Method for producing biomass which has a high exopolysaccharide content

Abstract

According to the invention, it was found that culturing of cells of the taxon Labyrinthulomycetes in a high content of sulphate makes it possible to obtain a biomass having a high EPS content, which biomass can in addition be advantageously further processed into a feedstuff.

Claims

1. A biomass dried comprising: exopolysaccharides (EPSs) of microorganisms of the taxon Labyrinthulomycetes, wherein the biomass has and a sulphate content, based on the dry mass, of 25 to 60 g/kg.

2. The biomass of claim 1, wherein said biomass has a sulphate content, based on the dry mass, of 25 to 40 g/kg.

3. The biomass of claim 1, wherein the microorganisms of the taxon Labyrinthulomycetes are of the family Thraustochytriaceae.

4. The biomass of claim 3, wherein the microorganisms of the taxon Labyrinthulomycetes are of the genus Althomia, Aplanochytrium, Elnia, Japonochytrium, Schizochytrium, Thraustochytrium, Aurantiochytrium, Oblongichytrium or Ulkenia.

5. The biomass of claim 4, wherein the microorganisms are of the genus Aurantiochytrium.

6. The biomass of claim 5, wherein the microorganisms are of species Aurantiochytrium limacinum.

7. A feedstuff, comprising the biomass of claim 1.

8. The feedstuff of claim 7, wherein said feedstuff comprises: 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; and d) a biomass content of 2 to 22% by weight.

9. The feedstuff of claim 8, wherein said feedstuff comprises: e) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight.

10. The feedstuff of claim 8, wherein said feedstuff comprises: f) an omega-3 fatty acid content of 1 to 6% by weight.

11. The feedstuff of claim 8, wherein said feedstuff comprises: g) a DHA content of 0.5 to 3% by weight.

12. The feedstuff of claim 9, wherein said feedstuff comprises: f) an omega-3 fatty acid content of 1 to 6% by weight; and g) a DHA content of 0.5 to 3% by weight.

13. The feedstuff of claim 7, wherein said feedstuff comprises: 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 biomass content of 8 to 18% by weight.

14. The feedstuff of claim 13, further comprising one or more of the following: e) a polyunsaturated fatty acid (PUFA) content of 5 to 8% by weight; f) an omega-3 fatty acid content of 2.5 to 4% by weight; g) a DHA content of 1.2 to 2.0% by weight.

15. The biomass of claim 1, comprising a sulphate content, based on the dry mass, of 25 to 30 g/kg.

16. The biomass of claim 15, wherein the microorganisms of the taxon Labyrinthulomycetes are of the family Thraustochytriaceae.

17. The biomass of claim 15, wherein the microorganisms are cells of the species Aurantiochytrium limacinum.

18. The feedstuff of claim 8, wherein said biomass comprises a sulphate content, based on the dry mass, of 25 to 30 g/kg.

19. The feedstuff of claim 18, wherein said feedstuff comprises: a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight.

20. The feedstuff of claim 19, wherein said feedstuff comprises an omega-3 fatty acid content of 1 to 6% by weight.

Description

WORKING EXAMPLES

Example 1

Producing Biomass by Fermentation of Aurantiochytrium limacinum SR21 in Media of Differing Sodium Sulphate Content

(1) 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)

(2) The composition of the starting media was as follows:

(3) 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.

(4) Medium 2: As per medium 1 plus 8 g/kg sodium sulphate

(5) Medium 3: As per medium 1 plus 12 g/kg sodium sulphate

(6) Medium 4: As per medium 1 plus 16 g/kg sodium sulphate

(7) 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).

(8) 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/I (medium 2), 114 g/l (medium 3), 116 g/l (medium 4). The viscosity of the resulting fermentation broth distinctly increased with increasing sulphate content, and this is evidence of the increase in the EPS content in the particular resulting fermentation broth.

(9) 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

(10) 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.

(11) 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

(12) 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.

(13) Spray drying was carried out in each case using a Bchi 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

(14) 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.

(15) 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%

(16) The determination of the sulphur content confirms that, with increasing sulphate content in the fermentation medium, more and more sulphate was also incorporated into the obtainable biomass, and this is further evidence of the greatly increased formation of EPS.

Example 4

Determining the Caking Tendency

(17) 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 cell stability of the cells in the biomasses concerned that is increased as a result of increased EPS formation.

Example 5

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

(18) 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

(19) 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

(20) Labyrinthulea biomasses and also fish oil as a currently still customary source of omega-3 fatty acids were tested for comparison.

(21) The feedstuff mixtures were each produced by mixing of the componentswith the exception of the oilsusing a double-helix mixer (model 500L, TGC Extrusion, France). The mixtures thus obtained were then comminuted to particle sizes below 250 m using a hammer mill (model SH1, Hosokawa-Alpine, Germany).

(22) 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

(23) 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.

(24) 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.

(25) TABLE-US-00003 TABLE 3 Energy inputs relating to producing pellets having the desired oil load capacity Barrel Barrel Amount 1 2 Feeder Rotational of Temp Temp 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

(26) In this connection, the variable SME is the specific mechanical energy. This is calculated as follows:

(27) $\mathrm{SME}\left(\mathrm{Wh}\text{/}\mathrm{kg}\right)=\frac{UI\cos \frac{\mathrm{Test}\mathrm{SS}}{\mathrm{Max}\mathrm{SS}}}{\mathrm{Qs}}$

(28) where

(29) U: operating voltage of the motor (here 460 V)

(30) I: current of the motor (A)

(31) cos : theoretical performance of the extruder motor (here 0.95)

(32) Test SS: test speed (rpm) of the rotating screws

(33) Max SS: maximum speed (267 rpm) of the rotating screws

(34) Q.sub.s: inlet flow rate of the mash (kg/h)

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

(36) 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

(37) 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 percent of sample remaining in the filter screen. The test was carried out with three samples and then the mean was determined.

(38) 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 percent of the dry weight of the sample used before the incubation with water.

(39) The results are shown in Table 4 below.

(40) 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

(41) It can be seen that a feedstuff according to the invention which contains a biomass according to the invention having a high EPS content 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.