USE OF LIPID EMULSIONS FOR THE BIO-ENCAPSULATION OF HIGHLY UNSATURATED FATTY ACIDS IN THE BRINE SHRIMP Artemia
PhD
Thesis by Kyung-Min Han
Faculty of Agricultural and Applied Biological Sciences, Ghent University,
Belgium, 2001, 190 pp.
Summary:
The final objective of this thesis is to better understand the HUFA
incorporation in Artemia nauplii by examining the effect of specific factors
susceptible to influence the lipid emulsion enrichment process.
With each of the present enrichment trials, the level of exogenously
supplied n-3 HUFA was clearly reflected in the body lipids of the nauplii,
confirming the efficacy of the present lipid emulsion enrichments. The use
of an emulsion containing 95% DHA (% total fatty acids) resulted in a
maximal DHA concentration in the A. franciscana nauplii of only 26 mg/g DW,
not very different from the value of 20.2-23.1 mg/g DW found when supplying
the same dose (0.2 g/l) of an emulsion mechanism that limits DHA
incorporation in the body lipids of the Artemia, which may be related to the
intrinsic property of Artemia to retroconvert DHA to EPA. Also the total n-3
HUFA incorporation levelled of or even declined with high dietary HUFA doses
as seen when supplying 0.4 g/l of emulsion instead of the lower doses of 0.2
or 0.3 g/l or with an emulsion containing 95% n-3 HUFA (emulsion DHA/EPA).
In chapter III, we evaluated the incorporation efficiencies and the
possible interactions or competitions among the three essential HUFA during
enrichment and subsequent starvation of A. franciscana nauplii. The
comparison of emulsions containing only one of the three HUFA clearly showed
a lower incorporation efficiency of DHA as compared to EPA and AA and
allowed to demonstrate the metabolic conversions of DHA to EPA by the
nauplii. No competitive interactions of EPA or of AA on DHA incorporation
were observed, when offering HUFA together. During the subsequent starvation
of EPA- or DHA- enriched Artemia, relative EPA and DHA losses were similarly
high in both emulsion. The general conclusion of a more important catabolism
of DHA as compared to EPA should therefore be attenuated.
In chapter IV, we examined a possibly new strategy to stabilize the HUFA
after enrichment. DHA-enriched nauplii (Karbogaz-gol, Turkmenistan) were
starved for 24-h at two different levels of dissolved oxygen. The results
indicated that the catabolism of DHA by the Artemia after enrichment was
indeed prevented by storage at low oxygen (< 2 ppm) levels during the
first 12 h of starvation. It is suggested that the low oxygen storage can be
used as an alternative or a complement to cold storage for reducing DHA
losses.
In chapter V and VI, we compared the fatty acid profile of A. franciscana
nauplii with that of three other bisexual strains, A. sinica, A. persimilis
and A. tibetiana and one parthenogenetic strain (A. parthenogenetica). The
newly-hatched nauplii of each of the five species were practically devoid of
DHA (less than 0.3 mg/g DW). Two ‘marine type’ species of Artemia could
be distinguished: A. parthenogenetica (Tanggu, P.R. China) and A. tibetiana
(Lagkor Co, Tibet, P.R. China), with a very high 20:5n-3 and low 18:3n-3
content. After enrichment, DHA was clearly incorporated into each Artemia
species, but the levels varied from 12.2 mg/g DW in A. persimilis to 37 mg/g
DW in A. sinica. The latter value represents the highest DHA enrichment
found in the present study. After 24 h enrichment, most Artemia nauplii had
developed into instar-II and –III. As the developmental status between the
strain with the highest (A. sinica) and the lowest (A. persimilis) DHA level
was similar, physiological mechanisms of DHA incorporation may differ
between Artemia strains. During starvation, DHA rapidly decreased in all
strains. Present data did not confirm previous findings of a particularly
high DHA retention in A. sinica.
In chapter I, despite the use of ICES emulsions in several of present
trials and despite the fact that A. franciscana, of the same origin and
batch, were hatched, enriched, sampled and analysed under identical
conditions, important variations were still noted between three consecutive
trials. In chapter II, we therefore compared the particle size distribution
in two ICES emulsions as a function of the preparation method and the
storage time. The 50% HUFA emulsion (ICES 50, ethyl ester based) contained
much smaller micelles than 30% HUFA emulsion (ICES 30, triacylglycerol
based) of which 90% were smaller than 12.3 µm as compared to only 2.3 µm
in ICES 50. In both emulsions, the distribution of the particle sizes
remained however markedly stable during the one week of storage, suggesting
the absence of micelle agglomeration. On the other hand, the blending method
highly affected particle sizes. Ultrasonic blending of both ICES 30 and 50
(1.1 and 0.6 µm, respectively) gave significantly smaller particles than
hand shaking (12.6 and 2.4 µm, respectively). The fatty acid composition of
24-h enriched Artemia was however independent of observed differences in
particle size diameters.
In chapter VII, the effect of supplementing various dietary fatty acids on
fatty acid incorporation, growth and reproduction characteristics in Artemia
franciscana was evaluated. The results indicated that fatty acids supplied
by a lipid emulsion as a supplement to the algal diet are well incorporated
in the Artemia. Apart from being an extra source of energy, these emulsions
may function as source of HUFA, which may play an essential role for growth
and reproduction (fecundity) of Artemia.
The results of this thesis indicated that the levels of n-3 HUFA in
Artemia vary not only depending
on enrichment protocol, such as dose, feeding time or frequency and fatty
acid composition of the emulsion, but also depending on the Artemia strain.
In addition, storage of enriched Artemia nauplii at low oxygen levels can
ameliorate the DHA retention which is beneficial for the culture of marine
fish and shrimp.