SOPHOROLIPID-CONTAINING COMPOSITIONS
20170044586 ยท 2017-02-16
Inventors
Cpc classification
International classification
Abstract
A process to produce a sophorolipid composition is disclosed, the steps including obtaining a sophorolipid containing composition having a pH of less than 5, adding 6 percent by weight or less of a free fatty acid to the composition, and thereafter adjusting the pH of the composition to a pH greater than 5. In some embodiments, the sophorolipid composition initially comprises from 4 to 80 percent by weight dry solids.
Claims
1. A process to produce a sophorolipid composition, the process comprising: (a) obtaining a sophorolipid containing composition comprising about 4-80 percent by weight total dry solids, comprising at least one sophorolipid, wherein the sophorolipid containing solution exhibits a pH of less than 5, (b) adding about 6 percent by weight or less of a least one free fatty acid to the sophorolipid containing composition to provide a fatty acid adjusted sophorolipid composition, and (c) adjusting the pH of the fatty acid adjusted sophorolipid composition to a pH greater than 5 to provide the sophorolipid composition.
2. The process of claim 1, wherein the total dry solids of (a) comprises from about 40 to about 99 percent by weight total sophorolipids based on the total dry solids.
3-9. (canceled)
10. The process of claim 13, wherein the sophorolipid containing composition of (a) comprises less than 75 percent by weight water.
11-12. (canceled)
13. The process of claim 1, wherein the sophorolipid containing composition of (a) comprises 25-96 percent by weight water.
13. (canceled)
14. The process of claim 1, wherein separating the sophorolipid layer from an aqueous layer comprises heating a fermentation broth containing sophorolipid to a temperature of about 70-75 C. to provide a heated broth, cooling the heated broth to an ambient temperature, and decanting a higher density sophorolipid layer from a lower density aqueous layer.
15. The process of claim 1, wherein the at least one sophorolipid comprises ester-form sophorolipid and acidic-form sophorolipid.
16. The process of claim 15, wherein a ratio of ester-form sophorolipid to acidic-form sophorolipid is at least 1:1.
17-18. (canceled)
19. The process of claim 16, wherein a ratio of ester form sophorolipid to acidic-form sophorolipid is not greater than 9:1.
20. The process of claim 19, wherein the fatty acid adjusted sophorolipid composition exhibits a pH of 12.5 or less.
21. (canceled)
22. The process of claim 1, wherein the fatty acid adjusted sophorolipid composition exhibits a pH of from about 6 to about 9.5.
23. The process of claim 1, wherein sufficient free fatty acid is added to obtain an adjusted sophorolipid composition having a formulated free fatty acid content from about 0.1 to about 6 percent by weight of the fatty acid adjusted sophorolipid composition.
24. (canceled)
25. The process of claim 1, wherein f sufficient free fatty acid is added to obtain an adjusted sophorolipid composition having a formulated free fatty acid content from about 0.5 to about 2.5 percent by weight of the fatty acid adjusted sophorolipid composition.
26-37. (canceled)
38. The process of claim 1, wherein the added free fatty acid comprises oleic acid.
39. The process of claim 1, wherein oleic acid comprises a majority of the added free fatty acid.
40. The process of claim 1, wherein the added free fatty acid comprises a free fatty acid, a neutralized fatty acid salt, a free fatty acid anion distributed in an aqueous sophorolipid containing composition, or mixtures thereof.
41. The process of claim 1, wherein the added free fatty acid is obtained from a fatty acid distillate derived from plant-based oil, animal fat or fish oil.
42. The process of claim 1 further comprising adding a biocide to the fatty acid adjusted sophorolipid composition.
43. The process of claim 1 further comprising adding a biocide effective at a pH of about 5-12.
44. The process of claim 42, wherein amounts of effective biocides are about at least 10 ppm (0.001 percent by weight).
45. The process of claim 24, wherein amounts of effective biocides are about less than 1 percent by weight (10000 ppm), less than 0.5 percent by weight (5000), or less than 0.05 percent by weight (500 ppm).
46-90. (canceled)
Description
EXAMPLES
Analytical Methods and Materials Utilized
[0038] Pourability is determined by adding sample solution to a 50 mL centrifuge tube and placing it into a freezer at the appropriate temperature (for example, if the pourability at 20 C. is being determined the freezer is set at 20 C./4 F.). After twenty four hours in the freezer, the tubes are tilted to check for pourability. If the material moves in the tube then the sample result is reported as flows. If the material does not move then the sample is considered frozen. The same method is used to determine the pourability of the samples at longer periods of time, with the sample being indicated to flow or not after the desired test period.
[0039] The ratio of ester-form sophorolipids to acidic-form sophorolipids in a formulated composition is determined according to the method described in Example 4, below,
[0040] Fatty Acid Distillate: is a mixture of free fatty acids derived from animal sources and typically comprises 73% oleic acid, 8% linoleic acid, 6% paimitoleic acid, and 1% linolenic acid (CAS# 112-80-1); available from Bremitag Great Lakes under the product name Emersol 213 NF.
[0041] 95 Dextose: A concentrated dextrose with a minimum dextrose concentration of 94% dextrose and a pH of 5 with a dry solid content of 70.5-71.5 percent by weight available from Cargill, Incorporated.
[0042] OHLY-KAT: Yeast extract, available from OHLY Americas.
[0043] Solulys 095E: Spray-dried corn steep with 24 wt % lactic acid, 44 wt % protein, 18 wt % ash, 1 wt % sugars, 13% other elements, available from Roquette Chemicals & Bio-Industries.
[0044] Magnesium Sulfate Heptahydrate: available from J.T. Baker under the product designation 2505-07, VWR.
[0045] Ammonium Phosphate Dibasic: available from J.T. Baker under the product designation 0784-07, VWR.
[0046] Ammonium Sulfate: available from J.T. Baker under the product designation 0792-07, VWR.
[0047] Ferrous Sulfate Heptahydrate: available from Fisher Scientific under the product designation 1146-500.
[0048] Manganous Sulfate Monohydrate: available from Midland Scientific under the product designation 2550-01, J.T. Baker.
[0049] Zinc Sulfate Hep ydrate: available from Fisher Scientific under the product designation Z76-500.
[0050] USP Glycerol: 99.0 wt % glycerol available from Baker Baker under the product designation 4043-00.
[0051] Crude Glycerol: 84% Glycerol, 11% Water, 2% NaCl, 1% Methanol, 2% Organic Residue, available from Cargill, Incorporated.
[0052] Ultra-Pure Water; 18 megohm resistivity water made using a water purification system available from Hydro Service and Supplies.
[0053] Tap Water: Cl 6.5 ppm; Cu 0.117 ppm; K 0.01 ppm; Mg 0.002 ppm; Mn 0.279 ppb; Na 0.998 ppm; P 0.014 ppm; and Zn 0.21 ppm, in aqueous solution available from the Rathbun Regional Water Association.
[0054] Hard Water: an aqueous solution containing CaCl.sub.2-2H.sub.2O 1.03 wt %; MgCl.sub.2-6H.sub.2O 0.56 wt %; and NaCl 3.76 wt %.
[0055] 2% KCl Solution: an aqueous solution is prepared by accurately weighing 20g KCl and adding it to 980 g of Tap Water. KCl available from Midland Scientific under the product designation 3040-05, J.T. Baker.
[0056] All the percentages listed are weight percentages (wt %), unless otherwise indicated to the contrary.
Example 1
Sophorolipids from Fermentation
[0057] Starmerella bombicola NRRL Y-17069 was obtained from the Agricultural Resource Service (ARS) Culture Collection. The original culture is plated for purity on a potato dextrose agar (PDA) plate. A single colony is selected from the plate and used to inoculate a 250 ml shake flask containing 50 ml of sterilized Yeast Mold (YM) broth. The shake flask is placed in a shaker incubator overnight (25 C. and 250 rpm). Following overnight incubation, sterile 80% glycerol is added to the seed broth to make a glycerol seed stock at a final glycerol concentration of 20%. One ml aliquots are added to cryo-vials and stored in a 80 C. freezer.
[0058] Pre-cultures are prepared by inoculating a 250 ml shake flask containing 50 ml of autoclaved YM broth with a single cryo-vial (1 ml glycerol stock) and incubating it in a shaking incubator (25 C. and 250 rpm) for 24 hours. The 50 ml culture is used to inoculate a 14 L New Brunswick fermenter containing 10 L of autoclaved Sophorolipid (SL) Seed medium at an OD600 of 0.02. The SL seed medium consists of 30 g/L dextrose, 48 g/L OHLY-KAT yeast extract and trace minerals (10 mg/L ferrous sulfate (heptahydrate), 2 mg/L manganous sulfate (monohydrate), 15 mg/L zinc sulfate (heptahydrate). The seed fermentor temperature is controlled at 25 C. Agitation begins at 550 rpm and is cascaded to a maximum of 1100 rpm to maintain a minimum % dissolved oxygen of 40 throughout the fermentation. The pH is not maintained. The seed culture is harvested at or near the peak oxygen uptake rate (OUR) (typically 115-130 at 29-30 hours).
[0059] The main fermentation Sophorolipid medium consists of 4 g/L dry basis nitrogen source (either raw light steep water or Solulys 095E), 1.65 g/L ammonium sulfate, 1.06 g/L ammonium phosphate (dibasic), 0.5 g/L magnesium sulfate (heptahydrate) and 2 mg/L thiamine-HCl. The starting dextrose concentration is 100 g/L (+/20) and the starting lipid source concentration is 30 g/L (+/10).
[0060] Main fertnentors are inoculated to an OD600 of 2.8 with S. bombicola 10 L seed culture. Salts, dextrose feeds and oil feeds are sterilized separately. The initial pH of the media is approximately 5.2 and is allowed to naturally drop and is maintained at 3.5 for the remainder of the fermentation with 2N NaOH. The fermentation temperature is maintained at 30 C., aeration is set at one volume of air per volume of medium per minute (VVM) based on initial volume. Agitation is maintained at a level that allows for a peak oxygen uptake rate (OUR) of 50 (+/5) mmol 1.sup.1 h.sup.1 following exponential cell growth and slowly trends down as the fermentation progresses due to increased fermentor volume and gradual slowing of cellular metabolism within an OUR range of 31 (+/5) mmol 1.sup.1 h.sup.1.
[0061] For the feed media, two addition vessels are utilized. One contains the sterilized lipid source and the other contains sterilized 600 g/L 95 Dextrose. 95 Dextrose is fed into the fermentor to maintain a fermentation broth concentration of 25 g/L (+/20) after an initial drop from the starting concentration of 100 g/L (+/20). The lipid source is fed into the fermenter between 9 and 40 hours of elapsed fermentation time. A total of 200 g/L of the lipid source is added (based on starting fermentation volume). As the lipid source is nearing depletion the dextrose feed is reduced or stopped to allow for both levels to reach near 0 g/L at the end of fermentation (EOF). EOF is determined by neutral lipid depletion (based on hexane extraction) and a free fatty acid content of <2.5 g/L (as measured by high pressure liquid chromatography with an evaporative light scattering detector (HPLC/ELSD). The final dextrose concentrations should be 5 g/L or less.
Example 2
Method for Obtaining a Fraction Enriched in Sophorolipids
[0062] Heat treatment of the sophorolipid fermentation broth is conducted by heating the vessel to 70 to 75 C. along with minimal agitation to facilitate an adequate heat transfer. Once the fermentation broth reaches 70 to 75 C., the agitation is stopped. The fermentation broth is then allowed to naturally cool as the sophorolipid product layer physically separates from the aqueous layer within the fermentation broth due to differences in density. The broth is allowed to gravity settle for a minimum of 30 minutes. The organic phase containing enriched crude sophorolipids is collected from the bottom of the vessel and the aqueous phase is left in the vessel.
[0063] The ratio of ester-form sophorolipids to acidic-form sophorolipids for various crude sophorolipid fermentation samples that have been prepared by the process related to this example is provided in Table 1. Samples 2-1 through 2-6 exhibit a formulated free fatty acid content of from 0.14 to 1.6 percent by weight of the sample.
TABLE-US-00001 TABLE 1 Ratio of Ester-From to Acidic-Form Sophorolipids Crude Sophorolipid Fermentation Ratio of Ester-form sophorolipid Sample (No.) Sample (No.) to Acidic-form sophorolipid 2-1 1-1 1.50 2-2 1-2 1.61 2-3 1-3 1.23 2-4 1-4 1.15 2-5 1-5 1.08 2-6 1-6 Not Analyzed
Example 3
Formulation with Pour Point Depressant
[0064] Crude sophorolipid (containing 48-57% dry solids, 50% water and exhibiting a pH of 15 to 3.8) measures of 464 g (230 g dry weight) of samples similar to 2-1 to 2-6 are each mixed with 14 g Ultra-Pure water. Each of the resulting solutions is neutralized using 10 g of 50% sodium hydroxide to achieve a pH of 6.9-7.1. USP grade glycerol, 522 g (520 g dry weight), is added to each of the solutions. The formulated solutions contain 23% dry weight as crude sophorolipid, 52% dry weight as glycerol and 25% as water. The characteristics of the formulated sophorolipid are detailed in Table 2.
TABLE-US-00002 TABLE 2 Characteristics of Formulated Pour Point Depressant Samples Crude Sophorolipid Sample (No.) 2-1 2-2 2-3 2-4 2-5 Formulated Sample(No.) 3-1 3-2 3-3 3-4 3-5 Density (g/mL) 1.18 1.19 1.19 1.19 1.19 Refractive Index 1.46 1.45 1.44 1.44 Conductivity (S) 485 504 428 426 Viscosity at 30 C. 154 123 171 183 pH 7.12 7.07 7.01 7.07 7.12 CMC (mg/L) 214 308 370 353 320
Example 4
Method to Determine Ratio of Esterform Sophorolipids to Acidic-Form Sophorolipids
[0065] The ratio of ester-form to acidic-form sophorolipids is determined on representative samples from Example 3 using an LCMS-based method. Samples are diluted in 50% acetonitrile and analyzed using a Dionex Summit HPLC System equipped with a Waters XBridge C18, 5 m, 2.1 ID150 mm column at a flow rate of 0.4 mL/min using a gradient shown in Table 3. Mass is detected using a Thermo Exactive mass spectrometer with a negative scan mode, scan range of 150-2000 mass-to-charge ratio (m/z), scan time of 30 min, electrospray ionization mode with a spray voltage of 4.0 kV, and a capillary temperature of 200 C. The mass spectrum is then filtered to only display masses of 500-750 m/z, which is the typical mass range for sophorolipids. The acidic-form fraction is defined as all peaks eluting between around 10-14 minutes with the first peak having a ink of 595 and last peak having a m/z of 707. Likewise, the ester-form fraction is defined as all peaks eluting between around 18-26 minutes with the first peak having a m/z of 603 and the last peak having a m/z of 689 (Chart 1). The peaks for the other ester-form fractions can be determined by utilizing the appropriate peaks in the LC method by procedures known to one of skill in the art. The ratio of ester-form to acidic form sophorolipid is defined as the ratio between the total area under the ester-form fraction peaks to the total area under the acidic-form fraction peaks on the chromatogram.
TABLE-US-00003 TABLE 3 LCMS gradient profile. Time (min) Solvent A (%) Solvent B (%) 0 5 95 20 70 30 23 70 30 25 5 95 30 5 95 Solvent A = Acetonitrile (LCMS grade) with 0.1% NH.sub.4OH, Solvent B = Water (LCMS grade) with 0.1% NH.sub.4OH
[0066] Chart 1: An exemplary MS spectrum when filtered from m/z 500-750 is set out below illustrating the acidic-form fraction and the ester-form fraction.
[0067] For the examples set forth in this application, the ratio of ester-form to acidic-form is an approximation based on the ratio of lactonic sophorolipid to acidic-form sophorolipid determined by using the method set forth above regarding identifying the ratio of lactonic sophorolipid and acidic-form sophorolipid. It is believed that any additional ester-form. sophorolipids present could be readily identified by appropriate determination of the peaks associated with such additional ester-form sophorolipids. It is believed that for the examples set forth below, the amount of additional ester-form sophorolipids present is small and therefore would only slightly increase the ratio of ester-form sophorolipids to acidic-form sophorolipids from the values listed.
Example 5
Determining Flowback Number
Sample Preparation
[0068] The following flowback method is used to determine the flowback numbers associated with the sophorolipid formulations of this invention. For flowback number determinations, aqueous solutions containing the neutralized sophorolipid samples are prepared at 0.1 percent by weight in 2% KCl Solution or 0.1 percent by weight in Hard Water. The same method can be used for measuring flowback numbers in other aqueous solutions such as Ultra-Pure Water. The resulting aqueous solution(s) are tested to determine the flowback numbers in accordance with the method described below. The flowback numbers are reported together with the aqueous solution that was utilized to conduct the test.
Apparatus
[0069] The flowback column consists of a clear polyacrylic column (8-inch length and 1-inch inner diameter), a Teflon bottom cap with 2 O-rings and 1 screen and a Teflon top cap with 2 O-rings and 1 screen. The top Teflon cap is differentiated from the bottom by a small hole drilled into the top of the cap. An outlet tube is attached to the top of the column. A 3-way valve is attached to the bottom of the column to control nitrogen flow to the column.
Packing the Column and Loading a Sample
[0070] An 80-100 g neutralized sophorolipid sample (Flowback fluid) is prepared as describe above and 190 g Unimin. Unifrae 20/40 white sand is weighed out. The bottom column cap with a capped compression fitting is screwed on to the column. About 35 g flowback fluid is slowly added into the column through the end of the column. The Unifrac sand is slowly added into the column under mild vortexing (about 1300 rpm). When the level of the sand is just below the level of the fluid, more flowback fluid is added in 0.5 to 3 mL increments by syringe. The addition of sand and flowback fluid is continued until the level of sand is just above the top of the column. Once the column is filled, an O-ring is placed on top of the column followed by a screen and the second O-ring on top of the screen. Then the column top cap containing a compression fitting and a hole for air bubble elimination that will fit the tip of a 1 ml syringe is screwed on to the column. The flowback fluid is added through the top compression fitting until the liquid level is just below the top of the syringe hole. The column is placed on the vortex (about 1300 rpm) to remove any air bubbles. Additional fluid is added until the fluid level in the syringe hole no longer drops. The hole is then plugged with a 1 mL syringe or suitable plug. Additional flowback fluid is added via syringe to the top of the column such that the fluid level is over the lip of the compression fitting. The column is placed on the vortex to remove any remaining air bubbles. If the liquid level no longer drops, place the syringe needle on the lip of the compression fitting and remove excess liquid. A compression fitting cap is placed on the top of the column.
[0071] The 3-way valve with the flow switched away from the column is attached to the bottom of the column. The outlet tube is attached to the top of the column. The remaining sand and flowback fluid is weighed.
Calibration of Nitrogen Flow
[0072] The nitrogen flow to the flowback column must he calibrated before each analysis. Tap Water (1000 mL) is added to a 1 L filter flask equipped with a rubber stopper that is fitted with a piece of plastic tubing. The tubing is inserted into a 1000 mL graduated cylinder above the 700 mL mark. The same nitrogen line used for flowback determination is connected to the filter flask and the nitrogen flow is turned on to the flask. The time it takes to displace 550 mL Tap Water out of the filter flask and into the graduated cylinder is measured using a stopwatch. The measured time period begins when the level reaches the 100 mL mark and ends at the 650 ml. mark. Nitrogen flow is adjusted until it takes between 49.5 and 50.5 seconds to collect 550 mL of Tap Water which corresponds to a nitrogen flow of 11 mL/sec.
[0073] Nitrogen flow rate is calculated as follows:
Flowback Data Collection
[0074] After the calibration of nitrogen flow the plastic tube attached to the column outlet is inserted into an empty graduated cylinder and is placed on a balance and tared. The three way valve is turned on to the column so that the gas flow is now passing through the column. The flowback start time is recorded. The weight of the fluid recovered is shown on the balance and fluid is recovered until the weight increase is less than 0.4 g per 10 minutes. The three way valve is then turned off and the weight of graduated cylinder with the recovered fluid is recorded.
[0075] The flowback number is calculated as follows:
[0076] Hard Water is tested to ensure proper column standardization. The expected flowback number for Hard Water is 54.8 +/3 (i.e. 51.8-57.8 respectively). The column and/or operation of the column should be adjusted if the flowback number for Hard Water is outside the 51.8-57.8 range.
Example 6
Testing Flowback Samples with Pour Point Depressant and No added Free Fatty Acid
[0077] Crude sophorolipids of samples 2-2, 2-3, 2-4, 2-5 and 2-6 are mixed with the glycerol as indicated in Table 5 and Ultra-Pure Water in a sample tube. The sample tube is placed on a shaker for 5 minutes and the pH of the mixture is adjusted with 50% NaOH to the pH indicated in Table 5 under stirring followed by shaking for another 15 minutes. The neutralized composition contains the dry solids content, glycerol content and water content as indicated in Table 5. The resulting aqueous solutions are prepared and tested to determine the flowback numbers in accordance with the method described in Example 5. The results of the testing are set forth below in Table 5.
TABLE-US-00004 TABLE 5 Flowback Number of Formulated Samples with Pour Point Depressant and no added Free Fatty Acid Crude Dry solid Formulated Sophorolipid content Neutralized 2% KCl Sample Sample excluding USP Glycerol composition Water Flowback (No.) (No.) glycerol (%) (%) Final pH (%) Number 6-1 2-2 23 52 7.1 25 80 6-2 2-3 22 53 7.2 25 83 6-3 2-3 5 70 7.4 25 63 6-4 2-3 10 65 7.1 25 76 6-5 2-4 23 52 7.0 25 85 6-6 2-5 10 60 7.1 25 76 6-7 2-6 10 65 7.5 25 69
Example 7
Flowback Number with Pour Point Depressant and Added Free Fatty Acid
[0078] Crude sophorolipid of samples 2-3, 2-4 and 2-5 are mixed with the glycerol, fatty acid distillate, and Ultra-Pure Water in a sample tube to enable the creation of formulated samples as indicated in Table 6 (and further described below). The sample tube is placed on a shaker for 5 minutes and the pH of the mixture is adjusted with 50% NaOH to the pH indicated in Table 6 under stirring followed by shaking for another 15 minutes. The resulting aqueous solutions are prepared and tested to determine the flowback numbers in accordance with the method described in Example 5. The results of the testing are set forth below in Table 6.
TABLE-US-00005 TABLE 6 Flowback Number of Formulated Samples with Pour Point Depressant and added Free Fatty Acid. Dry solid content %, Formulated Hard Crude excluding Free Fatty 2% KCl Water Formulated Sophorolipid glycerol Acid Flowback Flowback Sample Sample and free Content *Glycerol Water Number Number (No.) (No.) fatty acid (%) (%) (%) pH (%) (%) 7-1 2-3 4.0 1.5 70.0 25.0 7.0 65 7-2 2-3 9.0 1.0 65.0 25.0 7.0 84 7-3 2-3 13.5 2 60.0 25.0 7.4 86 7-4 2-4 4.5 0.6 70.0 25.0 8.0 70 66 7-5 2-4 3.6 0.5 71.0 25.0 8.8 68 57 7-6 2-4 17.4 0.8 57.03 25.0 8.4 75 7-7 2-4 16.9 1.3 57.02 25.0 8.7 88 78 7-8 2-5 17.5 0.8 56.9 25.0 8.3 89 77 7-9 2-5 17.0 1.3 56.9 25.0 8.0 75 7-10 2-5 4.5 0.6 70.0 25.0 7.6 67 63 7-11 2-5 3.6 0.5 71.0 25.0 8.8 66 58 *USP Glycerol is used for Samples 7-1 to 7-3; Crude Glycerol is used for Samples 7-4 to 7-11
Example 8
Pourability of Formulated Sophorolipid Samples with Pour Point Depressant and No Added Free Fatty Acid
[0079] Crude sophorolipid of samples 2-2 and 2-3 are mixed with the glycerol as indicated in Table 7 and Ultra-Pure Water in a sample tube. The sample tube is placed on a shaker for 5 minutes and the pH of the mixture is adjusted with 50% NaOH to the pH indicated in Table 7 under stirring followed by shaking for another 15 minutes. The sample is tested to determine its pourability in accordance with the procedure described in the analytical methods. The results of the testing are set forth below in Table 7. Formulated sophorolipid samples exhibit pourability at 20 C. (i.e., flows) for a minimum of 24 hours, all the samples exhibit pourability up to 8 weeks, sample 8-4 is pourable for 12 weeks, and sample 8-5 is pourable for 13 weeks.
TABLE-US-00006 TABLE 7 Pourability of Formulated Sophorolipid Samples with Pour Point Depressant and no added Free Fatty Acid at 20 C. Formulated Sample (No.) 8-1 8-2 8-3 8-4 8-5 Crude Sophorolipid Sample (No.) 2-2 2-2 2-2 2-3 2-3 Dry solid content %, 22.8 18.8 20.8 22.7 22.7 excluding glycerol and free fatty acid *Glycerol (%) 51.5 55.5 53.5 51.4 51.4 Water (%) 25.7 25.7 25.7 25.9 25.9 pH 7.1 7.1 7.1 6.8 7.0 24 hrs Flows Flows Flows Flows Flows Week 1 Flows Flows Flows Flows Flows Week 2 Flows Flows Flows Flows Flows Week 3 Flows Flows Flows Flows Flows Week 4 Flows Flows Flows Flows Flows Week 5 Flows Flows Flows Flows Flows Week 6 Flows Flows Flows Flows Flows Week 7 Flows Flows Flows Flows Flows Week 8 Flows Flows Flows Flows Flows Week 9 Flows Flows Week 10 Flows Flows Week 11 Flows Flows Week 12 Flows Flows Week 13 Frozen Flows *USP Glycerol is used for Samples 8-1, 8-2, 8-3, 8-5; Crude Glycerol is used for Sample 8-4 **Formulated samples 8-1, 8-2, and 8-3 were not evaluated past 8 weeks.
Example 9
Pourability of Formulated Sophorolipid Samples with Pour Point Depressant and with Added Free Fatty Acid
[0080] Crude sophorolipid of samples 2-4 and 2-5 are mixed with the glycerol and fatty acid distillate and Ultra-Pure Water in a sample tube to enable the creation of Formulated Samples as indicated in Table 8 (and as further described below). The sample tube is placed on a shaker for 5 minutes and the pH of the mixture is adjusted with 50% NaOH to the pH indicated in Table 8 under stiffing followed by shaking for another 15 minutes. The sample is tested to determine its pourability in accordance with the procedure described in the analytical methods. The results of the testing are set forth below in Table 8. Formulated sophorolipid samples exhibit pourability at 20 C. (i.e. flows) for a minimum of 24 hours.
TABLE-US-00007 TABLE 8 Pourability of Formulated Sophorolipid Samples with our Point Depressant and added Free Fatty Acid at 20 C. Dry solid content %, Formulated Crude excluding Free Fatty Formulated Sophorolipid glycerol Acid Sample Sample and free Content Crude Glycerol Water Pourability (No.) (No.) fatty acid (%) (%) (%) pH at 24 hrs 9-1 2-4 4.5 0.6 70.0 25.0 8.0 Flows 9-2 2-4 3.6 0.5 71.6 25.0 8.8 Flows 9-3 2-4 18.2 0.8 57.0 25.0 8.4 Flows 9-4 2-4 18.2 1.3 57.0 25.0 8.7 Flows 9-5 2-5 18.2 0.8 56.9 25.0 8.3 Flows 9-6 2-5 18.2 1.3 56.9 25.0 8.0 Flows 9-7 2-5 4.5 0.6 70.0 25.0 7.6 Flows 9-8 2-5 3.6 0.5 71.0 25.0 8.8 Flows
Example 10
Flowback Number with No Added Pour Point Depressant and with Added Free Fatty Acid
[0081] Crude sophorolipid of samples 2-4 and 2-5 are mixed with fatty acid distillate and Ultra-Pure Water in a sample tube to enable the creation of Formulated Samples as indicated in Table 9. The sample tube is placed on a shaker for 5 minutes and the pH of the mixture is adjusted with 50% NaOH to the pH indicated in Table 6 under stirring followed by shaking for another 15 minutes. The resulting aqueous solutions are prepared and tested to determine the flowback numbers in accordance with the method described in Example 5. The results of the testing are set forth below in Table 9.
TABLE-US-00008 TABLE 9 Flowback Number of Formulated Samples with no added Pour Point Depressant and with added Free Fatty Acid. Dry solid content (%), Formulated Crude excluding Free Fatty Formulate Sophorolipid glycerol Acid 2% KCl Hard Water Sample Sample and free Content Water Flowback Flowback (No.) (No.) fatty acid (%) (%) pH Number Number 10-1 2-4 8.9 1.1 89.9 7.1 83 61 10-2 2-4 4.5 0.6 94.9 8.1 70 61 10-3 2-4 3.6 0.5 95.9 8.1 70 61 10-4 2-5 8.9 1.1 89.9 7.2 84 62 10-5 2-5 8.5 1.6 89.9 7.1 77 10-6 2-5 4.5 0.6 94.9 7.3 70 59 10-7 2-5 3.6 0.5 96.0 7.2 67 61