METHOD FOR PRODUCING BIOMASS COMPRISING PROTEIN AND OMEGA-3 FATTY ACIDS FROM SINGLE MICROALGAE, AND BIOMASS PRODUCED THEREBY

Abstract

The present application relates to a method for producing biomass comprising protein and omega-3 fatty acids from single microalgae, and biomass produced thereby, the method for producing biomass according to one embodiment supplying the cultivation stage continuously with a nitrogen source to allow producing single microalgae-derived biomass having high protein and omega-3 fatty acid content, and as such, biomass produced thereby can be effectively used as the single microorganism source of protein and omega-3 fatty acids.

Claims

1. A method of preparing a biomass derived from Thraustochytrid microalgae, wherein the method comprising: culturing, a single strain of the Thraustochytrid microalgae in a medium; and continuously supplying a nitrogen source into the medium during the culturing.

2. The method according to claim 1, wherein the nitrogen source is supplied from immediately after inoculating the microalgae into the medium to the end of the culture.

3. The method according to claim 1, wherein the nitrogen source is continuously supplied such that the total nitrogen concentration in the culture medium is 300 ppm or more.

4. The method according to claim 1, wherein the medium comprises a carbon source and a nitrogen source.

5. The method according to claim 1, wherein the nitrogen source is i) any one or more organic nitrogen sources selected from the group consisting of a yeast extract, a beef extract, peptone, and tryptone, or ii) any one or more inorganic nitrogen sources selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea, monosodium glutamate (MSG), and ammonia.

6. The method according to claim 4, wherein the carbon source is any one or more selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, and glycerol.

7. The method according to claim 1, wherein the Thraustochytrid microalgae is microalgae of Thraustochytrium sp., Schizochytrium sp., Aurantiochytrium sp., or Thraustochytriidae sp.

8. The method according to claim 1, further comprising recovering the biomass from the strain, a cultured product of the strain, a dried product of the cultured product, or a pulverized product of the dried product.

9. The method according to claim 1, wherein the biomass comprises 50 wt % or more of proteins and 37 wt % or less of fats, based on the total weight of the biomass.

10. The method according to claim 1, wherein the biomass comprises 50 wt % or more of proteins, and omega-3 fatty acids, based on the total weight of the biomass.

11. The method according to claim 10, wherein the omega-3 fatty acid comprises any one or more of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

12. The method according to claim 10, wherein the biomass comprises 15 wt % to 60 wt % of omega-3 fatty acids, based on the weight of the total fatty acids.

13. A biomass derived from single Thraustochytrid microalgae, which is prepared by a method of preparing the biomass, wherein the method comprising: culturing a single strain of the Thraustochytrid microalgae in a medium; and continuously supplying a nitrogen source into the medium during the culturing.

14. The biomass according to claim 13, wherein the biomass comprises 50 wt % or more of proteins and 37 wt % or less of fats, based on the total weight of the biomass.

15. The biomass according to claim 13, wherein the biomass comprises 50 wt % or more of proteins and omega-3 fatty acids, based on the total weight of the biomass.

16. The biomass according to claim 15, wherein the omega-3 fatty acid comprises any one or more of docosahexaenoic acid and eicosapentaenoic acid.

17. The biomass according to claim 15, wherein the biomass comprises 15 wt % to 60 wt % of omega-3 fatty acids, based on the weight of the total fatty acids.

Description

MODE FOR INVENTION

[0054] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating one or more specific embodiments, and the scope of the present disclosure is limited by these exemplary embodiments.

Example 1. Examination of Production Amounts of Fats and Proteins According to Method of Culturing Microalgae of Thraustochytriidae sp.

[0055] 1-1. Preparation and Seed Culture of Microalgae

[0056] The microalgae of the Thraustochytriidae sp. CD01-6003 (Accession No: KCTC14346BP) was seeded in a sterile MJW01 medium (30 g/L of glucose, 3.0 g/L of MgSO.sub.4.Math.7H.sub.2O, 10 g/L of Na.sub.2SO.sub.4, 1.0 g/L of NaCl, 9.0 g/L of yeast extract, 1.0 g/L of MSG.Math.1H.sub.2O, 1.0 g/L of NaNO.sub.3, 0.1 g/L of KH.sub.2PO.sub.4, 0.5 g/L of K.sub.2HPO.sub.4, 0.5 g/L of CaCl.sub.2), and 10 mL/L of a vitamin mixed solution), and cultured in a 250 mL flask under conditions of 20° C. to 35° C. and 100 rpm to 300 rpm for 10 hours to 30 hours.

[0057] 1-2. Main Culture

1-2-1. Comparative Example (1)

[0058] The seed culture prepared in Example 1-1 was dispensed in a 5 L fermentor containing a sterile MJW01 medium supplemented with 6 g/L of ammonium sulfate ((NH.sub.4).sub.2.Math.SO.sub.4) and 5 g/L of yeast extract as nitrogen sources, and cultured under conditions of 20° C. to 35° C., 100 rpm to 500 rpm, and 0.5 vvm to 1.5 vvm for a total of 101 hours until the volume of the culture in the fermentor reached 2.8 L. Immediately after starting the culturing with additional nitrogen sources, ammonia gas was continuously supplied to the fermentor for 14 hours such that the total nitrogen concentration in the culture medium was 300 ppm or more, and then, the supply of ammonia gas was stopped. During the entire culturing process, a feed containing a carbon source was continuously supplied such that the concentration of the carbon source in the culture medium was maintained below 5%.

1-2-2. Example (1)

[0059] Culturing was performed in the same manner as in 1-2-1 for a total of 86 hours, except that, during the entire culturing process, ammonia gas was continuously supplied to the fermentor such that the total nitrogen concentration in the culture medium was 300 ppm or more.

1-2-3. Example (2)

[0060] Culturing was performed in the same manner as in 1-2-1 for a total of 75 hours, except that, during the entire culturing process, ammonia gas was continuously supplied to the fermentor such that the total nitrogen concentration in the culture medium was 300 ppm or more, and only 18 g/L of ammonium sulfate as the nitrogen source was added to the sterile MJW01 medium.

1-2-4. Example (3)

[0061] Culturing was performed in the same manner as in 1-2-1 for a total of 55 hours, except that, during the entire culturing process, ammonia gas was continuously supplied to the fermentor such that the total nitrogen concentration in the culture medium was 300 ppm or more, and 10 g/L of ammonium sulfate as the nitrogen source was further added to the feed containing the carbon source.

1-2-5. Example (4)

[0062] Culturing was performed in the same manner as in 1-2-1 for a total of 92 hours, except that, during the entire culturing process, ammonia gas was continuously supplied to the fermentor such that the total nitrogen concentration in the culture medium was 300 ppm or more, only 6 g/L of ammonium sulfate as the nitrogen source was added to the sterile MJW01 medium, and 12 g/L of ammonium sulfate as the nitrogen source was further added to the feed containing the carbon source.

[0063] 1-3. Analysis of Fat Content

[0064] Each of the microalgae cultures cultured in 1-2-1 to 1-2-5 was collected and centrifuged. Then, the obtained cells were washed with PBS three times, and dried at 60° C. for 16 hours. A hydrolysis reaction was allowed at 80° C. by adding an 8.3 M hydrochloric acid solution to 2 g of dried cells. Thereafter, 30 mL of ethyl ether and 20 mL of petroleum ether were added to the reaction product, and mixing for 30 seconds and centrifugation were repeated three times or more. A separated solvent layer was transferred to a pre-weighed round flask, and then dried in a container, from which the solvent and residual moisture were removed, through nitrogen (N2) purging. The total oil content was calculated by measuring the weight of oil which remained after drying the solvent. The content of omega-3 fatty acids (DHA and EPA) in the oil was measured by gas chromatography after being pretreated with 0.5 N methanolic NaOH and 14% trifluoroborane methanol (BF.sub.3-MeOH).

TABLE-US-00001 TABLE 1 Omega- Total fatty EPA/Total DHA/Total 3/Total Omega- acid/Biomass fatty fatty fatty EPA/Biomass DHA/Biomass 3/biomass Conditions (%) acid(%) acid(%) acid(%) (%) (%) (%) Comparative 43.42 0.49 10.06 10.55 0.21 4.37 4.58 Example(1) Example(1) 28.90 2.96 24.39 27.35 0.86 7.05 7.90 Example(2) 14.97 2.54 26.58 29.12 0.38 3.98 4.36 Example(3) 26.13 0.86 16.75 17.61 0.22 4.38 4.60 Example(4) 17.91 2.71 36.12 38.83 0.49 6.47 6.95

[0065] As a result, as shown in Table 1, the total fatty acid (TFA) content in the biomass was more reduced in Examples (1) to (4), in which culturing was performed while continuously supplying the nitrogen sources, as compared with Comparative Example(1). However, the EPA, DHA, and omega-3 contents in the total fatty acids in Examples (1) to (4) were equivalent to or higher than those of Comparative Example (1).

[0066] 1-4. Analysis of Protein Content

[0067] With respect to each 0.5 g to 1 g of the dried cells obtained in the same manner as in 1-3, the nitrogen content in the sample was quantitatively analyzed using an elemental analyzer. The weight ratio (TN %) of nitrogen present in each sample was multiplied by 6.25 and calculated as the total protein content in the sample.

TABLE-US-00002 TABLE 2 Conditions Total protein/Biomass(%) Comparative Example(1) Up to about 43.58 * Example(1) Up to about 58.10 * Example(2) 55.7 Example(3) 58.0 Example(4) 57.5 (* estimate)

[0068] As a result, as shown in Table 2, all of Examples (2) to (4) showed the total protein content of 55% or more, and Example (1) showed the total fatty acid content of 28.9%, as measured in 1-3. Considering that the microalgae biomass generally has the carbohydrate content of about 2% to about 3%, and the ash content of about 8% to about 10%, it may be estimated that the total protein content may reach up to about 58.10%. In addition, Comparative Example (1) showed the total fatty acid content of 43.42%, as measured in 1-3, estimating that the maximum total protein content is merely about 43.58%. These results confirmed that when the microalgae are cultured under the condition of continuously supplying the nitrogen source, proteins may be produced with a high content of 55% or more.

Example 2. Examination of Production Amounts of Fats and Proteins According to Method of Culturing Microalgae of Schizochytrium sp.

[0069] 2-1. Preparation and Seed Culture of Microalgae

[0070] Seed culture of the microalgae of the Schizochytrium sp. CD01-5004 (Accession No: KCTC14345BP) was performed in the same manner as in Example 1-1.

[0071] 2-2. Main Culture

2-2-1. Comparative Example (2)

[0072] The seed culture prepared in Example 2-1 was dispensed in a 5 L fermentor containing a sterile MJW01 medium, and cultured under conditions of 20° C. to 35° C., 100 rpm to 500 rpm, and 0.5 vvm to 1.5 vvm for a total of 105 hours until the volume of the culture in the fermentor reached 2.8 L. Immediately after starting the culturing with additional nitrogen sources, ammonia gas was continuously supplied to the fermentor for 10 hours such that the total nitrogen concentration in the culture medium was 300 ppm or more, and then, the supply of ammonia gas was stopped. During the entire culturing process, a feed containing a carbon source was continuously supplied such that the concentration of the carbon source in the culture medium was maintained below 5%.

2-2-2. Example (5)

[0073] Culturing was performed in the same manner as in 2-2-1 for a total of 84 hours, except that, during the entire culturing process, ammonia gas was continuously supplied to the fermentor such that the total nitrogen concentration in the culture medium was 300 ppm or more, and 6 g/L of ammonium sulfate and 10 g/L of yeast extract as the nitrogen sources were further added to the sterile MJW01 medium.

2-2-3. Example (6)

[0074] Culturing was performed in the same manner as in 2-2-2, except that culturing was performed for a total of 53 hours.

2-2-4. Examples (7) and (8)

[0075] Culturing was performed in the same manner as in 2-2-2, except that culturing was performed for a total of 50 hours.

2-2-5. Example (9)

[0076] Culturing was performed in the same manner as in 2-2-2, except that culturing was performed for a total of 47 hours.

2-2-6. Example (10)

[0077] Culturing was performed in the same manner as in 2-2-2 for a total of 47 hours, except that 5 g/L of MSG as the nitrogen source was further added to the sterile MJW01 medium.

[0078] 2-3. Analysis of Fat Content

[0079] Each of the microalgae cultures cultured in 2-2-1 to 2-2-6 was collected, and the fat contents thereof were analyzed in the same manner as in Example 1-3.

TABLE-US-00003 TABLE 3 Omega- Total fatty EPA/Total DHA/Total 3/Total Omega- acid/Biomass fatty fatty fatty EPA/Biomass DHA/Biomass 3/Biomass Conditions (%) acid(%) acid(%) acid(%) (%) (%) (%) Comparative 41.91 2.53 39.18 41.71 1.06 16.42 17.48 Example(2) Example(5) 24.39 4.25 42.92 47.17 1.04 10.47 11.50 Example(6) 20.12 3.90 37.81 41.70 0.78 7.60 8.39 Example(7) 20.95 3.68 39.54 43.22 0.77 8.28 9.05 Example(8) 19.99 4.50 38.84 43.34 0.90 7.76 8.66 Example(9) 24.90 3.98 39.27 43.25 0.99 9.78 10.77 Example(10) 25.04 5.07 42.02 47.09 1.27 10.52 11.79

[0080] As a result, as shown in Table 3, the total fatty acid (TFA) content in the biomass was more reduced in Examples (5) to (10), in which culturing was performed while continuously supplying the nitrogen sources, as compared with Comparative Example (2). However, the EPA, DHA, and omega-3 contents in the total fatty acids in Examples (5) to (10) were equivalent to or higher than those of Comparative Example (2).

[0081] 2-4. Analysis of Protein Content

[0082] Each of the microalgae cultures cultured in 2-2-1 to 2-2-6 was collected, and the protein contents thereof were analyzed in the same manner as in Example 1-4.

TABLE-US-00004 TABLE 4 Conditions Total protein/Biomass(%) Comparative Example(2) Up to about 45.1* Example(5) 62.4 Example(6) 64.9 Example(7) 65.6 Example(8) 66.3 Example(9) 60.7 Example(10) 58.7 [0083] (*: estimate)

[0084] As a result, as shown in Table 4, all of Examples (5) to (10) showed the total protein content of 58% or more, and Comparative Example (2) showed the total fatty acid content of 41.91%, as measured in 2-3. Considering that the microalgae biomass generally has the carbohydrate content of about 2% to about 3%, and the ash content of about 8% to about 10%, it may be estimated that the total protein content may reach up to about 45.1%. These results confirmed that when the microalgae are cultured under the condition of continuously supplying the nitrogen source, proteins may be produced with a high content of 58% or more.

[0085] 2-5. Analysis of Amino Acid Content and Composition

[0086] The microalgae culture of Example (10) cultured in 2-2-6 was collected, and the dry cells were obtained in the same manner as in 1-3, and 0.5 g to 1 g of the dry cells were subjected to acid hydrolysis. Thereafter, the resultant was subjected to liquid chromatography, and the total amino acid content and the contents of individual amino acids were analyzed. The concentrations of individual amino acids in the sample were normalized to the amount of dry cells used to calculate the contents (%) of individual amino acids relative to the weight of the dry cells, and the total amino acid content (%) relative to the weight of the dry cells was calculated by summing the contents of all detected amino acids.

TABLE-US-00005 TABLE 5 Content Content Content in in in biomass biomass biomass (%) (%) (%) Total 43.09 Alanine 2.50 Tyrosine 1.24 amino acids Aspartic 3.62 Cysteine 0.56 Phenylalanine 1.63 acid Threonine 1.80 Valine 2.06 Lysine 2.27 Serine 2.02 Methionine 0.82 Histidine 0.78 Glutamate 12.47 Isoleucine 1.36 Arginine 6.11 Glycine 0.00 Leucine 2.77 Proline 1.07

TABLE-US-00006 TABLE 6 Content Content Content in total in total in total amino amino amino acids acids acids (%) (%) (%) Total 100.00 Alanine 5.81 Tyrosine 2.88 amino acids Aspartic 8.39 Cysteine 1.30 Phenylalanine 3.79 acid Threonine 4.18 Valine 4.78 Lysine 5.27 Serine 4.69 Methionine 1.90 Histidine 1.82 Glutamate 28.93 Isoleucine 3.15 Arginine 14.19 Glycine 0.00 Leucine 6.43 Proline 2.48

[0087] As a result, as shown in Tables 5 and 6, Example (10), in which the microalgae were cultured under conditions of continuously supplying the nitrogen sources, showed the total protein content of 43.09% in the biomass, and had the highest content of glutamate among the individual amino acids, and its content ratio of glutamate to arginine was 2.04.

[0088] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.