WO2005009140A1

WO2005009140A1 - Use of anthocyanins in fish food

Info

Publication number: WO2005009140A1
Application number: PCT/GB2004/003120
Authority: WO
Inventors: Rune Blomhoff; George Alexander
Original assignee: Biochem Laboratories As; Beacham, Annabel, Rose
Priority date: 2003-07-17
Filing date: 2004-07-16
Publication date: 2005-02-03
Also published as: GB0316757D0; NO20060388L

Abstract

A solid fish feed or fish feed supplement contains one or more extracted or synthetic anthocyanins. One or more extracted or synthetic anthocyanins are used in the manufacture of a solid fish feed or fish feed supplement. A method of aquaculture comprises administering such fish feed or feed supplements to fish, or administering anthocyanins to fish.

Description

Use of anthocyanins in fish food

This invention relates to the use of anthocyanins in fish food, to anthocyanin-containing fish food and to the preparation of anthocyanin-containing fish food. Anthocyanins are a group of flavonoid glycosides. Each anthocyanin comprises three component parts: a hydroxylated core (the aglycone) ; the saccharide unit(s); and a counterion. Anthocyanins occur naturally as pigments in many fruit, flowers and vegetables and individual anthocyanins are available commercially, generally as chloride salts, e.g. from Polyphenols Laboratories AS, Sandnes, Norway. Contemporary aquacultural practices necessitate the rearing of fish in conditions of relative confinement. Fish such as Atlantic salmon {Salmo salar) and rainbow trout { Oncorhynchus mykiss) are subject to increased physiological stresses due to crowding, lack of short- distance migration, exposure to diseased cohorts, inability to escape rapid water temperature changes or contamination, handling stress and various psycho- physical factors. These stresses can be manifested as reduction in growth rate, decreased immune response, alteration in food conversion, alteration of the parr- smolt transformation and cataract development. These effects are described for example by Pickering et al in Gen.Comp.Endocrinol. 82:86-93 (1991), by Martinez et al in Arch. Int. Physiol .Biochem Biophys . 100:247-250 (1992) and by Jorgensen et al . in Aquat. Toxicol. 54:179-193 (2001) . Over the past few years, the episodes of cataract development in northern latitude European fish farms have been a cause for concern for a number of commercial enterprises. As these episodes of cataract development are highly associated with short-term increases in water temperature and the farmed fish are not able to escape to cold water refuge, the fish experience temporary increases in oxidative stress as their physiology has not sufficiently acclimated to the new temperature. As the enzymes present in the fish are appropriate for a colder water temperature, we believe that temperature- related overactivity of these enzymes may be inducing stresses within all tissues of the fish. We have now found that aquacultured fish benefit from the inclusion within their diet of anthocyanins. More particularly the use of anthocyanins had the effect of increasing the oxidative stress buffering capacity in liver, gill and muscle tissue. The increase in liver tissue would be of benefit to the fish as, during events that are particularly taxing to the liver such as metabolizing contaminants, the fish would be better able to perform such physiological tasks. Similarly, gill tissue has a significant non-respiratory role in maintaining plasma salt levels in anadromous fish such as salmon. As the gills undergo significant changes during the parr-to-smolt transformation, an increase in oxidative buffering capacity would be of value during this critical time. With respect to muscle tissue, salmonids are cruise swimmers, utilizing a relatively tiny band of muscles located along their midline. Burst swimming tasks are performed by the gross musculature evident in the fish. As these bursts are essentially anaerobic, the muscles can experience extreme changes in physiological state. Again, an increase in oxidative stress buffering capacity serves to reduce the potential for damage to the musculature if fish are physically taxed. This may take place during times of crowding or rapid temperature increases . At these times salmonids tend to swim with increased agitation and incorporate the use of their burst musculature more often. Negative effects associated with this sea-cage associated behaviour may also be reduced. It is also believed that the use of anthocyanins may have a positive effect on the cataract development seen recently in Scandinavian and North Sea fish farms. Thus viewed from one aspect the invention provides the use of one or more extracted or synthetic anthocyanins, preferably one or more essentially lipid- free anthocyanins, in the manufacture of a solid fish feed or fish feed supplement. Viewed from a further aspect the invention provides a method of aquaculture comprising administering a solid fish feed or feed supplement to fish, characterized in that said feed or supplement contains one or more extracted or synthetic anthocyanins or in that one or more anthocyanins is also administered to said fish as a feed supplement . Viewed from a still further aspect the invention also provides a solid fish feed or feed supplement, characterized in that said feed or feed supplement contains one or more extracted or synthetic anthocyanins, preferably one or more anthocyanins which are not lipid bound. By solid is meant herein that the feed or supplement is in a shape-retaining form, i.e. it is not in liquid form. The feed or supplement may be rigid or deformable (e.g. gel-like) . By extracted or synthetic is meant herein that the anthocyanin is either chemically synthesized, i.e. ex vivo synthesized, or that it has been synthesized in vivo in an organism (e.g. a plant) and then extracted from the cells of the organism. The feed or feed supplement thus does not include materials which are simply plants, parts of plants or the juice of plants or parts of plants. The fish feed according to the invention preferably comprises, as well as the anthocyanin, protein, lipid and optionally also vitamins, carbohydrates, and minerals. The protein is preferably fish, animal (e.g. mammal), plant or microorganism protein, e.g. fish meal or single cell protein of yeast or bacterial origin. The lipid is preferably a fish, animal (e.g. mammal), or plant oil, e.g. a triglyceride, especially preferably one containing omega-6 and/or omega-3 fatty acid residues (e.g. eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, etc). The use of fish oil, e.g. from marine fish waste, is especially preferred. The carbohydrate, if present, is preferably a plant starch, e.g. flour of the seeds of a grass-like plant, for example wheat, oats or barley. The nutrient, i.e. non- anthocyanin, fraction of the fish feed according to the invention may thus be any conventional fish feed. The fish feed according to the invention is especially preferably an extruded and pelletized feed. The anthocyanin may be incorporated into the fish feed as a pure anthocyanin or anthocyanin mixture, as an anthocyanin/spray drying adjuvant mixture or, less preferably, as an anthocyanin-containing plant extract (e.g. one prepared by contacting plant material with a solvent, especially preferably an alkanol solvent, or by crushing plant material) . In order that the bioavailability of the anthocyanin is high, the anthocyanin is preferably essentially free of lipid from the plant from which the anthocyanin derives, i.e. in its isolation, the anthocyanin is preferably freed from plant lipids and preferably also from other non-anthocyanin plant compounds, in particular sugars. As anthocyanins are generally extremely water- soluble, in the feeds and feed supplements of the invention the anthocyanin is preferably formulated to prevent its rapid dispersal into the surrounding water when the feed or supplement is administered. This may be achieved for example by encapsulation of the anthocyanin, e.g. with a physiologically tolerable coating agent, within liposomes or other fragmented liquid crystalline structures (e.g. cubic or hexagonal phase structures) , or by molecular admixture with an adjuvant which is poorly soluble in the ambient water (but which is more soluble in the gastrointestinal fluid of the recipient fish) . In one particularly preferred embodiment, the anthocyanin is dissolved or dispersed within the lipid component of the fish feed, e.g. a fish or plant oil . The anthocyanin used according to the invention may be a single anthocyanin or a mixture of at least two anthocyanins. In general it is preferred to use a mixture of at least two, e.g. up to 30, more preferably 3 to 16, anthocyanins. Where a mixture of anthocyanins is used, it is preferred to use anthocyanins having at least two different aglycone units and having at least two different saccharide units. More particularly, the anthocyanins used preferably include ones in which the aglycone units are two or more of cyanidin, peonidin, delphinidin, petunidin, malvidin and pelargonidin, especially three or more and more especially four or more. Moreover the anthocyanins used preferably include at least one monosaccharide anthocyanin, and more preferably in addition at least one disaccharide anthocyanin. Particularly preferably at least one of the anthocyanins used is a monosaccharide in which the saccharide is arabinose and especially preferably at least one of the anthocyanins used is a disaccharide in which the disaccharide is rutinose (i.e. 6-rhamnosyl- glucose) . Example of anthocyanins suitable for use in the invention include: cyanidin-3-0-β-glucoside; cyanidin-3- O-β-galactoside; cyanidin-3 -O-α-arabinoside,- cyanidin-3- O-β-xyloside,- cyanidin-3-0- (6" -O-α-rhamnosyl-β- glucoside) ; cyanidin-3-0- (2"-0-β-glucosyl-β- galactoside) ; cyanidin-3-0- (2"-0-β-glucosyl-β- glucoside); cyanidin-3-0- (2" -O-β-xylosyl-β-glucoside) ; cyanidin-3-0- (2"-0-β-xylosyl-β-galactoside) ; cyanidin- 3 , 5-di-O-β-glucoside,- cyanidin-3-0-β-galactoside-5-0-β- glucoside; cyanidin-3 -O-α-arabinoside-5-0-β-glucoside; cyanidin-3-0- (2 " -O-β-xylosyl-β-glucoside) -5-O-β- glucoside; cyanidin-3-0- (2" -0-β-xylosyl-6"-0-β-glucosyl- β-galactoside) ; pelargonidin-3-0-β-glucoside; pelargonidin-3-0- (6" -O-α-rhamnosyl-β-glucoside) ; pelargonidin-3 , 5-di-0-β-glucoside; peonidin-3-0-β- glucoside; peonidin-3-O-α-arabinoside; peonidin-3 , 5-di- O-β-glucoside; delphinidin-3-O-β-glucoside; delphinidin- 3-0- (6" -O-α-rhamnosyl-β-glucoside) ; delphinidin-3-0- (2"- O-β-xylosyl-β-glucoside) ; petunidin-3-O-β-glucoside; malvidin-3 -O-β-glucoside; malvidin-3, 5-di-0-β-glucoside; and malvidin-3 -O-α-arabinoside-5-O-β-glucoside . If desired, one or more hydroxy groups, especially on the saccharide unit, in the anthocyanins may be acylated, e.g. carrying a C₁_₁₂ , more especially a C₃.₉ saturated or unsaturated acyl group, for example a mono- or dicarboxylic acid residue, e.g. a malonyl, p-coumaryl or feruloylyl group . Thus for example such acylated compounds include cyanidin-3-0- (6" -0- (E-p-coumaryl) -2"- O-β-xylosyl-β-glucoside); cyanidin-3-0- (6" -0- (E-p- coumaryl) -2" -O-β-xylosyl-β-glucoside) -5-0-β-glucoside; cyanidin-3-0- (2"-0-β-xylosyl-6"-0- (E-feruloyl-β- glucosyl) -β-galactoside) ; cyanidin-3-0- (2" -O-β-xylosyl- 6"-0- [E-coumaryl-β-glucosyl] -β-galactoside) ; and petunidin-3-0- (6"-0- (4" ' -O-E-coumaryl) -α-rhamnosyl-β- glucoside) -5-0-β-glucoside . The counterion in the anthocyanins in the product of the invention may be any physiologically tolerable counteranions, e.g. chloride, succinate, fumarate, malate, maleate, citrate, etc. Preferably however the counterion is a fruit acid anion, in particular citrate, as this results in the products having a particularly pleasant taste. Particularly suitable sources for the anthocyanins are fruits such as cherries, bilberries, blueberries, blackcurrants, redcurrants, grapes, cranberries, pomegranate, strawberries, and apples, rosehips and vegetables such as red cabbage. Bilberries, in particular Vaccinium myrtillus, and blackcurrants, in particular Ribes nigrum, are especially suitable. The berries of V. myrtillus contain fifteen monosaccharide anthocyanins, namely the aglycone : saccharide combinations of cyanidin, peonidin, delphinidin, petunidin and malvidin and glucose, galactose and arabinose. The currants of R. nigrum contain four anthocyanins, namely the 3-glucosides and 3-rutinosides of cyanidin and delphinidin. The disaccharide anthocyanins are more water- soluble than the monosaccharides; moreover cyanidin and delphinidin anthocyanins are amongst the most water- soluble anthocyanins. Besides the anthocyanins, the feeds and feed supplements of the invention may desirably contain further beneficial or inactive ingredients, e.g. vitamins (in particular vitamin C) , flavones, isoflavones, anticoagulants (e.g. maltodextrin, silica, etc.), desiccants, etc. Desirably however the anthocyanins constitute 0.1 to 95% wt, preferably 1 to 50% wt, especially 5 to 30% wt of the product compositions, excluding any coating material. The anthocyanins used according to the invention may conveniently be extracted from their plant source (s) using a process as described in WO 03/039569 the entire contents of which are incorporated herein by reference. In such a process, spray dried anthocyanin is produced. This may contain a single anthocyanin or a mixture of anthocyanins. The use of a mixture of anthocyanins is preferred as noted above but single anthocyanins may be used if desired. Where a single anthocyanin is to be used and its plant source contains a plurality of anthocyanins, these may be separated during the process after lipid removal and before spray-drying, e.g. using chromatographic techniques. Spray-drying is preferably effected by spraying into an inert atmosphere, e.g. a nitrogen atmosphere, with inlet temperatures of 130 to 160°C and flow rates of 5 to 12 L/hour. If the spray- dried product is sticky, then increasing flow rate and inlet temperature and/or increasing anticoagulant content and/or increasing atomiser rotation rate will result in a non-sticky product. The optimum temperature, flow-rate, etc. can be determined in this way for each separate anthocyanin source . The anthocyanin solution to be spray-dried desirably is an aqueous solution containing anthocyanins at 5 to 15% wt, more preferably 8 to 12% wt, dry solids basis. Where anthocyanins from more than one plant source are to be included in the products, they are preferably mixed after spray-drying. Thus, for example, the product compositions preferably contain anthocyanins from V. myrtillus and R. nigrum mixed after spray- drying, preferably in a weight ratio of 0.5:1 to 1:0.5, especially about 1:1. Besides anthocyanins, the products will generally also contain an anticoagulant, e.g. maltodextrin, lactose or silica, added to the anthocyanin solution to be spray dried to prevent coagulation in the spray drying process. Typically the anticoagulant will be present at 0.3 to 0.8g, especially about 0.5g, per gram anthocyanin. The feed according to the invention is preferably produced by mixing the feed components to form a moist mass that is extruded and pelletized. Generally most or all of the lipid component of the feed, e.g. fish oil, is added to the extruded feed before or after pelletization. If desired however the feed components including lipid may be processed to form an emulsion which is then extruded and heat treated, e.g. microwaved, to form a solid product. If desired, the anthocyanins, e.g. in solution for example as a berry juice or juice concentrate, may simply be impregnated into a preformed feed. Typically the feed will comprise 10 to 80% wt protein, especially 20 to 60% wt; 10 to 60% wt lipid, especially 20 to 50% wt; and 1 to 20% wt carbohydrate, especially 2 to 10% wt . (The Atlantic HP50-65A that was used in Example 1 below contained: 49% protein; 27% fat; 8.1% carbohydrate; 13 g/kg of D,L-methionine; 30.9 g/kg L-lysine; 2.3 IE/gm vitamin D; 243.7 mg/kg vitamin E; 7.2 IE/gm vitamin A; 8.2 mg/kg copper; and 65.0 mg/kg astaxanthin) . The feed may be produced with a density lower, higher or more preferably similar to that of the water into which it is to be introduced, e.g. seawater for marine fish aquaculture. Low density feed is useful for example in rearing catfish as the taste of the fish is improved. The feed will normally be administered to the fish in an amount corresponding to the nutritional needs of the fish being fed, e.g. in conventional quantities. The precise amount used and the frequency of feed or feed supplement administration will, as is conventional, depend on the age and species of the fish, and the total fish mass in the volume (e.g. cage) to which the feed or feed supplement is administered. A feed consumption of 5% of bodyweight per day is not unusual. Typically the daily anthocyanin dosage may be 1 to 100 mg/kg bodyweight . The feed or feed supplement according to the invention may be administered to fish at any growth stage or to adult fish. However particularly preferably the anthocyanins are administered during metamorphosis stages, e.g. eye migration or gill transformation stages, at high temperature periods (e.g. Summer), when cages are crowded, at migration stages, or when contamination of the ambient water is detected or suspected. The fish receiving the fish feed or food supplement according to the invention may be any form of fish or shellfish; preferably however they are marine or fresh water vertebrate fish (e.g. salmon, trout, cod, haddock, hake, halibut, whiting, turbot, flounder, etc) or shrimp, prawn, lobster, langoustine, crab, mussel, or clam. Especially preferably the fish is a marine vertebrate. The invention will now be described further by way of illustration with reference to the following non- limiting Examples and Figures.

Figure 1 is a graph showing normalized FRAP activity (determined according to the assay referred to in Example 2) of liver of fish according to Example 2.

Figure 2 is a graph showing normalized FRAP activity (determined according to the assay referred to in Example 2) of gill of fish according to Example 2.

Figure 3 is a graph showing normalized FRAP activity (determined according to the assay referred to in

Example 2) of muscle of fish according to Example 2.

In each of Figures 1 to 3 , the following key is used: cont = control; sel = injected with sodium selenite; blu(e) = blueberry enhanced feed. The upper darker- coloured region of each bar represents the standard error of the mean.

Example 1 Feed

335 mL Of sugar-free liquid blueberry extract (available from Helios, Oslo, Norway) was lyophilized and the resultant anthocyanin-containing powder was resuspended in 100 mL of a 3% wt solution of gelatin in water at

50°C. The resulting warm solution was sprayed (atomised) over 3kg of fish food (available from Skretting Fiskfor, Norway as Atlantic HP50-65A in 3.0 mm pellets) spread out over a lm² surface. The treated feed was placed in a freezer at -20°C for storage before use.

Example 2

Feeding and Experimental Methods

Juvenile Atlantic salmon (mean bodyweight 36±4 g) from AquaNiva, Sollergstrand, Norway were placed 10 per tank into separate 150 L glass aquarium tanks with a through flow of dechlorinated municipal water, either at 9+l°C or 12+1°C. The tanks were illuminated on a 12 hours on, 12 hours off basis. After 3 weeks acclimatization, each group was then fed ad lib. twice per day with the formulation of Example 1 or the equivalent untreated commercial feed. After 20 days, all fish were anaesthetized with 80 mg/L MS222 with one blueberry treated feed group and one normal feed group also being injected i.p. with 19 μmol/kg bodyweight sodium selenite in teleost Ringer's solution the sodium selenite induces oxidative stress. The remaining fish were injected with teleost Ringer's solution alone. Feeding continued for a further ten days before the fish were sacrificed by a blow to the cranium and immediate decapitation (a method approved as humane by the Norwegian Animal Welfare

Authority) . The eyes were removed and rinsed in ice- cold phosphate buffered saline. The right eye was dissected along the orra serrata and the lens was removed and placed in ice-cold artificial aqueous humor for digital imaging. The left eye was left intact and placed in an 1.5 mL Eppendorf tube and snap frozen in liquid nitrogen. Gill, liver and muscle tissue was removed, rinsed in ice-cold phosphate buffered saline placed in 1.5 mL Eppendorf tubes and snap frozen. The eyes were imaged under darkroom conditions at 25X with a microscope equipped with a 2.5 megapixel digital camera. Pinhole illumination was provided by a 2 cm diameter tube with a pinhole perforation administered to aluminium foil placed at both ends of the tube. Light was provided by a dissection light source using a fibre optic light guide and the apparatus was held in place using chemical apparatus clamps. The pinhole outlet was placed at a 45° angle above the lens, projecting through the entire lens. Lens imperfections were measured as the backscatter from the pinhole source, as measured by the pixel intensity from the backscatter. Tissue and plasma antioxidant potential was determined using the ferric reducing antioxidant power (FRAP) assay (see Benzie et al . Anal .Biochem. 239 -.10 - 16 (1996) and Methods Enzymol. 299:15-27 (1999)). Briefly, thawed tissue was place wet-weighed and placed in lysis buffer and homogenized. The homogenate was extracted with methanol and FRAP was measured spectrophotometrically with a Technicon RA-1000 analyzer.

Results The fish consumed the approximate equivalent of 5 percent bodyweight per day (ca. 2 gms feed day) . The blueberry-enhanced fish feed consumed per fish per day was the equivalent of 0.22 mis of original blueberry extract. The fish gained, on average, about 4 gm over the 30 day feeding period. No weight gain differences in treatment groups were seen. As shown in Figure 1, the blueberry-enhanced feed showed a clear positive effect on liver FRAP in fish that were both injected with sodium selenite and those not so injected. The injection of sodium selenite did not have a significant effect on non-enhanced feed, but did have a FRAP-reducing effect on enhanced-feed fish, although the

FRAP increasing effect of the blueberry-enhanced feed was still seen. As shown in Figure 2, the blueberry-enhanced feed showed a positive effect on gill FRAP activity in fish that were fed the enhanced meal, with selenite reducing the extent of FRAP activity. As shown in Figure 3, the blueberry-enhanced feed had a significant effect on FRAP activity in muscle tissue with almost double the FRAP activity seen in control fish. Selenite did have the effect of reducing FRAP activity. The whole eye of salmon did not appear to exhibit any major changes in FRAP activity as the result of feeding fish a blueberry extract-enhanced feed, however the selenite treatment did appear to effectively diminish the FRAP activity of control-fed fish whereas feeding enhanced-feed appeared to prevent the FRAP- reducing effect of selenite.

Claims

Claims :

1. The use of one or more extracted or synthetic anthocyanins, preferably one or more essentially lipid- free anthocyanins, in the manufacture of a solid fish feed or fish feed supplement.

2. A method of aquaculture comprising administering a solid fish feed or feed supplement to fish, characterized in that said feed or supplement contains one or more extracted or synthetic anthocyanins or in that one or more anthocyanins is also administered to said fish as a feed supplement.

3. A solid fish feed or feed supplement, characterized in that said feed or feed supplement contains one or more extracted or synthetic anthocyanins, preferably one or more anthocyanins which are not lipid bound.

4. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the fish feed or feed supplement also comprises a fish, animal, plant or microorganism protein and a fish, animal or plant oil.

5. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the fish feed or feed supplement is in extruded and pelletized form.

6. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the anthocyanins comprise at least two different aglycone units and at least two different saccharide units.

7. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the anthocyanins include ones in which the aglycone units are two or more of cyanidin, peonidin, delphinidin, petunidin, malvidin and pelargonidin.

8. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the anthocyanins include at least one monosaccharide anthocyanin.

9. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the anthocyanins include at least one disaccharide anthocyanin.

10. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein at least one of the anthocyanins is a monosaccharide in which the saccharide is arabinose.

11. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein at least one of the anthocyanins is a disaccharide in which the disaccharide is rutinose (6-rhamnosyl-glucose) .

12. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein one or more of the anthocyanins is selected from cyanidin-3-O-β- glucoside; cyanidin-3-O-β-galactoside,- cyanidin-3-0-α- arabinoside; cyanidin-3-O-β-xyloside,- cyanidin-3-0- (6"- O-α-rhamnosyl-β-glucoside) ; cyanidin-3-0- (2" -O-β- glucosyl-β-galactoside) ; cyanidin-3-0- (2"-0-β-glucosyl-β- glucoside) ; cyanidin-3-0- (2 "-O-β-xylosyl-β-glucoside) ; cyanidin-3-0- (2" -O-β-xylosyl-β-galactoside) ; cyanidin- 3 , 5-di-0-β-glucoside; cyanidin-3-0-β-galactoside-5-0-β- glucoside,- cyanidin-3 -O-α-arabinoside-5-O-β-glucoside ; cyanidin-3-0- (2"-0-β-xylosyl-β-glucoside) -5-0-β- glucoside; cyanidin-3-0- (2"-0-β-xylosyl-6" -O-β-glucosyl- β-galactoside) ; pelargonidin-3-O-β-glucoside,- pelargonidin-3-0- (6" -O-α-rhamnosyl-β-glucoside) ; pelargonidin-3, 5-di-O-β-glucoside,- peonidin-3-O-β- glucoside; peonidin-3-O-α-arabinoside,- peonidin-3 , 5-di- O-β-glucoside; delphinidin-3-O-β-glucoside; delphinidin- 3-0- (6" -O-α-rhamnosyl-β-glucoside) ; delphinidin-3-O- (2"- O-β-xylosyl-β-glucoside) ; petunidin-3-O-β-glucoside,- malvidin-3 -O-β-glucoside; malvidin-3 , 5-di-O-β-glucoside,- and malvidin-3 -O-α-arabinoside-5-O-β-glucoside .

13. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein one or more hydroxy groups in the anthocyanins is acylated.

14. A use, method, or fish feed or feed supplement as claimed in claim 13 wherein one or more hydroxyl groups in the anthocyanins is acylated with a C^^ saturated or unsaturated acyl group.

15. A use, method, or fish feed or feed supplement as claimed in claim 13 wherein one or more of the anthocyanins is selected from cyanidin-3-0- (6"-0- (E-p- coumaryl) -2" -O-β-xylosyl-β-glucoside) ; cyanidin-3-0- (6"- 0- (E-p-coumaryl) -2 " -O-β-xylosyl-β-glucoside) -5-0-β- glucoside; cyanidin-3-0- (2 " -O-β-xylosyl-6" -0- (E- feruloyl-β-glucosyl) -β-galactoside) ; cyanidin-3-0- (2"-0- β-xylosyl-6"-0- [E-coumaryl-β-glucosyl] -β-galactoside) ; and petunidin-3-0- (6"-0- (4" ' -O-E-coumaryl) -α-rhamnosyl-β- glucoside) -5-0-β-glucoside.

16. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the counter ion in one or more of the anthocyanins is a fruit acid anion.

17. A use, method, or fish feed or feed supplement as claimed in claim 16 wherein the fruit acid anion is citrate .

18. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the source of one or more of the anthocyanins is a fruit or vegetable.

19. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the source of one or more of the anthocyanins is bilberries (Vaccinium myrtillus) or blackcurrants (Ribes nigrum) .

20. A use, method, or fish feed or feed supplement as claimed in any preceding claim wherein the anthocyanins constitute 0.1 to 95% wt of the fish feed or feed supplement, excluding any coating material.

21. A use, method, or fish feed or feed supplement as claimed in claim 20 wherein the anthocyanins constitute 5 to 30% wt of the fish feed or feed supplement, excluding any coating material .

22. A method of aquaculture as claimed in any preceding claim wherein the fish are marine or freshwater vertebrate fish.

23. A method of aquaculture as claimed in any preceding claim wherein the daily anthocyanin dosage is 1 to 100 mg/kg bodyweight .

24. Use as claimed in any of claims 1 to 21 wherein the anthocyanin in incorporated into the fish feed as an anthocyanin/spray drying adjuvant mixture.