Use of beta-1,3-glucan for modulating immune function and treating intestinal inflammation

Abstract

To provide beta-1,3-glucan, and to provide uses thereof for regulating immunity in the human body, which are methods for treating and / or preventing high cholesterol, diabetes, and allergies, and methods for treating and / or preventing intestinal inflammation.SOLUTION: Provided is a composition for oral administration for alleviating symptoms associated with intestinal inflammation in humans, the composition containing an effective amount of beta-1,3-glucan dried to a moisture content of about 40% or less from Euglena grown by fermentation, the beta-1,3-glucan not containing beta-1,6-glycosidic bonds, the effective amount being between 0.1 mg of beta-1,3-glucan / kg body weight and 100 mg of beta-1,3-glucan / kg body weight.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 247,629, filed October 28, 2015, and U.S. Provisional Patent Application No. 62 / 305,875, filed March 9, 2016, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Technical Field The present application relates to beta-1,3-glucan and its use for regulating immunity in the human body. Further, methods for treating and / or preventing high cholesterol, diabetes, and allergies are provided. The present application also relates to beta-1,3-glucan and its use for enhancing immune function in humans with intestinal inflammation. Further, methods for treating and / or preventing intestinal inflammation are provided. [Background technology]

[0003] Beta-glucan is a polymer of D-glucose linked by beta-glycosidic bonds produced by a variety of organisms, including yeast, fungi, bacteria, algae, oats, barley, and kelp. Different organisms produce beta-glucans with different branching structures, average molecular weights, solubilities, and / or tertiary structures. For example, beta-glucans from yeast are generally insoluble and have both beta-1,3- and 1,6-glycosidic bonds (beta-1,3- / 1,6-glucans). On the other hand, beta-glucans from oats are typically more soluble and have both beta-1,3- and 1,4-glycosidic bonds (beta-1,3- / 1,4-glucans). In contrast, beta-glucans from Euglena, etc. Beta-glucans from any algae contain almost exclusively 1,3-glycosidic bonds and no 1,6-glycosidic bonds. The specific glycosidic bonds of the various beta-glucan forms affect the properties of these molecules.

[0004] Some beta-glucans have been recognized to have health-beneficial properties. Because beta-glucans are typically associated with the surface of pathogenic microorganisms, the immune system of higher organisms has evolved to recognize beta-glucans and enhance immune responses. For example, it has been shown that yeast-derived beta-glucans can affect immune function by binding to complement receptor 3 or dectin-1 on macrophages (see Brown et al, Journal of Experimental Medicine, vol.196(3), pp.407-412 (2002)). At the physiological level, beta-glucans interact with cell surface receptors to initiate a series of events including phagocytosis and the production of certain cytokines. By introducing certain beta-glucans, the immune system can be primed, resulting in a stronger immune system response to actual disease challenge.

[0005] Modulating an individual's immune function to combat disease represents an alternative to administering conventional medicines. Modulated immune function can effectively treat or prevent the onset of disease in an individual. Modulating or enhancing an individual's immune function to combat disease also represents an alternative to administering conventional medicines. Modulated or enhanced immune function can effectively treat or prevent the onset of disease in an individual. Many conventional medicines cause undesirable side effects in patients. Furthermore, antibiotic-resistant strains of bacteria further increase health risks. Thus, there is a need for alternative disease treatments that have fewer, if any, side effects. There is also a need for more natural ways to prevent the onset of disease.

[0006] While beta-glucans derived from yeast and oats have been extensively studied, the health benefits resulting from beta-1,3-glucans derived from algae such as Euglena have received less attention. Described herein are methods for modulating immune function in an individual by administering beta-1,3-glucans derived from Euglena. Certain diseases can be treated and / or prevented by modulating immune function in an individual.

[0007] Current treatments for intestinal inflammation include dietary and / or lifestyle changes, medications, and surgery. However, there is a need for more effective treatments for intestinal inflammation and treatments with fewer, if any, side effects. Described herein is a method for enhancing immune function in individuals with intestinal inflammation by administering Euglena-derived beta-1,3-glucan. Summary of the Invention

[0008] The present disclosure provides a method of treating a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer in a human having said disease, the method comprising orally administering to the human an effective amount of a composition comprising Euglena beta-1,3-glucan grown using fermentation. In one embodiment, the disease is selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease.

[0009] In one embodiment, the disease is hyperlipidemia. In one embodiment, administration of the composition reduces cholesterol levels in a human. In one embodiment, the effective amount of the composition comprises between 0.1 mg beta-1,3-glucan / kg body weight and 100 mg beta-1,3-glucan / kg body weight. In one embodiment, the effective amount of the composition comprises between 0.1 mg beta-1,3-glucan / kg body weight and 50 mg beta-1,3-glucan / kg body weight. In one embodiment, the composition is administered in combination with a statin, nicotinic acid, bile acid resin, fibric acid derivative, or cholesterol absorption inhibitor.

[0010] In one embodiment, the disease is selected from the group consisting of inflammatory bowel disease, colitis, Crohn's disease, and colon cancer. In one embodiment, the disease is selected from the group consisting of inflammatory bowel disease, colitis, and Crohn's disease. In one embodiment, the disease is colitis. In one embodiment, the disease is inflammatory bowel disease. In one embodiment, the disease is Crohn's disease. In one embodiment, the disease is colon cancer. In one embodiment, the effective amount of the composition comprises between 0.1 mg beta-1,3-glucan / kg body weight and 100 mg beta-1,3-glucan / kg body weight. In one embodiment, administering the composition increases anti-inflammatory cytokine production. In one embodiment, the composition is administered in combination with an anti-inflammatory drug, an immunosuppressant, or an antibiotic.

[0011] In some embodiments, the Euglena is Euglena gracilis. In some embodiments, the Euglena is grown heterotrophically. In some embodiments, the beta-1,3-glucan is present in the form of paramylon. In some embodiments, the beta-1,3-glucan does not contain beta-1,6-glycosidic bonds. In some embodiments, the beta-1,3-glucan is purified from Euglena. In some embodiments, the composition comprises a Euglena biomass, the Euglena biomass comprising beta-1,3-glucan. In some embodiments, the Euglena biomass is dried to a moisture content of 40% or less. In some embodiments, the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0012] In some embodiments, the composition is administered as a single dose daily. In some embodiments, the composition is administered as multiple divided doses daily.

[0013] In one embodiment, the composition contains alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, astaxanthin, beta carotene, lutein, milk The composition further comprises an additional ingredient selected from the group consisting of acid bacteria probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake, reishi, or shiitake mushrooms, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof. In one embodiment, the additional ingredient is zinc.

[0014] In some embodiments, the composition further comprises a metal. In some embodiments, the metal comprises a member selected from the group consisting of iron, magnesium, lithium, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, manganese, selenium, and combinations thereof. In some embodiments, the beta-(1,3)-glucan and the metal form a complex. In some embodiments, the complex comprises a zinc beta-(1,3)-glucan complex.

[0015] In certain embodiments, the composition further comprises an additional component selected from the group consisting of Haematococcus pluvialis, astaxanthin, and colostrum.

[0016] In some embodiments, the composition is administered as a solid, hi some embodiments, the composition is administered as a suspension.

[0017] The present disclosure provides that the methods disclosed herein may also be adapted for corresponding uses.

[0018] The present disclosure provides a composition comprising Euglena beta-1,3-glucan grown using fermentation for use in treating a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer. In one embodiment, the disease is selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease.

[0019] The present disclosure provides for the use of a composition comprising Euglena beta-1,3-glucan grown using fermentation for the manufacture of a medicament for the treatment of a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer. In one embodiment, the disease is selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease.

[0020] In one aspect, the present application discloses a method for modulating immune function in a human comprising orally administering to the human in need thereof an effective amount of a composition comprising beta-1,3-glucan derived from fermentation-grown Euglena.

[0021] In some embodiments, an effective daily amount of the composition is between 0.1 mg of beta-1,3-glucan per kg of body weight and 50 mg of beta-1,3-glucan per kg of body weight.

[0022] In some embodiments, administration of the composition modulates an autoimmune response, blood sugar levels, an infection, or inflammation. In one embodiment, the inflammation is associated with allergies. In another embodiment, the autoimmune response is associated with diabetes. In another embodiment, the infection is a bacterial infection, a fungal infection, Or a viral infection.

[0023] In some embodiments, Euglena grows heterotrophically. In one embodiment, the beta-1,3-glucan comprises paramylon. In another embodiment, the beta-1,3-glucan does not comprise beta-1,6-glycosidic bonds. In a further embodiment, the beta-1,3-glucan is purified from Euglena.

[0024] In some embodiments, the composition comprises a Euglena biomass, and the Euglena biomass comprises beta-1,3-glucan. In some embodiments, the Euglena biomass is dried. In further embodiments, the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0025] In one embodiment, the composition is administered as a single dose daily, hi another embodiment, the composition is administered as multiple divided doses daily.

[0026] In some embodiments, the composition comprises alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, astaxanthin, beta carotene, lutein. The composition further comprises an additional ingredient selected from the group consisting of lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake mushroom, reishi mushroom, or shiitake mushroom, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof. In one embodiment, the composition is administered as a solid. In another embodiment, the composition is administered as a suspension.

[0027] In another aspect, the present application provides a method for modulating immune function in a human comprising orally administering to the human having or at risk for having elevated cholesterol an effective amount of a composition comprising beta-1,3-glucan derived from Euglena grown using fermentation. In some aspects, administration of the composition reduces cholesterol levels in the human.

[0028] In one embodiment, the effective amount of the composition is between 0.1 mg beta-1,3-glucan / kg body weight and 50 mg beta-1,3-glucan / kg body weight. In another embodiment, the Euglena grows heterotrophically. In a further embodiment, the beta-1,3-glucan comprises paramylon. In yet another embodiment, the beta-1,3-glucan does not comprise beta-1,6-glycosidic bonds. In another embodiment, the beta-1,3-glucan is purified from Euglena.

[0029] In some embodiments, the composition comprises a Euglena biomass, and the Euglena biomass comprises beta-1,3-glucan. In further embodiments, the Euglena biomass is dried. In some embodiments, the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0030] In one embodiment, the composition is administered as a single dose daily, hi another embodiment, the composition is administered as multiple divided doses daily.

[0031] In one embodiment, the composition comprises alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, astaxanthin, beta carotene, lutein, The composition further comprises an additional component selected from the group consisting of lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake mushroom, reishi mushroom, or shiitake mushroom, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof. In other embodiments, the composition is administered in combination with a statin, nicotinic acid, bile acid resin, fibric acid derivative, or cholesterol absorption inhibitor. In a further embodiment, the composition is administered as a solid. In another embodiment, the composition is administered as a suspension.

[0032] The present disclosure provides that the methods disclosed herein may also be adapted for corresponding uses.

[0033] The present disclosure provides a composition comprising a beta-1,3-glucan derived from Euglena grown using fermentation for use in modulating immune function in a human in need thereof. In some embodiments, the composition is administered orally to the human. The present disclosure provides a use of a composition comprising a beta-1,3-glucan derived from Euglena grown using fermentation for use in the manufacture of a medicament for modulating immune function in a human in need thereof. In some embodiments, the composition is administered orally to the human.

[0034] The present disclosure provides a composition comprising beta-1,3-glucan derived from Euglena grown via fermentation for use in modulating immune function in a human having or at risk for high cholesterol. In some embodiments, the composition is administered orally to the human. The present disclosure provides a use of a composition comprising beta-1,3-glucan derived from Euglena grown via fermentation for use in the manufacture of a medicament for modulating immune function in a human having or at risk for high cholesterol. In some embodiments, the composition is administered orally to the human.

[0035] Also described herein are methods for enhancing immune function in humans with intestinal inflammation by administering Euglena-derived beta-1,3-glucan, which are useful for treating diseases such as inflammatory bowel disease, colitis, and Crohn's disease.

[0036] In one aspect, the present application discloses a method for enhancing immune function in a human suffering from intestinal inflammation, comprising orally administering to the human an effective amount of a composition comprising beta-1,3-glucan derived from fermentation-grown Euglena.

[0037] In some embodiments, an effective daily amount of the composition is between 0.1 mg of beta-1,3-glucan / kg body weight and 100 mg of beta-1,3-glucan / kg body weight.

[0038] In some embodiments, the intestinal inflammation is inflammatory bowel disease. In one embodiment, the intestinal inflammation is colitis. In another embodiment, the intestinal inflammation is Crohn's disease.

[0039] In some embodiments, the Euglena grows heterotrophically. In one embodiment, the beta-1,3-glucan comprises paramylon. In another embodiment, the beta-1,3-glucan does not comprise beta-1,6-glycosidic bonds. In a further embodiment, the beta-1,3-glucan does not comprise beta-1,6-glycosidic bonds. ,3-Glucan is purified from Euglena.

[0040] In some embodiments, the composition comprises a Euglena biomass, and the Euglena biomass comprises beta-1,3-glucan. In some embodiments, the Euglena biomass is dried. In further embodiments, the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0041] In one embodiment, the composition is administered as a single dose daily, hi another embodiment, the composition is administered as multiple divided doses daily.

[0042] In some embodiments, the composition comprises alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, astaxanthin, beta carotene, lutein. The composition further comprises an additional ingredient selected from the group consisting of lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake mushroom, reishi mushroom, or shiitake mushroom, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof. In one embodiment, the composition is administered as a solid. In another embodiment, the composition is administered as a suspension.

[0043] In some embodiments, administration of the composition increases anti-inflammatory cytokine production, hi further embodiments, the composition is administered in combination with an anti-inflammatory drug, an immunosuppressant, or an antibiotic.

[0044] The present disclosure provides that the methods disclosed herein may also be adapted for corresponding uses.

[0045] The present disclosure provides compositions comprising beta-1,3-glucan from fermentation-grown Euglena for use in enhancing immune function in a human with intestinal inflammation. These uses are useful for treating diseases such as inflammatory bowel disease, colitis, and Crohn's disease. In some embodiments, the compositions are administered orally to the human. The present disclosure provides uses of compositions comprising beta-1,3-glucan from fermentation-grown Euglena for use in the manufacture of a medicament for enhancing immune function in a human with intestinal inflammation. These methods are useful for treating diseases such as inflammatory bowel disease, colitis, and Crohn's disease. In some embodiments, the compositions are administered orally to the human. [Brief description of the drawings]

[0046] [Figure 1] Figure 1A shows the structure of a beta-1,3-glucan chain, such as that from Euglena, and Figure 1B shows the structure of a beta-1,3-glucan backbone with beta-1,6-glucan side chains, such as that from yeast. [Diagram 2] FIG. 2 shows a schematic diagram for forming beta-1,3-glucan from Euglena grown using fermentation according to one embodiment of the present invention. [Diagram 3] FIG. 3 shows a first embodiment of a system for culturing Euglena according to the present invention. [Figure 4] Figure 4A shows a schematic diagram of one embodiment of Euglena grown fermentatively by a repeated batch method, and Figure 4B shows a graph of Euglena concentration obtained from a repeated batch method according to one embodiment of the invention. [Diagram 5] FIG. 5 shows a second embodiment of a system for culturing Euglena according to the present invention. [Figure 6] FIG. 6 shows a third embodiment of a system for culturing Euglena according to the present invention. [Figure 7-1]FIG. 7A graphically depicts the phagocytic index of mouse neutrophils taken from peripheral blood on day 14 of beta-glucan treatment. The negative control received no beta-glucan and had a response of 29.8±0.37%. Bars represent the mean±standard error (n=3 mice per treatment group). FIG. 7B graphically depicts the natural killer cell (NK) cell activity of mouse splenocytes taken on day 14 of beta-glucan treatment. The negative control received no beta-glucan and had a response of 11.6±0.28%. Bars represent the mean±standard error (n=3 mice per treatment group). [Figure 7-2] Figure 7C graphically depicts antibody formation after mice were given ovalbumin injections and daily beta-glucan treatment for 23 days. Negative controls received ovalbumin but no beta-glucan and had a response of 49.1 ± 4.6. Bars represent the mean ± SEM (n = 3 mice per treatment group). [Figure 8] Figure 8 graphically depicts survival of mice after E. coli injection on day 0. Starting 2 days prior to E. coli injection (day -2), mice were orally fed dried algae (i.e., Euglena), purified algal beta-1,3-glucan, and yeast-derived beta-glucan by gavage at doses equivalent to 0.01% of the daily food allotment for 5 days. The PBS control group received PBS gavage only, whereas the antibiotic treatment groups received 13 mg / kg ampicillin orally on days 0-4. Each treatment group consisted of n=10 mice. The light grey dashed line represents ampicillin, and the highest to lowest lines looking to the right of the graph (i.e., days 7-10) represent purified algal beta-1,3-glucan, dried algae, yeast beta-glucan, and PBS control, respectively. [Figure 9-1] Figure 9A graphically illustrates the effect of beta-glucan on cytokine production by mouse dendritic cells after seven days of treatment with purified beta-1,3-glucan from Euglena. Figure 9B graphically illustrates the effect of beta-glucan on cytokine production by mouse macrophages after seven days of treatment with purified beta-1,3-glucan from Euglena. [Figure 9-2]Figure 9C graphically illustrates the effect of beta-glucan on cytokine expression by mouse NK cells after 7 days of treatment with purified beta-1,3-glucan from Euglena. In Figures 9A-9C, the doses of beta-1,3-glucan are 0 mg / kg (A), 5 mg / kg (B), 20 mg / kg (C), and 200 mg / kg (D). [Figure 10] FIG. 10 graphically depicts (A) serum cholesterol levels and (B) serum triglyceride levels in mice fed a high cholesterol diet without beta-glucan for 14 days, followed by a normal cholesterol level diet containing a beta-glucan product (or a control diet) for an additional 30 days. Beta-glucan was given at a dose of 20 mg / kg. Serum was collected from mice after 12 hours of fasting. The dotted line represents the PBS control, and the solid line represents the purified beta-glucan from Euglena. [Figure 11] FIG. 11 graphically depicts (A) serum cholesterol levels and (B) serum triglyceride levels in mice fed a high cholesterol diet containing beta-glucan (or a control diet) for 14 days, followed by a normal cholesterol level diet containing a beta-glucan product (or a control diet) for an additional 30 days. Beta-glucan was given at a dose of 20 mg / kg. Serum was collected from mice after 12 hours of fasting. The dotted line represents the PBS control, and the solid line represents the purified beta-glucan from Euglena. [Figure 12] FIG. 12A graphically illustrates the effect on body weight of administering PBS ("None") to mice with DSS-induced colitis. FIG. 12B graphically illustrates the effect on body weight of administering purified beta-1,3-glucan (>99% by weight) ("AG") to mice with DSS-induced colitis. FIG. 12C graphically illustrates the effect on body weight of administering dried Euglena gracilis containing approximately 50% by weight beta-1,3-glucan ("A50") to mice with DSS-induced colitis. In FIGS. 12A-12C, each line represents the results for an individual mouse. [Figure 13]Figure 13 graphically illustrates the effect of beta-1,3-glucan on body weight in mice with DSS-induced colitis. The graph shows a summary of the results for mice treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50") based on the percentage of initial body weight as a function of time. Bars represent the mean ± standard error (n=10 mice per treatment group). [Figure 14] FIG. 14 graphically illustrates the effect of beta-1,3-glucan on body weight in mice with DSS-induced colitis. The graph summarizes the results for mice treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50") based on the percentage of initial body weight as a function of time. Body weight loss scores were calculated as follows: 0 (no loss), 1 (1-5%), 2 (5-10%), 3 (10-20%), and 4 (>20%). Bars represent the mean ± standard error (n=10 mice per treatment group). [Figure 15] FIG. 15 graphically illustrates the effect of beta-1,3-glucan on stool consistency in mice with DSS-induced colitis. The graph shows a summary of the results for mice treated with PBS ("none"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Stool consistency was scored as 0 (normal solid), 2 (semi-solid / soft), 3 (loose stool), and 4 (watery / diarrhea). Bars represent the mean ± standard error (n=10 mice per treatment group). [Figure 16] Figure 16 graphically illustrates the effect of beta-1,3-glucan on stool blood in mice with DSS-induced colitis. The graph shows a summary of the results for mice treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Bars represent the mean ± standard error (n=10 mice per treatment group). [Figure 17]Figure 17 graphically illustrates the effect of beta-1,3-glucan on colon length in mice with DSS-induced colitis. The graph shows colon length at day 10 in mice that received PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Bars represent the mean ± standard error (n=7 mice per treatment group). [Figure 18] FIG. 18A shows representative images of colon inflammation on day 10 in mice treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Images were obtained under a light microscope using a 10x objective. FIG. 18B graphically illustrates the effect of beta-1,3-glucan on the severity of colon inflammation in mice with DSS-induced colitis treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Bars represent the mean ± standard error (n=3 mice per treatment group). [Figure 19] Figure 19A shows representative FACS analysis images of day 10 T helper cell frequency in mice treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Figure 19B graphically depicts the effect of administration of PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50") to mice with DSS-induced colitis on cytokine positive cell production. Bars represent the mean ± standard error (n=3 mice per treatment group). [Figure 20] Figure 20 graphically illustrates the effect of beta-1,3-glucan on cytokine production by colonic immune cells in mice with DSS-induced colitis. Bars represent mean ± SEM (n=3 mice per treatment group). Graph shows cytokine profile of mice treated with PBS ("None"), purified beta-1,3-glucan ("AG"), or dried Euglena gracilis ("A50"). Bars represent mean ± SEM (n=3 mice per treatment group). [Figure 21]FIG. 21 graphically illustrates the reduction in basophil counts in individuals after daily intake of 250 mg of Euglena-derived beta-1,3-glucan. [Figure 22] FIG. 22 graphically illustrates the effect on urgent bowel movements or diarrhea in individuals after daily intake of 250 mg of Euglena-derived beta-1,3-glucan. [Diagram 23] FIG. 23 graphically illustrates the effect on levels of the enzyme superoxide dismutase (SOD) in an individual after daily intake of 250 mg of Euglena-derived beta-1,3-glucan. [Figure 24] FIG. 24 graphically illustrates the effect on malondialdehyde levels in an individual after daily intake of 250 mg of Euglena-derived beta-1,3-glucan. [Diagram 25] FIG. 25 is a schematic diagram of an embodiment of a fermentation process for preparing a complex of beta-glucan and trace metals. [Figure 26] FIG. 26 is a schematic diagram of another embodiment of a fermentation process for preparing a complex of beta-glucan and trace metals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present disclosure provides a method for treating a disease in a human having a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer, the method comprising orally administering to the human an effective amount of a composition comprising Euglena beta-1,3-glucan grown using fermentation. In an embodiment, the disease is selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease. In an embodiment, the composition comprising Euglena beta-1,3-glucan grown using fermentation can comprise Euglena biomass, Euglena-derived beta-1,3-glucan, or purified Euglena-derived beta-1,3-glucan, or a combination thereof. The composition can be used to treat a disease in a human having a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer. In some embodiments, the composition can be used to treat a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease in a human having said disease. In some embodiments, the composition can be used to treat hyperlipidemia. In some embodiments, the composition can be used to treat metabolic syndrome. In some embodiments, the composition can be used to treat inflammatory bowel disease. In some embodiments, the composition can be used to treat colitis. In some embodiments, the composition can be used to treat Crohn's disease. In some embodiments, the composition can be used to treat colon cancer.

[0048] The present application is directed to Euglena-derived beta-1,3-glucan and uses thereof. Euglena-derived beta-1,3-glucan can modulate immune function in an individual. The present application is also directed to Euglena-derived beta-1,3-glucan and uses thereof to modulate or enhance immune function in humans with intestinal inflammation. Euglena-derived beta-1,3-glucan can modulate or enhance immune function in individuals with intestinal inflammation. Euglena-derived beta-1,3-glucan can be administered orally as an edible composition containing either Euglena biomass or beta-1,3-glucan purified from Euglena. Also, Euglena-derived purified beta-1,3-glucan can be administered as a pharmaceutical formulation that can be administered orally or intravenously.

[0049] The inventors have discovered that administration of Euglena-derived beta-1,3-glucan grown by fermentation can be used to promote immune system health in animals, including humans, and to treat and / or prevent disease. For example, Euglena-derived beta-1,3-glucan can be used to regulate autoimmune responses, blood sugar levels, infections, or inflammation. In addition, Euglena-derived beta-1,3-glucan grown by fermentation can be used to promote immune system health in animals, including humans, and to treat and / or prevent disease. Administration of can results in a reduction in serum cholesterol or a reduction in serum triglycerides in animals, including humans.

[0050] The inventors have also surprisingly shown that administration of Euglena-derived beta-1,3-glucan grown using fermentation can be used to promote immune health and treat and / or prevent intestinal inflammation in animals, including humans. For example, Euglena-derived beta-1,3-glucan can be used to regulate or enhance immune function in humans with inflammatory bowel disease, colitis, or Crohn's disease. Additionally, administration of Euglena-derived beta-1,3-glucan grown by fermentation results in increased production of anti-inflammatory cytokines in animals, including humans.

[0051] There are several advantages to using Euglena-derived beta-1,3-glucan. First, beta-1,3-glucan is efficiently produced by Euglena grown using fermentation. Euglena grown by fermentation as described herein accumulates beta-1,3-glucan to about 30% to about 70% of the total Euglena cell mass. In contrast, for example, yeast-derived beta-glucan accounts for less than 15% of the total yeast cell mass. Second, Euglena-derived beta-1,3-glucan can be easily extracted and purified. In contrast, yeast-derived beta-glucan is bound to the yeast cell wall, making extraction difficult and costly. As discussed below, beta-1,3-glucan accumulates in Euglena granules. Its accumulation in granules facilitates the purification process, since Euglena-derived beta-1,3-glucan is not bound to cell wall components that may be difficult to separate. Third, Euglena-derived beta-1,3-glucan is readily bioavailable with little or no processing of the Euglena cells. This allows Euglena-derived beta-1,3-glucan to be effectively administered as Euglena biomass without the need for purification. Furthermore, if purified beta-1,3-glucan is desired, for example in a pharmaceutical formulation, it can be readily purified using the safe and cost-effective methods described herein. In contrast, beta-glucan made by oats or yeast must be extracted from the cell wall, requiring the use of hazardous chemicals and expensive enzymes.

[0052] Euglena produces a unique beta-glucan that is particularly useful for regulating immune function or for treating or prophylactically treating hyperlipidemia. Euglena also produces a unique beta-glucan that is particularly useful for treating or prophylactically treating intestinal inflammation. A significant portion of the beta-1,3-glucan produced by Euglena is located in the pyrenoids or cytoplasm of the organism as paramylon bodies, which are water-insoluble granules of beta-1,3-glucan. This unique beta-glucan is commonly referred to as paramylon. Compared to many other microbial, e.g., oat or fungal beta-glucans, the beta-glucan produced by Euglena is substantially more linear and contains primarily beta-1,3-linkages. The beta-1,3-glucan form is particularly effective for regulating immune function and for lowering serum cholesterol and triglyceride levels. The beta-1,3-glucan form is also particularly effective for treating or preventing intestinal inflammation.

[0053] definition When used in the context of use of a compound or composition, the term "effective amount" refers to an amount of a compound or composition that produces or achieves an intended result, whether that result relates to the prevention or treatment of an infection or disease state, or otherwise as described herein.

[0054] The terms "modulate," "modulating," "modulation," "enhancing," "enhancing," and "enhancement" are used synonymously herein and may be used to describe any human or animal The improved ability to mount an immune response in animals (including but not limited to dogs, cats, rodents, horses, sheep, cows, pigs, goats, donkeys, chickens, or rabbits) is described.

[0055] The term Euglena is understood to mean any species or strain of the genus Euglena, unless otherwise specified. In a preferred embodiment, the Euglena is Euglena gracilis, although other species of the genus Euglena are contemplated.

[0056] The term "biomass" is used to describe Euglena cell material. The product may be processed or unprocessed. For example, Euglena biomass may be dried or provided as wet cell mass.

[0057] The term "derived from" refers to a compound or material that is derived from a particular source. For example, beta-1,3-glucan derived from Euglena indicates that the beta-1,3-glucan originates from Euglena. The beta-1,3-glucan may be bound to Euglena or may be purified and therefore separated from Euglena.

[0058] As used herein, the term "pharmaceutical acceptable" means that a compound or composition is suitable for administration to a subject, including a human patient, to accomplish the treatment described herein without undue adverse side effects considering the severity of the disease or the need for treatment.

[0059] The terms "subject," "patient," and "individual" are used synonymously herein to describe any human or animal, including but not limited to, a dog, cat, rodent, horse, sheep, cow, pig, goat, donkey, chicken, or rabbit. In some embodiments, the terms "subject," "patient," and "individual" are used synonymously herein to describe any human or animal, including but not limited to, a dog, cat, rodent, horse, sheep, cow, pig, goat, donkey, llama, fish, chicken, or rabbit.

[0060] The terms "treat," "treating," and "treatment" are used synonymously herein and refer to any action benefiting a patient at risk for or suffering from a disease state or disease, including reducing, inhibiting, suppressing, or eliminating at least one symptom, slowing the progression of a disease, or inhibiting a disease.

[0061] The term "prophylactic administration" or "prophylactic treatment" refers to any action prior to the onset of a disease in order to reduce the likelihood of the onset of a disease or any action that reduces the likelihood of subsequent onset of a disease in a subject.

[0062] The term "about" includes and describes the value or parameter itself. For example, "about x" includes and describes "x" itself. In certain embodiments, when used in connection with a measurement or when used to modify a value, unit, constant, or range of values, the term "about" refers to a variation of ±1-10%. In some embodiments, when used in connection with a measurement or when used to modify a value, unit, constant, or range of values, the term "about" refers to a variation of ±5%. In some embodiments, when used in connection with a measurement or when used to modify a value, unit, constant, or range of values, the term "about" refers to a variation of ±10%.

[0063] The term "and / or" includes both the alternative and the jointly-inclusive. For example, "x and / or y" includes "x or y" and "x and y".

[0064] It will be understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0065] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0066] Properties of beta-1,3-glucan derived from Euglena Beta-1,3-glucan derived from Euglena is structurally different from beta-glucans produced by other microorganisms in terms of its carbohydrate branching structure, three-dimensional structure, solubility, and bioavailability.

[0067] Branched carbohydrate structure Beta-glucans produced by different organisms can differ substantially in the carbohydrate branching structure of the polymer. For example, beta-glucans derived from algae such as Euglena have almost exclusively 1,3-glycosidic bonds and no 1,6-glycosidic bonds (Figure 1A). In contrast, beta-glucans derived from yeast have a beta-1,3-glucan backbone that contains a mixture of beta-1,3- and beta-1,6-glycosidic bonds, typically with beta-1,6-side chains (2–3 glucose units long) every 10–30 glucose monomers (Figure 1B). Beta-glucans derived from oats and barley have a mixture of beta-1,3- and beta-1,4-glycosidic bonds. Beta-glucans derived from kelps (e.g., Laminaria spp.) have a mixture of beta-1,3- and beta-1,6-glycosidic bonds.

[0068] A significant portion of the beta-glucan produced by Euglena is located as paramylon bodies in the cytoplasm of the algae and is commonly referred to as "paramylon." Paramylon derived from Euglena has a linear structure that contains almost exclusively beta-1,3-glucan and no beta-1,6-side chain branches. The unbranched nature of paramylon is an important distinction compared to other beta-glucan sources when considering its use in immune support applications.

[0069] Studies of the branched structure of paramylon revealed its unique structure and are disclosed in U.S. Patent Application Publication No. 2013 / 0216586. After isolating paramylon from whole Euglena cells, linkage analysis was performed to determine the relative amounts of each bond type between glucose monomers. The results are summarized in Table 1.

[0070] [Table 1] The linkage analysis indicates that both paramylon samples are composed of 3-linked glucopyranosyl residues, with negligible amounts of 3,6-linked, terminal, and 2,3,4-linked glucopyranosyl residues, in addition to small amounts of 4-linked and 2,3-linked glucopyranosyl residues. These data support the idea that paramylon is composed primarily of linear, unbranched beta-1,3-glucan.

[0071] Beta-1,3-glucan is a form of beta-glucan that primarily binds to receptors on the surface of immune system cells, such as dectin-1 (macrophage receptor) and complement receptor 3. Beta-1,3-glucan can also be fermented by the microflora in the gut of an individual, resulting in the production of beneficial metabolites, such as short-chain fatty acids, that can impact the health of an animal. Beta-1,3-glucan can also be fermented by the microflora in the gut of an individual, resulting in the production of beneficial metabolites, such as short-chain fatty acids, that can impact the health of an individual.

[0072] Beta-1,3-glucans derived from fermentatively grown Euglena useful in the methods described herein contain about 85% or more beta-1,3-glycosidic linkages, about 87% or more beta-1,3-glycosidic linkages, about 90% or more beta-1,3-glycosidic linkages, about 91% or more beta-1,3-glycosidic linkages, about 92% or more beta-1,3-glycosidic linkages, about 93% or more beta-1,3-glycosidic linkages, or about 94% or more beta-1,3-glycosidic linkages.

[0073] three dimensional structure The three-dimensional structure and folding of beta-1,3-glucans can affect their bioavailability, surface area, and overall effectiveness in immune stimulating applications. In linear beta-1,3-glucan chains, the structure is dominated by the glycosidic linkage moiety. The glucopyranosyl chair rings are fairly rigid, so most of the flexibility of the glucan chain is due to rotation around the glycosidic bond. X-ray crystallography and spectroscopy show that linear glucans have a triple helical backbone in the solid state. Paramylon, made by Euglena, is considered one of the structurally simpler beta-glucans, with few glycosyl side chains. This is in stark contrast to laminaran, lentinan, scleroglucan, schizophyllan, or yeast-derived beta-glucans, which have 1,4-linked or 1,6-linked side chains exposed toward the outside of the helical structure.

[0074] The triple helix structure is stable over a wide temperature range at neutral pH and is insoluble in water. The hydrogen bonds result in a polymer. However, the hydrogen bonds can be destabilized by various means to change the conformation of the paramylon polymer. For example, paramylon can be dissolved in alkaline solutions (e.g., 0.2M NaOH or higher), aprotic polar solvents such as DMSO, in the presence of strong chaotropic agents (e.g., urea) or by raising the temperature above the triple helix melting temperature (-135°C). Various immunological effects can be obtained that are related to the native state conformation, the denatured conformation, or the denatured and restored beta-1,3-glucan conformation. Beta-1,3-glucan in any of these three conformations can serve as building blocks for further reactions that add or improve its functionality. Some of these modifications to produce functionalized beta-1,3-glucans and some of the uses of each of these glucans are discussed herein. The conformation of the beta-glucan and the resulting solubility can also affect how the beta-glucan is delivered. For example, water-soluble beta-1,3-glucan can be injected intravenously.

[0075] particle size Particle size is a factor that affects the function and bioavailability of beta-1,3-glucan particles. Euglena-derived beta-1,3-glucan particles are useful in the methods described herein. Generally, Euglena-derived beta-1,3-glucan particles have an average diameter of about 7 microns or less, about 6 microns or less, about 5 microns or less, about 4 microns or less, about 3 microns or less, about 2.5 microns or less, about 2 microns or less, about 1.5 microns or less, about 1 micron or less, about 0.5 microns or less, about 0.4 microns or less, about 0.3 microns or less, about 0.2 microns or less, or about 0.1 microns or less.

[0076] Beta-1,3-Glucan Purity Levels The purity level of the beta-glucan compound has been found to have an effect on efficacy, possibly due to the presence of other substances that inhibit the interaction between beta-glucan and immune cells. Using the methods described herein, beta-1,3-glucan can be easily isolated in the form of granules from Euglena cells. As a result, the purity of beta-1,3-glucan from Euglena is much higher than that of common preparations of beta-glucan from yeast and other organisms. For example, beta-1,3-glucan from Euglena is not bound to cell wall components as is the case with beta-glucan from yeast and other organisms. More precisely, beta-1,3-glucan from Euglena is mainly extracted in the form of water-insoluble granules. Thus, the purification of beta-1,3-glucan from Euglena is not complicated by the binding of beta-glucan to cell wall components. Using the methods described herein, purity levels of more than 95% by weight can be obtained as received. In some embodiments, purity levels of more than 99% by weight can be obtained as received. In comparison, the best yeast-derived beta-glucans rarely achieve 90% or higher purity, with most only around 70-80% purity. Moreover, because of the ease of separation due to the lack of cell walls in Euglena and the ease of recovery of beta-1,3-glucan granules, it is more cost-effective to produce high-purity beta-1,3-glucan from Euglena than from yeast-derived glucan. Finally, because no harsh chemicals (e.g., strong acids and bases or solvents) are required to recover Euglena-derived beta-1,3-glucan, the beta-1,3-glucan can be recovered in its native form without altering its chemical composition and structure. For one thing, the use of pure, unmodified beta-1,3-glucan from Euglena is advantageous compared to solubilized modified beta-1,3-glucans obtained from other organisms, which undergo modification during the extraction and / or purification process, since the bioavailability of Euglena-derived beta-1,3-glucan is higher. In some embodiments, the purified beta-1,3-glucan from Euglena is greater than 85% pure, greater than 90% pure, greater than 92% pure, or more pure. purity greater than 94%, purity greater than 95%, purity greater than 96%, purity greater than 97%, purity greater than 98%, or purity greater than 99%.

[0077] Methods for Treating Hyperlipidemia Oral administration of beta-glucan derived from Aureobasidium pullulans was recently found to prevent the development of high-fat diet (HFD)-induced fatty liver in mice (Aoki, S. et al. Scientific Reports 2015, 5, 10457). Beta-glucan derived from M. pullulans consists of both beta-1,3-glycosidic and beta-1,6-glycosidic linkages. In that study, the rate-limiting enzyme for cholesterol synthesis, HMGR, and cholesterol degradation, CYP7A1, were significantly upregulated in mice. The authors of the study suggested that an increase in CYP7A1 mRNA expression was the possible molecular basis for the observed improvement in fatty liver, liver damage, and cholesterol levels. Specifically, they pointed out the upregulation of IL-6. Induction of IL-6 was also involved in the prevention of HFD-induced fatty liver in mice. Another study involving yeast-derived beta-glucan proposed that the reduction of cholesterol plasma levels in mice was mediated by macrophages (Vetvicka, V. et al. J. of Immunotoxicology, 2009, 6, 30-35). Clearly, the exact molecular mechanism by which beta-glucan regulates the immune system and, in turn, cholesterol levels is complex and may depend on the exact structure of the beta-glucan.

[0078] Hyperlipidemia, or abnormally high blood cholesterol or triglyceride levels, increases the risk of heart attacks and cardiovascular disease. Hyperlipidemia can result from genetic factors or certain health or lifestyle factors, including a high-fat or high-cholesterol diet, obesity, or lack of regular exercise. Hyperlipidemia includes any disease that results in elevated blood cholesterol (i.e., hypercholesterolemia) or blood triglyceride (hypertriglyceridemia) levels. Cholesterol and triglycerides are bound to lipoproteins, including low-density lipoproteins (LDL) and high-density lipoproteins (HDL). LDL, often referred to as "bad" cholesterol, collects in the walls of blood vessels and can cause plaque growth and atherosclerosis. In contrast, HDL (often referred to as "good" cholesterol) mobilizes fats from cells, artery walls, and tissues through the bloodstream. Increasing the concentration of HDL particles reduces the buildup of atherosclerosis in the artery walls. Beta-1,3-glucan derived from Euglena grown using fermentation (e.g., Euglena biomass, Euglena beta-1,3-glucan, or Euglena purified beta-1,3-glucan, or a combination thereof) can be administered to a subject, including a human, to treat hyperlipidemia or prophylactically to a subject at risk for hyperlipidemia. Beta-1,3-glucan derived from Euglena grown using fermentation (e.g., Euglena biomass, Euglena beta-1,3-glucan, or Euglena purified beta-1,3-glucan, or a combination thereof) can be administered to a subject, including a human, to lower LDL. Beta-1,3-glucan derived from Euglena grown using fermentation (e.g., Euglena biomass, Euglena beta-1,3-glucan, or Euglena purified beta-1,3-glucan, or a combination thereof) can be administered to a subject, including a human, to increase HDL. Those at risk for hyperlipidemia include, but are not limited to, those who have previously been diagnosed with hyperlipidemia, those whose diet is high in fat or cholesterol, or those who have a parent or both parents with hyperlipidemia.

[0079] Beta-1,3-glucan derived from Euglena grown using fermentation (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to subjects, including humans, to treat nonalcoholic fatty liver disease (NAFLD) or prophylactically to subjects at risk for NAFLD. NAFLD is a condition associated with obesity and It is closely related to type 2 diabetes and is known to be a major risk factor for cardiovascular disease.

[0080] Beta-1,3-glucan derived from Euglena grown using fermentation (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to subjects, including humans, to treat metabolic syndrome or can be administered prophylactically to subjects at risk for metabolic syndrome. Metabolic syndrome refers to a set of conditions, including elevated blood pressure, high blood sugar levels, excess body fat, and abnormal cholesterol levels, that occur together and increase the risk of heart disease, stroke, and diabetes.

[0081] Administration of beta-1,3-glucan derived from Euglena grown using fermentation as described herein (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) has been shown to lower blood cholesterol and triglyceride levels (see Example 7). Such administration can be oral, for example, by administering a edible composition or oral pharmaceutical formulation, or can be intravenous injection, for example, by administering an intravenous pharmaceutical formulation. Alternative routes of administration, such as inhalation, are also contemplated. Oral edible compositions or oral pharmaceutical formulations include either purified or unpurified beta-1,3-glucan derived from Euglena grown using fermentation as described herein. In general, intravenous pharmaceutical formulations include purified beta-1,3-glucan derived from Euglena grown using fermentation. Typically, pharmaceutical formulations suitable for inhalation include purified beta-1,3-glucan derived from Euglena grown using fermentation and can be administered, for example, by nasal spray. The dietary composition or pharmaceutical formulation may be administered in combination with one or more of a statin, nicotinic acid, bile acid resin, fibric acid derivative, or cholesterol absorption inhibitor to enhance the treatment of hyperlipidemia.

[0082] Fermented Euglena useful for making beta-1,3-glucan for the treatment of hyperlipidemia generally rely on a growth medium to provide nutrients for growth and production of beta-1,3-glucan. Although it is contemplated that Euglena may grow with full or partial light exposure, it is generally preferred that Euglena grown using fermentation be grown heterotrophically. During fermentation, the growing Euglena efficiently produces beta-1,3-glucan. In some embodiments, the Euglena cells accumulate beta-1,3-glucan at about 30% to about 70% by weight, about 30% to about 40% by weight, about 40% to about 50% by weight, about 60% to about 70% by weight, about 40% to about 70% by weight, or about 50% to about 70% by weight. In some embodiments, the Euglena cells accumulate beta-1,3-glucan at about 50% to about 60% by weight, or about 50% to about 55% by weight.

[0083] Beta-1,3-glucan from fermentation-grown Euglena is administered to a subject in purified or unpurified form to treat hyperlipidemia. For example, fermentation-grown Euglena biomass can be orally administered to a subject to treat hyperlipidemia. Because fermentation-grown Euglena contains an effective amount of beta-1,3-glucan, the Euglena biomass is effective in lowering blood cholesterol levels or blood triglyceride levels without the need to purify the beta-1,3-glucan. This is due in part to the high bioavailability of beta-1,3-glucan made by fermentation-grown Euglena. In contrast, beta-glucan made by yeast is tightly bound to the yeast cell wall, resulting in a lower amount of bioavailable beta-glucan.

[0084] For example, the Euglena biomass is provided in the form of an orally administered edible composition for lowering blood cholesterol and / or blood triglyceride levels. The edible composition may be a food or a dietary supplement. To aid in intake, the Euglena biomass is processed into a sheet, paste, cream, powder, capsule, tablet, or other edible solid or liquid (such as a suspension) and can be consumed alone or in combination with another food. For example, in some embodiments, the Euglena biomass is administered in the form of a beverage, such as a nutritional shake, a nutritional bar, a baked good, or a cereal.

[0085] In one exemplary embodiment, the Euglena biomass is dried to prepare it for oral administration. For example, the Euglena biomass can be dried to a moisture content of about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less by freeze-drying the biomass, heating the biomass in an oven, double drum dryer, or similar drying device, or applying a vacuum to the biomass. Optionally, before or after drying the Euglena biomass, the biomass is processed into a powder. For example, Euglena cells are suspended in a solution, ground in a shear mixer, and dried to produce a powder. In some embodiments, the powdered Euglena biomass is processed to an average particle size of about 1000 microns or less, about 900 microns or less, about 800 microns or less, about 700 microns or less, about 600 microns or less, about 500 microns or less, about 400 microns or less, about 300 microns or less, about 250 microns or less, about 200 microns or less, about 100 microns or less, about 50 microns or less, about 25 microns or less, about 15 microns or less, about 10 microns or less, about 5 microns or less, about 3 microns or less, about 2 microns or less, about 1 micron or less, or about 0.5 microns or less. The dried and / or powdered Euglena biomass can then be orally administered to a subject, either directly or by mixing with another edible composition, to reduce the subject's blood cholesterol levels and / or blood triglyceride levels.

[0086] Orally administered Euglena biomass may further contain nutritional supplements in addition to beta-1,3-glucan. The nutritional supplements may be pre-existing in the Euglena biomass or may be added to the Euglena biomass. Exemplary additional supplements include alpha-tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, and the like. Acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake, reishi, or shiitake mushrooms, manganese, quercetin, bromelain, olive leaf, elderberry, umuka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and their derivatives, and pterostilbene. These additional supplements can function independently or synergistically with beta-1,3-glucan derived from fermentation-grown Euglena to lower blood cholesterol levels and / or blood triglyceride levels. Additional ingredients include either Haematococcus pluvialis, astaxanthin, and colostrum.

[0087] Orally administered Euglena biomass may further contain a pharma- ceutically acceptable excipient. Examples of pharma-ceutically acceptable excipients include fillers, binders, coating agents, preservatives, lubricants, flavors, sweeteners, colorants, surfactants, solvents, buffers, chelating agents, or stabilizers. Examples of pharma-ceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, malic acid, and the like. Examples of pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, and cellulose. Examples of pharmaceutically acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, and gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, and sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, and stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, and sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharma- ceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0088] Beta-1,3-glucan derived from Euglena grown using fermentation can also be administered to a subject in a purified form to lower blood cholesterol and / or blood triglyceride levels. In some embodiments, the beta-1,3-glucan is purified from Euglena to be greater than 85% pure, greater than 90% pure, greater than 92% pure, greater than 94% pure, greater than 95% pure, greater than 96% pure, greater than 97% pure, greater than 98% pure, or greater than 99% pure. Generally, beta-1,3-glucan is extracted by lysing Euglena cells and isolating the beta-1,3-glucan. Euglena cells can be lysed using sonication or high pressure homogenization. Optionally, additional chemicals are included during the lysis step to aid in lysis, although such chemicals are not necessary. Exemplary additional chemicals included during the lysis step include detergents (such as sodium dodecyl sulfate), enzymes, bases (such as sodium hydroxide), or acids (such as acetic acid or hydrochloric acid). Beta-1,3-glucan can be isolated from the lysed Euglena cells using filtration or gravity separation (such as gravity settling or centrifugation). To obtain a higher level of purity, the isolated beta-1,3-glucan is washed, for example, with an aqueous solution or ethanol.

[0089] Additionally, purified beta-1,3-glucan can be modified to enhance its effectiveness in lowering serum cholesterol or triglyceride levels. For example, beta-1,3-glucan can be sulfated, conjugated with a pyridinium moiety, or conjugated with a cationic moiety, such as dimethylethanolamine (DMAE).

[0090] Similar to Euglena biomass, purified beta-1,3-glucan from Euglena can be orally administered in the form of an edible composition, such as a food or dietary supplement, for lowering blood cholesterol and / or blood triglyceride levels. The purified beta-1,3-glucan can be provided as a paste, powder, capsule, tablet, or liquid (such as a suspension) and can be administered alone or mixed with another food. For example, the purified beta-1,3-glucan is orally administered in the form of a beverage, such as a nutritional shake, a nutritional bar, a baked good, or a cereal.

[0091] The orally administered purified beta-1,3-glucan in the composition may be supplemented with one or more additional supplements, such as alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha -Linolenic acid, astaxanthin, beta carotene, lutein, probiotic lactobacillus The composition may further comprise, for example, sucrose, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake mushroom, reishi mushroom, or shiitake mushroom, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, and pterostilbene. Additional ingredients include Haematococcus pluvialis, astaxanthin, and colostrum.

[0092] The purified beta-1,3-glucan can also be used in the form of a pharmaceutical preparation administered to a subject, such as a human, to reduce blood cholesterol and / or blood triglyceride levels. The pharmaceutical preparation can further include a pharma- ceutical acceptable excipient. Exemplary pharma-ceutical acceptable excipients include fillers, binders, coatings, preservatives, lubricants, flavors, sweeteners, colorants, surfactants, solvents, buffers, chelating agents, or stabilizers. Examples of pharma-ceutical acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, pregelatinized starch, corn starch, and potato starch. Examples of pharma-ceutical acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, and cellulose. Examples of pharma-ceutically acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, and gelatin. Examples of pharma-ceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, and sodium starch glycolate. Examples of pharma-ceutically acceptable lubricants include polyethylene glycol, magnesium stearate, and stearic acid. Examples of pharma-ceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, and sorbic acid. Examples of pharma-ceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharma-ceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0093] The pharmaceutical preparation can be administered orally or intravenously to a subject to lower blood cholesterol and / or blood triglyceride levels. Orally administered pharmaceutical preparations can be administered as solids or liquids, such as tablets, capsules, or syrups. For intravenously administered pharmaceutical preparations, the purified beta-1,3-glucan from Euglena is suspended or dissolved in an aqueous solution, such as saline.

[0094] Administration of an effective amount of a edible composition or pharmaceutical preparation containing beta-1,3-glucan derived from Euglena grown using fermentation can reduce serum cholesterol levels after the course of the administration regimen. For example, the reduction in serum cholesterol levels can be measured after 7 days, 14 days, 21 days, 30 days, or 60 days. In some embodiments, administration of beta-1,3-glucan derived from Euglena grown using fermentation results in a reduction in serum cholesterol levels of about 5% or more after 30 days of the effective amount, a reduction in serum cholesterol levels of about 10% or more after 30 days of the effective amount, a reduction in serum cholesterol levels of about 15% or more after 30 days of the effective amount, a reduction in serum cholesterol levels of about 20% or more after 30 days of the effective amount, a reduction in serum cholesterol levels of about 25% or more after 30 days of the effective amount, a reduction in serum cholesterol levels of about 30% or more after 30 days of the effective amount, or a reduction in serum cholesterol levels of about 35% or more after 30 days of the effective amount.

[0095] Similarly, beta-1,3-glucan derived from Euglena grown by fermentation Administration of an effective amount of a edible composition or pharmaceutical formulation containing the Euglena beta-1,3-glucan can reduce serum triglyceride levels after the course of the administration regimen. In some embodiments, administration of the Euglena beta-1,3-glucan results in a reduction of serum triglyceride levels of about 5% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 10% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 15% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 20% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 25% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 30% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 35% or more after 30 days of the effective amount, a reduction of serum triglyceride levels of about 40% or more after 30 days of the effective amount, or a reduction of serum triglyceride levels of about 45% or more after 30 days of the effective amount.

[0096] Each of the orally administered compositions containing beta-1,3-glucan derived from fermentation-grown Euglena, the orally administered pharmaceutical preparations containing beta-1,3-glucan derived from fermentation-grown Euglena, and the intravenously administered pharmaceutical preparations containing beta-1,3-glucan derived from fermentation-grown Euglena are administered in a dosage effective to lower serum cholesterol levels and / or serum triglyceride levels. Such dosage regimens are generally understood as the amount of beta-1,3-glucan per kg body weight for each of the compositions or pharmaceutical preparations. In some embodiments, the composition or pharmaceutical formulation is administered to a subject in an effective amount of about 0.1 mg or more of beta-1,3-glucan per kg body weight, about 0.25 mg or more of beta-1,3-glucan per kg body weight, about 0.5 mg or more of beta-1,3-glucan per kg body weight, about 1 mg or more of beta-1,3-glucan per kg body weight, about 2 mg or more of beta-1,3-glucan per kg body weight, about 5 mg or more of beta-1,3-glucan per kg body weight, about 10 mg or more of beta-1,3-glucan per kg body weight, about 15 mg or more of beta-1,3-glucan per kg body weight, about 25 mg or more of beta-1,3-glucan per kg body weight, or about 50 mg or more of beta-1,3-glucan per kg body weight. In some embodiments, an effective amount of the composition or pharmaceutical composition is about 0.1 mg beta-1,3-glucan per kg body weight to about 50 mg beta-1,3-glucan per kg body weight, about 0.1 mg beta-1,3-glucan per kg body weight to about 25 mg beta-1,3-glucan per kg body weight, about 0.2 mg beta-1,3-glucan per kg body weight to about 15 mg beta-1,3-glucan per kg body weight, about 0.5 mg beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight, or about 1 mg beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight. As described herein, an effective amount of the composition or pharmaceutical formulation can be administered to a subject once a day.In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0097] In some embodiments, an effective amount of the composition or pharmaceutical composition is from about 0.1 mg beta-1,3-glucan per kg body weight to about 100 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 50 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 25 mg beta-1,3-glucan per kg body weight, from about 0.2 mg beta-1,3-glucan per kg body weight to about 15 mg beta-1,3-glucan per kg body weight, from about 0.5 mg beta-1,3-glucan per kg body weight, beta-1,3-glucan to about 10 mg beta-1,3-glucan per kg body weight, about 1 mg beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight, about 50 mg beta-1,3-glucan per kg body weight to about 100 mg beta-1,3-glucan per kg body weight, about 50 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight, or about 25 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight. As described herein, an effective amount of the composition or pharmaceutical formulation can be administered to a subject once a day. In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0098] An effective amount of a composition or pharmaceutical preparation containing beta-1,3-glucan derived from Euglena grown using fermentation can be administered to a subject once a day to reduce serum cholesterol levels and / or serum triglyceride levels. In some embodiments, an effective amount of a edible or pharmaceutical composition containing beta-1,3-glucan derived from Euglena grown using fermentation is administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a edible or pharmaceutical composition containing beta-1,3-glucan derived from Euglena grown using fermentation is administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0099] Methods for modulating immune function Euglena-derived beta-1,3-glucan (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to a subject, such as a human, to modulate immune function. Administration of Euglena-derived beta-1,3-glucan to a subject results in a measurable increase in cytokine production, antibody titers, and immune system cell activity (e.g., rates of phagocytosis and natural killer cell cytotoxicity), indicating modulation of immune function. Subjects administered Euglena-derived beta-1,3-glucan also exhibit enhanced responses to infections.

[0100] Beta-1,3-glucan derived from Euglena useful for modulating immune function is grown by fermenting Euglena by the methods described herein. Fermented Euglena generally rely on a growth medium to provide nutrients for growth and production of beta-1,3-glucan. Although it is contemplated that Euglena may grow with full or partial light exposure, it is generally preferred that Euglena grown using fermentation are grown heterotrophically. During fermentation, the growing Euglena efficiently produces beta-1,3-glucan. In some embodiments, the Euglena cells accumulate beta-1,3-glucan at about 30% to about 70% by weight, about 30% to about 40% by weight, about 40% to about 50% by weight, about 60% to about 70% by weight, about 40% to about 70% by weight, or about 50% to about 70% by weight. In some embodiments, the Euglena cells accumulate beta-1,3-glucan at about 50% to about 60% by weight, or about 50% to about 55% by weight. Accumulates beta-1,3-glucan.

[0101] Beta-1,3-glucan derived from Euglena and grown by fermentation can be administered to a subject in purified or unpurified form to modulate immune function. For example, Euglena biomass grown by fermentation can be orally administered to a subject to modulate immune function. Because Euglena grown by fermentation contains effective amounts of beta-1,3-glucan, the Euglena biomass is effective without purifying the beta-1,3-glucan. This is due in part to the high bioavailability of the beta-1,3-glucan produced by Euglena. In contrast, beta-glucan produced by yeast is tightly bound to the yeast cell wall and has a lower amount of readily bioavailable beta-glucan.

[0102] The Euglena biomass is provided in the form of an edible composition that is orally administered to regulate immune function. The edible composition may be a food or a dietary supplement. To aid in intake, the Euglena biomass is processed into a sheet, paste, cream, powder, capsule, tablet, or other edible solid or liquid (such as a suspension) and can be consumed alone or in combination with some other food. For example, in some embodiments, the Euglena biomass is administered in the form of a beverage, such as a nutritional shake, a nutritional bar, a baked good, or a cereal.

[0103] In one exemplary embodiment, the Euglena biomass is dried to prepare it for oral administration. For example, the Euglena biomass can be dried to a moisture content of 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less by freeze-drying the biomass, heating the biomass in an oven, or applying a vacuum to the biomass. Optionally, before or after drying the Euglena biomass, the biomass can be processed into a powder. For example, Euglena cells are suspended in a solution, ground in a shear mixer, and dried to produce a powder. The powdered Euglena biomass can be processed to have an average particle size of about 1000 microns or less, about 900 microns or less, about 800 microns or less, about 700 microns or less, about 600 microns or less, about 500 microns or less, about 400 microns or less, about 300 microns or less, about 250 microns or less, about 200 microns or less, about 100 microns or less, about 50 microns or less, about 25 microns or less, about 15 microns or less, about 10 microns or less, about 5 microns or less, about 3 microns or less, about 2 microns or less, about 1 micron or less, or about 0.5 microns or less. The dried and / or powdered Euglena biomass can then be orally administered to a subject, either directly or by mixing with another edible composition, to modulate the immune function of the subject.

[0104] Orally administered Euglena biomass may further contain nutritional supplements in addition to beta-1,3-glucan. The nutritional supplements may be pre-existing in the Euglena biomass or may be added to the Euglena biomass. Exemplary additional supplements include alpha-tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, and the like. Acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake mushroom, reishi mushroom, or shiitake mushroom, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha-lipoic acid, essential oils, fish oils, spices and their derivatives, and pterostilbene. These supplemental supplements can function independently or synergistically with beta-1,3-glucan derived from fermentation-grown Euglena to modulate immune function. Additional ingredients include Haematococcus pluvialis, astaxanthin, and colostrum.

[0105] Orally administered Euglena biomass may further include a pharma- ceutically acceptable excipient. Exemplary pharma-ceutically acceptable excipients include fillers, binders, coating agents, preservatives, lubricants, flavors, sweeteners, colorants, surfactants, solvents, buffers, chelating agents, or stabilizers. Examples of pharma-ceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, pregelatinized starch, corn starch, and potato starch. Examples of pharma-ceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, and cellulose. Examples of pharma-ceutically acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, and gelatin. Examples of pharma- ceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, and sodium starch glycolate. Examples of pharma-ceutically acceptable lubricants include polyethylene glycol, magnesium stearate, and stearic acid. Examples of pharma-ceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, and sorbic acid. Examples of pharma-ceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharma-ceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0106] Beta-1,3-glucan derived from Euglena grown using fermentation can also be administered to a subject in a purified form to modulate function. In some embodiments, the beta-1,3-glucan is purified from Euglena to be greater than 85% pure, greater than 90% pure, greater than 92% pure, greater than 94% pure, greater than 95% pure, greater than 96% pure, greater than 97% pure, greater than 98% pure, or greater than 99% pure. Generally, beta-1,3-glucan is extracted by lysing Euglena cells and isolating the beta-1,3-glucan. Euglena cells can be lysed using sonication or high pressure homogenization. Optionally, additional chemicals are included during the lysis step to aid in lysis, although such chemicals are not necessarily required. Exemplary additional chemicals included during the lysis step include detergents (such as sodium dodecyl sulfate), enzymes, bases (such as sodium hydroxide), or acids (such as acetic acid or hydrochloric acid). Beta-1,3-glucan can be isolated from the lysed Euglena cells using filtration or gravity separation (such as gravity settling or centrifugation). To obtain a higher level of purity, the isolated beta-1,3-glucan is washed, for example, with an aqueous solution or ethanol.

[0107] Additionally, purified beta-1,3-glucan can be modified to enhance its efficacy as a regulator of immune function. For example, beta-1,3-glucan can be sulfated, conjugated with a pyridinium moiety, or conjugated with a cationic moiety, such as dimethylethanolamine (DMAE).

[0108] Similar to Euglena biomass, purified beta-1,3-glucan from Euglena can be administered orally in the form of edible compositions, such as foods and dietary supplements, to regulate immune function. The purified beta-1,3-glucan can be provided as a paste, powder, capsule, tablet, or liquid (such as a suspension) and can be administered alone or mixed with another food. For example, the purified beta-1,3-glucan can be administered orally in the form of a beverage, such as a nutritional shake, a nutritional bar, a baked good, or a cereal.

[0109] The orally administered purified beta-1,3-glucan in the composition may be supplemented with one or more additional supplements, such as alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha- It may further comprise linoleic acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake mushroom, reishi mushroom, or shiitake mushroom, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and their derivatives, and pterostilbene. Additional ingredients include Haematococcus pluvialis, astaxanthin, and colostrum.

[0110] The purified beta-1,3-glucan can also be used in the form of a pharmaceutical preparation administered to a subject, such as a human, to modulate immune function. The pharmaceutical preparation may further include a pharma- ceutical acceptable excipient. Exemplary pharma-ceutical acceptable excipients include fillers, binders, coating agents, preservatives, lubricants, flavorings, sweeteners, colorants, surfactants, solvents, buffers, chelating agents, or stabilizers. Examples of pharma-ceutical acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, pregelatinized starch, corn starch, and potato starch. Examples of pharma-ceutical acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, and cellulose. Examples of pharma-ceutical acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, and gelatin. Examples of pharma- ceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, and sodium starch glycolate. Examples of pharma-ceutically acceptable lubricants include polyethylene glycol, magnesium stearate, and stearic acid. Examples of pharma-ceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, and sorbic acid. Examples of pharma-ceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Examples of pharma-ceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0111] The pharmaceutical preparation can be administered orally to a subject or intravenously to a subject to modulate immune function. Alternative routes of administration, such as inhalation, are also contemplated. Orally administered pharmaceutical preparations can be administered as solids or liquids, such as tablets, capsules, or syrups. For intravenously administered pharmaceutical preparations, the purified beta-1,3-glucan from Euglena is suspended or dissolved in an aqueous solution, such as saline. Pharmaceutical preparations suitable for inhalation can be administered, for example, by nasal spray. The edible composition or pharmaceutical preparation can be administered in combination with one or more drugs to enhance the treatment of allergies, diabetes, or infectious diseases. For example, the edible composition or pharmaceutical preparation can be administered in combination with an antihistamine, insulin, or antibiotic.

[0112] By administering an effective amount of beta-1,3-glucan derived from Euglena grown using fermentation, for example, by administering a edible composition or pharmaceutical formulation described herein. Modulating immune function in a subject through the administration of beta-1,3-glucan from Euglena provides many health benefits. Compositions and formulations containing Euglena-derived beta-1,3-glucan can be administered to modulate immune function for the treatment or prophylactic treatment of disease. For example, administration of the composition or pharmaceutical formulation can be used to modulate an autoimmune response in a subject, modulate blood glucose levels in a subject, modulate an infection in a subject, or modulate inflammation in a subject.

[0113] A composition or pharmaceutical formulation containing beta-1,3-glucan derived from Euglena (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to a subject, including a human, to modulate an autoimmune response associated with diabetes, Crohn's disease, rheumatoid arthritis, fibromyalgia, systemic lupus erythematosus, glomerulonephritis, scleroderma, or multiple sclerosis. In some embodiments, the edible composition or pharmaceutical composition is administered prophylactically to reduce the progression of diabetes, Crohn's disease, rheumatoid arthritis, fibromyalgia, systemic lupus erythematosus, glomerulonephritis, scleroderma, or multiple sclerosis.

[0114] Also, a composition or pharmaceutical formulation containing beta-1,3-glucan derived from Euglena grown using fermentation as described herein (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to a subject to regulate blood glucose levels in the subject. After administration of a composition or pharmaceutical formulation containing beta-1,3-glucan, postprandial blood glucose is generally lower than in the absence of administration of beta-1,3-glucan. Regulation of blood glucose, particularly postprandial blood glucose, is important for general diabetes treatment and management in both type 1 and type 2 diabetes patients. Blood glucose levels can be measured using an A1C test, which reflects the average blood glucose level over the past 2-3 months. Specifically, the A1C test measures the percentage of hemoglobin that is coated with sugar (i.e., glycated). Thus, the compositions and pharmaceutical compositions containing beta-1,3-glucan derived from Euglena described herein are useful for treating hyperglycemia in diabetic patients. In some embodiments, a dietary composition or pharmaceutical formulation containing Euglena-derived beta-1,3-glucan described herein is administered prophylactically to a subject to reduce hyperglycemia.

[0115] Also, beta-1,3-glucan derived from Euglena grown using fermentation in a composition or pharmaceutical formulation (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to a subject to regulate an infectious disease, such as a bacterial infection, a fungal infection, or a viral infection in the subject. Administration of a composition or pharmaceutical formulation containing the beta-1,3-glucan described herein results in increased activity of both innate and adaptive immune functions. For example, administration of the composition or pharmaceutical formulation results in increased neutrophil phagocytosis, natural killer cell cytotoxicity, and antibody production. Each of these regulated immune functions has an effect on infectious diseases. A composition or pharmaceutical formulation containing beta-1,3-glucan derived from Euglena grown by fermentation can be administered to a subject with an infectious disease to treat the infection, or can be administered prophylactically to a subject to limit the risk of an infectious disease. These advances in therapeutic or prophylactic treatment of infections are particularly important for bacterial infections because of the risk of antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA).

[0116] Administration of a composition or pharmaceutical formulation comprising beta-1,3-glucan derived from Euglena grown using fermentation as described herein (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or combinations thereof) can be administered to a subject, including a human, to regulate inflammation in the subject. The administered beta-1,3-glucan can inhibit the production of inflammatory cytokines. , resulting in modulation of the inflammatory response in the subject. In some embodiments, the inflammation is associated with allergy or asthma. A composition or pharmaceutical formulation comprising a beta-1,3-glucan derived from Euglena described herein can be administered to a subject to treat inflammation, such as allergy or asthma. Additionally, a composition or pharmaceutical formulation comprising a beta-1,3-glucan derived from Euglena described herein can be administered prophylactically to a subject to suppress inflammation, such as allergy or asthma.

[0117] Each of the orally administered compositions containing beta-1,3-glucan derived from fermentation-grown Euglena, the orally administered pharmaceutical preparations containing beta-1,3-glucan derived from fermentation-grown Euglena, and the intravenously administered pharmaceutical preparations containing beta-1,3-glucan derived from fermentation-grown Euglena are administered in a dosage effective to modulate immune function. Such dosage regimens are generally understood to be the amount of beta-1,3-glucan per kg of body weight for each of the compositions or pharmaceutical preparations. In some embodiments, the composition or pharmaceutical formulation is administered to a subject in an effective amount of about 0.1 mg or more of beta-1,3-glucan per kg body weight, about 0.25 mg or more of beta-1,3-glucan per kg body weight, about 0.5 mg or more of beta-1,3-glucan per kg body weight, about 1 mg or more of beta-1,3-glucan per kg body weight, about 2 mg or more of beta-1,3-glucan per kg body weight, about 5 mg or more of beta-1,3-glucan per kg body weight, about 10 mg or more of beta-1,3-glucan per kg body weight, about 15 mg or more of beta-1,3-glucan per kg body weight, about 25 mg or more of beta-1,3-glucan per kg body weight, or about 50 mg or more of beta-1,3-glucan per kg body weight. In other embodiments, an effective amount of a composition or pharmaceutical composition used to regulate a subject's immune function, treat a disease, or for prophylactic administration can be about 0.1 mg beta-1,3-glucan per kg body weight to about 50 mg beta-1,3-glucan per kg body weight, about 0.1 mg beta-1,3-glucan per kg body weight to about 25 mg beta-1,3-glucan per kg body weight, about 0.2 mg beta-1,3-glucan per kg body weight to about 15 mg beta-1,3-glucan per kg body weight, about 0.5 mg beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight, or about 1 mg beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight. As described herein, an effective amount of a composition or pharmaceutical formulation can be administered to a subject once a day.In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0118] In some embodiments, an effective amount of the composition or pharmaceutical composition is from about 0.1 mg beta-1,3-glucan per kg body weight to about 100 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 50 ... ~About 25 mg of beta-1,3-glucan per kg of body weight, about 0.2 mg of beta-1,3-glucan per kg of body weight ~About 15 mg of beta-1,3-glucan per kg of body weight, about 0.5 mg of beta-1,3-glucan per kg of body weight ~About 10 mg of beta-1,3-glucan per kg of body weight, about 1 mg of beta-1,3-glucan per kg of body weight ~About 10 mg of beta-1,3-glucan per kg of body weight, about 50 mg of beta-1,3-glucan per kg of body weight About 100 mg of beta-1,3-glucan per kg of body weight, about 50 mg of beta-1,3-glucan per kg of body weight to about 75 mg of beta-1,3-glucan per kg of body weight, or about 25 mg of beta-1,3-glucan per kg of body weight to about 75 mg of beta-1,3-glucan per kg of body weight. As described herein, an effective amount of the composition or pharmaceutical formulation can be administered to a subject once a day. In some embodiments, an effective amount of the composition or pharmaceutical formulation can be administered to a subject as multiple daily doses, for example, twice a day or more frequently, three times a day or more frequently, or four times a day or more frequently. In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject once or more often per week, twice or more often per week, three times or more often per week, four times or more often per week, five times or more often per week, or six times or more often per week.

[0119] An effective amount of a composition or pharmaceutical preparation containing beta-1,3-glucan derived from Euglena grown using fermentation can be administered to a subject once a day to regulate immune function. In some embodiments, an effective amount of a edible or pharmaceutical composition containing beta-1,3-glucan derived from Euglena grown using fermentation is administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a edible or pharmaceutical composition containing beta-1,3-glucan derived from Euglena grown using fermentation is administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0120] How to treat intestinal inflammation Inflammation may be regulated by beta-1,3-glucans. A recent study found that oral administration of paramylon, a beta-1,3-glucan derived from Euglena, inhibited the development of atopic dermatitis-like skin lesions in mice (J. Vet. Med. Sci. 2010, 72(2), 755-763). In humans, atopic dermatitis is a common skin disease characterized by chronic and recurrent inflammation of the skin, resulting in itching, redness, swelling, and cracking of the skin. The exact cause of atopic dermatitis is unknown, but possible factors include genetics, immune system dysfunction, and environmental exposures. The authors of the mouse study found that oral administration of paramylon reduced serum IL-4 and IFN-γ levels, as well as IL-18 and IL-12 levels in skin lesions. These results suggest that purified beta-1,3-glucan from Euglena can suppress atopic dermatitis-like skin lesions in mice by activating certain cytokine pathways. However, the exact mechanism by which beta-1,3-glucan regulates inflammation in general remains to be elucidated. In addition, beta-1,3-glucan may regulate inflammation in various body organs through various pathways.

[0121] Intestinal inflammation can affect all or part of the digestive tract. Intestinal inflammation is a type of autoimmune disease and refers to inflammation of the small intestine, large intestine, intestine, or any combination thereof. Intestinal inflammation symptoms include severe or chronic abdominal pain, diarrhea, sudden weight loss, loss of appetite, and rectal bleeding. In addition, individuals with intestinal inflammation may also have symptoms unrelated to the digestive tract, such as joint pain, skin rash, sore eyes, sore mouth, and fever. Intestinal inflammation can not only cause life-threatening complications, but also increase an individual's risk of colon cancer. Although the exact underlying physiological cause of intestinal inflammation is unknown, it is generally believed that genetic and environmental factors shift an individual's immune response, resulting in an abnormal immune response.

[0122] Inflammatory bowel disease is a group of inflammatory disorders of the large and small intestine that includes colitis and Crohn's disease. Crohn's disease affects the entire thickness of the intestinal wall, whereas colitis is limited to the epithelial lining of the colon. Specific examples of colitis include ulcerative colitis, macroscopic colitis, and rheumatoid arthritis. These include lymphocytic colitis, collagenous colitis, diversion colitis, chemical colitis, ischemic colitis, and infectious colitis. As discussed herein, the use of beta-1,3-glucan to treat intestinal inflammation also refers to the use of beta-1,3-glucan to treat inflammatory bowel disease, colitis, and Crohn's disease. Also, as discussed herein, the use of beta-1,3-glucan to treat intestinal inflammation also refers to the use of beta-1,3-glucan to prophylactically treat intestinal inflammation. Similarly, as discussed herein, the use of beta-1,3-glucan to prophylactically treat intestinal inflammation also refers to the use of beta-1,3-glucan to prophylactically treat inflammatory bowel disease, colitis, and Crohn's disease.

[0123] Ulcerative colitis is one of the major forms of inflammatory bowel disease (IBD). It is a chronic disease that causes inflammation and ulcers in the lining of the colon. While the other major form is Crohn's disease, which most often affects the end part of the small intestine (ileum) and the beginning part of the colon, ulcerative colitis is typically limited to the colon. Ulcerative colitis progresses slowly in most cases and can worsen over time. Symptoms can range from mild to severe. Most people have periods of remission that can last for weeks or years. There is no ideal animal model for studying human colitis, but among the various chemically induced colitis animal models, the dextran sulfate sodium (DSS)-induced colitis model is the most widely used, mainly due to its simplicity and similarity to human ulcerative colitis (Comp. Clin. Pathol. 2010, 19, 235-239).

[0124] DSS primarily affects the large intestine, especially the middle and distal thirds of the colon. Disruption of the intestinal epithelial barrier, resulting in the entry of luminal bacteria or bacterial antigens into the mucosa and immune activation, are major events in human colitis. A similar phenomenon occurs in DSS colitis, where sulfated polysaccharides, which are directly harmful to the colonic epithelium, cause epithelial cell damage and the resulting immune response alters the mucosal barrier function of the entire colonic epithelium. Brief administration of DSS in drinking water to mice induces a highly reproducible acute inflammation that is limited to the colon and is characterized by ulcers, loss of crypts, and infiltration of granulocytes. The DSS colitis model is also widely used to study colon cancer, which develops in association with colonic inflammation, such as occurs in patients with long-standing ulcerative colitis. DSS-induced colitis is primarily caused by the destruction of the epithelium and the activation of macrophages and neutrophils in the absence of adaptive immunity, although T cell responses can exacerbate the inflammatory reaction. In fact, strong T cell response is involved in human ulcerative colitis.Therefore, the positive therapeutic results of the composition or medicine tested in rodents using DSS model can suggest therapeutic value for the treatment of human ulcerative colitis.Importantly, if a drug has anti-inflammatory properties and provides protection from DSS-induced colitis, it may be therapeutically valuable in other IBD.

[0125] Beta-1,3-glucan derived from Euglena grown using fermentation (e.g., Euglena biomass, Euglena-derived beta-1,3-glucan, or Euglena-derived purified beta-1,3-glucan, or a combination thereof) can be administered to a subject, including a human, to treat intestinal inflammation or can be administered prophylactically to a subject at risk for intestinal inflammation. Beta-1,3-glucan derived from Euglena grown using fermentation can be administered to a subject, including a human, to treat inflammatory bowel disease. Beta-1,3-glucan derived from Euglena grown using fermentation can be administered to a subject, including a human, to treat colitis. Beta-1,3-glucan derived from Euglena grown using fermentation can be administered to a subject, including a human, to treat Crohn's disease. People at risk for intestinal inflammation include, but are not limited to, people who have previously been diagnosed with intestinal inflammation or who have one or both parents with intestinal inflammation.

[0126] As described herein, administration of beta-1,3-glucan derived from fermentatively grown Euglena provides protection from chemically induced colitis (see Examples 12-19). Such administration can be oral, for example, by administering a edible composition or oral pharmaceutical formulation, or can be intravenous, for example, by administering an intravenous pharmaceutical formulation. Alternative routes of administration, such as inhalation, are also contemplated. The oral edible composition or oral pharmaceutical formulation comprises either purified or unpurified beta-1,3-glucan derived from fermentatively grown Euglena as described herein. Intravenous pharmaceutical formulations generally comprise purified beta-1,3-glucan derived from fermentatively grown Euglena. Typically, pharmaceutical formulations suitable for inhalation comprise purified beta-1,3-glucan derived from fermentatively grown Euglena and can be administered, for example, by nasal spray. The edible composition or pharmaceutical formulation can be administered in combination with one or more of an anti-inflammatory drug, an immunosuppressant, or an antibiotic to enhance treatment of intestinal inflammation. Examples of suitable drugs include, but are not limited to, mesalazine, budesonide, cyclosporine, and infliximab.

[0127] Fermented Euglena useful for making beta-1,3-glucan for the treatment of intestinal inflammation generally rely on a growth medium to provide nutrients for growth and production of beta-1,3-glucan. Although it is contemplated that Euglena may grow with full or partial light exposure, it is generally preferred that Euglena grown using fermentation be grown heterotrophically. During fermentation, the growing Euglena efficiently produces beta-1,3-glucan. In some embodiments, the Euglena cells accumulate beta-1,3-glucan at about 30% to about 70% by weight, about 30% to about 40% by weight, about 40% to about 50% by weight, about 60% to about 70% by weight, about 40% to about 70% by weight, or about 50% to about 70% by weight. In some embodiments, the Euglena cells accumulate beta-1,3-glucan at about 50% to about 60% by weight, or about 50% to about 55% by weight.

[0128] Euglena-derived, fermentation-grown beta-1,3-glucan is administered to a subject in purified or unpurified form to treat intestinal inflammation. For example, fermentation-grown Euglena biomass can be orally administered to a subject to treat intestinal inflammation. Because fermentation-grown Euglena contains an effective amount of beta-1,3-glucan, the Euglena biomass is effective in treating intestinal inflammation without the need to purify the beta-1,3-glucan. This is due in part to the high bioavailability of beta-1,3-glucan produced by fermentation-grown Euglena. Specifically, beta-1,3-glucan from Euglena accumulates in water-insoluble granules and is not bound to cell wall components. In contrast, beta-glucan produced by yeast is tightly bound to the yeast cell wall, resulting in a lower amount of bioavailable beta-glucan. In some embodiments, dried Euglena biomass containing unpurified beta-1,3-glucan is more effective at treating intestinal inflammation in a subject using the methods described herein than purified beta-1,3-glucan.

[0129] The Euglena biomass is provided in the form of an edible composition that is orally administered to treat intestinal inflammation. The edible composition may be a food or a dietary supplement. To aid in intake, the Euglena biomass is processed into a sheet, paste, cream, powder, capsule, tablet, or other edible solid or liquid (such as a suspension) that can be consumed alone or in combination with some other food. For example, in some embodiments, the Euglena biomass is added to beverages such as nutritional shakes, nutritional bars, baked goods, or cereals. It is administered in the form of

[0130] In one exemplary embodiment, the Euglena biomass is dried to prepare it for oral administration. For example, the Euglena biomass can be dried to a moisture content of 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less by freeze-drying the biomass, heating the biomass in an oven, double drum dryer or similar drying device, or applying a vacuum to the biomass. Optionally, during or after drying the Euglena biomass, the biomass can be processed into a powder. For example, Euglena cells are dried and then ground in a shear mixer or hammer mill to produce a powder. In some embodiments, the powdered Euglena biomass is processed to an average particle size of about 1000 microns or less, about 900 microns or less, about 800 microns or less, about 700 microns or less, about 600 microns or less, about 500 microns or less, about 400 microns or less, about 300 microns or less, about 250 microns or less, about 200 microns or less, about 100 microns or less, about 50 microns or less, about 25 microns or less, about 15 microns or less, about 10 microns or less, about 5 microns or less, about 3 microns or less, about 2 microns or less, about 1 micron or less, or about 0.5 microns or less. In some embodiments, the Euglena cells are disrupted or lysed before drying the Euglena biomass. For example, the Euglena cells can be suspended in an aqueous solution and exposed to high shear forces that disrupt the cells before drying, and then dried. Optionally, the dried biomass can be further ground as described above to achieve an average particle size of about 1000 microns or less. The dried and / or powdered Euglena biomass can then be orally administered to a subject, either directly or by mixing with another edible composition, to treat the subject's intestinal inflammation.

[0131] Orally administered Euglena biomass may further include nutritional supplements in addition to beta-1,3-glucan. The nutritional supplements may be pre-existing in the Euglena biomass or may be added to the Euglena biomass. For example, additional supplements may include metals, amino acids, enzymes, probiotics, fatty acids, oligosaccharides, vitamins, and immune stimulants. Exemplary additional supplements include alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, astaxanthin, beta-carotene, glyceryl stearate ... These supplements include rotin, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake, reishi, or shiitake mushrooms, manganese, quercetin, bromelain, olive leaf, elderberry, umuka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and their derivatives, and pterostilbene. These supplements can function independently or synergistically with beta-1,3-glucan derived from fermentation-grown Euglena to treat intestinal inflammation. Additional ingredients include either Haematococcus pluvialis, astaxanthin, and colostrum.

[0132] Orally administered Euglena biomass may further include a pharma- ceutically acceptable excipient. Exemplary pharma-ceutically acceptable excipients include fillers, binders, coating agents, preservatives, lubricants, flavors, sweeteners, colorants, surfactants, solvents, buffers, chelating agents, or stabilizers. Examples of pharma-ceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, pregelatinized starch, corn starch, and potato starch. Examples of pharma-ceutically acceptable binders include polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyvinyl ether, polyvinyl alcohol ... Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, and cellulose. Examples of pharmaceutically acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, and gelatin. Examples of pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, and sodium starch glycolate. Examples of pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, and stearic acid. Examples of pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, and sorbic acid. Examples of pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, and sorbitol. Examples of pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, and tartrates.

[0133] Beta-1,3-glucan derived from Euglena grown using fermentation can also be administered to a subject in a purified form to treat intestinal inflammation. In some embodiments, the beta-1,3-glucan is purified from Euglena to be greater than 85% pure, greater than 90% pure, greater than 92% pure, greater than 94% pure, greater than 95% pure, greater than 96% pure, greater than 97% pure, greater than 98% pure, or greater than 99% pure. Generally, beta-1,3-glucan is extracted by lysing Euglena cells and isolating the beta-1,3-glucan. Euglena cells can be lysed using sonication or high pressure homogenization. Optionally, additional chemicals are included during the lysis step to aid in lysis, although such chemicals are not necessarily required. Exemplary additional chemicals included during the lysis step include surfactants (such as sodium dodecyl sulfate), enzymes, bases (such as sodium hydroxide), or acids (such as acetic acid or hydrochloric acid). Beta-1,3-glucan can be isolated from the lysed Euglena cells using filtration or gravity separation (such as gravity settling or centrifugation). To obtain a higher level of purity, the isolated beta-1,3-glucan is washed, for example, with an aqueous solution or ethanol.

[0134] Additionally, purified beta-1,3-glucan can be modified to enhance its efficacy in modulating intestinal inflammation. For example, beta-1,3-glucan can be sulfated, conjugated with a pyridinium moiety, or conjugated with a cationic moiety, such as dimethylethanolamine (DMAE).

[0135] Similar to Euglena biomass, purified beta-1,3-glucan from Euglena can be orally administered in the form of edible compositions such as foods and dietary supplements to treat intestinal inflammation. The purified beta-1,3-glucan can be provided as a paste, gel, powder, capsule, tablet, or liquid (such as a suspension) and can be administered alone or mixed with another food. For example, the purified beta-1,3-glucan can be orally administered in the form of a beverage such as a nutritional shake, a nutritional bar, a baked good, or a cereal.

[0136] The orally administered purified beta-1,3-glucan in the composition may be supplemented with one or more additional supplements, such as alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha- Linolenic acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, maitake, reishi, or shiitake mushroom extracts, It may further include manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and their derivatives, and pterostilbene.Additional ingredients include Haematococcus pluvialis, astaxanthin, and colostrum.

[0137] The purified beta-1,3-glucan can also be used in the form of a pharmaceutical preparation administered to a subject, such as a human, to treat intestinal inflammation. The pharmaceutical preparation can further include a pharma- ceutical acceptable excipient. Exemplary pharma-ceutical acceptable excipients include fillers, binders, coating agents, preservatives, lubricants, flavorings, sweeteners, colorants, surfactants, solvents, buffers, chelating agents, or stabilizers. Examples of pharma-ceutical acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltitol, pregelatinized starch, corn starch, and potato starch. Examples of pharma-ceutical acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, and cellulose. Examples of pharma- ceutically acceptable coating agents include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, and gelatin. Examples of pharma-ceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, and sodium starch glycolate. Examples of pharmacologically acceptable lubricants include polyethylene glycol, magnesium stearate, and stearic acid. Examples of pharma-ceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, and sorbic acid. Examples of pharma-ceutically acceptable sweeteners include sucrose, saccharin, aspartame, and sorbitol. Examples of pharma-ceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, and tartrates.

[0138] The pharmaceutical preparation can be administered orally to a subject or intravenously to a subject to treat intestinal inflammation. Orally administered pharmaceutical preparations can be administered as solids or liquids, such as tablets, capsules, or syrups. For intravenously administered pharmaceutical preparations, the purified beta-1,3-glucan from Euglena is suspended or dissolved in an aqueous solution, such as saline.

[0139] Administration of an effective amount of a dietary composition or pharmaceutical preparation containing beta-1,3-glucan derived from Euglena grown by fermentation can treat intestinal inflammation after the course of the administration regimen. For example, a reduction in symptoms associated with intestinal inflammation can be measured after 7 days, 14 days, 21 days, 30 days, or 60 days. Reduction in symptoms associated with intestinal inflammation includes reduced abdominal pain, reduced diarrhea, reduced weight loss, reduced anorexia, and reduced rectal bleeding. These symptoms can be measured by weight, stool consistency, stool blood, and colon length. Reduction in symptoms associated with intestinal inflammation is also measured by reduced immune cell infiltration and damage to the colon as determined by pathology. As described herein, administration of beta-1,3-glucan derived from Euglena grown by fermentation results in reduced abdominal pain, reduced diarrhea, reduced weight loss, reduced anorexia, and / or reduced rectal bleeding after 30 days of an effective amount. In some embodiments, administration of beta-1,3-glucan derived from Euglena grown using fermentation results in about a 5% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 10% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 15% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 20% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 25% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 30% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 35% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 40% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose, about a 45% or greater reduction in symptoms associated with intestinal inflammation after 30 days of an effective dose. a reduction in symptoms associated with intestinal inflammation by about 50% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 55% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 60% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 65% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 70% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 75% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 80% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 85% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 90% or more after 30 days of effective dosage, a reduction in symptoms associated with intestinal inflammation by about 95% or more after 30 days of effective dosage, or a reduction in symptoms associated with intestinal inflammation by about 99% or more after 30 days of effective dosage.

[0140] Additionally, administration of an effective amount of a dietary composition or pharmaceutical formulation containing beta-1,3-glucan derived from fermentatively grown Euglena can increase anti-inflammatory cytokine production over the course of a dosing regimen. For example, IL-10 is involved in suppressing and terminating inflammatory responses (Asadullah, K. et al., Pharm. Rev. 2003, 55, 241-269). Additional anti-inflammatory cytokines include IL-1 receptor antagonist, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10-associated inflammatory cytokines, and IL-10-specific inflammatory cytokines. -6, and IL-11, and IL-13. Specific cytokine receptors for IL-1, tumor necrosis factor-α, and IL-18 may function as proinflammatory cytokine inhibitors (Opal, SM et al. CHEST 2000, 117, 1162-1 172).

[0141] The increase in anti-inflammatory cytokine production can be measured, for example, after 7 days, 14 days, 21 days, 30 days, or 60 days. The increase in anti-inflammatory cytokine production in an individual can be measured, for example, by analysis of blood or tissue samples obtained from the individual. In some embodiments, administration of beta-1,3-glucan derived from Euglena grown using fermentation results in an effective amount of about 5% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 10% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 15% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 20% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 25% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 30% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 35% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 40% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 45% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 50% or more increase in anti-inflammatory cytokine production after 30 days, an effective amount of about 5 ... the effective amount of the compound results in an increase of about 50% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 55% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 60% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 65% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 70% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 75% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 80% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 85% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 90% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, about 95% or more in anti-inflammatory cytokine production after 30 days of administration of the compound, or about 100% or more in anti-inflammatory cytokine production after 30 days of administration of the compound.

[0142] The orally administered composition containing beta-1,3-glucan derived from fermentation-grown Euglena, the orally administered pharmaceutical preparation containing beta-1,3-glucan derived from fermentation-grown Euglena, and the intravenously administered pharmaceutical preparation containing beta-1,3-glucan derived from fermentation-grown Euglena are each administered in a dosage effective to treat intestinal inflammation. Such dosage regimens are generally understood as the amount of beta-1,3-glucan per kg body weight for each of the compositions or pharmaceutical preparations. In some embodiments, the compositions or pharmaceutical preparations contain at least about 0.1 mg of beta-1,3-glucan per kg body weight, at least about 0.25 mg of beta-1,3-glucan per kg body weight, at least about 0. ... The subject is administered an effective amount of about 1 mg or more of beta-1,3-glucan per kg of body weight, about 2 mg or more of beta-1,3-glucan per kg of body weight, about 5 mg or more of beta-1,3-glucan per kg of body weight, about 10 mg or more of beta-1,3-glucan per kg of body weight, about 15 mg or more of beta-1,3-glucan per kg of body weight, about 25 mg or more of beta-1,3-glucan per kg of body weight, about 50 mg or more of beta-1,3-glucan per kg of body weight, about 75 mg or more of beta-1,3-glucan per kg of body weight, or about 100 mg or more of beta-1,3-glucan per kg of body weight. In some embodiments, an effective amount of the composition or pharmaceutical composition is from about 0.1 mg beta-1,3-glucan per kg body weight to about 100 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 50 mg beta-1,3-glucan per kg body weight, from about 0.1 mg beta-1,3-glucan per kg body weight to about 25 mg beta-1,3-glucan per kg body weight, from about 0.2 mg beta-1,3-glucan per kg body weight to about 15 mg beta-1,3-glucan per kg body weight. beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight, about 1 mg beta-1,3-glucan per kg body weight to about 10 mg beta-1,3-glucan per kg body weight, about 50 mg beta-1,3-glucan per kg body weight to about 100 mg beta-1,3-glucan per kg body weight, about 50 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight, or about 25 mg beta-1,3-glucan per kg body weight to about 75 mg beta-1,3-glucan per kg body weight. As described herein, an effective amount of the composition or pharmaceutical formulation can be administered to a subject once a day.In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a composition or pharmaceutical formulation can be administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0143] An effective amount of a composition or pharmaceutical preparation containing beta-1,3-glucan derived from Euglena grown using fermentation can be administered to a subject once a day to treat intestinal inflammation. In some embodiments, an effective amount of a edible or pharmaceutical composition containing beta-1,3-glucan derived from Euglena grown using fermentation is administered to a subject as multiple daily doses, for example, twice or more frequently per day, three or more frequently per day, or four or more frequently per day. In some embodiments, an effective amount of a edible or pharmaceutical composition containing beta-1,3-glucan derived from Euglena grown using fermentation is administered to a subject once or more frequently per week, twice or more frequently per week, three or more frequently per week, four or more frequently per week, five or more frequently per week, or six or more frequently per week.

[0144] Complexes with trace metals In some embodiments, beta glucan can be complexed with trace metals to create complexes that can be used simultaneously to increase overall immune system activity while increasing trace metal bioavailability. Trace metals include copper, zinc, iron, cobalt, magnesium, molybdenum, manganese, lithium, chromium, nickel, vanadium, selenium, and combinations thereof. The beta glucan-trace metal complex can be the result of complexation of beta glucan with soluble inorganic trace metal salts in solution.

[0145] Beta-glucan polysaccharides are derived from paramylon granules present in dry or wet whole-cell algae suspensions. The polysaccharide can be either a bioavailable form of beta-glucan, such as beta-glucan present in grains or dried or wet whole cell yeast, or an extracted source of beta-glucan from algae, yeast, or other organisms. The polysaccharide can consist of a suspension or paste of Euglena gracilis grown heterotrophically in one or more sterile bioreactors. Euglena can also be grown in an optimal manner such that the beta-glucan portion of the algal product constitutes more than 20% of the algal biomass, measured on a dry weight basis. Examples of processes for growing and producing such products are shown in Figures 5 and 6.

[0146] Referring to FIG. 25, an embodiment of a fermentation process is shown. Algal biomass is produced in a fermenter (1) in a synthetic medium under sterile conditions. After a desired time in the fermenter (1), the fermenter fermentation broth is transferred to a centrifuge (2) which dewaters the fermentation broth to produce two process streams: wet algae meal, which contains about 75% moisture, and spent medium. The wet algae meal contains a mixture of whole algae cells, algae cell fragments, and polysaccharide granules. The wet algae meal can be a polysaccharide solution containing more than 50% beta-glucan, a non-digestible polysaccharide, by dry weight. The wet algae meal is transferred to a mixer (3), such as a mixing tank or any device that allows mixing (e.g., a ribbon blender). Optionally, the pH of the polysaccharide solution can be adjusted by the addition of an acid or base (A).

[0147] ZnSO 4 -H 2A concentrated solution of a soluble metal salt (B), such as 0, can be added to the mixer (3) and vigorously mixed with the polysaccharide solution for 1 to 120 minutes. Any water-soluble metal salt (B) can be used. For example, the metal salt (B) can be mixed with the beta-glucan such that the final product is a copper polysaccharide complex, a zinc polysaccharide complex, an iron polysaccharide complex, a cobalt polysaccharide complex, a magnesium polysaccharide complex, a manganese polysaccharide complex, and combinations thereof. Preparation of the soluble metal salt (B) solution can include heating a mixture of the metal salt (B) in water while mixing. Optionally, the mixer (3) can be heated or cooled. Optionally, the mixer (3) can be heated to a temperature necessary to pasteurize the material and inactivate enzyme activity. During mixing of the polysaccharide solution and the metal salt (B) solution, some amount of complex formation will occur between the metal ions and the polysaccharides present in the wet algae meal, such that the final product can be considered a metal polysaccharide complex.

[0148] After mixing the desired amount, the mixture is transferred to a dewatering device (4), which can be any device capable of drying a material. For example, the dewatering device (4) can be a tray dryer, belt dryer, rotary drum dryer, etc. Once the material contains less than 10% moisture, it is transferred to a mill (5) where its particle size is reduced to less than 500 μm. More preferably, the particle size is reduced to less than 250 μm. Once the material is milled, it is packaged (6) in appropriately sized containers and labeled. Optionally, the addition of the metal salt (B) solution to the wet algal food may be omitted, with the resulting product being algal food.

[0149] Referring to FIG. 26, another embodiment of the fermentation process is shown. Algal biomass is produced in a fermenter (7) with a synthetic medium under sterile conditions. Optionally, algal biomass can be produced in a culture tank under non-sterile conditions using any medium that contains only feed grade materials and is free of harmful substances (e.g., heavy metals, toxins, dangerous chemicals). After the desired time in the fermenter or culture tank (7), the fermenter fermentation broth is transferred to a mixer (8), such as a mixing tank or any device that allows mixing (e.g., a ribbon blender). The fermenter fermentation broth contains a mixture of whole algal cells, algal cell fragments, and polysaccharide granules. In the case of non-sterile culture tanks, low levels of non-algal biomass may also be present.

[0150] Optionally, the pH of the fermenter broth is adjusted by adding acidic or basic chemicals (C) to the mixer (8) to lyse the cells, thereby releasing most of the polysaccharide granules from within the cells. This can be accomplished by adding a base (e.g., NaOH) to the fermenter broth. Optionally, the fermentation broth may be mechanically processed through a high pressure homogenizer or ultrasonic cell disrupter to lyse the cells. Optionally, the fermentation broth may be adjusted to an alkaline pH and then neutralized before centrifugation. After sufficient time has passed for most, if not all, of the cells to be lysed, the resulting mixture is transferred to a centrifuge (9) and the fermentation broth is dewatered to produce two process streams: a crude polysaccharide solution (D) and a mixture of other biomass materials (E).

[0151] The crude polysaccharide solution (D) is transferred to a mixer (10), such as a mixing tank or any device that allows mixing (e.g., a ribbon blender). Optionally, the crude polysaccharide solution (D) may be washed with water or a suitable alcohol (ethanol, isopropanol) to remove non-polysaccharide material. Further washing may be performed using any chemical suitable for removing non-polysaccharide material. The pH of the crude polysaccharide solution (D) may optionally be adjusted with an acid or base (F).

[0152] ZnSO 4 H 2A concentrated solution of a soluble metal salt (G), such as O, is prepared and added to a mixing tank (10) and vigorously mixed with the polysaccharide solution for 1-120 minutes. Any water-soluble metal salt can be used such that the final product can be, for example, a copper polysaccharide complex, a zinc polysaccharide complex, an iron polysaccharide complex, a cobalt polysaccharide complex, a magnesium polysaccharide complex, or a manganese polysaccharide complex. Preparation of the soluble metal salt solution can include heating a mixture of metal salts in water while mixing. Optionally, the mixer (10) can be heated or cooled. Optionally, the mixer (10) can be heated to a temperature required to pasteurize the material and inactivate enzyme activity. As the polysaccharide solution and the metal salt solution are being mixed, some amount of complex formation will occur between the metal ions and the polysaccharides present, such that the final product can be considered a metal polysaccharide complex.

[0153] After mixing the desired amount, the mixture is transferred to a dewatering device (11), which can be any device capable of drying a material. For example, the dewatering device (11) can be a tray dryer, belt dryer, rotary drum dryer, etc. Once the material contains less than 10% moisture, it is transferred to a mill (12) where its particle size is reduced to less than 500 μm, more preferably less than 250 μm. Once the material is ground, it is placed into appropriately sized bags (13) and labeled.

[0154] The non-polysaccharide material (E), which contains the partially hydrolyzed proteins and amino acids, is transferred to a mixer 14, such as a mixing tank or any device that allows mixing (e.g., a ribbon blender). The pH of the non-polysaccharide material (E) can optionally be adjusted with an acid or base (H). ZnSO 4 H 2A concentrated solution of a soluble metal salt (I), such as O, is prepared and added to the mixer (14) and vigorously mixed with the amino acid-rich material for 1-120 minutes. Any water-soluble metal salt may be used such that the final product may be, for example, copper proteinates, zinc proteinates, iron proteinates, cobalt proteinates, magnesium proteinates, manganese proteinates, and combinations thereof. Preparation of the soluble metal salt solution may include heating a mixture of metal salts in water while mixing. Optionally, the mixer (14) may be heated or cooled. Optionally, the mixer (14) may be heated to a temperature necessary to pasteurize the material and inactivate enzyme activity. As the non-polysaccharide solution and the metal salt solution are being mixed, some amount of complex formation occurs between the metal ions and the partially hydrolyzed proteins and amino acids present, such that the final product may be considered a metalloprotein.

[0155] After the desired amount of mixing, the mixture is transferred to a dewatering device (15), which is any device capable of drying materials. For example, the dewatering device (15) can be a tray dryer, belt dryer, rotary drum dryer, multiple effect evaporator, etc. Once the material contains less than 10% moisture, the mill (16) where the particle size is reduced to less than 500 μm. More preferably, the particle size is reduced to less than 250 μm. Once the material is ground, it is placed in appropriately sized bags (17) and labeled. Optionally, the addition of the metal salt solution to each of the process streams (D, E) may be omitted, and the resulting product will be a relatively pure polysaccharide and partially hydrolyzed protein meal.

[0156] The advantages of complexing trace metals with beta-glucan include increased bioavailability of trace metals coupled with the immune system modulating properties of beta-glucan, which is indigestible in the intestine and prevents trace metals from binding to agonists until they are released, for example, in the intestine.

[0157] Furthermore, since some trace elements such as zinc are typically required in feed for optimal immune system function, combination with immune enhancing compounds such as beta-glucan may be more preferable in animal feed or vitamin premix mixtures than combination with other sources of the same trace metals that can be provided as separate products, such as amino acids or proteins. The method of the present invention demonstrates the ability of Euglena-derived beta-glucan to bind or absorb sufficiently high concentrations of zinc and other trace metals to deliver significant concentrations of the trace metals in animal feed.

[0158] Some embodiments of the metal beta glucan complex include a member selected from the group consisting of copper beta glucan complex, zinc beta glucan complex, iron beta glucan complex, cobalt beta glucan complex, magnesium beta glucan complex, molybdenum beta glucan complex, manganese beta glucan complex, and combinations thereof.

[0159] Any trace metal-containing inorganic salt may be used, and some examples of salts include those that are already commercially used as feed ingredients. Examples of such inorganic salts include, but are not limited to, metal sulfates, metal oxides, metal chlorides, hydrated metal salts, metal acetates, metal bromides, metal iodides, metal phosphates, metal selenites, and combinations thereof. The salt moieties include, but are not limited to, iron, magnesium, lithium, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, manganese, selenium, tungsten, iodine, and combinations thereof.

[0160] In some embodiments, the resulting metal polysaccharide complex contains 3% to 25% by weight metal and at least 25% by weight beta glucan. In certain cases, the polysaccharide portion of the product can be composed of at least 50% by weight beta glucan. In certain cases, the polysaccharide portion of the product can be composed of about 50% to 60% by weight beta glucan. Zinc sulfate or zinc oxide can be used as the trace metal-containing salt to make the zinc beta glucan complex, which can contain at least 1% by weight zinc on a dry weight basis and can be administered at less than 3% by weight of the total content in the animal feed.

[0161] In certain cases, the polysaccharide portion of the product can be comprised of about 30% to about 70% beta-1,3-glucan by weight, about 30% to about 40% beta-1,3-glucan by weight, about 40% to about 50% beta-1,3-glucan by weight, about 60% to about 70% beta-1,3-glucan by weight, about 40% to about 70% beta-1,3-glucan by weight, or about 50% to about 70% beta-1,3-glucan by weight. In some embodiments, the polysaccharide portion of the product can be comprised of about 50% to about 60% beta-1,3-glucan by weight, or about 50% to about 55% beta-1,3-glucan by weight.

[0162] Method for Producing a Composition Containing Euglena-Derived Beta-1,3-Glucan Euglena is a genus of green algae that naturally grows and reproduces under photosynthetic conditions and is therefore dependent on sunlight for survival. However, large-scale cultivation of photosynthetically grown Euglena is difficult and not cost-effective. Moreover, photosynthetically grown Euglena produces low amounts of beta-glucan (i.e., less than 20% of the total Euglena cell mass). Thus, Euglena useful in the methods and compositions described herein are cultured by fermentation in large fermentation tanks. Generally, fermented Euglena cultures grow heterotrophically with little or no ambient light and rely on provided nutrients to synthesize beta-1,3-glucan and other cellular components. Fermentatively grown Euglena can be cultured to higher cell densities than naturally occurring or photosynthetic Euglena cultures, thereby producing greater amounts of beta-1,3-glucan. The fermented Euglena described herein can be obtained using the methods described in U.S. Patent Application Publication No. 2013 / 0303752.

[0163] Preferably, Euglena useful in the methods disclosed herein are cultured in an environment that is controlled to keep Euglena as the predominant microorganism in that environment. It is difficult to control the growth of any one organism because many contaminating organisms can compete for the same biological resources (e.g., nutrients, micronutrients, minerals, and / or organic energy). Many of these organisms have high growth rates and can out-compete Euglena, which lacks some of the controlled growth mechanisms that favor Euglena. These growth processes can include one or more of the following methods: employing a growth medium that favors Euglena, operating at temperatures that favor Euglena, pH levels that favor Euglena, adding compounds that are toxic to competing organisms other than Euglena, and selectively filtering or isolating Euglena. Each of these methods affects the growth rate and the ability of Euglena to convert energy into beta-1,3-glucan. Generally, Euglena grown in an uncontrolled environment will not exhibit the same beneficial properties, such as high beta-1,3-glucan concentrations, fast growth rates, and efficient Euglena growth, that beta-1,3-glucan produced in a more controlled growth environment will exhibit.

[0164] To achieve cost-effective, large-scale Euglena cultures that efficiently produce beta-1,3-glucan, the organisms are typically cultivated in large aerobic fermentation tanks. The growth medium provides a carbon source, a nitrogen source, and other growth nutrients for Euglena growth and beta-1,3-glucan production. The culture medium, harvest schedule, and fermentation conditions are carefully controlled to ensure optimal beta-1,3-glucan production. In some embodiments, the production method produces large amounts of Euglena having about 30% to about 70% beta-1,3-glucan by weight, about 30% to about 40% beta-1,3-glucan by weight, about 40% to about 50% beta-1,3-glucan by weight, about 50% to about 60% beta-1,3-glucan by weight, about 60% to about 70% beta-1,3-glucan by weight, about 40% to about 70% beta-1,3-glucan by weight, or about 50% to about 70% beta-1,3-glucan by weight.

[0165] Efficient production of beta-1,3-glucan from fermentation-grown Euglena reduces the cost of beta-1,3-glucan production in several ways. First, the beta-1,3-glucan made by Euglena is not contained in the cell wall of the organism and does not require elaborate and / or expensive fractionation or extraction methods as are necessary for other organisms known to make beta-glucan. Second, Euglena organisms are relatively large and can be separated from water relatively quickly by using centrifuges, filters, or other separation devices. Third, individual Euglena cells are composed of a larger proportion of beta-1,3-glucan (as a percentage of total cell mass) compared to other organisms, resulting in easier recovery of the beta-1,3-glucan. In some embodiments, Euglena growth is supplemented by light exposure.

[0166] Fermentation and growth of Euglena Euglena-derived beta-1,3-glucan useful in the compositions and methods described herein is made by growing Euglena using fermentation. Generally, a culture medium is provided to the Euglena so that the culture grows heterotrophically. However, it is contemplated that Euglena can be cultured at least partially in light. When Euglena is fermented heterotrophically rather than grown photosynthetically, large-scale production of beta-1,3-glucan is substantially more cost-effective due in part to the large-scale establishment of photosynthetic growth conditions for the algae and the cell densities that can be achieved during fermentative growth.

[0167] Exemplary methods for growing Euglena using fermentation are described herein and in U.S. Patent Publication No. 2013 / 0303752. These efficient and cost-effective methods allow for the cultivation of Euglena useful in the methods and compositions described herein, including the production of Euglena-derived beta-1,3-glucan that can be administered as Euglena biomass or further purified. Euglena biomass can be used, for example, in edible compositions. Euglena-derived purified beta-1,3-glucan can be administered as an edible composition or as a pharmaceutical formulation that can be administered orally or intravenously.

[0168] Euglena grown using fermentation are cultured using a growth medium. The growth medium provides nutrients to the growing Euglena culture, including a carbon source, a nitrogen source, and other micronutrients. The growth medium also contains a buffer to maintain the pH of the growth culture. To prevent the growth of undesirable organisms (such as bacteria), the growth medium is sterilized before being added to the fermentation tank. The growth medium can be sterilized, for example, by using a filter, steam, autoclave, or a combination thereof. Optionally, different components of the medium are kept in separate storage locations to prevent formation of the complete growth medium and contamination of the growth medium during storage.

[0169] Fermenting Euglena depends on the carbon source present in the growth medium. Examples of carbon sources include glucose, dextrose, or other sugars, acetate, or ethanol. In some embodiments, Euglena is cultured in a growth medium that contains a carbon source at about 50 g / L or less, about 40 g / L or less, about 30 g / L or less, about 25 g / L or less, about 20 g / L or less, about 15 g / L or less, about 10 g / L or less, about 5 g / L or less, about 4 g / L or less, about 3 g / L or less, about 2 g / L or less, about 1 g / L or less, about 0.5 g / L or less, or about 0.1 g / L or less. Optionally, the growth medium is supplemented with additional carbon source during the growth process. For example, the carbon source can be added to the growth medium two or more times, three or more times to the growth medium, or four or more times to the growth medium during the growth process of the Euglena culture. The carbon source can be added semi-continuously. The carbon source can also be added continuously to the growth medium.

[0170] Growth media useful for growing Euglena by fermentation also include a nitrogen source, such as ammonium hydroxide, ammonia gas, ammonium sulfate, or glutamic acid. In some embodiments, the growth medium includes from about 0.1 g / L to about 3 g / L of the nitrogen source, from about 0.2 g / L to about 2 g / L of the nitrogen source, or from about 0.5 g / L to about 1 g / L of the nitrogen source. Preferably, the nitrogen source is ammonium hydroxide.

[0171] The growth medium further contains additional nutrients necessary for Euglena culture. For example, the growth medium may contain potassium phosphate (about 0.25 g / L to about 5 g / L potassium phosphate, about 0.5 g / L to about 4 g / L potassium phosphate, or about 1 g / L to about 3 g / L potassium phosphate, etc.), magnesium sulfate (about 0.25 g / L to about 5 g / L magnesium sulfate, about 0.5 g / L to about 4 g / L magnesium sulfate, or about 1 g / L to about 3 g / L magnesium sulfate, etc.), calcium chloride (about 0.005 g / L to about 0.5 g / L calcium chloride, about 0.01 g / L to about 0.4 g / L calcium chloride, or 0.1 g / L to about 0.25 g / L calcium chloride, or about 0.25 g / L to about 0.4 ... g / L of calcium chloride), or a trace metals stock solution containing micronutrients.

[0172] Maintaining the pH of the growth medium allows for efficient beta-1,3-glucan production, Euglena cell growth, and helps limit the growth of undesirable bacteria. A pH of about 3 to about 4 favors Euglena but provides suboptimal growth conditions for most bacteria. In some embodiments, the pH of the growth medium is about 2 to about 7, about 2 to about 6, about 3 to about 5, about 3 to about 4, or about 3 to about 3.5. To maintain the pH of the growth medium in the desired range, a buffer, such as citrate and / or citric acid, can be included in the growth medium.

[0173] The desired pH of the growth medium can be achieved or maintained in several ways. The pH of the growth medium can be manually monitored and acid or base can be manually added periodically to achieve the desired pH of the growth medium. Alternatively or additionally, the pH of the growth medium can be measured with a pH sensor connected to an automatic control system, which controls a pump, hopper, or other device that automatically adds acid or base to achieve the desired pH of the growth medium programmed into the automatic control system. In some embodiments, the metabolic processes of the Euglena are sufficient to regulate the pH of the growth medium within the desired range.

[0174] To provide sufficient oxygen to the Euglena during fermentation, the growth medium can be optionally oxygenated, for example, to about 0.5 mg / L to about 4 mg / L of oxygen, about 1 mg / L to about 3 mg / L of oxygen, or about 2 mg / L of oxygen. The cell culture medium can be oxygenated prior to being charged to the fermentation tank, or the fermenting Euglena culture can be mixed with ambient oxygen to facilitate dissolution into the growth medium.

[0175] A system for fermenting Euglena can include one or more bioreactors. Euglena cultures are grown in bioreactors to a particular cell density or a particular length of time, after which the cultures are harvested or used to inoculate a larger bioreactor. Optionally, a portion of the Euglena culture can be left in the bioreactor to inoculate fresh growth medium added to the bioreactor. Euglena grown using fermentation can be grown in a multi-stage process, which may require two or more, three or more, or four or more bioreactors, with the contents of an earlier bioreactor being transferred to and diluted in a later bioreactor. In another example of fermenting Euglena, Euglena cell cultures are grown using a fed-batch method, where fresh growth medium or specific medium components are continuously added to the bioreactor as the Euglena culture grows. Repeated batch methods can also be used to ferment Euglena, where the Euglena culture is harvested at regular intervals or continuously and replaced with fresh growth medium.

[0176] In one example, Euglena is cultured in a single bioreactor or fermentation tank. Cell growth medium is added to the bioreactor and inoculated with Euglena culture. The Euglena culture can be, for example, Euglena colonies selected from cultures or growth plates from different bioreactors. In some embodiments, the single bioreactor is about 100 liters or more, about 200 liters or more, about 300 liters or more, about 500 liters or more, about 750 liters or more, or about 1000 liters or more. In some embodiments, the single bioreactor is about 100 liters or more, about 200 liters or more, about 300 liters or more, about 500 liters or more, about 750 liters or more, about 1,000 liters or more, about 5,000 liters or more, about 10,000 liters or more, about 15,000 liters or more, or about 20,000 liters or more. Pre-harvest Euglena fermentation in bioreactor.

[0177] Euglena cultures can also be grown in a multi-stage fermentation process in which multiple bioreactors are used in sequence, with each bioreactor having a larger bioreactor volume than the bioreactor in the preceding bioreactor. The Euglena culture is first cultured to reach a certain cell density. This culture is then used to inoculate the next subsequent bioreactor.

[0178] FIG. 2 shows an overview of an exemplary embodiment for forming beta-1,3-glucan from Euglena grown by fermentation in a multi-stage process useful in the methods and compositions disclosed herein. FIG. 3 shows an exemplary bioreactor that can be used to grow Euglena by fermentation in a multi-stage process. At 200, growth medium is added to the first bioreactor stage and Euglena cells are inoculated. The first bioreactor stage can be any small vessel useful for starter cultures, such as an Erlenmeyer flask or slant medium oxygenated by surface diffusion on a rotating or orbital shaker table or magnetic stir bar. FIG. 3 shows one embodiment of the first bioreactor stage, labeled "slant medium" in this figure. This first bioreactor can be heated using a heated table. The first bioreactor can also have an environmentally controlled atmosphere. A heated shaker table, such as the New Brunswick Scientific Innova 4000 Heated Desktop Incubator, can be used to heat and vigorously agitate the surface of the growth medium. This is an example of an apparatus that combines the ability to provide sufficient movement to oxygenate the flask. After inoculation of the growth medium, the Euglena culture is cultured in the first bioreactor for a predetermined time point or until a predetermined cell density is achieved. In some embodiments, the Euglena is cultured in the first bioreactor for about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, about 60 hours or more, about 72 hours or more, about 84 hours or more, about 96 hours or more, about 108 hours or more, or about 120 hours or more. In some embodiments, Euglena are grown in the bioreactor until a cell density of about 20 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 50 grams dry weight per liter or more, about 75 grams dry weight per liter or more, about 100 grams dry weight per liter or more, about 125 grams dry weight per liter or more, about 150 grams dry weight per liter or more, about 175 grams dry weight per liter or more, or about 200 grams dry weight per liter is reached. In some embodiments, Euglena are grown in the bioreactor until a cell density of about 10 grams dry weight per liter to about 200 grams dry weight per liter, about 15 grams dry weight per liter to about 150 grams dry weight per liter, about 20 grams dry weight per liter to about 100 grams dry weight per liter, about 20 grams dry weight per liter to about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter is reached. If necessary or desired, the first bioreactor is replenished with fresh growth medium while the Euglena are growing.

[0179] In step 205, the contents of the first bioreactor or a portion of the contents of the first bioreactor is transferred to a second bioreactor. Optionally, in step 210, the contents of the first bioreactor or a portion of the contents of the first bioreactor is harvested for further processing. A portion of the contents of the first bioreactor can be retained as inoculum for a different culture. Optionally, the contents of the first bioreactor stage are filtered at 200 or the Euglena are otherwise concentrated before the Euglena are transferred to the second stage at 210.

[0180] The Euglena culture from the first bioreactor is used to inoculate the growth medium of the second bioreactor to initiate further cultivation of Euglena. Generally, the second bioreactor is larger than the first bioreactor. FIG. FIG. 1 shows an example of one of the Euglena stages, labeled "Flask" in this diagram. The contents of the first bioreactor are diluted in the second bioreactor such that the concentration of Euglena in the second bioreactor after dilution is from about 0.1 grams dry weight per liter to about 10 grams dry weight per liter. The second bioreactor can be up to about 10 times larger than the first bioreactor stage, up to about 20 times larger than the first bioreactor stage, up to about 30 times larger than the first bioreactor stage, up to about 50 times larger than the first bioreactor stage, up to about 75 times larger than the first bioreactor stage, or up to about 100 times larger than the first bioreactor stage. In some embodiments, Euglena is grown by fermentation in a second bioreactor for about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, about 60 hours or more, about 72 hours or more, about 84 hours or more, about 96 hours or more, about 108 hours or more, or about 120 hours or more. In some embodiments, Euglena is fermented in the bioreactor until a cell density of about 20 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 50 grams dry weight per liter or more, about 75 grams dry weight per liter or more, about 100 grams dry weight per liter or more, about 125 grams dry weight per liter or more, about 150 grams dry weight per liter or more, about 175 grams dry weight per liter or more, or about 200 grams dry weight per liter is reached. In some embodiments, Euglena is fermented in the bioreactor until a cell density of about 10 grams dry weight per liter to about 200 grams dry weight per liter, about 15 grams dry weight per liter to about 150 grams dry weight per liter, about 20 grams dry weight per liter to about 100 grams dry weight per liter, about 20 grams dry weight per liter to about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter is reached.If necessary or desired, the second bioreactor is replenished with fresh growth medium while the Euglena are growing.

[0181] As further illustrated in FIG. 2, the contents or a portion of the contents of the second bioreactor are transferred to a third bioreactor in step 215. FIG. 3 shows one embodiment of a first bioreactor stage, labeled in this figure as a "Seed Fermenter." Optionally, the contents or a portion of the second bioreactor are harvested from the second bioreactor in step 220. Generally, the third bioreactor is about 1 to about 100 times larger than the second bioreactor and can be about 100 liters or more, about 250 liters or more, about 500 liters or more, about 1000 liters or more, about 2500 liters or more, about 5000 liters or more, about 7500 liters or more, about 10,000 liters or more, about 15,000 liters or more, about 25,000 liters or more, about 50,000 liters or more, or about 100,000 liters or more. In some embodiments, the Euglena is grown by fermentation in the third bioreactor for about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, about 60 hours or more, about 72 hours or more, about 84 hours or more, about 96 hours or more, about 108 hours or more, or about 120 hours or more to reach a cell density of about 20 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 50 grams dry weight per liter or more, about 75 grams dry weight per liter or more, about 100 grams dry weight per liter or more, about 125 grams dry weight per liter or more, about 150 grams dry weight per liter or more, about 175 grams dry weight per liter or more, or about 200 grams dry weight per liter. In some embodiments, the Euglena is cultured in a third bioreactor at a dry weight of about 10 grams per liter to about 200 grams per liter, at a dry weight of about 15 grams per liter to about 150 grams per liter, at a dry weight of about 20 grams per liter to about 100 grams per liter, at a dry weight of about 20 grams per liter to about 2 ... The third bioreactor is fermented until a cell density of about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter is reached. If necessary or desired, the third bioreactor is replenished with fresh growth medium while the Euglena are growing.

[0182] As further illustrated in Figure 2, the contents or a portion of the contents of the third bioreactor are transferred to a fourth bioreactor in step 225. Figure 3 shows one embodiment of the fourth bioreactor stage, labeled "Production Fermenter" in this figure. Optionally, the contents or a portion of the third bioreactor are harvested from the third bioreactor in step 230. Typically, the fourth bioreactor has a volume that is about 1 to about 100 times larger than the third bioreactor stage, with a volume of at least about 100 liters, at least about 250 liters, at least about 500 liters, at least about 1000 liters, at least about 2500 liters, at least about 5000 liters, at least about 7500 liters, at least about 10,000 liters, at least about 15,000 liters, at least about 25,000 liters, at least about 50,000 liters, at least about 100,000 liters, at least about 200,000 liters, at least about 400,000 liters, at least about 600,000 liters, at least about 800,000 liters, or at least about 1,000,000 liters. In some embodiments, the Euglena is grown by fermentation in the fourth bioreactor for about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, about 60 hours or more, about 72 hours or more, about 84 hours or more, about 96 hours or more, about 108 hours or more, or about 120 hours or more to reach a cell density of about 20 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 50 grams dry weight per liter or more, about 75 grams dry weight per liter or more, about 100 grams dry weight per liter or more, about 125 grams dry weight per liter or more, about 150 grams dry weight per liter or more, about 175 grams dry weight per liter or more, or about 200 grams dry weight per liter.In some embodiments, the Euglena are fermented until a cell density of about 10 grams dry weight per liter to about 200 grams dry weight per liter, about 15 grams dry weight per liter to about 150 grams dry weight per liter, about 20 grams dry weight per liter to about 100 grams dry weight per liter, about 20 grams dry weight per liter to about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter is reached. The fourth bioreactor can be replenished with fresh growth medium while the Euglena are growing. While FIG. 2 illustrates that the contents of the fourth bioreactor are harvested in step 240, it is further contemplated that a subsequent bioreactor may be used. FIG. 3 illustrates one method of harvesting using a centrifuge.

[0183] Repeated batch methods can also be used to cultivate Euglena using fermentation. In the repeated batch method, a portion of the Euglena grown in a bioreactor is harvested and / or transferred to a different bioreactor, and a portion of the Euglena remains in the bioreactor as inoculum for subsequent Euglena growth. The inoculum is then diluted by adding fresh growth medium to the bioreactor. FIG. 4A illustrates one exemplary embodiment of Euglena grown using fermentation by the repeated batch method. In step 400, growth medium is added to the bioreactor and Euglena cells are inoculated. The Euglena is grown in a bioreactor by fermentation such that the Euglena cell density is greater than or equal to about 20 grams dry weight per liter, greater than or equal to about 30 grams dry weight per liter, greater than or equal to about 40 grams dry weight per liter, greater than or equal to about 50 grams dry weight per liter, greater than or equal to about 75 grams dry weight per liter, greater than or equal to about 100 grams dry weight per liter, greater than or equal to about 125 grams dry weight per liter, greater than or equal to about 150 grams dry weight per liter, greater than or equal to about 175 grams dry weight per liter, or greater than or equal to about 200 grams dry weight per liter. The Euglena can be grown until it reaches a predetermined cell density above the dry weight. In some embodiments, the Euglena is fermented in the bioreactor until it reaches a cell density of about 10 grams dry weight per liter to about 200 grams dry weight per liter, about 15 grams dry weight per liter to about 150 grams dry weight per liter, about 20 grams dry weight per liter to about 100 grams dry weight per liter, about 20 grams dry weight per liter to about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter. Growth of the Euglena can continue for about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, or about 60 hours or more to reach the desired cell density.

[0184] In step 410, a first portion of the bioreactor contents is harvested. A second portion of the bioreactor contents remains in the bioreactor to inoculate the next culture. Thus, after the first portion of the bioreactor contents is harvested, fresh medium is added to the bioreactor in 420 to dilute the cell density. The process can continue until a desired amount of Euglena production is reached or until contaminating organisms eventually occupy a significant portion of the bioreactor, providing reason to empty and sanitize the bioreactor. An example of repeated batch harvest of Euglena growth versus time is shown in FIG. 4B. As seen in FIG. 4B, once Euglena ferment to a predetermined cell density, a portion of the contents is harvested and the remainder is diluted with fresh medium to inoculate a new culture.

[0185] Fed-batch methods, such as constant flow fed-batch methods, can also be used to culture Euglena using fermentation. In fed-batch methods, Euglena cultures are grown in an inoculated growth medium until a desired cell density is reached. This initial growth phase is generally referred to as the "batch phase." The batch phase can be about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, or about 60 hours or more. During that time, the Euglena culture reaches a cell density of about 20 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 50 grams dry weight per liter or more, about 75 grams dry weight per liter or more, about 100 grams dry weight per liter or more, about 125 grams dry weight per liter or more, about 150 grams dry weight per liter or more, about 175 grams dry weight per liter or more, or about 200 grams dry weight per liter or more. For example, Euglena can grow in a bioreactor during the batch phase until it reaches a cell density of about 10 grams dry weight per liter to about 200 grams dry weight per liter, about 15 grams dry weight per liter to about 150 grams dry weight per liter, about 20 grams dry weight per liter to about 100 grams dry weight per liter, about 20 grams dry weight per liter to about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter. After the batch phase is completed, the Euglena cells are continuously harvested and replaced with fresh growth medium. This harvesting and replacement process is referred to as the continuous phase. During the continuous phase, nutrient levels, pH, dissolved oxygen, or other parameters are monitored, for example, using one or more sensors.

[0186] In one embodiment, the repeated batch process is carried out for up to 30 days. In another embodiment, the repeated batch process is carried out for up to 60 days. In yet another embodiment, the repeated batch process is carried out for up to 90 days. In yet another embodiment, the repeated batch process is carried out for up to 120 days.

[0187] Any one of the bioreactors (whether it is the first bioreactor, the second bioreactor, the third bioreactor, the fourth bioreactor, or a subsequent bioreactor in a multi-stage process, the bioreactor in a repeated batch process, or the bioreactor in a fed-batch process) is used to contain the Euglena during growth. The bioreactor can be a metal or glass fermentation tank of any of the above types. Figures 5 and 6 illustrate exemplary bioreactors useful for growing Euglena by fermentation.

[0188] Typically, the bioreactor is closed to the atmosphere during operation, except for one or more controlled vents, one or more inputs for aeration or oxygen supply, or one or more inputs for supplying additional growth medium. This allows for better control of fermentation conditions and limits contamination. The inputs for supplying growth medium can include a sterilizing filter, e.g., a filter with a pore size of less than 0.2 microns, to prevent unwanted non-euglena microorganisms from entering the chamber. To further limit contamination, any one of the bioreactors can be cleaned or sanitized between batches. Cleaning or sanitizing the bioreactor can include steam, heat, or a sanitizing agent such as ethanol, bleach, or other chemicals.

[0189] To help stimulate Euglena growth and beta-1,3-glucan production during fermentation, the growth medium can be oxygenated or mixed. For example, additional oxygen can be provided to the Euglena cultures by aerating the growth medium or by receiving additional liquid in which pressurized oxygen has been pre-dissolved in the liquid. The growth medium can be aerated, for example, by supplying oxygen-enriched air to the growth medium during growth or by mixing the culture with one or more mixing devices to agitate the culture.

[0190] One or more of the bioreactors used to ferment Euglena can include an airlift or bubble column system to mix the Euglena or aerate the growth medium. Figure 6 shows an example of a bioreactor for growing Euglena using airlift fermentation. Airlift reactors can provide sufficient mixing and aeration with less shear stress on the growing cells.

[0191] One or more bioreactors can include a mechanical agitator with a mixing blade to mix or aerate the Euglena during growth. Figure 5 shows an exemplary bioreactor that includes a mechanical agitator, e.g., a low shear mixing blade, such as a marine blade or a fixed angle blade. The low shear mixing blade limits damage to the Euglena cells during growth.

[0192] The Euglena are harvested or transferred after growth in the last bioreactor, whether it is the first bioreactor, the second bioreactor, the third bioreactor, the fourth bioreactor, or a subsequent bioreactor. In some embodiments, the Euglena are harvested or transferred to a new bioreactor after fermenting in the bioreactor for about 12 hours or more, about 24 hours or more, about 36 hours or more, about 48 hours or more, about 60 hours or more, about 72 hours or more, about 84 hours or more, about 96 hours or more, about 108 hours or more, or about 120 hours or more. For example, the Euglena can be cultured for about 12 to about 120 hours before being harvested or transferred to a new bioreactor, e.g., about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours to about 60 hours before being harvested or transferred to a new bioreactor. In harvesting the Euglena cell culture, the entire contents of the final bioreactor may be taken, or a portion of the cell culture may be set aside, which can be used as inoculum in a different bioreactor or in the same bioreactor.

[0193] In some embodiments, the method of growing Euglena using fermentation comprises growing Euglena at a concentration of about 20 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 50 grams dry weight per liter or more, about 75 grams dry weight per liter or more, about 80 grams dry weight per liter or more, about 90 grams dry weight per liter or more, about 100 grams dry weight per liter or more, about 150 grams dry weight per liter or more, about 160 grams dry weight per liter or more, about 170 grams dry weight per liter or more, about 180 grams dry weight per liter or more, about 190 grams dry weight per liter or more, about 20 grams dry weight per liter or more, about 21 grams dry weight per liter or more, about 22 grams dry weight per liter or more, about 23 grams dry weight per liter or more, about 24 grams dry weight per liter or more, about 25 grams dry weight per liter or more, about 26 grams dry weight per liter or more, about 27 grams dry weight per liter or more, about 28 grams dry weight per liter or more, about 29 grams dry weight per liter or more, about 30 grams dry weight per liter or more, about 31 grams dry weight per liter or more, about 32 grams dry weight per liter or more, about 33 grams dry weight per liter or more, about 34 grams dry weight per liter or more, about 35 grams dry weight per liter or more, about 36 grams dry weight per liter or more, about 37 grams dry weight per liter or more, about 38 grams dry weight per liter or more, about 39 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 40 grams dry weight per liter or more, about 40 grams dry weight per liter The method produces Euglena at a cell density of 100 grams dry weight or more, about 125 grams dry weight or more, about 150 grams dry weight or more, about 175 grams dry weight or more, or about 200 grams dry weight or more. In other embodiments, the method for growing Euglena produces Euglena at a cell density of about 10 grams dry weight per liter to about 200 grams dry weight per liter, about 15 grams dry weight per liter to about 150 grams dry weight per liter, about 20 grams dry weight per liter to about 100 grams dry weight per liter, about 20 grams dry weight per liter to about 60 grams dry weight per liter, or about 40 grams dry weight per liter to about 60 grams dry weight per liter. In yet other embodiments, the Euglena has about 30% to about 70% beta-1,3-glucan by weight, such as about 30% to about 40% beta-1,3-glucan, about 40% to about 50% beta-1,3-glucan, about 50% to about 60% beta-1,3-glucan, or about 60% to about 70% beta-1,3-glucan, about 40% to about 70% beta-1,3-glucan, or about 50% to about 70% beta-1,3-glucan by weight.

[0194] After the Euglena are harvested from the bioreactor, the Euglena biomass is separated from the growth medium by, for example, centrifugation, tangential flow filtration, filter press, belt press, or other solid-liquid separation device. Typically, the Euglena biomass is separated from the growth medium until the composition reaches about 20% by weight or more solids, about 25% by weight or more solids, about 30% by weight or more solids, about 35% by weight or more solids, or about 40% by weight or more solids.

[0195] The harvested Euglena biomass can then be dried before being further processed. The Euglena biomass can be dried using a belt dryer, spray dryer dryer, drum dryer, oven, or by spreading the biomass over a large surface area and utilizing evaporative drying. Heat or vacuum can be applied to aid the drying process. The Euglena biomass can also be freeze-dried to produce a dry biomass. The Euglena biomass can be dried until it reaches a moisture content of 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less.

[0196] The dried Euglena biomass can be administered as an edible composition for reducing serum cholesterol levels or serum triglyceride levels or for regulating immune function in a subject. The Euglena biomass can also be further processed to purify Euglena-derived beta-1,3-glucan for use in an edible composition of a pharmaceutical formulation administered to reduce serum cholesterol levels or serum triglyceride levels or for regulating immune function in a subject.

[0197] The dried Euglena biomass can be administered as an edible composition to treat intestinal inflammation in a subject, and the Euglena biomass can be further processed to purify Euglena-derived beta-1,3-glucan for use in an edible composition of a pharmaceutical formulation administered to treat intestinal inflammation in a subject.

[0198] Purification of beta-1,3-glucan derived from fermentatively grown Euglena Beta-glucan can be extracted from Euglena by liquid / solid separation, physical separation methods, or other methods. A significant portion of the beta-1,3-glucan produced by Euglena is in the form of paramylon. Paramylon is generally present in Euglena in the form of water-insoluble granules of about 0.5 to about 2 microns, and is located within the Euglena cells. Thus, beta-1,3-glucan is generally extracted by lysing the Euglena cells and extracting the beta-1,3-glucan. The beta-1,3-glucan is purified by isolating the beta-1,3-glucan from the residual biomass. Optionally, the beta-1,3-glucan is purified using methanol. Preferably, the beta-1,3-glucan is purified without the use of chloroform.

[0199] Beta-1,3-glucan from Euglena is extracted by lysing the cells and isolating the beta-1,3-glucan. Euglena cells can be lysed using sonication or high pressure homogenization. Optionally, a lysis chemical is included in the lysis step. However, it is possible to lyse Euglena cells without adding a lysis chemical. Exemplary lysis chemicals that can be included in the lysis step include detergents (such as sodium dodecyl sulfate), enzymes, bases (such as sodium hydroxide), or acids (such as acetic acid or hydrochloric acid). After lysing the Euglena cells, the beta-1,3-glucan is isolated using filtration or gravity separation (such as gravity sedimentation or centrifugation). To obtain higher purity, the isolated beta-1,3-glucan can then be washed, for example, with an aqueous solution or ethanol.

[0200] After purification of Euglena-derived beta-1,3-glucan, further processing steps can modify the purified beta-1,3-glucan. Modified beta-1,3-glucan exhibits increased binding affinity to immune system receptors such as Dectin-1, a protein that has been identified as a beta-glucan receptor. For example, sulfated polysaccharides have been demonstrated to exhibit anti-HIV activity (e.g., U.S. Patent No. 5,861,383). In one exemplary method for preparing sulfated beta-1,3-glucan, purified beta-1,3-glucan is dissolved in dimethyl sulfoxide and mixed with a mixture of dry pyridine and chlorosulfonic acid. The mixture is then heated and the supernatant is decanted. Distilled water or methanol can then be added to the supernatant to precipitate the beta-1,3-glucan pyridinium sulfate, which can then be collected by filtration. Alternatively, sodium chloride can be added to the supernatant to raise the pH to 9 and sodium beta-1,3-glucan sulfate can be precipitated in an acetone solution (see Sakagami et al, In vivo 3:243-248 (1989)).

[0201] Beta-1,3-glucan derived from Euglena can also be modified to be cationic. Cationic beta-glucan can be more biologically active as an immunomodulator because it has increased binding affinity with beta-glucan receptors such as Dectin-1 and Complement Receptor 3 (see Sakagami et al, Antiviral Research, 21:1-14 (1993)). Beta-1,3-glucan derived from Euglena grown by fermentation can be modified with dimethylethanolamine (DMAE) to make cationic beta-1,3-glucan. One exemplary method of producing DMAE beta-1,3-glucan includes dissolving Euglena-derived beta-1,3-glucan in a basic solution (such as a solution containing NaOH) and adding DMAE chloride (as a solution or dry powder). The resulting reaction produces DMAE beta-1,3-glucan.

[0202] Exemplary embodiments Embodiment I-1. The present disclosure provides a method for regulating immune function in a human comprising orally administering to a human in need thereof an effective amount of a composition comprising beta-1,3-glucan derived from Euglena grown using fermentation.

[0203] Embodiment I-2. The present disclosure provides the method of embodiment I-1, wherein the effective amount of the composition is from 0.1 mg of beta-1,3-glucan / kg body weight to 50 mg of beta-1,3-glucan / kg body weight.

[0204] Embodiment I-3. The present disclosure provides the method of embodiment I-1, wherein administration of the composition modulates an autoimmune response, blood sugar levels, infection, or inflammation.

[0205] Embodiment I-4. The present disclosure provides the method of embodiment I-3, wherein the inflammation is associated with allergy.

[0206] Embodiment I-5. The present disclosure provides the method of embodiment I-3, wherein the autoimmune response is associated with diabetes.

[0207] Embodiment I-6. The present disclosure provides the method of embodiment I-3, wherein the infection is a bacterial infection, a fungal infection, or a viral infection.

[0208] Embodiment I-7. The present disclosure provides the method of embodiment I-1, wherein the Euglena is cultured heterotrophically.

[0209] Embodiment I-8. The present disclosure provides the method of embodiment I-1, wherein the beta-1,3-glucan comprises paramylon.

[0210] Embodiment I-9. The present disclosure provides the method of embodiment I-1, wherein the beta-1,3-glucan does not contain beta-1,6-glycosidic bonds.

[0211] Embodiment I-10. The present disclosure provides the method of embodiment I-1, wherein the beta-1,3-glucan is purified from Euglena.

[0212] Embodiment I-11. The present disclosure provides the method of embodiment I-1, wherein the composition comprises Euglena biomass, and the Euglena biomass comprises said beta-1,3-glucan.

[0213] Embodiment I-12. The present disclosure provides the method of embodiment I-11, wherein the Euglena biomass is dried.

[0214] Embodiment I-13. The present disclosure provides the method of embodiment I-12, wherein the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0215] Embodiment I-14. The present disclosure provides the method of embodiment I-1, wherein the composition is administered as a single dose daily.

[0216] Embodiment I-15. The disclosure provides the method of embodiment I-1, wherein the composition is administered as multiple divided doses daily.

[0217] EMBODIMENT I-16. The present disclosure provides the method of embodiment I-1, wherein the composition is selected from the group consisting of alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linole ... The composition further comprises additional ingredients selected from the group consisting of: hydroxypropyl cellulose, ...

[0218] Embodiment I-17. The disclosure provides the method of embodiment I-1, wherein the composition is administered as a solid.

[0219] Embodiment I-18. The disclosure provides the method of embodiment I-1, wherein the composition is administered as a suspension.

[0220] Embodiment I-19. The present disclosure provides a method for regulating immune function in a human comprising orally administering to said human having or at risk for having high cholesterol an effective amount of a composition comprising beta-1,3-glucan derived from Euglena grown using fermentation.

[0221] Embodiment I-20. The present disclosure provides the method of embodiment I-19, wherein administering the composition reduces the level of cholesterol in a human.

[0222] Embodiment I-21. The present disclosure provides the method of embodiment I-19, wherein the effective amount of the composition is from 0.1 mg of beta-1,3-glucan / kg body weight to 50 mg of beta-1,3-glucan / kg body weight.

[0223] Embodiment I-22. The present disclosure provides the method of embodiment I-19, wherein the Euglena is grown heterotrophically.

[0224] Embodiment I-23. The present disclosure provides the method of embodiment I-19, wherein the beta-1,3-glucan comprises paramylon.

[0225] Embodiment I-24. The present disclosure provides the method of embodiment I-19, wherein the beta-1,3-glucan does not contain beta-1,6-glycosidic bonds.

[0226] Embodiment I-25. The present disclosure provides the method of embodiment I-19, wherein the beta-1,3-glucan is purified from Euglena.

[0227] Embodiment I-26. The present disclosure provides the method of embodiment I-19, wherein the composition comprises Euglena biomass, and the Euglena biomass comprises beta-1,3-glucan.

[0228] Embodiment I-27. The present disclosure provides the method of embodiment I-26, wherein the Euglena biomass is dried.

[0229] Embodiment I-28. The present disclosure provides the method of embodiment I-27, wherein the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0230] Embodiment I-29. The present disclosure provides the method of embodiment I-19, wherein the composition is administered as a single dose daily.

[0231] Embodiment I-30. The present disclosure provides the method of embodiment I-19, wherein the composition is administered as multiple divided doses daily.

[0232] Embodiment I-31. The present disclosure provides the method of embodiment I-19, wherein the composition comprises alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava, turmeric, spinach, thyme, thyme extract, thyme glycerol ... Lulina, Chlorella, Stevia, Calcium D-Glucarate, Coenzyme Q10, Peptides, Dimethylglycine, Docosahexaenoic Acid, Eicosapentaenoic Acid, Alpha-Rhino The composition further comprises additional ingredients selected from the group consisting of: oleic acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake, reishi, or shiitake mushrooms, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof.

[0233] Embodiment I-32. The disclosure provides the method of embodiment I-19, wherein the composition is administered in combination with a statin, nicotinic acid, a bile acid resin, a fibric acid derivative, or a cholesterol absorption inhibitor.

[0234] Embodiment I-33. The present disclosure provides the method of embodiment I-19, wherein the composition is administered as a solid.

[0235] Embodiment I-34. The present disclosure provides the method of embodiment I-19, wherein the composition is administered as a suspension.

[0236] Embodiment II-1. The present disclosure provides a method for enhancing immune function in a human having intestinal inflammation, comprising orally administering to the human an effective amount of a composition comprising beta-1,3-glucan derived from Euglena grown using fermentation.

[0237] Embodiment II-2. The present disclosure provides a method of embodiment II-1, wherein the effective amount of the composition is from 0.1 mg of beta-1,3-glucan / kg body weight to 100 mg of beta-1,3-glucan / kg body weight.

[0238] Embodiment II-3. The present disclosure provides the method of embodiment II-1, wherein the intestinal inflammation is inflammatory bowel disease.

[0239] Embodiment II-4. The present disclosure provides the method of embodiment II-1, wherein the intestinal inflammation is colitis.

[0240] Embodiment II-5. The present disclosure provides the method of embodiment II-1, wherein the intestinal inflammation is Crohn's disease.

[0241] Embodiment II-6. The present disclosure provides the method of embodiment II-1, wherein the Euglena is grown heterotrophically.

[0242] Embodiment II-7. The present disclosure provides the method of embodiment II-1, wherein the beta-1,3-glucan comprises paramylon.

[0243] Embodiment II-8. The present disclosure provides the method of embodiment II-1, wherein the beta-1,3-glucan does not contain beta-(1,6)-glycosidic bonds.

[0244] Embodiment II-9. The present disclosure provides the method of embodiment II-1, wherein the beta-1,3-glucan is purified from Euglena.

[0245] Embodiment II-10. The present disclosure provides the method of embodiment II-1, wherein the composition The product includes Euglena biomass, and the Euglena biomass includes the beta-1,3-glucan.

[0246] Embodiment II-11. The present disclosure provides the method of embodiment II-10, wherein the Euglena biomass is dried.

[0247] Embodiment II-12. The present disclosure provides the method of embodiment II-11, wherein the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0248] Embodiment II-13. The present disclosure provides a method of embodiment II-1, wherein the composition is administered as a single dose daily.

[0249] Embodiment II-14. The present disclosure provides a method of embodiment II-1, wherein the composition is administered as multiple divided doses daily.

[0250] EMBODIMENT II-15. The present disclosure provides the method of embodiment II-1, wherein the composition comprises alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-lysine, glyceryl stearate ... The composition further comprises additional ingredients selected from the group consisting of norenic acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake, reishi, or shiitake mushrooms, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof.

[0251] Embodiment II-16. The present disclosure provides a method of embodiment II-1, wherein the composition is administered as a solid.

[0252] Embodiment II-17. The present disclosure provides the method of embodiment II-1, wherein the composition is administered as a suspension.

[0253] Embodiment II-18. The present disclosure provides a method of embodiment II-1, wherein administering the composition increases anti-inflammatory cytokine production.

[0254] Embodiment II-19. The present disclosure provides the method of embodiment II-1, wherein the composition is administered in combination with an anti-inflammatory drug, an immunosuppressant, or an antibiotic.

[0255] Embodiment III-1. The present disclosure provides a method for treating a disease in a human having a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer, the method comprising orally administering to the human an effective amount of a composition comprising fermentatively grown Euglena beta-1,3-glucan.

[0256] Embodiment III-2. The present disclosure provides a method for treating a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease in a human having said disease, the method comprising orally administering to the human an effective amount of a composition comprising Euglena beta-1,3-glucan grown using fermentation.

[0257] Embodiment III-3. The present disclosure provides the method of any one of embodiments III-1 to III-2, wherein the disease is hyperlipidemia.

[0258] Embodiment III-4. The present disclosure provides a method of embodiment III-3, wherein administering the composition reduces the level of cholesterol in a human.

[0259] Embodiment III-5. The present disclosure provides a method of any one of embodiments III-3 to III-4, wherein the effective amount of the composition comprises 0.1 mg of beta-1,3-glucan / kg body weight to 100 mg of beta-1,3-glucan / kg body weight.

[0260] Embodiment III-6. The present disclosure provides a method of any one of embodiments III-3 to III-4, wherein the effective amount of the composition comprises 0.1 mg of beta-1,3-glucan / kg body weight to 50 mg of beta-1,3-glucan / kg body weight.

[0261] Embodiment III-7. The present disclosure provides a method of any one of embodiments III-3 to III-6, wherein the composition is administered in combination with a statin, nicotinic acid, bile acid resin, fibric acid derivative, or cholesterol absorption inhibitor.

[0262] Embodiment III-8. The present disclosure provides the method of embodiment III-1, wherein the disease is selected from the group consisting of inflammatory bowel disease, colitis, Crohn's disease, and colon cancer.

[0263] Embodiment III-9. The present disclosure provides the method of embodiment III-1, wherein the disease is selected from the group consisting of inflammatory bowel disease, colitis, and Crohn's disease.

[0264] Embodiment III-10. The present disclosure provides the method of any one of embodiments III-8 to III-9, wherein the disease is colitis.

[0265] Embodiment III-11. The present disclosure provides the method of any one of embodiments III-8 to III-9, wherein the disease is inflammatory bowel disease.

[0266] Embodiment III-12. The present disclosure provides the method of any one of embodiments III-8 to III-9, wherein the disease is Crohn's disease.

[0267] Embodiment III-13. The present disclosure provides the method of embodiment III-8, wherein the disease is colon cancer.

[0268] Embodiment III-14. The present disclosure provides a method of any one of embodiments III-8 to III-13, wherein the effective amount of the composition comprises 0.1 mg of beta-1,3-glucan / kg body weight to 100 mg of beta-1,3-glucan / kg body weight.

[0269] Embodiment III-15. The present disclosure provides the method of any one of embodiments III-8 to III-14, wherein administering the composition increases anti-inflammatory cytokine production.

[0270] Embodiment III-16. The present disclosure provides the method of any one of embodiments III-8 to III-15, wherein the composition is administered in combination with an anti-inflammatory drug, an immunosuppressant, or an antibiotic.

[0271] Embodiment III-17. The present disclosure relates to any one of embodiments III-1 to III-16. The method includes the steps of:

[0272] Embodiment III-18. The present disclosure provides a method of any one of embodiments III-1 to III-17, wherein the Euglena is grown heterotrophically.

[0273] Embodiment III-19. The present disclosure provides the method of any one of embodiments III-1 to III-18, wherein the beta-1,3-glucan is present in the form of paramylon.

[0274] Embodiment III-20. The present disclosure provides the method of any one of embodiments III-1 to III-19, wherein the beta-1,3-glucan does not contain beta-1,6-glycosidic bonds.

[0275] Embodiment III-21. The present disclosure provides the method of any one of embodiments III-1 to III-20, wherein the beta-1,3-glucan is purified from Euglena.

[0276] Embodiment III-22. The present disclosure provides the method of any one of embodiments III-1 to III-20, wherein the composition comprises Euglena biomass, and the Euglena biomass comprises said beta-1,3-glucan.

[0277] Embodiment III-23. The present disclosure provides the method of embodiment III-22, wherein the Euglena biomass is dried to a moisture content of 40% or less.

[0278] Embodiment III-24. The present disclosure provides the method of embodiment III-23, wherein the Euglena biomass is processed to have an average particle size of 1000 microns or less.

[0279] Embodiment III-25. The present disclosure provides a method of any one of embodiments III-1 to III-24, wherein the composition is administered as a single dose daily.

[0280] Embodiment III-26. The present disclosure provides the method of any one of embodiments III-1 to III-24, wherein the composition is administered as multiple divided doses per day.

[0281] EMBODIMENT III-27. The present disclosure provides the method of any one of embodiments III-1 to III-26, wherein the composition is selected from the group consisting of alpha tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid, astaxanthin, beta carotene, lutein ... The composition further comprises additional ingredients selected from the group consisting of: lactic acid bacteria probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of maitake, reishi, or shiitake mushrooms, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof.

[0282] Embodiment III-28. The present disclosure provides the method of embodiment III-27, wherein the additional component is zinc.

[0283] Embodiment III-29. The present disclosure provides the method of any one of embodiments III-1 to III-27, wherein the composition further comprises a metal.

[0284] Embodiment III-30. The present disclosure provides the method of embodiment III-29, wherein the metal comprises a member selected from the group consisting of iron, magnesium, lithium, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, manganese, selenium, and combinations thereof.

[0285] Embodiment III-31. The present disclosure provides the method of embodiment III-30, wherein the beta-(1,3)-glucan and the metal form a complex.

[0286] Embodiment III-32. The present disclosure provides the method of embodiment III-31, wherein the complex comprises a zinc beta-(1,3)-glucan complex.

[0287] Embodiment III-33. The present disclosure provides the method of any one of embodiments III-1 to III-26, wherein the composition further comprises an additional component selected from the group consisting of Haematococcus pluvialis, astaxanthin, and colostrum.

[0288] Embodiment III-34. The present disclosure provides the method of any one of embodiments III-1 to III-33, wherein the composition is administered as a solid.

[0289] Embodiment III-35. The present disclosure provides the method of any one of embodiments III-1 to III-33, wherein the composition is administered as a suspension.

[0290] Embodiment III-36. The present disclosure provides a composition comprising fermentatively grown Euglena beta-1,3-glucan for use in treating a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer.

[0291] Embodiment III-37. The present disclosure provides a composition comprising fermentatively grown Euglena beta-1,3-glucan for use in treating a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease.

[0292] Embodiment III-38. The present disclosure provides for the use of a composition comprising Euglena beta-1,3-glucan grown using fermentation for the manufacture of a medicament for the treatment of a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, Crohn's disease, and colon cancer.

[0293] Embodiment III-39. The present disclosure provides for the use of a composition comprising Euglena beta-1,3-glucan grown using fermentation for the manufacture of a medicament for the treatment of a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease.

[0294] The following examples further illustrate embodiments of the present application. These examples are intended only to illustrate embodiments of the present application and should not be construed as limiting the present application.

[0295] Working Example Immune Response / Hyperlipidemia Example 1: Oral administration of Euglena biomass containing beta-1,3-glucan Euglena is cultivated in a bioreactor using fermentation with the repeated batch method. In the step, Euglena are cultured to a cell density of about 40 grams dry weight / liter to about 80 grams dry weight / liter and harvested. Euglena cells have a beta-1,3-glucan content of about 70% by weight. The Euglena cell culture is filtered using tangential flow filtration, the spent growth medium is discarded, and the cells are washed with water. The resulting Euglena biomass is rolled into thin sheets and dried to a moisture content of less than 10%. The Euglena biomass is then orally administered to humans to modulate immune function.

[0296] Example 2: Oral administration of Euglena biomass containing beta-1,3-glucan as a dietary supplement Euglena are cultured in a bioreactor using fermentation with a repeated batch process. In the final step, Euglena are cultured to a cell density of about 40 grams dry weight / liter to about 80 grams dry weight / liter and harvested. Euglena cells have a beta-1,3-glucan content of about 70% by weight. The Euglena cell culture is filtered using tangential flow filtration, the spent growth medium is discarded, and the cells are washed with water. The Euglena biomass is then dried in a drum dryer to a moisture content of less than 5%. A hammer mill is used to process the dried flakes into a powder with an average particle size of less than 250 microns. The powder is then formed into tablets for oral administration to modulate immune function, treat hyperlipidemia, or prophylactically treat hyperlipidemia. Alternatively, the powder is mixed with food and orally administered with the food to modulate immune function. Alternatively, the powder is placed in a capsule for oral administration to modulate immune function, treat hyperlipidemia, or prophylactically treat hyperlipidemia.

[0297] Example 3: Purification of beta-1,3-glucan from Euglena In one exemplary embodiment, beta-1,3-glucan is purified from Euglena grown by fermentation as described herein by heating the Euglena in a 1% sodium dodecyl sulfate solution, centrifuging the solution, and washing the pellet with water and ethanol. Approximately 1 part Euglena biomass (on a dry weight basis) is suspended in 5 parts 1% (wt / vol) sodium dodecyl sulfate solution. The suspension is mixed and then heated to about 100° C. for about 30 minutes. The solution is then cooled and centrifuged at >500 RCF for about 5 minutes. The supernatant is discarded and the pellet is washed by resuspending in 10 parts water, mixing vigorously, and centrifuging at >500 RCF for 5 minutes. This washing process can be repeated two more times with 10 parts 95% ethanol to reach a beta-glucan pellet with 95% purity. The pellet can be further dried to a white powder.

[0298] In another exemplary embodiment, Euglena grown by fermentation as described herein is purified from beta-1,3-glucan by pumping through a high pressure homogenizer to lyse the cells. The lysed mixture is then centrifuged to recover the beta-1,3-glucan. The supernatant is discarded and the pellet is washed by resuspending in water, mixing vigorously, and centrifuging at >500 RCF. The washing process can be repeated two more times with 10 parts water to reach a 95% pure beta-1,3-glucan pellet. The pellet can be further dried to a white powder.

[0299] The methods described herein are less toxic than some previously described methods for the extraction of beta-1,3-glucan and may have the added advantage of receiving the safety and all-natural product certifications required for the production of food grade or functional food grade products. The resulting purified beta-1,3-glucan is then formed into tablets for oral administration or mixed with food for oral administration. Alternatively, the purified beta-1,3-glucan can be mixed with an aqueous solution as a pharmaceutical composition. The pharmaceutical composition may further comprise a pharmacologically acceptable excipient. The pharmaceutical composition can then be administered intravenously.

[0300] Example 4: Immune response of mice administered Euglena-derived beta-1,3-glucan Euglena biomass containing beta-1,3-glucan was grown using the fermentation process described herein. Whole cell products and purified beta-glucan extracts were tested in mouse studies. Whole cell products were made from Euglena cells grown with glucose as the organic carbon source. The whole cell product contained approximately 50% beta-1,3-glucan by weight and was dried directly after centrifugation without further processing. The whole cell product was fractionated to isolate beta-1,3-glucan, and the beta-glucan fraction was then repeatedly washed to remove non-beta-glucan cellular components to obtain a purified sample of beta-1,3-glucan. The purified sample contained approximately 93% beta-1,3-glucan by weight. A commercially available yeast-derived beta-glucan was also compared.

[0301] Whole cell Euglena biomass samples, purified beta-1,3-glucan samples, and yeast-derived beta-glucan products were dried and ground to a particle size of less than 500 microns. These dried powders were then mixed with PBS buffer and diluted to appropriate concentrations before being administered by oral gavage to BALB / c mice. Blood was collected from each mouse to measure non-specific immune system activity. The following parameters were assessed: phagocytic activity (ability of macrophages to ingest foreign particles), natural killer (NK) cell activity (ability of NK cells to destroy foreign or infected cells), and antibody titers.

[0302] Phagocytosis Phagocytosis is a response by the immune system to capture and destroy potentially harmful particles (e.g., bacteria). The phagocytic index is measured as the percentage of neutrophils that actively capture and phagocytose the labeled particles (Figure 7A). Nine BALB / c mice were assigned to each treatment group and fed daily by oral gavage with the beta-glucan product (20 mg / kg mouse). The control group received only oral gavage with PBS. Three mice from each treatment group were sacrificed on days 1, 7, and 14 to collect material for analysis. The phagocytic index is measured as the percentage of neutrophil cells that actively capture and phagocytose the labeled particles at the assigned time. Mice that received only the PBS control had a phagocytic index of 30%, while mice that received the highest dose of purified beta-glucan from Euglena showed nearly double the phagocytic activity (59%). The group that received the purified algal beta-glucan product phagocytosed best at each dose level. Mice fed whole Euglena biomass and yeast-derived beta-glucan at the two lowest dose levels showed similar phagocytic activity, whereas mice fed yeast-derived beta-glucan at the two highest dose levels showed slightly higher phagocytic activity.

[0303] Natural killer cell cytotoxicity Natural killer (NK) cell activity (the ability of NK cells to destroy foreign or infected cells) was measured to determine the degree of immune function modulation due to the administered beta-glucan (Figure 7B). NK cell cytotoxicity is an indicator of a nonspecific immune response by NK cells to kill potentially pathogenic organisms. Nine BALB / c mice were assigned to each treatment group and fed daily by oral gavage with the beta-glucan product (20 mg / kg mouse). The control group received only oral gavage with PBS. On days 1, 7, and 14, three mice from each treatment group were sacrificed to collect material for analysis. While mice fed the PBS control showed a cytotoxicity index of 12%, mice fed with either whole Euglena biomass or purified Euglena beta-glucan at a low dose of 10 mg / kg showed more than three-fold higher cytotoxicity index (36-50%). At doses of 20 mg / kg and above, both the whole Euglena biomass treatment and the purified Euglena beta-glucan treatment induced stronger cytotoxic responses than the yeast-derived beta-glucan product.

[0304] Antibody titers Antibody formation: beta-glucan can act as a vaccine adjuvant Three BALB / c mice were assigned to each treatment group and were orally administered daily doses of beta-glucan products equivalent to 4, 10, 20, and 40 mg / kg starting on day 0. Antigen (ovalbumin) was given by intraperitoneal injection on days 3 and 16, and antibody titers were measured using an ELISA assay on day 23. Oral gavage of PBS was used as a negative control (Figure 7C). The significant increase in antibody titers indicates that products such as beta-glucan may act as vaccine adjuvants (enhancers). All of the beta-glucan treatment groups induced increased antibody production compared to the negative control, and this effect was enhanced at higher doses. Treatment with purified algal beta-glucan produced the most antibodies at each of the treatment levels, closely followed by the whole Euglena biomass treatment group. Compared to those administered moderate doses of Euglena-derived beta-1,3-glucan (10 and 20 mg / kg), mice fed the yeast beta-glucan product showed substantially lower antibody titers (15-50% lower), whereas mice fed the whole Euglena biomass product at the highest dosage rate showed significantly lower antibody titers (15-50% lower). The titer was similar to that of

[0305] summary Both specific immune responses (i.e., antibody production) and non-specific immune responses (NK cell cytotoxicity and phagocytic activity) were significantly increased in treatment groups given either of the beta-glucan products. For all immune metrics, purified algal beta-1,3-glucan induced the strongest immune responses at all treatment levels. Whole Euglena biomass and purified algal beta-1,3-glucan induced very strong antibody responses that were several fold higher than the titers seen in the negative control. These data indicate the potential for these products to act as adjuvants.

[0306] Whole Euglena biomass performed as well as or better than the yeast-derived beta-glucan product at nearly every treatment level in both antibody production and NK cell cytotoxicity assays. In most cases, whole Euglena biomass induced nearly the same or better responses at only one-quarter to one-half the dosage levels compared to the yeast-derived product.

[0307] The yeast-derived beta-glucan product elicited a lower phagocytic response than the purified algal beta-glucan, but performed as well as or better than all Euglena biomass products in this category. In general, the overall effect of all beta-glucan products on phagocytosis was muted compared to NK cell cytotoxicity and antibody production.

[0308] Example 5: E. coli challenge of mice administered Euglena-derived beta-1,3-glucan Euglena was cultured in a sterile fermenter as described herein. Once the Euglena biomass reached a target density in the fermenter, the cells were centrifuged and the resulting paste was stored frozen at -20°C. The frozen paste was thawed and dried at 65°C until dry flakes formed, then ground to a particle size of less than 250 microns to produce a composition comprising Euglena biomass containing Euglena-derived beta-1,3-glucan. Purified beta-1,3-glucan from Euglena was produced by lysing Euglena cells and isolating beta-1,3-glucan through a method that results in an extract containing >90% beta-glucan and a particle size of less than 250 microns. To compare samples of Euglena-derived beta-1,3-glucan with samples of purified beta-glucan from yeast, each dried product was mixed with phosphate buffered saline (PBS), diluted to the appropriate concentration, and then given by oral gavage to mice at the prescribed dosage levels.

[0309] Ten BALB / c mice were assigned to each treatment group and administered a nominal lethal dose (3 × 10 7 ) of E. coli was administered by intramuscular injection on day 0. Mice were orally administered ampicillin (13 mg / kg) by oral gavage daily starting 2 days before E. coli injection (day -2) until 2 days after injection (day +2). The control group received PBS gavage only, whereas the antibiotic treatment group received oral ampicillin (13 mg / kg) on ​​days 0, 1, 2, 3, and 4. Mice were evaluated daily until day 10.

[0310] Figure 8 summarizes the results of the E. coli challenge experiment. All mice in the control group that received only PBS died within 7 days of E. coli injection. In contrast, mortality at day 10 was reduced by at least 40% in all treatment groups. Notably, 70% of mice that received purified beta-1,3-glucan from Euglena survived 10 days after E. coli injection. This treatment group and the group that received ampicillin showed very similar survival rates over time, suggesting that the algal beta-1,3-glucan treatment provided protection against bacterial infection in mice similar to the common antibiotic ampicillin. Mice that received the Euglena biomass product, which contains approximately 50% beta-glucan, also showed a significant reduction in mortality compared to the control group. 50% of the mice in this treatment group survived 10 days after E. coli injection, compared to 40% in the group that received the yeast-derived beta-glucan extract.

[0311] Example 6: Cytokine expression in mice administered beta-1,3-glucan derived from Euglena Euglena biomass containing beta-1,3-glucan was cultivated using fermentation methods as described herein. The whole cell product was fractionated to isolate beta-glucan, and the beta-glucan fraction was then repeatedly washed to remove non-beta-1,3-glucan cellular components. The purified sample contained approximately 93% beta-1,3-glucan by weight. The purified beta-glucan product was used to evaluate the effect of beta-1,3-glucan on cytokine expression. Cytokines play an important role in the immune system and are involved in the host response to infection and inflammation.

[0312] BALB / c mice (6 mice / group) were fed purified beta-1,3-glucan products by oral gavage for 7 days. Mice were dosed at 0 mg / kg (A), 5 mg / kg (B), 20 mg / kg (C), and 200 mg / kg (D). On day 8, dendritic cells (DCs; CD11c+ cells) were isolated from collagenase-digested small intestines by magnetic sorting and cultured overnight. Supernatants were tested for various cytokines and chemokine factors by Luminex multiplex assay. As shown in Figure 9A, increasing amounts of beta-1,3-glucan increased the production of IL-1b, TNF-a, IL-10, IL-18, IP-10, and MCP-1. The dose effects of Rantes and MIP-1a were less pronounced, as levels of these chemokines were high and relatively unchanged for beta-1,3-glucan administered at doses of 5, 20, and 200 mg / kg. Expression of IFN-g, IL-12, IL-6, IL-9, IL-2, IL-23, GMCSF, IL-4, and IL-5 was undetectable.

[0313] BALB / c mice (6 per group) were fed purified beta-1,3-glucan products by oral gavage for 7 days. Mice were dosed at 0 mg / kg (A), 5 mg / kg (B), 20 mg / kg (C), and 200 mg / kg (D). On day 8, macrophages (F4 / 80 cells) were isolated from collagenase-digested small intestines by magnetic sorting and cultured overnight. Supernatants were analyzed for various cytokines and chemokine factors by Luminex multiplex assay. Figure 9B shows that increasing amounts of beta-1,3-glucan increased IL-6, IL-1b, TNF-a, IL-10, IL-18, Rantes, MIP-1a, IP-10, and MCP-1. Expression of IFN-g, IL-17, IL-22, IL-4, IL-9, IL-18, IL-2, IL-23, and GMCSF was undetectable.

[0314] BALB / c mice (6 per group) were fed purified beta-1,3-glucan products by oral gavage for 7 days. Mice received doses of 0 mg / kg (A), 5 mg / kg (B), 20 mg / kg (C), and 200 mg / kg (D). On day 8, NK1.1+ cells (NK cells) were isolated from collagenase-digested small intestines by magnetic sorting and cultured overnight. Supernatants were analyzed for various cytokine and chemokine factors by Luminex multiplex assay. Figure 9C shows that increasing amounts of beta-1,3-glucan increased IFNg, IL-1b, TNF-a, IL-10, IL-22, Rantes, and IL-17. In contrast, the effect of beta-1,3-glucan on the expression of MIP-1a, IP-10, and MCP-1 appears to be relatively independent of the dose of beta-glucan administered. As the amount of beta-1,3-glucan increased, the production of IL-23 decreased. The expression of IL-6, IL-4, IL-9, IL-2, and GMCSF was undetectable.

[0315] Example 7: Treatment of hyperlipidemia in mice by administering Euglena-derived beta-1,3-glucan Euglena were grown in sterile fermentors as described herein. Once the Euglena biomass reached a target density in the fermentor, the Euglena were filtered, lysed, and the beta-1,3-glucan was purified. The purified beta-1,3-glucan from Euglena was used in the mouse experiments described below. Phosphate buffered saline was administered as a control.

[0316] In the first experiment, Balb / c mice were fed a high cholesterol diet (about 16 mg cholesterol daily) without beta-glucan for 14 days. After this period, the mice were fed a diet containing Euglena-derived beta-1,3-glucan or a control with normal cholesterol levels (about 110 mg / dL) for an additional 30 days. Serum was collected after 12 hours of fasting, and blood cholesterol and triglycerides were measured. As illustrated in FIG. 10A, the serum cholesterol levels of the mice increased from an average of about 115 mg / dL to about 265 mg / dL during the high cholesterol feeding. In control mice that were placed back on a diet containing normal amounts of cholesterol and then administered phosphate-buffered saline, cholesterol levels remained elevated at least until day 34, decreasing to about 181 mg / dL by day 44. In contrast, mice that received Euglena-derived beta-1,3-glucan showed an immediate and steady decrease in blood cholesterol levels. Similar results were obtained for triglyceride levels. As seen in FIG. 10B, serum triglyceride levels rose from about 105 mg / dL to about 138 mg / dL while the mice were on the high cholesterol diet. Control mice only had a slight decrease in serum triglyceride levels after 30 days on a normal cholesterol diet. In contrast, mice treated with beta-glucan showed an immediate and sustained decrease in serum triglyceride levels, which were lower at day 44 than at the beginning of the experiment.

[0317] In a second experiment, Balb / c mice were fed a high cholesterol diet (about 16 mg cholesterol daily) for 14 days along with purified Euglena-derived beta-1,3-glucan or a control. After 14 days, the mice were fed a normal cholesterol diet (about 110 mg / dL) and administered the same beta-glucan product or a control for an additional 30 days. Serum was collected after 12 hours of fasting and blood cholesterol and triglycerides were measured. As illustrated in FIG. 11A, serum cholesterol in mice fed only the control rose the most over the 14 days with the high cholesterol diet, rising from about 118 mg / dL to about 275 mg / dL. Mice fed purified beta-1,3-glucan from Euglena (Euglena) showed a small increase in serum cholesterol from about 118 mg / dL to about 230 mg / dL. Similar results were obtained for triglyceride levels. As seen in FIG. 11B, serum triglyceride levels in control mice increased the most during the high cholesterol diet (from about 105 mg / dL to about 145 mg / dL). There was only a slight decrease in serum triglyceride levels after 30 days on the cholesterol diet. In contrast, mice treated with beta-glucan showed an immediate and sustained decrease in serum triglyceride levels after being placed back on a normal cholesterol diet, which were lower at day 44 than at the start of the study.

[0318] Example 8: Clinical trial to evaluate the effect of ingesting Euglena-derived beta-1,3-glucan on an individual's metabolic health The trial will assess the impact of consuming Euglena-derived beta-1,3-glucan on cholesterol levels, blood sugar, infections, and inflammation. Participants will be recruited after written informed consent (approved by a registered Institutional Review Board) and a preliminary selection process. The study will be four weeks in duration, with assessments at baseline, 2, and 4 weeks of beta-1,3-glucan consumption. Euglena-derived beta-1,3-glucan will be administered in capsule form, giving each individual a daily dose of 250 mg.

[0319] The effect of beta-glucan on metabolic health is measured using one or more of fasting blood levels of markers of glucose and lipid metabolism, a general health and wellness assessment, and blood pressure and blood chemistry measurements that may include protein carbonyl content (a marker of oxidative stress), acute phase inflammatory markers (e.g., C-reactive protein, alpha-1 acid glycoprotein, fibrinogen, platelet factor 4), metabolic biomarkers (e.g., C-peptide, cortisol, pancreatic polypeptide, proinsulin, peptide YY), cytokine panel, and antibody analysis.

[0320] Intestinal inflammation Example 9: Oral administration of Euglena biomass containing beta-1,3-glucan Euglena are cultured using fermentation in a bioreactor using a repeated batch method. In the final step, Euglena are cultured to a cell density of about 40 grams dry weight / liter to about 80 grams dry weight / liter and harvested. Euglena cells have a beta-1,3-glucan content of about 50% by weight. The Euglena cell culture is filtered using tangential flow filtration, the spent growth medium is discarded, and the cells are washed with water. The resulting Euglena biomass is rolled into thin sheets and dried to a moisture content of less than 10%. The dried biomass is pulverized to less than 1000 microns, and the Euglena biomass is then orally administered to humans to treat intestinal inflammation.

[0321] Example 10: Oral administration of Euglena biomass containing beta-1,3-glucan as a dietary supplement Euglena are cultured using fermentation in a bioreactor using a repeated batch process. In the final step, Euglena are cultured to a cell density of about 40 grams dry weight / liter to about 80 grams dry weight / liter and harvested. Euglena cells have a beta-1,3-glucan content of about 50% by weight. The Euglena cell culture is filtered using tangential flow filtration, the spent growth medium is discarded, and the cells are washed with water. The Euglena biomass is then dried in a drum dryer to a moisture content of less than 5%. The dried flakes are processed into a powder with an average particle size of less than 250 microns using a hammer mill. The powder is then formed into a tablet for oral administration to treat intestinal inflammation. Alternatively, the powder is mixed with food and orally administered with the food to treat intestinal inflammation. Alternatively, the powder is placed into a capsule for oral administration to treat intestinal inflammation.

[0322] Example 11: Purification of beta-1,3-glucan from Euglena In one exemplary embodiment, beta-1,3-glucan is extracted from Euglena by heating Euglena grown by fermentation as described herein in a 1% sodium dodecyl sulfate solution, centrifuging the solution, and washing the pellet with water and ethanol. Purification: Approximately 1 part Euglena biomass (on a dry weight basis) is suspended in 5 parts 1% (wt / vol) sodium dodecyl sulfate solution. The suspension is vortexed and then heated to approximately 100°C for approximately 30 minutes. The solution is then cooled and centrifuged at >500 RCF for approximately 5 minutes. The supernatant is discarded and the pellet is washed by resuspending in 10 parts water, vortexing vigorously, and centrifuging at >500 RCF for 5 minutes. This washing process can be repeated two more times with 10 parts 95% ethanol to reach a 95% pure beta-glucan pellet. The pellet can be further dried to a white powder.

[0323] In another exemplary embodiment, Euglena grown by fermentation as described herein is purified from beta-1,3-glucan by pumping through a high pressure homogenizer to lyse the cells. The lysed mixture is then centrifuged to recover the beta-1,3-glucan. The supernatant is discarded and the pellet is washed by resuspending in water, mixing vigorously, and centrifuging at >500 RCF. The washing process can be repeated two more times with 10 parts water to reach a 95% pure beta-1,3-glucan pellet. The pellet can be further dried to a white powder.

[0324] The methods described herein are less toxic than some previously described methods for the extraction of beta-1,3-glucan and may have the added advantage of receiving the safety and all-natural product certifications required for the production of food grade or functional food grade products. The resulting purified beta-1,3-glucan is then formed into tablets for oral administration or mixed with food for oral administration. Alternatively, the purified beta-1,3-glucan can be mixed with an aqueous solution as a pharmaceutical composition. The pharmaceutical composition may further comprise a pharmacologically acceptable excipient. The pharmaceutical composition can then be administered intravenously.

[0325] Example 12: In vivo study to determine the effect of pretreatment with Euglena-derived beta-1,3-glucan on chemically induced acute colitis Euglena biomass containing beta-1,3-glucan was grown using a fermentation process as described herein. Dried Euglena biomass and purified beta-glucan extracts were tested in mouse studies. Dried Euglena biomass was produced from Euglena cells grown with glucose as the organic carbon source. The Euglena biomass contained approximately 50% beta-1,3-glucan by weight and was centrifuged and then dried without further processing. The Euglena biomass product was fractionated to isolate beta-1,3-glucan, and the beta-glucan fraction was then repeatedly washed to remove non-beta-glucan cellular components to yield a purified sample of beta-1,3-glucan. The purified sample contained approximately 95% beta-1,3-glucan by weight. Phosphate buffered saline (PBS) was administered as a negative control.

[0326] Dried Euglena biomass samples and purified beta-1,3-glucan samples were dried and ground to a particle size of less than 500 microns. These dried powders were then mixed with PBS buffer and diluted to the appropriate concentration before being fed by oral gavage to C57BL / 6J mice. For the duration of the study (15 days), mice were treated once a day, after 2 hours of fasting, with either (i) PBS ("None"), (ii) dried Euglena biomass ("A50"; 250 μg / mouse / day), or (iii) purified beta-1,3-glucan ("AG"; 125 μg / mouse / day). This dosing ensured that mice receiving dried Euglena biomass received a comparable dose of beta-1,3-glucan as mice receiving purified beta-1,3-glucan. Ten mice were used for each treatment group. After 5 days (day 0), mice were administered a 2.5% solution of dextran sulfate sodium (DSS), a compound known to induce acute colitis, in drinking water until day 5, after which they were returned to normal water. Body weights and visual observations were performed daily. On day 10, mice were euthanized. Colon samples were processed and immune cells were isolated for cytokine analysis. The effect of Euglena-derived beta-1 on chemically induced acute colitis was determined using various parameters. The effect of ,3-glucan pretreatment is described in Examples 13-19.

[0327] Example 13: Effect of pretreatment with Euglena-derived beta-1,3-glucan on body weight in mice with chemically induced acute colitis Mice were treated as described in Example 12. The weight of each mouse was recorded every day during the experiment. A loss of body weight is considered a sign of colitis. The initial body weight was considered as 100% to calculate the percentage of body weight at subsequent time points. The percentage of the initial body weight was measured as a function of time after DSS treatment (Figures 12A-12C). The statistical significance of the data is shown in Table 2.

[0328] [Table 2] Mice treated with either phosphate-buffered saline (FIG. 12A) or purified beta-1,3-glucan (FIG. 12B) showed a rapid loss of body weight during the first 5 days after DSS treatment. By day 10, the body weight of PBS-treated mice had fallen to about 75% of their initial body weight. Mice treated with purified beta-1,3-glucan showed a similar weight loss from days 5 to 8, falling to about 80% of their initial body weight. However, by days 9 and 10, the body weight of mice treated with purified beta-1,3-glucan had increased to about 83% and 85%, respectively. This data suggests that treatment with purified beta-1,3-glucan is effective in early recovery from DSS-induced acute colitis, especially when considered in conjunction with other factors (such as stool consistency and the presence of blood in the stool).

[0329] Mice treated with dried Euglena biomass did not show a significant loss in body weight up to day 6 after DSS treatment, at which point a gradual loss in body weight was observed (Figure 12C). By day 9, the body weight of mice treated with dried Euglena biomass had decreased to about 92% of their initial body weight. However, the body weight of mice treated with dried Euglena biomass increased slightly to about 95% on day 10. This data indicates that treatment with dried Euglena biomass is effective in preventing or minimizing the severity of acute colitis induced by DSS in mice.

[0330] The combined results for mice treated with PBS, purified beta-1,3-glucan, or dried Euglena biomass are shown in Figure 13. The combined results for mice treated with PBS, purified beta-1,3-glucan, or dried Euglena biomass are also presented as a function of weight loss score over time (Figure 14). Weight loss scores were calculated as follows: 0 (no loss), 1 (1-5%), 2 (5-10%), 3 (10-20%), and 4 (>20%). The statistical significance of this data is shown in Table 3.

[0331] [Table 3] Example 14: Euglena-derived vegetative proliferation in mice with chemically induced acute colitis Effect of pretreatment of tert-1,3-glucan on stool consistency Mice with chemically induced acute colitis were treated as described in Example 12. The stool consistency of each mouse was recorded daily throughout the experiment. Weight loss was considered a sign of colitis. Stool consistency was scored as 0 (normal solid), 2 (semi-solid / soft), 3 (loose stool), and 4 (watery / diarrhea). Stool consistency was determined as a function of time after DSS treatment (Figure 15). The statistical significance of the data is shown in Table 4.

[0332] [Table 4] Mice treated with PBS had normal stool consistency on days 0-3. Mice treated with PBS began to produce loose stools from day 4. Mice treated with purified beta-1,3-glucan had normal stool consistency on days 0-4 and began to produce loose stools from day 5. Mice treated with dried Euglena biomass produced semi-solid / soft stools on day 5, with normal stool consistency up until that point. This data indicates that treatment with dried Euglena biomass is particularly effective in preventing or minimizing the severity of DSS-induced acute colitis in mice.

[0333] Example 15: Effect of pretreatment with Euglena-derived beta-1,3-glucan on stool blood in mice with chemically induced acute colitis Mice with chemically induced acute colitis were treated as described in Example 12. The degree of visible stool blood / rectal bleeding level for each mouse was recorded every day throughout the experiment. Stool blood was scored as follows: 0 (no blood), 1 (redness on the fecal pellet), 2 (occasional blood on the feces), 3 (blood-covered feces / blood-stained feces), and 4 (gross bleeding / blood around the anus). Stool blood scores were recorded as a function of time after DSS treatment (Figure 16). The statistical significance of this data is shown in Table 5.

[0334] [Table 5] Mice treated with PBS did not have blood in their stools on days 0-3. Mice treated with PBS produced blood in their stools starting on day 4. Mice treated with purified beta-1,3-glucan did not have blood in their stools on days 0-4 and began to have blood in their stools on day 5. The amount of blood in the stool produced in mice treated with purified beta-1,3-glucan was significantly less than that produced in mice treated with PBS. Mice treated with dried Euglena biomass did not produce blood in their stools until day 6, at which point there was a small amount of blood evident. The amount of blood in the stool produced in mice treated with dried Euglena biomass was less than that produced in mice treated with purified beta-1,3-glucan. This data indicates that treatment with dried Euglena gracilis or purified beta-1,3-glucan is effective in preventing or minimizing the severity of acute colitis induced by DSS in mice.

[0335] Example 16: Effect of cerebrospinal fluid on colon length in mice with chemically induced acute colitis Effect of pretreatment with Euglena-derived beta-1,3-glucan Mice with chemically induced acute colitis were treated as described in Example 12. On day 10, the mice were euthanized and the colon length of each individual mouse was subsequently measured (7 mice / treatment group). The data are shown in FIG. 17. The colon lengths of mice treated with PBS or purified beta-1,3-glucan are within the margin of error, but the colon length of mice treated with dried Euglena biomass is significantly increased. Specifically, the colon length of mice treated with PBS is approximately 5.5 cm, whereas the colon length of mice treated with dried Euglena gracilis is approximately 6.5. This data indicates that treatment with dried Euglena biomass is particularly effective in preventing or minimizing acute colitis induced by DSS in mice.

[0336] Example 17: Effect of pretreatment with Euglena-derived beta-1,3-glucan on colonic inflammation in mice with chemically induced acute colitis Mice with chemically induced acute colitis were treated as described in Example 12. The extent of colonic inflammation was measured after euthanasia of the mice on day 10 (3 mice / treatment group). Distal colonic pieces were snap frozen, cryosectioned (6 μm sections), and subjected to hematoxylin / eosin staining. Representative images from each treatment group are shown in Figure 18A. The severity of colonic inflammation in individual mice was also graded (0-4) by a pathologist based on immune cell infiltration and damage to the overall structure of the mucosa and mucosa submucosa (Figure 18B). A total of 15 sections (500 μm apart each) were examined for 3 mice.

[0337] Of the three treatment groups, mice treated with PBS showed the highest level of colon inflammation (graded at approximately 3.4). Mice treated with purified beta-1,3-glucan showed a slightly reduced level of colon inflammation (graded at approximately 2.8), and mice treated with dried Euglena gracilis showed the lowest level of colon inflammation (graded at approximately 1.8). This data indicates that treatment with dried Euglena gracilis or purified beta-1,3-glucan is effective in preventing or minimizing the severity of DSS-induced acute colitis in mice.

[0338] Example 18: Effect of pretreatment with Euglena-derived beta-1,3-glucan on T-helper cell responses in mice with chemically induced acute colitis Mice with chemically induced acute colitis were treated as described in Example 12. The frequency of cytokines associated with T helper cell function was analyzed in individual mice on day 10 (3 mice / treatment group). Mesenteric lymph node cells were activated with PMA / ionomycin, stained for the presence of cytokines associated with T helper cell function, and analyzed by FACS (Figure 19A). The percentage of cytokine-positive cells is shown in Figure 19B.

[0339] Mice treated with dried Euglena biomass had significantly lower frequencies of Th1 (IFN-γ), Th17 (IL-17 and IL-22), and Th9 (IL-9) producing CD4+ T cells compared to mice treated with PBS. Mice treated with purified beta-1,3-glucan had relatively lower levels of Th1 (IFN-γ) producing CD4+ T cells compared to mice treated with PBS. The statistical significance of this data is shown in Table 6.

[0340] [Table 6] Example 19: Effect of pretreatment with Euglena-derived beta-1,3-glucan on T-helper cell cytokine profile in mice with chemically induced acute colitis Mice with chemically induced acute colitis were treated as described in Example 12. Cytokine production by colonic immune cells was analyzed in individual mice on day 10 (3 mice / treatment group). Immune cells were isolated from colonic tissue using magnetic separation and equal numbers of cells from each mouse were cultured for 24 hours. Levels of spontaneously released (T helper cell specific) cytokines were measured in triplicate using a multiplex assay (Figure 20).

[0341] The data show that Th1 (IFN-γ) and Th17 (IL-17) cytokines were suppressed in mice treated with dried Euglena biomass. Higher levels of both pro-inflammatory (IL-22, IL-9 (Th9)) and anti-inflammatory (IL-10, IL-4 (Th2)) cytokines were produced by colonic immune cells in mice treated with purified beta-1,3-glucan. The particularly striking protection of mice from colitis by treatment with dried Euglena biomass is likely due to increased production of the cytokine IL-10 and inhibition of recruitment of inflammatory immune cells.

[0342] Example 20: Clinical study to evaluate the effect of consuming Euglena-derived beta-1,3-glucan on intestinal inflammation in individuals The trial will evaluate the effect of consuming Euglena-derived beta-1,3-glucan on the treatment of intestinal inflammation. Participants will be recruited after written informed consent (approved by a registered Institutional Review Board) and a preliminary selection process. The study will be four weeks in duration, with assessments at baseline, two and four weeks of beta-1,3-glucan consumption. Euglena-derived beta-1,3-glucan will be administered in capsule form, giving each individual a daily dose of 250 mg.

[0343] The effect of beta-glucan on intestinal inflammation is measured using one or more of the following: severity of abdominal pain, occurrence of diarrhea, body weight, appetite level, presence or absence of rectal bleeding, general health and wellness assessment, and blood pressure and blood chemistry measurements that may include protein carbonyl content (a marker of oxidative stress), acute phase inflammatory markers (such as C-reactive protein, alpha-1 acid glycoprotein, fibrinogen, and platelet factor 4), cytokine panel, and antibody assays.

[0344] As shown in Figure 21, oral intake of Euglena-derived beta-1,3-glucan reduces the number of inflammatory basophils in an individual. This data indicates that Euglena-derived beta-1,3-glucan regulates the immune system function of an individual.

[0345] As shown in Figure 22, oral intake of Euglena-derived beta-1,3-glucan affects the number of urgent bowel movements or diarrhea in an individual. The data shows a statistically significant decrease in intestinal disorders from weeks 0 to 2 (p<0.05, *) and an increase in intestinal disorders reaching a statistical trend from weeks 2 to 4 (p<0.1, (*)). The data indicates that Euglena-derived beta-1,3-glucan contributes to an individual's intestinal health.

[0346] Oral intake of Euglena-derived beta-1,3-glucan may also modulate an individual's response to oxidative stress and contribute to inflammation. Superoxide dismutase (SOD) is an enzyme involved in neutralizing superoxide ions, which are highly reactive and damaging free radicals. SOD plays a very important role in reducing oxidative stress. As shown in FIG. 23, oral intake of Euglena-derived beta-1,3-glucan increases an individual's levels of SOD. A significant increase in average SOD levels was observed during the first two weeks of oral intake of Euglena-derived beta-1,3-glucan, followed by a mild decrease during the next two weeks. Despite a mild decrease over the final two weeks of the study, the overall improvement over the four weeks was highly significant for all participants (p<0.01, **).

[0347] Another measure of free radical stress in the body is lipid peroxidation. Lipid peroxidation is one of the major mechanisms of cellular damage induced by oxidative stress. Specifically, malondialdehyde is a marker of free radical stress in the body and is a measure of oxidative damage to lipids in the bloodstream. A significant decrease in lipid peroxidation was observed during the first two weeks of oral intake of Euglena-derived beta-1,3-glucan, followed by a mild increase during the next two weeks (Figure 24). Despite a mild increase over the final two weeks of the study, the overall improvement over four weeks was highly significant (p<0.01, **).

[0348] The above description includes several numerical ranges in the text and figures. Since the embodiments of the present invention can be practiced through the disclosed numerical ranges, the numerical ranges provide any range or value within the disclosed numerical ranges, although the exact range limits are not literally stated in the specification.

[0349] The above description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiment will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. The entire disclosures of the patents and publications mentioned in this application are incorporated herein by reference. Finally, the present invention may be construed according to the claims and their equivalents. [1] A composition for oral administration for treating a disease selected from the group consisting of hyperlipidemia, metabolic syndrome, inflammatory bowel disease, colitis, and Crohn's disease in a human having said disease, comprising: The composition comprises a Euglena biomass containing 50% to 70% by weight of beta-1,3-glucan, The composition contains an effective amount of beta-1,3-glucan that does not contain beta-1,6-glycosidic bonds from Euglena grown by fermentation, the effective amount being between 0.1 mg of beta-1,3-glucan / kg body weight and 100 mg of beta-1,3-glucan / kg body weight. composition. [2] The composition described in [1], wherein the disease is hyperlipidemia. [3] The composition described in [2], wherein administration of the composition reduces the cholesterol level of the human. [4] The composition described in [2] or [3], wherein the effective amount is between 0.1 mg of beta-1,3-glucan / kg body weight and 50 mg of beta-1,3-glucan / kg body weight. [5] The composition according to any one of [1] to [4], which is administered in combination with a statin, nicotinic acid, bile acid resin, fibric acid derivative, or cholesterol absorption inhibitor. [6] The composition described in [1], wherein the disease is colitis. [7] The composition described in [1], wherein the disease is inflammatory bowel disease. [8] The composition described in [1], wherein the disease is Crohn's disease. [9] The composition described in any one of [6] to [8], wherein administration of the composition increases production of anti-inflammatory cytokines.

[10] The composition described in any one of [6] to [9], which is administered in combination with an anti-inflammatory drug, an immunosuppressant, or an antibiotic.

[11] The composition described in any one of [1] to

[10] , wherein the Euglena is Euglena gracilis.

[12] The composition according to any one of [1] to

[11] , wherein the Euglena grows heterotrophically.

[13] The composition described in any one of [1] to

[12] , wherein the beta-1,3-glucan is present in the form of paramylon.

[14] The composition described in any one of [1] to

[13] , wherein the beta-1,3-glucan does not contain beta-1,6-glycosidic bonds.

[15] The composition described in any one of [1] to

[14] , wherein the Euglena biomass is dried to a moisture content of 40% or less.

[16] The composition described in

[15] , wherein the Euglena biomass is processed to have an average particle size of 1,000 microns or less.

[17] The composition of any one of [1] to

[16] , administered as a single dose daily.

[18] The composition of any one of [1] to

[16] , administered as multiple divided doses per day.

[19] Alpha-tocopherol, cholecalciferol, zinc, chromium, selenium, arginine, ascorbic acid, alkylglycerols, caffeine, kava kava, turmeric, spirulina, chlorella, stevia, calcium D-glucarate, coenzyme Q10, peptides, dimethylglycine, docosahexaenoic acid, eicosapentaenoic acid, alpha- The composition according to any one of [1] to

[18] , further comprising an additional ingredient selected from the group consisting of linoleic acid, astaxanthin, beta-carotene, lutein, lactobacillus probiotics, bifidobacterium probiotics, mannooligosaccharides, fructooligosaccharides, astragalus, echinacea, esberitox, garlic, glutathione, kelp, L-arginine, L-ornithine, lecithin granules, extracts of Maitake, Reishi, or Lentinula edodes, manganese, quercetin, bromelain, olive leaf, elderberry, umka, pantothenic acid, quercetin, alpha lipoic acid, essential oils, fish oils, spices and derivatives thereof, pterostilbene, and combinations thereof.

[20] The composition described in

[19] , wherein the additional component is zinc.

[21] The composition according to any one of [1] to

[19] , further comprising a metal.

[22] The composition of

[21] , wherein the metal comprises a member selected from the group consisting of iron, magnesium, lithium, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, manganese, selenium, and combinations thereof.

[23] The composition described in

[22] , wherein the beta-(1,3)-glucan and the metal form a complex.

[24] The composition described in

[23] , wherein the complex comprises a zinc beta-(1,3)-glucan complex.

[25] The composition described in any one of [1] to

[18] , further comprising an additional component selected from the group consisting of Haematococcus pluvialis, astaxanthin, and colostrum.

[26] The composition described in any one of [1] to

[25] , which is administered as a solid.

[27] The composition described in any one of [1] to

[25] , which is administered as a suspension.

Claims

1. 1. A composition for oral administration for reducing symptoms associated with intestinal inflammation in a human, comprising: The composition contains an effective amount of beta-1,3-glucan obtained by drying Euglena grown by fermentation to a moisture content of about 40% or less, The beta-1,3-glucan does not contain beta-1,6-glycosidic bonds; The effective amount is between 0.1 mg of beta-1,3-glucan / kg body weight and 100 mg of beta-1,3-glucan / kg body weight. composition.

2. The composition of claim 1 , wherein the Euglena is Euglena gracilis.

3. The composition of claim 1 , wherein the Euglena grows heterotrophically.

4. The composition of claim 1, wherein the beta-1,3-glucan is present in the form of paramylon.

5. 2. The composition of claim 1, wherein the beta-1,3-glucan is purified from Euglena.

6. The composition of claim 1 , administered as a single dose daily.

7. 1. A composition for oral administration for modulating the immune system of an individual, comprising: The composition contains an effective amount of beta-1,3-glucan obtained by drying Euglena grown by fermentation to a moisture content of about 40% or less, The beta-1,3-glucan does not contain beta-1,6-glycosidic bonds; The effective amount is between 0.1 mg of beta-1,3-glucan / kg body weight and 100 mg of beta-1,3-glucan / kg body weight. composition.

8. The composition of claim 7, wherein the individual is experiencing intestinal inflammation.

9. 9. The composition of claim 8, wherein the intestinal inflammation is measured by the severity of abdominal pain, occurrence of diarrhea, body weight, appetite level, presence or absence of rectal bleeding, or an assessment of general health and wellness.

10. 9. The composition of claim 8, wherein administration of an effective amount reduces symptoms associated with intestinal inflammation.

11. The composition of claim 7 , wherein the individual is at high risk of intestinal inflammation.

12. The composition of claim 7 , wherein the Euglena grows heterotrophically.

13. The composition of claim 7 , administered as a single dose daily.

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