Modified Starch

Abstract

Described herein is an octenyl succinic acid modified starch (OSA modified starch) degraded by at least one enzyme capable of cleaving 1,4-linkages of a starch molecule from the non-reducing ends to produce short chain saccharides, wherein the content of non-covalently bound, free octenyl succinic acid in the OSA modified starch is less than about 0.50% by weight, based on total weight of the modified starch, and wherein content of alpha-1,6-glycosidic linkages is higher than 12%, a method of preparing same, and an encapsulation agent comprising same as well as a method of encapsulating an active agent with said encapsulation agent.

Claims

1. An OSA modified starch comprising a starch molecule degraded by at least one enzyme capable of cleaving the 1,4-linkages of the starch molecule from the non-reducing ends to produce short chain saccharides, wherein the content of non-covalently bound, free octenyl succinic acid in the OSA modified starch is less than about 0.50% by weight, based on the total weight of the OSA modified starch and wherein the content of the alpha-1,6-glycosidic linkages is higher than 12%.

2. The OSA modified starch according to claim 1, wherein the OSA modified starch has been degraded to a dextrose equivalent of higher than about 20.

3. The OSA modified starch according to claim 1, wherein the OSA modified starch has been degraded to a dextrose equivalent of about 30 to about 40.

4. The OSA modified starch according to claim 1, wherein the content of the covalently bound octenyl succinic acid in the OSA modified starch is from about 0.1% to 10% by weight, based on the total weight of the OSA modified starch.

5. The OSA modified starch according to claim 1, wherein the content of the covalently bound octenyl succinic acid in the OSA modified starch is from about 0.5% to about 5% by weight, based on the total weight of the OSA modified starch.

6. The OSA modified starch according to claim 1, wherein the content of covalently bound octenyl succinic acid in the OSA modified starch is from about 2% to about 3% by weight, based on the total weight of the OSA modified starch.

7. The OSA modified starch according to claim 1, wherein the OSA modified starch is an OSA modified waxy starch.

8. The OSA modified starch according to claim 1, wherein the OSA modified starch is an OSA modified waxy corn starch.

9. The OSA modified starch according to claim 1, wherein the enzyme is selected from the group consisting of -amylase, glucoamylase, pullulanase, maltogenase, exo-alpha-1,4-glucosidase, exo-1,4-alpha-D-glucan maltotetrahydrolase and exo-1,4-alpha-D-glucan-maltohexahydrolase.

10. The OSA modified starch according to claim 1, wherein the enzyme is -amylase or glucoamylase.

11. The OSA modified starch according to claim 1, wherein 5% by weight aqueous solution of the OSA modified starch has a pH of less than about 7.

12. (canceled)

13. The OSA modified starch according to claim 1, wherein a 5% by weight aqueous solution of the OSA modified starch has a pH of less than about 4.7.

14. The OSA modified starch according to claim 1, wherein a 0.5% by weight aqueous solution of the OSA modified starch has a surface tension of less than about 40 mN/m.

15. A method for the preparation of an OSA modified starch comprising the steps of: a) providing an OSA modified starch, the OSA modified starch comprising a starch molecule degraded by at least one enzyme capable of cleaving the 1,4-linkages of the starch molecule from the non-reducing ends to produce short chain saccharides, and b) tempering the OSA modified starch at a temperature of about 50 C. to about 180 C. for about 0.1 h to about 8 h.

16. The method for the preparation of an OSA modified starch according to claim 15, wherein the OSA modified starch in step b) is tempered at a temperature of about 80 C. to about 160 C. for about 0.2 h to about 5 h.

17. The method for the preparation of an OSA modified starch according to claim 15, wherein a 5% by weight aqueous solution of the OSA modified starch has a pH of less than about 7.

18. (canceled)

19. The method for the preparation of an OSA modified starch according to claim 15, wherein a 5% by weight aqueous solution of the OSA modified starch has a pH of less than about 4.7.

20. An OSA modified starch obtainable by the method of claim 15.

21. An encapsulation agent comprising the OSA modified starch according to claim 1.

22. A method of encapsulating an active agent comprising the encapsulation agent according to claim 21, the method comprising the steps of: a) forming a solution or dispersion of the encapsulation agent; and b) at least one of emulsifying, dispersing and precipitating the active agent in the solution or dispersion formed in step a).

23-24. (canceled)

Description

EXAMPLES

[0174] The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard.

[0175] The OSA modified starches used as starting products in the Example can be characterized as follows:

TABLE-US-00001 Degradation of the starch Funnel viscosity [sec] OSA Starch 1 enzymatic 7.5-9.5 at 19.0% solids determined according to method A OSA Starch 2 enzymatic 9.0-10.0 at 19.0% solids, determined according to method A OSA Starch 3 enzymatic 12.0-22.0 at 19.0% solids, determined according to method A OSA Starch 4 acid catalyzed 14.0-18.0 (Comparative Example) at 8.5% solids, determined according to method B OSA Starch 5 acid catalyzed 16.0-25.0 (Comparative Example) at 8.5% solids, determined according to method C OSA Starch 6 Acid catalyzed 28.0-37.0 (Comparative Example) At 19.0% solids, Determined according to method D

[0176] The viscosities were measured according to the following methods A, B, C or D.

Method A:

Equipment:

[0177] 1. Stainless steel Stormer cup.

[0178] 2. Dual scale thermometer.

[0179] 3. Cooling bath.

[0180] 4. BF Funnel (obtained from QMAS Lab).

[0181] 5. Timer or stop watch.

[0182] 6. Ring stand and ring to hold BF funnel.

[0183] 7. 100 mL graduated cylinder.

[0184] 8. Boiling water bath.

[0185] 9. Magnetic stirrer.

[0186] 10. General Electric AF 72 silicone anti-foam emulsion.

Procedure:

[0187] 1. The stainless steel Stormer Cup and thermometer is tared.

[0188] 2. 38.0 grams (anhydrous basis) of sample (19% solids) are added into the cup.

[0189] 3. Purified water is added until the total weight of starch and water is 200.0 grams.

[0190] 4, One drop silicone emulsion is added.

[0191] 5. The sample is allowed to mix long enough to dissolve any lumps.

[0192] 6. The sample solution is heated or cooled to about 22.2 C. (72 F.).

[0193] 7. 100 mL of the sample solution is run through BF funnel @ 22.2 C. (72 F.) and the flow time for 100 mL is recorded.

Method 13:

Equipment:

[0194] 1. Stainless steel beaker, 600 mL.

[0195] 2. Thermometer, general purpose, capable of accurately measuring 22.2 C. (72 F.).

[0196] 3. Cool and/or warm water bath (as required), used to adjust the test sample to 22.2 C. (72 F.).

[0197] 4. ABF funnel, calibrated, obtained from QMAS Lab.

[0198] 5, Support stand and 3-3 ring, used to hold ABF funnel.

[0199] 6. Extension clamp, round jaws, used to hold graduated cylinder in an inverted position above funnel.

[0200] 7. Graduated cylinder, 100 mL.

[0201] 8. Magnetic stir plate and 1-2 stir bar.

[0202] 9. Balance, top loading, capable of 0.01 gram accuracy.

[0203] 10. Weighing boat or smooth surface weighing paper.

[0204] 11. Magnetic stir bar retriever.

[0205] 12. Laboratory FilmParafilm.

[0206] 13. Stopwatch or timer.

Reagents:

[0207] 1. pH 6.0 buffer Solution:

[0208] Preparation of 1 kilogram of pH 6.0 Buffer Solution: 7.74 grams of citric acid, monohydrate and 17.93 grams of sodium phosphate, dibasic, anhydrous are dissolved in 974.33 grams of distilled or deionized water. The suspension is mixed with a magnetic stir bar and stir plate until the citric acid and sodium phosphate have dissolved. The pH of the buffer solution shall be 6.00.1. The pH 6.0 buffer solution is transferred from the beaker into a clean, dry container.

Procedure:

[0209] 1. 17.00.05 grams (anhydrous basis) of sample are weighed on an appropriate size weighing paper.

[0210] 2. The stainless steel beaker is tared.

[0211] 3. The necessary amount of pH 6.0 buffer solution for a total charge (starch+buffer solution) of 200.0 grams is weighed into the beaker (Weight of pH 6.0 buffer solution is calculated as 200.0weight of as is starch).

[0212] 4. A magnetic stir bar is added and the beaker is placed on a magnetic stirrer before mixing is started.

[0213] 5. While mixing, the pre-weighed starch sample is slowly added into the beaker. The speed of the mixer is adjusted to obtain a good vortex.

[0214] 6. After addition of all starch, the beaker and its contents are weighed, and recorded for the total weight.

[0215] 7. The beaker is covered with parafilm and mixing is continued until the sample is fully dispersed.

[0216] 8. After the sample Is fully dispersed, the sample Is taken off the stir plate and the parafilm is removed.

[0217] 9. The sample is adjusted for moisture loss by adding purified water, to bring the sample to the total weight recorded in step #6, and mixed again to obtain a homogenous sample.

[0218] 10. The stir bar is removed.

[0219] 11. A support stand with ring and extension clamp is set up so that the top of the ABF funnel is 12 above the base of the support stand when placed in a 3 to 3 ring. The extension clamp should be set so that the cylinder mouth is 2-2 above the top of the funnel when hung inverted in the extension clamp.

[0220] 12. The sample is heated or cooled to 22.2 C.0.3 C. (72 F.1 F.) in a warm or cool water bath. The sample is stirred to temperature using the thermometer.

[0221] 13. 100 mL of the sample are transferred to a graduated cylinder.

[0222] 14. The contents of the cylinder is poured into the funnel while holding a finger over the tip orifice. The air is removed from the funnel stem and tip by allowing a small amount of sample to run through the tip back into the graduated cylinder. This small amount of sample is poured back to the funnel, the cylinder is hung upside down in the extension clamp, and allowed to drain into the funnel.

[0223] 15. Using a stopwatch or timer, the time for the 100 mL of 22.2 C.0.3 C. (72 F.1 F.) solution to flow through the funnel back into the beaker is recorded. The time is measured din seconds until the sample flows to the apex of the stem.

Method C:

Equipment:

[0224] 1. Stainless steel Stormer cup, 300 mL capacity.

[0225] 2. Thermometer, general purpose, capable of accurately measuring 22.2 C. (72 F.).

[0226] 3. Cooling bath or warm water bath, used to adjust the test sample to 22.2 C. (72 F.).

[0227] 4. ABF funnel, calibrated (obtained from QMAS Lab).

[0228] 5. Support stand and 3-3 ring (used to hold ABF funnel).

[0229] 6. Extension clamp, round jaws, used to hold graduated cylinder in an inverted position above funnel.

[0230] 7. Graduated cylinder, 100 mL.

[0231] 8. Magnetic stir plate and 1-2 stir bar.

[0232] 9. Balance, top loading, capable of 0.01 gram accuracy.

[0233] 10. Magnetic stir bar retriever.

[0234] 11. Stopwatch or timer.

Procedure:

[0235] 1, A support stand with ring and extension clamp is set up so that the top of the ABF funnel is 12 above the base of the support stand when placed in a 3 to 3 ring. The extension clamp should be set so that the cylinder mouth is 2-2 above the top of the funnel when hung inverted in the extension clamp.

[0236] 2. The moisture content of the sample being tested is determined.

[0237] 3. A stainless steel Stormer cup is tared.

[0238] 4. 25.5 grams (anhydrous basis) of sample is added into the cup.

[0239] 5, Purified water is added until the total weight of starch and water is 300.0 grams.

[0240] 6. A magnetic stir bar iss added and placed on a magnetic stirrer. The sample is mixed until thoroughly dispersed.

[0241] 7. The stir bar is removed.

[0242] 8. The sample is heated or cooled to 22.2 C. (72 F.) using a thermometer to gently stir the sample to temperature.

[0243] 9, 100 mL of solution is transferred to a graduated cylinder.

[0244] 10. The contents of the cylinder are poured into the funnel while holding a finger over the tip orifice. The air is removed from the funnel stem and tip by allowing a small amount to run through the tip back into the graduate. This is returned back to the funnel and the cylinder is hung upside down in the extension clamp, allowing it to drain into the funnel.

[0245] 11. A stopwatch or timer is used to record the time for the 100 mL of 22.2 C. (72 F.) solution to flow through the funnel back into the stainless steel cup. The time is measured until the solution flows to the apex of the stem.

Method D:

Equipment:

[0246] 1. Waring two-speed commercial blender equipped with a 1-liter stainless steel container.

[0247] 2, Variable voltage transformer (Variac), 120/140 volt (Fisher Scientific #09-521-110 or equivalent).

[0248] 3. Top loading balance capable of 0.01 gram accuracy (Ohaus GT4800 or equivalent).

[0249] 4. Weighing paper (or equivalent).

[0250] 5. Small spatula (or equivalent).

[0251] 6. 1000 ml fat separator (soup skimmer), type for kitchen use, available at retail stores (optional).

[0252] 7. Beaker, 400 mL stainless steel, or equivalent.

[0253] 4. Dual scale thermometer.

[0254] 5. Cooling bath capable of cooling to 22.2 C. (72 F.).

[0255] 6. BF Funnel, calibrated (obtained from QMAS Laboratory).

[0256] 7. Electric timer or stop watch.

[0257] 8. Ring stand, ring and finger clamp to hold the BF funnel and graduated cylinder.

[0258] 9. 100 mL graduated cylinder.

Procedure:

[0259] 1. 57.0 grams (anhydrous) of starch are weighed onto weighing paper.

[0260] 2. The required amount of water is weighed directly into the stainless steel blender container to bring the total charge of starch and water to 300 grams.

[0261] 3. The blender container is placed onto the blender base and connect the blender to a Variac. With the blender on low speed setting, the Variac is increased until a vortex is formed.

[0262] 4. Two drops of defoamer are added to the blender container.

[0263] 5. The starch sample is added rapidly but uniformly to the blender. If necessary, the speed is increased by turning up the Variac to just maintain a vortex. The sides of the blender cup are scraped down with a spatula to incorporate all the starch into the dispersion.

[0264] 6. The sample is continuously mixed for about 3 to 5 minutes, until all visible particles are dispersed.

[0265] 7. The contents are transferred to a fat separator or a 400 mL beaker, and the sample is allowed to sit undisturbed for about 30 minutes.

[0266] 8. After 30 minutes, either the defoamed sample is poured from the fat separator into a 400 mL beaker or any foam that has come to the top of the beaker is removed with a spoon or spatula and discarded.

[0267] 9. The temperature of the sample is adjusted to 22.2+/0.3 C. (72+/0.5 F.).

[0268] 10. 100 mL of the mixture is transferred from the beaker to a 100 mL graduated cylinder and:

[0269] a) A finger is placed over the orifice of the BF funnel. 100 mL is poured into the funnel.

[0270] The finger is removed slightly allowing some of the mixture back into the graduate, clearing the stem of any foam or trapped air. The graduate is placed in an inverted position above the funnel, allowing it to drain while the test sample flows through the funnel back into the beaker.

[0271] b) The stopwatch is started and the finger simultaneously removed. The watch is stopped when the liquid surface (not foam) reaches the apex of the funnel (where the stem begins). The time is recorded in seconds.

[0272] The OSA modified starches according to the present invention were prepared as follows:

[0273] In each case of the OSA modified, enzymatically degraded starches (OSA Starches 1 to 3) as well as of acid hydrolyzed starches (OSA Starches 4 to 6) as comparative examples (all purchased from Ingredion), about 0.5 to 1 kg was weighed in a tray.

[0274] Subsequently, each starch was tempered in the tray in a Memmert drying cabinet (Memmert GmbH & Co KG) for the specified time and the specified temperature. The air pressure was not modified during drying.

[0275] During tempering, the OSA modified starches were mixed and stirred about every 30 min to prevent clumping and clogging.

[0276] After tempering the tempered starch was sieved through a conventional household sieve to remove bigger clumps.

Example 1: Analysis of the OSA Modified Starches According to the Present Invention

[0277] A) Analysis of Non-Covalently Bound, Free Octenyl Succinic Acid and of the Covalently Bound Octenyl Succinic Acid in the OSA Modified Starches Prior and after the Tempering.

[0278] The portion of non-covalently bound, free octenyl succinic acid is determined as follows by HPLC.

[0279] Sample preparation for determining the content of non-covalently bound, free octenyl succinic acid:

[0280] In each case 125 mg of the OSA modified starch is weighed into a 20 mL vial, to which is added 15.0 mL of methanol. The vial is sealed and mixed for 18 h. the sample Is diluted with water to yield a 2:1 water:methanol solution. The solution is filtered through a PVDF 0.45 m filter and analyzed

[0281] Sample preparation for determining the total content of octenyl succinic acid:

[0282] In each case 20 mg starch is weighed into a 20 mL vial, to which is added 15.0 mL 0.05N KOH solution. The vial is crimp capped and heated at 75 C. for 3 h or until the starch is completely dissolved. The sample is allowed to cool, filtered through a filter, and collected for analysis.

[0283] Preparation of a standard/reference material for the analysis of the OSA modified starches:

[0284] In each case 20 mg of reference material, octenyl succinic acid anhydride (CAS No.: 42482-06-4), is weighed into a 20 mL vial to which is added 20 mL 0.1N KOH solution. The reference is heated at 75 C. for 3 h until the octenyl succinic acid anhydride is completely dissolved, Secondary standards are prepared by diluting stock solution using 33/67 methanol/water producing standards in the range of 0.2 to 20 m/mL OSA.

[0285] Analysis was carried out using Empower-HPLC [0286] Mobile phase: Channel A, 1.0% phosphoric acid aqueous solution [0287] Channel B, acetonitrile [0288] Flow Rate: 0.3 mL/min [0289] Column: Cortecs C18 2.7 m, 2.1100 mm [0290] Column Temperature: 40 C. [0291] UV Detection Wavelength: 205 nm [0292] Analysis Time: 20 min [0293] Injection Volume: 5.0 L [0294] Retention time: 5.5 min and 6.0 min

[0295] The content of covalently bound octenyl succinic acid is calculated as the difference between the total content of octenyl succinic acid and the content of non-covalently bound, free octenyl succinic acid.

[0296] The results are given in Table 1.

TABLE-US-00002 TABLE 1 Analysis of free and covalently bound octenyl succinic acid (OSA) Covalently Total Free bound OSA Degradation OSA OSA (%) of the starch Tempering (%) (%) (calculated) OSA Starch 1 enzymatic 2.44 0.52 1.92 3 h @150 C. 2.52 0.44 2.08 OSA Starch 2 enzymatic 2.50 0.69 1.80 2 3 h @150 C. 2.52 0.41 2.11 OSA Starch 3 enzymatic 2.60 0.75 1.85 3 h @150 C. 2.56 0.40 2.16

[0297] Table 1 shows that the content of free octenyl succinic acid can be reduced with tempering.

B) Analysis of Non-Covalently Bound, Free Octenyl Succinic Acid and Covalently Bound Octenyl Succinic Acid in the OSA Modified Starches According to the Present Invention after Tempering for Different Times.

[0298] The samples were prepared and the analysis was performed as described in Example 1A.

[0299] The results are given in Table 2.

TABLE-US-00003 TABLE 2 Analysis of non-covalently bound, free and covalently bound octenyl succinic acid at different times of tempering. Total Free Covalently OSA OSA bound OSA Tempering (%) (%) (%) (calculated) OSA Starch 1 2 h @150 C. 2.78 0.47 2.31 OSA Starch 1 4 h @150 C. 2.76 0.45 2.31 OSA Starch 1 6 h @150 C. 2.79 0.42 2.37 OSA Starch 1 8 h @150 C. 2.73 0.10 2.63 OSA Starch 2 2 h @150 C. 2.80 0.44 2.36 OSA Starch 2 4 h @150 C. 2.81 0.39 2.42 OSA Starch 2 6 h @150 C. 2.79 0.36 2.40 OSA Starch 2 8 h @150 C. 2.74 0.17 2.57

[0300] The results in Table 2 show that tempering reduces over time the content of non-covalently bound, free octenyl succinic acid. Yet, the viscosity of the starches increases if tempered for more than 6 h so that the emulsions and/or dispersions of the starches need to be diluted, because they need to be dried with a lower dry solids content.

C) Analysis of the DE-Values

[0301] The dextrose equivalent was determined by the titration method for total sugars #-10-9070 under the NF 22 monograph far noncrystallizing sorbitol solution. In duplicate 0.05 or 0.10 g starch was accurately weighed into an Erlenmeyer flask and brought to a weight of 50 g with deionized water, to which was added 50 mL cupric sulfate iodide. The solution was gently refluxed on a hot plate for 5 min and then cooled to room temperature. Thereafter 25 mL 5N sulfuric acid was added slowly with constant stirring. The solution was then titrated with 0.1N sodium thiosulfate and starch indicator to reach a sky blue equivalent point. Reducing sugar (%) was calculated by subtracting the volume of titrant in the blank form from the volume of titrant in the sample, converting the mL titrant into mg of sugar from the #5-10-9070 reference table, and dividing the mg sugar by 10 times the grams of sample used.

[0302] The results are shown in Table 3.

TABLE-US-00004 TABLE 3 DE values Tempering DE-value OSA Starch 1 36.5 3 h @150 C. 35.3
D) Analysis of the Content of alpha-1,6-glycosidic Linkages

[0303] The content of alpha-1,6-glycosidic linkages is determined using high-temperature .sup.1H-NMR.

[0304] The solid samples were dissolved in D.sub.2O/TSP-d.sub.4 on a steam bath and .sup.1H-NMR spectra were measured at 90 C. using a Bruker Avance III HD 400 MHz NMR instrument at 400 MHz, TSP-d.sub.4 was added as internal standard.

[0305] The results are shown in Tables 4 and 5.

TABLE-US-00005 TABLE 4 Alpha-1,6-glycosidic linkages alpha-1,6- glycosidic Degradation of linkages the starch Temerping (%) OSA Starch 1 enzymatic 11.4 3 h @150 C. 13.1 OSA Starch 2 enzymatic 11.0 3 h @150 C. 13.3 OSA Starch 3 enzymatic 10.6 3 h @150 C. 13.9 OSA Starch 4 acid catalyzed 5.1 (comparative 3 h @150 C. 4.6 example) OSA Starch 6 acid catalyzed 3 h @150 C. 5.9 (comparative example) OSA Starch 5 acid catalyzed 4.8 (comparative 3 h @150 C. 5.6 example)

TABLE-US-00006 TABLE 5 Alpha-1,6-glycosidic linkages at different time points during tempering alpha-1,6- glycosidic linkages Tempering (%) OSA Starch 1 11.4 OSA Starch 1 2 h @150 C. 12.8 OSA Starch 1 4 h @150 C. 15.6 OSA Starch 1 6 h @150 C. 16.6 OSA Starch 1 8 h @150 C. 18.7 OSA Starch 2 11.0 OSA Starch 2 2 h @150 C. 12.2 OSA Starch 2 4 h @150 C. 14.2 OSA Starch 2 6 h @150 C. 14.7 OSA Starch 2 8 h @150 C. 17.5

[0306] The enzymatically degraded starches naturally have a higher content of alpha-1,6-glycosidic linkages, which can be increased during tempering. Yet, the viscosity of the starches increases if tempered for more than 6 h so that the emulsions and/or dispersions of the starches need to be diluted, because they need to be dried with a lower dry solids content.

E) Analysis of the Surface Tension of a Solution Comprising the OSA Modified Starch According to the Present Invention

[0307] Samples of the starch were suspended in water and stirred over-night with a magnetic stirrer. On the day of measurement, further dilutions of the stock solution were prepared and the surface tension was determined with a Kruess-tensiometer K11 MK3 using the plate method at 20 C. Values were recorded every 60 s for 10 min. Samples were measured up to four times. Differences between the samples were analyzed with a single factor variance with post-hoc test according to Fisher. Missing values were extrapolated for evaluation.

[0308] The results are shown in Table 6.

TABLE-US-00007 TABLE 6 Surface tension of solutions comprising the OSA modified starches OSA Starch 1 OSA Starch 2 OSA Starch 1 OSA Starch 2 Tempering Concentration 3 h @150 C. 3 h @150 C. 0.10% 58.97 1.27 57.15 1.63 43.45 2.76 42.40 0.25% 52.05 1.11 50.95 0.64 39.20 36.35 1.20 0.50% 43.07 2.63 46.17 3.27 34.50 0.42 35.40 0.85 0.75% 43.35 0.78 43.30 35.18 0.47 35.50 1.40 1.00% 43.13 1.46 38.90 2.43 34.40 0.38 33.95 2.62 2.00% 39.15 1.91 36.85 0.21 33.10 3.00% 37.45 0.21 30.80 1.84 31.80 0.68 30.85 1.06

[0309] Analysis of the surface tensions shows that solutions containing the OSA modified starch according to the present invention have a reduced surface tension, which leads to a better emulsifying activity as well as a better wetting behavior and accordingly to an optimized colloidal protection and/or stabilizing activity as well as dispersion stabilizing activity.

F) Analysis of the pH of a 5% by Weight Aqueous Solution Comprising the OSA Modified Starches Prior and after Tempering

[0310] The results are shown in Table 7.

TABLE-US-00008 TABLE 7 pH-values pH (5% by weight modified Tempering starch in water) OSA Starch 1 4.23 3 h @150 C. 4.23 OSA Starch 2 4.18 3 h @150 C. 4.18 OSA Starch 3 4.21 3 h @150 C. 4.15 OSA Starch 5 4.21 (comparative example) 3 h @150 C. 4.15

[0311] Tempering does not substantially influence the pH of an aqueous solution measured at ambient temperatures (15 C. to 25 C.) comprising 5% by weight of the OSA modified starches.

G) Analysis of the Molecular Weight of the Starch Portion of the OSA Modified Starches Prior and after the Tempering

[0312] The OSA modified starch was dissolved in DMSO over-night and subsequently heated at about 95 to 100 C. for 1 h followed by cooling of the solution.

[0313] The obtained solution was filtered through a 0.45 m PP membrane and the molecular weight was determined by gel permeation chromatography using the following parameters. [0314] Column set: Phenogel 10 m 100 , 10.sup.3 , 10.sup.5 [0315] Guard column: Phenogel 10 m [0316] Injection volume: 100 10 L [0317] Number of Injections: 2/sample [0318] Standards: Pullulan (788 K-180 Da) [0319] Sample concentration: 10.3-10.8 mg/4 mL [0320] Detector: RID-10A from Shimadzu [0321] Column Temperature: 60 C. [0322] Mobile-phase: DMSO+0.03 M NaNO.sub.3 [0323] Flow rate: 1 mL/min [0324] Run time: 45 min

[0325] The results are shown in Table 8.

TABLE-US-00009 TABLE 8 Molecular weight M.sub.w [Da] M.sub.n [Da] M.sub.w [Da] M.sub.n [Da] starch starch low MW low MW Tempering portion portion portion portion OSA Starch 1 90554 16278 451 364 3 h @150 C. 114480 18899 558 397 OSA Starch 2 152318 20938 452 363 3 h @150 C. 211872 24783 598 407 OSA Starch 5 2087236 107922 (comparative 3 h @150 C. 207371 15485 example) (M.sub.w: mass average of the molar mass; M.sub.n: number average of the molar mass)

[0326] The molecular weight of the OSA modified starches according to the present invention increases during tempering. The molecular weight of OSA modified starches not according to the present invention decreases during tempering.

H) Analysis of Viscosities of Solutions of OSA Modified Starches Before and after Tempering

TABLE-US-00010 TABLE 9 Analysis of viscosities Viscosity [MPas] Viscosity [MPas] of a 28.57% of a 15% by by weight weight aqueous aqueous solution solution of the of the OSA OSA modified Tempering modified starch starch OSA Starch 1 54 2 h @150 C. 62 3 h @150 C. 62 4 h @150 C. 65 6 h @150 C. 75 OSA Starch 2 74 3 h @150 C. 94 OSA Starch 5 246 (comparative example) 3 h @150 C. 46

[0327] The viscosity was measured on a Brookfield RV viscometer with spindle 3 at 100 rpm at 20 C.

[0328] The viscosity of an aqueous solution of the OSA modified starch according to the invention increases with the tempering. The viscosity of an aqueous solution of an OSA modified starch not according to the invention (OSA Starch 5's degradation is catalyzed by an acid) decreases with the tempering.

I) Analysis of the C.I.E.-LAB Color Values of the OSA Modified Starch Before and after Tempering

TABLE-US-00011 TABELLE 10 C.I.E-LAB-Farbwerte Tempering L-value a-value b-value Hi-Cap 100 L 96.3 0.2 2.1 Hi-CAP 100 L 2 h @150 C. 89.4 0.4 12.9 Hi-CAP 100 L 3 h @150 C. 84.2 1.9 16.6 Hi-CAP 100 L 4 h @150 C. 79.3 3.4 20.5 Hi-CAP 100 L 5 h @150 C. 74.3 5.2 22.0 Hi-CAP 100 L 6 h @150 C. 68.0 7.8 22.5 Hi-CAP 100 H 95.8 0.3 2.4 Hi-CAP 100 H 2 h @150 C. 84.4 1.8 15.9 Hi-CAP 100 H 3 h @150 C. 79.8 3.5 18.7 Hi-CAP 100 H 4 h @150 C. 74.2 5 21.1 Hi-CAP 100 H 6 h @150 C. 72.1 5.9 21.7 Hi-CAP 100 H 8 h @150 C. 61.9 9.3 21.4

[0329] The L-value gives the brightness, the -value gives the red-green content and the b-value gives the yellow-blue content of a color. The L-values are generally positive and lie between 0 for ideal black colors and 100 for ideal white colors.

Example 2: Analysis of the OSA Modified Starches According to the Present Invention in an Emulsion

[0330] Comparison of tempered (according to the invention) and not tempered (not according to the invention) enzymatically degraded OSA Starch 1 or land a comparative starch that was degraded under acid catalysis as emulsifier in an oil/water emulsion.

[0331] An oil in water emulsion was prepared with different OSA modified starches as follows.

[0332] 59.75% by weight water, 0.2% by weight citric acid and 0.05% by weight sodium benzoate, were added to a flask and mixed with a propeller stirrer until all ingredients were dissolved. Then 24% by weight starch were added. To this solution 16.0% by weight Delios V Oil (MCI Oil purchased from BASF) were added and the solution was further stirred for 3 min at 20 C.

[0333] Subsequently, the emulsion was tested for its thermostability. Samples were incubated in a water bath at 65 C. for 1 h, 3 h and 1 d, before the average particle diameter was determined. Additionally, the viscosities of the emulsions were determined.

[0334] The average particle diameter vias measured on a Light scattering particle size analyzer, LS 13320, Beckman Coulter.

[0335] Viscosities were measured on a Brookfield RV viscometer with spindle 3 at 100 rpm at 20 C.

[0336] Results are shown in Tables 11, 12 and 13.

TABLE-US-00012 TABLE 11 Viscosities and particle diameter d(90) after incubation of the oil in water emulsions comprising OSA modified starches for 1 h, 3 h and 1 d, at 20 C. or 65 C. OSA OSA Starch 5 Starch 5 OSA OSA (comparative (comparative Starch 1 Starch 1 example) example) Tempering 3 h @150 C. 3 h @150 C. Viscosity after 1 d 102 143 558 64 @20 C. [mPas] Viscosity after 1 d 46 59 182 30 @65 C. [mPas] D90 [m] fresh 0.81 0.51 1.25 1.40 D90 [m] after 0.82 0.51 1.31 1.38 1 d @20 C. D90 [m] after 20.6 0.51 1.28 1.39 1 h @65 C. D90 [m] after 3 h 2.85 0.51 1.32 1.38 @65 C. D90 [m] after 1 d 4.63 0.51 1.29 1.38 @65 C.

TABLE-US-00013 TABLE 12 Particle diameter D(4, 3) after incubation of the oil in water emulsion comprising OSA modified starches for 1 h, 3 h and 1 d, at 65 C. Average D(4, 3) Standard Tempering particle diameter deviation OSA Starch 1 0.506 0.269 Fresh emulsion OSA Starch 1 0.944 0.924 after 1 h @65 C. OSA Starch 1after 3 h 1.280 1.240 @65 C. OSA Starch 1 2.140 1.730 after 1 d @65 C. OSA Starch 1 3 h @150 C. 0.322 0.151 Fresh emulsion OSA Starch 1 3 h @150 C. 0.322 0.150 after 1 h @65 C. OSA Starch 1after 3 h 3 h @150 C. 0.333 0.148 @65 C. OSA Starch 1after 1 d 3 h @150 C. 0.410 0.148 @65 C.

[0337] The OSA modified starches that were tempered for 3 h at 150 C. and are thus according to the present invention, have a significantly smaller and more constant particle size in the emulsion compared to starches that were not tempered and are thus not according to the invention. Also a starch not according to the invention because it was degraded under acid catalysis (OSA Starch 5) does not show these small particle sizes.

[0338] Further, the standard deviation of the average particle diameter D(4,3) is much smaller for the emulsions comprising the OSA modified starches according to the present invention.

[0339] FIG. 1 shows the particle diameter distribution for the tempered OSA Starch 1, and FIG. 2 for the not tempered OSA Starch 1. FIG. 3 shows the particle diameter distribution for the tempered OSA Starch 5 and FIG. 4 for the not tempered OSA Starch 5 (FIGS. 3 and 4 are comparative examples).

[0340] A comparison of the data presented in these Figures show a significantly uniform, sharper, particle diameter distribution which is further more stable for the OSA modified starch based emulsions according to the present invention (OSA Starch 1), also when incubated at higher temperatures over a longer period of time.

TABLE-US-00014 TABLE 13 Viscosities of aqueous starch/oil emulsions Concentration Viscosity Viscosity starch/oil in the at 20 C. at 65 C. emulsion Tempering [mPas] [mPas] OSA Starch 1 24%/16% 102 46 OSA Starch 1 24%/16% 2 h @150 C. 120 52 OSA Starch 1 24%/16% 3 h @150 C. 142 62 OSA Starch 1 24%/16% 4 h @150 C. 168 64 OSA Starch 1 24%/16% 6 h @150 C. 235 68 OSA Starch 1 16.8%/11.2% 8 h @150 C. 452 75 OSA Starch 2 24%/16% 272 76 OSA Starch 2 24%/16% 3 h @150 C. 572 120

[0341] Viscosities were determined on a Brookfield RV viscometer with spindle 3 at 100 rpm at 20 C. and 65 C., respectively. The viscosities of the emulsions increase with increasing tempering time.

Example 3: Analysis of the OSA Modified Starches as Encapsulating Agents

[0342] An emulsion consisting of 60% by weight demineralized water, 31.86% by weight OSA Starch 1, 0.14% by weight ascorbic acid, 5.03% by weight Delios MCT Oil (BASF), 2.67% by weight of a 30% natural beta-carotene dispersion and 0.3% by weight alpha-tocopherol was prepared as described in Example 2, wherein the beta-carotene was dispersed and dissolved in the oil phase. The emulsion was then incubated in a water bath as described in Example 2.

[0343] Moreover, a sample of the emulsion was spray-dried after incubation for 1 d at 65 C. and reconstituted in water.

[0344] The spray-drying was performed in a Bchl B290 lab spray-dryer using the following settings [0345] Inlet temperature; 180 C. [0346] Outlet temperature: 80-90 C. [0347] Temperature of the emulsion: 50-60 C. [0348] Pump speed: 60-65% [0349] Aspirator: 80-85% [0350] Air pressure: 12 bar [0351] Air flow rate: 45%

[0352] All particle diameters were determined as described for Example 2. The results are shown in Tables 14 and 15.

TABLE-US-00015 TABLE 14 Particle diameters (D90) OSA Starch 1 + OSA Starch 1 + -carotene -carotene Tempering 3 h @150 C. D90 [m] frisch 0.57 0.36 D90 [m] nach 1 d 0.59 0.36 @20 C. D90 [m] nach 1 h 0.63 0.38 @65 C. D90 [m] 1.03 0.41 after 3 h @65 C. D90 [m] 1.23 0.39 after 6 h @65 C. D90 [m] 2.03 0.38 after 1 d @65 C. D90 [m] 1.28 0.38 after reconstitution of the spray-dried powder

TABLE-US-00016 TABLE 15 Particle diameters (D(4, 3)) Average D(4, 3) Standard particle deviation Tempering diameter [m] [m] OSA Starch 1 + 0.388 0.125 -carotene fresh emulsion OSA Starch 1 + 1.030 1.570 -carotene after 1 d @65 C. OSA Starch 1 + 0.591 0.453 -carotene after reconstitution of the spray-dried powder OSA Starch 1 + 3 h @150 C. 0.186 0.128 -carotene fresh emulsion OSA Starch 1 + 3 h @150 C. 0.208 0.130 -carotene after 1 d @65 C. OSA Starch 1 + 3 h @150 C. 0.231 0.126 -carotene after reconstitution of the spray-dried powder

[0353] The OSA modified starches according to the present invention, which were tempered for 3 ft at 150 C., show a significantly smaller and more uniform particle size in the emulsion compared to starches not tempered. This result was also found for the spray-dried powder after reconstitution.

[0354] Further, the standard deviation of the average particle diameter D(4,3) of the OSA modified starches according to the present invention is much smaller.

[0355] FIG. 5 shows the particle size distribution for the not tempered OSA Starch 1. FIG. 6 shows the particle size distribution for the tempered OSA Starch 1. With this comparison the more uniform and more stable particle size distribution of the tempered OSA Starch 1 is clearly shown.

[0356] FIG. 7 shows the particle size distribution of the not tempered OSA Starch 1+-carotene after reconstitution, and FIG. 8 shows the particle size distribution of the tempered OSA Starch 1+-carotene after reconstitution. Also this comparison shows the more uniform and more stable particle size distribution of the tempered OSA Starch 1+-carotene, even after reconstitution.

Example 3: Analysis of the OSA Modified Starches According to the Present Invention in Comparison with a Mixture of OSA Modified Starches and Free OSA-Maltose

[0357] Emulsions were prepared as described for Example 2 and the following tests have been carried out.

A) Blend of OSA Starch 1 or 2, which was not Tempered and OSA-Maltose:

[0358] In order to study the effect of the addition of OSA-maltose, i.e. partly replacing OSA Starch 1 or 2, which was not tempered with OSA-maltose on the emulsion stability, blends as set forth in Table 16 were prepared and the emulsion test conducted as described above.

[0359] 5% and 20%, respectively, of the OSA Starch 1 or 2 were replaced by OSA-maltose. The emulsion prepared from these blends were tested at ambient (20 C.) and elevated (65 C.) temperatures and the particle size distribution was investigated as described above. Comparison was made against OSA Starch 1 or 2, which was not tempered and the OSA Starch 1 or 2 which was tempered for 3 hours at 150 C.

TABLE-US-00017 TABLE 16 Fineness of emulsion (with added OSA-maltose)- D90 [m] of emulsions stored at elevated temperatures 95% OSA 80% OSA 95% OSA Starch 2 80% OSA Starch 2 Starch 1 not not Starch 1 not not tempered tempered tempered tempered 5% OSA-maltose 20% OSA-maltose fresh emulsion 0.79 0.54 0.64 0.95 3 h @ 65 C. 2.85 1.69 3.26 1.80 1 d @ 65 C. 4.56 3.13 4.16 2.56

[0360] FIG. 9 shows the particle size distribution for the mixture of 95% not tempered OSA Starch 1 plus 5% OSA-maltose.

[0361] FIG. 10 shows the particle size distribution for the mixture of 95% not tempered OSA Starch 2 plus 5% OSA-maltose,

[0362] FIG. 11 shows the particle size distribution for the mixture of 80% not tempered OSA Starch 1 plus 20% OSA-maltose.

[0363] FIG. 12 shows the particle size distribution for the mixture of 80% not tempered OSA Starch 2 plus 20% OSA-maltose.

[0364] There is no effect of OSA-maltose addition to OSA Starch 1 or 2 which is not tempered. Neither 5% nor 20% OSA-maltose addition (blending OSA-maltose with OSA Starch 1 or 2) revealed significant effects on emulsion stability compared with the OSA Starch 1 or 2 which was not tempered alone. Compared with the particle size/emulsions stability improvements of the tempered OSA Starch 1 or 2 respectively, the samples to which OSA-maltose was added show clearly inferior results. For example after 3 hours at 65 C. the D90 of the mixture of 80% not tempered OSA Starch 1 plus 20% OSA-maltose is 3.26, whereas it is 0.51 for tempered OSA Starch 1 according to the invention (see Table 11).

B) Blend of Tempered OSA Starch 2 and OSA-Maltose:

[0365] In order to study the effect of replacing 20% OSA Starch 2, which was tempered for 3 hours at 150 C. with OSA-maltose on the emulsion stability, blends as set forth in table 17 were prepared and the emulsion test was conducted as described above. The emulsion was exposed to ambient (20 C.) and elevated (65 C.) temperatures and the particle size distribution was monitored over time as described above.

TABLE-US-00018 TABLE 17 D90 [m] of emulsions stored at elevated temperatures 100% OSA Starch 2 80% OSA Starch 2 tempered for tempered for 3 h@150 C. 3 h@150 C. 0% OSA-maltose 20% OSA-maltose fresh emulsion 0.48 0.51 3 hours @ 65 C. 0.48 0.61 1 day @ 65 C. 0.49 0.62

[0366] FIG. 13 shows the particle size distribution of 100% OSA Starch 1 which was tempered for 3 h @150 C.

[0367] FIG. 14 shows the particle size distribution for the mixture of 80% OSA Starch 1 which was tempered for 3 h at 150 C. plus 20% OSA-maltose.

[0368] There was no positive impact after exchanging 20% of the OSA Starch 1 which was tempered for 3 h at 150 C. by OSA-maltose. The particle size distribution remained as stable as in the reference emulsion.

C) Emulsion Stability of Pure OSA-Maltose

[0369] Under the assumption that the used OSA-material had reacted sufficiently with maltose to yield OSA-modified-maltose (OSA-maltose) and to compare its emulsion stabilizing properties as such a further emulsion test with OSA-maltose as sole emulsifier was conducted.

[0370] This test is mimicking a typical formulation with 24% OSA Starch 1 or 2 solids that is, 24% solids OSA-maltose was used, to fully replace the OSA modified starch.

TABLE-US-00019 TABLE 18 D90 [m] of emulsions stored at elevated temperature 24% OSA-maltose solids containing 0.72% OSA (added as 40% solution) 0.72% OSA fresh emulsion 3.91 217.00 3 hours @65 C. 149.00 208.00 1 day @ 65 C. 185.00 171.00

[0371] An emulsion was obtained with a D90 of 3.91 m. When stored at elevated temperatures of 65 C., however the particle size increased considerably (D90=185 m), indicating much inferior emulsion stability compared to the emulsions made with OSA Starch 1 or 2 which was not tempered. The considerable increase in oil droplet size upon storage conditions at 65 C. however reveals that OSA-maltose is not suitable for emulsion stabilization at elevated temperatures in general.

[0372] A further emulsion produced for comparison made with 0.72% pure OSA (same OSA content as in 24% OSA-maltose containing 3% OSA) revealed that pure OSA is not as effective as the OSA-maltose for emulsion stabilization.

[0373] FIG. 15 shows the particle size distribution of 24% OSA-maltose solids containing 0.72% OSA.

[0374] FIG. 16 shows the particle size distribution of 0.72% pure OSA.