Method for reducing the self-heating tendency of biomass

Abstract

Disclosed herein are LC-PUFA biomass compositions having a reduced tendency to self-heat and thus having reduced packaging requirements for shipping. Also disclosed are methods for producing such biomass compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing dates of U.S. Provisional Patent Application Nos. 62 / 718,549, filed on August 14, 2018, and 62 / 876,076, filed on July 19, 2019, the disclosures of which are hereby incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for reducing the autothermal tendency of biomass containing significant amounts of polyunsaturated fatty acids. Background Art

[0004] Lipids containing polyunsaturated fatty acids (PUFAs) are of great interest in the feed, food, and pharmaceutical industries. Fatty acids are classified based on the length and saturation characteristics of the carbon chain. Based on the number of carbons present in the chain, fatty acids are referred to as short-chain, medium-chain, or long-chain fatty acids. When there are no double bonds between the carbon atoms, fatty acids are referred to as saturated fatty acids. When double bonds are present, fatty acids are referred to as unsaturated fatty acids. When there is only one double bond, unsaturated long-chain fatty acids are monounsaturated. When there are more than one double bond, unsaturated long-chain fatty acids are polyunsaturated.

[0005] PUFAs can be produced by microorganisms during fermentation. The biomass of PUFA-containing microorganisms is collected and then processed to extract the PUFA oils it contains. The PUFA-containing microbial biomass can also be used directly as a product, particularly in the feed industry.

[0006] It has been found that compositions containing PUFAs are susceptible to self-heating. For example, during storage or transportation, the temperature of the biomass in a container or packaging can spontaneously increase, sometimes leading to unexpected explosions and fires.

[0007] In order to ensure the safety of combustible materials (such as self-heating biomass) during transport, appropriate packaging is required. The United Nations (UN) Classification of Self-Heating Substance is a widely accepted standard for classifying combustible materials. See Figure 1. In this classification, several self-heating tests are designed. Based on the test results, packaging requirements will be determined. For example, if biomass material is subjected to dangerous self-heating when it is heated in an oven at 100°C for a period of 24 hours in a 100mm sample cube, then according to the UN standard, it is recommended that such material be packaged in materials of packing group III and be classified as self-heating material and in transport hazard category 4.2. In another example, if a biomass material does not undergo dangerous self-heating when tested in a 25 mm sample cube at 140°C for a period of 24 hours, and it does not undergo dangerous self-heating when tested in a 100 mm sample cube at 120°C for a period of 24 hours, it would not be labeled as a self-heating material if it is transported in a package having a volume not exceeding 3 cubic meters. Different packaging requirements are also used as an alternative method of classifying the self-heating tendency of biomass.

[0008] Several attempts have been made in the past to reduce the self-heating tendency of biomass. For example, WO 2011 / 054800 describes a method in which the moisture content of the biomass is controlled during the drying step to reduce the self-heating tendency of the biomass. WO 2018 / 005856 describes the use of antioxidants to enhance the oxidative stability of algal biomass.

[0009] However, self-heating remains a challenging issue in the transportation and storage of biomass containing large amounts of PUFAs. Therefore, there is a need to identify new methods that can effectively reduce self-heating in biomass. Summary of the Invention

[0010] The present invention now provides a biomass composition having a reduced tendency to self-heat. This composition comprises cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the composition does not undergo dangerous self-heating when tested at 120°C in a 100 mm sample cube. In the self-heating test, the sample cube is suspended in an oven and the oven temperature is maintained at 120°C for 24 hours. If the sample temperature spontaneously rises to 60°C or more above the oven temperature (which is 180°C), the sample is classified as a self-heating material.

[0011] The present invention also provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the composition does not undergo dangerous self-heating when tested at 100°C in a 100 mm sample cube. In this self-heating test, the sample cube is suspended in an oven, and the oven temperature is maintained at 100°C for 24 hours. If the sample temperature spontaneously rises to 60°C or more above the oven temperature (160°C), the sample is classified as a self-heating material.

[0012] The present invention further provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the composition does not undergo dangerous self-heating when tested at 140°C in a 25 mm sample cube, but still undergoes dangerous self-heating when tested at 100°C in a 100 mm sample cube.

[0013] Biomass compositions according to the present invention have the advantage of a reduced tendency to self-heat and, therefore, can be shipped more safely than biomass compositions that have not been treated by the methods disclosed herein. Packaging requirements for biomass compositions treated by the methods disclosed herein can be reduced by one or more levels compared to compositions that have not been treated in this manner. A further advantage of the compositions of the present invention is that, even after treatment, the quality of the PUFAs contained therein does not deteriorate. The methods disclosed herein do not negatively impact the quality of the PUFAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a diagram showing the United Nations classification of self-heating substances.

[0015] Figure 2 shows the temperature profiles of samples D1 and D2 in 25 mm cubes tested at 140° C. In Figures 2-25 , the right-hand graph represents an enlarged view of the peak region shown in the left-hand graph.

[0016] FIG3 shows the temperature profiles of samples D1 and D2 in a 100 mm cube tested at 100° C.

[0017] FIG4 shows the temperature profiles of samples S1-S5 in 25 mm cubes tested at 140°C.

[0018] FIG5 shows the temperature profiles of samples S1 - S5 in a 100 mm cube tested at 120°C.

[0019] FIG6 shows the temperature profiles of samples D2 and D3 in 25 mm cubes tested at 140°C.

[0020] FIG7 shows the temperature profiles of samples D2 and D3 in a 100 mm cube tested at 100° C.

[0021] FIG8 shows the temperature profiles of samples D3 and D4 in 25 mm cubes tested at 140°C.

[0022] FIG9 shows the temperature profiles of samples D3 and D4 in a 100 mm cube tested at 100° C.

[0023] FIG10 shows the temperature profiles of samples D4 and D5 in 25 mm cubes tested at 140°C.

[0024] FIG. 11 shows the temperature profiles of samples D4 and D5 in a 100 mm cube tested at 100° C.

[0025] FIG12 shows the temperature profiles of samples D5 and D6 in 25 mm cubes tested at 140°C.

[0026] FIG13 shows the temperature profiles of samples D5 and D6 in a 100 mm cube tested at 120°C.

[0027] FIG14 shows the temperature profiles of samples D4 and D7 in 25 mm cubes tested at 140°C.

[0028] FIG15 shows the temperature profiles of samples D4 and D7 in a 100 mm cube tested at 100°C.

[0029] FIG. 16 shows the temperature profiles of samples D7 and D8 in 25 mm cubes tested at 140° C.

[0030] FIG. 17 shows the temperature profiles of samples D7 and D8 in a 100 mm cube tested at 120° C.

[0031] FIG. 18 shows the temperature profiles of samples D8 and D9 in 25 mm cubes tested at 140° C.

[0032] FIG. 19 shows the temperature profiles of samples D8 and D9 in a 100 mm cube tested at 120° C.

[0033] FIG. 20 shows the temperature profiles of samples D9 and D10 in 25 mm cubes tested at 140° C.

[0034] FIG. 21 shows the temperature profiles of samples D9 and D10 in a 100 mm cube tested at 120° C.

[0035] FIG. 22 shows the temperature profiles of samples D4 and D11 in 25 mm cubes tested at 140° C.

[0036] FIG. 23 shows the temperature profiles of samples D4 and D11 in a 100 mm cube tested at 100° C.

[0037] FIG. 24 shows the temperature profiles of samples S6-S9 in 25 mm cubes tested at 140°C.

[0038] FIG. 25 shows the temperature profiles of samples S6-S9 in a 100 mm cube tested at 100°C.

[0039] Figure 26 shows the maximum temperatures reached for samples with varying percentages of PUFA at 140°C in 25 mm cubes and at 120°C in 100 mm cubes. DETAILED DESCRIPTION

[0040] It is known that dried PUFA-containing oily biomass undergoes oxidation and can spontaneously heat itself. This self-heating problem is particularly pronounced in microbial cells containing long-chain polyunsaturated fatty acids (LC-PUFA). To identify means of reducing the self-heating tendency of biomass, different conditions such as fermentation length, pasteurization, drying method, addition of inert ingredients, and addition of antioxidants were experimentally examined. Table 1 shows a chart of the samples prepared and tested in this application.

[0041] Table 1

[0042]

[0043] The self-heating material classification test sample is tested using the United Nations 49CFR 173.124-Class 4, Part 4.2 (see Figure 1). In the self-heating test, the sample cube is suspended in an oven and the oven temperature is maintained at a given temperature for 24 hours. If the sample temperature spontaneously rises to 60°C or higher than the ambient internal temperature (set point) of the oven, the sample is considered to be subject to dangerous self-heating. Tests are conducted at temperatures within the range of 100°C-140°C in 25mm and 100mm cubes. A series of tests are conducted to determine the classification of any given self-heating material. For example, if a material does not undergo dangerous self-heating as defined above when tested at 140°C in a 100mm sample cube, the material is considered to be a non-self-heating material in Part 4.2 of the United Nations standard. If a material self-heats under the above conditions, further testing will be performed in a 25mm sample cube at 140°C. If a substance self-heats under new conditions, it will be classified as a dangerous self-heating material according to Section 4.2 of the UN standard and will require packaging materials of Packing Group II for transportation. In the present invention, the corresponding samples are compared and analyzed based on the temperature characteristic curve shown in Figure 1 to determine whether a reduction in oxidation and self-heating characteristics has been achieved.

[0044] It is known that the sensitivity of biomass increases with increasing PUFA content. In particular, PUFAs with 20 or more carbon atoms have a higher sensitivity to self-heating. It is also known that the sensitivity of biomass increases with increasing numbers of double bonds in PUFAs. In particular, PUFAs with 3 or more double bonds have a higher sensitivity to self-heating.

[0045] It has been identified in the present invention that reducing the fermentation length, eliminating the pasteurization step (batch or in-line after fermentation), changing the drying method, adding inert ingredients and adding antioxidants will help reduce the autothermal sensitivity of the biomass.

[0046] By using one or more of the above methods, a biomass composition is produced that has a reduced tendency to self-heat.

[0047] In one embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein when the composition is placed in a 100 mm sample cube and heated in an oven at 120° C. for 24 hours, the composition does not spontaneously self-heat in the test oven, i.e., defined as the temperature of the composition does not rise to 60° C. or more above the oven temperature of 120° C. In one embodiment, the above composition further comprises an effective amount of at least one added antioxidant to provide oxidative stability.

[0048] Based on the United Nations 49CFR 173.124 - Class 4, Part 4.2 classification, compositions falling within the above description can be classified as exempt substances if they are transported in packages with a volume not exceeding 3 cubic meters. See Figure 1.

[0049] In one embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs and is classified as an exempt substance based on the United Nations 49 CFR 173.124 - Class 4, Part 4.2 classification when the composition is shipped in a package having a volume of no more than 3 cubic meters. In one embodiment, the above composition further comprises an effective amount of at least one added antioxidant to provide oxidative stability.

[0050] In another embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein when the composition is placed in a 100 mm sample cube and heated in an oven at 100° C. for 24 hours, the composition does not spontaneously self-heat in the test oven, i.e., defined as the temperature of the composition does not rise to 60° C. or more above the oven temperature of 100° C. In one embodiment, the above composition further comprises an effective amount of at least one added antioxidant to provide oxidative stability.

[0051] Based on the United Nations 49CFR 173.124 - Class 4, Part 4.2 classification, compositions falling within the above description can be classified as exempt substances if they are transported in packages with a volume not exceeding 450 liters. See Figure 1.

[0052] In one embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs and is classified as an exempt substance based on the United Nations 49 CFR 173.124 - Class 4, Part 4.2 classification when the composition is shipped in a package with a volume of no more than 450 liters. In one embodiment, the above composition further comprises an effective amount of at least one added antioxidant to provide oxidative stability.

[0053] In another embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the composition does not spontaneously self-heat in the test oven, i.e., defined as the temperature of the composition does not rise to 60°C or more above the oven temperature of 140°C, when placed in a 25 mm sample cube and heated in an oven at 140°C for 24 hours, but the composition still undergoes hazardous self-heating in the test oven, i.e., defined as the temperature of the composition does not rise to 60°C or more above the oven temperature of 100°C, when placed in a 100 mm sample cube and heated in an oven at 100°C for 24 hours. In one embodiment, the above composition further comprises an effective amount of at least one added antioxidant to provide oxidative stability.

[0054] Compositions that fall within the above description may avoid the labeling requirements of Packing Group II but still need to use Packing Group III packaging materials and be labeled according to the United Nations 49 CFR 173.124 - Class 4, Part 4.2 classification. See Figure 1.

[0055] In one embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs and is not subject to the labeling requirements of Packing Group II based on the United Nations classification of 49 CFR 173.124 - Class 4, Part 4.2.

[0056] In one embodiment, the present invention provides a composition comprising cells containing one or more types of polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs and is only required to have the labeling requirements of Packing Group III based on the United Nations 49 CFR 173.124 - Class 4, Part 4.2 classification.

[0057] The compositions listed above with reduced self-heating tendency can be obtained based on the teachings provided by the present invention as described below.

[0058] It has been found that the tendency of the biomass to self-heat can be reduced by harvesting cells from the fermentation broth earlier than normal to obtain young cells. In one embodiment, the tendency of the biomass to self-heat can be reduced if the fermentation process is terminated before the 5th day of fermentation. In one embodiment, the tendency of the biomass to self-heat can be reduced if the fermentation process is terminated before the 6th day of fermentation. In one embodiment, the tendency of the biomass to self-heat can be reduced if the fermentation process is terminated before the 7th day of fermentation.

[0059] It has also been found that the self-heating tendency of the biomass can be reduced if the biomass is dried by a drum drying process rather than a freeze drying process.

[0060] It has also been found that the self-heating tendency of biomass can be reduced by adding two different types of antioxidants. If a natural antioxidant is used in combination with a synthetic antioxidant, the reduction in the self-heating tendency of biomass is surprisingly good. In one embodiment, the natural antioxidant can be lecithin or Rosen. In another embodiment, the synthetic antioxidant can be one of ethoxyquin, TAP1010, or TBHQ.

[0061] It has also been found that the self-heating tendency of biomass can be reduced by adding an inert ingredient to the dry biomass. It has been found that adding sugar to the fermentation broth after the broth is pasteurized can help reduce the self-heating tendency of biomass. In one embodiment, the inert ingredient can be any composition that does not react with the cellular biomass. In a specific embodiment, the inert ingredient is a sugar. In one embodiment, the sugar can be selected from dextrose, fructose, sucrose and maltose. By finishing the fermentation before the sugar source is completely consumed by the microorganism, such as in the example of the earlier harvest mentioned above, the same effect can be achieved.

[0062] In one embodiment, the self-heating tendency of the biomass can be further reduced if the treatment is performed in a method that combines any two or more of the above methods.

[0063] In a preferred embodiment, the composition according to the invention has an oil content and PUFAs as described below.

[0064] The biomass composition of the present invention has a tendency to self-heat before processing because it contains a reasonable level of polyunsaturated fatty acids. In one embodiment, the composition comprises oil, and the oil accounts for at least 20wt.%, such as at least 25wt.%, such as at least 30wt.%, such as at least 35wt.%, such as at least 40wt.%, such as at least 45wt.%, such as at least 50wt.%, such as at least 55wt.%, such as at least 60wt.%, such as at least 65wt.%, such as at least 70wt.%, such as at least 75wt.%, such as at least 80wt.%, such as at least 90wt.%, such as at least 95wt.%. In another embodiment, the composition comprises oil, and the oil accounts for between 30-70wt.%, such as between 40-60wt.%, between 45-55wt.%, of the weight of the composition. In one embodiment, the weight of the composition is referred to as the dry cell weight of the biomass. Such biomass can be algae cells or any other microbial cells containing PUFAs.

[0065] In embodiments of the present invention, the composition comprises PUFAs, particularly LC-PUFAs. In one embodiment, the composition is biomass. In another embodiment, the composition is dried biomass. In another embodiment, the composition is dried microbial cell biomass. In another embodiment, the composition is dried algal cell biomass.

[0066] In one embodiment, the composition comprises at least 20 wt.%, e.g., at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.%, at least 55 wt.%, at least 60 wt.%, at least 65 wt.%, at least 70 wt.%, at least 75 wt.%, at least 80 wt.%, at least 85 wt.%, at least 90 wt.%, at least 95 wt.%, at least 100 wt.% of PUFAs with at least 3 double bonds relative to the total fatty acids in the oil. In one embodiment, the weight of the composition is referred to as the dry cell weight of the biomass.

[0067] In one embodiment, the composition comprises at least 20 wt.%, e.g., at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.%, at least 55 wt.% of PUFAs having at least 3 double bonds, relative to the weight of the composition. In another embodiment, the composition comprises between 20-55 wt.%, between 20-40 wt.%, between 20-30 wt.%, or between 20-25 wt.% of PUFAs having at least 3 double bonds, relative to the weight of the composition. In one embodiment, the weight of the composition is referred to as the dry cell weight of the biomass.

[0068] In one embodiment, the invention is directed to a method for reducing the self-heating tendency of a composition comprising cells comprising one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises limiting the length of the fermentation process to less than 6 days.

[0069] In one embodiment, the invention relates to a method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises removing the pasteurization step after fermentation.

[0070] In one embodiment, the present invention is directed to a method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises a drum drying step instead of a freeze drying step.

[0071] In one embodiment, the invention relates to a method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises adding at least one type of natural antioxidant and at least one type of synthetic antioxidant to the fermentation broth at the end of fermentation.

[0072] In one embodiment, the natural antioxidant is lecithin or Roseen, and wherein the synthetic antioxidant is ethoxyquin, TAP1010, or TBHQ.

[0073] In one embodiment, the invention is directed to a method for reducing the autothermal tendency of a composition comprising cells comprising one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises causing the fermentation broth to comprise at least 50 g / L of sugars at the end of the fermentation.

[0074] In one embodiment, the sugar is one or more types selected from the group consisting of glucose, fructose, sucrose, and maltose.

[0075] In one embodiment, the composition recited in the above method comprises at least 20 wt.%, e.g., at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.%, at least 55 wt.% of PUFAs having at least 3 double bonds, relative to the weight of the composition. In another embodiment, the composition comprises between 20-55 wt.%, between 20-40 wt.%, between 20-30 wt.%, or between 20-25 wt.% of PUFAs having at least 3 double bonds, relative to the weight of the composition. In one embodiment, the weight of the composition is referred to as the dry cell weight of the biomass.

[0076] In one embodiment, the weight of the composition is referred to as the dry cell weight of the biomass.Such biomass may be algae cells or any other microbial cells containing PUFAs.

[0077] In embodiments of the present invention, the compositions recited in the above methods comprise PUFAs, particularly LC-PUFAs. In one embodiment, the composition is biomass. In another embodiment, the composition is dried biomass. In another embodiment, the composition is dried microbial cell biomass. In another embodiment, the composition is dried algal cell biomass.

[0078] In one embodiment, the composition is biomass. In another embodiment, the biomass is microbial cells. The microbial cells can be of the genus Mortierella, Schizochytrium, or Crypthecodinium.

[0079] In one embodiment, the PUFAs are one or more types of long-chain PUFAs. In another embodiment, the PUFAs are ω-3 or ω-6 PUFAs. In another embodiment, the PUFAs are one or more PUFAs selected from the group consisting of dihomo-gamma-linolenic acid (DGLA, 20:3ω-6), arachidonic acid (ARA, 20:4ω-6), eicosapentaenoic acid (EPA, 20:5ω-3), docosahexaenoic acid (DHA, 22:6ω-3), and docosapentaenoic acid (DPA 22:5ω-3 or DPA 22:5ω-6).

[0080] The LC-PUFAs described herein are fatty acids that contain at least 3 double bonds and have a chain length of 20 or more carbons. Polyunsaturated fatty acids (PUFAs) are classified based on the position of the first double bond from the methyl end of the fatty acid; ω-3 (n-3) fatty acids contain the first double bond at the third carbon, while ω-6 (n-6) fatty acids contain the first double bond at the sixth carbon. For example, docosahexaenoic acid (DHA) is an ω-3 long-chain polyunsaturated fatty acid (LC-PUFA) with a chain length of 22 carbons and 6 double bonds, commonly designated "22:6n-3". In one embodiment, the PUFA is selected from ω-3 fatty acids, ω-6 fatty acids, and mixtures thereof. In another embodiment, the PUFA is selected from LC-PUFA. In yet further embodiments, the PUFA is selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (ARA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), stearidonic acid (SDA), and mixtures thereof. In another embodiment, the PUFA is selected from the group consisting of DHA, ARA, and mixtures thereof. In further embodiments, the PUFA is DHA. In yet further embodiments, the PUFA is ARA.

[0081] As used herein, "cell" refers to a biological material containing oil, such as a biological material derived from an oily microorganism. The oil produced by a microorganism or obtained from a microbial cell is referred to as a "microbial oil." In one embodiment, microbial oil refers to a crude oil extracted from the biomass of a microorganism without further processing. The oil produced by algae and / or fungi is also referred to as algae oil and / or fungal oil, respectively.

[0082] As used herein, "microorganism" refers to organisms such as algae, bacteria, fungi, yeast, protists, and combinations thereof, for example, single-cell organisms. In some embodiments, the microbial cell is a eukaryotic cell. Microbial cells include, but are not limited to, golden algae (e.g., microorganisms of the order Stramenopiles); green algae; diatoms; flagellates (e.g., microorganisms of the order Dinophyceae, including members of the genus Crypthecodinium, such as, for example, Crypthecodinium cohnii or C. cohnii); microalgae of the order Thraustochytriales; yeasts (Ascomycetes or Basidiomycetes); and fungi of the genera Mucor, Mortierella (including, but not limited to, Mortierella alpina and Mortierella sect, schmuckeri), and Pythium (including, but not limited to, Pythium insidiosum).

[0083] In one embodiment, the microorganism is from the genus Mortierella, Crypthecodinium, or Thraustochytriales. In yet further embodiments, the microbial cell is from Crypthecodinium cohnii. In yet even further embodiments, the microbial cell is selected from Crypthecodinium cohnii, Mortierella alpina, Thraustochytrium, Schizochytrium, and mixtures thereof.

[0084] In yet further embodiments, the microorganism includes but is not limited to a microorganism belonging to: Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium, and Saprolegnia. In another embodiment, the ARA is obtained from a microbial cell, and the microbial cell is from: Mortierella, including but not limited to: Mortierella elongata, Mortierella exigua, Mortierella hygrophila, Mortierella alpina, Mortierella schmuckeri, and Mortierella minutissima. In yet further embodiments, the microbial cell is from Mortierella alpina.

[0085] In an even further embodiment, the microbial cell is from a microalgae of the order Thraustochytriales, which includes but is not limited to Thraustochytrium (species include arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, striatum); Schizochytrium (species include aggregatum, limnaceum, mangrovei, minutum, octosporum); Ulkenia (species include amoeboidea, kerguelensis, minuta, profunda, radiate, sailens, sarkariana, schizochytrops, visurgensis, yorkensis); Aurantiacochytrium; Oblongichytrium; Sicyoidochytium; Parientichytrium; Botryochytrium; and combinations thereof. In another embodiment, the microbial cell is from the Thraustochytriales order. In yet another embodiment, the microbial cell is from the genus Thraustochytriales. In yet a further embodiment, the microbial cell is from the genus Schizochytrium. In yet a further embodiment, the microbial cell is selected from the genus Thraustochytriales, Schizochytrium, or a mixture thereof.

[0086] Example

[0087] Example 1

[0088] In this example, Schizochytrium sp. was cultured in a fermentation vessel. Harvesting the fermentation broth early to obtain younger cells (D1) eliminated the self-heating behavior when compared to harvesting the broth at the end of fermentation (D2). Both samples were dried by lyophilization. See Table 2. Each of the two samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 100°C. The temperature profiles for each test can be seen in Figures 2 and 3. Self-heating was clearly seen in D2, where the temperature rose to >60°C above the oven temperature set point, while no self-heating was observed in D1: when both were compared using 100 mm cubes at 100°C, the sample temperature never rose above the oven temperature. Therefore, according to Figure 1, D2 would be classified in Packing Group III, and D1 would not be classified therein.

[0089] Table 2

[0090]

[0091]

[0092] Next, the harvest broth was tested at different time points to determine when self-heating characteristics occurred. Samples were tested at the following time points: 2 (S1), 3 (S2), 4 (S3), 5 (S4), and 6 (S5) days. See Table 2. Each of the two samples was tested twice: a 25mm cube at 140°C and a 100mm cube at 120°C. The temperature profiles of these tests can be seen in Figures 4 and 5. Self-heating is clearly seen in samples S4 and S5 (5- and 6-day fermentation samples, respectively). For the 25mm cube at 140°C, S4 reached 14°C above the set point, while S5 reached 24°C above the set point. For the 100mm cube at 120°C, S4 exceeded the set point by 30°C, while S5 exhibited dangerous self-heating, reaching a temperature of 240°C above the set point. Therefore, ending fermentation on day 5 can help reduce product self-heating. If samples S1-S4 are shipped in packaging with a volume not exceeding 3 cubic meters, they can be classified as exempt according to UN standards.

[0093] Example 2

[0094] In this example, the same Schizochytrium species strain as in Example 1 was used. Comparison of unpasteurized cells with pasteurized cells revealed unexpected and surprising results. Drying the unpasteurized fermentation broth (D2) helped improve the tendency toward self-heating compared to the broth dried after pasteurization (D3). See Table 3. Both samples were also dried by freeze-drying. Each sample was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 100°C. The temperature profiles for each test can be seen in Figures 6 and 7. For the 25 mm cubes at 140°C, D3 experienced dangerous self-heating, reaching a maximum temperature 60°C higher than D2. For the 100 mm cubes at 100°C, D3 reached a maximum temperature 50°C higher than D2, with both rising above the ambient oven temperature. Therefore, based on Figure 1, D3 would be classified as Packaging Group II, while D2 would be classified as Packaging Group III.

[0095] Table 3

[0096]

[0097]

[0098] Example 3

[0099] In this example, the same Schizochytrium species strain as in Example 1 was used. When the drying methods were investigated, a surprising discovery was made. When the whole-cell biomass (D4) was dried by rotary drum drying, the self-heating properties were improved when compared to the whole-cell biomass (D3) dried by freeze drying. See Table 4. This is surprising when the difference between the residual moisture contents is not significant. Each of these samples was tested twice: a 25 mm cube at 140°C and a 100 mm cube at 100°C. The temperature profiles for each test can be seen in Figures 8 and 9. For the 25 mm cube at 140°C, the time taken for each sample to reach the maximum temperature was approximately the same, but the maximum temperature reached by D4 was 48°C lower than the maximum temperature of D3. For the 100 mm cube at 100°C, the opposite was observed, with the maximum temperature being approximately the same (approximately 230°C), but D4 took 1.7 hours longer to reach this temperature than D3. Based on this data, D3 would be classified in packing group II and D4 in packing group III (see Figure 1).

[0100] Table 4

[0101] sample Culture age (days) Pasteurized? Drying method Fat% PUFA% D3 6 yes Freeze-dried 50.4％ 46.8％ D4 6 yes Drum drying 50.6％ 48.0％

[0102] Example 4

[0103] In this example, the same Schizochytrium species strain as in Example 1 was used. Several different experiments were conducted to test the effectiveness of different antioxidants and their combinations. Adding the antioxidant ethoxyquin to the pasteurized fermentation broth (D5) helped improve the self-heating performance when compared to the broth without antioxidants (D4). See Table 5. The following examples show that combinations of antioxidants can have unexpected results. Both samples were dried by drum drying. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 100°C. The temperature profiles for each test can be seen in Figures 10 and 11. For the 25 mm cubes at 140°C, the maximum temperature for D5 was reduced by 28°C when compared to D4, and the time taken to reach the temperature increased by 12.5 hours. For the 100 mm cubes at 100°C, D4 experienced dangerous self-heating, while no self-heating was observed in D5. Both results confirm the effectiveness of ethoxyquin in reducing the chance of spontaneous heating. Therefore, based on Figure 1, D4 would be classified in Packing Group III, while D5 would not be classified in either Packing Group III or Packing Group II.

[0104] Table 5

[0105]

[0106] Example 5

[0107] In this example, the same Schizochytrium species strain as in Example 1 was used. Adding the antioxidant ethoxyquin along with lecithin to the pasteurized fermentation broth (D6) helped improve self-heating performance when compared to a broth containing only ethoxyquin (D5). Both samples were dried by drum drying. See Table 6. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 120°C. The temperature profiles for each test can be seen in Figures 12 and 13. For the 25 mm cubes at 140°C, the maximum temperature in D6 was 7°C lower than that in D5. For the 100 mm cubes at 120°C, both samples followed similar temperature profiles, although D6 remained approximately 5°C lower than D5 throughout the test. The results of both tests show that adding lecithin along with ethoxyquin can help reduce the chance of self-heating.

[0108] Table 6

[0109]

[0110] Example 6

[0111] In this example, the same Schizochytrium species strain as in Example 1 was used. Adding the antioxidant Roseen to the pasteurized fermentation broth (D7) helped to improve self-heating when compared to a broth without the antioxidant (D4). See Table 7. Both samples were dried by drum drying. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 100°C. The temperature profiles for each test can be seen in Figures 14 and 15. For the 25 mm cubes at 140°C, the maximum temperature for D7 was reduced by 18°C and the time taken to reach that temperature increased by 1.1 hours when compared to D4. For the 100 mm cubes at 100°C, D7 did not exhibit self-heating until the end of the test window, while D4 exhibited dangerous self-heating 6.2 hours into the test. The results of both tests according to Figure 1 would place D4 in packing group III, whereas D7 would be exempt from packaging and marking according to packing group III if it were transported in a volume not exceeding 450 litres.

[0112] Table 7

[0113]

[0114] Example 7

[0115] In this example, the same Schizochytrium species strain as in Example 1 was used. Adding the antioxidant Roseen together with lecithin to the pasteurized fermentation broth (D8) helped improve self-heating when compared to a broth containing only Roseen (D7). Both samples were dried by drum drying. See Table 8. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 120°C. The temperature profiles for each test can be seen in Figures 16 and 17. For the 25 mm cubes at 140°C, although the maximum temperature of D8 was 14°C higher than that of D7, it took D8 0.2 hours longer to reach that temperature than D7. For the 100 mm cubes at 120°C, in D8, the maximum temperature was reduced by 52°C and the time to reach that temperature was increased by 1.1 hours compared to D7. According to Figure 1, D7 would be exempt from packaging and labeling according to Packing Group III if transported in a volume < 450 L, while more testing would be required to classify D8.

[0116] Table 8

[0117]

[0118] Example 8

[0119] In this example, the same Schizochytrium species strain as in Example 1 was used. Adding the antioxidants Roseen and TAP1010 along with lecithin to the pasteurized fermentation broth (D9) helped improve self-heating when compared to a broth containing only Roseen and lecithin (D8). Both samples were dried by drum drying. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 120°C. The temperature profiles for each test can be seen in Figures 18 and 19. For the 25 mm cubes at 140°C, the maximum temperature for D9 was 37°C lower than for D8, and the time it took to reach that temperature increased by 3 hours. For the 100 mm cubes at 120°C, although the maximum temperatures reached by both samples were similar, D9 took 6.5 hours longer to reach that temperature than D8. The results of both tests showed that adding TAP1010 further reduced the chance of self-heating.

[0120] Table 9

[0121]

[0122] Example 9

[0123] In this example, the same Schizochytrium species strain as in Example 1 was used. Adding the antioxidants Roseen, TAP1010, and TBHQ along with lecithin to the pasteurized fermentation broth (D10) helped improve autothermal performance compared to a broth containing only Roseen, TAP1010, and lecithin (D9). See Table 10. Both samples were drum-dried. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 120°C. The temperature profiles for each test can be seen in Figures 20 and 21. For the 25 mm cubes at 140°C, the maximum temperature for D10 decreased by 6°C compared to D9, and the time taken to reach that temperature increased by 1.1 hours. For the 100 mm cubes at 120°C, although D10 had a maximum temperature 53°C higher than D9, it took 2.4 hours longer to reach that temperature. The results of both tests showed that the addition of TBHQ can help delay the onset of self-heating.

[0124] Table 10

[0125]

[0126]

[0127] Example 10

[0128] In this example, the same Schizochytrium species strain as in Example 1 was used. Experiments were also conducted to test the effectiveness of including inert ingredients in the biomass. Including dextrose in the pasteurized fermentation broth (D11) helped improve self-heating when compared to a broth with low residual glucose (D4). See Table 11. Both samples were dried by drum drying. Each of these samples was tested twice: 25 mm cubes at 140°C and 100 mm cubes at 100°C. The temperature profiles for each test can be seen in Figures 22 and 23. For the 25 mm cubes at 140°C, when compared to D4, the maximum temperature was reduced by 50°C, although the time taken to reach the maximum temperature for D11 was reduced by 0.4 hours. For the 100 mm cubes at 100°C, when compared to D4, the maximum temperature was reduced by 110°C, although the time taken to reach the maximum temperature for D11 was reduced by 2.9 hours. Therefore, according to Figure 1, D4 would be classified in packing group III and D11 would not need to be packaged and labeled according to packing group III or packing group II.

[0129] Table 11

[0130]

[0131] Then the addition of different sugars (fructose, sucrose and maltose) was used to experiment. When compared with the broth (S6) of low residual glucose, adding these sugars to the pasteurized fermentation broth (S7-9) helped to improve self-heating. These samples were all dried by freeze drying. Each of these samples was tested twice: 25mm cubes at 140°C and 100mm cubes at 100°C. The temperature characteristic curves of each test can be seen in Figures 24 and 25. For the 25mm cubes at 140°C, all samples containing sugar (S7-9) reached a maximum temperature lower than the control (S6). However, each sugar reduced the temperature by different amounts: fructose (S7) -24°C, sucrose (S8) -14°C and maltose (S9) -9°C. Similarly, for the 100mm cubes at 100°C, all samples containing sugar (S7-9) reached a maximum temperature lower than the control (S6). For the sample containing fructose (S7), the maximum temperature was 48°C lower than the control, but it took about two hours longer to reach the temperature. For the sample containing sucrose (S8), the maximum temperature was 35°C lower than the control, and it took 2.5 hours longer to reach the temperature. For the sample containing maltose (S9), the maximum temperature was 36°C lower than the control, but it took about 0.6 hours longer to reach the temperature. Therefore, the sugar in the fermentation broth can help to reduce the self-heating properties. The presence of sugar can be achieved by adding sugar to the completed fermentation or by residual amount in the case where the fermentation is completed before all sugars from one or more raw materials are consumed.

[0132] Example 11

[0133] In this example, the same Schizochytrium species strain as in Example 1 was used. Harvesting the fermentation broth at different time points resulted in dried samples with varying amounts of PUFAs (polyunsaturated fats), with earlier samples having the least PUFAs and later samples having the most PUFAs. See Table 12. In the following graph (Figure 26), the PUFA percentages in samples S1-S5 (previously discussed in Example 1) are plotted against the maximum temperatures reached by the samples during oven testing (25 mm cubes at 140°C and 100 mm cubes at 120°C). For the 25 mm cubes at 140°C, the maximum temperature reached increased only slightly with increasing PUFA percentage. However, for the 100 mm cubes at 120°C, the maximum temperature increased significantly when the PUFA percentage in the biomass exceeded 20%. Therefore, keeping the PUFA percentage of the dry biomass below 20% can help reduce product self-heating.

[0134] Table 12

[0135]

Claims

1. A method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises limiting the length of a fermentation process to obtain the composition to less than 6 days, and wherein the composition is from the genus Schizochytrium.

2. A method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises not performing a pasteurization step after the fermentation from which the composition is obtained, and wherein the composition is from the genus Schizochytrium.

3. A method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises a drum drying step instead of a freeze drying step, and wherein the composition is from the genus Schizochytrium.

4. A method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises adding at least one type of natural antioxidant and at least one type of synthetic antioxidant to the fermentation broth at the end of the fermentation from which the composition is obtained, and wherein the composition is from the genus Schizochytrium.

5. The method of claim 4, wherein the natural antioxidant is lecithin or Roseen, and wherein the synthetic antioxidant is ethoxyquin, TAP1010, or TBHQ.

6. A method for reducing the self-heating tendency of a composition comprising cells containing one or more polyunsaturated fatty acids (PUFAs) having at least 20 carbon atoms and at least three double bonds, wherein the composition has at least 20 wt% PUFAs, and wherein the method comprises causing the fermentation broth to contain at least 50 g / L of sugars at the end of the fermentation from which the composition was obtained, and wherein the composition is from the genus Schizochytrium.

7. The method according to claim 6, wherein the sugar is one or more types selected from the group consisting of: Glucose, fructose, sucrose, and maltose.

8. The method of any one of claims 1 to 7, wherein the composition is biomass.

9. The method of claim 8, wherein the biomass is microbial cells.

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