2. Culturing Haematococcus algae (Fig. 1)

The life cycle of Haematococcus algae dictates how production of natural astaxanthin is optimized. At Mera Pharmaceuticals, the algae are grown in three phases:

• Small-volume stock cultures maintained in the laboratory are scaled up in increasingly larger containers to produce inoculum for outdoor mass culture;

• Continuous culture of macrozooids in large, outdoor tubular closed photobioreactors or modules under nutrient-replete conditions;

• Reddening in ponds under nutrient-poor conditions.

In the first two phases, the emphasis is on obtaining high concentrations of Haematococcus cells over very short periods of time. Rapid cell proliferation is maintained in Mera Pharmaceuticals Growth Modules (MGMs) by periodically harvesting part of the culture into ponds and replacing this with fresh nutrient medium.

In ponds, on the other hand, conditions are controlled to accelerate cell encystment and astaxanthin production. The ability to grow large concentrations of pure cultures of Haematococcus pluvialis in AGMs and to stock ponds with those high concentrations ensures a very rapid and synchronized reddening, minimizing the risk of contamination and maximizing astaxanthin production.

3. Processing of algal meal (Fig. 1)

• When a pond culture has properly reddened, it is harvested, dewatered and rinsed.

• Cells in the resulting slurry are then mechanically broken to improve the release of the pigment. This process improves bioavailability of astaxanthin when ingested, since intact cyst walls are inefficiently digested (Sommer et al., 1994).

• Cell-broken product is subsequently pasteurized and dried at moderate temperature to minimize degradation of astaxanthin and other nutrients and to reduce bacterial levels to levels safe for human consumption (Table 2).

• The resulting dry flakes are then ground up to a powder and mixed with an approved food-grade natural antioxidant, which helps stabilize the pigment during storage.

• The algal meal is then packed in air-tight aluminium-lined polyethylene bags and stored until further processing.

4. Algal meal composition (Table 2)

Haematococcus pluvialis algal meal is a good source of protein, fat, astaxanthin and other micronutrients. Haematococcus has been employed as a constituent of feeds for a variety of animals, with no negative effects observed on growth, survival, behavior, physiology or biochemistry (Nakazoe et al. 1984; Schiedt et al. 1986; Storrebakken et al. 1987; Ito et al. 1989; Sommer et al. 1991, 1992; Choubert & Heinrich 1993). Haematococcus pluvialis has been fed at levels up to 6% in trout diets without any negative effects (Choubert & Heinrich 1993).

There is no indication that Haematococcus spp. contain any compounds that may have a deleterious effect on other organisms. Safety studies conducted by Mera Pharmaceuticals in a representative animal model and with human volunteers have demonstrated that astaxanthin supplements formulated with Haematococcus pluvialis, grown and processed under Mera Pharmaceuticals proprietary technology, are safe for human consumption.

Table 1. Possible synonyms for Haematococcus pluvialis Flotow, 1844 (Almgren, 1966).

Fig. 1. Mera Pharmaceuticals' production process for the algal meal prepared from the green algae: Haematococcus pluvialis.

 

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Table 2. Typical composition of Nutraxan™ Asta dried Haematococcus pluvialis algal meal.

Proximate analysis Crude protein 18.08 % Crude fat 19.43 % Ash 3.28 % Crude fiber 4.30 % Moisture 3.54 % Energy Calories 470 per 100 g Calories from fat 175 per 100 g Calories from saturated fat 28 per 100 g Carbohydrates 55.67 % Dietary fiber 26.5 % Insoluble fiber 25.2 % Soluble fiber 1.3 % Sugars 0.77 % Cholesterol 0 % Amino acids Alanine 7.48 % Arginine 5.54 % Aspartic acid 7.62 % Cystine/Cysteine 0.90 % Glutamic acid 9.60 % Glycine 5.01 % Histidine 1.52 % Isoleucine 3.55 % Leucine 7.73 % Lysine 4.33 % Methionine 1.5 % Phenylalanine 3.56 % Proline 4.83 % Serine 4.77 % Threonine 4.99 % Tryptophan 1.60 % Tyrosine 2.92 % Valine 5.28 % Met + Cys 2.39 % Met + tyr 6.48 % Micro-organisms Aerobic plate count < 1000 CFU/g E. coli < 10 CFU/g Salmonella Negative/25 g Heavy metals Lead <0.5 ppm Mercury <0.1 ppm Cadmium <0.5 ppm Arsenic <0.5 ppm

Carotenoids Total carotenoids 10.54 % Total astaxanthin 2.18 % Free astaxanthin 3.6 % Astaxanthin monoester 87.2 % Astaxanthin diester 14.6 % Fatty acids Caprylic acid C-8:0 <0.01 % Capric acid C-10:0 <0.01 % Lauric C-12:0 <0.01 % Myristic C-14:0 0.09 % C-14:1 <0.01 % Palmitic C-16:0 2.82 % Palmitoleic C-16:1 0.11 % Stearic C-18:0 0.17 % Oleic C-18:1 3.28 % Linoleic C-18:2 3.11 % g linolenic w -6 C-18:3 1.89 % Octadecatetraenoic C-20:0 0.04 % Gadoleic C-20:1 0.03 % Total saturated fat: 3.12 % Tot.monosaturated fat: 3.42 % Tot.polyunsaturated fat: 5.00 % Vitamins Vitamin A 22000 IU/100 g Alpha tocopherol 412 mcg/g* Vitamin B6 0.14 mg/100 g Vitamin B12 0.04 mg/100 g Thiamine (B1) 0.09 mg/100 g Riboflavin (B2) 0.26 mg/100 g Niacin 0.45 mg/100 g Folic acid 0.39 mg/100 g Pantothenic acid 2.47 mg/100 g Vitamin C 0 mg/100 g (* before addition of any antioxidant) Minerals Calcium 890 ppm Phosphorous 3900 ppm Potassium 2300 ppm Sodium 2400 ppm Magnesium 1500 ppm Iron 880 ppm Cobalt 0.58 ppm Nickel 5.7 ppm Selenium <0.5 ppm Molybdenum <0.5 ppm Zinc 49 ppm Chromium 5.1 ppm

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References

1. Almgren K. (1966). Ecology and distribution in Sweden of algae belonging to Haematococaceae. Svensk Botanisk Tidskrift. BD.60, H.1, 49-73.

2. Blochmann, F. 1886: Uber eine neue Haematococcusart. Verh. Naturhist.-Med. Vereins, Heidelberg, N.F. 3, pp441-462.

3. Boussiba S., L. Fan, and A. Vonshak (1993). Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. Methods in enzomology, 213: 386-

4. Choubert G. and O. Heinrich (1993). Carotenoid pigments of the green alga Haematococcus pluvialis : assay on rainbow trout Onchorhynchus mykiss, pigmentation in comparison with synthetic astaxanthin and cantaxanthin.

5. Cohn F. 1850: Nachtrage zur Naturgeshichte des Protococcus pluvialis Kützing. - Nov.Acta Leop.-Carol., pp607-764.

6. Droop M.R. (1953). On the ecology of flagellates from some brackishand fresh water rockpools of Finland. Acta Botanica Fennica 51, Ed. By: Societas Pro Fauna et Flora Fennica. 52pp.

7. Droop M.R. (1961). Haematococcus pluvialis and its allies. III organic nutrition. - Ibid. 5:4, 247-259.

8. Elliot A.M. (1934). Morphology and life history of Haematococcus pluvialis. Archiv. Protistekunde, 82:250-272.

9. Engelmamm T.W.(1882). Uber assimilation von Haematococcus. - Bot. Zeit. 40:39, 664-669.

10. Goodwin T.W., M. Jamikorn (1954). Studies in carotegenesis. II. Carotenoid synthesis in the alga Haematococcus pluvialis. Biochemical Journal, 57:376-381.

11. Hazen, T.E. (1899). The life history of Sphaerella lacustris. Mem. Torrey Bot. Club 6(3)n: 211-247.

12. Kobayashi M., T. Kakizono, K. Yamaguchi, N. Nishio and S. Nagai (1992a). Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. Journal of fermentation and Bioengineering, 74:61-63.

13. Nakazoe J., S. Ishii, M. Kamimoto and M. Takeuchi (1984). Effects of supplemental carotenoid pigments on the carotenoid accumulation in young red seabream (Chrysophyrys major). Bull. Tokai. Reg. Fish. Res. Lab., 113:29-41.Proctor V.E. (1957): Some controlling factors in the distribution of Haematococcus pluvialis. - Ecology, 457-462.

14. Schiedt K., M. Vecchi and E. Glinz (1986). Astaxanthin and its metabolites in wild rainbow trout Onchorhynchus mykiss . Aquaculture, 94:79-88.

15. Sommer T.R., W.T. Potts and N.M. Morissy (1991). Utilisation of microalgal astaxanthin by rainbow trout (Onchorhynchus mykiss). Aquaculture, 94:79-88.

16. Sommer T.R., F.M.L.D. Souza and N.M. Morissy (1991). Pigmentation of adult rainbow trout, Onchorhynchus mykiss, using the green alga Haematococcus pluvialis. Aquaculture, 106:63-74.

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