BIODELTA (PTY) LTD

2002/004847/07

PO Box 100 Simondium 7670 South Africa TEL: +27 (0)21- 874 2936 FAX: +27 (0)21- 874 2946 Ema il: inf o @b io del ta .ne t

BioBiotic
Source: Revised:

TM

Dr. Len Dekker (PhD Biotechnology) 03/05/2004

The probiotic potential of BioBiotic for surviving an antibiotic ban in animal feeds
TM

Consumer demand for natural organic products is resulting in the systematic removal of antibiotic growth promoters from commercial animal feeds. In 1986 Sweden was the first country to ban the use of antibiotics and the European Union has recently approved the phasing out of antibiotics as animal growth promoters. The feed business has become to rely on the use of antibiotics in order to maintain economic viability in a very competitive industry, and these growth promoters are included at low enough levels to allow the survival of natural intestinal microflora, while suppressing pathogenic bacterial growth. The problem is that antibiotic-resistance is on the increase, which could result in a health crisis for both animals and consumers. Manufacturers are now challenged to formulate feeds containing natural alternatives to antibiotics in order to comply with consumer demands for a transition from cheap food to safe food. A probiotic approach is steadily growing with the strategy to develop enhanced immune systems in animals to resist disease by using high quality natural feeds that do not require antibiotics, while retaining exceptional growth rates. Why BioBiotic? BioBiotic contains a carefully selected strain of Spirulina platensis with enhanced levels of natural immune stimulating phytochemicals. Biodelta developed a unique cultivation process for Spirulina that enhances the biosynthesis and accumulation of specific immune stimulating compounds within the organism. BioBiotic is a natural growth stimulant and focus on immune enhancement with the potential to replace antibiotics in animal feeds. It is a registered animal feed supplement in South Africa with the following benefits: It stimulates the immune system by increasing the macrophage function and antibody response. Inhibits the growth of pathogens in the digestive tract, including clearance of E. coli and Staphylococcus in the blood.

The unique Polysaccharides in BioBiotic enhance the growth rate of Lactobacillus species thereby ensuring a healthy intestinal population. BioBiotic is supplied in a granulated, larger particle size for ease of application.

Recommended Use:

Poultry - Broilers, Layers and Breeders. Ostriches Exotic Birds Fish Farming and Koi Breeding Crocodiles Pigs - Weaners Horses

Trials conducted at the University of Stellenbosch show that BioBiotic results in a significant weight gain and reduced mortality in broilers. Biodelta (Pty) Ltd is proud to be associated with a world-class Spirulina production facility and quality control is assured on-site by technicians who conduct chemical and microbiological assays to guarantee the purity of the ponds and final product. At Biodelta a specially designed plastic tunnel individually covers each growth pond for optimum environmental control in order to produce a product of consistent quality. Each pond is therefore completely isolated and harvested separately for quality control purposes. This configuration also allows for the manipulation of selected ponds in order to produce Spirulina biomass with enhanced characteristics that will suite specific customer needs. These enhanced characteristics include selective increases in protein, chlorophyll, phycocyanin, polysaccharides, or trace elements (such as selenium, chromium, zinc and cobalt).

An individually covered Spirulina growth pond at the Biodelta organic farm near Franschhoek, South Africa.

Immune enhancement potential of Spirulina Spirulina has been shown to enhance immune function, reproduction and increase growth. Studies conducted in vitro have shown that treatment of chicken macrophages with a water extract of Spirulina resulted in immune stimulation, resulting in significantly increased macrophage function, antibody response and phagocytosis. Remarkably, the macrophage cultures exposed to the extract produced a factor in the culture medium with tumoricidal potential and other enhanced effector functions (Quereshi et al., 1995a; Quereshi et al., 1994). In complementary studies, it was found that broiler chicks on 1% dietary Spirulina mounted a higher anti-sheep red blood cell response compared to control chicks. Two additional immunological endpoints, cutaneous basophilic hypersensitivity (CBH) and bacterial clearance potential, were then examined in white leghorn chicks fed a Spirulina diet. In preliminary trials, feeds were made at Spirulina levels of 0, 0.1 and 1%, and were then repeated in twofold increments from 0.025% to 1.6% with a 0% control group. Chicks were placed on the diets from day one after hatching and were randomly assigned to multiple pens. Pens were thermostatically controlled and feeds and water was available for ad libitum consumption. At 14 days, chicks were injected in the toe web with 100 g of the mitogenic compound, phytohemagglutinin-P (PHA-P), to examine the in vivo T-lymphocyte proliferative response. The injections of PHA-P mediate a localised cellular response, indicative of T-cell activity. This response is manifested by swelling triggered by the T -lymphocytes which are the key contributors. The swelling at the site of injection was measured after 24 and 36 hours with a constant tension caliper to quantify the response. It was shown that dietary Spirulina of 0.1% elicited an approximately two-fold higher CBH response after PHA-P injection, a significant improvement over the chicks fed the control diet. Chicks fed Spirulina diets of 0.4 to 1.6% elicited T-cell responses nearly four-fold greater than the controls. The effects of dietary Spirulina on chicken macrophage phagocytic function and nitrite production were also examined (Al-Batshan et al., 2001). Day old broiler chicks were randomly assigned to various pens and dietary treatment groups included a basal diet with 0, 0.5, 1.0 and 2.0 % dietary Spirulina. Feed and water were provided for ad libitum consumption from one day of age. Sephadex-elicited macrophages were harvested at 14, 35 and 42 days of age. Phagocytosis assay was performed by coincubating sheep red blood cells (SRBC) with the adherent macrophage monolayers. For nitrite quantification, macrophage cultures from various dietary treatment groups were stimulated in the presence or absence of 1 g/ml of E. coli lipopolysaccharide (LPS), which is a known inducer of nitrite production. These culture supernatant fractions were then tested for nitrite levels using the Greiss reagent technique. All Spirulina dietary group macrophages exhibited an enhanced phagocytic activity in terms of overall phagocytic percentage (range = 28 to 39% versus 24 to 25% in the control group) and the average number of SRBC per phagocytic macrophage (range = 2.2 to 3.6 versus 1.8 to 2.5 in the control group). This increase was linear with each incremental in crease of dietary Spirulina. While LPS-induced nitrite levels in macrophages from basal diet group ranged from 60 to 278 M over the three development stages, these levels in all Spirulina dietary groups were significantly higher (0.5% group range = 198 to 457 M; 1.0% group range = 161 to 359 M; and 2.0% group range = 204 to 420 M). These date clearly show that Spirulina feeding upregulates macrophage phagocytic as well as metabolic pathways leading to increased nitric oxide synthase activity. This would certainly have a positive 3

immunomodulatory effect since antimicrobial effects of nitric oxide, produced by macrophages, have been well documented against pathogenic intracellular microorganisms including bacteria, viruses and protozoa (Boockvar et al., 1994; Kreil and Eibl, 1996; Evans et al., 1996). Previous studies have also shown a direct involvement of macrophage-produced nitrite in the resolution of coccidiosis in chickens (Qureshi et al., 2000). Anti-bacterial properties of Spirulina Bacterial clearance was also assayed by injecting each chick with Escherichia coli or Staphylococcus aureus suspensions via the brachial vein at 3 weeks of age. Immediately after injection, 500 l of blood was drawn as a time zero post injection. Subsequent blood samples were drawn at each 15 minutes for one hour. All chicks were sacrificed after final bleeding and spleens were collected for bacteriological analysis. Chicks in every dietary group exhibited bacterial clearance from the blood over the 1-hour period. However, the diet with 0.1% of Spirulina showed a significantly enhanced amount of bacterial clearance compared to the control or other concentrations. In the 0.1% Spirulina group, bacterial clearing started immediately after injection and by 30 minutes post-injection the bacterial count was almost negligible in the blood. This heightened bacterial clearance indicates that Spirulina supplementation improves the activity of phagocytic cells, namely monocytes, macrophages, heterophils and thrombocytes in chickens. This bacterial clearance is entailed by entrapment of bacteria by cells of the mononuclear phagocytic system residing in the liver. Apparently, the bacterial clearing within the microenvironment of these organs is increased by the immonopotentiating effect of Spirulina due to elevated mononuclear phagocytosis (Quereshi et al., 1995b). In corresponding experiments, white leghorn chicks were fed a diet containing 1% Spirulina and challenged with a single dose of Staphylococcus aureus and the splenic bacterial load was quantified. After a period of 80 minutes, the splenic load of bacteria was 5 000 for the control group and 1 250 for the group fed the 1% Spirulina. In similar challenge tests with E. coli, the optimal concentration for bacterial clearing was found to be 0.1% of Spirulina. At 0.1% Spirulina inclusion rate, chicks had splenic bacterial counts of less than 500 whereas the control group without Spirulina had counts of 4 000 after 80 minutes. Therefore, it can be implied that dietary Spirulina improves both cell-mediated (T-cell) and mononuclear phagocytic system potential in chickens, allowing them to better resist disease encounters (Quereshi et al., 1995b). Anti-viral properties of Spirulina Other researchers have discovered a water-soluble extract of Spirulina that was shown to inhibit viral cell-penetration and replication of the Herpes Simplex Virus Type 1 (HSV-1) in cultured HeLa cells in a dose-dependent manner indicating that Spirulina may prevent herpetic encephalitis by feeding in the diet. At just 1 mg/ml, the extract was shown to inhibit viral protein synthesis without suppressing host cell functions. Hamsters fed the extract and then challenged with the HSV-1 had prolonged survival times and higher survival rates, while all of the control animals without the extract died (Hayashi et al., 1993). Further research identified this antiviral extract as a novel sulfated polysaccharide termed Calcium Spirulan, which has been shown to inhibit 4

replication of many enveloped viruses by inhibition of viral penetration into target cells without host toxicity. Presently, Calcium Spirulan has shown activity against human cytomegalovirus, measles virus, mumps virus, influenza A virus, human immunodeficiency virus (HIV-1) as well as HSV-1 (Hayashi and Hayashi, 1996). Intestinal microflora enhancement The probiotic activity of some bacterial strains with the ability to colonise the intestinal epithelium contributing to stabilise the intestinal microflora, especially after antibiotic treatment, has been described in several reports (Gasson, 1993). Because the gut microbiota can play a major role in health, there is currently some interest in functional feed ingredients that may stimulate beneficial lactic acid bacteria. It was previously reported that Spirulina contains growth promoters, in the form of unique polysaccharides that enhances the growth of lactic acid bacteria (Parada et al., 1998). Feeding rats a diet with 5% spirulina for 100 days (compared to a control group not fed spirulina) revealed: 1. the weight of the caecum increased 13%; 2. lactobacillus increased 327%; 3. vitamin B1 inside the caecum increased 43%. Since spirulina did not supply this additional B1, it improved overall B1 absorption. The study suggests eating spirulina increases lactobacillus and may increase efficient absorption of Vitamin B1 and other vitamins from the entire diet (Tokai et al., 1997). Other potential benefits The blue-green algae, Spirulina platensis, has been used for hundreds of years as a food source for humans and animals due to the excellent nutritional profile and high carotenoid content. Spirulina is relatively high in protein with values ranging from 5565% and includes all of the essential amino acids (Clement et al., 1967; Bourges et al., 1971; Anusuya Devi et al., 1981). The available energy has been determined to be 2.5 3.29 kcal/gram and phosphorus availability 41% (Yoshida and Hoshii, 1980). Although Spirulina powder appears as a bluish-green colour, in fact it contains one of the highest levels of carotenoids of any natural food source when properly cultivated and processed (Matsuno et al., 1974; Tanaka et al., 1974; Nells and De Leenheer, 1983; Miki et al., 1986). Carotenoids are a family of over 600 natural lipid-soluble pigments that are primarily produced within phytoplankton, algae and plants. Some fungal and bacterial species can also synthesise carotenoids, but animals cannot produce them de novo. Within the various classes of natural pigments, the carotenoids are the most widespread and structurally diverse pigmenting agents. Carotenoids are responsible for a wide variety of colours in nature, the most notable are the brilliant yellow to red colours of fruits and leaves of plants. In combination with proteins, carotenoids also contribute to the wide range of blue, green, purple, brown and reddish colours of fish, insect, bird and crustacean species. These natural pigments help protect cells against light damage, but the pigments have broader functions in various organisms as precursors to vitamin A, antioxidant activity in quenching oxygen radicals, immune enhancement, hormone regulation, and additional roles in growth, reproduction and maturation. The major carotenoids of Spirulina are carotene, -cryptoxanthin and zeaxanthin. Japanese quail are commonly used in studies to evaluate pigmentation of egg yolks since the absorption and deposition on xanthophylls in egg yolk are similar to those in Leghorn hens (Nelson, 1966). In one study conducted with Japanese quail, a total of 5

120 animals were placed on a carotenoid-free diet at 8 weeks of age, this barley-based diet also served as a control throughout the trial. Experimental diets were formulated by inclusion of Spirulina at concentrations of 0.25, 0.5, 1, 2 and 4%, and were balanced for methionine, calcium and phosphorus and further balanced to be isocaloric and isonitrogenous. After a period of 4 weeks on the control diet (at 12 weeks of age) the animals were randomly divided into 12 groups of 10 birds each which allowed for two replicates of each treatment, and instated on the Spirulina test diets for 21 days. Eggs were collected daily and yolk colour was determined a Roche egg yolk colour fan, and statistically analysed using one-way analysis of variance and Duncans multiple range test. Within 3 days there were significant differences in yolk colours between each level of Spirulina inclusion. Pigmentation was at a maximum and stable after 7 days on the diets. The optimal level of yolk colour (between 8 and 9 on the Roche egg yolk fan) was achieved with 1 1.5 % Spirulina diet and the colour levels of the egg yolks remained stable as long as the supplementation continued. When the diets returned to the carotenoid-free feeds (without Spirulina) the egg yolks gradually returned back to the control levels of 2 on the colour scale (Anderson et al., 1991). In a complementary study, at all levels of Spirulina inclusion from 1.5%, fertility increased from 87% to over 96% (Ross and Dominy, 1990). Another trial involved White Leghorn hens in which a control diet was first fed for 12 days to deplete carotenoids, and thereafter 14 groups were assigned to different dietary treatments. Various levels of Spirulina, yellow maize or dehydrated berseem meal was included in the feeds and hens were fed the diets ad libitum for a period of thirty days. Dietary pigments were apparent in the yolks after 3 days and reached a maximum plateau at 7 days. The results demonstrated that Spirulina produced markedly higher pigmentation scores of 13-14.8 compared to birds fed yellow maize (4.7-8.0) and berseem meal (4.8-5.6). The Spirulina diets gave the highest scores at all levels tested and produced a much deeper yolk colour than produced by even the highest level of the conventional carotenoid sources. The diet containing 3% Spirulina scored 13.3 on the Roche colour fan with deep orange yolks. A similar trend was also observed in the boiled eggs, Spirulina provided substantially higher deposition and pigmentation. Slightly lower scores were recorded after cooking. It can be speculated that the higher colour values in the case of raw eggs might be due to the brightness of the vitelline membrane, higher level of pigment deposition at the periphery rather than towards the central region of the yolks, or due to the overall stability of the particular carotenoids. Sensory evaluation, including flavour, taste, colour and overall acceptance was judged by an independent panel. The results revealed that eggs from hens fed Spirulina had significantly higher scores than the yellow maize, berseem meal or indigenous eggs for each of the sensory tests (Saxena et al., 1982). Since the key to pigmentation of egg yolks with Spirulina is a result of the carotenoids, it is critical to utilise a supply that contains the highest concentrations to maintain consistency and optimal coloration. Spirulina is produced worldwide but the quality varies considerably, the source should contain total carotenoid levels of 3.0 grams/kg of algae or greater to attain satisfactory results. Poor growth conditions, manufacturing or packaging of Spirulina leads to oxidation and a low carotenoid level, which can often be detected by a prevailing blue colour of the algae. The blue colour is the result of degradation of the chlorophyll and carotenoids such that the blue phycocyanin pigments predominate. Furthermore, many manufacturers grow Spirulina as a by-product of other operations such as domestic raw sewage treatment; 6

this is most likely in India, China or Asian countries. In these operations, Spirulina is exposed to pathogenic coliforms, protozoa, viruses and intestinal parasites, all of which may cause infections and disease to livestock and man (Saxena et al., 1983). Spirulina has also a very high affinity for heavy metals when grown in contaminated growth mediums, such as tannery wastewaters (Dunn, 1997). Quality Spirulina is grown in dedicated ponds with only food grade nutrients, appropriate drying, and packaged in foil laminate bags to protect it from light, heat and oxygen. Mild conditions in processing are necessary to preserve carotenoids and other sensitive components (Anderson et al., 1991). A supporting study compared the use of freezedried and extruded Spirulina as pigmenting agents for egg yolks. It was shown that egg yolk colour scores from quail fed freeze-dried Spirulina were higher than scores of eggs from quail fed the extruded feed. This can be attributed to a loss of carotenoids during the manufacturing process. There was no adverse effect of the Spirulina on egg production, feed per egg, egg weight, final body weight, or mortality in any of the studies (Ross et al., 1994). Care should therefore be taken to prevent heat damage when mixing Spirulina into commercial feeds, and proper testing should be conducted in order to evaluate each feed manufacturing process prior to making the necessary adjustments if required. Blue-green algae and microcystin toxins The microalgae industry has developed to its current status by providing a safe and nutritious product for the human supplement market as well as the animal and aquaculture feed markets. The vast majority of this microalgae biomass is produced from Spirulina, Chlorella, and Aphanizomenon flos-aqua. Concern over the discovery of toxic algae blooms in Klamath Lake underscore vital differences between cultured Spirulina and species of wild lake-grown algae. Scientists believe there are over 30,000 species of microscopic algae. The immense range of species includes nutritious varieties like Spirulina and Chlorella, as well as potentially dangerous species such as the Microcystis strains identified in Upper Klamath Lake. In this way, microalgae are similar to mushrooms common cultured table mushrooms are absolutely safe and healthful while others, such as toadstools can be poisonous. The same situation occurs in the bacterial group some like the lactobacilli are essential for good digestion while others, such as Salmonella can cause disease. Cultured Spirulina can be grown free of contaminating algae for several reasons. The growing conditions for Spirulina are unique as it is cultivated and thrives in high salinity and very alkaline conditions. Competitor algae and other contaminants simply cannot compete and grow under these harsh conditions. Ponds are monitored and analysed every day and carefully controlled to maintain a balanced chemistry. Samples are carefully scrutinised on a daily basis via microscopic testing to assure purity and cleanliness. Water runoff from outside sources cannot enter the raised raceways that cultivate Spirulina. Thus, there is no possibility for agricultural waste such as pesticides and herbicides to contaminate the cultures. Finally, as a precautionary measure, lots are periodically screened for cyanotoxins. Cyanotoxins have long been recognised as a water-based disease that causes animal illness and death. The biotoxins, microcystin and nodularin, have been implicated in causing irreversible hepatotoxicity and tumour promoting reactions in laboratory rats. Evidence in China suggests a correlation between microcystins in drinking water and primary liver cancer. Australia, Canada, and Great Britain are moving to establish 7

maximum acceptable concentration (MAC) of microcystins in drinking water, which are in parts per billion. In Australia a limit of 1 ppb (1 g/litre) has been proposed. The June 1994 Criteria Document on Microcystin-LR for Canadian drinking water recommends a MAC of 0.5 ppb (0.5 g/litter) for microcystin-LR and 1 ppb (1.0 g/litre) for total microcystins. An even lower level of 0.01 ppb (0.01 g/litre) has been proposed for Chinese waters based on research and levels of microcystins found in drinking local water. Because of the risk for cancer and liver damage, further research is needed to elucidate the dose dependent effects of microcystins. Excellent reviews of cyanotoxins, microcystins, and nodularins have been published recently (Carmichael; 1992, 1994, 1997). Despite the controlled conditions, precautions, quality control, and proven safety of Spirulina, consumers often confuse or associate Spirulina with wild algae which are vulnerable to microcystin toxin contamination. Trace elements Biodelta also developed the ability to enhance the trace element concentration in BioBiotic. Selected trace elements are added as inorganic chelates at specific stages in the Spirulina growth cycle. These elements are then metabolised and converted into organic complexes within the organism. Selenium levels can be enhanced to a final concentration of at least 200 mg/kg. Zinc levels can be enhanced to a final concentration of at least 500 mg/kg. After determining the optimum inclusion rate of Spirulina in a specific feed formulation, levels of specific trace elements can be enhanced in the Spirulina product to comply with the final trace element concentration required for a specific feed application. Heavy metal contamination Biodelta evaluated different commercial Spirulina products for heavy metal contamination. Many of these products contain aluminium levels as high as 400 ppm, which is usually not specified in the product analysis. Aluminium is a potentially toxic heavy metal and can result in a variety of serious health problems. BioBiotic is regularly screened for the presence of all heavy metals and the aluminium content is less than 30 ppm.

Summary The probiotic potential of Spirulina has been presented in numerous international scientific publications. The most important characteristic of Spirulina is its immune enhancement and anti-viral properties. Dietary Spirulina of 0.1% elevates the cutaneous basophil hypersensitivity response (T-cell) and macrophage-based bacterial killing response. This leads to improved cell-mediated and mononuclear phagocytic system potential in chickens, allowing them to better resist disease encounters. Spirulina is also an excellent source of nutrition and provides a superior natural source of carotenoids that are extremely effective in colouring egg yolks and skin and feathers of many fish and bird species. An inclusion rate of 0.5-1.5% in feeds provides the enhanced pigmentation benefits, but it is critical to utilise quality Spirulina that consistently contains at least 3.0 grams of total carotenoids per kilogram. Eggs from hens that are fed with Spirulina algae have favoured taste, flavour, colour and overall acceptance compared to conventional pigmentation sources. BioBiotic contains enhanced levels of the active immune stimulating phytochemicals found in Spirulina. The value of BioBiotic does not only depend on the identification and presence of individual phytochemicals, but rather on the combined synergistic effect of all these substances together.

GENERAL PRODUCT COMPOSITION

BioBiotic
GENERAL ANALYSIS Protein Energy Carbohydrates Lipids Ash Moisture Bulk density Particle size 58 19.18 15 0.5 12 7 0.6 <800 % MJ/kg % % % % kg/L micron AMINO ACIDS (values in g/100g) Arginine Serine Aspartic acid Glutamic acid Glycine Threonine Alanine Tyrosine Proline Methionine Valine Phenylalanine Isoleucine Leucine Histidine Lysine Cysteine Tryptophan 3.78 2.77 5.13 6.94 3.03 2.58 3.63 3.14 2.47 1.50 3.06 2.71 2.90 4.52 1.52 2.83 1.74 1.04

PIGMENTS & VITAMINS -carotene -cryptoxanthin Zeaxanthin Xanthophylls Chlorophylla Phycocyanin Vitamin B12 Vitamin E 1 255 72 523 1 183 10 150 128.2 0.4 109 mg/kg mg/kg mg/kg mg/kg mg/kg g/kg mg/kg mg/kg

MINERALS (values in mg/kg) Magnesium Calcium Phosphorus Potassium Sodium Chloride Iron Cobalt Chromium Arsenic Lead Mercury Cadmium Nickel Manganese Molybdenum Copper Aluminium *Zinc *Selenium 8 040 5 370 10 100 19 500 12 600 1 080 986 15 5.1 1.7 1.7 <0.05 0.2 23 71 5.9 9.5 19 59 1.4

MICROBIAL ANALYSIS Entero bacteria Coliform E-coli Salmonella Shigella Yeast & moulds Staphylococcus negative negative negative negative negative <100 CFU/g negative

* Zinc and Selenium concentration can be increased to 500 mg/kg and 200 mg/kg respectively.

Packaging in 25-kg cardboard containers fitted with inner HDPET liners for protection against oxygen, light and moisture. 10

REFERENCES
Anderson D.W., C. Tang and E. Ross. 1991. The xanthophylls of Spirulina and their effect on egg yolk pigmentation. 1991. Poultry Science. 70:115-119. Anusuya Devi M., Subbulakshimi G., Madhavi Devi K. and Venkataram L.V. 1981. Studies on the proteins of mass-cultivated, blue-green alga (Spirulina platensis). J. Agric. Food Chem. 29: 522-525. Boockvar K.S., Granger D.L., Poston R.M., Maybodi M., Washington M.K., Hibbs Jr. J.B. and Kurlander R.L. 1994. Nitric oxide produced during murine listeriosis is protective. Infe. Immun., 62: 1089. Bourges H., Sotomayor A., Mendoza E. and Chavez A. 1971. Utilization of the algae Spirulina as a protein source. Nutr. Rep. Int. 4:31-43. Carmichael, W.W. 1992. Cyanobacterial secondary metabolites-the cyanotoxins. J. Appl. Bacterial. 72:445-459. Carmichael, W.W. 1994. The toxins of Cyanobacteria. Sci Am. Jan Vol. 270:78-86. Carmichael, W.W. 1997. The Cyanotoxins. In, Advances in Botanical Research Vol. 27: 211-255. Academic Press Ltd. Clement G., Giddey C. and Menzi R. 1967. Amino acid composition and nutritive value of the algae Spirulina maxima. J. Sci. Food Agric. 18:497-501. Dunn K.M. (1997) The biotechnology of high rate algal ponding systems in the treatment of saline tannery wastewaters. PhD thesis, Rhodes University, Grahamstown, South Africa. Evans T.G., Reed S.S. and Hibbs Jr. J.B. 1996. Nitric oxide production on murine Leishmaniasis correlation of progressive infection with increasing systemic synthesis of nitric oxide. Amer. J. Trop. Med., 54: 486. Gasson M.J. 1993. Progress and potential in the biotechnology of lactic acid bacteria. FEMS Microbiol. Rev. 12: 3-19. Hayashi K., Hayashi T. and Morita N. 1993. An extract from Spirulina platensis is a selective inhibitor of Herpes Simplex Virus Type 1 penetration into HeLa cells. Phytotherapy Res. 7:76-80. Hayashi T. and Hayashi K. 1996. Calcium Spirulan, an inhibitor of enveloped virus replication, from blue-green alga Spirulina platensis. J. Nat. Prod. 59:83-87. Kreil T.R. and Eibl M.M. 1996. Nitric oxide and viral infection: NO antiviral activity against flavivirus in vitro, and evidence for contribution in vivo. Virology, 219: 304. Matsuno T., Nagata S., Iwahashi M., Koike T. and Okada M. 1974. Intensification of color of fancy red carp with zeaxanthin and myxoxanthophyl, major carotenoid constituents of Spirulina. Buul. Jpn. Soc. Sci. Fish. 45: 627-632. Miki W., Yamaguchi K. and Konosu S. 1986. Carotenoid composition of Spirulina maxima. Bull. Jpn. Sco. Sci. Fish. 52(7): 1225-1227. Nells H.J.C.F. and De Leenheer A.P. 1983. Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids. Anal. Chem. 55: 27-275. Nelson T.S. 1966. Feed pigments 1. The Japanese quail as an assay animal for feed pigments. Poultry Science. 45: 747-753. Parada J.L., Zulpa de Caire G. 1998. Lactic acid bacteria growth promoters from Spirulina platensis. Intern. J. Food Microbiol. 45: 225-228.

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Qureshi M.A., Garlich D., Kidd M.T. and Ali R.A. 1994. Immune enhancement potential of Spirulina platensis in chickens. Poultry Science. 73: 46. Qureshi M.A., Heggen C.L. and Hussain I. 2000. Avian macrophage: effector functions in health and disease. Develop. Comp. Immunol., 24: 103. Qureshi M.A., Kidd M.T. and Ali R.A. 1995a. Spirulina platensis extract enhances chicken mavrophage functions after in vitro exposure. Journal of Nutritional Immunology. 3(4): 35-45. Qureshi M.A., Ali R.A. and Hunter R. 1995b. Immunomodulatory effects of Spirulina platensis supplementation in chickens. Proc. 44th Western Poultry Disease Conference, Sacramento, California. 117-121. Ross E. and Dominy W. 1990. The nutritional value of dehydrated, blue-green algae (Spirulina platensis) for poultry. Poultry Science. 69: 794-800. Ross E., Puapong D.P., Cepeda F.P. and Patterson P.H. 1994. Comparison of freeze-dried and extruded Spirulina platensis as yolk pigmentation agents. Poultry Science. 73: 1282. Saxena P.N., Ahmad M.R., Shyam M.R. and Amla D.V. 1983. Cultivation of Spirulina in sewage for poultry feed. Experientia. 39: 1077. Saxena P.N., Shyam M.R., Srivastava R., Doval H.K.P. and Sinha D. 1982. Effect of feeding sewagegrown Spirulina on yolk pigmentation of White Leghorn eggs. Avian Research. 66: 41-46. Tanaka Y., Matsuguchi H. and Katayama T. 1974. Comparative biochemistry of carotenoids in algaeIV: Carotenoids in Spirulina platensis. Mem. Fac. Fisch. Kagoshima Univ. 23: 111-115. Tokai Y., et al. 1987. Effects of spirulina on caecum content in rats. Chiba Hygiene College Bulletin. Feb. 1987 Vol. 5, No. 2. Japan. Yoshida M. and Hoshii H. 1980. Nuritive value of Spirulina, green algae, for poultry feed. Japan Poultry Sci. 17: 27-30.

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