Summary

 

SYNOPSIS OF THE PROJECT:

 ‘‘Innovative research for the production of European sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) of high biological and dietetic value’’

 Acronym: ‘‘I C H T H I A S I S’’

 09ΣΥΝ-24-1069

 
The vision and the main objective of the present research was to contribute to the differentiation of existing Greek fish production, by implemented a culture of gilthead sea bream and European sea bass of high biological and dietetic value. The cultured fish will incorporate micro-nutrients, which they will positively influence consumer health by having favorable effects in specific operations of human organism. Such a “functional product” will open new markets, improve the competitiveness of enterprises of the aqua-sector and contribute to the sustainable and viable development of aquaculture in Greece and in general in the European Union.

 Thus, the current project aimed the planning, development and production of enriched fish with natural biological components (trace elements, polyphenols resveratrol, and ω3 fatty acids) through feeding, targeting to enforce the health benefits of consumer.

 The ICHTHIASIS consortium was composed by four partners: 1. Plagton SA, 2. Medical School, University of Ioannina (Laboratory of Physiology), 3. Hellenic Centre for Marine Research (Nutrition lab) and 4. Department of Biology, University of Patras (Unit of Microbiology).

 

The project was consisted of 5 Work packages (WP).


In WP1, 3 extruded diets for sea bass and sea bream were produced containing 3 levels (low, intermediate and high) of each of the selected trace elements, Zn (150, 200 and 250 mg/kg feed), Cu (5, 10, και 20 mg/kg feed) and Se (0,2 , 0,5 και 1,0 mg/kg feed). The low levels of the aforementioned trace elements were considered as the control group. 18.000 sea bass and gilthead sea bream fish (initial fish weight 15-20gr) were distributed in 6 sea cages and fed the 3 feeds in duplicate for 12 months. The results showed that the incorporation of the high levels of the selected trace elements improved the growth performance of the fish.

 
The analysis of the concentration of Zn/ Cu/ Se in fish muscles fed with low (control), medium and high trace element enriched diets, showed significant differences between control and medium (p<0.01) and control and high levels, fed fish groups (p<0.001).


The anticholesterinemic effects of enriched fish was evaluated by the addition of powderized fish muscle tissue in the feed of hypercholesterinemic rats (subjected to high fat diet-HFD , WT-control rats and Tg-transgenic rats overexpressing HSP70, a protective protein against stress), for 6 months. The results showed that total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, HDL and mainly the antiatherogenic activity were significantly increased, in hypercholesterinemic rats fed on fish which were fed on medium Zn dietary level, compared to fish fed control or high Zn dietary levels.


In WP2, based on the above data sea bream and sea bass fish, weighting, 252 ± 15 gr, were fed a feed with medium trace metals enrichment, for 1 month.

 
Fish muscle tissue analyses showed that: 1. metal transfer factors -Tf (ratio of metal concentration in fish muscle tissue to metal concentration in fish feed) of both fish species were Tf<<1, accordingly metal bioaccumulation risk is diminished 2. trace metal bio-availability was in general restricted, 3. metal concentrations were higher (NS) in fish fed enriched diets in metals compared to control and dietary metal enriched groups 4. Zn showed the best bio-availability in sea bream and a significant concentration increase (p<0.001) found in the metal enriched fish group. 5. the antioxidant molecule α-tocoferol (Vit.E), revealed significant concentration differences  between control and metal enriched fish in muscle tissue of sea bass (p<0.01) and in liver tissue of sea bream (p<0.001) and 6. Hsp70 levels were increased in tissue samples from sea bream and sea bass fish, fed on medium metal concentrations enriched diets, compared to control group, suggesting the enhancement of fish antistress protection. 

 

A CLINICO-LABORATORY STUDY (effects of trace element, Zn/Cu/Se, enriched-fish on human consumers) was performed on 12 healthy adult volunteers subjected to control-fish meals (fish weight 252+/-15gr), 3 times per week, for 1 month. Then, for 30 days any fish meals were excluded from their diet and right after they started again consuming 3 times per week, for 1 month, sea bream meals (weight 252 ± 15gr), fed on medium metal ( Cu/Zn/Se) concentration enriched feed.


All volunteers a day before starting and a day after ending the study were submitted for complete hematological and biochemical tests, evaluation of plasma total antioxidant capacity (TAC) and platelet aggregation reactivity (EC50), as well as serum HSP70 levels estimation. The findings of the clinic-laboratory study were: 1. no significant differences on volunteers’ body weight or body mass index (BMI) were registered before or after consumption of either control or metal enriched fish meals, 2. no significant differences on volunteers hematological and biochemical tests, namely blood glucose, uric acid, creatinin, urea, K, Na, Ca, P, Mg, hepatic enzymes ,LDL ,HDL, atheromatic index, thyroid hormones, were registered, before or after consumption of either control or metal enriched fish meals, 3. serum HSP70 levels were significantly increased in volunteer consumers of either control or metal enriched fish (
Ρ<0000). This increase was significantly higher in volunteers fed on metal enriched fish (Ρ<0.00002) and even higher in female compared to male consumers. Increased HSP70 levels exert protection against DNA damages, increase TAC and reveal much enhanced in metal- enriched fish consumers, 4. significant decrease of serum cholesterol levels in consumers fed metal- enriched fish, was registered, at the end of the study compared to cholesterol levels at the beginning, while none cholesterole decrease was observed in control fish consumers, 5. significant increase of PAF’s IC50 for platelet aggregation was registered in volunteers fed on metal enriched fish  (Ρ<0.00000), or on control fish(Ρ<0.0000) but this increase was significantly stronger for the volunteers fed on metal enriched fish (Ρ<0.000). Results on platelet aggregation agonists (PAF, AA, ADP) revealed that fish consumers develop resistance only to PAF which is significantly stronger to subjects fed on metal enriched fish. Resistance to PAF protects from thrombosis and negative heart effects and many other pathological conditions (over 50) that PAF is involved in, 6. total Antioxidant Capacity (TAC) of volunteers revealed that fish consumption increases significantly this blood index (control fish and metal enriched fish consumers, Ρ< 0.001 and Ρ<0.000, correspondingly ), while diet based on metal enriched fish induces highly significant TAC increase (Ρ<0.000). In conclusion, fish consumers develop significant TAC increase which enables them to resist to oxidative stress involved in very many pathological conditions.

 
In WP3, the effects of the dietary incorporation of grape pomace on the growth performance and health of gilthead sea bream were evaluated.
Wine grape pomace is an abundant by-product of the wine-industry in Greece. It contains resveratrol (3,5,4'-trihydroxy-trans-stilbene), a type of natural phenol located in the skin of red grapes, which has been reported to have many benefits in human health. Four iso-nitrogenous (CP 44%) and iso-energetic (22 MJ/kg) pelleted diets were manufactured in the experimental facilities of HCMR (Ag. Kosmas, Athens), containing four levels of dry grape pomace’ inclusion: 0% (control), 2%, 4% and 8%. The diets were fed to sea bream (average weight of 115 g) reared in experimental sea cages (1.1 x 1.0 x 1.5 m), with 20 fish per cage, 3 cages per diet, for a period of nine weeks at an average sea water temperature of 27.8 ο C ± 1.32. The results showed that all growth performance indices indicated that grape pomace can be incorporated to sea bream juvenile diets up to 4%, without compromising significantly fish growth, feed utilization and the integrity of hepatocytes and of the intestinal epithelium cells. The incorporation of grape pomace in sea bream diets seems that did not induced any effects on the organoleptic characteristics of fish.

 
Muscle tissue analyses, of raw or grilled sea bream fed on control or enriched diet incorporated 8% grape pomace, showed that resveratrol concentration were significantly higher in raw fish fed control or 8% enriched feed compared to grilled (p<0.001) and significantly higher in 8% enriched feed cultivated fish compared to control fish. Total Antioxidant Capacity (TAC) profile was similar to resveratrol’s. On the contrary ascorbic acid (Vit.C),
α-tokoferol (Vit.E) and HSP 70 presented no differences between tested fish groups.

 
A second CLINICO-LABORATORY STUDY (effects consuming sea bream fed on a diet incorporating 8% grape pomace (enriched fish) on human consumers) was performed on 12 healthy adult volunteers who followed the same protocol corresponding to formerly applied clinico-laboratory study.

 
The findings of the clinic-laboratory study were: 1.Total Antioxidant Capacity (TAC) of volunteers showed that fish consumption increases significantly this blood index for both control fish and fish fed the diet incorporating 8% grape pomace (
Ρ< 0.001 and Ρ<0.0001, correspondingly), but profoundly diet based on enriched fish surpasses diet based on control fish, improving significantly thus the antioxidant potency of human body. 2. significant increase of PAF’s IC50 for platelet aggregation was registered in volunteers fed on enriched fish (Ρ<0.0001), or on control fish (Ρ<0.001) but this increase was significantly higher for the volunteers fed on enriched fish (Ρ<0.000). Obviously feeding based on enriched fish surpasses diet based on control fish, by ameliorating PAF effects and protecting from atheromatosis.

 
In Wp4, the ability of various microalgal strains (both commercial and new isolates) to accumulate lipids (TAGs) rich in polyunsaturated fatty acids (PUFAs) was studied. The obtained results showed that the lipids of at least some of these microalgae contained PUFAs of high pharmaceutical and neutraceutical interest. Specifically, the lipids of Nannochloropsis sp., N. salina and Asterionella sp. S2 contained eicosapentaenoic acid (ΕΡΑ) in percentages up to 35% w/w in total lipids, the lipids of the new isolates Amphidinium sp. S1, Prymnesium parvum S2, Prorocentrum minimum S1 and Prorocentrum triestinum S1 were characterized by high percentage (up to 27% w/w) of docosahexaenoic acid (DHA), whereas the lipids of Chlorella sp., Asterionella sp. S1 and S2 were rich in α-linolenic acid (ALA) (up to 38% w/w).

 
Among the various microalgae, some strains were chosen to be further cultivated in both lab- and industrial- scale bioreactors due to their fatty acid composition and their application in aquaculture. Specifically, the microalgae N. salina and Chlorella sp. were cultivated in lab-scale bioreactors and accumulated up to 26.6 and 38.1% lipids in dry biomass, respectively, and 31.3 and 33.0% polysaccharides. These lipids were rich in PUFAs (i.e. linoleic acid, ALA and EPA). The microalgae Tetraselmis sp. and Chlorella sp. were initially cultivated in lab-scale and these cultures were used as inoculum for the bioreactors of 300 L established in PLAGTON S.A. Tetraselmis sp. and Chlorella sp. accumulated 2.33 and 2.44% w/w lipids, respectively. The lipids of the above mentioned strains contained higher percentages of neutral lipids, glycolipids plus sphingolipids (G+S), while phospholipids were found in lower quantities. Furthermore, PUFAs were mainly located in G+S.

 
The microalgae Chlorella sp. and Nannochloropsis oculata and the rotifer Brachionus plicatilis were cultivated in the industrial-scale bioreactors in the facilities of PLAGTON S.A.. These microalgal strains and the bakers’ yeast were used as feed for the rotifer. Analysis of fatty acid composition of the lipids of the rotifer B. plicatilis showed that the lipids of B. plicatilis were enriched with PUFAs when consuming PUFAs-rich feed.

 
Moreover, a feeding trial was performed in the HCMR’s facilities in Ag. Kosmas, Athens. Four experimental pelleted diets containing dry microalgae Spirulina platensis at four inclusion concentrations: 0% (control), 2%, 4% and 8% were produced. The diets were fed to sea bream with initial weight 7 g. The feeding trial was lasted for 3 months. No differences were found among fish groups fed the experimental diets in terms of growth performance, feed utilization and the integrity of hepatocytes and of the intestinal epithelium cells.

 
In WP5, a significant number of University dissertations, presentations in international scientific peer-review journals and symposiums were accomplished. A web page (www. ichthiasis.gr) was devoted in the project’s results.