PROCESS FOR PRODUCING A PUFA-CONTAINING BIOMASS WHICH HAS HIGH CELL STABILITY

Abstract

According to the invention, it was found that culturing of PUFA-producing cells in a content of sulphate makes it possible to obtain a biomass having high cell stability and thus PUFAs protected against oxidation in a sustained manner, which biomass can in addition be advantageously further processed into a feedstuff.

Claims

1-15. (canceled)

16. A PUFA-containing biomass, comprising a sulphate content, based on the dry biomass, of 25 to 60 g/kg, and wherein the biomass contains cells of the taxon Labyrinthulomycetes.

17. The PUFA-containing biomass of claim 16, wherein said biomass has a sulphate content, based on the dry biomass, of 25 to 40 g/kg.

18. The PUFA-containing biomass of claim 16, wherein said biomass has a sulphate content, based on the dry mass, of 25 to 30 g/kg.

19. The PUFA-containing biomass of claim 16, wherein the cells of the taxon Labyrinthulomycetes (Labyrinthulea, slime nets) are of the family Thraustochytriaceae.

20. The PUFA-containing biomass of claim 19, wherein the cells are of a genus selected from the group consisting of: Althomia; Aplanochytrium; Elnia; Japonochytrium; Schizochytrium; Thraustochytrium; Aurantiochytrium; Oblongichytrium; and Ulkenia.

21. The PUFA-containing biomass of claim 20, wherein the cells are of the species Aurantiochytrium limacinum.

22. A feedstuff, comprising a PUFA-containing biomass, wherein said PUFA-containing biomass has a sulphate content, based on the dry mass, of 25 to 60 g/kg, and wherein the biomass contains cells of the taxon Labyrinthulomycetes.

23. The feedstuff of claim 22, comprising the following properties: a) a total protein content of 30 to 60% by weight; b) a total fat content of 15 to 35% by weight; c) a total starch content of at most 25% by weight; d) a content of biomass of 2 to 22% by weight.

24. The feedstuff of claim 23, wherein the biomass comprises cells of a genus selected from the group consisting of: Althomia; Aplanochytrium; Elnia; Japono-chytrium; Schizochytrium; Thraustochytrium; Aurantiochytrium; Oblongichytrium; and Ulkenia.

25. The feedstuff of claim 24, wherein the biomass comprises cells of the species Aurantiochytrium limacinum.

26. The feedstuff of claim 23, comprising one or more of the following properties: e) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight; f) an omega-3 fatty acid content of 1 to 6% by weight; g) a DHA content of 0.5 to 3% by weight.

27. The feedstuff of claim 23, comprising at least two of the following properties: e) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight; f) an omega-3 fatty acid content of 1 to 6% by weight; g) a DHA content of 0.5 to 3% by weight.

28. The feedstuff of claim 23, comprising all of the following properties: g) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight; f) an omega-3 fatty acid content of 1 to 6% by weight; g) a DHA content of 0.5 to 3% by weight.

29. The feedstuff of claim 22, comprising the following properties: a) a total protein content of 40 to 50% by weight; b) a total fat content of 20 to 30% by weight; c) a total starch content of 5 to 15% by weight; d) a content of Thraustochytriaceae biomass of 10 to 16% by weight.

30. The feedstuff of claim 29, comprising one or more of the following properties: e) a polyunsaturated fatty acid (PUFA) content of 3 to 10% by weight; f) an omega-3 fatty acid content of 2 to 4.5% by weight; g) a DHA content of 1.2 to 2.2%.

31. The feedstuff of claim 29, comprising at least two of the following properties: e) a polyunsaturated fatty acid (PUFA) content of 3 to 10% by weight; f) an omega-3 fatty acid content of 2 to 4.5% by weight; g) a DHA content of 1.2 to 2.2%.

32. The feedstuff of claim 29, comprising all of the following properties: e) a polyunsaturated fatty acid (PUFA) content of 3 to 10% by weight; f) an omega-3 fatty acid content of 2 to 4.5% by weight; g) a DHA content of 1.2 to 2.2%.

33. The feedstuff of claim 32, wherein the biomass comprises cells of a genus selected from the group consisting of: Althomia; Aplanochytrium; Elnia; Japono-chytrium; Schizochytrium; Thraustochytrium; Aurantiochytrium; Oblongichytrium; and Ulkenia.

34. The feedstuff of claim 32, wherein the biomass comprises cells of the species Aurantiochytrium limacinum.

35. A method for farming animals, comprising feeding said animals the feedstuff of claim 22.

Description

WORKING EXAMPLES

Example 1

Producing Biomass by Fermentation of Aurantiochytrium limacinum Sr21 in Media of Differing Sodium Sulphate Content

[0163] The cells were cultured for about 75 h in a feed process using a steel fermenter having a fermenter volume of 2 litres with a total starting mass of 712 g and an attained total final mass of 1.3-1.5 kg. During the process, a glucose solution (570 g/kg glucose) was metered in (fed-batch process)

[0164] The composition of the starting media was as follows:

[0165] Medium 1: 20 g/kg glucose; 4 g/kg yeast extract; 2 g/kg ammonium sulphate; 2.46 g/kg magnesium sulphate (heptahydrate); 0.45 g/kg potassium chloride; 4.5 g/kg potassium dihydrogen phosphate; 0.1 g/kg thiamine (HCl); 5 g/kg trace element solution.

[0166] Medium 2: As per medium 1 plus 8 g/kg sodium sulphate

[0167] Medium 3: As per medium 1 plus 12 g/kg sodium sulphate

[0168] Medium 4: As per medium 1 plus 16 g/kg sodium sulphate

[0169] The composition of the trace element solution was as follows: 35 g/kg hydrochloric acid (37%); 1.86 g/kg manganese chloride (tetrahydrate); 1.82 g/kg zinc sulphate (heptahydrate); 0.818 g/kg sodium EDTA; 0.29 g/kg boric acid; 0.24 g/kg sodium molybdate (dihydrate); 4.58 g/kg calcium chloride (dihydrate); 17.33 g/kg iron sulphate (heptahydrate); 0.15 g/kg copper chloride (dihydrate).

[0170] Culturing was carried out under the following conditions: Culture temperature 28° C.; aeration rate 0.5 vvm, stirrer speed 600-1950 rpm, control of pH in the growth phase at 4.5 using ammonia water (25% v/v). The following biomass densities were achieved: 100 g/l (medium 1), 111 g/l (medium 2), 114 g/l (medium 3), 116 g/l (medium 4).

[0171] After the culturing process, the fermentation broths were heated to 60° C. for 20 minutes in order to prevent further cellular activity.

Example 2

Determining the DHA Content of the Biomasses

[0172] After inactivation, the biomasses obtained were subjected to a fatty acid analysis. To this end, 0.2-0.5 ml of each fermentation broth was admixed with 1 ml of internal standard and topped up with 9 ml of a methanol/chloroform solution (1:2; v/v). The samples were treated for 10 min in an ultrasonic bath. Subsequently, the samples were concentrated to dryness under a nitrogen blanket at 50° C. in a thermal block. 2 ml of 0.5 N KOH were added to each of the residues of drying and incubated at 100° C. for 15 min. Subsequently, the samples were cooled down to room temperature, admixed with, in each case, 2 ml of 0.7 N HCl and 1 ml of boron trifluoride solution (14% BF3 in methanol) and incubated at 100° C. for a further 15 min. After cooling down to room temperature, the samples were each extracted with a mixture composed of 3 ml of water and 2 ml of heptane. After centrifugation for 1 min at 2000 rpm, 1 ml from each upper phase was transferred to a GC vial and analysed by gas chromatography.

[0173] The analysis revealed that all four biomasses contained a DHA fraction of more than 32% by weight with regard to the total amount of fatty acids present.

Example 3

Drying the Biomasses Obtained

[0174] The cooled-down biomass-containing fermentation broths having different contents of Na.sub.2SO.sub.4 according to Example 1 were each separately subjected to spray drying.

[0175] Spray drying was carried out in each case using a Büuchi mini spray dryer B-290 (diameter of nozzle tip: 0.7 mm; flow rate of spray air: 742 L/h; flow rate of aspirator: 35 m.sup.3/h; temperature of inlet air: 220° C.; temperature of outlet air: 80° C.).

Example 4

Determining the Sulphate and DHA Contents of the Spray-Dried Samples

[0176] The samples obtained by spray drying the fermentation broths having different contents of Na2SO4 were subjected to a sulphate or sulphur determination and a DHA concentration determination. DHA determination was carried out as described under Example 2. Sulphur content was determined in accordance with DIN EN ISO 11885.

TABLE-US-00001 TABLE 1 Analysis of spray-dried samples Starting medium of the fermentation broth used for spray drying Property Medium 1 Medium 2 Medium 3 Medium 4 Sulphur in 3.60 g/kg 7.72 g/kg 10.0 g/kg 11.0 g/kg accordance with DIN EN ISO 11885 DHA content 16.6% 15.6% 16.0% 16.0%

Example 4

Determining the Caking Tendency

[0177] The fermentation broths obtained after fermentation in media having different contents of sodium sulphate exhibited distinct differences in the spray drying process and the spray-dried material obtained exhibited varying caking tendency, the basis of this being released oil. The biomasses obtained by fermentation in media 3 and 4 showed a distinctly lower caking tendency than the biomasses obtained by fermentation in media 1 and 2. This is evidence of the increased cell stability of the cells in the biomasses concerned.

Example 5

Drying the Sulphate-Rich Biomass from Example 1 for the Purpose of Feedstuff Production

[0178] The biomass from Example 1 obtained in the sulphate-rich medium 4 was subjected to a two-stage drying process for the purpose of producing feedstuffs: Firstly, the fermentation broth was concentrated by evaporation to a dry mass of about 20% by weight. This was followed by spray drying of the concentrated fermentation broth using a Production Minor™ spray dryer (GEA NIRO) at a drying air inlet temperature of 340° C. By means of spray drying, a powder having a dry mass of more than 95% by weight was thus obtained.

Example 6

Producing a Feedstuff by Extrusion

[0179] The feedstuff mixtures shown in Table 3 were produced. Besides the biomass to be used according to the invention as per Example 5, two further commercially available Labyrinthulea biomasses and also fish oil as a currently still customary source of omega-3 fatty acids were tested for comparison.

[0180] The feedstuff mixtures were each produced by mixing of the components—with the exception of the oils—using a double-helix mixer (model 500L, TGC Extrusion, France). The mixtures thus obtained were then comminuted to particle sizes below 250 μm using a hammer mill (model SH1, Hosokawa-Alpine, Germany).

TABLE-US-00002 TABLE 2 Feedstuff compositions used in the extrusion process (data in % by weight) Ingredient M1 M2 M3 M4 Fish meal 10.00 10.00 10.00 10.00 Soya protein concentrate 23.10 23.20 23.10 20.27 Pea protein concentrate 15.00 15.00 15.00 15.00 Wheat gluten 9.90 9.90 9.90 9.90 Wheat meal 18.12 10.82 10.55 16.46 Fish oil 10.00 — — — Biomass from Example 1 — 16.00 — — Commercially available biomass 1 — — 16.74 — Commercially available biomass 2 — — — 13.52 Rape oil 10.00 11.00 11.00 11.00 Vitamin/mineral premix 1.00 1.00 1.00 1.00 DCP 2.00 2.00 2.00 2.00 Yttrium oxide 0.03 0.03 0.03 0.03 DL-Methionine 0.35 0.36 0.33 0.33 Aquavi Lys 0.17 0.35 0.08 0.19 TrypAmino 0.09 0.09 0.08 0.09 L-Histidine 0.24 0.25 0.19 0.21

[0181] For the extrusion process, use was made in each case of 140 kg per feedstuff. The extrusion process was carried out using a twin-screw extruder (CLEXTRAL BC45) having a screw diameter of 55.5 mm and a maximum flow rate of 90-100 kg/h. Pellets of 4.0 mm in size (diameter and length) were extruded. To this end, the extruder was equipped with a high-speed cutter in order to convert the product to the intended pellet size.

[0182] Various extrusion parameters were then tested in order to find out under what extrusion conditions it is possible to obtain an optimal oil load capacity of the extrudate obtained. In this connection, it became apparent that, surprisingly, an optimal oil load capacity can be achieved with a very low energy input. In this connection, the energy input was distinctly lower than when using fish oil. Furthermore, the optimal energy input in the case of an algae biomass to be preferably used according to the invention was again distinctly lower than in the case of commercially available algae biomasses. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Energy inputs relating to producing pellets having the desired oil load capacity Barrel 1 Barrel 2 Rotational Amount of Temp Temp Feeder rate speed water Current SME Diet (° C.) (° C.) (kg/h) (rpm) (0-10) (A) (Wh/kg) M1 31 116-118  112 215 9 11 34.6 M2 32 98-104 141 253 5 7 20.6 M3 32 97-102 136 255 5 8 24.6 M4 31 99-107 133 253 5 8 24.9

[0183] In this connection, the variable “SME” is the specific mechanical energy. This is calculated as follows:

[00001]$\mathrm{SME}.Math..Math.\left(\mathrm{Wh}.Math.\text{/}.Math.\mathrm{kg}\right)=\frac{UI\cos .Math..Math.\Phi .Math.\frac{\mathrm{Test}.Math..Math.\mathrm{SS}}{\mathrm{Max}.Math..Math.\mathrm{SS}}}{\mathrm{Qs}}$

[0184] where

[0185] U: operating voltage of the motor (here 460 V)

[0186] I: current of the motor (A)

[0187] cos φ: theoretical performance of the extruder motor (here 0.95)

[0188] Test SS: test speed (rpm) of the rotating screws

[0189] Max SS: maximum speed (267 rpm) of the rotating screws

[0190] Qs: inlet flow rate of the mash (kg/h)

[0191] After extrusion, the extrudate was dried in a vibrating fluidized bed dryer (model DR100, TGC Extrusion, France).

[0192] This was followed, after the extrudate had cooled down, by an oil coating process by means of vacuum coating (vacuum coater PG-10VCLAB, Dinnisen, the Netherlands).

Example 7

Ascertaining the Abrasion Resistance and Water Stability of the Feedstuffs from Example 6

[0193] Abrasion resistance was ascertained as follows: Before being loaded with oil, the dried extrusion product was exposed to a mechanical load using the Holmen pellet tester (Borregaard Lignotech, Hull, UK). Before carrying out the test, the samples were screened in order to remove any adherent fine particles. The processed samples (100 g) were subsequently introduced into the pellet tester using a 2.5 mm filter screen. The pellets were subsequently conveyed through a pipe having right-angled pipe bends at high air velocity for 30 seconds. Subsequently, abrasion was determined by weighing. Abrasion resistance was specified as PDI (Pellet Durability Index), defined as the amount in per cent of sample remaining in the filter screen. The test was carried out with three samples and then the mean was determined.

[0194] Water stability was carried out using the oil-loaded samples. The method was essentially carried out as described by Baeverfjord et al. (2006; Aquaculture 261, 1335-1345), with slight modifications. 10 g samples were introduced into metallic infusion baskets having a mesh size of 0.3 mm. The infusion baskets were subsequently introduced into a plastic trough containing water, and so the samples were completely covered with water. The trough was subsequently exposed for 30 minutes to a shake-agitation of 30 shake units per minute. Thereafter, the samples were carefully dried with blotting paper and then weighed before and after they had been subjected to oven-drying at a temperature of 105° C. for 24 hours. Water stability was calculated as the difference in the dry weight of the sample before and after the incubation in water and specified in per cent of the dry weight of the sample used before the incubation with water.

[0195] The results are shown in Table 4 below.

TABLE-US-00004 Sample M1 M2 M3 M4 Abrasion 90.0 93.3 88.3 85.2 resistance [%] Water stability [%] 95.7 98.5 93.8 90.2

[0196] It can be seen that a feedstuff according to the invention which contains a biomass according to the invention has a distinctly higher abrasion resistance and water stability than feedstuffs which contain a commercially available Labyrinthulea biomass or fish oil as a source of omega-3 fatty acids.

Example 8

Producing Feedstuffs for Feeding Experiments

[0197] Feedstuffs each containing 42.5% by weight of total protein and 24% by weight of total lipid, based on the dry mass, and having a pellet size of 3 mm were produced by extrusion of the biomass from Example 5.

[0198] Three different feedstuff formulations in total were produced (Diet 1, 2 and 3). The control formulation “Diet 1” contained 11.0% by weight of fish oil. In the formulation “Diet 2”, the fish oil was partly (about 50%) replaced by Aurantiochytrium biomass, this being done by adding 9.1% by weight of biomass and, for that reason, reducing the amount of fish oil to 5.5% by weight. In the formulation “Diet 3”, the fish oil was completely replaced by Aurantiochytrium biomass, this being done by adding 16% by weight of biomass and, at the same time, increasing the amount of rape oil from 8.2 to 9.9% by weight. Differences in the total weight were balanced out by the amount of wheat added.

[0199] The individual components of the feedstuff are shown in the table below.

TABLE-US-00005 TABLE 5 Formulations used for farming Components (g kg.sup.−1) Diet 1 Diet 2 Diet 3 Aurantiochytrium biomass 0.0 91.6 160.0 SPC 229.0 229.0 229.0 Fish meal 150.0 150.0 150.0 Wheat 147.6 111.0 80.5 Fish oil 110.0 55.0 0.0 Wheat gluten 100.0 100.0 100.0 Pea protein concentrate 100.0 100.0 100.0 Rape oil 82.0 82.0 99.1 Monosodium phosphate 20.0 20.0 20.0 Vitamin mixture 20.0 20.0 20.0 Soya lecithin 10.0 10.0 10.0 L-Lysine (50% by weight) 10.0 10.0 10.0 Betafine 9.4 9.4 9.4 Mineral mixture 5.2 5.2 5.2 L-Histidine (98% by 4.2 4.2 4.2 weight) DL-Methionine (99% by 2.0 2.0 2.0 weight) Carop. Pink (10% by 0.50 0.50 0.50 weight) Yttrium Oxide 0.10 0.10 0.10

[0200] The individual components were—with the exception of the oils—mixed intimately with each other and then an extrudate was produced using a twin-screw extruder (Wenger Tex. 52, Wenger, USA) through use of an outlet nozzle having a diameter of 2 mm. The extrudates were dried for about 1 hour in a carousel dryer (Paul Klöckner, Verfahrenstechnik GmbH, Germany) at 65° C. to a water content of 7 to 8% by weight. The extrudates were then dried overnight at room temperature before the oils were applied by vacuum coating (Dinnissen, Sevenum, the Netherlands).

Example 9

Feeding Experiments Using the Formulations from Example 8

[0201] The feeding experiments were carried out by feeding each of these formulations for a total of 12 weeks to each of three tanks containing smolts having a mean weight of 83.6 g and a total salmon weight of 4 kg per tank.

[0202] Over this period, the total salmon weight per tank increased from 4 kg to 15-17 kg per tank. In this connection, the fish consumed 8 to 11 kg of feed per tank, corresponding to a feed conversion rate (FCR) of 0.8 to 0.9 kg of feed per kg of fish.

[0203] The results of the feeding experiments are shown in the table below.

TABLE-US-00006 TABLE 6 Diet-dependent fish weight gain Diet Final weight [g] 1 331 2 362 3 339

[0204] Altogether, it was established that it was possible to achieve an increase in salmon growth both in the case of complete and in the case of partial replacement of the fish oil by an Aurantiochytrium biomass according to the invention.

[0205] Interestingly, partial replacement of the fish oil by the Aurantiochytrium biomass achieved a higher salmon growth than complete replacement by the Aurantiochytrium biomass.

[0206] In this connection, it was established that the fish fed with the control formulation Diet 1, having a mean final weight of 331 g, had a distinctly lower final weight than the fish fed with the formulations Diet 1 or 2. In this connection, the fish fed with the formulation Diet 2 performed the best: they achieved a distinctly increased mean final weight of 362 g.

Example 10

Fatty Acid Utilization by the Fish

[0207] Fatty acid utilization was ascertained by lipid detection using the Bligh & Dryer extraction method and subsequent fatty acid analysis in accordance with AOCS Ce 1b-89. Both muscle samples and total salmon samples were analysed. In this connection, the results shown in the tables below were obtained (displayed in each table is the amount of ascertained fats at the start and end of the diet in grams, based in each case on 100 g of total fat).

TABLE-US-00007 TABLE 7 Diet-dependent fatty acid profile of salmon muscle samples Diet PUFAs Omega-3 fatty acids DHA Start 41.6 32.7 22.1 1 31.8 19.0 10.1 2 35.3 21.7 14.2 3 38.0 22.8 16.4

TABLE-US-00008 TABLE 8 Diet-dependent fatty acid profile of total salmon samples Diet PUFAs Omega-3 fatty acids DHA Start 32.5 22.4 12.5 1 29.9 17.2 9.0 2 32.6 18.8 11.5 3 35.2 19.6 13.1

[0208] It can be observed that it was already possible to achieve a distinct increase in the content of PUFAs, omega-3 fatty acids and DHA in the case of partial replacement of the fish oil by an Aurantiochytrium biomass according to the invention. In the case of complete replacement of the fish oil by the Aurantiochytrium biomass, the increase in the content of PUFAs is accordingly higher.

Example 11

Determining the Fat Content in Salmon Liver

[0209] Each of 3 smolts were fed for 9 weeks in each case with the different formulations Diet 1, 2 and 3 and the livers of the salmons were subsequently removed for determination of the fat content. Fat was extracted according to the method by Folch (1957; J. Biol. Chem., 226 (1), 497-509). Fat content was then determined by a gravimetric method.

[0210] It became apparent that it was possible to significantly reduce the fat content in the liver from 8% by weight to 4-5% by weight by virtue of the presence of the biomass according to the invention in comparison with feeding without the biomass.

[0211] Fat deposition in the liver is considered to be a sign of an imbalance in food metabolism and, in particular, also an indication of oxidative stress. The distinct reduction in the proportion of fat in the liver is thus a clear indication of the reduction of stress and thus of the improvement in the physical condition of the salmon.