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Beta-carotene studies in Spirulina
Bioresource Technology 38 ( 1991 ) l 11-113
Beta-Carotene Studies in
Spirulina
C. V. Seshadri, B. V. Umesh & R. Manoharan Shri AM M Murugappa Chettiar Research Centre (MCRC), Algal Division, Saveriyarpuram, India 622 505
Abstract The increasing importance of natural beta-carotene in fighting xerophthalmia and cancer has given special importance to algal sources of beta-carotene. The susceptibility to quick degradation of this valuable nutrient in oxygen atmosphere, light or heat calls for specific attention to processing and storage practices. In the case of Spirulina it was found that initial losses of beta-carotene on spray drying were between 7 and 10%. On storage in coloured bottles containing air, more than 50% was lost in less than 45 days. The particle size of the dried material seems to have an influence. Flakes (about 20 mesh+) retained 52% of the original beta-carotene level while the spray-dried fine powder (100 mesh-), retained only 34% of the original level. This is" explainable in terms of surface area available" for active reaction which is higher in the powder than in flakes. This questions the suitability ~f using spray drying for Spirulina drying. In this paper, data will be presented to substantiate the behaviour of beta-carotene on drying and storage by various methods'. Key words: Spirulina alga, fi-carotene, spray drier, storage and preservation, temperature effects. INTRODUCTION The fact that dietary beta-carotene has a strong influence on serum beta-carotene which is in turn inversely associated with the risk of cancer (Suda et al., 1986) has generated extensive research interests in utilising various sources of beta-carotene and evaluation of their efficacy. This leads to the other question of how to preserve the betacarotene that is present in the natural product as it is prone to rapid degradation in an atmosphere of heat, light, oxygen and humidity (Goodwin, 1980). Algal sources, Spirulina or Dunaliella, offer a wide scope for production of large quantities of natural beta-carotene directly in an edible state in a
shorter duration compared with any other natural sources. This has given a considerable impetus to the algal industry. Unless required for very special applications it is simpler to consume the carotene in its natural state without resorting to expensive extraction methods. Hence the algal product brought into the retailer's shop must be capable of retaining most of the original beta-carotene over a period of time if the full benefits are to be derived from the product. Our experiments are conducted with this in mind to achieve the best possible protection during processing as well as during storage and shelf use.
METHODS As our pilot plant for Spirulina production is equipped to dry the alga by spray drying as well as by solar drying, the studies are confined to the dry material available by these two drying methods. The following are the two major areas in which some preliminary studies have been done: ( 1 ) loss of beta-carotene while spray drying; (2) preservation of beta-carotene in the finished or dried product. The method of extraction used in these studies was the cold saponification process (AOAC, 1975). Sample size in dry material was 100 mg and in wet slurry 1000 mg. All results are computed on a bone dry basis. In the chromatographic separation the betacarotene was separated by petroleum ether through a bed of alumina and absorbence was measured at 450 nm. As the standard beta-carotene is highly unstable for long term studies, the content of beta-carotene was computed using the extinction coefficient method (Goodwin, 1980).
RESULTS Drying losses Fresh algal samples immediately after harvest and washing were analysed for beta-carotene levels on
111 Bioresource Technology 0960-8524/91/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
112
C. 1/. SeshadrL B. W. Umesh, R. Manoharan
different production days from different ponds. When the same batch of samples was dried in the spray drier, a fine powder with a residual moisture content of 3 - 4 % was obtained. W h e n analysed for beta-carotene levels after due correction for moisture content the loss of beta-carotene due to drying was found to vary from 7% to 11%. The drier is generally operated at 180°C inlet and 90-100°C outlet of the spray chamber. Table 1 gives our observations.
Influence of drying temperature There is a marked difference in beta-carotene loss with the varying outlet temperatures of the spray drier when compared to the fresh undried sample. It is presumed that the dried particles are in equilibrium temperature with that of the exhaust air. This is presented in Fig. 1. It is interesting to note that while there is a steady loss in betacarotene, the total carotenoids remains unaltered over the same temperature range. The significance is that the beta-carotene is identical to a dimeric structure of Vitamin A and capable of giving two Vitamin A molecules, whereas other members of the carotene group are capable of contributing only one. Thus one potential molecule of Vitamin A is lost in the course of drying.
Table
Storage practices
1
Date of analysis
Beta-carotene in fresh sample (mg/kg)
Beta-carotene after spray drying (mglkg)
% Loss
17.7.89 18.7.89 20.7.89 28.7.89
1841 1988 2288 2725
1683 1845 2027 2525
9 7 11 7
25
o
o ~o 2 0
o
"5 15 o o~lQ
[3_
/ / ~° 0
/,,Total carotenoids ~--
5 0 90
Influence of particle size During the above investigation it was also found that coarser particles -- generated during spray drier operation and subsequently removed from the bottom of the main chamber -- showed higher values of beta-carotene than the finer ones collected from the cyclone separator belonging to the same batch. However, the operational problems of the shift schedules became a constraint in studying this aspect in detail. A similar phenomenon took place in solar dried Spirulina. The thinner flakes that had dried in about 3 h of exposure to radiation contained 12% less total carotenes than the ones that were fairly thick -- which needed 10 h of solar radiation. This led to a postulation -- very much in vogue with physical chemists and chemical engineers -- that for a given mass, the larger the surface area, the faster would be the reaction. To elaborate, the finer particles having larger surface area provide more active sites for reaction with surrounding oxygen than the coarser ones which have less area exposed. Thus the betacarotene present in the inner core of the particles (which is greater in larger particles) escapes the process of oxidation. W h e n compared on a unit weight basis the large particles show a higher content than the finer particles.
I 95
[
100
I 110
I 120
Outlet t e m p e r a t u r e (°C)
Fig. 1. Percentageloss of carotenoids against outlet temperature.
If the above hypothesis is to be true for drying, it should equally hold good wlfile in storage. Then it was decided to analyse the phenomena by collecting dry samples belonging to the same harvest batch both from the spray drier as well as the solar drier. Samples of 100 g were stored in dark tinted bottles and were analysed once in 2 weeks over a period of 6 weeks. This total duration was arrived at after some preliminary results indicating that the highest rate of degradation occurs in the initial weeks immediately after packaging. Samples were analysed in duplicate each time. The particle size of the spray-dried powder was through 100 mesh while that of the solar dried was 20 mesh retained. The loss of beta-carotene determined at scheduled intervals during storage in both samples is presented in Fig. 2. The inconsistency of results in the case of powder samples possibly arises due to improper settlement of the powder material giving room for cavities within the body of the material stored. Since such a possibility is very unlikely in the case of granular flakes, the duplicate values are more
Beta-carotene studies in Spirulina 7:)
1oo
~- 8,0
o 8 6o ¢; o) 40
o Spray powder I
200 ~
15
~x 30
I
45
Days of s t o r a g e
Fig. 2. l.oss of beta-carotene during storage for solar flakes and spra5 powder. 100
• 90
A Storage under antioxidant o Control
~
©
e g r0
113
sample size used in the experiment. If the container volume is too large in comparison to the volume of material stored, the oxygen availability is far in excess of the actual requirement for degradation of beta-carotene present in the sample. This would cause a very steady oxidation of the carotenoids. On the other hand, if packed full with minimum headroom a reverse situation arises. After some initial loss the rate of degradation slows down due to nonavailability of oxygen from the surroundings. (1) One should use identical containers and quantities for any storage study on beta-carotene preferably to the same degree of compaction of the material inside the container. (2) For retail selling the size could be such that, once opened on a daily intake basis one exhausts the contents within 15 days of purchase. Such a container should be packed well with minimum access for air/oxygen. If needs be, a permissible antioxidant can be added.
g_ 60 -
5')
CONCLUSIONS I 5
I [ I 10 15 20 Days of s t o r a g e
I 25
Fig. 3. Loss of beta-carotene during storage with and without antioxidant.
consistent in the case of solar-dried samples of SpirulitTa. Preservation attempts In pharmacology, antioxidants are widely employed to protect the oxygen sensitive components from the oxygen atmosphere. Sodium metabisulphite, an antioxidant, is recommended at levels between 0.1 and 1.0% in the products to be preserved (Blacow, 1972). The usefulness of such an antoxidant is evident in controlling the beta-carotene loss as seen in Fig. 3.
The losses of beta-carotene involved while drying the alga can be minimized by lowering the drying temperature. (2 It may be preferable to produce the dry alga in the form of coarse granules or flakes as the beta-carotene in them is less susceptible to degradation. (3 It is possible to retard the degradation rate of beta-carotene in Spirufina by using antioxidants. (4) In case of objection to antioxidant usage, smaller packages with tightly packed powder can alleviate the problem of degradation to a considerable extent. Alternatively if vacuum packaging is available, this can be made use of for smaller size packages.
DISCUSSION REFERENCES In Figs 2 and 3 the nature of the degradation of beta-carotene has been presented differently. In Fig. 2 it is projected as linear while in Fig. 3 it is shown to be an exponential decay. Which is the true behaviour? This raises an important and interesting problem for packaging of Spirulina. Both types of behaviour are observable depending on the container size and on the
Association of Official Analytical Chemists (1975). Official Methods of Analysis, 12th Edn. AOAC, Washington, DC. Blacow, N. W. (ed.) (1972). Martindale: The Extra Pharmacopoeia, 26th Edn. The Pharmaceutical Press, London. Goodwin, T. W. (1980). The Biochemistry of the Carotenoids, Vol. 1, 2nd Edn. Chapman and Hall, New York. Suda, D., Schwartz, J. & Shklar, G. (1986). Inhibition of experimental oral carcinogenesis by topical beta-carotene. Carcinogenesis, 7 (5), 71 l - 15.
Beta-Carotene Studies in
Spirulina
C. V. Seshadri, B. V. Umesh & R. Manoharan Shri AM M Murugappa Chettiar Research Centre (MCRC), Algal Division, Saveriyarpuram, India 622 505
Abstract The increasing importance of natural beta-carotene in fighting xerophthalmia and cancer has given special importance to algal sources of beta-carotene. The susceptibility to quick degradation of this valuable nutrient in oxygen atmosphere, light or heat calls for specific attention to processing and storage practices. In the case of Spirulina it was found that initial losses of beta-carotene on spray drying were between 7 and 10%. On storage in coloured bottles containing air, more than 50% was lost in less than 45 days. The particle size of the dried material seems to have an influence. Flakes (about 20 mesh+) retained 52% of the original beta-carotene level while the spray-dried fine powder (100 mesh-), retained only 34% of the original level. This is" explainable in terms of surface area available" for active reaction which is higher in the powder than in flakes. This questions the suitability ~f using spray drying for Spirulina drying. In this paper, data will be presented to substantiate the behaviour of beta-carotene on drying and storage by various methods'. Key words: Spirulina alga, fi-carotene, spray drier, storage and preservation, temperature effects. INTRODUCTION The fact that dietary beta-carotene has a strong influence on serum beta-carotene which is in turn inversely associated with the risk of cancer (Suda et al., 1986) has generated extensive research interests in utilising various sources of beta-carotene and evaluation of their efficacy. This leads to the other question of how to preserve the betacarotene that is present in the natural product as it is prone to rapid degradation in an atmosphere of heat, light, oxygen and humidity (Goodwin, 1980). Algal sources, Spirulina or Dunaliella, offer a wide scope for production of large quantities of natural beta-carotene directly in an edible state in a
shorter duration compared with any other natural sources. This has given a considerable impetus to the algal industry. Unless required for very special applications it is simpler to consume the carotene in its natural state without resorting to expensive extraction methods. Hence the algal product brought into the retailer's shop must be capable of retaining most of the original beta-carotene over a period of time if the full benefits are to be derived from the product. Our experiments are conducted with this in mind to achieve the best possible protection during processing as well as during storage and shelf use.
METHODS As our pilot plant for Spirulina production is equipped to dry the alga by spray drying as well as by solar drying, the studies are confined to the dry material available by these two drying methods. The following are the two major areas in which some preliminary studies have been done: ( 1 ) loss of beta-carotene while spray drying; (2) preservation of beta-carotene in the finished or dried product. The method of extraction used in these studies was the cold saponification process (AOAC, 1975). Sample size in dry material was 100 mg and in wet slurry 1000 mg. All results are computed on a bone dry basis. In the chromatographic separation the betacarotene was separated by petroleum ether through a bed of alumina and absorbence was measured at 450 nm. As the standard beta-carotene is highly unstable for long term studies, the content of beta-carotene was computed using the extinction coefficient method (Goodwin, 1980).
RESULTS Drying losses Fresh algal samples immediately after harvest and washing were analysed for beta-carotene levels on
111 Bioresource Technology 0960-8524/91/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
112
C. 1/. SeshadrL B. W. Umesh, R. Manoharan
different production days from different ponds. When the same batch of samples was dried in the spray drier, a fine powder with a residual moisture content of 3 - 4 % was obtained. W h e n analysed for beta-carotene levels after due correction for moisture content the loss of beta-carotene due to drying was found to vary from 7% to 11%. The drier is generally operated at 180°C inlet and 90-100°C outlet of the spray chamber. Table 1 gives our observations.
Influence of drying temperature There is a marked difference in beta-carotene loss with the varying outlet temperatures of the spray drier when compared to the fresh undried sample. It is presumed that the dried particles are in equilibrium temperature with that of the exhaust air. This is presented in Fig. 1. It is interesting to note that while there is a steady loss in betacarotene, the total carotenoids remains unaltered over the same temperature range. The significance is that the beta-carotene is identical to a dimeric structure of Vitamin A and capable of giving two Vitamin A molecules, whereas other members of the carotene group are capable of contributing only one. Thus one potential molecule of Vitamin A is lost in the course of drying.
Table
Storage practices
1
Date of analysis
Beta-carotene in fresh sample (mg/kg)
Beta-carotene after spray drying (mglkg)
% Loss
17.7.89 18.7.89 20.7.89 28.7.89
1841 1988 2288 2725
1683 1845 2027 2525
9 7 11 7
25
o
o ~o 2 0
o
"5 15 o o~lQ
[3_
/ / ~° 0
/,,Total carotenoids ~--
5 0 90
Influence of particle size During the above investigation it was also found that coarser particles -- generated during spray drier operation and subsequently removed from the bottom of the main chamber -- showed higher values of beta-carotene than the finer ones collected from the cyclone separator belonging to the same batch. However, the operational problems of the shift schedules became a constraint in studying this aspect in detail. A similar phenomenon took place in solar dried Spirulina. The thinner flakes that had dried in about 3 h of exposure to radiation contained 12% less total carotenes than the ones that were fairly thick -- which needed 10 h of solar radiation. This led to a postulation -- very much in vogue with physical chemists and chemical engineers -- that for a given mass, the larger the surface area, the faster would be the reaction. To elaborate, the finer particles having larger surface area provide more active sites for reaction with surrounding oxygen than the coarser ones which have less area exposed. Thus the betacarotene present in the inner core of the particles (which is greater in larger particles) escapes the process of oxidation. W h e n compared on a unit weight basis the large particles show a higher content than the finer particles.
I 95
[
100
I 110
I 120
Outlet t e m p e r a t u r e (°C)
Fig. 1. Percentageloss of carotenoids against outlet temperature.
If the above hypothesis is to be true for drying, it should equally hold good wlfile in storage. Then it was decided to analyse the phenomena by collecting dry samples belonging to the same harvest batch both from the spray drier as well as the solar drier. Samples of 100 g were stored in dark tinted bottles and were analysed once in 2 weeks over a period of 6 weeks. This total duration was arrived at after some preliminary results indicating that the highest rate of degradation occurs in the initial weeks immediately after packaging. Samples were analysed in duplicate each time. The particle size of the spray-dried powder was through 100 mesh while that of the solar dried was 20 mesh retained. The loss of beta-carotene determined at scheduled intervals during storage in both samples is presented in Fig. 2. The inconsistency of results in the case of powder samples possibly arises due to improper settlement of the powder material giving room for cavities within the body of the material stored. Since such a possibility is very unlikely in the case of granular flakes, the duplicate values are more
Beta-carotene studies in Spirulina 7:)
1oo
~- 8,0
o 8 6o ¢; o) 40
o Spray powder I
200 ~
15
~x 30
I
45
Days of s t o r a g e
Fig. 2. l.oss of beta-carotene during storage for solar flakes and spra5 powder. 100
• 90
A Storage under antioxidant o Control
~
©
e g r0
113
sample size used in the experiment. If the container volume is too large in comparison to the volume of material stored, the oxygen availability is far in excess of the actual requirement for degradation of beta-carotene present in the sample. This would cause a very steady oxidation of the carotenoids. On the other hand, if packed full with minimum headroom a reverse situation arises. After some initial loss the rate of degradation slows down due to nonavailability of oxygen from the surroundings. (1) One should use identical containers and quantities for any storage study on beta-carotene preferably to the same degree of compaction of the material inside the container. (2) For retail selling the size could be such that, once opened on a daily intake basis one exhausts the contents within 15 days of purchase. Such a container should be packed well with minimum access for air/oxygen. If needs be, a permissible antioxidant can be added.
g_ 60 -
5')
CONCLUSIONS I 5
I [ I 10 15 20 Days of s t o r a g e
I 25
Fig. 3. Loss of beta-carotene during storage with and without antioxidant.
consistent in the case of solar-dried samples of SpirulitTa. Preservation attempts In pharmacology, antioxidants are widely employed to protect the oxygen sensitive components from the oxygen atmosphere. Sodium metabisulphite, an antioxidant, is recommended at levels between 0.1 and 1.0% in the products to be preserved (Blacow, 1972). The usefulness of such an antoxidant is evident in controlling the beta-carotene loss as seen in Fig. 3.
The losses of beta-carotene involved while drying the alga can be minimized by lowering the drying temperature. (2 It may be preferable to produce the dry alga in the form of coarse granules or flakes as the beta-carotene in them is less susceptible to degradation. (3 It is possible to retard the degradation rate of beta-carotene in Spirufina by using antioxidants. (4) In case of objection to antioxidant usage, smaller packages with tightly packed powder can alleviate the problem of degradation to a considerable extent. Alternatively if vacuum packaging is available, this can be made use of for smaller size packages.
DISCUSSION REFERENCES In Figs 2 and 3 the nature of the degradation of beta-carotene has been presented differently. In Fig. 2 it is projected as linear while in Fig. 3 it is shown to be an exponential decay. Which is the true behaviour? This raises an important and interesting problem for packaging of Spirulina. Both types of behaviour are observable depending on the container size and on the
Association of Official Analytical Chemists (1975). Official Methods of Analysis, 12th Edn. AOAC, Washington, DC. Blacow, N. W. (ed.) (1972). Martindale: The Extra Pharmacopoeia, 26th Edn. The Pharmaceutical Press, London. Goodwin, T. W. (1980). The Biochemistry of the Carotenoids, Vol. 1, 2nd Edn. Chapman and Hall, New York. Suda, D., Schwartz, J. & Shklar, G. (1986). Inhibition of experimental oral carcinogenesis by topical beta-carotene. Carcinogenesis, 7 (5), 71 l - 15.