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Relative stabilities of microalgal carotenoids in microalgal extracts, biomass and fish feed: effect of storage conditions
Innovative Food Science and Emerging Technologies 4 (2003) 227–233
Relative stabilities of microalgal carotenoids in microalgal extracts, biomass and fish feed: effect of storage conditions Luisa Gouveiaa,*, Jose´ Empisb ¸ do Lumiar, Edifıcio ´ INETI-DER—Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada do Paco G, 1649-038 Lisboa, Portugal b ´ ´ ´ Centro de Engenharia Biologica e Quımica, Instituto Superior Tecnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
a
Received 5 April 2001; accepted 2 April 2002
Abstract Carotenoids, whose functional importance has become the object of much attention in the last years, are a source of vitamin A and food colouring agents for both animals andyor humans. As consumer demand for natural carotenoids increases, there is a natural barrier to their utilisation insofar as their low stability to oxidative environments is concerned. The aim of this work was to test stability of carotenoids present in microalgal biomass, such as Chlorella vulgaris (Cv) and Haematococcus pluvialis (Hp), already proven to be efficient colouring agents and of their acetone extracts, both as such and in formulated feeds, under different storage conditions, namely at room temperature under light exposure, at room temperature in the dark, frozen at y18 8C, with added antioxidant (0.01% ascorbic acid at room temperature) and stored under vacuum or nitrogen atmosphere. The best storage conditions for microalgal dry biomass carotenoids were under vacuum in both microalgae, when retention totaled 80 and 90%, respectively, for Cv and Hp, even after 1.5 years. Carotenoid extract stabilities were found to be much shorter, and loss of carotenoid pigments was almost total after 15 and 30 days, respectively, for Cv and Hp. In formulated diets, carotenogenic biomass revealed stability during the maximum storage period of six months. As a conclusion, both microalgal dry biomasses may constitute natural, encapsulated and relatively concentrated forms of edible carotenoids, which exhibit good preservation without any special storage conditions, both as such or in finished fish feed. 䊚 2003 Elsevier Science Ltd. All rights reserved. Keywords: Pigments; Food colourings; Carotenoids; Stability Industrial relevance: With an estimated annual production of 100 million tons of carotenoids in nature they are key natural coloring agents and antioxidants not only for food products but also for animal feed purposes. Consequently their storage stability is essential. Interestingly this work reveals that carotenoids are quite stable in their natural matrix such as dried microalgae but also in formulated fish feed pellets. Improved stability could be achieved with exclusion of oxygen while extracts where unstable. These data offer the potential to store products rich in carotenoids for up to 1.5 years without major losses.
1. Introduction Carotenoids are responsible for the beautiful colours of many fruits (pine-apple, citrus, tomatoes, mango, paprika) and flowers (Eschscholtzia, Narcissus), as well as the colour of many birds (flamingo, ibis, canary), insects (ladybird) and marine animals (crustaceans, salmonids). The total carotenoid production in nature has been estimated at approximately 100 000 000 t a year. Algae are a rich source of carotenoids, providing these to the aquatic food chain. *Corresponding author. Tel.: q351-21-712-7215; fax: q351-21712-7195. E-mail address: [email protected] (L. Gouveia).
Carotenoids and other essential antioxidants are receiving considerable public attention. A high daily intake of carotenoids has been recognised to reduce risk of cardiovascular disease (Neuman, Nahum & BenAmotz, 1999) and certain types of cancer, such as breast and lung cancer (De Stefani et al., 1999; Tavani et al., 1999), atherosclerosis, cataracts, macular degeneration and other major degenerative diseases (Cooper, Eldridge & Peters, 1999) and their contribution to enhance immune resistances to viral, bacterial, fungal and parasitic infections. Some carotenoids are also important in human nutrition as a source of vitamin A (e.g. from bcarotene).
1466-8564/03/$ - see front matter 䊚 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1466-8564(03)00002-X
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L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
Table 1 Ingredients and chemical composition of the experimental diets to rainbow trout
Table 2 Ingredients and chemical composition of the experimental diets to gilthead seabream
Basal mixture (gykg)
Basal mixture (gykg)
Control
Fishmeal Wheat meal Fish protein concentrate (CPSP) Fish oil Vitamin mixa Mineral mixb Choline chloride
480 360 100 45 10 5 3
Total carotenoid source (mgykg) Chlorella vulgarisc Haematococcus pluvialisc
Control
Control
Fishmeal Soybean meal Maize gluten Soybean oil Cod liver oil Fish protein concentrate (CPSP) Wheat Wheat middlings Vitamin mixa Mineral mixb Choline chloride
350 150 50 35 15 70 150 140 5 10 5
Total carotenoid source (mgykg) Chlorella vlgarisc Haematococcus pluvialisc
Control
Proximate composition Dry matter (DM) (%) Crude protein (% DM) Crude fat (% DM) Ash (% DM)
Cv diet
Hp diet
80.0 80.0 90.3 46.5 11.4 10.6
91.2 46.1 11.5 10.2
91.0 46.3 11.0 10.4
a (mg kgy1 diet): vit. E: 20; vit. K: 35; vit. B1: 5; vit. B2: 5; vit. B3: 10; vit. B5: 100; vit. B6: 5; vit. B9: 2; vit. B12: 0.05; vit. H (biotin): 0.5; ascorbic acid: 200; p-aminobenzoic acid: 50; inositol: 500; (UI kgy1 diet): vit. A: 10 000; vit. D3: 2,000. b (mg kgy1 diet): Co2q: 0.4; Cu2q: 5.0; Fe2q: 40; F2q: 1.0; I2q: 0.6; Mg2q: 100; Mn2q: 10. c In total carotenoid content basis.
Carotenoids have also been utilised by the food industry to colour foods and beverages (e.g. orange juice) and in aquaculture to impart adequate colour to farmed products. In addition, carotenoids are precursors of many important chemicals responsible for the flavour of foods and the fragrance of flowers. Despite the importance of carotenoids, these compounds are very unstable and their oxidative degradation can be triggered by light, temperature or extreme pH in the presence of oxygen. The main purpose of this work was to evaluate the storage stability of carotenoids derived from microalgae (as extracts, as microalgal dry biomass and as biomass in formulated fish diets), under different storage conditions. 2. Materials and methods 2.1. Microalgae Two carotenogenic microalgae were used in this study: Chlorella vulgaris (INETI 58) was cultivated in a Sorokin and Krauss growth medium (Vonshak, 1986) and carotenogenesis was performed according Gouveia et al. (1996a). The total carotenoid pigments was 0.4% (afdw); Haematococcus pluvialis (INETI 33) was cultivated in Bold Basal Medium (Richmond, 1986) in
Proximate composition Dry matter (DM) (%) Crude protein (% DM) Crude fat (% DM) Ash (% DM)
Cv diet
Hp diet
40.0 40.0 91.8 48.6 12.8 10.0
91.6 47.7 12.7 10.2
91.1 48.1 12.9 10.0
a (mg kgy1 diet): vit. E: 20; vit. K: 35; vit. B1: 5; vit. B2: 5; vit. B3: 10; vit. B5: 100; vit. B6: 5; vit. B9: 2; vit. B12: 0.05; vit. H (biotin): 0.5; ascorbic acid: 200; p-aminobenzoic acid: 50; inositol: 500; (UI kgy1 diet): vit. A: 10 000; vit. D3: 2,000. b (mg kgy1 diet): Co2q: 0.4; Cu2q: 5.0; Fe2q: 40; F2q: 1.0; I2q: 0.6; Mg2q: 100; Mn2q: 10. c In total carotenoid content basis
airlifts at low light conditions (150 mE my2 sy1). Carotenogenesis was induced by nutrient starvation and sodium chloride addition (2%) at high luminosity, (1000 mE my2 sy1). Harvesting was made without prior flocculation by simply removing agitation. The total amount of carotenoid pigments was 1.9% (afdw). 2.2. Analysis Total carotenoid pigments were determined for dry biomass (at intervals, during 1.5 years); after acetone extraction (during 6 months) and in dry biomass incorporated into fish diets, both before and after pelleting (during 6 months). Duplicate samples were taken for each determination. Two fish diets were tested, namely for trout and gilthead seabream cultures, with microalgal biomass carotenoid incorporation of 80 and 40 mg pigmentsykg diet, respectively (Table 1 and Table 2) which described ingredients and proximate composition of the diets. Dry biomass sampling was performed at weeks: 1, 2, 4 (1 month), 10, 40, 52 (1 year) and 78 (1.5 year). Total carotenoid content in the algae was determined spectrophotometrically after extraction with acetone (Choubert & Storebakken, 1989). Carotenoids are expressed using extinction coefficients (E1% 1 cm ) of 2150 for algal pigments at their absorption maximum in acetone (Gouveia, Gomes & Empis, 1997). Carotenoid extract samples were taken at days: 1, 6, 15, 30 (1 month), 75 (2.5 month) and 180 (6 month).
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
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Table 3 Dry biomass stability of Chlorella vulgaris. Total carotenoid content of dry Chlorella vulgaris biomass, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time
Storage conditions
0 1 week 1 month 2.5 month 1 year 1.5 year *
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
320.5"0.9 315.0"2.8 312.8"3.5 300.1"1.6 280.9"2.0 140.2"2.2
320.5"0.9 318.1"1.4 318.4"1.9 295.2"2.8 290.8"3.1 225.5"0.8
320.5"0.9 319.5"2.3 310.7"1.7 295.1"4.1 230.4"5.1 212.3"2.1
320.5"0.9 316.4"0.5 300.3"1.7 270.7"2.5 235.1"4.1 125.4"2.5
320.5"0.9 318.2"0.4 319.4"1.3 315.9"2.2 315.0"0.8 305.3"3.1
320.5"0.9 317.8"2.3 314.2"3.1 300.5"2.6 298.2"1.6 285.7"2.4
Values are means of two determinations"S.D.
Table 4 Total carotenoid content loss of dry Chlorella vulgaris biomass at different time periods (%)
Table 6 Total carotenoid content loss of Haematococcus pluvialis dry biomass at different time periods (%)
Time
Time
1 week 1 month 2.5 month 1 year 1.5 year
Storage conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
1.5 6.2 28.0 86.0 89.0
0.6 7.8 9.4 56.0 57.0
0.3 7.8 28.0 34.0 40.0
1.2 15.6 26.5 62.5 70.0
0.6 1.5 1.5 6.0 6.0
0.9 6.2 6.9 12.5 20.0
Carotenoid extract from fish diets were taken at 1, 2.5 and 6 month. 2.3. Storage conditions Dry biomass microalgal (Chlorella vulgaris and Haematococcus pluvialis) was maintained during 1.5 year under the following conditions: a. At room temperature under light exposure; b. At room temperature in the dark; c. Frozen at y18 8C in the dark; d. With antioxidant (0.01% ascorbic acid) at room temperature in the dark; e. Under a vacuum (P-0.05 atm) in the dark; f. Under a nitrogen atmosphere in the dark. Carotenoid pigment acetone extracts of both algae (Chlorella vulgaris and Haematococcus pluvialis), were
Storage conditions*
1 week 1 month 2.5 month 1 year 1.5 year
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
1.3 4.8 26.0 73.0 70.0
0.8 6.8 7.0 44.0 55.0
0.4 5.0 18.0 36.0 40.0
0.6 3.2 13.0 42.0 52.0
0.4 0.9 1.8 8.2 9.1
0.7 1.2 4.3 7.1 10.0
maintained under the same storage conditions a–f above, during 6 months. Chlorella vulgaris and Haematococcus pluvialis biomass, incorporated into fish diets, was stored refrigerated at 4 8C, in the dark, during 6 months. 3. Results The total amount of carotenoid pigments of Chlorella vulgaris biomass, under different storage conditions, during the 1.5 year long trial period, is shown in Table 3. Even at room temperature and in presence of light, depletion on carotenoid content occurred only after approximately 1 month. Without any atmosphere modification, just by maintaining the biomass in the dark, carotenoid contents are approximately constant during
Table 5 Dry biomass stability of Haematococcus pluvialis. Total carotenoid content of dry Haematococcus pluvialis biomass, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time
0 1 week 1 month 2.5 month 1 year 1.5 year *
Storage conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
1500.2"1.6 1480.1"1.8 1460.2"4.3 1428.9"3.9 1110.6"2.8 850.3"5.1
1500.2"1.6 1488.7"4.6 1470.3"6.2 1398.2"3.5 1395.0"2.0 1000.1"1.0
1500.2"1.6 1494.7"2.7 1480.6"4.2 1425.8"4.6 1230.2"4.1 1100.5"1.3
1500.2"1.6 1491.0"1.4 1472.2"1.5 1452.6"4.8 1305.6"0.7 980.0"4.0
1500.2"1.6 1494.1"1.7 1490.6"7.2 1486.7"2.1 1473.4"2.9 1430.4"1.7
1500.2"1.6 1489.5"4.5 1485.1"3.4 1482.4"2.5 1435.3"5.6 1415.2"2.0
Values are means of two determinations"S.D.
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
230
Table 7 Acetone extract stability of Chlorella vulgaris. Total carotenoid content of Chlorella vulgaris acetone extract, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time (days)
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
0 1 6 15 30 75 180
320.5"0.9 176.0"7.1 39.4"4.2 8.0"1.9 0.0"0.0 0.0"0.0 0.0"0.0
320.5"0.9 267.8"4.6 263.7"5.1 263.4"3.0 230.4"2.8 217.6"1.9 153.6"3.5
320.5"0.9 276.5"5.6 271.4"3.4 254.4"1.2 252.2"0.5 236.8"0.1 198.4"2.7
320.5"0.9 275.8"6.0 262.4"4.9 242.9"2.1 182.4"5.6 125.8"1.3 102.4"4.5
320.5"0.9 313.6"0.4 310.1"1.2 297.9"4.7 285.8"5.0 280.6"2.7 278.4"1.5
320.5"0.9 314.2"5.2 306.2"2.6 293.8"1.8 288.3"3.4 282.2"4.7 278.1"3.7
*
Storage conditions
Values are means of two determinations"S.D.
Table 8 Total carotenoid content loss of Chlorella vulgaris acetone extract at different time periods (%)
Table 10 Total carotenoid content loss of Haematococcus pluvialis acetone extract at different time periods (%)
Time
Time
1 day 6 day 15 day 1 month 2.5 month 6 month
Storage conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
45.0 87.7 97.5 100 100 100
16.3 17.6 17.7 28.0 32.0 52.0
13.6 15.2 20.5 21.2 26.0 38.0
13.8 18.0 24.1 43.0 60.7 68.0
2.0 3.1 6.9 10.7 12.3 13.0
1.8 4.3 8.2 9.9 11.8 13.1
the first 2.5 months (10% loss, approximately) (Table 4). After 1 year carotenoid amount was reduced to half. The best storage condition found was under a vacuum, with total loss of carotenoids of only 6% even after 1.5 year, closely followed by storage under nitrogen atmosphere, where a loss of 12,5% of total carotenoids after 1 year was registered, and only 20% after 1.5 years (Table 4). The storage stability of Haematococcus pluvialis biomass parallels that of Chlorella vulgaris (Table 5). The best conditions were also found to be under a vacuum and under nitrogen atmosphere (losses in the same range of magnitude). Under those conditions carotenoid content remained 90% of the initial amount even after 1.5 years (Table 6).
1 day 6 day 15 day 1 month 2.5 month 6 month
Stored conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
16.6 42.7 68.0 100 100 100
0 24.4 52.6 58.2 60.0 63.0
2.0 10.4 12.8 14.2 15.9 29.0
2.4 7.8 10.2 18.9 29.3 40.0
0,4 2.3 4.2 9.1 9.8 12.5
0.3 1.9 5.2 8.7 10.0 14.2
Acetone extracts of Chlorella vulgaris and Haematococcus pluvialis carotenoid pigments are much more unstable than the corresponding dry biomass, which is thus shown to constitute a natural oxidation barrier. Carotenoids disappear between 15 and 30 days from both extracts, at room temperature in the presence of light (Table 7 and Table 9). Loss of acetone extracts carotenoid content for Chlorella vulgaris and Haematococcus pluvialis are present in Table 8 and Table 10. Chlorella and Haematococcus carotenoids extracts present almost the same behaviour with respect to storage conditions and the best conditions were again found to be under vacuum and under nitrogen atmosphere, as for dry biomass.
Table 9 Acetone extract stability of Haematococcus pluvialis. Total carotenoid content in Haematococcus pluvialis acetone extract, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time (days)
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
0 1 6 15 30 75 180
1500.2"1.6 1251.0"3.9 859.5"4.1 480.0"0.9 0.0"0.0 0.0"0.0 0.0"0.0
1500.2"1.6 1500.0"3.8 1134.0"0.9 711.0"5.7 627.0"4.6 600.0"4.1 555.0"1.8
1500.2"1.6 1470.0"2.3 1344.0"2.8 1308.0"4.6 1287.0"5.0 1261.5"1.0 1065.0"3.4
1500.2"1.6 1464.0"0.6 1383.0"5.9 1347.0"1.7 1216.5"2.7 1060.5"4.2 900.0"3.5
1500.2"1.6 1494.0"3.5 1465.5"4.8 1437.0"1.5 1363.5"0.2 1353.0"3.1 1312.5"5.0
1500.2"1.6 1495.5"3.8 1471.5"5.6 1422.0"4.1 1369.5"3.8 1350.0"5.0 1287.0"0.4
*
Storage conditions
Values are means of two determinations"S.D.
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233 Table 11 Total carotenoid content loss of Chlorella vulgaris and Haematococcus pluvialis biomass, incorporated in formulated fish diets for rainbow trout, at different time periods (%)* Time (month)
Chlorella vulgaris diet
Haematococcus pluvialis diet
1 2.5 6
6.4 8.5 24.0
7.8 8.9 18.8
*
Stored at 4 8C, in the dark.
Without any special atmosphere conditions during storage, but simply maintaining the samples in the dark, the period needed for a carotenoid loss of 50% is approximately 30 days for the extracts, compared to 1.5 year for dry biomass. In diets formulated as fish feed, carotenoid content remained quite stable, without significant loss during the trial period, for both microalgal biomass sources of carotenoids (Table 11 and Table 12). Pelleting process of the diets (with a pelleting machine without steam) didn’t affect carotenoid content of the diets (temperature above 50 8C). 4. Discussion The importance of carotenoid pigments has been recognised in the last years for their roles as antioxidants, sources of vitamin A and colouring agents in foods and beverages. In the marine environment they are probably best known for eliciting the pink–red hue to the flesh of salmonids, as well the diverse colours found in shrimp, lobsters, crayfish and ornamental fishes. Carotenoids have nevertheless also been reported to yield a general enhancement of performance of specific reproduction and metabolism, namely growth and survival, both in eggs, larval and fish and crayfish development (Thompson, Fletcher, Houlihan & Secombes, 1994; Torrissen & Christiansen, 1995; Paripatananont, Tangtrongpairoj, Sailasuta & Chansue, 1999). Decreasing natural catches and increasing world dependence on fish as a food source, as well as technological improvements, had led to increased aquacultural production. Total aquaculture production in 1995 was over 25 million tons and this industry is projected to grow at a rate of 8% per year for the foreseeable future (Tacon, 1998). In aquacultural practice, astaxanthin remained the best pigment due to it effectiveness in colouring, and to the commercially acceptable nature of the food products obtained. The major market for astaxanthin is as a pigmentation supplement in aquaculture, primarily for salmon and trout, but also for shrimp, and its market value is ;US$2.500ykg with an annual worldwide market estimated at US$200 million (Lorenz & Cysewki, 2000).
231
Other works have shown that astaxanthin can also protect skin from the damaging effects of ultraviolet radiation, ameliorate age-related macular degeneration, protect against chemically induced cancers, increase high-density lipoproteins and enhance the immune system (Lorenz & Cysewki, 2000). Epidemiological studies have also demonstrated a correlation between increased carotenoid intake and reduced incidence of coronary heart disease and certain cancers, macular degeneration as well as an increased resistance to viral, bacterial, fungal and parasitic infections (Lorenz & Cysewki, 2000). As an antioxidant, astaxanthin surpasses the benefits of others; the antioxidant activities of astaxanthin have been shown to be approximately 10 times greater than those of b-carotene, lutein, zeaxanthin, cantaxanthin, and over 500 times greater than that shown by atocoferol. The name ‘super vitamin E’ has been proposed for astaxanthin (Miki, 1991). Actually, a fruit- and vegetable- rich diet is recommended to increase the dietary amounts of carotenoids; however, it has been shown that the bioavailability of carotenoids in fruits and vegetables is significantly lower than that of algae-derived supplements (Werman, BenAmotz & Mokady, 1999). It has also been proved that carotenoid extracts have higher anti-oxidant properties than synthetic ones (Levin, Yeshurun & Mokady, 1997). Although consumer demand for natural products is increasing, over 95% of the market needs are met by synthetic astaxanthin, and this provides an opportunity for competitive production of natural astaxanthin by Haematococcus pluvialis and Chlorella vulgaris. Both algae can be utilised as whole biomass, without extraction, as was successfully proven for Chlorella vulgaris for egg pigmentation (Gouveia et al., 1996b) and in aquaculture products (Gouveia, Gomes & Empis, 1996c; Gouveia et al., 1997; Gouveia, Choubert, Gomes, Rema & Empis, 1998; Gouveia, Veloso & Empis, 1999a; Gouveia & Empis, 1999b; Gouveia et al., 2002) comparable to products obtained using synthetic dies. Also Haematococcus pluvialis can be an effective pigment source for aquacultural products as it is an efficient source of relatively concentrated astaxanthin (Gomes et al., 2002) Table 12 Total carotenoid content loss of Chlorella vulgaris and Haematococcus pluvialis biomass, incorporated in formulated fish diets for gilthead seabream, at different time periods (%)* Time (month)
Chlorella vulgaris diet
Haematococcus pluvialis diet
1 2.5 6
5.0 7.6 22.0
4.1 7.0 18.6
*
Stored at 4 8C, in the dark.
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L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
Compared with Chlorella vulgaris, Haematococcus pluvialis revealed a greater stability, probably due to the depth of the cyst walls that surround the cell in the carotenogenic stage. The absence of an antioxidant effect in presence of added ascorbic acid can only be interpreted by assuming that the limiting factor in the oxidative degradation is a textural, rather than a bulk effect. In this work, stability of biomass of both algae seems to be predominantly affected by light, and in second order by oxygen content, whereas the storage temperature in only important to a lesser extent. pH influence was not studied in this work. Extract microalgal pigments are much more unstable than pigment contained in dry biomass or in biomass incorporated in fish feed diets, which is crucial, because many processing steps can be bypassed, e.g. extraction, encapsulation or emulsion and drying. The use of microalgal biomass as a pigment source of finish fish feed is very promising in terms of stability of carotenoids because feedstuffs should, as far as possible, be stored for a minimum length of time. Feed are composed of perishable biological material and the maximum permissible storage time is 6 months (Silva & Anderson, 1995), a period during which no great loss of pigment for both microalgae in both fish diets tested, was observed. A microalgal carotenoid loss is similar in both fish diets, instead of different carotenoid and fat content of the diets. For use as a feed ingredient in aquaculture, microalgae should also be valued as sources of some micronutrients, such as vitamins and minerals, but the contents and variation of these potentially valuable constituents were not determined in this work. In this study it is concluded that both microalgae may constitute natural, concentrated and encapsulated forms of edible carotenoids, which can be kept during appropriate periods without any special storage conditions, both as dry biomass, or in prepared diets. Acknowledgments This work was supported by FCT (Portugal) through the project PDCTMyMARy15237y1999. References Choubert, G., & Storebakken, T. (1989). Dose response to astaxanthin and canthaxanthin pigmentation of rainbow trout fed various dietary carotenoid concentrations. Aquaculture, 81, 69 –77. Cooper, D. A., Eldridge, A. L., & Peters, J. C. (1999). Dietary carotenoids and certain cancers, heart disease, and age-related macular degeneration: A review of recent research. Nutrition Review, 57, 201 –214. De Stefani, E., Boffetta, P., Deneo-Pellegrini, H., Mendilaharsu, M., Carzoglio, J. C., Ronco, A., & Olivera, L. (1999). Dietary antioxidants and lung cancer risk: A case-control study in Uruguay. Nutrition Cancer, 34, 100 –110.
Gomes, E., Dias, J., Silva, P., Valente, L., Empis, J., Gouveia, L., Bowen, J., & Young, W. (2002). Utilisation of natural and synthetic sources of carotenoids in the skin pigmentation of gilthead seabream (Sparus aurata). European Food Research Technology, 214, 287 –293. Gouveia, L., Veloso, V., Reis, A., Fernandes, H. L., Empis, J., & Novais, J. M. (1996a). Evolution of the pigments in Chlorella vulgaris during carotenogenesis. Bioresources Technology, 57, 157 –163. Gouveia, L., Veloso, V., Reis, A., Fernandes, H. L., Empis, J., & Novais, J. M. (1996b). Chlorella vulgaris used to colour egg yolk. Journal of Food Science and Technology, 70, 167 –172. Gouveia, L., Gomes, E., & Empis, J. (1996c). Potential use of a microalgal (Chlorella vulgaris) in the pigmentation of rainbow trout (Oncorhynchus mykiss) muscle. Lebensmittel-Untersuchung and Forschung, 202, 75 –79. Gouveia, L., Gomes, E., & Empis, J. (1997). Use of Chlorella vulgaris in diets for rainbow trout to enhance pigmentation of muscle. Journal of Applied Aquaculture, 7(2), 61 –70. Gouveia, L., Choubert, G., Gomes, E., Rema, P., & Empis, J. (1998). Use of Chlorella vulgaris as a carotenoid source for salmonids: Effect of dietary lipid content on pigmentation, digestibility and muscular retention. Aquaculture International, 6(4), 269 –279. Gouveia, L., Veloso, V., & Empis, J. (1999a). Natural pigments from Chlorella vulgaris and Haematococcus pluvialis microalgae. Biotechnological production and use as ingredients in the food chain. In Proceedings of 1st International Congress on Pigments in Food ¸ I.S.B.N.: 84-699Technology, Sevilha, Espanha, 24–26 Marco. 0185-0. Gouveia, L., & Empis, J. (1999b). Functional food Chlorella vulgaris microalga as an ingredient for food chain pigmentation. In Proceedings of Euro Food Chem 10, Conference on Functional FoodsA New Challenge for the Food Chemist, September 22–24, Budapest, Hungary, I.S.B.N. Gouveia, L., Choubert, G., Gomes, E., Pereira, N., Santinha, J., & Empis, J. (2002). Pigmentation of gilthead seabream, Sparus aurata (L.1875) using Chlorella vulgaris (Chlorophyta, Volvocales) microalga. Aquaculture Research, 33, 987 –993. Levin, G., Yeshurun, M., & Mokady, S. (1997). In vivo peroxidative effect of 9-cis-b-carotene compared with that of the all-trans isomer. Nutrition Cancer, 27, 293 –297. Lorenz, R., & Cysewki, G. (2000). Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Tibtech, vol.18, April 2000. Miki, W. (1991). Biological functions and activities of carotenoids. Pure Applied Chemistry, 63, 141 –146. Neuman, I., Nahum, H., & Ben-Amotz, A. (1999). Prevention of exercise-induced asthma by a natural isomer mixture of betacarotene. Annual Allergy Ashtma Immunology, 82, 549 –553. Richmond, A. (1986). In: A. Richmond, CRC Handbook of Microalgal Mass Culture. Boca Raton: CRC Press. Silva, S., & Anderson, T. (1995). Diet preparation. In S. Silva, T. Anderson, Fish Nutrition in Aquaculture (pp. 196 –202). Chapman & Hall Aquaculure series 1 Chapman & Hall. Tacon, A. (1998). Private global trends in aquaculture and aquafeed production 1984–1995. World Aquaculture Society Annual Meeting, February, Las Vegas, NV, p. 531. Tavani, A., Gallus, S., La Vecchia, C., Negri, E., Montella, M., Dal Maso, L., & Franceschi, S. (1999). Risk factors for breast cancer in women under 40 years. European Journal of Cancer, 35, 1361 –1367. Thompson, I., Fletcher, T., Houlihan, D., & Secombes, C. (1994). Effect of dietary vitamin A intake on the immunocompetence of
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233 Atlantic salmon (Salmo salar L.). Fish Physiology Biochemistry, 12, 513 –5123. Torrissen, O., & Christiansen, R. (1995). Requirements for carotenoids in fish diets. Journal of Applied Ichthyology, 11, 225 –230. Paripatananont, T., Tangtrongpairoj, J., Sailasuta, A., & Chansue, N. (1999). Effect os astaxanthin on the pigmentation of goldfish Carassius auratus. Journal of World Aquaculture Society, 30(4), 454 –460.
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Vonshak, A. (1986). Laboratory techniques for the cultivation of microalgae. In A. Richmond, CRC Handbook of Microalgal Mass Culture (pp. 117 –143). Boca Raton, USA: CRC Press. Werman, M. J., Ben-Amotz, A., & Mokady, S. (1999). Availability and antiperoxidative effects of beta-carotene from Dunaliella bardawil in alcohol-drinking rats. Journal of Nutrition Biochemistry, 10, 449 –454.
Relative stabilities of microalgal carotenoids in microalgal extracts, biomass and fish feed: effect of storage conditions Luisa Gouveiaa,*, Jose´ Empisb ¸ do Lumiar, Edifıcio ´ INETI-DER—Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada do Paco G, 1649-038 Lisboa, Portugal b ´ ´ ´ Centro de Engenharia Biologica e Quımica, Instituto Superior Tecnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
a
Received 5 April 2001; accepted 2 April 2002
Abstract Carotenoids, whose functional importance has become the object of much attention in the last years, are a source of vitamin A and food colouring agents for both animals andyor humans. As consumer demand for natural carotenoids increases, there is a natural barrier to their utilisation insofar as their low stability to oxidative environments is concerned. The aim of this work was to test stability of carotenoids present in microalgal biomass, such as Chlorella vulgaris (Cv) and Haematococcus pluvialis (Hp), already proven to be efficient colouring agents and of their acetone extracts, both as such and in formulated feeds, under different storage conditions, namely at room temperature under light exposure, at room temperature in the dark, frozen at y18 8C, with added antioxidant (0.01% ascorbic acid at room temperature) and stored under vacuum or nitrogen atmosphere. The best storage conditions for microalgal dry biomass carotenoids were under vacuum in both microalgae, when retention totaled 80 and 90%, respectively, for Cv and Hp, even after 1.5 years. Carotenoid extract stabilities were found to be much shorter, and loss of carotenoid pigments was almost total after 15 and 30 days, respectively, for Cv and Hp. In formulated diets, carotenogenic biomass revealed stability during the maximum storage period of six months. As a conclusion, both microalgal dry biomasses may constitute natural, encapsulated and relatively concentrated forms of edible carotenoids, which exhibit good preservation without any special storage conditions, both as such or in finished fish feed. 䊚 2003 Elsevier Science Ltd. All rights reserved. Keywords: Pigments; Food colourings; Carotenoids; Stability Industrial relevance: With an estimated annual production of 100 million tons of carotenoids in nature they are key natural coloring agents and antioxidants not only for food products but also for animal feed purposes. Consequently their storage stability is essential. Interestingly this work reveals that carotenoids are quite stable in their natural matrix such as dried microalgae but also in formulated fish feed pellets. Improved stability could be achieved with exclusion of oxygen while extracts where unstable. These data offer the potential to store products rich in carotenoids for up to 1.5 years without major losses.
1. Introduction Carotenoids are responsible for the beautiful colours of many fruits (pine-apple, citrus, tomatoes, mango, paprika) and flowers (Eschscholtzia, Narcissus), as well as the colour of many birds (flamingo, ibis, canary), insects (ladybird) and marine animals (crustaceans, salmonids). The total carotenoid production in nature has been estimated at approximately 100 000 000 t a year. Algae are a rich source of carotenoids, providing these to the aquatic food chain. *Corresponding author. Tel.: q351-21-712-7215; fax: q351-21712-7195. E-mail address: [email protected] (L. Gouveia).
Carotenoids and other essential antioxidants are receiving considerable public attention. A high daily intake of carotenoids has been recognised to reduce risk of cardiovascular disease (Neuman, Nahum & BenAmotz, 1999) and certain types of cancer, such as breast and lung cancer (De Stefani et al., 1999; Tavani et al., 1999), atherosclerosis, cataracts, macular degeneration and other major degenerative diseases (Cooper, Eldridge & Peters, 1999) and their contribution to enhance immune resistances to viral, bacterial, fungal and parasitic infections. Some carotenoids are also important in human nutrition as a source of vitamin A (e.g. from bcarotene).
1466-8564/03/$ - see front matter 䊚 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1466-8564(03)00002-X
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L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
Table 1 Ingredients and chemical composition of the experimental diets to rainbow trout
Table 2 Ingredients and chemical composition of the experimental diets to gilthead seabream
Basal mixture (gykg)
Basal mixture (gykg)
Control
Fishmeal Wheat meal Fish protein concentrate (CPSP) Fish oil Vitamin mixa Mineral mixb Choline chloride
480 360 100 45 10 5 3
Total carotenoid source (mgykg) Chlorella vulgarisc Haematococcus pluvialisc
Control
Control
Fishmeal Soybean meal Maize gluten Soybean oil Cod liver oil Fish protein concentrate (CPSP) Wheat Wheat middlings Vitamin mixa Mineral mixb Choline chloride
350 150 50 35 15 70 150 140 5 10 5
Total carotenoid source (mgykg) Chlorella vlgarisc Haematococcus pluvialisc
Control
Proximate composition Dry matter (DM) (%) Crude protein (% DM) Crude fat (% DM) Ash (% DM)
Cv diet
Hp diet
80.0 80.0 90.3 46.5 11.4 10.6
91.2 46.1 11.5 10.2
91.0 46.3 11.0 10.4
a (mg kgy1 diet): vit. E: 20; vit. K: 35; vit. B1: 5; vit. B2: 5; vit. B3: 10; vit. B5: 100; vit. B6: 5; vit. B9: 2; vit. B12: 0.05; vit. H (biotin): 0.5; ascorbic acid: 200; p-aminobenzoic acid: 50; inositol: 500; (UI kgy1 diet): vit. A: 10 000; vit. D3: 2,000. b (mg kgy1 diet): Co2q: 0.4; Cu2q: 5.0; Fe2q: 40; F2q: 1.0; I2q: 0.6; Mg2q: 100; Mn2q: 10. c In total carotenoid content basis.
Carotenoids have also been utilised by the food industry to colour foods and beverages (e.g. orange juice) and in aquaculture to impart adequate colour to farmed products. In addition, carotenoids are precursors of many important chemicals responsible for the flavour of foods and the fragrance of flowers. Despite the importance of carotenoids, these compounds are very unstable and their oxidative degradation can be triggered by light, temperature or extreme pH in the presence of oxygen. The main purpose of this work was to evaluate the storage stability of carotenoids derived from microalgae (as extracts, as microalgal dry biomass and as biomass in formulated fish diets), under different storage conditions. 2. Materials and methods 2.1. Microalgae Two carotenogenic microalgae were used in this study: Chlorella vulgaris (INETI 58) was cultivated in a Sorokin and Krauss growth medium (Vonshak, 1986) and carotenogenesis was performed according Gouveia et al. (1996a). The total carotenoid pigments was 0.4% (afdw); Haematococcus pluvialis (INETI 33) was cultivated in Bold Basal Medium (Richmond, 1986) in
Proximate composition Dry matter (DM) (%) Crude protein (% DM) Crude fat (% DM) Ash (% DM)
Cv diet
Hp diet
40.0 40.0 91.8 48.6 12.8 10.0
91.6 47.7 12.7 10.2
91.1 48.1 12.9 10.0
a (mg kgy1 diet): vit. E: 20; vit. K: 35; vit. B1: 5; vit. B2: 5; vit. B3: 10; vit. B5: 100; vit. B6: 5; vit. B9: 2; vit. B12: 0.05; vit. H (biotin): 0.5; ascorbic acid: 200; p-aminobenzoic acid: 50; inositol: 500; (UI kgy1 diet): vit. A: 10 000; vit. D3: 2,000. b (mg kgy1 diet): Co2q: 0.4; Cu2q: 5.0; Fe2q: 40; F2q: 1.0; I2q: 0.6; Mg2q: 100; Mn2q: 10. c In total carotenoid content basis
airlifts at low light conditions (150 mE my2 sy1). Carotenogenesis was induced by nutrient starvation and sodium chloride addition (2%) at high luminosity, (1000 mE my2 sy1). Harvesting was made without prior flocculation by simply removing agitation. The total amount of carotenoid pigments was 1.9% (afdw). 2.2. Analysis Total carotenoid pigments were determined for dry biomass (at intervals, during 1.5 years); after acetone extraction (during 6 months) and in dry biomass incorporated into fish diets, both before and after pelleting (during 6 months). Duplicate samples were taken for each determination. Two fish diets were tested, namely for trout and gilthead seabream cultures, with microalgal biomass carotenoid incorporation of 80 and 40 mg pigmentsykg diet, respectively (Table 1 and Table 2) which described ingredients and proximate composition of the diets. Dry biomass sampling was performed at weeks: 1, 2, 4 (1 month), 10, 40, 52 (1 year) and 78 (1.5 year). Total carotenoid content in the algae was determined spectrophotometrically after extraction with acetone (Choubert & Storebakken, 1989). Carotenoids are expressed using extinction coefficients (E1% 1 cm ) of 2150 for algal pigments at their absorption maximum in acetone (Gouveia, Gomes & Empis, 1997). Carotenoid extract samples were taken at days: 1, 6, 15, 30 (1 month), 75 (2.5 month) and 180 (6 month).
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
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Table 3 Dry biomass stability of Chlorella vulgaris. Total carotenoid content of dry Chlorella vulgaris biomass, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time
Storage conditions
0 1 week 1 month 2.5 month 1 year 1.5 year *
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
320.5"0.9 315.0"2.8 312.8"3.5 300.1"1.6 280.9"2.0 140.2"2.2
320.5"0.9 318.1"1.4 318.4"1.9 295.2"2.8 290.8"3.1 225.5"0.8
320.5"0.9 319.5"2.3 310.7"1.7 295.1"4.1 230.4"5.1 212.3"2.1
320.5"0.9 316.4"0.5 300.3"1.7 270.7"2.5 235.1"4.1 125.4"2.5
320.5"0.9 318.2"0.4 319.4"1.3 315.9"2.2 315.0"0.8 305.3"3.1
320.5"0.9 317.8"2.3 314.2"3.1 300.5"2.6 298.2"1.6 285.7"2.4
Values are means of two determinations"S.D.
Table 4 Total carotenoid content loss of dry Chlorella vulgaris biomass at different time periods (%)
Table 6 Total carotenoid content loss of Haematococcus pluvialis dry biomass at different time periods (%)
Time
Time
1 week 1 month 2.5 month 1 year 1.5 year
Storage conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
1.5 6.2 28.0 86.0 89.0
0.6 7.8 9.4 56.0 57.0
0.3 7.8 28.0 34.0 40.0
1.2 15.6 26.5 62.5 70.0
0.6 1.5 1.5 6.0 6.0
0.9 6.2 6.9 12.5 20.0
Carotenoid extract from fish diets were taken at 1, 2.5 and 6 month. 2.3. Storage conditions Dry biomass microalgal (Chlorella vulgaris and Haematococcus pluvialis) was maintained during 1.5 year under the following conditions: a. At room temperature under light exposure; b. At room temperature in the dark; c. Frozen at y18 8C in the dark; d. With antioxidant (0.01% ascorbic acid) at room temperature in the dark; e. Under a vacuum (P-0.05 atm) in the dark; f. Under a nitrogen atmosphere in the dark. Carotenoid pigment acetone extracts of both algae (Chlorella vulgaris and Haematococcus pluvialis), were
Storage conditions*
1 week 1 month 2.5 month 1 year 1.5 year
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
1.3 4.8 26.0 73.0 70.0
0.8 6.8 7.0 44.0 55.0
0.4 5.0 18.0 36.0 40.0
0.6 3.2 13.0 42.0 52.0
0.4 0.9 1.8 8.2 9.1
0.7 1.2 4.3 7.1 10.0
maintained under the same storage conditions a–f above, during 6 months. Chlorella vulgaris and Haematococcus pluvialis biomass, incorporated into fish diets, was stored refrigerated at 4 8C, in the dark, during 6 months. 3. Results The total amount of carotenoid pigments of Chlorella vulgaris biomass, under different storage conditions, during the 1.5 year long trial period, is shown in Table 3. Even at room temperature and in presence of light, depletion on carotenoid content occurred only after approximately 1 month. Without any atmosphere modification, just by maintaining the biomass in the dark, carotenoid contents are approximately constant during
Table 5 Dry biomass stability of Haematococcus pluvialis. Total carotenoid content of dry Haematococcus pluvialis biomass, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time
0 1 week 1 month 2.5 month 1 year 1.5 year *
Storage conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
1500.2"1.6 1480.1"1.8 1460.2"4.3 1428.9"3.9 1110.6"2.8 850.3"5.1
1500.2"1.6 1488.7"4.6 1470.3"6.2 1398.2"3.5 1395.0"2.0 1000.1"1.0
1500.2"1.6 1494.7"2.7 1480.6"4.2 1425.8"4.6 1230.2"4.1 1100.5"1.3
1500.2"1.6 1491.0"1.4 1472.2"1.5 1452.6"4.8 1305.6"0.7 980.0"4.0
1500.2"1.6 1494.1"1.7 1490.6"7.2 1486.7"2.1 1473.4"2.9 1430.4"1.7
1500.2"1.6 1489.5"4.5 1485.1"3.4 1482.4"2.5 1435.3"5.6 1415.2"2.0
Values are means of two determinations"S.D.
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233
230
Table 7 Acetone extract stability of Chlorella vulgaris. Total carotenoid content of Chlorella vulgaris acetone extract, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time (days)
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
0 1 6 15 30 75 180
320.5"0.9 176.0"7.1 39.4"4.2 8.0"1.9 0.0"0.0 0.0"0.0 0.0"0.0
320.5"0.9 267.8"4.6 263.7"5.1 263.4"3.0 230.4"2.8 217.6"1.9 153.6"3.5
320.5"0.9 276.5"5.6 271.4"3.4 254.4"1.2 252.2"0.5 236.8"0.1 198.4"2.7
320.5"0.9 275.8"6.0 262.4"4.9 242.9"2.1 182.4"5.6 125.8"1.3 102.4"4.5
320.5"0.9 313.6"0.4 310.1"1.2 297.9"4.7 285.8"5.0 280.6"2.7 278.4"1.5
320.5"0.9 314.2"5.2 306.2"2.6 293.8"1.8 288.3"3.4 282.2"4.7 278.1"3.7
*
Storage conditions
Values are means of two determinations"S.D.
Table 8 Total carotenoid content loss of Chlorella vulgaris acetone extract at different time periods (%)
Table 10 Total carotenoid content loss of Haematococcus pluvialis acetone extract at different time periods (%)
Time
Time
1 day 6 day 15 day 1 month 2.5 month 6 month
Storage conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
45.0 87.7 97.5 100 100 100
16.3 17.6 17.7 28.0 32.0 52.0
13.6 15.2 20.5 21.2 26.0 38.0
13.8 18.0 24.1 43.0 60.7 68.0
2.0 3.1 6.9 10.7 12.3 13.0
1.8 4.3 8.2 9.9 11.8 13.1
the first 2.5 months (10% loss, approximately) (Table 4). After 1 year carotenoid amount was reduced to half. The best storage condition found was under a vacuum, with total loss of carotenoids of only 6% even after 1.5 year, closely followed by storage under nitrogen atmosphere, where a loss of 12,5% of total carotenoids after 1 year was registered, and only 20% after 1.5 years (Table 4). The storage stability of Haematococcus pluvialis biomass parallels that of Chlorella vulgaris (Table 5). The best conditions were also found to be under a vacuum and under nitrogen atmosphere (losses in the same range of magnitude). Under those conditions carotenoid content remained 90% of the initial amount even after 1.5 years (Table 6).
1 day 6 day 15 day 1 month 2.5 month 6 month
Stored conditions Light
Dark
Frozen
Antiox
Vacuum
Nitrog
16.6 42.7 68.0 100 100 100
0 24.4 52.6 58.2 60.0 63.0
2.0 10.4 12.8 14.2 15.9 29.0
2.4 7.8 10.2 18.9 29.3 40.0
0,4 2.3 4.2 9.1 9.8 12.5
0.3 1.9 5.2 8.7 10.0 14.2
Acetone extracts of Chlorella vulgaris and Haematococcus pluvialis carotenoid pigments are much more unstable than the corresponding dry biomass, which is thus shown to constitute a natural oxidation barrier. Carotenoids disappear between 15 and 30 days from both extracts, at room temperature in the presence of light (Table 7 and Table 9). Loss of acetone extracts carotenoid content for Chlorella vulgaris and Haematococcus pluvialis are present in Table 8 and Table 10. Chlorella and Haematococcus carotenoids extracts present almost the same behaviour with respect to storage conditions and the best conditions were again found to be under vacuum and under nitrogen atmosphere, as for dry biomass.
Table 9 Acetone extract stability of Haematococcus pluvialis. Total carotenoid content in Haematococcus pluvialis acetone extract, storage under different conditions during the trial period (total carotenoids mgykg dry alga)* Time (days)
Light
Dark
Frozen
Antiox
Vacuum
Nitrog
0 1 6 15 30 75 180
1500.2"1.6 1251.0"3.9 859.5"4.1 480.0"0.9 0.0"0.0 0.0"0.0 0.0"0.0
1500.2"1.6 1500.0"3.8 1134.0"0.9 711.0"5.7 627.0"4.6 600.0"4.1 555.0"1.8
1500.2"1.6 1470.0"2.3 1344.0"2.8 1308.0"4.6 1287.0"5.0 1261.5"1.0 1065.0"3.4
1500.2"1.6 1464.0"0.6 1383.0"5.9 1347.0"1.7 1216.5"2.7 1060.5"4.2 900.0"3.5
1500.2"1.6 1494.0"3.5 1465.5"4.8 1437.0"1.5 1363.5"0.2 1353.0"3.1 1312.5"5.0
1500.2"1.6 1495.5"3.8 1471.5"5.6 1422.0"4.1 1369.5"3.8 1350.0"5.0 1287.0"0.4
*
Storage conditions
Values are means of two determinations"S.D.
L. Gouveia, J. Empis / Innovative Food Science and Emerging Technologies 4 (2003) 227–233 Table 11 Total carotenoid content loss of Chlorella vulgaris and Haematococcus pluvialis biomass, incorporated in formulated fish diets for rainbow trout, at different time periods (%)* Time (month)
Chlorella vulgaris diet
Haematococcus pluvialis diet
1 2.5 6
6.4 8.5 24.0
7.8 8.9 18.8
*
Stored at 4 8C, in the dark.
Without any special atmosphere conditions during storage, but simply maintaining the samples in the dark, the period needed for a carotenoid loss of 50% is approximately 30 days for the extracts, compared to 1.5 year for dry biomass. In diets formulated as fish feed, carotenoid content remained quite stable, without significant loss during the trial period, for both microalgal biomass sources of carotenoids (Table 11 and Table 12). Pelleting process of the diets (with a pelleting machine without steam) didn’t affect carotenoid content of the diets (temperature above 50 8C). 4. Discussion The importance of carotenoid pigments has been recognised in the last years for their roles as antioxidants, sources of vitamin A and colouring agents in foods and beverages. In the marine environment they are probably best known for eliciting the pink–red hue to the flesh of salmonids, as well the diverse colours found in shrimp, lobsters, crayfish and ornamental fishes. Carotenoids have nevertheless also been reported to yield a general enhancement of performance of specific reproduction and metabolism, namely growth and survival, both in eggs, larval and fish and crayfish development (Thompson, Fletcher, Houlihan & Secombes, 1994; Torrissen & Christiansen, 1995; Paripatananont, Tangtrongpairoj, Sailasuta & Chansue, 1999). Decreasing natural catches and increasing world dependence on fish as a food source, as well as technological improvements, had led to increased aquacultural production. Total aquaculture production in 1995 was over 25 million tons and this industry is projected to grow at a rate of 8% per year for the foreseeable future (Tacon, 1998). In aquacultural practice, astaxanthin remained the best pigment due to it effectiveness in colouring, and to the commercially acceptable nature of the food products obtained. The major market for astaxanthin is as a pigmentation supplement in aquaculture, primarily for salmon and trout, but also for shrimp, and its market value is ;US$2.500ykg with an annual worldwide market estimated at US$200 million (Lorenz & Cysewki, 2000).
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Other works have shown that astaxanthin can also protect skin from the damaging effects of ultraviolet radiation, ameliorate age-related macular degeneration, protect against chemically induced cancers, increase high-density lipoproteins and enhance the immune system (Lorenz & Cysewki, 2000). Epidemiological studies have also demonstrated a correlation between increased carotenoid intake and reduced incidence of coronary heart disease and certain cancers, macular degeneration as well as an increased resistance to viral, bacterial, fungal and parasitic infections (Lorenz & Cysewki, 2000). As an antioxidant, astaxanthin surpasses the benefits of others; the antioxidant activities of astaxanthin have been shown to be approximately 10 times greater than those of b-carotene, lutein, zeaxanthin, cantaxanthin, and over 500 times greater than that shown by atocoferol. The name ‘super vitamin E’ has been proposed for astaxanthin (Miki, 1991). Actually, a fruit- and vegetable- rich diet is recommended to increase the dietary amounts of carotenoids; however, it has been shown that the bioavailability of carotenoids in fruits and vegetables is significantly lower than that of algae-derived supplements (Werman, BenAmotz & Mokady, 1999). It has also been proved that carotenoid extracts have higher anti-oxidant properties than synthetic ones (Levin, Yeshurun & Mokady, 1997). Although consumer demand for natural products is increasing, over 95% of the market needs are met by synthetic astaxanthin, and this provides an opportunity for competitive production of natural astaxanthin by Haematococcus pluvialis and Chlorella vulgaris. Both algae can be utilised as whole biomass, without extraction, as was successfully proven for Chlorella vulgaris for egg pigmentation (Gouveia et al., 1996b) and in aquaculture products (Gouveia, Gomes & Empis, 1996c; Gouveia et al., 1997; Gouveia, Choubert, Gomes, Rema & Empis, 1998; Gouveia, Veloso & Empis, 1999a; Gouveia & Empis, 1999b; Gouveia et al., 2002) comparable to products obtained using synthetic dies. Also Haematococcus pluvialis can be an effective pigment source for aquacultural products as it is an efficient source of relatively concentrated astaxanthin (Gomes et al., 2002) Table 12 Total carotenoid content loss of Chlorella vulgaris and Haematococcus pluvialis biomass, incorporated in formulated fish diets for gilthead seabream, at different time periods (%)* Time (month)
Chlorella vulgaris diet
Haematococcus pluvialis diet
1 2.5 6
5.0 7.6 22.0
4.1 7.0 18.6
*
Stored at 4 8C, in the dark.
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Compared with Chlorella vulgaris, Haematococcus pluvialis revealed a greater stability, probably due to the depth of the cyst walls that surround the cell in the carotenogenic stage. The absence of an antioxidant effect in presence of added ascorbic acid can only be interpreted by assuming that the limiting factor in the oxidative degradation is a textural, rather than a bulk effect. In this work, stability of biomass of both algae seems to be predominantly affected by light, and in second order by oxygen content, whereas the storage temperature in only important to a lesser extent. pH influence was not studied in this work. Extract microalgal pigments are much more unstable than pigment contained in dry biomass or in biomass incorporated in fish feed diets, which is crucial, because many processing steps can be bypassed, e.g. extraction, encapsulation or emulsion and drying. The use of microalgal biomass as a pigment source of finish fish feed is very promising in terms of stability of carotenoids because feedstuffs should, as far as possible, be stored for a minimum length of time. Feed are composed of perishable biological material and the maximum permissible storage time is 6 months (Silva & Anderson, 1995), a period during which no great loss of pigment for both microalgae in both fish diets tested, was observed. A microalgal carotenoid loss is similar in both fish diets, instead of different carotenoid and fat content of the diets. For use as a feed ingredient in aquaculture, microalgae should also be valued as sources of some micronutrients, such as vitamins and minerals, but the contents and variation of these potentially valuable constituents were not determined in this work. In this study it is concluded that both microalgae may constitute natural, concentrated and encapsulated forms of edible carotenoids, which can be kept during appropriate periods without any special storage conditions, both as dry biomass, or in prepared diets. Acknowledgments This work was supported by FCT (Portugal) through the project PDCTMyMARy15237y1999. References Choubert, G., & Storebakken, T. (1989). Dose response to astaxanthin and canthaxanthin pigmentation of rainbow trout fed various dietary carotenoid concentrations. Aquaculture, 81, 69 –77. Cooper, D. A., Eldridge, A. L., & Peters, J. C. (1999). Dietary carotenoids and certain cancers, heart disease, and age-related macular degeneration: A review of recent research. Nutrition Review, 57, 201 –214. De Stefani, E., Boffetta, P., Deneo-Pellegrini, H., Mendilaharsu, M., Carzoglio, J. C., Ronco, A., & Olivera, L. (1999). Dietary antioxidants and lung cancer risk: A case-control study in Uruguay. Nutrition Cancer, 34, 100 –110.
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