Spirulina in combating Protein Energy Malnutrition ( PEM ) and Protein Energy Wasting ( PEW )-A review

Spirulina, is a simple extract of blue-green algae, which is now used worldwide as a food product and as a dietary supplement. It contains, essential amino acids, lipids, vitamins, minerals and anti-oxidants and can be considered as a wholesome food supplement. Spirulina contains, approximately, 65% to 71% protein by dry weight and is claimed to be non toxic nutritious food with exceptional properties. A large amount of scientific literature available about Spirulina and its usage in treatment of child malnutrition, nutrition rehabilitation of HIV-infected, cancer patients, hepato-protective effects etc. However, there is no specific review available which gives more emphasis on the protein and energy content and its effects. In the present work, we reviewed several papers and reports and paid more attention on protein content, which is the major constituent of Spirulina and its effect on various disease conditions and possibility of using Spirulina in combating against Protein Energy Malnutrition (PEM) and Protein Energy Wasting (PEW). This work is of certain significance for nutritionists, doctors and public health workers involved in combating malnutrition. The risks involved and optimal intake level for humans and animals are discussed in detail. Keywords: Protein Energy Malnutrition (PEM):Protein Energy Wasting (PEW):Spirulina¸ Toxicity.


Introduction
It is not accurately known when people started using microalgae as food source or food supplement but the first recorded evidence is from Bernal Diaz del Castillo, a member of Heman Cortez"s (Spanish conquistador) troops, reported in 1520, in Tenochtitlan (today Mexico City):that S. maxima was harvested from the lake Texcoco, dried and sold for human consumption.Native Mexicans called it as Tecuitlalt, meaning "excrements of stones".The topic of the Tecuitlalt, which was earlier discovered in 1520, was not mentioned again until 1940, the French phycologist Pierre Dangeard mentioned about a cake called "dihe", consumed by Kanembu tribe, African Lake Chad, Kanem (Chad, Africa).Dangeard studied the "dihe" samples and found that it is like a puree of spring form blue algae (Sánchez et al. 2003).
The first unialgal culture achieved by Beijerinck in 1890 and cultivation only started in 1919 by Otto Warburg"s (Richmond 2008).Otto Warburg is well known for his work on Chlorella and his aim is to understand and use them as a model for physiology and photosynthesis research and not as a potential food source.He used microalgae, Chlorella, because of its fast growing, nonmotile and simple life cycle properties.Warburg's study was important because he was able to understand the number of quantas required to possess photosynthesis and he also proposed the concept of light utilization efficiency and conversion of light to chemical energy (Richmond 2008).During 1964 and1965, the botanist Jean Leonard, during his Belgian Trans-Sabaran Expedition, confirmed that dihe is made up of Spirulina and thus chemical analysis was started on Spirulina (Sanchez, 2003).During that time, Léonard received a request from Sosa-Texcoco Ltd, Mexico to study a bloom of algae in their sodium hydroxide production facility.As a result, the first systematic and detailed study of the growth requirements and physiology of Spirulina was performed.This study, which was a part of Ph.D. thesis by Zarrouk (1966):was the basis for establishing the first large-scale production plant of Spirulina (Habib et al. 2008).
A single cell protein, which is nothing but a protein derived from culture of single celled organisms, has gained popularity as alternative food source during World War 1 and World War II.After the formation of United Nations in the post war period, hunger and malnutrition problems were highlighted by the Food and Agriculture Organization of United Nations and has introduced the concept of protein gap and reported that 25% of world"s population has a deficiency of protein intake in their diet.Many research projects on yeast, chlorella, Spirulina, some bacteria and moulds for large scale production of "single cell proteins" were launched.In 1950, the United States and Japan began the experimental cultivations of this microorganism to investigate its chemical composition and industrial applications.Studies were accelerated after the release of the book, Algal Culture from Laboratory to Pilot Plant (Burlew, 1953):which triggered research work around the globe (Richmond, 2008).Japan was the first country to produce Chlorella based diet food (Sanchez, 2003).Spirulina, in 1967 was established as a "wonderful food source" by the International Association of Applied Microbiology.
The first pilot plant which produced 150 tonnes of dry Spirulina biomass per year started production in 1973; its production capacity was thereafter raised to 300 tonnes of medium-grade product per year from 12.0 hectares of natural ponds by Sosa-Texcoco Ltd, Mexico.The annual value of Spirulina production represented a third of the company's income from the manufacture of powdered soda from the lake deposits.In 1995, Sosa-Texcoco ceased production of Spirulina.The only remnant today, Lake Texcoco, still has a living algae Spirulina population.
From 1970, the nutritional and medicinal studies on Spirulina have been extensively studied along with its applications in water treatment.According to the national report received by Food and Agriculture Organization (FAO):United Nation, the production of algae culture was greater than 68000 tons in 2008 and major contribution from China and Chile.China started to produce Spirulina through factories in 1990 and there were more than 80 factories by 1997 (Habib et al. 2008).Spirulina is produced in at least 30 countries (Australia, Bangladesh, Benin, Brazil, Burkina Faso, Chad, Chile, China, Costa Rica, Côte d'Ivoire, Cuba, Ecuador, France, India, Israel, Italy, Japan, Madagascar, Martinique, Mexico, Myanmar, Philippines, Peru, Portugal, Spain, Thailand, Togo, United States of America and Vietnam) (Habib et al. 2008).

Benefits of Spirulina
Spirulina does not need fertile land and has an advantage of having rapid growth within 20 days, takes less energy input and less water per kilo than soya, corn proteins and is environmental friendly as there is possibility of recycling water after harvesting and produce more oxygen than trees per acre by consuming carbon dioxide (H.E.Remigio M. Maradona 2008).
Very few studies were found to describe the actual respiration rate of Spirulina sp. and in the below analysis, we could able to collect some information to prove Spirulina produces more oxygen than trees per acre.The notations used to measure trees size is "dbh".dbh refers to the tree diameter measured at 4.5 feet above the ground and based on a study conducted on trees and their oxygen release rates by David et al. (2007):trees with 1 -3 inch -dbh produce 2.9 kg O2/per year; 9-12 inch dbh: 22.6 kg O2/year; 18-21 inch dbh: 45.6 kg O2/year; 27-30 inch dbh; 91.1 kg O2/year and greater than 30 inch dbh 110.3 kg/O2 year.With reference to the data reported by Dinesh et al. (2010):after calculations, 42 ft 2 area with 1000 L can produce approx.20.717 kg oxygen per year with cell concentration varying from 1 * 10 5 to 5 * 10 5 cells per ml and 200 ft 2 area with 4000 L, can produce approx.100 kg oxygen per year, if cell concentration per ml increases, still increase in the rate of oxygen release can be achieved.With reference to the respiration rate reported by Rym (2012)

Spirulina and its nutritional composition
The common name, Spirulina, refers to the dried biomass of Arthrospira platensis, (Gershwin and Belay 2007):which belongs to the oxygenic photosynthetic bacteria that cover the groups Cyanobacteria and Prochlorales.These photosynthetic organisms, Cyanobacteria, were first considered as alge until 1962 and for the first time, these blue green algae were added to prokaryote kingdom and proposed to call these microorganisms as Cyanobacteria (Stanier and Van Neil 1962):where algae is considered to be a very large and diverse group of eukaryotic organisms.This designation was accepted and published in 1974 by the Bergey's Manual of Determinative Bacteriology, which is worldwide considered as a bible for biologists (Sánchez et al. 2003).Scientifically, there is a quite distinction between This common difference between scientists and customers is difficult to change (Sánchez et al. 2003).These Arthrospira genus, constitute a helical trichomes of varying size and with various degree of coiling including tightly coiled morphology to even straight uncoiled form.The filaments are solitary and reproduce by binary fission and the cells of the trichomes vary from 2 µm to 12 µm and can sometime reach up to 16 µm.Species of the genus Arthrospira have been isolated from alkaline brackish and saline waters in tropical and subtropical regions.Among the various species included in the genus Arthrospira, A. platensis is the most widely distributed and is mainly found in Africa but also in Asia.Arthrospira maxima is believed to be found in California and Mexico (Gershwin 2007).
Since, 1970, Spirulina was analyzed physically and chemically and numerous properties were evaluated.Based on the nutrition profile listing of various multi-national global players (Table 1), Spirulina contains approximately 65 to 70% proteins, 15 to 25% carbohydrates, 4 to 9 % fats and remaining vitamins, minerals, pigments and very few toxic contaminations.In this article, the main focus is review of all the works carried out on protein content for combating against Protein Energy Malnutrition (PEM) and Protein Energy Wasting (PEW) around the globe is discussed in detail.

Comparision of protein content in Spirulina with other foods
Spirulina contains more natural proteins when compared with other natural foods (Table 2).The true protein digestibility and the biological activity of Spirulina protein calculated by Narasimha et al. (1982) is 75.5 and 68 respectively.The Recommended Dietary Allowance (RDA) for protein consumption is 0.8g/kg body weight and for athletes, RDA ranges from 1.2 to 1.4 g/kg/day (Otten et al. 2006).The advantage of Spirulina protein can withstand without denaturating up to 67 0 C (Sánchez et al. 2003).

Risk assessment of Spirulina
Extensive care should be taken while consuming or prescribing Spirulina as a protein source.Spirulina has more vitamin A, in terms of beta carotenes, when compared with any natural foods and care should be taken while administrating Spirulina that it should not exceed the Recommended Dietary Allowance (RDA) of 400 mcg to 900 mcg for normal adult male or female, where as lactation stage, it can go up to 1300 mcg per day.Daily intake of Vitamin A >25,000 IU for >6 years an >100,000 IU for >6 months are considered toxic (Penniston and Tanumihardjo 2006;Fairfield and Fletcher 2002).100 grams of Spirulina, contains greater than 353000 IU (Gershwin and Belay 2007) and based on the recommended dietary allowance of Vitamin A, it is recommended to consume only 7 grams of Spirulina per day which contains 25000 IU and children less than 6 years to 6 months can take 25 grams of Spirulina per day.
Excessive consumption of vitamin K, up to 0.2 g/kg body weight does not show any toxic effect on rats (Molitor and Robinson 1940) and recommended RDA for vitamin K is >19 years, 90 mcg per day and >6 months to 12 months, 2.5 mcg per day and it varies between 30 mcg to 60 mcg for 1 to 19 years.With reference to these values, <1 year to 6 months kids, it is recommended to give 4 grams of Spirulina per day for adults which contributes about 90 mcg vitamin K and < 2 grams for 1 year to 6 months kids.Based on the composition, other vitamins are within RDA limit per 10 grams of Spirulina powder.After observing, RDA for adults for minerals, calcium (1300 mg):iron (10 mg):iodine (150mcg): phosphorus (700mg):Magnesium (420 mg):Zinc (11 mg):Selenium (0.055 mg):Copper (0.9 mg):Manganese (2.3 mg):Boron (1000 to 10000 mcg):Germanium (1.5 mg) (Schauss 1991):Potassium (4700 mg) and Sodium (1500 mg):most of them are within the range for consumption of 100 grams of Spirulina.Based on RDA values for chromium (35 mcg) and molybdenum (45 mcg) are safe for 10 grams of Spirulina per day.Coming to the pigments, phycocyanin at 0.25 to 5.0 g/kg body weight have not shown any toxic effect in rats (Naidu et al. 1999) and GLA can be consumed up to 1.6 g per day.RDA recommends 30 to 45 grams of carbohydrates per meal and maximum of 195 grams per day and maximum of 40 % of calories coming from carbohydrates and fats about 20 to 60 g per day for an average adult (Dietary Reference Intakes, 2004).
Overall, based on the complete nutritional assessment, normal healthy adult can take < 4 grams of Spirulina per day.But, it is found that there is no toxic effect on rats, when consumed greater than 0.8 g/kg of pure Spirulina powder (Krishnakumari et al. 1981).Based on the nutritional facts and composition and RDA values, it is recommended to take < 4 grams of Spirulina per day for healthy adult due to the presence of excess Vitamin A and not more than 10 grams as it exceeds the RDA values of heavy metals (Table 3).
The black color in the Table 3 shows that the particular nutrient exceeds the RDA value.It was not colored black at 4 grams at Vitamin A and Vitamin B, even though they are exceeding the RDA values as the Vitamin A, upper limit is 7500 mcg and Vitamin B, there is no upper limit (Dietary Reference Intakes, 2004).Chromium and vitamin A are exceeding RDA values at 10 grams of Spirulina and at 15 grams consumption, molybdenum, vitamin K is exceeding and at 25 grams, Iron was exceeding RDA value and at 100 grams, protein, manganese, phosphorous, vitamin B2 are exceeding and at 200 grams, vitamin E, B3, B6, copper, iodine and magnesium are exceeding and at 500 grams, calcium, vitamin B1, potassium and sodium are exceeding RDA and it is strongly recommended to consume less than 4 grams per day for an average healthy adult to avoid any toxic effect based on the above scientific data.Care should be taken while consuming and data related to cobalt and fluorine in water used for Spirulina production was not reported by many companies and these contents will also affect the quality of Spirulina along with arsenic, lead, cadmium, mercury and silicon concentrations.
We believe that the above Table 3 will give basic idea about Spirulina and risks involved in consumption and industries should take care while preparing formulations or food fortification products using Spirulina.

Protein Energy Malnutrition (PEM)
The World Health Organization (WHO) defines malnutrition as ""the cellular imbalance between the supply of nutrients and energy and the body"s demand for them to ensure growth, maintenance, and specific functions" and Protein Energy Malnutrition (PEM) refers to a form of malnutrition, where there is inadequate calorie or protein intake.Malnutrition is present in both developed as well as under developing nations.Due to lack of adequate food supply caused by socio-economical, political and environmental factors, malnutrition was prevalent in developing countries and in developed countries 6 to 51% of hospitalized children were found to be malnourished (Grover and Ee 2009).

Protein Energy Wasting (PEW)
Wasting refers to the process by which a debilitating diseases cause"s muscle and fat tissue to waste "away" and it is also referred to acute malnutrition.Protein Energy Wasting (PEW): term was coined by the "International Society of Renal Nutrition and Metabolism (ISRNM)" to address the syndromes of muscle wasting, malnutrition and inflammation during Chronic Kidney Diseases (CKD) orAcute Kidney Injury (AKI) and it also means loss of body protein mass and fuel reserves (Fouque et al. 2008).

Spirulina: PEM and PEW
The idea of using Spirulina to combat against malnutrition i.e. hunger alleviation, was conceived in 1984 by Fox RD, in his work on fighting against malnutrition with Spirulina with various available technologies.He also promoted villagers to grow Spirulina from recycled village wastes and proposed to use Spirulina as concentrated nutritional food supplement to increase immuno-resistance against infectious diseases (Fox 1985).Henrikson (1989):in his book on "Earth food Spirulina", explained various nutritional properties, clinical studies and in recent updated edition released in 2010, he has also included chapters on alge for bio-fuel production and interesting recipes.S. platensis is described as a good source of complementary diet to prevent malnutrition in developing countries (Kim 1990).The idea of cultivation of Spirulina or any other alternative protein source, for reducing the incidents off hunger, starvation, and malnutrition was suggested by Rodulfo (1990).Bucaille (1990) studied the effectiveness of Spirulina algae as food for children with protein-energy malnutrition.Many experiments were done on rats to prove the renoprotective properties of Spirulina (Table 10).

France
The use of Spirulina as a possible feed for aqua culture was demonstrated in the study by growing tilapia, small pelagic fishes, shrimp, and mollusks in a series of artificial canals.
Aqua culture Fox 1999

Kenya
Five spirulina cultivation sites were selected and the possibility for further development and promotion were evaluated in the project.It is found that is Significant efforts need to be made to improve the scope of production, bringing nutrition experts and NGO"s at national level.

Production Harris 2010
Brazil Spirulina was proved to be an adequate protein source for recovery of body weight and muscle protein of protein malnourished rats.

Rats Voltarelli 2011
India Value added extruded product with 5% Spirulina + 95% Wheat flour + 5% Corn flour was developed and sensory parameters like taste, odour, texture, color, appearance were found to be at acceptable level.
Different formulations of cassava cake were developed varying the concentration of Spirulina platensis and cassava bran.Based on the sensory tests, the product received excellent acceptance level.

Iran
The Spirulina platensis and Chlorella vulgaris were incorporated into probiotic fermented milks to increase the functional properties.

Product development Beheshtipour 2013 Indonesia
The aim of the work is to develop a product for local production of Spirulina in Indonesia and promoting amoung uneducated Indonesian fishermen.

Production Van Koolwijk 2014 Egypt
Sixteen food formulas were prepared for as complementary food babies (1-3 years age) by using spirulina at 0, 2.5 0.5 and 7.5% for the production of two types of baby food one of them is ready to eat by using some fruits and vegetables and evaluated.

Algeria
The product formulation composed by 2/3 of jujube syrup, 1/6 Spirulina water extract and 1/6 natural lemon juice was found to be best and further analysis of nutrients was done.The formulation reveals satificatory microbiological quality and also allows exploration of the Ziziphus jujuba fruit which is in extinction in Algeria.

Benahmed et al. 2014
Arab League Five blends were prepared with one control and other blends with varying spirulina concentration from 2.5 to 10% and properties like taste, texture, odour, nutritional composition, physical and functional properties and microbiological properties were evaluated.

Morsy et al. 2014
France The phenomenon of social conversion by farmers from traditional agricultural systems to Spirulina production was explained in detail along with the impact on conversion.

Production Stéfanini 2015
India High saline (0.4M NaCl) and low nitrogen (<0.01 M NaNO3) significantly increased the carotenoid production in Spirulina platensis, which may be resulted due to excessive formation of free radicals under stress.

PEM due to hunger
There are two types of hungers, one is hunger due to lack of food and other is lack of micronutrients, "hidden hunger" (Bindu and Channarayappa, 2014).Lack of adequate food sources in under developing countries and lack of proper nutritional awareness in developed countries, resulted in micronutrient deficiency and malnutrition.The self sufficiency in some developing countries was achieved by increasing the production of cereal crops but it resulted in decrease in the production of pulses which are main sources of protein (Babu and Rajasekaran 1991).This may be the prime cause of malnutrition in developing countries.
Spirulina can be considered as a best source of protein in terms of gram protein per cultivatable land ratio but major problem faced in developing countries is the acceptance level of this super food into daily recipes.Various studies from 1991 on social acceptance of algal supplements as alternative food, cost effectiveness in growing Spirulina in developing countries, field and clinical studies on human population were done by many researchers (Table 4 and Table 5) to understand and alleviate hidden hunger and malnutrition (Babu and Rajasekaran 1991) and provide food security.Few studies on each and every micro nutrient were tabulated in Table 6 and Spirulina as an alternative cheap feed for animals and aqua culture was also tabulated in Table 7.
In 1989, Earthrise Farms, USA, tried popularizing granular bars.Mass cultivation, process optimization and economic analysis for growing Spirulina was done at Santaigo production facility, Chile (Valderrama et al. 1987)  The experiments done on rats showed that the biological values of alga were acceptable and also established in his work that dried spirulnia contain more β-carotene (pro-vitamin A).

Rats
France Clement et al. 1967 Spirulina (0 to 26.7%) was fed to male rats for 6 weeks.At low levels of Spirulina feed (<2.7) and high level (>10.7%)caused reduction in plasma, liver and heart α-tocopherol.Liver retinoid levels decreased when fed with >10.7% and < 10.7 % there is in increase in retinoids.
Spirulina was found to significantly alter the storage and utilization of Vitamin A.
Rats USA Mitchell et al. 1990 The absorption of β-carotene in Spirulina fed rats was found to be low when compared with rats fed with synthetic β-carotene.Spirulina fed rats have not shown dosage related increase in Vitamin A in liver and serum but the vitamin A storage was found to be much higher than expected.
Rats India Annapurna et al. 1991 The initial loss of beta-carotene on spray drying were between 7 to 10% and on storage in colored bottles containing air, more than 50% loss was observed in <45 days.Flakes (>20 mesh size) retain 52% and spray dried fine powder (100 mesh) retained 34%.The authors recommended producing dry alga in the form of flakes or granules to retain beta carotene.Sodium metabisulphite (0.1 to 1.0%) as an antioxidant can be used to retard degradation rate of beta carotene.
The opened containers should be exhausted within 15 days of purchase and minimum air/oxygen access is required to retain betacarotene.

India Seshadri et al 1991
Spirulina based on β -carotene level (equivalent to 60 µg/d and 120 µg/d) was fed to vitamin A depleted rats for 10 days.The Spirulina fed group showed better growth.
Rats India Kapoor and Mehta 1993 Hexachlorocyclohexane (HCH) (1000 ppm) was mixed with Spirulina (0.0628% and 3.18%) and fed to male albino rats for 7 weeks.Growth rate reduced but body weight increased at the end of seventh week.
The ameliorating effects of alga on the dietary toxicity of HCH in retinol deficient albino rats were established.

Human
The Netherlands Dagnelie et al. 1991 No difference in body weight gain, relative liver, or relative kidney weight could be found in male weaning Wistar rats fed with Spirulina for four weeks.The rats were initially feed for 6 weeks, vitamin B-12 deficient diet.These data illustrate that cobalamins from algae are indeed absorbed by the rat but distribution pattern over liver and kidneys indicates that at least part of the cobalamins, measured by a specific radioassay, may actually be analogues.

Rats The Netherlands
Van den Berg 1991 Vitamin E Spirulina (1500 mg/kg/day) and Vitamin E (50mg/kg/day) was fed to adult female albino rats of wistar strain weighting between 180 and 220 grams for 6 weeks.The lens soluble protein, glutathione and water content profiles show the preventive role of Spirulina and Vitamin E in naphthalene-induced cataract in rats.

Rats India Haque and Gilani 2005
Calcium Novel sulfated polysaccharide (named calcium spirulan (Ca-SP)) alga Spirulina platensis was extracted using hot water by bioactivitydirected fractionation.The unique polysaccharide was composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate, and calcium.Ca-SP was found to inhibit the replication of several enveloped viruses, including Herpes simplex virus type 1, human cytomegalovirus, measles virus, mumps virus, influenza A virus, and HIV-1.
Inhibitor Japan Hayashi et al. 1996 These results suggest that Ca-SP, a novel sulfated polysaccharide, could reduce the lung metastasis of B16-BL6 melanoma cells, by inhibiting the tumor invasion of basement membrane probably through the prevention of the adhesion and migration of tumor cells to laminin substrate and of the heparanase activity.
Inhibitor Japan Lee et al. 1998 Ca-SP at 20 μg/ml or less may retard the repair process of damaged vascular endothelium through inhibition of vascular endothelial cell proliferation by induction of a lower ability to respond to stimulation by endogenous basic fibroblast growth factor.
Inhibitor Japan Kaji et al. 2002 Iron Ingestion of daily dose of Spirulina (10 g) recommended for human consumption by the commercial source would provide up to 1.5 to 2 mg absorbed iron.

USA Johnson and Shubert 1986
The absorption of iron from Spirulina was significantly lower than that of ferrous sulphate and whole egg but significantly greater than that from whole wheat.

Rats India Kapoor and Mehta 1992
Spirulina might promote the growth rate of Iron Deficiency Anemia (IDA) rats and there was an repletion effect of Spirulina on IDA rats.

Rats
China Jiangming et al. 1994 The pregnant and lactating rats fed with Spirulina + wheat gluten (22% protein equivalent) showed significant higher iron storage and hemoglobin content then casein + wheat gluten diet.

Rats
India Kapoor and Mehta 1998 In vitro digestion/Caco-2 cell culture system was used to measure the iron Spirulina availability.6.5-fold increase in iron content using Spirulina digest in comparison with meat was observed.

Iron Availability
France Puyfoulhoux et al. 2001 Spirulina (3 grams/day) is supplemented for 12 weeks to 40 people with >50 years age both male and female.Increase in hemoglobin is found after 12 weeks and increase in Complete cell count (CCC) and indoleamine 2,3-dioxygenase (IDO) enzyme activity was observed.Spirulina may ameliorate anemia and immunosenescence in older subjects.

Human (>50 years)
USA Selmi et al. 2011 Iodine Spirulina was grown in 10 -8 to 10 -4 g/l Potassium Iodide (KI) and 0.5 to 15 mg/L Selenious acid (H2SeO3) and bioaccumulation was observed for pharmaceutical formulation purpose.The increase of selenium and iodine accumulation is observed at maximum 13 mg/L and 500 mg/L concentrations respectively and polynomial equation to explain the accumulation was also developed.If iodine content in medium is 500mg/L, then iodine concentration in biomass 2 mg/L, total lyophilized biomass is 0.8 g/l and iodine enrichment coefficient is 0.4%.(Branger et al. 2003) (Table 5).
Spirulina was administered up to 90 g/day to under nourished children at the Hôpital Bichat, France and in spite of heavy dosage, absorption levels of proteins were found good (Santillan 1974).Spirulina was administered 2 to 3 grams per kg body weight to 5 to 10 month"s old children and observed that the protein retention rate is high (Proteus Inc. 1975).1.5 grams of Spirulina mixed with 12 g baked barley sprout, vitamin B1 and zinc recovered children from diarrhea and constipation (Miao 1987).Many such studies on children with protein energy diseases (Bucaille 1990):malnutrition (Simpore et al. 2006):Vitamin A deficiency (Seshadri 1993):nutrition for healthy children (Ramesh et al. 2013;Masuda et al. 2014;Dewan 2014) were done along with special studies like using C 13 stable isotope along with Spirulina to understanding the kinetics of protein breakdown (IAEA 1992).Still further studies on Spirulina are required to establish the optimal concentration level for each disease conditions and malnutrition.Various studies on micronutrients and role of Spirulina in human body is listed in Table 6.
According Since 1967, many researchers tried establishing Spirulina as a major source of vitamin A with highest bioavailability of betacarotene in rats (Clement et al. 1967;Mitchell et al. 1990;Annapurna et al. 1991;Kapoor and Mehta 1993;Venkataraman et al. 1994; Garbuzova-Davis and Bickford 2010):humans (Wang et al. 2008;Li et al. 2012) and aqua culture (Regunathan and Wesley 2006).The studies on loss of beta-carotene during storage, spray drying and other unit operations was done by Seshadri et al (1991).The studies for increasing the productivity of beta-carotene by changing the medium composition of Spirulina were also done (Sujatha and Nagarajan 2013).
Based on their studies on bioavailability of Vitamin B12 in humans (Herbert and Drivas (1982) and Dagnelie et al. (1991)) it is suggested not to use it as a source of vitamin B12 for children with B12 deficiencies as it contain non-cobalamin Vitamin B-12 analogues that are unavailable to humans.Similar results were also supported by Van den Berg (1991).Studies on Vitamin K, were not available and studies on Vitamin E are found to be less (Haque and Gilani 2005;Mendiola et al. 2008).Calcium spirulan (Ca-SP)) from Spirulina platensis was isolated in Japan in 1998 and since then enormous amount of research was done on it, against various diseases including HIV, measles virus, mumps virus, influenza A virus, and cancer (Hayashi et al. 1996;Mishima et al. 1998;Lee et al. 1998;Kaji et al. 2002).Research on Spirulina as a source of iron for healthy and anemic rats were done since 1986 (Johnson and Shubert 1986;Kapoor and Mehta 1992;Jiangming et al. 1994;Kapoor and Mehta 1998;Puyfoulhoux et al. 2001) and aged population in 2011 (Selmi et al. 2011) and exact bioavailability values were also established (Table 6).
Magnesium fortification of Spirulina does not improve the magnesium bioavailability (Planes et al 2002) but further studies on this area are required and bioenrichment of Spirulina with high concentrations of iodine (Mosulishvili et al. 2002):zinc and selenium (Wang and Songgang 1998) significantly improved the mineral concentrations.Spirulina with zinc can be used for treatment of chronic arsenic poisoning with meelanosis and keratosis (Misbahuddin et al. 2006).The bioavailability values of selenium were available in Table 6 (Cases et al. 2002).Purified selenium from Spirulina can be used as strong antioxidant and have potential applications in chemoprevention (Chen and Wong 2009).Large corporations are required to promote research on Spirulina for establishing exact bioavailability values of micronutrients so that Spirulina can be used for enteral nutrition in malnourished patients.Overall, based on the above data, Spirulina can be used as a source for alleviating hidden hunger or micronutrient deficiencies in children but care should also be taken while administrating Spirulina to the patients by referring to Table 3.

Spirulina as an alternative feed for animals and aquaculture
Spirulina as an alternative feed for animals and aqua was reported in Table 7. Cyanobacteria as a source of food for aqua culture were established by Fox (1999).A system including Spirulina, Artemia and mangrove fauna was used for producing tilapia, small pelagic fishes, shrimp, and mollusks in artificial canals with circulating filtered sea water.Spirulina was used as a feed for poultry, pig, cattle and many other animals and aqua culture.Various studies on Spirulina as an alternative feed for animals and aquaculture were listed in Table 7.
After considering the listed facts in Table 7, Spirulina can be fed up to 10 % for poultry (Ross and Dominy 1990) and less than 4% for Quail (Ross et al. 1994).Increase in the Spirulina content up to 40g/kg for 16 d in 21 day old boiler male chicks, resulted in yellow and red coloration of flesh and this may be due to the accumulation of the yellow pigment, zeaxanthin (Toyomizu et al. 2001).Pigs (Nedeva et al. 2014):rabbits (Peiretti and Meineri 2008) and lambs (Holman et al. 2012) can receive up to 10% of the feed and increase in the Spirulina content in cattle resulted in increase in milk yield and weight (Stanley and Jones 1976;Kulpys et al. 2009;Heidarpour et al 2011).Spirulina as an alternative feedstock and immune booster for various types of fish including big mouth buffalo, (Stanley and Jones 1976):milk fish (Santiago et al. 1989):cultured striped jack (Shigeru et al. 1991):carp (Ayyappan 1992;Ramakrishnan et al. 2008):red sea bream (Mustafa et al. 1994):tilapia (Olvera-Novoa et al.Spirulina (0 to 8 %) in pelletized form was fed to shrimps.The growth, survival and pigmentation were considerably more when compared with single cell ingredient sources like lactic yeast.

Shrimp
Cuzon et al. 1981 The Philippines Wild milkfish fry (90/m 2 ):were fed with Spirulina and formulated diet.The stocking rate was 92.5/m 2 after 7 weeks and the Spirulina fed fish gave weight increment (0.881±0.140 g).

Milkfish
Santiago et al. 1989 USA Spirulina (0 to 20%) was fed to poultry and found that day old chicks, after 3 weeks, the growth rate reduced for 10% and 20% spirulina feed and >12% diet receiving Hubbard male boiler chicks after 41 days, a slight decrease in growth was observed but there is no significant difference due to the Spirulina and concentration level up to 10% except increase in color of yolk and fertility rate.

Poultry
Ross and Dominy 1990

Japan
The levels of carotenoids were increased by supplementation of S. maxima 5 to 10% diet to Cultured striped Jack Caranx delicatissimus.The red sea bream were fed with 2% Spirulina for 95 days and elevated protein assimulation and increased stromal fraction was observed.

Mustafa et al. 1994 USA
There was a consistent increase in yolk color with increase in concentration from 0% to 4% of freeze dried spirulina in quails fed for 8 weeks.Yolk color increased more in freeze-dried Spirulina when compared with extruded Spirulina.
Quail Ross et al. 1994 USA Spirulina and its potential applications as an animal feed were reviewed.Aqua Culture

Belay et al. 1996 South Africa
Juvenile Haliotis midae, Port Alfred, South Africa, were fed red alga (Pfocamium corallorhiza) for 3 months before the experiment.Spirulina based diet (19% protein) was fed for 124 days along with 4 other feeds (casein, fishmeal, soya oil cake and torula yeast).fishmeal and Spirulina spp.algae are found to be most suitable proteins for inclusion in practical diets for H. midae.

Abalone
Britz 1996 México Spirulina (20 to 100%) diet along with animal protein was given 6% of their body weight to tilapia in a closed recirculating system.After a 9 week feeding period, the growth rate and protein utilization increased in 20% and 40% spirulina diet.Further increase in Spirulina, decreased the growth.

Lithuania
The Lithuanian black and white cows in their early lactation period were fed with 200 g of spirulina per day for 90 days.The cow"s receiving Spirulina became 8.5 to 11 % fatter and gave 34 kg milk per day in the beginning of their lactation and it is found to be 6 kg more than those of the control group.
Cow Kulpys et al. 2009 Taiwan White shrimp (Litopenaeus vannamei):were given Spirulina (6 to 20 µg/g):which is earlier hot water extracted and compared with normal spirulina (200 to 600 mg/L) for 24 to 96 hours.Shrimp that received the hot-water extract of S. platensis had enhanced innate immunity and increased resistance against V. alginolyticus infection.

Shrimp Tayag et al 2010
Iran Twenty four Holstein calves were given Spirulina platensis 0 g to 25 g per day for 57 days.The results showed that treatment effect was not significant on the final weight, daily gain; daily feed intake, feed efficiency and digestibility coefficient below 25 g.Increase in the level up to 25 grams, decrease in digestibility in terms of crude protein, dry matter, neutral detergent fiber, and organic matter were observed.However, reduction in plasma cholesterol, LDL, HDL concentration was found and there is no effect on other blood parametters like BUN, albumin and globulin.

Catfish
Promya and Chitmanat 2011 Turkey Spirulina (0 to 10%) was fed to fish (3.75±0.02g)for 12 weeks.The specific growth rate, feed intake, total egg production, hatching rate of eggs was found to be higher.The yellow and blue coloration of the yellow tail cichlid and carotenoid in skin was enhanced.
Yellow tail Güroy et al. 2012 Australia 24 weaned lambs (purebred Merino dams sired by Dorset, White Suffolk, Black Suffolk and Merino rams lamb) weighing 37.5±5.2kg, 42 days old were fed with 0 to 20 wt/vol Spirulina for 6 weeks.Lambs on Spirulina levels of 10% recorded the highest mean live weight of 41.9 ± 0.7 kg and lambs with 20% did not significantly improve when compared to the control group (0%).
lamb Holman et al. 2012 France Spirulina platensis was given as a sole food for zebrafish broodstock, egg production was found to be lower but survival rate (73%) was higher when compared with commercial feed (55%).No difference in egg and larval weight and size was observed and the larval survival rate of 69% at 31 days post fertilization was observed.The spirulina based diet is recommended for zebrafish larvae in the first few days of life.

Zebrafish Geffroy 2013
Bangladesh Studies were done on the growth performance, feed utilization and body composition of fiingerlings of stinging cat fish and effect of spirulina.

Cat fish Ali 2014
Bulgaria 48 Danube white pigs, weighing 12.15 to 12.471 kg were given 2 to 3 g per day for 47 days.The weight increased to 30.9 to 33.9 kg and significant increase in growth intensity from 12.50% to 14.25% was observed.The number of erythrocytes and hemoglobin are 15% and 13 % higher in 3 g fed pigs.There are relatively small number of sick animals (<2.40%) when compared with control group (5.40%).

PEM in cancer patients
Protein calorie under nutrition is seen in advanced cancer patients with loss of adipose tissue, visceral protein and skeletal muscle varying unpredictably from patient to patient.Good nutrition may increase the survival rate of the patients (Nixon et al. 1980).
Table 8 shows the list of clinical studies on Spirulina and its benefits for alleviating protein energy malnutrition in cancer patients.
Spirulina algae extract, when applied tropically for 3 times per week for 28 weeks along with 0.1% 7, 12-dimethylbenz[a]anthracene in mineral oil, removed tumors in hamsters (Joel Schwartz 1988).This study promoted many scientists to explore the antitumor immunotherapy potential of Spirulina.Extensive studies including human trails (Babu et al 1995):cell lines (Konícková et al. 2014) and on rats (Zhang et al. 2001;Khan et al. 2005;Chamorro-Cevallos et al. 2008;Akao et al. 2009 andIsmail et al. 2009) were done to prove the cancer inhibitory properties of Spirulina.

PEM in HIV patients
After the discovery of calcium spirulan (Ca-Sp) by Hayashi et al. (1996):extensive research has been done to inhibit the replication of several enveloped viruses, including (human immunodeficiency virus) HIV-1.Studies on peripheral blood mononuclear cells (Ayehunie et al 1998) and humans (Simpore et al 2005;Yamani et al. 2009;Azabji-Kenfack 2011) were done along with structural modification studies on calcium spirulan (Lee et al. 2001).Spirulina was fed up to 25 g/day to HIV patients and considerable improvement in weight loss, anaemia, karnofsky score, CD4 cell count was observed along with decrease in the HIV viral load (Simpore et al 2005;Yamani et al. 2009;Azabji-Kenfack 2011) (Table 9).

Protein Energy Wasting
Protein energy wasting describes the increase of mechanisms causing syndromes of wasting, malnutrition, inflammation, and their interrelationships in individuals with chronic kidney disease (CKD) or acute kidney injury (AKI) (Fouque et al. 2008).Studies on abrupt loss of kidney function due to exposure to mercury to the kidney and its failure in rats was studied by Fukino et al. (1990).Spirulina was administered along with mercury to rats which were alive up to 10 days, where the control group which were fed only mercury died within 4 days.The study confirms the protective effect of Spirulina against renal failures and reduction of general renal dysfunctions.Extensive research was done on rats but very few studies were available on human population (Table 10).Stoilov et al. (1999) proposed the idea of using 5 to 30% protein hydrolysate from Spirulina, fish and macroronus along with natural bee honey can be used to patients with chronic renal insufficiency.High Spirulina diet should be avoided for patients with renal stone deposition problems (Farooq et al. 2005).

Conclusions
The present review had revealed that significant studies were done on Spirulina to establish its potential use as a food supplement, food additive, animal or aqua feed and to combat against all forms of Protein Energy Malnutrition (PEM) and Protein Energy Wasting (PEW).But many studies are required on human population to find the exact clinical dosage of this super food to patients with different protein-calorie or renal disease conditions.The above review also suggests to use Spirulina not greater than 4 grams per day for normal healthy adults, <25 Rats Kuhad et al. 2006 India Spirulina (1,000 mg/kg) was administered orally for 8 days and Cisplatin treatment was given on day 4 and Nephrotoxicity was assessed after 6 days.There is decrease in the levels of superoxide dismutase, catalase and glutathione peroxidase and increase in lipid peroxidation, plasma urea, creatinine, urinary β-NAG, plasma and kidney tissue malondialdehyde.Spirulina significantly protected the Cisplatin induced nephrotoxicity through its antioxidant properties.
Rats Mohan et al. 2006 Saudi Arabia Rats were fed with 0.75% ethylene glycol in drinking water for three weeks and after that they were fed with Spirulina (20 mg/kg body weight) for another three weeks.The rats fed with spirulina recovered from nephrolithiasis or renal stone disease and completely from hepatotoxicity induced by ethylene glycol.

Rats Al-Attar 2010
India Oral pretreatment with Spirulina to rats, prevented 4-nitroquinoline-1-oxide induced hepato and nephrotoxicity.The antioxidant properties mediated by Spirulina in eliminating reactive free radicals were established.
Rats Viswanadha et al. 2011 Mexico Phycobiliproteins and C-phycocyanin extracted from Spirulina were fed to rats with mercury (5 mg/Kg Intraperitoneal).All doses of phycobiliprotein and C-phycocyanin prevented enhancement of oxidative markers and protected against mercuric cholirde caused cellular damage in the kidneys.grams/day for HIV patients, <2 % for aqua culture and <10% for poultry and animal feed.Exact dosage has to be developed for cancer patients and patients with renal problems.Spirulina is not recommended as a source of vitamin B12 for vitamin B12 deficiency children (Herbert and Drivas 1982;Dagnelie et al. 1991) and low dosage of Spirulina (5g/day) does not showed any significant increase in weight in malnourished children (Branger et al. 2003)

Rats
the Spirulina and its development in Chad since 1988 and initiatives of the BIEP (Interminestrial Bureau of Studies and Programing):in collaboration with the BECMA (Bureau of Studies and Culture of Microalgae) were outlines in this paper along with the production technology.ProductionHalawlaw 2013 Ohira et al. (1998)ear and as perOhira et al. (1998):5.676kg O2 per year per 150 grams dry weight Spirulina, can be It is quite evident based on the above scientific data that Spirulina produce more oxygen than trees per acre.
Spirulina and Arthrospira genus.Stizenberger, in 1852 gave the name Arthrospira based on the septa presence, helical form and multicellular structure and Gomont in 1892, confirmed aseptate form of the Spirulina genus.Geitler in 1932, reunified both members designating them as Spirulina without considering the septum.The worldwide research on microalgae was carried out in the name of Spirulina, but the original species exploited as food with excellent health properties belongs to genus Arthrospira.

Table 3 :
Risk assessment of spirulina based on US-RDA values for healthy adults (19 to 50) years and the black color indicates excess than RDA

Table 4 :
Production of Spirulina and its social acceptance, cost effectiveness studies for hunger alleviation in developing countries Spirulina platensis culture was bought from France to Institute of Biology, Hanoi, Vietnam.In 1977, a pilot pond of 12 m 2 was started in Thuanha, Vietnam and in 1980, it is expanded to 3000 m 2 and all process parameters were optimized to suite Vietnam atmosphere.Spiruilna cultivation in pilot plant was started by Applied Botany Section, Biological Research Division, Bangladesh Council of Scientific and Industrial Research Laboratories (BCSIR):Dhaka.A technology was developed to suite Bangladesh environment.

Table 5 :
Clinical studies for combating malnutrition using Spirulina in developed and in under developing countries.Adsorption of Spirulina protein was found to be good and also observed that in spite of heavy dosage, there is no noteworthy increase in blood uric acid.Spirulina fusiformis was given every day as nutritional supplement to 5000 pre-school children for a period of 6 to 13 months and clinical parameters were evaluated.Based on the survey at the end of the study, 4% reduction in incidence of Bitot's spots was observed.Spirulina to 182 children for 90 days, suffering from malnutrition does not resulted in any change in weight gain.Misola (millet, soja, peanut) based food was given to 550 undernourished children suffering from malnutrition at the Centre Medical St. Camille, Uagadougou, Burkina Faso.This study confirms that Spirulina plus Misola are good food supplements for undernourished children.(30 days old) were fed for 60 days with 17% protein from Spirulina and compared to rats fed 17% protein casein.The body weight, length, soleus muscle total protein, protein degradation and DNA were similar in both groups but The muscle protein synthesis rates were increased in rats fed with Spirulina diet.Spirulina daily intake and other group without Spirulina intake were monitored for 9 months and the Spirulina treated children showed larger improvement in height for age Z score.Shimla were divided into equal groups and one group was given 1 gram Spirulina + 40 grams wheat basan ladoo (an Indian recipe) for 6 days a week for two months and other group was given placebo for the same period.The group with Spirulina supplementation showed less prevalence of common ailments (paleness of skin, conjunctiva, dental caries, fatigue) when compared with other group.

Table 6 :
Various studies on micronutrients and role of Spirulina in human health to IIMSAM (Intergovernmental Institution for the use of Micro-algae Spirulina against Malnutrition):recognized by United Nations (UN) and consultative observer of The United Nations Economic and Social Council (ECOSOC):UN, there are 178 million globally estimated stunned children.IIMSAM works to promote the use of Spirulina against acute malnutrition and global food security by promoting its mandate and collaborating with various countries (H.E.Remigio M. Maradona 2008).

Table 7 :
Various studies on Spirulina as an alternative feed for animals and aquaculture for alleviating hunger and for providing food security Valenciennes):was fed with 29 g dry weight per kg body weight Spirulina for 28 days and an increase of 14 g/Kg body weight was observed.

Table 8 :
Various studies on Spirulina for nutrition rehabilitation patients suffering from cancer , 12-dimethylbenz[a]anthracene in mineral oil when applied tropically for 3 times per week for 28 weeks found to remove gross tumors in Hamster.However, microscopic sections of the buccal pouch in the Spirulina fed group showed localized areas of dysplasia and early carcinoma-in-situ undergoing destruction.β carotene and mineral oil fed group of hamsters also showed considerable decrease in tumors.by injecting cyclophosphamie annd 60 Co- irradiation.30 and 60 mg/kg increased the level of white cells in blood and nucleated cells and DNA in bone marrow but no effect in red cells in mice but 12 mg/kg increased the level of red cells, white cells and hemoglobin in blood and nucleated cells in bone marrow in dogs.Spirulina has chemoprotective and radio protective capabilities.versus B16 melanoma) were fed with spirulina to elucidate the mechanism of raising antitumor NK (Natural Killer) activation.Orally administered Spirulina enhances tumoricidal NK activation through the MyD88 pathway.Spirulina and BCG-cell wall skeleton synergistically augmented IFN-γ production and antitumor potential in the B16D8 versus C57BL/6 system.We infer from these results that NK activation by Spirulina has some advantage in combinational use with BCG-cell wall skeleton for developing adjuvant-based antitumor immunotherapy.
Daim et al. 2013)was established in rats and still human trails are required to find the exact dosage of Spirulina.The above summary of results, presented in the Table9, from various sources confirms that Spirulina can be used to combat against protein energy wasting.

Table 10 :
Farooq et al. 2005 spirulina and protein energy wasting in renal problems protein hydrolysate prepared from protein source Spirulina, fish, and macroronus with 70 to 95% of natural bee honey can be used to patients with chronic renal insufficiency or other protein metabolic disorders.IndiaEffect of urinary oxalate and uric acid level on high spirulina diet and risk of nephrolithiasis was found by Spirulina (1500 mg/kg and 0;75% in drinking water) for 4 weeks.The crystal deposition and damage in renal cells was observed.During hyperoxaluric conditions the Spirulina diet must possibly be avoided and can be considered in normal subjects checked for family history of renal stone deposition.RatsFarooq et al. 2005India Renoprotective potential of Spirulina (500 too 1500mg/Kg) against Gentamicin (100mg/kg) was evaluated on rats.Treatment with Spirulina significantly restored renal functions, reduced lipid peroxidation and enhanced reduced glutathione levels, superoxide dismutase and catalase activities.

Table 9 :
Rodríguez- Sánchez et al.  2012   EgyptHepatonephroprotective and antioxidant potential of Spirulina against deltamethrin toxicity in rats was assessed.Spirulina normalized the elevated serum levels of AST, ALT, APL, uric acid, urea and creatinine.Furthermore, it reduced deltamethrin-induced lipid peroxidation and oxidative stress in a dose dependent manner.Various studies on Spirulina for nutrition rehabilitation patients suffering from HIV patients Country Summary of Results Subject Reference USA Spirulina (0.3 to 1.2 µg/ml):reduced viral production by approximatly 50% in peripheral blood mononuclear cells (PBMC).Fractionation of the extract revealed antiviral activity in the polysaccharide fraction and also in a fraction depleted of polysaccharides and tannins.JapanCalcium ion binding with the anionic part of the molecule was replaced with sodium and potassium ion in Calcium Spirulan.The replacement of calcium ions with sodium and potassium maintained the antiviral activity but divalent and trivalent metal ions decreased the antiviral activity.25 g/day was fed to 84 children with HIV infection for 8 weeks and compared with 86 undernourished children.Level of anaemia decreased during the study and 81.8% of undernourished children and 63.6% of HIV infected children were recuperated.Spirulina can be effective for weight loss and anaemia for HIV and HIV negative undernourished children.HIV were given 10 grams of Spirulina per day for six months.No difference was found in patients receiving Spirulina and the control group but there was an increase in protidemia, creatinemia and Karnofsky score.calculated as per 1.5 g/Kg body weight proteins and spirulina (25%) was given to malnourished HIV infected adults with age 18 to 35 years for 12 weeks.HIV viral load significantly decreased and increase in CD4 cell count was observed at the end of the study.
and it is also not recommended for patients with family history of renal stone depositions.Extensive studies on Vitamin K, Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B6, improvement of cobalamin Vitamin B-12 in Spirulina strains using genetic modification, clinical potential of calcium-spirulan against HIV and other viral diseases, antitumor and renoprotective properties of Spirulina on human population are required for further understanding the clinical potential of Spirulina to combat against PEM and PEW.Specific mechanisms should be developed by industries to remove vitamin A, vitamin K, vitamin B12, molybdenum and chromium to increase the consumption level up to 100 grams of Spirulina per day.Development of various Spirulina fortified foods are required to create nutritional awareness and increase the acceptance level in developing countries.