of enzyme inhibitors or activators, and agitation. As an example, in some cases the method includes adjusting the temperature of the suspension to a working temperature of the enzyme mixture (e.g., a temperature at which the enzymes in the enzyme mixture all function, or at which one or more of the enzymes function optimally, or a temperature that optimizes functionality of all the enzymes together). In some cases, the temperature is between 4°C and 80°C, or any subrange thereof (e.g., 20-60°C, 50°C ±5°C, etc.). As another example, in some cases the method includes adjusting the pH of the suspension to a working pH of the enzyme mixture (e.g., a pH at which the enzymes in the enzyme mixture all function, or at which one or more of the enzymes function optimally, or a pH that optimizes functionality of all the enzymes together). In some cases, the pH is between 3 and 12, or any subrange thereof (e.g., 5-8, 5.8 ±0.5, etc..

[0017] According to any of the above, some implementations of treating the suspension with the enzyme mixture includes maintaining one or more parameters of the suspension at one or more of the working parameters of the enzyme mixture (e.g., maintaining the temperature, pH, etc. at the working temperature, pH, etc. of the enzyme mixture) for a period of time. In some cases, the period of time is at least 10 minutes, but in many cases is substantially longer (e.g., at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, etc.). In some cases, treating the suspension includes deactivating the enzyme mixture after the time period has elapsed. In this regard, the enzyme mixture can be deactivated in any suitable manner, such asby heating (to a temperature that denatures or otherwise inactivates the enzymes), changing the pH (to a pH that is hostile to the enzymes), or otherwise denaturing or inactivating the enzymes.

[0018] The method according to any of the above can include extracting a high-protein concentrate from the suspension. In some implementations, extracting the high-protein concentrate from the suspension involves separating the suspension into a solid phase and a liquid phase using centrifugation, filtration decanting, or any other method for separating the suspension into a solid phase and a liquid phase. In some cases, due to the processes used (e.g., the enzyme treatment), the liquid phase contains proteins separated from other portions of the organic matter (such as carbohydrates, lipids, and other components). Congruently, in some cases, extracting the high- protein concentrate from the suspension includes drying the liquid phase to evaporate water (or other solvent) while leaving behind the high-protein concentrate solute. In some cases, the drying is effectuated by spray drying, but in some cases it is done through vacuum drying, oven drying, freeze drying, fluid bed drying, or any other method of drying.

[0019] The method according to any of the above includes, in some cases, incorporating the high-protein concentrate into a food product. The food product can be any food product in which an alternative protein would be useful, such as protein powder, imitation meat, supplements, or any other food product.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The objects and features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only typical embodiments of the disclosed systems and methods and are, therefore, not to be considered limiting of its scope, the systems and methods will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0021] Figure 1 shows a flow chart illustrating a method for recovering proteins using enzyme extraction, in accordance with a representative embodiment of the disclosed systems and methods;

[0022] Figure 2 shows a flow chart illustrating a method of creating an organic matter suspension, in accordance with a portion of Figure 1;

[0023] Figure 3 shows a flow chart illustrating a method of treating a suspension with an enzyme mixture, in accordance with a portion of Figure 1; and

[0024] Figure 4 shows a flow chart illustrating a method of extracting a high-protein concentrate from a treated suspension, in accordance with a portion of Figure 1.

[0025] Figure 5 shows gelification of the product when the sample is hydrolyzed with proteases and lack of gelification when the sample is hydrolyzed with carbohydrases.

[0026] Figure 6 shows the comparison of soluble protein after enzymatic extraction from agar-washed and unwashed seaweed by-product using different carbohydrases.

[0027] Figure 7 shows the protein concentration (mg / mL) after enzymatic extraction of seaweed by-product, a) individual enzymes and b) combination of enzymes.

[0028] Figure 8 shows the protein concentration after enzymatic extraction of by-product after different pre-treatments of the initial biomass.

[0029] Figure 9 shows the protein concentration (mg / mL) after using only carbohydrase, only protease, or both (sequentially or combined).

[0030] Figure 10 shows the solubility of proteins from seaweed by-product at different pH.

[0031] Figure 11 shows a schematic representation of chemical extraction methods in combination with enzymatic methods.

[0032] Figure 12 shows the effect of the acid used for adjusting the pH on the final protein concentration (mg / mL).

[0033] Figure 13 shows the protein concentration (mg / mL) and yield (%) after enzymatic extraction (enzyme Beta glucanase 1 (Viscozyme®)) from different seaweed sources. Yield = mass of dry powder obtained from 100 g of initial dry seaweed.

[0034] Figure 14 shows the protein concentration (mg / mL) and yield (%) after enzymatic extraction with different carbohydrases (Cellulase 4 (TS 23 001), Cellulase 6 (Ultraflo® XL), Cellulase 5 (Shearzyme®), Cellulase 3 (Cellulyve®), Beta glucanase 1 (Viscozyme®), Cellulase 1 (Rohament®), Cellulase 2 (Celluclast®)) from an invasive seaweed species (Rugulopteryx okamurae). Yield = mass of dry powder obtained from 100 g of initial dry seaweed.DETAILED DESCRIPTION

[0035] There is a clear need to diversify protein sources for both human, pets and livestock consumption. Accordingly, disclosed herein are methods for extracting proteins from organic matter for use as food-grade protein products or in other useful protein applications. A description of embodiments will now be given with reference to the Figures. It is expected that the present systems and methods may take many other forms and shapes, hence the following disclosure is intended to be illustrative and not limiting, and the scope of the disclosure should be determined by reference to the appended claims. Indeed, while the Figures include flow charts that illustrate general processes shown in linear order, it is important to note that any portion of any method, embodiment, iteration, implementation, case, flow chart, or other part of this disclosure can be rearranged, modified, substituted, excluded, repeated, or combined with any other portion in any manner.

[0036] Referring to Figure 1 , a method 100 is provided for extracting proteins from organic matter. As shown by box 110, some embodiments of the method include obtaining the organic matter. While processes exist to extract proteins from many kinds of organic matter, the processes disclosed herein are particularly suitable for extracting protein from algae.

[0037] Accordingly, the main embodiments disclosed herein use organic matter that includes algae, seaweed and algae, and seaweed byproducts, in particular. In this regard, algae is a particularly useful source of organic matter for several reasons, such as its natural abundance, high protein content, nutritional value, low cost, ease of harvesting, and high availability. In some embodiments, the methods disclosed herein are configured to utilize one or more specific types of algae (e.g., types of algae with greater protein content, greater protein yield using the processes described herein, greater availability, or other advantages). Accordingly, while the algae could include green algae (Chlorophyta), in some cases the algae includes algae with a higher protein content, such as brown algae (Phaeophyta) or red algae (Rhodophyta). Some embodiments of the organic matter include algae with a protein content of 30% ±5% by dry weight, such as certain algae from the genus Porphyra. In some embodiments, the organic matter includes one or more of the following: Gracilaria (spp.), Gelidium (spp.), Cystoseira (spp.), Laminaria (spp.), and Rugulopteryx Okamurae. Indeed, in certain embodiments the methods for extracting proteins are specially configured (e.g., by utilizing specific concentrations of specific enzymes) to optimize the quality and quantity of the protein extracted from these particular varieties of seaweed.

[0038] In some embodiments, the organic matter includes byproducts from the processing of raw organic matter (including any of the organic matter listed above). As an example, the carbohydrate industry often uses certain species of seaweed (such as those listed above) to extract various carbohydrates (e.g., agar, alginate, carrageenan). After carbohydrate extraction, byproducts often remain, which can often have a considerable percentage of protein. These byproducts are often discarded, or sometimes go toward producing low-quality animal feed. Through the processes disclosed herein, protein can be extracted from these byproducts to produce high-quality, food-grade protein products that are fit (and desirable) for human consumption.

[0039] In accordance with the foregoing, some embodiments of the method 100 include obtaining algae byproducts, and some embodiments of the method include extracting carbohydrates from algae to form carbohydrate products and algae byproducts, and the algae byproducts may then be used as the organic matter from which proteins are extracted.

[0040] As shown in box 120 of Figure 1, some embodiments of the method include creating a suspension containing the organic matter. Figure 2 provides a more detailed breakdown of creating a suspension in accordance with box 120. In particular, Figure 2 illustrates that, in some embodiments, creating the suspension includes one or more of the following: suspending the organic matter in a homogenization medium (box 122); homogenizing the organic matter and the homogenization medium to form an organic mixture (box 124); (optionally) removing residual carbohydrates from the organic matter (box 125); separating the organic mixture into a supernatant and a precipitate (box 126); and suspending the precipitate in a suspension medium to hydrate the precipitate and form the suspension (box 128).

[0041] With respect to box 122, the homogenization medium can include any suitable medium, including one or more of water, salt water, a buffer, a non-aqueous medium (e.g., ethanol), or any other suitable liquid medium. That said, in some embodiments, water or salt water is used. Where salt water is used, any type of salt or combination of salts may be used (e.g., sodium chloride (NaCl), potassium chloride (KC1), sodium bicarbonate (NaHCCh), sodium carbonate (Na2COs), potassium sorbate (C6H7KO2), copper sulphate (CuSCh) or any other type of salt) the concentration of salt within the water can be any suitable concentration, such as between 0.01% and 15% w / v, or any subrange thereof (e.g., 1% ±0.5%, 2% ±1%, 3.5% ±1.5%, etc.). In some cases, the specific type of salt used is extremely beneficial in producing an organolepticallyacceptable protein product. In some embodiments, one or more additional additives is included in the homogenization medium, in any suitable concentration.

[0042] The organic matter can be suspended in the homogenization medium in any suitable concentration. In some embodiments, the concentration is between 1% and 50% w / v, or any subrange thereof (e.g., between 5% and 20%, between 8% and 12%, approximately 10% (±1%), or any other subrange).

[0043] With respect to box 124, some embodiments include homogenizing the organic matter and the homogenization medium to form an organic mixture (e.g., after suspending the organic matter in the homogenization medium, while adding the organic matter to the homogenization medium, or at any other suitable time). While the homogenization can occur in any suitable manner (e.g., using one or more of a mixer, a blender, a pulverizer, a grinder, an immersion blender, an agitator, or any another homogenization mechanism), some embodiments utilize a high-speed homogenizer. Additionally, in some embodiments, the homogenization need not result in a perfectly homogenous mixture. Rather, in some embodiments, a generally homogenous mixture (e.g., a generally consistent blend) is sufficient. That said, in some embodiments, a truly homogenous organic mixture is achieved.

[0044] With respect to box 125, some embodiments include removing residual carbohydrates from the organic matter (e.g., after homogenization, during homogenization, or at any other suitable time). In some cases, removing residual carbohydrates includes agitating the organic mixture. The agitation can be done at any temperature, but in some cases it is done at a temperature of between 70°C and 150 °C (or any subrange thereof), or at any temperature greater than about 70°C. Some implementations include agitation at a temperature of greater than (or equal to) 90°C, greater than (or equal to) 100°C, or at any set of temperature and pressure conditions that cause the organic mixture to boil or simmer. In some cases, the agitation is done for at least 5 minutes (and in some cases, for between 10-60 minutes, with 30 minutes being preferred). In some embodiments, agitation of the heated mixture can cause residual carbohydrates (e.g., agar, etc.) to leach from the organic matter solute and into the solvent, which can later be separated out into a supernatant and a precipitate (with the residual carbohydrates being dissolved in the supernatant).

[0045] To expound on the foregoing, box 126 shows that some embodiments include separating the organic mixture into a supernatant and a precipitate. The supernatant and precipitate can be separated in any suitable manner, including through the use of at least one of centrifuging,filtering, decanting, aspirating, or otherwise separating the precipitate from the supernatant. That said, in some cases, centrifugation or filtration may be particularly suited to removing the supernatant (along with residual carbohydrates dissolved therein). In some embodiments, the supernatant can subsequently be discarded or repurposed, and the precipitate (containing the desired protein) can be further processed for protein extraction.

[0046] In accordance with box 128, some embodiments include suspending the precipitate in a suspension medium to form a suspension. In some cases, this includes hydrating the precipitate. In some embodiments, hydrating the precipitate includes allowing the precipitate to soak in the suspension medium, or agitating the precipitate in the suspension medium, until a desired degree of hydration is achieved (e.g., until there are no lumps in the suspension, until the suspension has a constant shear, until a consistent consistency is achieved, or until a satisfactory degree of hydration is otherwise determined). In some embodiments, the hydrating includes measuring one or more parameters of the suspension (e.g., viscosity, shear rate, transparency, volatility, etc.) and continuing to hydrate until desired parameters are achieved.

[0047] While the suspension medium can include any liquid medium, water-based media may be particularly suited to hydrating the precipitate. Accordingly, some embodiments of the suspension medium include at least one of water, salt, and a buffer. As with the homogenization medium, where salt water is used, any salt may be used, and the concentration of salt can be any suitable concentration (e.g., between 0.01% and 15% w / v, or any subrange thereof).

[0048] In some embodiments, the precipitate is suspended in the suspension medium at a concentration of between 1% and 50% w / v, or any subrange thereof (e.g., between 5% and 20%, 8% and 12%, approximately 10% (±1%), or any other concentration or concentration range between 1% and 50%). In some embodiments, the hydration is carried out for at least 5 minutes (and in some cases, at least 10 minutes, at least 20 minutes, at least 30 minutes, or within any other timeframe required for satisfactory hydration).

[0049] Turning now to box 130 of Figure 1, some embodiments of the method include treating the suspension with an enzyme mixture. In this regard, some embodiments of the enzyme mixture include one or more active enzymes, such as carbohydrases (e.g., maltases, amylases, xylanases, mannases, glucanases, arabinoses, cellulases, beta-glucosidases, beta-glucanases, hemicellulases, etc.) or proteases (e.g., alkaline proteases, neutral proteases, papains, aminopeptidases, carboxypeptidases, glutaminases, transpeptidases, endoproteases, caseinproteases, or other types of carbohydrases or proteases.). In this regard, while some embodiments may contain only a single active enzyme, some embodiments include at least two different active enzymes, which may be employed simultaneously or sequentially. While the enzymes could be any enzymes capable of assisting the breakdown of organic structures to isolate proteins, it is important to note that this disclosure includes particular embodiments in which the enzymes are specifically chosen, concentrated, and otherwise configured for maximum efficacy (and minimal denaturation of proteins) in connection with the specific types of seaweed used. For example, in some cases the enzyme mixture is specifically targeted for assisting in protein extraction from Gracilaria, Gelidium, Cystoseira, Laminaria, and Rugulopteryx Okamurae. In some cases, at least one of the enzymes includes a carbohydrase (e.g., maltase, amylase, xylanase, mannase, glucanase, etc.). In some cases, at least one of the enzymes include a protease. Importantly, in some cases, at least one of the enzymes is cellulase, and in some cases, at least one of the enzymes is beta glucanase. In some such cases, at least one of the enzymes is cellulase, and in some such cases, at least one of the enzymes is beta glucanase. In some cases, the enzyme mixture includes approximately equal parts of cellulase and beta glucanase. In some cases the enzyme mixture includes approximately twice as much cellulase as beta glucanase. In some cases the enzyme mixture includes approximately twice as much beta glucanase as cellulase. Indeed, in some embodiments, the combination of cellulase and beta glucanase is key in obtaining a high-quality, organoleptically acceptable protein from Graciliaria, Gelidium, Cystoseira, Laminaria, and Rugulopteryx Okamurae.

[0050] In some embodiments, the enzyme mixture includes one or more additional or alternative enzymes. In this regard, using specific types of enzymes in specific concentrations, or even using specific enzyme species, is key to obtaining a high-quality, organoleptically acceptable protein from a particular type of seaweed or combination thereof. Accordingly, in some cases, the additional or alternative enzymes are selected from any of the following types of enzymes: arabinase, cellulase, beta glucosidase, endogalactouronase, hemicellulase, protease, papain, aminopeptidase, carboxypeptidase, glutaminase, transpeptidase, endoprotease, or other types of carbohydrases or proteases. In some cases, specific enzymes are used. Indeed, the enzyme mixture of some embodiments includes TS 23-001 (e.g., derived from aspergillus niger). Some embodiments include cellulyve 50L (e.g., derived from trichoderma reesei). Some embodiments include G016L: Beta-glucosidase 16L (e.g., derived from trichoderma sp.). Some embodimentsinclude D040L: depol 40L (e.g., derived from trichoderma sp. or aspergillus sp). Some embodiments include viscozyme (e.g., derived from aspergillus aculeatus). Some embodiments include celluclast (e.g., derived from trichoderma reesei). Some embodiments include ROHAMENT CL (e.g., derived from trichoderma reesei). Some embodiments include TS23.010 (e.g., derived from bacillus licheniformis). Some embodiments include prolyve BS2 liquide (e.g., derived from bacillus subtilis). Some embodiments include P144GL: promod 144GL (e.g., derived from carica papaya). Some embodiments include flavourzyme OOL (e.g., derived from aspergillus oryzae). Some embodiments include protana prime (e.g., derived from aspergillus oryzae). Some embodiments include protana uboost (e.g., derived from bacillus licheniformis). Some embodiments include F373MDP: FlavorPro 373MDP (e.g., drived from bacillus sp.). Some embodiments includes F750MDP: FlavorPro 750 MDP (e.g., derived from aspergillus sp. and bacillus sp.). Some embodiments include COROLASE 7089 (e.g., derived from bacillus amyloliquefaciens). More information about additional or alternative carbohydrases is included in Table 1 below, and more information about additional or alternative proteases is included in Table 2 below.

[0051] Table 1 : Carbohydrases

[0052] Table 2: Proteases

[0053] Figure 3 provides additional details regarding some embodiments of the treatment with the enzyme mixture. For example, box 132 shows that some embodiments of the method include adjusting the parameters of the suspension to fit the working parameters of the enzyme mixture. The parameters to be adjusted can include any parameter that might affect the function of an enzyme, such as pH, temperature, enzyme concentration, substrate concentration, product concentration, the presence of enzyme inhibitors or activators, and agitation. The term “working parameters” refers to any set of parameters at which one or more of the enzymes in the enzyme mixture can operate. For example, a working temperature for the enzyme mixture would be a temperature at which at least one of the enzymes in the mixture can carry out its intended function (e.g., by participating in a certain reaction, etc.).

[0054] Some embodiments include optimizing the parameters. For example, in some embodiments, the parameters are adjusted to match parameters at which all the enzymes in the enzyme mixture can function, or at which one or more of the enzymes function optimally, or at which the functionality of all the enzymes together is optimized. In some embodiments, different parameters can be optimized for different enzymes. In some embodiments, the method includes multiple optimization phases, where the parameters are optimized for different enzymes which are added either simultaneously or sequentially (e.g., two enzymatic hydrolysis steps take place wherein in the first step a first phase is optimized for a first enzyme and in the second step a second phase is optimized for a second enzyme).

[0055] It is worth noting that, in some cases, the parameters of the suspension may already fit the working parameters or optimized parameters of the enzyme mixture, in which case adjustment of the parameters may already be completed. In other cases, it may be desirable for the hydration of the precipitate to occur at different parameters than the treatment with the enzyme mixture, in which case the adjustment of parameters may be performed subsequent to the hydration.

[0056] By way of non-limiting illustration, the adjustment of parameters can include adjusting the temperature of the suspension to a working temperature (or an optimal temperature, or any temperature between a working temperature and an optimal temperature) of the enzyme mixture. In some cases, the temperature is between 4°C and 80°C, or any subrange thereof (e.g., 20-60°C, 50°C ±5°C, etc.). As another example, in some cases the method includes adjusting the pH of the suspension to a working pH (or an optimal pH, or any pH in between a working pH and an optimal pH) of the enzyme mixture. In some cases, the pH is between 3 and 12, or any subrange thereof (e.g., 5-8, 5.8 ±0.5, etc.).

[0057] With reference to box 134, some embodiments include adding the enzyme mixture to the suspension (e.g., after adjusting the parameters, while adjusting the parameters, or at any other suitable time or times during the performance of the method).

[0058] The concentration of the enzyme mixture in the suspension can be any concentration suitable for treating the suspension. In some embodiments, the enzyme mixture is added in amounts sufficient to cause the concentration of the enzyme mixture in the suspension to be approximately 0.01%-20%, or any suitable subrange thereof (e.g., approximately 0. l%-5%, 0.5%-3.5%, 2% ±0.5%, etc.). In some cases, after the enzyme mixture is added to the suspension, the concentration of each enzyme within the suspension is between approximately 0.01% and 20%, or any subrange thereof (e.g., approximately 1% ±0.5% cellulase, approximately 1% ±0.5% beta glucanase, etc.).

[0059] Regarding box 136, some embodiments of treating the suspension with the enzyme mixture include allowing the enzyme mixture to work (e.g., maintaining the temperature, pH, etc. at the working temperature, pH, etc. of the enzyme mixture) for a certain period of time. In some cases, the period of time is at least 10 minutes, but in many cases it is longer (e.g., at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, etc.). In some cases, a treatment of approximately 4 hours ±1 hour may be sufficient (depending on theparameters of the suspension and the enzyme mixture used) for effective protein extraction. In some embodiments, the parameters are adjusted and this portion of the method is repeated.

[0060] With reference to box 137, some embodiments of treating the suspension with the enzyme mixture include deactivating the enzyme mixture (e.g., after the time period has elapsed or the enzyme mixture has otherwise been allowed to carry out its intended function). In this regard, the enzyme mixture can be deactivated in any suitable manner, such as by heating (to a temperature that denatures or otherwise inactivates the enzymes), changing the pH (to a pH that is hostile to the enzymes), or otherwise denaturing or inactivating the enzymes. By way of nonlimiting illustration, some embodiments include heating the suspension to a temperature of greater than 90°C, greater than 100°C, or any other suitable deactivation temperature.

[0061] Turning now to box 140 of Figure 1, some embodiments of the method according to any of the above can include extracting a high-protein concentrate from the suspension. Although the high-protein concentrate can include any proteins that can be extracted from organic matter using the methods described herein, in some cases high-protein concentrates containing particular proteins may be achieved through specific applications of the disclosed methods. For example, in some embodiments, the high-protein concentrate includes one or more of the following: glycoproteins (e.g., lectins, which may have certain therapeutic properties); phycobiliproteins (e.g., phycocyanins / allophycocyanins, phycoerythrocyanins, phycoerythrins), which may have antioxidant, anticancer, and anti-inflammatory properties; and mycosporines (e.g., mycosporine-like amino acids, and RuBisCO enzymes (ribulose-l,5-bisphosphate carboxylase / oxygenase)).

[0062] Figure 4 further describes extracting a high- protein concentration, in accordance with some embodiments. As box 142 illustrates, in some embodiments extracting the high- protein concentrate from the suspension includes separating the suspension into a solid phase and a liquid phase. This separation may be done in any manner, such as through centrifugation, decanting, or any other method for separating the suspension into a solid phase and a liquid phase. In some cases, due to the processes used (e.g., the enzyme treatment, etc.), the liquid phase contains proteins, whereas other portions of the organic matter (such as carbohydrates, lipids, and other components) become part of the solid phase. Accordingly, in some embodiments, the liquid phase is further processed, while the solid phase is discarded or repurposed.

[0063] With reference to box 144, some embodiments include drying the liquid phase to evaporate water (or other solvent) while leaving behind the high-protein concentrate solute. In some cases, the drying is effectuated by spray drying, but in some cases it is done through vacuum drying, oven drying, freeze drying, fluid bed drying, or any other method of drying. In some embodiments, the high-protein concentrate is collected after drying.

[0064] With reference to box 145, the method can also include processing to increase the organoleptic acceptability of the high-protein concentrate. While the high-protein concentrate itself can be the subject of the processing, in some embodiments one or more other components is the subject of the processing, such as the organic matter, the suspension, the organic mixture, the precipitate, or another intermediary in the protein extraction process. In some embodiments, the processing includes filtration (e.g., filtering using activated carbon or another filter), using a drying aid, adding colors or flavors, removing denatured proteins, or other processing that may improve the texture, flavor, scent, appearance, or other properties of the high-protein concentrate relevant to use of the high-protein concentrate in food products.

[0065] Turning back to Figure 1, box 150 illustrates that some embodiments include incorporating the high-protein concentrate into a food product. The food product can be any food product in which an alternative protein would be useful, such as protein powder, imitation meat, supplements, or any other human, pet and livestock food product (including beverages).

[0066] The described methods can be modified in any suitable manner. For instance, some embodiments may include one or more additional protein extraction techniques. In some embodiments, isoelectric precipitation and subsequent solubilization of the protein in an alkaline medium is used, but this may be undesirable in some circumstances, as very extreme conditions can lead to denaturation of protein with a consequent loss of functionality and organoleptic acceptability. Some embodiments incorporate enzymatic hydrolysis, high-pressure homogenization, ultrasound, or microwave extraction techniques. Some embodiments include additional filtration or dialysis processes to increase the protein content of the high-protein extract.

[0067] The described methods can include several benefits over previous systems. For example, it is worth noting that due at least in part to the complexity and rigidity of the algae cell wall, the extraction of algae proteins is more difficult and complicated than extraction of other proteins, such as those of plant or animal origin. Thus, while prior techniques for extracting proteins do exist, the methods described herein are particularly well suited for efficiently andeffectively extracting proteins from algae, while causing minimal denaturation of the proteins and otherwise maximizing their organoleptic acceptability (e.g., pleasant taste, odor, appearance, etc.). Additionally, other protein extraction techniques can lead to large amounts of chemical waste and can be damaging to the environment. In contrast, the techniques described herein can produce high yield at low cost and with little waste, while providing a product with a high enough quality for human consumption.ExamplesExample 1 - Protein Extraction from Algae Byproduct

[0068] Obtain organic material comprising algae byproduct from algae carbohydrate extraction. Suspend the organic material in water to a concentration of 10% w / v. Homogenize the suspension using a high shear homogenizer (Ultraturrax type) at 15,000 RPM for 2 minutes. Place the suspension in a water bath at 90°C for 30 minutes. Centrifuge (or filter) the suspension to eliminate the supernatant (containing excess carbohydrate, e.g., agar, alginate or carrageenan). Resuspend the resulting pellet in water at a concentration of 10% w / v. Agitate the suspension for 30 minutes to achieve full hydration of the pellet. Adjust the pH of the suspension to 5.8 and the temperature to 50°C. Add enzymes to the suspension, including 1% cellulase and 1% beta glucanase. Maintain at pH 5.8, 50°C for 4 hours. Heat the suspension to 80°C for 20 minutes to pasteurize and deactivate enzymes. Filter (or centrifuge) to separate the suspension. Spray dry the supernatant to obtain protein concentrate in powder form.Example 2

[0069] This example concerns the effectiveness of protein extraction (especially from the subproduct of seaweed hydrocolloids production). This by-product is a residue from the agrifood industry that it is currently discarded. This by-product can be obtained from the different hydrocolloid’s providers worldwide, some examples are Hispanagar, Roko, Agar de Asturiasamong others. The objective is to reuse this by-product by extracting protein. The process can be used also with pristine seaweed (raw seaweed).

[0070] Seaweed by-products from different providers were characterised (Table 3). o By-product 1 (provided by Roko SL) and 2 (provided by Hispanagar SL) is Gelidium seaweed that has been subjected to a chemical method to extract agar. This method involves alkali / acid and heat treatments. o By-product 3 (provided by Vetik) is Furcellaria seaweed that has been subjected to a carrageenan extraction process.

[0071] Each by-product and pristine seaweed were analysed with the following standard test procedures: o Protein (%) - PNTA0135 Protein / Nitrogen by thermal conductivity (Dumas method) ( ISO 16634). o Carbohydrates (%) - PNTA0081 Moisture by gravimetry o Lipid (%) - PNTA0133 Fat by gravimetry o Ash (%) - PNTA0083 Ashes by gravimetry o Moisture (%) - PNTA0081 Moisture by gravimetryTable 3. Composition of the biomass (by-products and pristine seaweeds), nd: not determined.

[0072] From all the seaweed by-products, the inventors focused on by-product 1 because it presents the highest protein content, lowest ash content and is available at large amounts. All the following experiments have been carried out with by-product 1.A GAR WASHING

[0073] Before any enzymatic protease extraction, any residual agar was washed from the sample (by-product 1) to: a) avoid the gelification of the product. This step was only necessary when the sample was hydrolysed with proteases because proteases do not degrade agar (Figure 5). b) improve the yield of protein in the final product. Initially it was thought that removing the agar when using carbohydrase would decrease the amount of carbohydrates and therefore improve the protein content (see Figure 6).

[0074] Seaweed by-product 1 was washed with distilled water. First, distilled water was added to seaweed by-product 1 (ratio 2: 1) and the mixture was stirred for 30 min at room temperature using helix mixer at low speed (50 rpm). Then, the temperature was increased from room temperature to 90 °C for 5 min to solubilise the residual agar. After centrifugation at 2500xg for 15 min, the supernatant (containing agar) was discarded, and the pellet (washed seaweed) was used for protein extraction.If carbohydrases were used, there is no need to do the agar washing.ENZYME SELECTION

[0075] Initially, an enzyme pre-screening was made to ensure the use of enzyme on by product 1 was efficient. Distilled water was added to agar washed wet seaweed (ratio 1:5 to 1:7 (10 g of wet seaweed to 50 or 70 mL of water)) and the pH was adjusted to 5.0 - 6.0 (depending on the enzyme used). Then, the enzyme was added and stirred for 4 h. The amount of enzyme added was 3.33 mL for each 100 g of seaweed. After centrifuging at 2500 xg for 10 min, the liquid extract was obtained, and the residual seaweed was discarded. Six enzymes (4 cellulases and 2 beta glucanases (Depol®, Viscozyme®, Cellulyve®, Celluclast®, TS-23-001, and Rohament®)) were assayed individually (Figure 7a) or in combination (Figure 7b). The most promising enzymes were beta glucanase 1 (Viscozyme®) and beta glucanase 2 (Depol®) (individually or in combination), followed by cellulase 1 (Celluclast®) and cellulase 2 (Rohament®). The efficiency of the enzymes was tested by measuring the amount of protein in the liquid extract after the enzymatic reaction. The protein was measured using the BCA assay (Pierce™ BCA Protein Assay Kits, ThermoFisher).

[0076] The combinations beta glucanase 1 (Viscozyme®) and beta glucanase 2 (Depol®) and beta glucanase 1 (Viscozyme®) and cellulase 2 (Celluclast®) were chosen based on protein concentration in the liquid extract after individual enzymatic treatment and combined activities (each enzyme has a distinct activity).EFFECT OF INACTIVATION METHOD AND pH CONTROL

[0077] The pH of reaction was between 4 and 5, and the reaction temperature was between 40 and 50 °C. After the reaction, the enzyme was inactivated. The inventors compared the two methods for inactivating the enzyme: increase the pH to 7.0 or increase the temperature to 90°C (Table 5).

[0078] The inventors also tested if controlling and adjusting the pH during the enzymatic reaction affected the final protein in the extract, so the inventors compared “pH control” vs “pH free fall”. pH control: pH is measured continuously and adjusted if needed. pH free fall: pH is measured before starting the reaction and it is not further adjusted during the reaction (the pH naturally drops due to enzymatic action).

[0079] All the experiments were tested using by-product 1 with cellulase 1 (Rohament®). pH free fall resulted in a higher protein concentration than controlling the pH. No difference was observed between inactivation by heating and by increasing the pH for pH free fall (table 5).Table 5. Protein concentration (mg / mL) in the extracts using different extraction conditions (pH control vs. pH free fall) and inactivation methods (increasing pH vs increasing temperature).EFFECT OF INITIAL BIOMASS

[0080] Biomass, either cultivated seaweed or by-products, can be wet (fresh) or dried (at mild conditions or at high temperatures).o Wet seaweed: the suspension for enzymatic protein extraction was done with the fresh seaweed as it comes, without modification. o Dried, low temperature: seaweed was dried for 6 to 16 h at 50 °C. Then, the suspension for enzymatic protein extraction was carried out using the dried seaweed. o Dried, high temperature: seaweed was dried for more than 6 h at temperatures higher than 90 °C. Then, the suspension for enzymatic protein extraction was carried out using the dried seaweed.

[0081] We analysed the differences on protein extraction efficiency using by-product 1 in wet and dried forms. Seaweed was dispersed in distilled water, pH was adjusted to 5.0 and the enzyme beta glucanase 2 (Viscozyme®) was added. The reaction took place for 3 h at 40 - 60 °C. The extraction using the wet by-product (without heat treatment) presented the highest protein concentration. Protein was also obtained from dried seaweed at low temperatures; however, significantly lower concentrations were obtained (Figure 8). Drying the initial seaweed at high temperatures resulted in absence of protein after the extraction, so this pretreatment was discarded.EFFECT OF SCALE

[0082] A larger batch was made with the beta glucanase 1 (Viscozyme®) to start testing scalability. 200 g of wet seaweed (by-product 1) was added to 400 mb of distilled water and the pH was adjusted to 5.0. Then, beta glucanase 1 (Viscozyme®) was added to a final concentration of 0.7% and stirred for 3 h at 60 °C. An overstirrer stirring rod was used to simulate the mixing in a bioreactor. The obtained powder contained 15 % of protein, and the yield was 21% (similar to previous experiments performed at same or similar conditions with beta glucanase 1 (Viscozyme®) and by-product 1, but at a smaller scale (max. 30 g of seaweed)).COMBINATION OF CARBOHYDRASE WITH PROTEASE

[0083] The extraction process with carbohydrases was combined with a protease to increase the protein extraction. The protease was added simultaneously and sequentially to look for differences (Figure 9). The individual treatments refer to the addition of one enzyme for 1 h (beta glucanase 1 (Viscozyme®)) as the carbohydrase and Protease 1 ((Flavourzyme®) as the protease). The sequential treatment refers to the addition of beta glucanase 1 (Viscozyme®) for the first hour of reaction, followed by addition of protease 1 (Flavourzyme®) for the second hour of reaction. In the simultaneous treatment, beta glucanase 1 (Viscozyme®) and protease 1 (Flavourzyme®) were added at the same time and the reaction took place for 2 h. In all the tests, the enzyme was added to a final concentration of 0.7%. Sequential and combination procedures increased the protein concentration in comparison with using individual enzymes.SOL UBILITY OF PROTEINS A T DIFFERENT pH

[0084] The effect of a chemical extraction (alkaline-acid precipitation) was also evaluated using by-product 1. The chemical method consists of alkali solubilisation (protein solubility) of the proteins followed by acid precipitation. The solubility of proteins extracted was tested against different pH by adjusting the pH with NaOH or HC1. For each of the pH values, the sample was stirred for 30 minutes in an orbital shaker at 200 rpm. The samples were then centrifuged at 2000 xg for 20 min, and the protein content of the supernatant was measured spectrophotometrically using the BCA assay. Results are expressed in absorbance: higher absorbance means higher protein content. Proteins were more soluble at alkali pH, especially at pH 11. According to the results (Figure 10), solubilization pH was set at 11. Regarding precipitation, pH 2 presented the lowest absorbance (lowest protein solubility) and the highest protein precipitate weight, so precipitation pH was set at pH 2.COMBINATION OF ENZYMATIC AND CHEMICAL PROTOCOLS

[0085] The enzymatic methodology was combined with chemical precipitation of proteins to increase the purity of the final product (Figure 10). Seaweed by-product 1 was used. Enzymatic methodology refers to the use of carbohydrases to extract proteins (in this case, beta glucanase 1 (Viscozyme®)), while the chemical method is the alkali solubilisation of the proteins followed by acid precipitation, according to results reported in the previous section.

[0086] In protocol V2.0, the seaweed by-product was only treated with the chemical method (alkali solubilisation and acid precipitation). First, by-product 1 was resuspended in water and the pH was adjusted to 11 using NaOH or KOH to solubilise the proteins. After stirring for 30 min using an overhead stirrer, the mixture was separated by centrifugation for 20 min at 2000 xg, and the residual seaweed (pellet) was discarded. The supernatant was collected, and the pH was adjusted to 2 - 3 using HC1 to precipitate the proteins. The mixture was centrifuged to obtain the precipitated proteins (pellet). The protein concentration in the resulting precipitate had 0.9 mg / mL protein and the yield of the process was 0.4%.

[0087] In protocol V2.1, the seaweed by-product was previously treated with carbohydrases (beta glucanase 1 (Viscozyme®), 3 h, pH 5.0 as previously described) and the residual seaweed was separated from the protein extract by centrifugation for 20 min at 2000 xg. The resulting seaweed residue (pellet after centrifugation) was treated with the chemical method (alkali solubilisation at pH 11 followed by acid precipitation at pH 2 - 3). The protein concentration in the precipitate was 3.5 mg / mL and the yield of the process was 0.6%.

[0088] In protocol V2.2, the seaweed by-product was subjected to enzymatic treatment followed by chemical treatment. After the enzymatic extraction (Beta glucanase 1 (Viscozyme®), 3 h, pH 5.0, final concentration 0.7% as previously described), the pH of the mixture was increased to 11 and further stirred for 30 min using an overhead stirrer. The mixturewas then centrifuged for 20 min at 2000 xg, and the supernatant was subjected to acid precipitation by adjusting the pH to 2 - 3 with HC1. The protein concentration in the precipitate was 1.9 mg / mL and the yield of the process was 0.6%.EFFECT OF ACIDS

[0089] Different acids (hydrochloric acid, citric acid, tartaric acid and phosphoric acid) were used on by-product 1 in the enzymatic extraction in order to test possible differences when adjusting the pH for the enzymatic reaction. The seaweed byproduct was added to water to create a suspension, and a solution of IM hydrochloric acid, citric acid, tartaric acid or phosphoric acid were used to adjust the pH to 4 - 5. Then, Beta glucanase 1 (Viscozyme®) was added and the reaction took place at 40 °C for 3 h in an orbital shaker at 200 rpm. Citric acid and phosphoric acid resulted in the highest protein concentration (Figure 12). The inventors chose citric acid for adjusting the initial pH of the seaweed previous to enzymatic reaction, since it is a common acid in the food industry.EFFECT OF SEAWEED SPECIES

[0090] Enzymatic extraction with carbohydrases was tested using different seaweeds and by-products (Figure 13). 5 g of each of the dried seaweed and by products was hydrated with excess of distilled water (200 mL). The wet seaweed was added to 75 mL of water and the pH was adjusted to 5 with HC1. The enzyme Beta glucanase 1 (Viscozyme®) was added to a final concentration 0.7%, and the reaction was kept at 40 °C for 3 h while stirring in an orbital shaker at 200 rpm. The mixture was then centrifuged at 2000 xg for 10 min to obtain the protein extract. The protein concentration in the extract was measured spectrophotometrically using the BCA assay. The highest protein content was found in Palmaria palmata and by-product 2, while thehighest yield was by-product 1. These results demonstrated that the enzymatic extraction of protein is feasible in different seaweed species and by-products.EXTRA CTION OF PROTEIN FROM INVASIVE SEA WEED

[0091] Enzymatic extraction with different carbohydrases (Cellulase 4 (TS 23 001), Cellulase 6 (Ultraflo® XL), Cellulase 5 (Shearzyme®), Cellulase 3 (Cellulyve®), Beta glucanase 1 (Viscozyme®), Cellulase 1 (Rohament®), Cellulase 2 (Celluclast®)) was tested using an invasive seaweed species (Rugulopteryx okamurae). 5 g of dried Rugulopteryx okamurae were stirred in an orbital shaker at 200 rpm in 50 mL of distilled water at pH 5 and 50 °C for 3 h. Then, the mixture was centrifuged for 10 min at 2000 xg, and the protein concentration of the resulting supernatant (protein extract) was measured using the BCA assay (Pierce™ BCA Protein Assay Kits, ThermoFisher). The highest protein concentration was obtained with Cellulase 1 (Rohament®), Cellulase 2 (Celluclast®) and Beta glucanase 1 (Viscozyme®) (Figure 14).

[0092] Before concluding, it is worth noting that any of the components in any of the figures, embodiments, implementations, instances, cases, methods, applications, examples, iterations, and other parts of this disclosure can be combined in any suitable manner. Additionally, any component can be removed, separated from other components, modified with or without modification of like components, or otherwise altered together or separately from anything else disclosed herein.

[0093] As used herein, the singular forms “a”, “an”, “the” and other singular references include plural referents, and plural references include the singular, unless the context clearly dictates otherwise. For example, reference to an enzyme includes reference to one or more enzymes, and reference to proteins includes reference to one or more proteins. In addition, where reference is made to a list of elements (e.g., elements a, b, and c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and / or a combination of all of the listed elements. Moreover, the term “or” by itself isnot exclusive (and therefore may be interpreted to mean “and / or”) unless the context clearly dictates otherwise. Similarly, the term “and” by itself is not exclusive (and therefore may be interpreted to mean “and / or”) unless the context clearly dictates otherwise. Furthermore, the terms “including”, “having”, “such as”, “for example”, “e.g.”, and any similar terms are not intended to limit the disclosure, and may be interpreted as being followed by the words “without limitation”.

[0094] In addition, as the terms “on”, “disposed on”, “attached to”, “connected to”, “coupled to”, etc. are used herein, one object (e.g., a material, element, structure, member, etc.) can be on, disposed on, attached to, connected to, or otherwise coupled to another object — regardless of whether the one object is directly on, attached, connected, or coupled to the other object, or whether there are one or more intervening objects between the one object and the other object. Also, directions (e.g., “front”, “back”, “on top of’, “below”, “above”, “top”, “bottom”, “side”, “up”, “down”, “under”, “over”, “upper”, “lower”, “lateral”, “right-side”, “left-side”, “base”, etc.), if provided, are relative and provided solely by way of example and for ease of illustration and discussion and not by way of limitation.

[0095] The described methods may be embodied in other specific forms without departing from their spirit or essential characteristics. The described embodiments, examples, and illustrations are to be considered in all respects only as illustrative and not restrictive. The scope of the described methods is, therefore, indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. Moreover, any component and characteristic from any embodiments, examples, and illustrations set forth herein can be combined in any suitable manner with any other components or characteristics from one or more other embodiments, examples, and illustrations described herein.

Claims

CLAIMS1. A method of producing a concentrated protein product, the method comprising: obtaining a non-animal organic matter comprising at least one of (a) an algae, and (b) byproducts derived from the algae; creating a suspension containing the organic matter; treating the suspension with an enzyme mixture; and extracting a high-protein concentrate from the suspension.

2. The method as recited in claim 1, wherein the algae comprises at least one of the genus Gracilaria, Gelidium, Cystoseira, Laminaria and Rugulopteryx.

3. The method as recited in claim 2, wherein the enzyme mixture comprises at least one of cellulase and beta glucanase.

4. The method as recited in claim 1, wherein the organic matter comprises the byproducts derived from the algae, wherein the byproducts include carbohydrate-extraction byproducts.

5. The method as recited in claim 1, wherein creating the suspension comprises: suspending the organic matter in a homogenization medium; homogenizing the organic matter and the homogenization medium to form an organic mixture; separating the organic mixture into a supernatant and a precipitate; and hydrating the precipitate by suspending the precipitate in a suspension medium, thereby forming the suspension.

6. The method as recited in claim 5, wherein the homogenization medium comprises at least one of water, salt, and a buffer.

7. The method as recited in claim 6, wherein a concentration of the organic matter in the homogenization medium is between 5% and 20% w / v.

8. The method as recited in claim 5, wherein separating the organic mixture into a supernatant and a precipitate comprises at least one of centrifuging, filtering, and decanting the organic mixture.

9. The method as recited in claim 5, further comprising removing residual carbohydrates from the organic mixture.

10. The method as recited in claim 9, wherein the removing residual carbohydrates comprises agitating the organic mixture at a temperature of greater than 70°C for at least 5 minutes.

11. The method as recited in claim 5, wherein the suspension medium comprises at least one of water, salt, and a buffer.

12. The method as recited in claim 11, wherein a concentration of the precipitate in the suspension medium is between 5% and 20% w / v.

13. The method as recited in claim 5, wherein the hydrating the precipitate comprises suspending the precipitate in a hydration medium for at least 10 minutes.

14. The method as recited in claim 1 , wherein treating the suspension with the enzyme mixture comprises at least one of i) adjusting a temperature of the suspension to a working temperature of the enzyme mixture, and ii) adjusting a pH of the suspension to a working pH of the enzyme mixture.

15. The method as recited in claim 14, wherein the treating the suspension with the enzyme mixture further comprises maintaining the suspension at the working temperature of the enzyme mixture and at the working pH of the enzyme mixture for a time period of at least 30 minutes.

16. The method as recited in claim 1 , wherein treating the suspension with the enzyme mixture comprises at least one of i) adjusting a temperature of the suspension to a working temperature of a first one of the enzymes in the enzyme mixture, and ii) adjusting a pH of the suspension to a working pH of a first one of the enzymes in the enzyme mixture, maintaining the suspension at the working temperature and at the working pH of a first one of the enzymes in the enzyme mixture for a time period of at least 30 minutes, iii) adjusting a temperature of the suspension to a workingtemperature of a second one of the enzymes in the enzyme mixture, and iv) adjusting a pH of the suspension to a working pH of a second one of the enzymes in the enzyme mixture and maintaining the suspension at the working temperature and at the working pH of a second one of the enzymes in the enzyme mixture for a time period of at least 30 minutes.

17. The method as recited in claim 15, wherein the treating the suspension with the enzyme mixture further comprises deactivating the enzyme mixture after the time period has elapsed.

18. The method as recited in claim 1 , wherein extracting the high-protein concentrate from the suspension comprises separating the suspension into a solid phase and a liquid phase.

19. The method as recited in claim 18, wherein extracting the high-protein concentrate from the suspension further comprises drying the liquid phase to obtain the high-protein concentrate.

20. The method as recited in claim 1, further comprising processing at least one of the organic matter, the suspension, and the high-protein concentrate to increase an organoleptic acceptability of the high-protein concentrate.

21. The method as recited in claim 1, further comprising incorporating the high-protein concentrate into a food product.

Images