Photosynthetically controlled spirulina products with enhanced content and / or bioavailability of non-oppositionally active vitamin B12 - Patent Application 20100223333

Abstract

Spirulina products, biomass, extracts, various foods, dietary supplements, and methods of preparation and use of Spirulina products are provided. Spirulina products are prepared from Arthrospira sp. biomass cultivated under controlled ultra-high density conditions with intense UV irradiation and intense continuous mixing. Spirulina products may be made from algae biomass and / or wet / dried extracts thereof, and are characterized by high levels of unopposed B12, providing more bioavailable B12 than pseudo-B12. Spirulina products may have more bioavailable B12 than pseudo-B12 (e.g., at least two or three times more bioavailable B12 than pseudo-B12, and up to thousands of times more bioavailable B12), providing a large portion of the daily B12 intake required in a wide range of foods.

Description

[Technical field]

[0001] The present invention relates to the field of photosynthetically controlled Spirulina products, and more particularly to enhancing the content and / or bioavailability of B12 in Spirulina products. [Background technology]

[0002] Commercially available spirulina products are typically made from cyanobacteria grown in open ponds and / or other sunlight-based culture systems. Commercially available spirulina typically contains pseudo-B12 (also called cobamides, in which the molecule's lower ligand (5,6-dimethylbenzimidazole (DMB)) is replaced by adenine as the base), which opposes and reduces its bioavailability. Pseudovitamin B12 typically refers to compounds that are corrinoids with a structure similar to vitamin B12 but that do not have vitamin activity, and are therefore metabolically unavailable. For example, Sabrina et al., 2022 (Biologically active or just "pseudo"-vitamin B12 as predominant form in algae-based nutritional supplements?, Journal of Food Composition and Analysis, 10, 104464), incorporated herein in its entirety, teaches a simple and rapid ultra-performance liquid chromatography method with UV detection for measuring algae-based nutritional supplements containing both physiologically active vitamin B12 and its inactive pseudoform. The measurements show a large variability in the concentration of pseudovitamin B12 within all samples analyzed. The Chlorella product contained primarily physiologically active cobalamin, while pseudovitamin B12 was the predominant form in the Spirulina-labeled nutritional supplement.

[0003] Spirulina algae (Spirulina platensis) cultivated in geothermal-driven photobioreactors has been proposed as a potentially resource-efficient, zero-carbon, nutritional alternative to conventional beef, for example in Tzachor et al., 2022 (Environmental Impacts of Large-Scale Spirulina (Arthrospira platensis) Production in Hellisheidi Geothermal Park Iceland: Life Cycle Assessment, Marine Biotechnology 24:991-1001), which is incorporated herein in its entirety. Furthermore, Tzachor et al., 2022 suggests direct decarboxylation of food systems and diets by incorporating Spirulina from geothermal parks into diets as a beef replacement, as well as indirect decarboxylation of food systems by issuing, selling, and purchasing carbon credits between companies. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sabrina et al., Journal of Food Composition and Analysis, 2022, 10, 104464 [Non-Patent Document 2] Tzachor et al., Marine Biotechnology, 2022, 24:991-1001) Summary of the Invention [Means for solving the problem]

[0005] The following is a simplified summary to provide an initial understanding of the invention. This summary does not necessarily identify key elements, is not intended to limit the scope of the invention, and is merely intended to serve as an introduction to the description that follows.

[0006] One embodiment of the present invention provides a Spirulina product made from an aqueous extract of Arthrospira species cultured under photosynthetically controlled conditions to yield upregulated bioactive compounds, including unopposed B12, the Spirulina product having more bioavailable B12 than pseudo-B12.

[0007] One embodiment of the invention provides a method for preparing a Spirulina product, comprising culturing Arthrospira sp. cyanobacteria under photosynthetically controlled conditions to produce upregulated bioactive compounds including unopposed B12, preparing an aqueous extract of the cultured Arthrospira sp. cyanobacteria having more bioavailable B12 than pseudoB12, and optionally drying the extract to produce a Spirulina product having more bioavailable B12 than pseudoB12.

[0008] One aspect of the present invention provides food products and / or dietary supplements comprising any of the disclosed Spirulina biomass, products, extracts, water soluble and / or water insoluble components thereof, and / or combinations thereof, having more bioavailable B12 than pseudo-B12.

[0009] These, additional and / or other aspects and / or advantages of the present invention will be set forth in the following detailed description, and may possibly be inferred from the detailed description and / or learned by practice of the invention. [Brief description of the drawings]

[0010] For a better understanding of embodiments of the present invention and to show how it may be carried into effect, reference will now be made, purely by way of example, to the accompanying drawings in which like numerals indicate corresponding elements or sections throughout, and in which:

[0011] [Figure 1]1A-1F provide chromatograms with experimental results showing the difference between pseudo-B12 and active methylcobalamin (MeB12) in the disclosed extracts and prior art extracts. [Diagram 2] FIG. 2 is a high level schematic flow chart illustrating the cultivation, extraction and processing methods according to some embodiments of the present invention. [Figure 3A] 3A-3D are high level schematic diagrams of culture systems according to some embodiments of the present invention. [Figure 3B] 3A-3D are high level schematic diagrams of culture systems according to some embodiments of the present invention. [Figure 3C] 3A-3D are high level schematic diagrams of culture systems according to some embodiments of the present invention. [Figure 3D] 3A-3D are high level schematic diagrams of culture systems according to some embodiments of the present invention. [Figure 4(1)] FIG. 4 shows non-limiting examples of uses of the disclosed Spirulina biomass and / or dry / wet extracts in various food products according to some embodiments of the present invention. [Figure 4 (2)] This is a continuation of Figure 4(1).

[0012] These, additional and / or other aspects and / or advantages of the present invention will be set forth in the following detailed description, and may possibly be inferred from the detailed description and / or learned by practice of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the following description, various aspects of the present invention are described. For the purpose of explanation, specific configurations and details are shown to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be practiced without the specific details shown herein. Furthermore, well-known features may be omitted or simplified so as not to obscure the present invention. With specific reference to the drawings, it is emphasized that the specific details shown are exemplary and are presented for the purpose of merely explaining the present invention, and are presented for the purpose of providing what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention, and the description taken with the drawings will make clear to those skilled in the art how some forms of the present invention can be embodied in practice.

[0014] Before at least one embodiment of the invention is described in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is applicable to other embodiments that may be practiced or carried out in various ways, as well as to combinations of the disclosed embodiments. It is also to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0015] Some embodiments of the present invention provide efficient and economical methods and mechanisms for preparing Spirulina extracts and powders with high levels of unopposed active vitamin B12 (bioavailable B12 minus pseudoB12), thereby providing an improvement to the field of dietary supplements and improved bioavailable B12 content in foods and other products. Spirulina extracts are typically prepared from cyanobacteria such as Arthrospira species, although it should be noted that the term algae (as in blue-green algae) is also commonly used to refer to cyanobacteria due to their photosynthetic capabilities and chlorophyll content. In some of the results disclosed herein, bioavailable unopposed B12 was measured as methylcobalamin (MeB12), but the disclosed extracts also contain other forms of bioavailable unopposed B12 (as opposed to pseudoB12, which is a form that is not metabolically available), such as 5-deoxyadenosylcobalamin, another form of metabolically active vitamin B12.

[0016] Spirulina products, biomass, extracts, various food products, dietary supplements, and methods of preparing and using the Spirulina products are provided. The Spirulina products are prepared from Arthrospira sp. biomass that is cultured under controlled, ultra-high density conditions using intense UV irradiation and intense continuous mixing. Non-limiting examples of photosynthetically controlled conditions under which Arthrospira sp. are cultured include temperatures of 31±2° C., a pH of 10.8±0.2, and a density of 700-1,500 μmol / m 2 s, a subrange thereof, or even higher in some cases. Specifically, as disclosed below, irradiances of 70 to 150 μmol / m in ultra-high density cultures having densities of 3 g / l to 10 g / l are 2 It has been found that cultivation of Arthrospira species at a UV intensity of 100 s results in the disclosed characteristics for use in the disclosed Spirulina products.

[0017] Spirulina products may be made from algal biomass and / or wet / dry extracts thereof and are characterized by high levels of unopposed B12 (e.g., methylcobalamin, 5-deoxyadenosylcobalamin, or other metabolically active forms of B12), providing more bioavailable B12 than pseudo-B12. Spirulina products may have more bioavailable B12 than pseudo-B12 (e.g., at least two or three times more bioavailable B12 than pseudo-B12), providing a majority of the daily B12 intake required in a wide range of foods (e.g., 15% or more of the daily requirement, DV).

[0018] Spirulina products may be prepared from any of the aqueous algae extracts, dry extracts, spirulina biomass, dry biomass, spirulina paste, spirulina powder and / or spirulina liquid without further processing. Aqueous extracts may be prepared by aqueous extraction of Arthrospira spp. biomass cultivated under controlled ultra-high density conditions using intense UV irradiation and intense continuous mixing, and are characterized by high levels of unopposed B12 (e.g., methylcobalamin, 5-deoxyadenosylcobalamin or other metabolically active forms of B12), which is in contrast to prior art B12 supplements that contain high amounts of pseudo-B12. Aqueous spirulina extracts may be produced by aqueous extraction and freeze and thaw cycles applied to cultivated Arthrospira spp.

[0019] The Spirulina products may be used accordingly as food and / or dietary supplements or food fortifications - as a source of bioavailable B12. Various food products may include the disclosed Spirulina products, biomass, extracts, Spirulina powders, pastes and liquids, their water-soluble and / or water-insoluble components, and / or combinations thereof. Examples of food products may include meat analogs, baked goods, frozen foods, functional bars, gummies, various beverages (e.g., shakes, smoothies) and beverage additives, effervescent tablets, multi-layer capsules, e.g., for use in various beverages and drinks, and combinations thereof. Dietary supplements and additives may include the disclosed products and / or fractions thereof, and may be used as stand-alone supplements or may be added to various foods, for example, as disclosed herein. Furthermore, the disclosed Spirulina products, in addition to providing bioavailable B12 and additional nutrients, may be used in foods to directly decarb foods in the sense suggested by Tzachor et al., 2022. For example, bakeries may decarbonize their production lines by adding the disclosed Spirulina products to breads and baked goods, simultaneously enhancing the nutritional value of their products by providing bioavailable B12, and utilizing the negative carbon footprint of the disclosed Spirulina products to balance the carbon emissions from the bakery and reduce the carbon footprint of their products. Various types of food products may be enriched with the disclosed Spirulina products in any suitable form to enhance their nutritional content, particularly their bioavailable B12 content, and in some cases simultaneously reduce their carbon footprint.

[0020] Table 1 provides liquid chromatography by tandem mass spectrometry (LC-MS / MS) results for two Spirulina extracts grown and extracted as disclosed below, along with a commercially available prior art Spirulina extract derived from open pond grown algae. Measurement methods were as described, for example, in Sela et al., 2020, Wolffia globosa-Mankai Plant-based protein contains bioactive vitamin b12 and is well absorption in humans, Nutrients 12(10), 3067. The disclosed Spirulina extracts are dried to form a Spirulina powder that includes a water-soluble fraction (typically blue) and a water-insoluble fraction (typically green), which are present in a 1:1 ratio in the disclosed Spirulina extracts and powders. The data compares the content of active MeB12 with the content of pseudo-B12 molecules, which are physiologically inactive and even harmful in blocking B12 receptors, in ng / mg powder, and shows that the disclosed extracts and powders have a much higher content of bioavailable MeB12 compared to pseudo-B12, and a higher content of bioavailable MeB12 compared to prior art extracts and powders that contain only pseudo-B12. Note that in the disclosed extracts, the concentration per ml of extract is about 5 times that per mg of powder (the powders include the corresponding dried extracts). Further comparisons of total MeB12 per 100g serving are made, including a comparison with beef. The data clearly show an approximately 1000-fold higher content of bioavailable MeB12 in the disclosed products, which makes them useful as additives and supplements in a variety of applications (e.g., as dietary supplements and / or as additives to meat analogs due to their high levels of bioavailable MeB12; the lack of MeB12 is considered a drawback of prior art meat analogs).

[0021] [Table 1]

[0022] It should be noted that bioavailable MeB12 in higher amounts and / or concentrations than pseudoB12 may be present in both water-soluble and water-insoluble portions and may be used with or without disruption of the cell wall. Thus, various embodiments include foods and / or dietary supplements that include the disclosed Spirulina biomass, extracts (dry or wet), products and / or fraction compounds, for example, in the form of a biomass, extract, paste, liquid or powder, and have more bioavailable MeB12 in the powder or other dry product than pseudoB12, or at least 50 ng / ml of unopposed MeB12 in the biomass, extract or other liquid product. Various embodiments of foods include, for example, meat analogs, baked goods, frozen foods, ice cream, functional bars, gummies, beverages, shakes, beverage additives, effervescent tablets, etc., that include the disclosed Spirulina biomass, extracts, pastes, liquids, powders and / or fraction compounds.

[0023] In various embodiments, foods and / or dietary supplements comprising the disclosed Spirulina biomass, its extracts (dry or wet), products and / or fraction compounds, e.g., in the form of a biomass, extract, paste, liquid or powder, may have an unopposed B12 concentration (e.g., of methylcobalamin, 5-deoxyadenosylcobalamin or other metabolically active forms of B12) of at least 0.7 μg / 100g, at least 2 μg / 100g, at least 10 μg / 100g, at least 100 μg / 100g, at least 1000 μg / 100g, at least 2000 μg / 100g or at least 3000 μg / 100g of unopposed B12 (and more bioavailable B12 than pseudo-B12).

[0024] Figures 1A-1F provide chromatograms with experimental results showing the difference between pseudoB12 and bioavailable MeB12 in the disclosed extracts and prior art extracts. Specifically, Figures 1A and 1B provide chromatograms of bioavailable MeB12 and pseudoB12 in the water-soluble (blue) fraction of the disclosed extracts, respectively, and Figures 1C and 1D provide chromatograms of bioavailable MeB12 and pseudoB12 in the water-insoluble (green) fraction of the disclosed extracts, respectively, both pairs of graphs show the purity of MeB12 without any opposition in the disclosed extracts, while Figures 1E and 1F provide chromatograms of bioavailable MeB12 and pseudoB12 in prior art extracts, respectively, showing that these are mostly additional MeB12-like compounds called pseudoB12 that are not bioavailable and even oppose the bioavailability of MeB12 (see multiple peaks in Figure 1F). As is evident from the data, the disclosed extracts and fractions thereof result in much purer MeB12 than prior art extracts / powders and commercial products from algae grown in open ponds under different conditions than those currently disclosed. The inventors suggest that this high purity results from the growth and extraction conditions of the disclosed extracts and may be responsible for the high bioavailability of MeB12 in the disclosed extracts. For example, the inventors suggest that controlled light conditions (spectral composition, intensity, light modulation) may activate different metabolic pathways in Spirulina / Arthrospira cyanobacteria, which may enable or enhance the synthesis of unopposed methylcobalamin.

[0025] 2 is a high-level schematic flow chart illustrating a culture, extraction and processing method 300 according to some embodiments of the present invention. The method steps may be performed in conjunction with a culture system 101 described herein, which may be optionally configured to perform the method 300. The method 300 may be at least partially performed by at least one computer processor, for example, in a controller 103 including a corresponding processing unit. Certain embodiments include a computer program product including a computer readable storage medium having embodied thereon a computer readable program configured to perform the relevant steps of the method 300. The method 300 may include the disclosed steps in any order.

[0026] As shown generally in FIG. 2, a method 300 for preparing a Spirulina product may include culturing (step 310) an Arthrospira sp. cyanobacterium under photosynthetically controlled conditions, for example in a culture system 101, to yield upregulated bioactive compounds including unopposed methylcobalamin B12 (MeB12). Irradiation may be provided by at least one of the light sources 125 configured to emit UV light in both the UVA and UVB spectrums. In some embodiments, the ratio between the radiation intensities of the UVA / UVB radiation may be in the range of 10-15, for example, UBA / UVB of 10. In certain embodiments, the method 300 may be used to grow a cyanobacterium, for example, Arthrospira sp., from which a Spirulina product is produced. The method 300 may generate UV radiation in the range of 1,000-10,000 kJ / m 2 Strength, for example, 5000 kJ / m 2 , or any other intermediate value (step 312). For example, the controller 103 may be configured to control the supply of UV radiation using on / off radiation pulses. In some embodiments, each pulse may last 0.0099 seconds and may be provided at 1-100 such pulses per second, e.g., 10 times / second. 2It should be noted that irradiation with UV light provides approximately 10 times the intensity of solar UV radiation, altering the chemical composition of the algae and / or cyanobacteria resulting in the products, extracts and compositions disclosed herein.

[0027] Method 300 further includes harvesting the cyanobacteria (step 315), e.g., performing a continuous harvest and matching the harvest rate to the growth rate. Method 300 further includes preparing a Spirulina product from the harvested cyanobacteria, e.g., from the algal biomass and / or from a wet or dry extract thereof (step 320). In various embodiments, the Spirulina product may be prepared from at least one of an aqueous extract, a dry extract, a Spirulina biomass, a dry biomass, a Spirulina paste, a Spirulina powder, and / or a Spirulina liquid without further processing.

[0028] For example, method 300 may include preparing an aqueous extract of cultured Arthrospira sp. cyanobacteria to result in a Spirulina extract having at least 50 ng / ml of unopposed B12 and / or more bioavailable B12, e.g., at least two-fold or three-fold more bioavailable B12 (step 325). Unopposed or bioavailable B12 refers to metabolically active forms of vitamin B12, such as methylcobalamin and 5-deoxyadenosylcobalamin, as opposed to pseudoB12, which represents the metabolically inactive form.

[0029] Extraction may be performed by applying one or more freeze-thaw cycles to break down cell walls and increase the extractability of the suspension. For example, the harvested biomass may be rapidly frozen to -20°C and then thawed at 0-4°C until completely frost-free. The method 300 may further include drying the extract to obtain a spirulina powder having more bioavailable B12 than pseudo-B12 (step 330), e.g., at least two or three times more bioavailable B12. The product, biomass, extract (wet or dry), or derivatives thereof may be prepared as described herein.

[0030] Method 300 may further include preparing a dietary supplement, a pharmaceutical composition, a food product (e.g., a meat analog) and / or a food additive from the biomass, its extract (wet or dry), powder and / or fractions, and / or using the biomass or extract for these purposes (step 340), and / or using the Spirulina product, its biomass, extract (wet or dry) and / or fractions as a food product, such as a meat analog, baked goods, frozen food, ice cream, functional bars, gummies, beverages, shakes, beverage additives, effervescent tablets, etc. (step 350). The use of the Spirulina product in any of these products may also contribute to the reduction of their carbon footprint through the negative carbon footprint of growing and processing Spirulina.

[0031] In certain embodiments, the wet biomass of Spirulina may be suspended in pure water (hot or cold) to obtain a product with 10% dry matter by weight. Insoluble material may be removed by continuous centrifugation. The supernatant containing the soluble bioactive substances may be used as a plant extract, which may include water-soluble pigments (e.g., phycocyanin), proteins, nucleic acids, polysaccharides and ash. These compounds may be further fractionated (e.g., by chromatography and ethanol precipitation) to enhance the content of bioavailable (e.g., unopposed) B12. In some embodiments, the harvested biomass may be used directly, e.g., orally, but advantageously, using the extract and / or its fractionated compounds allows the use of smaller amounts for daily consumption.

[0032] The disclosed products, extracts and / or fraction compounds thereof may be used as such, for example in powder form, in dietary supplements having more bioavailable B12 (e.g., methylcobalamin or 5-deoxyadenosylcobalamin) than pseudo-B12. Note that bioavailable B12 is present in both water-soluble and water-insoluble portions and may be used with or without disruption of the cell wall. Alternatively or complementary, the disclosed extracts and / or fraction compounds thereof may be used in pharmaceutical compositions (by themselves or as additives to other pharmaceutical compositions), for example with a pharmaceutically acceptable carrier(s). Uses of the disclosed biomass, extracts and / or fraction compounds thereof include use as dietary supplements and / or as pharmaceutical compositions and / or as foods, for example as additives in meat analogs, shakes and / or ice cream, as well as baked goods (see example in FIG. 4), frozen foods, functional bars, gummies, various beverages and beverage additives, or combinations thereof. The Spirulina product may be used in the form of biomass, extract (wet or dry), paste, liquid, powder, or any other form. In various embodiments, the disclosed food products may include Spirulina extract, Spirulina powder, water-soluble and / or water-insoluble components thereof, and / or any combination thereof. Further examples of food products include Spirulina extract, phycocyanin-enhanced Spirulina extract, and effervescent tablets, including multi-layer capsules, for use in various beverages and drinks.

[0033] 3A-3D are high level schematic diagrams of a culture system 101 according to some embodiments of the present invention. The culture system 101 is configured to grow algae and / or cyanobacteria in one or more bioreactors 100 at high density and under high illumination intensity. The elements of FIGS. 3A-3D may be combined in any operable combination, and the illustration of certain elements in certain figures and not in others is merely for illustrative purposes and is non-limiting. Note that any disclosed values ​​may be modified by ±10% of the value.

[0034] FIG. 3A is a high-level schematic diagram of a cultivation system 101 having a bioreactor 100 and extraction equipment for extracting a plant extract 140 according to some embodiments of the present invention. The bioreactor 101 contains a growth medium 100A (e.g., water, optionally including additives) and Nannochloropsis algae 90 (e.g., N. oculata, N. australis, N. gaditana, N. granulate, N. limnetica, N. oceanica, N. salina, Nitzschia paleacea, Phaeodactylum tricornutum, Pavlova lutheri, Rebecca salina, etc.). salina, or any strain or combination thereof, or equivalent algae), and (i) an illumination system 120 including a plurality of intense light sources 125, e.g., Light Emitting Diodes (LEDs, e.g., between 400-700 nm, sub-ranges thereof, e.g., 400-500 nm, 500-600 nm, 600-700 nm, and / or specific wavelengths, e.g., 650 nm), which may be arranged in one or more horizontal and / or vertical panels (shown only diagrammatically and in part); and (ii) a gas supply (e.g., a CO2-rich air source, e.g., with a CO2 concentration of 30% or more). A bubbling system 110 associated with a gas supply (e.g., air or nitrogen) includes multiple spargers of at least two types: spargers (multiple) 110A with large nozzles (e.g., diameter >1 mm, possibly in the range of 1-5 mm or a subrange thereof) to generate large bubbles 115A that move quickly (e.g., 100 / min, which indicates the ratio of cumulative bubble volume to vessel volume, possibly within ±30%, 50-150 / min or subranges thereof) through the algal culture and mix it; and spargers (multiple) 110A with large nozzles (e.g., diameter >1 mm, possibly in the range of 1-5 mm or a subrange thereof) to generate large bubbles 115A that move quickly (e.g., 5 / min) through the algal culture and mix it.It further includes a bubbling system 110 including sparger(s) 110B with small nozzles (e.g., <1 mm in diameter, possibly in the range of 0.1-1 mm or a subrange thereof) to generate small bubbles 115B that move at a rate (ratio of cumulative bubble volume to vessel volume, possibly within ±30%, 2-30 / min or subranges thereof) that allow the CO to diffuse to the algal cells. In some cases, more than two types of nozzles may be used to control the mixing of the algal culture and delivery of sufficient concentrations of CO to the algae. Two or more types of spargers (each having multiple nozzles) may be distributed in one or more locations and may be configured to generate turbulent mixing of the algae within the culture vessel and provide CO2 to the algae to an extent that maximizes or optimizes its growth and / or its content and / or composition, such as to maximize the content and / or composition of non-opposing B12 (e.g., methylcobalamin or 5-deoxyadenosylcobalamin) and / or increase the bioavailability of B12.

[0035] As shown diagrammatically in the enlarged region of FIG. 3A, a very dense algal culture (e.g., having an algal density of at least 5 g / l, and possibly in the range or subrange of 5-15 g / l) and a localized powerful light source 125 creates illuminated and dark zones within the bioreactor 100, and powerful agitation of the algal culture by large gas bubbles 115B continuously mixes the liquid and moves the algal cells 90 between the dark and illuminated zones. In a non-limiting example, the light source 125 is irradiated with light having a length of, for example, 4 m with a light path of about 2.5 cm. 2 Use at least 24 LEDs over an area of ​​at least 700 µmol m -2 s -1 , at least 1000 µmol m -2 s -1 , at least 1200 µmol m -2 s -1, or intermediate values, respectively. It has been found that the strong heterogeneous illumination, dark zone periods, and high levels of CO2 supplied to the algal cells 90 by the small gas bubbles 115A result in high algal growth rates and also modify the biosynthetic pathways employed by the algae to form organic compounds. For example, in high density conditions, the illumination zones may extend up to several millimeters (e.g., 1-5 mm) from the point light source 125, while the dark zones between illumination zones may extend for tens of millimeters (e.g., 20-30 mm) between successive illumination zones, such that individual algal cells spend periods in the dark zones to assimilate CO2 using light energy absorbed in the illumination zones.

[0036] Growth conditions may be monitored to maintain optimal growth, for example, temperature may be kept constant (e.g., 15°C, 20°C, 25°C, 27°C, 12-17°C, 15-20°C, 20-25°C, 25-30°C, 20-40°C, 20-50°C, or any subranges thereof) or may be varied within a specific range, and chemical conditions such as pH, O2 and CO2 content and / or content of various ions or compounds may be kept constant or may be varied within a specific range (e.g., pH value of 6.7-7.2, or any subrange thereof). For example, organic carbon content may be maintained at or about 20% by weight. Alternatively or complementary, CO2 concentration may be kept below 40% and / or pH may be monitored to indicate and regulate CO2 concentration. Similarly, algae density may be kept constant or may be varied within a specific range. The flow rate of small bubbles 115B from the sparger 110B with small nozzle may be adjusted to increase or decrease CO2 levels (e.g., increased above 5 / min, e.g., up to 7-10 / min to increase CO2 levels, or decreased to 2 / min to decrease CO2 levels). In certain embodiments, levels of various nutrients may be monitored and additional nutrients may be provided via one or both bubble streams, e.g., phosphorus may be added to the gas supply if low P levels are detected or growth is inhibited.

[0037] Algal slurry may be removed from bioreactor 101 periodically or continuously to balance biomass growth, for example, as shown diagrammatically in FIG. 3A. The disclosed Spirulina products may include plant extract 140, which may be produced by mixing the algal slurry with ethanol (step 130) and then extracted from the mixture by one or more steps of, for example, phase separation (step 132) and thermal separation (step 134). The disclosed Spirulina products may constitute one or more of the separated phases. Non-limiting examples of separation process parameters include a volume ratio of at least 5:1 (or optionally within the range of 3.5:1 to 7:1 or intermediate ranges) between ethanol and algal slurry (which mixture may be stirred for 0.5 to 4 hours, e.g., 2 hours), use of centrifugation for phase separation, optionally phase separation under vacuum (e.g., at a pressure of 75 to 125 mbar), and a temperature range of 55° C. to 65° C. for thermal separation. In certain embodiments, up to 90% of the algal mass may be removed per day.

[0038] In particular, the inventors have surprisingly found that the extract 140 obtained by the separation steps 132, 134 contains more bioavailable B12 than pseudo-B12 (e.g., at least two or three times more bioavailable B12 than pseudo-B12). Thus, the plant extract 140 may be kept in a relatively pure form and high temperature and chemical modifications may be avoided.

[0039] As further shown diagrammatically in Figures 3B-3D, the culture system 101 includes at least one first sparging unit 110A having multiple nozzles and configured to distribute a first predetermined fluid 115A (e.g., air and / or nitrogen bubbles) into a water-filled algae culture vessel 100 (e.g., a bioreactor) at a first operating flow rate and allow mixing therein (shown diagrammatically by arrows 118). The culture system 101 may further include at least one second sparging unit 110B having multiple nozzles and configured to distribute a second predetermined fluid 115B (e.g., air bubbles with CO2, shown diagrammatically, and / or dissolved phosphorus for mass transfer) into the vessel 100 at a second operating flow rate. Fluids exiting the vessel 100, such as gas from the second predetermined fluid 115B, may be recycled 113 to fully utilize the remaining CO2 (shown diagrammatically) therein.

[0040] The cultivation system 101 may further include at least one controller 103 configured to communicate with the sparging unit 110 and control the first and second operating flow rates provided thereby. The controller 103 may include one or more processing units implementing computer code. For example, at least one nozzle of the first sparging unit 110A and / or at least one nozzle of the second sparging unit 110B may be configured to distribute fluids into the cultivation vessel 100 based on instructions from the at least one controller 103. In some embodiments, the first operating flow rate may be based on the second operating flow rate and / or at least one of the operating flow rates may be predetermined. In some embodiments, the first operating flow rate may be adapted to enable turbulent mixing of the algae in the cultivation vessel 100. In some embodiments, the second operating flow rate may be adapted to enable mass transfer and / or assimilation of substances in the liquid in the cultivation vessel 100. Information may flow between the controller 103 and the sparging unit 110, and between the controller 103 and other elements in the system, as indicated diagrammatically by arrows.

[0041] The second predetermined fluid 115B may include gas bubbles having a CO2 concentration of greater than 30%. The source of the first predetermined fluid and / or the second predetermined fluid may be external to the cultivation system 101, for example, a geothermal power plant may provide a source of dissolved carbon and / or sulfur for the second predetermined fluid.

[0042] A first operating flow rate (e.g., 100 ml / min) of at least one nozzle of the first sparging unit 110A may be different from a second operating flow rate (e.g., 5 ml / min) of at least one nozzle of the second sparging unit 110B. In some embodiments, at least one nozzle of the first sparging unit 110A may have a diameter greater than about 1 millimeter. In some embodiments, at least one nozzle of the second sparging unit 110B may have a diameter less than about 1 millimeter. In some embodiments, the nozzles of the first sparging unit 110A and the second sparging unit 110B may dispense the same fluid (e.g., air), with the nozzles of each sparging unit having different diameters. The larger opening of the first sparging unit 110A may be configured to provide large bubbles (e.g., of air and / or nitrogen) to the first predetermined fluid 115A to agitate and mix 118 the suspended biomass in the vessel 100, while the smaller opening of the second sparging unit 110B may be configured to provide small bubbles (e.g., of or including CO2) to the second predetermined fluid 115B to transfer the CO2 from a gas to a liquid accessible to the suspended biomass in the vessel 100. Advantageously, the difference in size of the delivered bubbles may prevent combination of bubbles in streams 115A, 115B and simultaneously provide mixing 118 from a high throughput of large, fast bubbles in stream 115A and effective CO2 supply from a low throughput of small, slow bubbles in stream 115B.

[0043] The culture system 101 may further include a physical barrier 104 configured to separate a first fluid distributed by the first sparging unit 110A and a second fluid distributed by the second sparging unit 110B within the culture vessel 100. In some embodiments, at least one nozzle of the first sparging unit 110A and / or the second sparging unit 110B may be embedded in the physical barrier 104 (not shown). In some embodiments, the physical barrier 104 may be adapted to allow flow from one side of the barrier 104 (having a first fluid distribution) to the other side of the barrier 104 (having a second fluid distribution) at predetermined (e.g., upper and lower) positions of the culture vessel 100 to generate a controlled flow within the vessel 100.

[0044] A culture vessel 100 having a physical barrier 104 may include at least one light source 125 embedded in the physical barrier 104 such that the vessel 100 may be illuminated from within from at least one light source 125 embedded in the physical barrier 104 (illumination is indicated diagrammatically by arrows 203). A culture vessel 100 may include multiple physical barriers 104, each including at least one light source 125, such that a modular system may be created in which algae and / or cyanobacteria grow between adjacent physical barriers 104, and at least one controller 103 may control illumination of all light sources 125 embedded in the physical barriers 104. In some embodiments, the physical barrier 104 may include an illumination system 120 with multiple powerful light sources 125.

[0045] In certain embodiments, the culture system 101 may be configured to reach very high densities of cultivated biomass, with a corresponding small optical depth resulting in a relatively thin illumination zone 116 and a much thicker dark zone 117 in the vessel 100, and the biomass is continuously stirred (e.g., by strong bubbling of fluids 115A and optionally 115B) such that individual cells of algae and / or cyanobacteria have a short residence time in the illumination zone 116 before returning to the dark zone 117 118. In a non-limiting example, the thickness of the illumination zone 116 may be configured to be between 0.1 cm and 1.5 cm depending on the density of the suspension and the illumination density, and may be controlled by the controller 103 and adjusted according to the specific requirements. Thus, the illumination 203 (and especially its UV component) may be set to a very high level, since the short residence time prevents radiation damage to individual cells.

[0046] The cultivation system 101 may further include at least one sensor 105 (e.g., a temperature sensor) coupled to the controller 103 and configured to detect at least one characteristic within the cultivation vessel 100. For example, the at least one sensor 105 may be configured to detect any of the pH level, temperature, and pressure conditions within the cultivation vessel 100 and / or sections thereof. In some embodiments, the at least one sensor 105 may also be configured to detect a parameter external to the cultivation vessel 100, for example, to measure the mass flow rate of gas emissions from the cultivation vessel 100 and determine the amount of material absorbed by the algal cells by subtracting the amount released from the amount inserted into the vessel (e.g., by the second sparging unit 102).

[0047] The culture system 101 may further include at least one database 106 (and / or memory unit) configured to store an algorithm for the operation of the controller 103, e.g., a database of operation speeds for each nozzle and / or each sparging unit. In some embodiments, the culture system 101 may further include a power supply 107 coupled to the controller 103 and configured to provide power to the culture system 101. The power supply 107 may be configured to supply power to the at least one first sparging unit 110A and the at least one second sparging unit 110B, e.g., to operate at different speeds.

[0048] Data collected by the at least one sensor 105 may be analyzed by the controller (or processor) 103 to detect whether an attribute exceeds a predetermined threshold, for example, a pH level and / or a temperature and / or a CO2 concentration threshold in the vessel 100. If the condition in the culture vessel 100 (e.g., as detected by the sensor 105) exceeds at least one threshold, the controller 103 may operate at least one nozzle of the first sparging unit 110A and / or at least one nozzle of the second sparging unit 110B at a different flow rate. For example, detecting a CO2 concentration in the vessel 100 of more than 40% (or detecting a low pH level) may cause at least one nozzle of the second sparging unit 110B to reduce the flow rate of the second sparging unit 110B to about 2 millimeters per minute. In some embodiments, the at least one nozzle of the second sparging unit 110B may only operate when it receives a signal from the sensor 105 that the attribute exceeds a predetermined threshold, and may not operate at a constant rate.

[0049] At least one nozzle of the first sparging unit 110A may be configured to operate only when a signal is received from the sensor 105 that the attribute exceeds a predetermined threshold, e.g., to increase the mixed flow 118 as the density of the algae population increases. At least one nozzle of the first sparging unit 101 and / or at least one nozzle of the second sparging unit 110B may operate at a constant speed, continuously, or possibly intermittently. At least one nozzle of the first sparging unit 110A and / or at least one nozzle of the second sparging unit 110B may operate at a non-constant speed, continuously or possibly intermittently.

[0050] The culture vessel 100 may include a bubble column configuration in which at least one first sparging unit 110A and at least one second sparging unit 110B are disposed on the same surface of the bubble column vessel. The culture vessel 100 may have an airlift configuration in which the at least one second sparging unit 110B is positioned at the bottom of a downcomer that may be distal to the sensor 105, thus increasing the residence time of bubbles from the at least one second sparging unit 110B.

[0051] Cultivation system 101 may be configured to allow for maintaining at least 20% organic carbon in vessel 100, calculated in addition to carbon provided as CO2 bubbles. In some embodiments, at least a portion of the algae in vessel 100 may include any photosynthetic microorganisms, such as algae and / or cyanobacteria, used to prepare Spirulina preparations, including, for example, Arthrospira platensis, A. fusiformis, and / or A. maxima.

[0052] In some embodiments, an optimized controlled supply of harmful UV radiation may allow for increasing the amount and / or bioavailability of B12 (e.g., methylcobalamin and / or 5-deoxyadenosylcobalamin) in the cultured Arthrospira spp. and extracted Spirulina while avoiding compromising the growing algae or growth rate. In some embodiments, the on / off nature of the radiation supply may allow for controlling the amount of harmful radiation supplied. Furthermore, the constant mixing and / or bubbling 118 of the suspension in the vessel (by the sparging unit 110) ensures only brief exposure of any individual algal or cyanobacterial cells to the intense radiation, preventing photoinhibition and damage to the cells. The controller 103 may be configured to control the degree of turbulence provided by the sparging unit 110, respectively, to avoid radiation damage to the cells. For example, the method 300 and culture system 101 may be configured to achieve the required UV light modulation in a thin film culture system by turning the UV light source on and off and / or creating shadow patterns that result in intermittent illumination of the algae and / or cyanobacteria. In a bubbled culture system 101, the relative velocity of the culture suspension and air bubble flow relative to the UV light source may be controlled to achieve a particular pattern of on / off UV exposure cycles.

[0053] 3C and 3D show schematic diagrams of an embodiment of a culture system 101 according to some embodiments of the present invention. The culture system 101 may include at least one illumination unit 120 coupled to a controller 103 for illuminating the culture vessel 100. The illumination unit 120 and the controller 103 (or another controller) may be included in a bioreactor illumination system for growing algae and / or cyanobacteria. The distance between the culture vessel 100 and the illumination unit 120 may be changed to control the illumination received by the culture vessel 100. For example, moving the illumination unit 120 closer to the culture vessel 100 increases the illumination of the culture in the culture vessel. The distance between the culture vessel 100 and the illumination unit 120 may be controlled, for example, by a controller 103 included in the illumination system. In addition to or instead of changing the distance of the illumination unit 120 from the culture vessel 100, the illumination intensity of the light source 125 in the illumination unit 120 may be controlled. The illumination unit 120 may include at least one light source 125 (e.g., LED), and each light source 125 may be separately controlled by the controller 103. In some embodiments, one or more light sources 125 may be controlled to illuminate at a different intensity than another light source 125. All light sources 125 may be controlled to vary illumination intensity manually or according to pre-set timing and / or sensed conditions within the culture vessel 100. At least some of the light sources 125 may be configured to emit light in the UV spectrum, e.g., both the UVA and UVB ranges. The ratio between the emitted radiation in UVA and the emitted radiation in UVB (UVA / UVB ratio) may be between 10-15, e.g., 10, 12, 14, 15 UVA / UVB, or may have intermediate values. The UV radiation may be between 1,000-10,000 kJ / m 2 , e.g., 2000 kJ / m 2 , 5000kJ / m 2 , 7000kJ / m 2 , 9000kJ / m 2, or any other intermediate value. The controller 103 may be configured to control the provision of the UV radiation using pulses of radiation, for example, each pulse may last for less than 0.01 seconds, for example 0.008 seconds, 0.009 seconds, 0.0095 seconds or 0.0099 seconds, or any intermediate value, and 1 to 100 of these pulses may be provided per second, for example 10 times per second. 1,000 kJ / m for about 0.01 seconds 2 It should be noted that irradiation with UV light provides approximately 10 times the intensity of solar UV radiation, which alters the chemical composition of the algae and / or cyanobacteria resulting in the extract compositions disclosed below. Some light sources 125 may be configured to emit light in the visible spectrum (e.g., wavelengths between 400-700 nm).

[0054] The amount of light delivered to the culture vessel 100 may be defined as the average light flux delivered to the surface of the culture vessel 100. The culture system 101 may be used to support ultra-high density cultures (e.g., up to 1, 5 or 10 g / l, or intermediate biomass densities), and the illumination unit 120 is configured to have a light distribution of the light sources 125 that provides an average light flux per algal / cyanobacteria cell that is comparable to the average light flux per cell of a low density culture that achieves a similar level of average illumination per cell. The light intensity within the culture vessel 100 may be measured with at least one sensor 105 and adjusted by the controller 103. For example, for ultra-high density cultures, a typical thickness of the illumination zone 116 may be in the range of, for example, 1 mm to 5 mm, while a typical thickness of the dark zone 117 may be in the range of, for example, 20 mm to 30 mm. The distance of the illumination unit 120 from the side of the vessel 100 may be adjusted relative to the prevailing optical thickness (or optical depth, OD) of the biomass suspension to avoid photoinhibition and / or photobleaching. For example, in early culture stages, when the culture density is relatively low, the illumination unit 120 may initially be kept at a certain distance from the side of the vessel 100 for biomass growth, but in later culture stages, as the OD increases, the illumination unit 120 may be moved closer to the side of the vessel 100 to promote biomass growth.

[0055] The ultra-high density culture is continuously and / or intermittently mixed and / or agitated by mechanical means and / or by strong bubbling of fluids 115A and optionally 115B to move the algae and / or cyanobacteria between the illumination zone 116 and the dark zone 117 and prevent damage to the cells (schematically indicated by arrows 118 in FIG. 3B), and short light passes provide illumination cycles (between the illumination zone 116 and the dark zone 117) of the algae / cyanobacteria. Ultra-high density cultures may be illuminated with various wavelengths, since at such biomass densities, short wavelength light passes may have little effect on growth. This is in contrast to the practice of illuminating algae with a specific wavelength (e.g., blue light) for normal growth, since they would respond differently to light. However, the inventors have experimentally found that illumination with any wavelength may be used for ultra-high density cultures.

[0056] Light penetration into the culture vessel 100 may correspond to at least one of light intensity, light wavelength, the particular algae strain, and / or algae culture density. It is noted that light penetration into the culture vessel 110 may determine the volume ratio between the illuminated zone 116 and the dark zone 117 within the culture vessel 110, and thus may affect the light intensity provided by the illumination unit 120, the gas flow rate through the first sparging unit 110A, the gas flow rate through the second sparging unit 110B, etc., each of which may be adjusted and optimized.

[0057] In some embodiments, the culture vessel 100 may be illuminated by the illumination unit 201 to provide a daily dose of more than 90% of maximum algae growth in the culture vessel 100. In some embodiments, the illumination unit 201 may be configured to illuminate the suspension in the vessel 100 using several high intensity light sources 120 that are heterogeneously, e.g., spaced apart, such that illumination of the cells is locally and temporally controlled (through agitation 118). The inventors have found that high intensity intermittent illumination actually enhances algae and / or cyanobacteria growth over the usual practice configuration with a homogenous distribution of low intensity light sources.

[0058] For example, the illumination photon flux density of at least one light source 120 is 1200 μmole / m 2 In various embodiments, the photon flux density may be 1000 to 1500 μmole / m -2 s or may have intermediate values. In some embodiments, the illumination unit 120 2 As an illustrative, non-limiting example, a 6 m 2 The illumination units 120 each have a surface area of ​​120 μmole / m2 and a light path (thickness of the illumination zone 116) of about 4 cm. -2 The vessel 100 may include 24 LED light sources 125 having a luminous flux of 100 s. In some embodiments, at least a portion of the cyanobacteria in the vessel 100 comprises Arthrospira species, which are used to prepare Spirulina extract.

[0059] The controller 103 may be configured to control the irradiation wavelength of the at least one light source 125, for example using a dedicated irradiation module adapted to change the wavelength of the emitted irradiation. In some embodiments, a constant temperature of 27° C. may be maintained within the container 100. In some embodiments, the controller 103 may be configured to control the at least one light source 125 to irradiate at a wavelength of 650 nanometers. It is noted that, according to common practice, algae are irradiated with a specific wavelength (e.g., blue light) for optimal growth, but experiments performed by the inventors have shown that irradiation with other wavelengths (e.g., red light) may be used for enhanced growth.

[0060] As shown diagrammatically in FIG. 3D, the culture system 101 may be configured to operate with an integrated sparging unit 110, which may include two or more types of nozzles integrating sparging units 110A, 110B. The integrated sparging unit 110 may be configured to distribute a predetermined fluid 115 to the culture vessel 100. The predetermined fluid 115 may include one or both of the predetermined fluids 115A, 115B, or various mixtures of portions thereof. For example, the integrated sparging unit 110 may include at least one nozzle for distributing a first predetermined fluid 115A and at least one nozzle (e.g., having different diameters) for distributing a second predetermined fluid 115B, which in combination are shown diagrammatically as a non-limiting predetermined fluid 115. The sparging unit 110 may be configured to generate turbulent mixing of the algae and / or cyanobacteria in the culture vessel 100 and to provide CO2 for assimilation by the algae and / or cyanobacteria, as disclosed, for example, with reference to FIGS. 3B and 3C. In certain embodiments, the culture system 101 may include a combination of dedicated sparging units 110A, 110B and an integrated sparging unit 110.

[0061] The inventors have found that the disclosed Spirulina extracts extracted from algae grown under the conditions disclosed above contain more bioavailable B12 than unopposed metabolically active B12 (e.g., methylcobalamin and / or 5-deoxyadenosylcobalamin), such as pseudo-B12. The extracts, biomass, or portions thereof may be used as additives to various food products to provide a significant portion of the daily requirement of B12 via unopposed methylcobalamin B12 without adding much mass to the product. This is because the concentration of unopposed B12 in Spirulina products is very high (see, e.g., Table 1, thousands of times higher than B12 in meat, for example). Thus, the addition of small amounts of the disclosed Spirulina products will not alter other nutritional or organoleptic properties of the food while still providing a significant amount of B12.

[0062] Long-term studies have shown that the content of bioavailable (e.g., unopposed) MeB12 under the disclosed growth conditions was consistent and provided a reliable source of bioavailable MeB12. Table 2 provides results for bioavailable MeB12 and pseudovitamin B12 in the disclosed Spirulina biomass tracked over a nine-month period. These results were consistent across six independent replicates, yielding similar results. 12 and Vitamin B 12 Both active forms of vitamin B1 and B2 were observed, with the active form making up the majority (>98%) of the composition. 12 Governed by, active B 12 Although the content of is net negative (see Table 1), the results obtained here indicate that the B 12 1 shows the effect of light management on enhancing native metabolic pathways to achieve

[0063] [Table 2]

[0064] Thus, the unopposed active vitamin B12 content was found to be statistically consistent over 9 months of continuous culture, resulting in a concentration of 1.64 μg / 100 g, and thus comparable or slightly superior to beef, which has a vitamin B12 concentration of 0.7-1.5 μg / 100 g. Furthermore, the disclosed Spirulina products also provide comparable concentrations of macro- and micronutrients (e.g., essential amino acids, α-linolenic acid (ω-3 fatty acid) and linoleic acid (ω-6 fatty acid), various minerals including calcium, potassium, magnesium and iron, and vitamins such as β-carotene), thus providing a good meat substitute product whose nutritional value is comparable to meat but without the adverse health effects associated with meat consumption, and furthermore with a negative carbon footprint. On the other hand, the disclosed Spirulina products are superior in their nutritional content, and especially in their bioavailable unopposed active vitamin B12 content, to traditional Spirulina or other plant-based meat substitutes.

[0065] For example, the disclosed Spirulina products contain high levels of unopposed active vitamin B12 (e.g., 1-3 μg / 100 g) and are grown with a slightly negative carbon footprint (-0.008 kg CO2 equivalents / kg, see Tzachor et al., 2022), a unique combination compared to both beef products and prior art Spirulina products. Compared to beef products, the disclosed Spirulina products contain comparable amounts of unopposed active vitamin B12, as well as comparable protein composition and other nutrients such as iron (see Tzachor et al., 2022), yet provide greater carbon credits relative to beef (e.g., 99.4 kg CO2 equivalents / kg for beef, minus 0.008 kg CO2 equivalents / kg for the disclosed Spirulina products, resulting in a carbon credit of 99.408 kg CO2 equivalents / kg). In comparison to prior art Spirulina products grown under natural or artificial light (rather than the disclosed high intensity growing conditions), the disclosed Spirulina products contain higher levels of unopposed active Vitamin B12, as described.

[0066] If a portion of the disclosed spirulina product is used, for example, as a meat or dairy replacement or as an additive in baked goods to provide unopposed active vitamin B12, only a portion of the negative carbon footprint may be allocated to the product. For example, if a disclosed spirulina product with 17 μg / kg of unopposed active vitamin B12 is used to replace meat (which has a carbon footprint of 99.4 kg CO2 equivalents / kg), each μg of unopposed active vitamin B12 may be associated with 5.85 kg CO2 equivalents / μg unopposed B12 (99.4 kg CO2 equivalents / kg / 17 μg / kg unopposed active vitamin B12), resulting in a reduction in the carbon footprint of the resulting product in addition to providing unopposed active vitamin B12. For bread, assuming typical values ​​of 1-1.5 kg CO2 equivalents / kg bread, the addition of the disclosed Spirulina products increases unopposed active vitamin B12 while simultaneously decreasing the carbon footprint of the bread (e.g., approximately 0.1 kg CO2 equivalents / μg unopposed B12).

[0067] Figure 4 shows non-limiting examples of the use of the disclosed Spirulina biomass and / or dry / wet extract in various food products according to some embodiments of the present invention. The examples illustrate, among others, the disclosed Spirulina in dough containing the disclosed Spirulina baked into bread and cookies, jellies, smoothies, and various types of beverages and shakes to provide natural bioavailable vitamin B12 and help reduce fatigue and lethargy in people who lack B12, for example due to nutritional limitations. In the baked products, 0.4 g of the disclosed dry Spirulina powder (0.04% by weight, this powder contained 30.0 mcg / g bioavailable B12) was added per kg of dough, resulting in 0.6 μg of B12 per 50 g of dough, which provides 25% of the Recommended Dietary Intake (RDI). The dough acquired a slight green hue due to the algae extract powder, which disappeared in the base bread. The dough was baked at 200°C for 20 min, losing 21±3% through evaporation, leaving 0.58±0.04 μg B12 per 50 g serving of bread (which is about 25% of the RDI), thus providing an excellent source of B12. The sensory properties of the bread were unchanged in terms of color, odor and taste compared to bread prepared without spirulina powder.

[0068] Similarly, the disclosed Spirulina biomass and / or dry / wet extracts may be used in a wide variety of products, such as meat analogs, various baked goods, frozen foods, various ice cream types, functional bars, gummies, beverages or shakes, beverage additives such as effervescent tablets, or combinations thereof. For example, the disclosed products may be formulated to provide a significant portion of the daily requirement for B12, such as 15% or more.

[0069] Additionally, foods that include Spirulina products may have a reduced, zero or negative carbon footprint achieved by the included Spirulina products, or may directly decarb the product in the sense suggested by Tzachor et al., 2022. For example, baked goods may include (or be enriched with) the water soluble fraction of the Spirulina product, the water insoluble fraction of the Spirulina product, and / or fraction compounds thereof, and in addition to providing bioavailable B12 and additional nutrients, may have a reduced, zero or negative carbon footprint resulting from adding the disclosed Spirulina products, which have a large negative carbon footprint, as explained above.

[0070] In the above description, an embodiment is an example or implementation of the invention. The various appearances of "one embodiment," "one embodiment," "particular embodiment," or "some embodiments" do not necessarily all refer to the same embodiment. Although various features of the invention may be described in the context of a single embodiment, the features may be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may be implemented in a single embodiment. A specific embodiment of the invention may include features from different embodiments disclosed above, and a specific embodiment may incorporate elements from other embodiments disclosed above. The disclosure of an element of the invention in the context of a specific embodiment is not to be considered as limiting its use only in the specific embodiment. Furthermore, it is to be understood that the invention can be implemented or embodied in various ways, and the invention can be embodied in specific embodiments other than those outlined in the above description.

[0071] The present invention is not limited to the above diagrams or corresponding descriptions. For example, the flow does not have to move through each box or state shown, and does not have to move in the exact same order as shown and described. The meanings of technical and scientific terms used herein are generally understood by those skilled in the art to which the present invention belongs, unless otherwise defined. Although the present invention has been described with respect to a limited number of embodiments, these should not be construed as limiting the scope of the present invention, but rather as illustrating some of the preferred embodiments. Other possible variations, modifications, and applications are also within the scope of the present invention. Therefore, the scope of the present invention should not be limited by what has been described above, but should be limited by the appended claims and their legal equivalents.

Claims

1. A spirulina product made from Arthrospira sp. cultivated under photosynthetically controlled conditions resulting in upregulated bioactive compounds including unopposed B12, the spirulina product having more bioavailable B12 than pseudo-B12.

2. 2. The Spirulina product of claim 1, wherein the Spirulina product has at least 0.7 μg / 100 g, at least 2 μg / 100 g, at least 10 μg / 100 g, at least 10 μg / 100 g, at least 100 μg / 100 g, at least 1000 μg / 100 g, at least 2000 μg / 100 g, or at least 3000 μg / 100 g of unopposed B12.

3. 3. The spirulina product of claim 1 or claim 2, wherein the spirulina product has at least two or three times more bioavailable B12 than pseudo-B12.

4. 4. The Spirulina product of any one of claims 1 to 3, comprising at least one of an aqueous extract without further processing, a dry extract, a Spirulina biomass, a dry biomass, a Spirulina paste, a Spirulina powder and / or a Spirulina liquid.

5. The photosynthetically controlled conditions included growing the Arthrospira sp. at 31±2° C., a pH of 10.8±0.2, and a pH of 700-1500 μmol / (m ２ The spirulina product according to any one of claims 1 to 4, comprising culturing the spirulina under an irradiance of 0.1 to 0.5 nm.

6. The photosynthetically controlled conditions include culturing the Arthrospira sp. at a density of 3 g / l to 10 g / l at a concentration of 70 to 150 μmol / m ２ 6. The Spirulina product according to claim 1 , further comprising culturing the Spirulina product under an ultraviolet light intensity of 0.1 s.

7. 7. The Spirulina product of any one of claims 1 to 6, produced from an aqueous extract produced by water extraction and freeze and thaw cycles applied to the cultured Arthrospira sp.

8. 8. A pharmaceutical composition comprising a fractionated compound of a Spirulina product according to any one of claims 1 to 7.

9. 7. Use of a Spirulina product or a fraction thereof according to any one of claims 1 to 6 as a dietary supplement.

10. 8. A dietary supplement comprising a Spirulina product and / or a fraction thereof according to any one of claims 1 to 7.

11. 11. The dietary supplement of claim 10 comprising at least one of a water soluble fraction of the Spirulina product and a water insoluble fraction of the Spirulina product.

12. 12. The dietary supplement of claim 10 or claim 11, further comprising a pharma- ceutically acceptable carrier.

13. A food product comprising the Spirulina product or a fraction thereof according to any one of claims 1 to 7.

14. 14. The food product of claim 13, comprising at least one of a meat analog, a baked good, a frozen meal, an ice cream, a functional bar, a gummy, a beverage, a shake, a beverage additive, an effervescent tablet, or a combination thereof.

15. 14. The food product of claim 13, comprising a water soluble fraction of the Spirulina product, a water insoluble fraction of the Spirulina product, and / or a meat analog comprising at least one of the fraction compounds thereof.

16. 14. The food product of claim 13, comprising a baked good comprising at least one of the water soluble fraction of the Spirulina product, the water insoluble fraction of the Spirulina product, and / or fraction compounds thereof.

17. 15. The food product of claim 13 or claim 14 comprising at least 0.7 μg / 100 g, at least 2 μg / 100 g, at least 10 μg / 100 g, at least 10 μg / 100 g, at least 100 μg / 100 g, at least 1000 μg / 100 g, at least 2000 μg / 100 g, at least 3000 μg / 100 g of unaltered B12.

18. 18. A food product according to any one of claims 13 to 17, having a reduced, zero or negative carbon footprint achieved by the Spirulina product contained therein.

19. 1. A method for preparing a Spirulina product comprising: Cultivating Arthrospira sp. cyanobacteria under photosynthetically controlled conditions to produce upregulated bioactive compounds, including unopposed B12; preparing a spirulina product from the cultured Arthrospira sp. cyanobacteria, the spirulina product having more bioavailable B12 than pseudo-B12; The method includes:

20. 20. The method of claim 19, wherein the spirulina product has at least 0.7 μg / 100 g, at least 2 μg / 100 g, at least 10 μg / 100 g, at least 10 μg / 100 g, at least 100 μg / 100 g, at least 1000 μg / 100 g, at least 2000 μg / 100 g, or at least 3000 μg / 100 g of unopposed B12.

21. 21. The method of claim 19 or claim 20, wherein the spirulina product has at least 2 or 3 times more bioavailable B12 than pseudo-B12.

22. 22. The method of any one of claims 19 to 21, further comprising drying the extract to yield the spirulina product.

23. 23. The method of any one of claims 19 to 22, further comprising preparing the Spirulina product from at least one of an aqueous extract, a dry extract, a Spirulina biomass, a dry biomass, a Spirulina paste, a Spirulina powder and / or a Spirulina liquid without further processing.

24. The Arthrospira spp. culture was carried out at 31±2° C., pH 10.8±0.2 and pH 700-1500 μmol / (m ２ 24. The method according to any one of claims 19 to 23, wherein the method is carried out under an irradiance of s).

25. The culture of Arthrospira spp. is cultured at a density of 70 to 150 μmol / m in ultra-high density culture having a density of 3 g / l to 10 g / l. ２ 25. The method of any one of claims 19 to 24, carried out under an ultraviolet intensity of 0.1 s.

26. 26. The method of any one of claims 19 to 25, wherein the Spirulina product is made from an aqueous extract prepared by water generation and freeze and thaw cycles applied to the cultivated Arthrospira sp.

27. 27. The method of any one of claims 19 to 26, further comprising using the Spirulina product and / or fractions thereof as a dietary supplement or additive and / or as a food product comprising at least one of a meat analogue, baked goods, frozen food, ice cream, functional bar, gummy, beverage, shake, beverage additive, effervescent tablet, or combinations thereof.

28. 28. The method of claim 27, wherein the dietary supplement, additive and / or food product comprises at least 0.7 μg / 100 g, at least 2 μg / 100 g, at least 10 μg / 100 g, at least 10 μg / 100 g, at least 100 μg / 100 g, at least 1000 μg / 100 g, at least 2000 μg / 100 g or at least 3000 μg / 100 g of unaltered B12.

29. 29. The method of claim 27 or claim 28, further comprising reducing the carbon footprint of the dietary supplement, additive and / or food product through use of the Spirulina product.

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