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Blue-green algae are a diverse group of microscopic plants rarely used for human food. The exception is Spirulina, which has become widely available as a food ingredient within the last twenty years. It is a nutraceutical food with rather unique phytonutrients and characteristics. Spirulina can be used in a variety of healthy food applications. Wild Spirulina platensis, (or Arthrospira as it is sometimes called), grows in sunny warm-water volcanic soda lakes throughout the world. This alga thrives in waters containing high amounts of sodium carbonate with pH values above ten. Where wild Spirulina is found there are often flocks of flamingos and other animals feeding on it. Spirulina has a distinctive spiral structure with filaments of cells being about 10 microns in diameter and up to 1,000 microns in length.

Spirulina is the only blue-green algae commercially cultivated for food use. Modern Spirulina farms have specially designed raceway type ponds using paddlewheel circulation for cultivation. The tiny spiral shaped algae are harvested by multi-stage filtration, rinsed and spray-dried. Spray drying evaporates the water and with minimal heating, preserving nutritive value. It is supplied as very fine dark-blue-green powder, usually packed in sealed oxygen barrier type shipping containers. Since time immemorial humans have eaten Spirulina The Spaniards observed the Aztecs utilizing it for food. Indigenous people around Lake Chad in Africa still eat 'dihe' as they call it. In the West it was first mass-produced in Mexico City where it was growing in a soda works. It was first sold commercially as food in the early 1980s, and since then it has become popular as a health food for "energy" and a dieting aid. Spirulina is today consumed as food in more than seventy-five countries worldwide.

Product Development
Considerable potential exists for Spirulina to be used in energy or granola bars, snacks, sports pastas, and specialty juices and liquid meal replacements. It is especially good for athletes, the aging and others with special health concerns and those needing extra immune system support. Health benefits are found in animal and human studies at feeding levels starting at an equivalent of about one-gram per day, assuming a one-hundred-sixty pound person. Pure Spirulina is a dark blue-green frne powder with a mild seaweed taste. It easily mixes with other dry powder ingredients. While not completely soluble, it suspends nicely in most liquids with gentle agitation. When used in light colored foods at levels of less than five percent it turns the food a pale green, at one percent or less it has little if any effect on color. When used at the lower levels it does not affect the taste of most foods. Commercially available blue pigment, (phycocyanin or DIC Lina-blue) extracted from is useful as a food coloring. While water soluble, it is not heat stable. In Japan it is used for coloring frozen foods and chewing gum.

Potential Benefits
Investigation is underway in regard to the anti-viral and anti-HW properties found with invitro studies of Spirulina extracts. Many in vitro and in vivo studies demonstrate even small amounts or Spirulina stimulate immune system functions, especially those mediated by macrophages. It accelerates immune system competence in young animals. Data also suggests there is synergy between Spirulina and Vitamin C. Spirulina may serve to extend Vitamin C in vivo. Other studies show Spirulina may have potential as a therapy for some kinds of cancers. In China it is used in hospitals to lower blood lipids, reduce fatigue and increase levels of IgG, IgA and IgM. It may have anti-radiation therapy potentiaL Recent work done in Macedonia reports hematological benefits for athletes in intensive training. Other studies show Spirulina may help with liver and kidney detoxification. Animals experimentally fed Spirulina were afforded significant protection from the effects of high doses of dioxin, gentamicin, cisplatin and organic mercury. Since Spirulina has a prokaryotic type of cell structure the cell wall components affect the immune system of man and animals. The thin cell wall is made of polysaccharides and muramyl peptides. Spirulina polysaccharides have both anti-oxidant and immune system stimulating properties. These polysaccharides are linked within the cell wall to muramyl peptides. Muramyl peptides are thought to be essential to humans and animals for good health. They help maintain the immune system in a state of optimal readiness and provide benefits for the central nervous system by stimulating metabolism while enhancing slow-wave sleep and may affect organ development. Chinese scientists have documented the afore mentioned phycocyanin as stimulating hematopoiesis, emulating the hormone erythropoetin. This is a potentially valuable therapy for persons suffering from certain forms of anemia or bone marrow damage.

Nutrition
Spirulina's cell wall contains no cellulose and is made of mucopolysaccharides. The cell wall is easily digested in the human gut. Most striking nutritionally is the high amino acid content, about sixty-two percent, and very high carotenoid pigment content, about 4 mg/gram. The carotenoids consist of about ten different kinds, mainly beta-carotene, zeaxanthin and myxoxanthophyll with small amounts of lutein and several others. Vitamin A activity is about 2,300 LU per gram. Spirulina derives its intense dark blue-green color from a mixture of pigments in addition to carotenoids. Green chlorophyll makes up about one percent while the briffiant hyacinth blue phycocyanins are at about a whopping fourteen percent. Phycocyanins are peptides, which may have health benefits. This alga contains only about seven percent fat and twenty percent of the fat is GLA, gamma-linolenic acid. GLA may have inflammation modulating properties and is itself a popular dietary supplement. Spirulina is usually a good source of iron, containing about one-mg per gram. This makes it an attractive supplement for women or athletes. It also may contain appreciable amounts of zinc, B- 12, riboflavin and thiamin.

Safety
The UN-FAO recognizes Spirulina as a potential weapon against malnutrition for the third world and has sponsored safety studies since the early 1980s. The results show Spirulina is safe and non-toxic. Testing reveals it has a no-adverse-effect level of at least l2gm[kg in mice. No acute or chronic problems, no reproductive toxicity and Spirulina is non-mutagenic.

Quality Assurance
As with mushrooms, care needs to be taken to ensure that the supplier has true Spirulina, not a potentially toxic species that looks similar. Algal toxins, heavy metals are a concern for wild-harvested lake algae. The best suppliers provide algal toxin test results and environmental contaminates like pesticides from agricultural run-off can be a for additional assurance. Visual microscopic examination together with GLA analysis can confirm authenticity. While visual identification of blue-green algae can be difficult, Spirulina is distinguished as the only filamentous blue-green algae with appreciable amounts of GLA fatty acid, (18: 3:6), about one percent by weight. GLA content should be at least 900mg/lOOgm to meet the accepted definition of Spirulina by the NNFA in the United States. Moisture should be less then seven percent. Bacterial or mold contamination are indicators of improper growing conditions, drying or storage. Color should be very dark blue-green. Carotenoid pigments should be at least 350mg/100gm. "Bright" green color indicates oxidized stale product with low carotenoid content.

RELEVENT STUDIES ON SPIRULINA

Spirulina Biomedical Abstracts

version 6129/98

#1-14 Hematopolesis and Immunomodulation #15-20 Anti-Cancer

#21-25 Anti-Viral #26-27 Kidney Function #28-45 Cholesterol & Misc. #46-63 Safety

 Hematopojesis and Immunomodulation

1. Baojiang G. et a', Second Asia-Pacific Conference on Algal Biotechnology, April 25-27,1994, p.24, "Study on Effect and Mechanism of Polysaccharides of Spirulina platensis on Body Immune Functions Improvement" Polysaccharides of Spirulina platensis, at the dosage of 15O-3OO mg/kg by injection or taking orally, can increase the phagocyte percentage and phagocytic index of abdominal macrophage, the percentage of T-lymphocyte and haemolysin content in the peripheral blood of mouse. The results demonstrate the ploysaccari des can improve both the nonspecific flinction of cellular immunity and the specific humoral immunity. The mechanism seems to be related to the fact that the polysaccharides can eithance the reproductive ability of marrowcyte, the growth of thymus and spleen, the biosynthesis of serum protein, and that polysaccharide can eliminate the inhibition effect of inhibitive circular phosphamide on immune system of body.

2. Besednova, N.N. et a', Zhurnal Mikrobiologii, Epidemiologli, Immunobiologii; 56 (12), 75-79 1979 'Immunostimulating activity of lipopolysaccharides from blue-green algae" The whole cells of blue-green algae and lipopolysaccarides isolated from these cells were shown to stimulate the production of macro-(malnly) and microglobulln anti-bodies in rabbits. The macro- and microphage indices in rabbits increased significantly after the injection of LPS isolated from blue-green algae 24-48 hours before injecting the animals with a virulent Y pseudotuberculosis strain. Besides, the inhibiting action of this strain on the migration of phagocytes to the site of infection was abolished immediately after the injection. The use of the indirect hemagglutination test allowed to provide the absence of close antigenic interrelations -between blue-green algae and the following organisms: Spirulina platensis, Microcystis aeruginosa, Phormidium africanum and P. uniccinatum.

3. Duncan P.L. and Kiesius P.H., Journal of Aquatic Animal Health 8: 308-313, 1996 "Effects of Feeding Spirulina on Specific and Nonspecific Immune Responses of Channel Catfish" Administration of various immunostimulants to fish has resulted in enhanced immune responses. The purpose of this study was to determine if feeding Spirulina, a processed form of the blue-green alga Spirulina platensis, enhanced specific and nonspecific immunity and resistance against Edwardsiella ictaluri infection in channel catfish Ictaluras punctatus. Peritoneal phagocytes from fish fed Spirulina showed enhanced phagocytosis to zymosan and increased chemotakis to E. ictaluri exoantigen. No significant difference in mortality due to E. ictaluri existed between fish fed Spirulina and fish fed a basal diet. No significant difference in antibody titer or in the percentage of fish positive for E. ictaluri antibody was found between the two groups after immunization with formalin-killed E. ictaluri. Spirulina -fed fish had significandy higher antibody titers to key hole limpet hemocyanin KLH) on day 22, and a greater percentage of these fish were positive for KLH antibody on days 15 and 36. Feeding Spirulina enhanced nonspecific cellular immune responses such as chemotaxis and phagocytosis but did not provide protection against infection with E. ictaluri. The use of Spirulina in feed resulted in enhanced antibody responses to KHL, a thymus-dependent antigen, but not to E. iclaluri, a thymus-independent antigen. These results indicate that stimulation of the nonspecific immune system of channel catfish does not provide enhanced protection from E. ictaluri. 

4. Evets L.B. et al, Grodenski State Medical University, January 15, 1994 Russian Federation Committee of Patents and Trade, Patent Number: (19) RU (11) 2005486 Cl (51) 5 A 61 K35/80 "Means to Normalize the levels of Immunoglobulin E. Using the food supplement Spirulina". IgE of children living in highly radioactive areas is greatly above normal. Studies in 270 children show that consumption of about 5 grams per day of Spirulina tablets normalized IgE within 6 weeks. Children not consuming Spirulina did not change level of IgE. No side effects were observed. Spirulina lowers the amount of IgE in the blood, which in turn normalizes the allergies in the body.

5. Hayashi 0., Katoh T., Okuwaki Y., J. Nutri. Sci. Vitaminol., 40, 431-441, 1994, "Enhancement of Antibody Production in Mice by Dietary Spirulina platensis". Mice fed a Spirulina platensis diet showed increased numbers of splenic antibody-producing cells in the primary immune response to sheep red blood cells (SRBC). However, immunoglobulin G (IgG)- antibody production in the secondary immune response was hardly affected. The percentage of phagocytic cells in peritoneal macrophages from the mice fed S platensis diet, as well as the proliferation of spleen cells by either concanavalin A (Con A) or phytoheagglutinin (PHA) was significantly increased. Addition of a hot-water extract of S. platensis (S HW) to an in vitro culture of spleen cells markedly increased proliferation of these cells, whereas culture of thymus cells was scarcely affected. The Spirulina extract also significantly enhanced interleukin-l (W-l) production from peritoneal macrophages. Addition to the in vitro spleen cell culture of SHAW as well as the supernatant of macrophages stimulated with SHAW resulted in enhancement of antibody production, that is, an increase of the number of PFC. These results suggest that Spirulina enhances the immune response, particularly the primary response, by stimulating macrophage functions, phagocytosis, and IL-1 production. 

6. Kolman, A. eta', Toxicology Letters, 48 (1989)165-169 Elsevier, "Radioprotective effect of extract from Spiruilna phuensis in mouse bone marrow cells studied by using the micronucleus test. The Radioprotective effect of an extract of Spirulina platensis has been studied using the micronucleus test in polychromatic crytlirocytes of bone marrow of mice. In this system the extract caused a significant reduction of the micronucleus frequencies induced by gamma-radiation.

7. Qureshi M.A. and R.A. ALl, IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 18(3), 457-463(1996) "SPIRULiNA PLATENSIS EXPOSURE ENHANCES MACROPHAGE PHAGOCYTIC FUNCTION IN CATS". Bronchoalveolar lavage macrophages isolated form cats were cultured on glass coversups. Macrophages were exposed to a water soluble extract of Spirulina platensis in concentration range of 0 to 60 mg per mL for two hours. Spirulina-extract exposure did not cause significant macrophage cytotoxicity over untreated controls. Macrophage monolayers from treated and control cultures were incubated with sheep red blood cells (SRBC) as well as viable Eschenchia coli. The percentages of phagocytic macrophages for both of these particulate antigens were higher (a two-fold increase in SRBC phagocytosis and over 10% increase in Escherichia coli uptake) in cultures treated with various concentrations of Spirulina-extracL However, the numbers of either types of particles internalized by phagocytic macrophage were not different between the control and treated cultures. These data which showed that Spirulina platensis extract enhances macrophage phagocytic function imply that dietary Spirulina supplementation may improve the disease resistance potential in cats.

8. Qureshi M.A., Garlich J.D., Kidd MT., and Ali R.A. Poultry Science V.73, 5.1 p.46 #136, Immune Enhancement Potential of Spirulina platensis in Chicken" The effects of Spirulina platensis (SP) on the immune function of chickens were examined. Sephadex-elicited macrophage cultures were exposed to 10, 20 and 40 mcg/mL (v/v) water soluble extract of SP for one hour. These doses were not cytotoxic to Macrophages. SP-treated Macrophages exhibited an activated phenotype in terms of morphological changes, enhanced phagocytosis and induced tumoricidal factor secretion in culture supernatant. No change was seen in NK~ell activity. In the second study, 0, 10, 100 and 10,000 (10k) PPM SP in corn-soy diet were fed to K-strain Leghorns. Chicks in all groups had comparable body weights. However, larger thymi, higher NK activity and CBH response were observed in 10k SP-treated chickens. Enhanced secondary anti-SRBC antibody response was seen in 10K SP, Log2 (6.8) over 0 PPM (5.5) Leghorn chicks. These data suggest that SP exposure improves immune performance of chickens without adversely affecting other performance characteristics.

9. Qureshi M. A., DVM, Ph.D., Kidd M.T., Ph.D., and Ali R.A., M.S., Journal of Nutritional Immunology, No.3 (4)1995, pages 35-45, "Spirulina pt('tensis Extracts Enhances Chicken Macrophage Functions Mter in vitro Exposure" The effects of Spirulina platensis (Sp) extract exposure on chicken macrophages were examined. Sephadex-elicited abdominal exudate macrophage monolayers were exposed to varying concentration (10 to 40~g/~) of Sp for 1 to 16 hours. Spirulina-treated macrophages exhibited phenotypic changes in terms of increased spreading and vacuolization with minimal cytotoxicity. Percentage of phagocytic macrophages for unopsonized sheep red blood cells (SRBC) and average number of internalized SRBC was significantly higher in Sp-treated macrophages as compared to the sham-treated controls. However, phagocytosis of opsonized SRBC was not affected by Sp-treatment. Macrophage cultures exposed to Sp produced a factor in their culture supernatant with tumoricidal po~ntial which was similar in reactivity to the one produced by macrophages after exposure to lipopolysaccharide. The ability of splenic natural killer cells to kill tumor cell targets was not affected by Sp treatment. These finding suggest that Spirulina exposure enhances selected effect or functions of cells of the chicken immune system after in-vitro exposure.

10. Qureshi M.A., Mi R.A., Hunter R.L. Proceedings of the Forty-fourth Western Poultry Disease Conference, Sacramento, CA, pages 117-121, March 1995, Veterinary Extension, University of California, Davis, CA, USA, "1mmunomodulatory Effects of Spiruilna p1~Uensis Supplementauon in Chickens" The cyanobacteria Spirulina platensis (Spirulina) is commercially produced for human consumption as well as agricultural feed ingredient. We have shown that chicken macrophages exposed to a water-soluble Spirulina extract show enhanced phagocytic activity in vitro suggesting activation of mononuclear phagocytic system function. Furthermore, dietary supplementation of Spirulina (1,000 PPM to 10,000 PPM) improved thymic weights, enhanced CBH response, increased tumor cell killing by NK cells and doubled the macrophage phagocytic potential over chickens fed a basal diet. Chicks on 1.000 PPM Spirulina diet cleared significantiy more E. Coli from circulation at 30 (P=.05) and 40 (P--.005) minutes post i.v. inoculation of Staph. aureus in chicks fed 10,000 PPM over controls (P=.0273). Additional studies were carried Out to titrate the levels of Spirulina for optimum immune functions. Chicks were fed seven different levels of Spirulina from 250 PPM to 16,000 PPM in two fold increments per level. Chicks in groups from 1,000 PPM to 16,000 PPM Spirulina treatments showed enhanced CBH response. Chicks in 1,000 PPM group exhibited enhanced E. coli clearance between 30 to 60 minutes and decreased splenic bacterial counts at 80 minutes post inoculation. Data from these studies imply that Spirulina enhances several immunological end points in chickens both during in vitro and in in-vivo exposures.

11. Qureshi M.A., J.D. GARLICH, and M.T. KIDD "DIETARY SPIRULINA PLATENSIS ENHANCES HUMORAL AND CELL-MEDIATED IMMUNE FUNCTIONS IN CHICKENS" Cornell K-strain White Leghorns and br~er chicks were raised to 7 wks and 3 wks of age respectively, with diets containing various levels (0, 10, 100, 1,000 and 10,000 PPM) of Spirulina platensis from day of hatch. Chicks in all treatment groups had comparable body weights. While bursal and splenic weights did not change, the K-strain chicks had larger thymuses (P~.05) over the controls (0 PPM group). No differences were observed in anti-sheep red blood cells antibodies during primary response. However, during secondary response, K-strain chicks in all Spirulina dietary groups had higher total anti-SRBC titers with 10,000 PPM group being the highest (6.8 Log2) versus the 0 PPM (5.5 Log2) group. In broiler chicks, a one Log increase in IgG (p<.05) was observed in 10,000 ppm group over the controls. Similarly, chicks in 10,000 ppm Spirulina groups had a higher PIIA-P-mediated lymphoproliferative response over the 0 PPM controls. Macrophages isolated from both had higher phagocytic potential than the 0 PPM groups. Spirulina supplementation at 10,000 PPM level also increased NK-cell activity by two fold over the controls. These studies show that Spirulina supplementation increases several immunological functions implying that a dietary inclusion of Spirulina at a level of 10,000 PPM may enhance disease resistance potential in chickens.

12. Thang C. et al, Second Asia-Pacific Conference on Algal Biotechnology, April 25-27, p.58, "The Effects of Polysaccharide and Phycocyanin from Spirulina platensis variety on Peripheral blood and Hematopoletic System of Bone Marrow in Mice". The C-phycocyanin and polysaccharide from filamentary alga Spirulina platensis var. Were isolated and purified by an improved procedure. Shown to be homogeneous by polyacrylamide gel or agarose electrophoresis. The C-phycocyanin with a purity of 4.71 (A620/A277) possesses absorption maximum at 620 nm. Under denaturation (by 12% SDSpolyacrylamide slab gels), C-phycocyanin yielded two coloured polypeptides with the molecular weights of 14.5 and 15.0 KD for its alpha and beta subunits, respectively. This amino acid analysis showed that C-phycocyanin contained 14 amino acids, absent of histidine (His), proline (pro) and methionine (Met). The isoelectric point of C-phycocyanin was 4.8. The components of polysaccharide were determined by thin layer and gas chromatography and found to be L-rhamose, D-xylose, D-mannose, D-galactose, D-glucose and glucuronic acid in the proportions of 24.4%, 28.0%, 9.9%, 3.6%, 38.7% and 20.6%. TR spectroscopy showed that the polysaccharide had a betaglycoside linkage. The estimated molecular weight of polysaccharide is 21950.

By using the techniques of colony forming unit-erythroid (CFU-E) culture of fetal liver -cells in mice in vitro, we found that C-phycocyanin possessed high erythropoietin ~PO) activity. Per I 2.Sng C-phycocyanin matched with 850 mu EPO. The specific activity of C-phycocyanin was 56000u/mg C-phycocyanin. The effects of polysaccharide and phycocyanin on the peripheral blood and bone marrow hematopoietic stem and progenitor cell in normal, irradiated and anemic mice were studied. After normal mice was ingested periteally with C-phycocyanin or polysaccharide for 9 days, the blood picture had no significant change. The C-phycocyanin and polysaccharide can augment the proliferation of colony forming unit-granulocyte and macrophage (CFU-GM), colony forming unit-erythroid (CFU-E), burst forming unit-erythroid (BFU-E) and colony forming unit-fibroblast (CFU-F), promote the hematopoiesis of bone marrow in normal. The C-phycocyanin and polysaccharide can markedly enhance the colony forming stimulating activity in normal mice serum. The prepared serum-free spleen cell conditioned medium stimulated by C-phycocyanin or polysaccharide in vivo and vitro provided powerfully increase in CFU-GM colony formation. After in vitro addition of C-phycocyanin or polysaccharide, there was pronounced increase in number of CFU-GM, CFU-E and BFU-E. Simultaneously, C-phycocyanin can induce CEU-E and BDU-E colony formation in absence of EPO. These studies demonstrate the unique capacity of C-phycocyanin and polysaccharide to influence the differentiation and proliferation of committed hematopojetic progenitor cell possibly by modulating the production of such factors required for hematopoietic differentiation and purification.

Stimulated recovery by C-phycocyanin and polysaccharide was observed in leukocyte and bone marrow nucleated cell counts and the number of CFU-GM colony formation in mice after single wholebody ⁶⁰Co gamma-ray irradiation. The C-phycocyanin and polysaccharide can lower the anemic degree of mice combined treatment of ⁶⁰Co gama-ray irradiation and benzohydrazine hydrochloric acid peritoneal injection

13. Portoni B., Smith N., Dixon B., Kawanigashi D., American Fisheries Society, Fish Health Section, Madison Wisconsin August 7, 1996 "Immune Response Activation in Channel Catfish Ictaluras punctatus Fed Spirulina platensis Enriched Artemia". The cyanobacteria Spirulina spp. is commercially produced for human consumption as well as agricultural feed ingredients. Recently, whole Spirulina and cell extracts were found to enhance immunity in animals by increasing phagocytic activity.

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