FEED SUPPLEMENT MATERIAL FOR USE IN AQUACULTURE FEED

Abstract

The invention concerns a method of sustainably producing an aquaculture meat product by feeding a fish over its dietary cycles an aquaculture feed composition, said method comprising the step of formulating an aquaculture feed composition by replacing all or part of fish oil in the composition with a single microbial source of eicosapentaenoic acid (“EPA”) and docosahex-aenoic acid (“DHA”). In a preferred embodiment, the microbial source comprising DHA and EPA derives from a microorganism/microbe of the genus Schizochytrium or Thraustochytrium.

Claims

1. A method of sustainably producing an aquaculture meat product by feeding a fish over its dietary cycles an aquaculture feed composition, said method comprising the step of formulating an aquaculture feed composition by replacing all or part of fish oil in the composition with a single microbial source of eicosapentaenoic acid (“EPA”) and docosahexaenoic acid (“DHA”).

2. The method of claim 1 wherein the aquaculture feed composition comprises a total amount of EPA and DHA that is at least about 0.8%, measured as a weight percent of the aquaculture feed composition.

3. The method of claim 1 wherein the ratio of concentration of EPA to concentration of DHA is at least 2:1, based on individual concentrations of EPA and DHA in the microbial source or in the aquaculture feed composition.

4. The method of claim 1, wherein the ratio of concentration of EPA to concentration of DHA is 1:1 or lower, based on individual concentrations of EPA and DHA in the microbial source or in the aquaculture feed composition.

5. The method of claim 1, wherein the microbial source is a microbial oil and wherein the microbial oil is provided in a form selected from the group consisting of: biomass, processed biomass, partially purified oil and purified oil, any of which is obtained from the microbial source.

6. The method of claim 1, wherein the microorganism from which the microbial source derives is an algae, fungi or yeast.

7. The method of claim 6, wherein the microorganism is a member of the genus Schizochytrium or Thraustochytrium.

8. The method of claim 7, wherein the microorganism has the characteristics of the species deposited under ATCC Accession No. PTA-10208 or PTA-10209 or PTA-10210 or PTA-10211 or PTA-10212 or PTA-10213 or PTA-10214 or PTA-10215.

9. The method of claim 6, wherein the microorganism is a mutant strain.

10. The method of claim 6, wherein the microorganism is a transgenic microbe genetically engineered for the production of polyunsaturated fatty acid containing microbial oil comprising EPA and DHA.

11. The method of claim 1, wherein the aquaculture meat product has a ratio of concentration of EPA to concentration of DHA that is equal to or greater than 2:1, based on the concentration of each in the aquaculture meat product.

12. The method of claim 1, wherein the aquaculture meat product has a ratio of concentration of EPA to concentration of DHA that is equal to or lower than 1:1, based on the concentration of each in the aquaculture meat product.

13. Feed additive composition for aquaculture feed comprising eicosapentaenoic acid (“EPA”) and docosahexaenoic acid (“DHA”) derived from a single microbial source.

14. Additive composition of claim 13, which is provided in a form selected from the group consisting of biomass, processed biomass, partially purified oil and purified oil, any of which is obtained from the microbial source.

15. Additive composition of claim 13, wherein the microorganism from which the microbial source derives is an algae, fungi or yeast.

16. Additive composition of claim 15, wherein the microorganism is a member of the genus Schizochytrium or Thraustochytrium.

17. Additive composition of claim 16, wherein the microorganism has the characteristics of the species deposited under ATCC Accession No. PTA-10208 or PTA-10209 or PTA-10210 or PTA-10211 or PTA-10212 or PTA-10213 or PTA-10214 or PTA-10215.

18. Additive composition of claim 13, which is a purified microbial oil form containing at least 40% w/w DHA & EPA, preferably about 50% w/w DHA & EPA.

19. Aquaculture feed comprising a single microbial source of eicosapentaenoic acid (“EPA”) and docosahexaenoic acid (“DHA”).

20. Aquaculture feed of claim 19 comprising a total amount of EPA and DHA that is at least about 0.08%, measured as a weight percent of the feed.

21. Aquaculture feed of claim 19 wherein the ratio of concentration of EPA to concentration of DHA is equal to or less than 1:1, based on individual concentrations of EPA and DHA in the microbial source or in the feed.

22. Aquaculture feed of claim 19 wherein the ratio of concentration of EPA to concentration of DHA is equal to greater than 2:1, based on individual concentrations of EPA and DHA in the microbial source or in the feed.

23. Aquaculture feed of any of claim 19, wherein the microbial source is a microbial oil and wherein the microbial oil is provided in a form selected from the group consisting of biomass, processed biomass, partially purified oil and purified oil, any of which is obtained from the microbial source.

24. Aquaculture feed of claim 19, wherein the microorganism from which the microbial source derives is an algae, fungi or yeast.

25. Aquaculture feed of claim 24, wherein the microorganism is a member of the genus Schizochytrium or Thraustochytrium.

26. Aquaculture feed of claim 25, wherein the microorganism has the characteristics of the species deposited under ATCC Accession No. PTA-10208 or PTA-10209 or PTA-10210 or PTA-10211 or PTA-10212 or PTA-10213 or PTA-10214 or PTA-10215.

Description

EXAMPLE 1 GROWTH CHARACTERISTICS OF THE ISOLATED MICROORGANISM DEPOSITED UNDER ATCC ACCESSION NO. PTA-10212

[0094] The isolated microorganism deposited under ATCC Accession No. PTA-10212 was examined for growth characteristics in individual fermentation runs, as described below. Typical media and cultivation conditions are shown in Table 3.

TABLE-US-00003 TABLE 3 PTA-10212 Vessel Media Ingredient concentration ranges Na.sub.2S0.sub.4 g/L 31.0 0-50, 15-45, or 25-35 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 KCl g/L 1.0 0-5, 0.25-3, or 0.5-2 MgS0.sub.4*7H.sub.20 g/L 5.0 0-10, 2-8, or 3-6 (NH.sub.4).sub.2S0.sub.4 g/L 0.44 0-10, 0.25-5, or 0.05-3 MSG*1H20 g/L 6.0 0-10, 4-8, 01-5-7 CaCl.sub.2 g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast extract) g/L 6.0 0-20, 0.1-10, or 1-7 KH.sub.2P0.sub.4 g/L 0.8 0.1-10, 0.5-5, or 0.6-1.8 Post autoclave (Metals) Citric acid mg/L 3.5 0.1-5000, 10-3000, or 3-2500 FeSO.sub.4*7H.sub.2O mg/L 10.30 0.1-100, 1-50, or 5-25 MnCl.sub.2*4H.sub.2O mg/L 3.10 0.1-100, 1-50, or 2-25 ZnS0.sub.4*7H.sub.2O mg/L 3.10 0.01-100, 1-50, or 2-25 CoCl.sub.2*6H.sub.2O mg/L 0.04 0-1, 0.001-0.1, or 0.01-0.1 Na.sub.2MoO.sub.4*2H.sub.2O mg/L 0.04 0.001-1, 0.005-0.5, or 0.01-0.1 CuSO.sub.4*5H.sub.2O mg/L 2.07 0.1-100, 0.5-50, or 1-25 NiSO.sub.4*6H.sub.2O mg/L 2.07 0.1-100, 0.5-50, or 1-25 Post autoclave (Vitamins) Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Vitamin B 12 mg/L 0.16 0.01-100, 0.05-5, or 0.1-1 Ca[1/2]-pantothenate mg/L 2.06 0.1-100, 0.1-50, or 1-10 Biotin mg/L 3.21 0.1-100, 0.1-50, or 1-10 Post autoclave (Carbon) Glycerol g/L 30.0 5-150, 10-100, or 20-50 Nitrogen Feed: MSG*1H.sub.2O g/L 17 0-150, 10-100, or 15-50

[0095] Typical cultivation conditions would include the following: [0096] pH 6.5-9.5, about 6.5-about 8.0, or about 6.8-about 7.8; [0097] temperature: 15-30 degrees Celsius, about 18-about 28 degrees Celsius, or about 21 to about 23 degrees Celsius; [0098] dissolved oxygen: 0.1-about 100% saturation, about 5-about 50% saturation, or about 10-about 30% saturation; and/or [0099] glycerol controlled @: 5-about 50 g/L, about 10-about 40 g/L, or about 15-about 35 g/L.

[0100] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl at 22.5° C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cell weight of 26.2 g/L after 138 hours of culture in a 10 L fermentor volume. The lipid yield was 7.9 g/L; the omega-3 yield was 5.3 g/L; the EPA yield was 3.3 g/L and the DHA yield was 1.8 g/L. The fatty acid content was 30.3% by weight; the EPA content was 41.4% of fatty acid methyl esters (FAME); and the DHA content was 26.2% of FAME. The lipid productivity was 1.38 g/L/day, and the omega-3 productivity was 0.92 g/L/day under these conditions, with 0.57 g/L/day EPA productivity and 0.31 g/L/day DHA productivity.

[0101] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl at 22.5° C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cell weight of 38.4 g/L after 189 hours of culture in a 10 L fermentor volume. The lipid yield was 18 g/L; the omega-3 yield was 12 g/L; the EPA yield was 5 g/L and the DHA yield was 6.8 g/L. The fatty acid content was 45% by weight; the EPA content was 27.8% of FAME; and the DHA content was 37.9% of FAME. The lipid productivity was 2.3 g/L/day, and the omega-3 productivity was 1.5 g/L/day under these conditions, with 0.63 g/L/day EPA productivity and 0.86 g/L/day DHA productivity.

[0102] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl at 22.5° C. with 20% dissolved oxygen at pH 6.8-7.7, PTA-10212 produced a dry cell weight of 13 g/L after 189 hours of culture in a 10 L fermentor volume. The lipid yield was 5.6 g/L; the omega-3 yield was 3.5 g/L; the EPA yield was 1.55 g/L and the DHA yield was 1.9 g/L. The fatty acid content was 38% by weight; the EPA content was 29.5% of FAME; and the DHA content was 36% of FAME. The lipid productivity was 0.67 g/L/day, and the omega-3 productivity was 0.4 g/L/day under these conditions, with 0.20 g/L/day EPA productivity and 0.24 g/L/day DHA productivity.

[0103] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm C at 22.5-28.5° C. with 20% dissolved oxygen at pH 6.6-7.2, PTA-10212 produced a dry cell weight of 36.7 g/L-48.7 g/L after 191 hours of culture in a 10 L fermentor volume. The lipid yield was 15.2 g/L-25.3 g/L; the omega-3 yield was 9.3 g/L-13.8 g/L; the EPA yield was 2.5 g/L-3.3 g/L and the DHA yield was 5.8 g/L-1 1 g/L. The fatty acid content was 42.4%-53% by weight; the EPA content was 9.8%-22% of FAME; and the DHA content was 38.1%-43.6% of FAME. The lipid productivity was 1.9 g/L/day-3.2 g/L/day, and the omega-3 productivity was 1.2 g/L/day-1.7 g/L/day under these conditions, with 0.31 g/L/day-0.41 g/L/day EPA productivity and 0.72 g/L/day-1.4 g/L/day DHA productivity.

EXAMPLE 2 APPARENT DIGESTIBILITY OF DHA AND EPA AS MICROBIAL BIOMASS IN ATLANTIC SALMON

[0104] Atlantic salmon of initial body weight ca 200 g were randomly distributed into 1.5 m tanks with 50 fish per tank. The water temperature was in the range of 10° C. Fish were adapted to the control diet for 2 weeks before starting feeding the experimental diets.

[0105] A 3-mm diet was produced by extrusion at Nofima in Bergen according to the formulation described in table 4. Only rapeseed oil was added to the diet and the basal levels of DHA and EPA in the control diet comes from the fish meal which has been included in the diet at a level of ca. 23%. NO fish oil was added to the diet.

[0106] As microbial source of DHA and EPA the biomass of a species of Schizochytrium sp. ATCC PTA-10208 also known as OvegaGold™ has been used. The biomass inclusions were 0, 0.9, 3.5 and 6.2% of the diet which corresponds to DHA levels of 0, 0.5, 1.0, 1.5%.

[0107] Fish were fed for at least 4 weeks and each dietary treatment was performed in triplicates. After 4 weeks of experimental feeding, feces were collected by stripping each fish from each individual tank. Muscle sample was taken out from five fish per tank.

[0108] Apparent digestibility coefficients were determined for the omega-3 fatty acids and for the nutrients such as dry matter, lipids and protein.

[0109] Statistical analysis was performed using Statbox Pro (one-way ANOVA).

TABLE-US-00004 TABLE 4 Diet formulation Major diet OvegaGold OvegaGold OvegaGold ingredients providing providing providing (%) Ctrl 0.5% DHA 1.0% DHA 1.5% DHA Fish meal 23 22 22.4 22 Soy protein conc. 17 17 17 17 Rapeseed oil 22 21 20 19 Wheat meal 11.4 10.9 8.7 7.4 Corn gluten 5 5 5 5 Wheat gluten 13 13 13 13 Pea protein 2 2 2 2 concentrate OvegaGold 0 2.5 5.2 8.0

[0110] Results of in-feed recovery of DHA and EPA are presented in Table 5. Results show a very good recovery of DHA and EPA after feed processing by extrusion and a clear dose response.

TABLE-US-00005 TABLE 5 In feed recovery Recovery (mg/g) Treatments EPA DHA EPA + DHA Total Ω-3 Control 2.21 4.32 6.53 25.81 OvegaGold providing 4.31 9.61 13.92 34.26 0.5% DHA From supplementation 2.1 5.3 6.4 OvegaGold providing 5.57 12.80 18.37 37.38 1.0% DHA From supplementation 3.4 8.5 11.9 OvegaGold providing 7.52 17.64 25.16 43.38 1.5% DHA From supplementation 5.3 13.3 18.6

[0111] Apparent digestibility coefficient was determined for DHA, EPA, DHA+EPA and omega-3 fatty acids. FIGS. 1, 2 and 3 present the digestibly of DHA+EPA, EPA and DHA, respectively. Results show that EPA and DHA provided via an algal source are highly digestible.

[0112] FIG. 1: Apparent digestibility of DHA in the diet supplemented with graded amounts of OvegaGold. Results of statistical analysis are presented in Table 6.

[0113] FIG. 2: Apparent digestibility of EPA in the diet supplemented with graded amounts of OvegaGold. Results of statistical analysis are presented in Table 6.

[0114] FIG. 3: Apparent digestibility of DHA in the diet supplemented with graded amounts of OvegaGold. Results of statistical analysis are presented in Table 6.

[0115] FIG. 4: Muscle deposition of omega-3 fatty acids following ca 4 weeks of feeding the experimental diets. Muscle content confirms the high bioavailability of omega-3 fatty acids from a microalgal source.

TABLE-US-00006 TABLE 6 Apparent digestibility coefficients as mean ± Sd ADC (%) Treatment EPA ±SD DHA ±SD EPA + DHA ±SD Total Ω 3 ±SD Control 97.35 0.05 94.18 0.23 95.25 0.17 98.36 0.07 OvegaGold 98.50 0.06 96.96 0.06 97.40 0.06 98.65 0.02 0.5% DHA OvegaGold 98.72 0.16 97.52 0.14 97.88 0.14 98.69 0.11 1.0% DHA OvegaGold 99.05 0.05 98.19 0.14 98.44 0.11 98.90 0.08 1.5% DHA

[0116] As a conclusion, DHA and EPA are highly bioavailable when provided as a microalgal biomass supplemented to a salmon diet.