A Method for the Fractionation of a Lipid Fraction and a Protein Fraction from a Lipid and Protein Containing Biomass

Abstract

The present invention relates to a method for simultaneously recovering in aqueous medium, from a lipid and protein containing biomass, of a lipid fraction and a protein fraction, wherein the method comprises an extraction step to extract the lipid and protein fraction from the biomass, characterised in that the refining step consists of contacting the biomass with a solution of at least one organic acid in water having a pH of between 1 and 6, wherein the concentration of the organic acid is at least 0.1 M, and in that the method further comprises subjecting the thus treated biomass to a phase separation and recovering a first lipid phase containing at least 65% with respect to the total weight of the lipids contained in the biomass and a second phase containing one or more precipitated proteins.

Claims

1. A method for simultaneously recovering in aqueous medium a lipid fraction and a protein fraction from a lipid and protein containing biomass comprising contacting the biomass with a solution of at least one organic acid in water at a concentration of at least 0.25 mol/l and having a pH of between 1 and 6, wherein the ratio of the weight of the aqueous organic acid solution to the weight of the biomass is between 3 to 15 to create treated biomass, and in that the method further comprises subjecting the thus treated biomass to a phase separation and recovering from that phase separation a first lipid phase containing at least 65% with respect to the total weight of the lipids contained in the biomass and a second protein phase containing dissolved proteins in the supernatant, one or more precipitated proteins and/or insoluble material.

2. The A method according to claim 1, wherein the biomass is contacted with the solution of at least one organic acid in water at a temperature between 10 and 40 C.

3. The method according to claim 1, wherein the at least one organic acid is a weak mono- or polycarboxylic acid with a pKa of between 1.0 and 6.0.

4. The A method according to claim 1 wherein the pH is between 3.0 and 6.0.

5. (canceled)

6. The method according to claim 1, wherein the ratio of the weight of the aqueous organic acid solution to the weight of the biomass is maximum 15.

7. The method according to claim 1, wherein the second phase of precipitated proteins comprises at least 25% of the proteins with respect to the total weight of the proteins contained in the biomass.

8. The method according to claim 1, wherein the lipid fraction has a purity in lipids of at least 70.0% based on the weight of the lipid fraction.

9. The method according to claim 1, wherein the precipitated protein fraction of the second phase has a purity in functional proteins of at least 25.0% based on the weight of the precipitated fraction.

10. The method according to claim 1, wherein the at least one organic acid is a monocarboxylic organic acid, a polycarboxylic organic acid, or an alfa hydroxyl acid, or a mixture of two or more of such organic acids.

11. A method according to claim 1, wherein the organic acids are of the formula
RCOOH wherein R is an aromatic moiety or a straight chain or branched alkane moiety, which may be saturated or which may contain one or more unsaturated bonds, wherein R may contain one or more substituents.

12. The method according to claim 1, wherein the organic acid is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, pentanoic acid, hexanoic acid, lactic acid, mandelic acid, glycolic acid, malic acid, citric acid, ascorbic acid, succinic acid, azelaic acid, barbituric acid, benzilic acid, cinnamic acid, glutaric acid, gluconic acid, malic acid, folic acid, propiolic acid, tannic acid, uric acid, gallic acid, acetylsalicylic acid, a C.sub.8-C.sub.12 fatty acid, benzoic acid, adipic acid, and trimellitic acid or a mixture of two or more of the afore mentioned acids.

13. The method according to claim 10, wherein the at least one organic acid is alpha hydroxyl acid selected from the group consisting of glycolic acid, lactic acid, citric acid, and mandelic acid or a mixture of two or more of these acids.

14. The method according to claim 1, wherein the at least one organic acid is lactic acid at a concentration of at least 0.25 mole/l.

15. The method according to claim 1, wherein the second phase contains proteins in a precipitated state, and a liquid phase containing one or more proteins in a dissolved state, and wherein the proteins dissolved in the second liquid phase are subjected to a further treatment to cause precipitation thereof.

16. The method according to claim 1, wherein in advance of being contacted with the organic acid, the biomass is subjected to a mechanic disruption treatment causing cell disruption of the biomass.

17. The method according to claim 16, wherein the mechanically treated biomass is separated into a fluid mass to be treated and a solid residue.

18. The method according to claim 1, further comprising the step of recovering the first lipid phase, a layer comprising supernatant with proteins dissolved in the aqueous solution of the organic acid, and a lower layer comprising precipitated proteins.

19. The method according to claim 1, further comprising the step of subjecting the mixture of the aqueous organic acid solution and biomass to mechanical separation and recovering the first lipid phase, a second layer of supernatant comprising proteins dissolved in the aqueous solution of the organic acid, a layer of precipitated proteins and/or a layer of insoluble material.

20. The method according to claim 16, additionally comprising the step of recovering a layer comprising solid biomass exoskeleton.

21. The method according to claim 1, wherein the lipid fraction contains at least 70% of the total amount of lipids present in the biomass on weight basis with respect to the weight of the lipid fraction.

22. The method according to claim 1, wherein the protein fraction contains at least 25% of the total amount of proteins present in the biomass on weight basis with respect to the weight of the protein fraction.

23. The method according to claim 1, wherein the biomass is of animal, vegetal, bacterial or fungal origin, or a mixture of two or more of the afore mentioned biomasses.

24. The method according to claim 23, wherein the biomass is of animal origin and is selected from the group consisting of insects, fish, poultry parts, and mammalian animal parts.

25. The method according to claim 23, wherein the biomass is a vegetal biomass selected from the group consisting of algae, soybeans, peas, chickpeas, beans, sprouts, mushrooms, various seeds such as sesame, line seed, chia seed, sunflower seed, cereals, bananas, and avocado, or a fungal biomass selected from the group consisting of fungi and yeasts, or a bacterial biomass.

26. The method according to claim 1 wherein the biomass is living biomass, dead biomass or a mixture hereof.

27. The method according to claim 1, wherein the biomass is fresh biomass, partly dehydrated biomass, fully dehydrated biomass, or is a biomass which has been subjected to re-hydration in advance of being contacted with the solution of the at least one organic acid in water.

28. The method according to claim 27, wherein the partly or fully dehydrated biomass is produced by freeze drying or a drying method at a temperature of maximum 60 C.

29. The method according to claim 27, wherein in advance of being contacted with the organic acid, the biomass is wetted with water or a solution of at least one organic acid in water.

30. A food or a feed composition containing a protein fraction, a lipid fraction or a combination thereof, obtained by the method of claim 1.

31. An adhesive composition comprising a protein fraction, a lipid fraction or a combination thereof, obtained by the method of claim 1.

32. The composition according to claim 30, wherein at least 20 wt. % of the proteins with respect to the total weight of the proteins are soluble in an aqueous solution having a pH of between about 4 and 6.

33. The composition according to claim 32, wherein at least 25 wt. % of the proteins with respect to the total weight of the proteins are soluble in an aqueous solution having a pH of 7.

Description

[0035] The present invention is further elucidated in the examples below.

[0036] FIG. 1 shows the mass fractions of an upper layer and a first and second pellet obtained when subjecting fresh crushed larvae of example 1 either to rendering or to the treatment of the present invention.

[0037] FIG. 2 shows the solubility of the protein fraction contained in the protein pellet 1 recovered from the larvae juice after lactate treatment and rendering as a function of the pH of the solution.

[0038] FIG. 3 gives an overview of the mass distribution of the different fractions with distribution of biomolecules (proteins and fats) when treating insect juice with different organic acids according to the invention and HCl.

[0039] FIG. 4 shows a scheme according to which biomass or more specific insect larvae or insects in general may be treated according to the present invention, in particular treatment of the larvae juice only (option 1), treatment of a mixture of the pellet containing the exoskeleton of the larvae and the larvae juice (option 2), and separate treatment of the larvae juice and the pellet containing the exoskeleton of the larvae (option 3).

[0040] FIG. 5 shows the dry weight percentages of fractions after centrifugation of several lactate tests on defrosted, crushed wet insect larvae. In the different tests, the temperature and solid:liquid ratio was varied as described in example 2.

EXAMPLE 1

[0041] Fresh larvae were mechanically pretreated to a larvae juice and a chitin rich fraction.

[0042] The larvae juice was mixed in a recipient with an aqueous solution of lactic acid 0.5 M and a pH of 1.8. The liquid to dry solid ratio was 13.5 on weight basis. The mixture was shaken at room temperature (21 C.) for 30 minutes on a horizontal shaker, at a rotation speed of 200 rpm. Thereafter phase separation was performed by subjecting the mixture to centrifugation (e.g. 4000 g during 30 minutes) or to settling. As a result of the phase separation three layers were formed: [0043] 1. An upper layer which mostly contained lipids, [0044] 2. followed by a layer of supernatant containing amongst others dissolved proteins. [0045] 3. The layer most below contained precipitated proteins, and will be referred to as pellet 1.

[0046] The results of the pellets and upper layer given below were determined gravimetrically and based on dry weight of the upper layer and the dried proteins.

[0047] The upper layer was recovered by pipetting and was dried at 105 C. for 48 h. The lipid extraction yield was determined gravimetrically.

[0048] The protein pellet (pellet 1) was freeze-dried until stable weight and the extraction yield was determined gravimetrically.

[0049] The dissolved proteins in solution in the second layer were recovered by pH precipitation with NaOH 1 M at pH 4.5 at 4 C. and centrifugation at 4000 g during 15 minutes. The precipitated proteins (pellet 2) were freeze-dried until stable weight and the extraction yield was determined gravimetrically.

[0050] The protein and lipid composition of the different fractions is shown in Table 1. As can be seen from table 1, the upper layer counted for 40 wt. % of the insect juice dry weight. The pellet 1 fraction was 22 wt. % of the insect juice dry weight, whereas the pellet 2 fraction was 12 wt. %. The upper layer contained 92 wt. % lipids with respect to the weight of the upper layer. The pellet fractions contained 32 to 38 wt. % lipids with respect to the weight of the pellet fractions.

[0051] The proteins contained in the pellet 1 fraction showed the lowest solubility (22-23% with respect to the total protein weight of pellet 1) at pH 4-6 and the highest solubility of 63% (with respect to the total protein weight of pellet 1) on weight basis at a pH of 10.

EXAMPLE 2

[0052] Defrosted, crushed, wet larvae referred to as Option 2 in 4 were treated with 0.5 M lactate during 1 hour at room temperature, at a liquid to dry solid ratio of respectively 3 and 11. After centrifuging for 15 minutes, 4 layers were formed, as follows: [0053] 1. An upper layer (1), which mostly contained lipids, [0054] 2. a second layer of supernatant (2) with dissolved proteins. These dissolved proteins were not precipitated in an after-treatment by changing the pH as described in FIG. 4 to obtain pellet 2. The pellet 2 proteins are therefore a part of the supernatant proteins in these tests. [0055] 3. The layer (3) underneath the second layer contained precipitated proteins (cfr. pellet 1), [0056] 4. and the bottom layer (4) contained the exoskeleton pellet.

[0057] The weight of the above-mentioned pellets and upper layer were gravimetrically and based on dry weight without biochemical analysis of the fractions. The mass of supernatant proteins was determined via total N analysis of the supernatant. The lipid fraction and both pellets were washed before drying and weighing to remove any remaining lactate and other water soluble compounds. The sum of all fractions represents the mass balance, which is below 100% due to the washing procedure and because the amount of carbohydrates and minerals present in the fractions was not measured.

[0058] This example aimed at reducing the amount of liquid, increasing the mass and purity of the upper lipid containing layer and decrease the mass of the exoskeleton pellet. This experiment also aimed at increasing the mass of pellet 1 which contained the precipitated proteins.

[0059] The results show that room temperature is a temperature which is sufficiently high to permit isolating the lipids, cleaning the exoskeleton pellet and achieving precipitation of proteins. From the results given in FIG. 5 it can be observed that by increasing the amount of solvent added and thus by increasing the liquid:solid ratio from 3 to 11, the separation of the afore-mentioned compounds may be improved.

[0060] When increasing the temperature to above room temperature, in this example 45 C., a dirty pellet was obtained, probably due to denaturation of proteins that clog onto the exoskeletons.

[0061] As can be seen from FIG. 5, optimal results in terms of the amount of lipid and proteins that could be isolated, was obtained at room temperature and a liquid over solid ratio of 11.

Comparative Experiment A

[0062] The set-up of example 1 was used, with the difference that larvae juice was mixed with water only, without addition of the organic acid, and shaken at 200 rpm at 100 C. during 18 hours to simulate rendering on laboratory scale. The results are shown in FIG. 1.

[0063] The protein and lipid composition of the different fractions is shown in Table 1. The results of table 1 are visualized in FIG. 1.

TABLE-US-00001 TABLE 1 Protein and lipid composition of upper layer and pellet fractions by the lactate treatment process, compared to rendering. Upper Protein Protein Mass layer fraction 1 fraction 2 balance Example 1 Mass fraction (wt. %) 40 22 12 74 Protein content (wt. ) 8 29 46 Lipid content (wt. %) 92 32 38 Comparative experiment A Mass fraction (wt. %) 10 57 1 68 Protein content (wt. %) <d.I. 33 40 Lipid content (wt. %) 96 46 39

[0064] The upper layer, obtained by rendering, was 10% of the insect juice dry weight, compared to an upper layer consisting of 40 wt. % when subjecting the biomass to the treatment with lactic acid. Hydrolysis of the lipid phase into fatty acids is probably causing the reduced yield of the upper lipid layer. The pellet 1 fraction, obtained by rendering was 57 wt. % with respect to the weight of the biomass. The pellet 2 fraction obtained using rendering was 1 wt. % with respect to the weight of the biomass. The upper layer contained 96% lipids. The pellet fractions contained 39 to 46 wt. % of lipids with respect to the weight of the respective pellet fractions.

[0065] The weight of pellet fraction 2 was low when compared to Example 1, indicating a low content of water-soluble proteins in pellet fraction 2, probably due to denaturation of proteins in the course of the temperature treatment at 100 C. This hypothesis was further explored by measurement of the water solubility of the different protein fractions as is shown in FIG. 2.

[0066] The solubility of the pellet fraction 1 was 10-11 wt. % with respect to the weight of pellet fraction 1 in a pH range between 2 and 10.

[0067] From the comparison of Comparative experiment A and Example 1, it can be concluded that extraction of proteins using rendering gave rise to a pellet fraction 1 with lower water solubility when compared to example 1, indicating denaturing of the proteins with loss of functionality in comparative experiment A.

Comparative Experiment B

[0068] Similar to example 2, defrosted, crushed, wet larvae referred to as Option 2 in Figure were treated with HCl at pH 2 during 1 hour. The pH was the same as in example 2. A liquid:solid ratio was used of respectively 3, 6 and 11 and the experiment was carried out at 45 C. A liquid:solid ratio was used of respectively 3 and 11 and the experiment was carried out at room temperature. In none of these experiments a lipid layer was formed, thus extraction of lipids from the biomass with HCl could not be achieved. The same experiment was repeated with insect juice instead of whole crushed larvae and described in the next paragraph (HCl at pH2) since a small upper lipid layer was formed with only the insect juice as resource, thus without the chitin fraction.

EXAMPLE 3-6: EFFECT OF THE NATURE OF THE ORGANIC ACID

[0069] An insect juice was prepared as described in Example 1 and subjected to fractionation by treatment of the juice at room temperature with lactic acid (example 3), acetic acid (example 4), citric acid (example 5). Additionally, an experiment was conducted with lactic acid where the pH was also kept at 2 by addition of HCl (example 6). The results are shown in FIG. 3 and table 2.

TABLE-US-00002 TABLE 2 Example 3 Example 4 Example 5 Comparitive ex. B Example 6 lactic acid acetic acid citric acid water/HCl pH 2 lactic acid pH 2 total weight of dried isolated fraction (g) (sum of lipids, proteins and organic acid per fraction for 100 g of dry resource) upper layer 45.69 40.70 63.26 26.48 59.11 pellet 1 30.39 43.27 29.71 59.61 20.82 pellet 2 6.13 2.22 12.50 1.40 16.97 supernatant 50.54 44.20 95.12 23.33 58.31 mass balances per component (%) or (g/100 g of resource) sum all lipids 61.17 63.38 60.51 60.79 69.55 sum all proteins 29.85 29.98 31.89 30.70 31.93 sum all others 42.31 38.38 108.19 20.21 53.78 sum all 133.33 130.74 200.58 111.70 155.26 sum proteins and lipids 91.02 92.36 92.40 91.49 101.48 mass balances per isolated fraction (%) (sum of % lipids and proteins per fraction) upper layer 101.27 100.86 93.77 103.31 100.09 pellet 1 90.04 93.71 60.06 92.97 88.71 pellet 2 85.51 24.86 49.10 34.18 91.98 extracton yield (%) (isolated weight of lipid or protein versus weight of lipid or protein in resource) upper layer-lipid yield 66.95 53.95 77.49 35.20 68.44 upper layer-protein yield 17.83 23.64 38.98 19.41 36.23 pellet 1 lipid yield 29.12 46.05 18.97 64.80 19.13 pellet 1 protein yield 32.00 39.21 19.94 64.80 16.18 pellet 2 protein yield 9.49 1.90 12.52 1.56 21.79 pellet 2 lipid yield 3.60 / 2.77 / 12.64 purity of isolated fractions (%) (weight of lipid or protein versus weight of fraction) upper layer-lipids 89.63 84.02 74.12 80.81 80.52 upper layer-proteins 11.65 16.83 19.65 22.51 19.57 pellet 1 lipids 58.61 67.45 38.64 66.07 63.90 pellet 1 proteins 31.43 26.26 21.41 26.89 24.81 pellet 2 proteins 46.23 24.86 31.96 34.18 41.00 pellet 2 lipids 39.25 / 17.14 / 50.98

[0070] The purity of the components contained in each fraction was determined by analysis of proteins and lipids. The rest (other) was calculated by subtraction of proteins and lipids. For example, the upper layer which constituted 59 wt. % of the biomasss was divided over 48% lipids and 12% proteins on weight basis. The sum of all dry weight fractions was higher than 100 due to contribution the dry weight of the organic acids added to induce fractionation. Table 2 also shows the mass balances where other were taken out of the calculations. This also allowed the calculation of products yields and purity of lipids or proteins in the isolated fractions.

[0071] The data shown in FIG. 3 and table 2 indicate that, with all treatments, the weight of the upper layer varied, and the upper layer contained a relatively pure lipid fraction. However, proteins are found in all fractions (upper layer, pellet 1, pellet 2 and supernatant). The nature and amount of protein contained in a certain fraction, depends on water solubility of the proteins, precipitation behavior of the proteins and protein interaction with lipids.

[0072] The overall goal of the treatment is to maximize the yield and purity of lipids in the upper layer and maximize the yield and purity of proteins in pellet 1.

[0073] Additionally, pellet 2 can increase the amount or yield of isolated proteins.

[0074] In table 2 it is seen that the maximum yield of lipids (in wt. % of lipid fraction with respect to the weight of the biomass) in the upper layer was obtained with citric acid (77.49%), followed by lactic acid at pH 2 (68.44%) and lactic acid as such (66.95%). The purity of lipids in the upper layer, however, was the highest with lactic acid as such (89.63%) while citric acid showed a significant lower purity (74.12%). The purity of the upper layer from the lactic acid treatment at pH 2 was intermediate (80.52%)

[0075] Another discriminator to evaluate the performance of the different acid treatments is the extraction yield and purity of proteins in pellet 1. The highest protein yield (in wt. % with respect to the weight of the biomass) was obtained with HCl at pH2 (52.22 wt. %), however, since the lipid yield was insufficient (35.20 wt. %) and protein denaturation might have caused the high protein yield, this treatment can be dismissed. The second highest protein yield was obtained with acetic acid (39.21%), followed by lactic acid as such (32.00 wt. %). The purity of proteins in the lactic acid pellet 1 on weight basis was higher than with acetic acid (31.43% versus 26.26%). The yield of lipids, however, was lower for acetic acid versus lactic acid (53.95 wt. % versus 66.95 wt. %), concluding that overall the lactic acid treatment is superior. Since citric acid and lactic acid at pH 2 obtained the highest lipid yields, the protein yields and purities are evaluated. Pellet 1 obtained with the citric acid treatment only yielded 19.94 wt. % of proteins with a protein purity of only 21.41%. Pellet 1 of the lactic acid treatment at pH only yielded 16.18 wt. % of proteins with a protein purity of 24.81%. These values are inferior to the protein yield and purity obtained with the lactic acid treatment (resp. 32.00 wt. % and 31.43%).

[0076] Based on all observations and goals, i.e. maximized yield and purity of proteins and lipids respectively in pellet 1 and the upper layer, the treatment with lactic acid is superior to all other treatments.

EXAMPLE 7. EXTRACTION OF LIPIDS AND PROTEINS FROM MINCED MEAT, BY TREATMENT WITH LACTIC ACID

[0077] Minced meat, as alternative resource, was subjected to fractionation by treatment with lactic acid according to the method described in example 1. The results of the treatment of minced meat with lactic acid are shown in table 3. The yield of the upper layer lipids was low compared to example 1 while the purity of lipids was higher. Apparently, separation of lipids was less effective with this resource since many lipids are present in both pellets. Additionally, the yield and purity of proteins in pellet 1 were higher with minced meat compared to example 1. This examples shows that the treatment must be fine-tuned according to the resource to be fractionated.

TABLE-US-00003 TABLE 3 Treatment of minced meat with lactic acid lactic acid total weight of dried isolated fraction (g) (sum of lipids, proteins and organic acid per fraction for 100 g of dry resource) upper layer 22.25 pellet 1 46.72 pellet 2 32.72 supernatant 27.83 mass balances per component (%) or (g/100 g of resource) sum all lipids 57.33 sum all proteins 38.09 sum all others 34.09 sum all 129.52 sum proteins and lipids 95.43 mass balances per isolated fraction (%) (sum of % lipids and proteins per fraction) upper layer 98.15 pellet 1 86.84 pellet 2 91.62 extracton yield (%) (isolated weight of lipid or protein versus weight of lipid or protein in resource) upper layer-lipid yield 36.38 upper layer-protein yield 2.57 pellet 1 lipid yield 33.83 pellet 1 protein yield 55.58 pellet 2 protein yield 33.86 pellet 2 lipid yield 29.79 purity of isolated fractions (%) (weight of lipid or protein versus weight of fraction) upper layer-lipids 93.75 upper layer-proteins 4.40 pellet 1 lipids 41.51 pellet 1 proteins 45.32 pellet 2 proteins 39.42 pellet 2 lipids 52.20

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