Water-soluble, high-molecular-weight chitosan powders

Abstract

The poor water solubility of high molecular weight chitosan has limited its use in areas such as food and beverage products, post-harvest treatments, cosmetic and pharmaceutical products, medical treatments, and environmental pollution treatments. Disclosed is a high-molecular-weight, solid-state chitosan powder that is completely water-soluble.

Claims

1. A solid-state salt of a chitosan and an acidic amino acid; wherein: (a) said chitosan has an average molecular weight at least 70 kDa; (b) said acidic amino acid comprises aspartic acid, glutamic acid, or both; (c) the mass of said acidic amino acid is at least 0.7 times the mass of said chitosan; and (d) said salt is a solid-state salt, that has the ability to completely dissolve in water at 25° C. to yield an aqueous solution comprising at least 1% chitosan by mass; wherein: (e) said solid-state salt is essentially free of mineral acid; (f) wherein said solid-state salt is essentially free of acetic acid, and said solid-state salt is essentially free of acetate salts; (g) said solid-state salt is essentially free of surfactant; and (h) said solid-state salt is not a coating on an agricultural product.

2. The solid-state salt of claim 1, wherein said salt has the ability to dissolve in a Tris-HCI aqueous buffer solution having an initial pH of 6.8-7.0 at a rate at least twice as fast, under otherwise comparable conditions, as the rate at which unmodified chitosan dissolves in a Tris-HCI aqueous buffer solution having an initial pH of 6.8-7.0 and containing the same acidic amino acid in the form of a free amino acid, to reach the same final concentrations of the dissolved chitosan and the dissolved free acidic amino acid.

3. The solid-state salt of claim 1, wherein said chitosan has an average molecular weight at least 100 kDa.

4. The solid-state salt of claim 1, wherein said chitosan has an average molecular weight at least 300 kDa.

5. The solid-state salt of claim 1, wherein said chitosan has an average molecular weight at least 700 kDa.

6. The solid-state salt of claim 1, wherein said chitosan has an average molecular weight at least 1000 kDa.

7. The solid-state salt of claim 1, wherein said acidic amino acid consists essentially of aspartic acid, glutamic acid, or both.

8. The solid-state salt of claim 1, wherein the mass of said acidic amino acid is at least 0.8 times the mass of said chitosan.

9. The solid-state salt of claim 1, wherein the mass of said acidic amino acid is at least 1.0 times the mass of said chitosan.

10. The solid-state salt of claim 1, wherein said solid-state salt has the ability to dissolve in water at 25° C. to yield a solution comprising at least 4% chitosan by mass.

11. The solid-state salt of claim 1, wherein said solid-state salt has the ability to dissolve in water at 25° C. to yield a solution comprising at least 5% chitosan by mass.

12. The solid-state salt of claim 1, wherein said solid-state salt has the ability to dissolve in water at 25° C. to yield a solution comprising at least 6% chitosan by mass.

13. The solid-state salt of claim 1, wherein said solid-state salt has the ability to dissolve in water at 25° C. to yield a solution comprising at least 7% chitosan by mass.

14. The solid-state salt of claim 1, wherein said solid-state salt has the ability to dissolve in water at 25° C. to yield a solution comprising at least 8% chitosan by mass.

15. The solid-state salt of claim 1, wherein said solid-state salt has the ability to form a gel upon dissolving in water.

16. The solid-state salt of claim 1, wherein said solid-state salt is essentially free of salts of mineral acids.

17. The solid-state salt of claim 1, wherein said solid-state salt is essentially free of plasticizer.

18. A process for making an aqueous chitosan solution, said process comprising dissolving the solid-state salt of claim 1 in water.

19. The process of claim 18, wherein the water is essentially free of acetic acid, essentially free of acetate salts, essentially free of mineral acids, and essentially free of salts of mineral acids.

Description

MODES FOR CARRYING OUT THE INVENTION

Example 1: Materials

(1) Chitosan powders, MW 789 kDa and 1017 kDa (Kitto Life, Korea), were kindly provided by the Department of Food Science and Technology, Catholic University of Daegu, Korea. Acetic acid was purchased from Aldrich Chemicals (Milwaukee, Wis., U.S.A.). Aspartic acid and glutamic acid were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All reagents and chemicals were of analytical grade.

Example 2: Preparation of Solutions of Chitosan with Amino Acids or Other Acids

(2) For each of the mixtures listed in Tables 1 & 2, the acid (viz., acetic acid, aspartic acid, glutamic acid, or combination of acids) was first dissolved in warm, distilled water (60° C.). Chitosan powder was then added to the acidic solution, which was subjected to continuous magnetic stirring. After the chitosan had dissolved, the resulting chitosan solutions were stored in screw-capped glass bottles until used or until analyzed. Solutions were dried either by air-drying a 2 mm deep solution at 25° C. in a plastic container under a fume hood, or by oven-drying at 80-90° C. On an industrial scale, spray-drying might be used instead. Dried powders were stored in a closed container at 4° C. until used. (As of the filing date of the present application, we have not measured data concerning the shelf life of these preparations.)

Example 3: Determination of Viscosity and pH

(3) Viscosity was measured with a viscometer (model DV-I+, Brookfield Engineering Labs Inc., Middleboro, Mass., USA) at 60 rpm using a T-F spindle, with triplicate measurements. The pH values of the prepared chitosan solutions were measured with a pH meter (Accumet AP61, Fisher Scientific, Pittsburgh, Pa., USA), with triplicate measurements.

Example 4: Centrifuging and Filtering the Chitosan Solutions

(4) Each chitosan solution was transferred into a 15 mL clean centrifuge tube, and then centrifuged at 5000 rpm for 20 min. The degree of solubilization was assessed by visual observation of any precipitated particles. To evaluate undissolved solids, a 1% chitosan solution was diluted with distilled water at a 1:2 ratio (v/v) chitosan solution:distilled water. The diluted mixture was passed through a filter paper under vacuum filtration. The filter paper with any residue was dried and weighed to calculate the residue percentage:

(5) $\mathrm{Residue}\mathrm{percentage}=\frac{\begin{array}{c}\mathrm{Weight}\mathrm{of}\mathrm{dried}\mathrm{filter}\mathrm{paper}\mathrm{after}\mathrm{filtration}-\\ \mathrm{Weight}\mathrm{of}\mathrm{dry}\mathrm{filter}\mathrm{paper}\mathrm{before}\mathrm{filtration}\end{array}}{\begin{array}{c}\mathrm{Total}\mathrm{weight}\mathrm{of}\mathrm{chitosan}\mathrm{and}\\ \mathrm{amino}\mathrm{acid}\mathrm{added}\mathrm{to}\mathrm{the}\mathrm{solution}\end{array}}\times 100\%$

Examples 5-22: Dissolution Times, and Re-Dissolution Times (after Drying) for Various Combinations of Chitosan with Aspartic Acid, Glutamic Acid, and Acetic Acid

(6) Dissolution times, and re-dissolution times (after drying) for various combinations of chitosan with aspartic acid, glutamic acid, and acetic acid are shown in Tables 1 and 2. Table 1 shows data for 789 kDa chitosan, and Table 2 shows data for 1017 kDa chitosan. The re-dissolution rates for the chitosan/amino acid combinations (after drying) were at least twice as fast as the original dissolution rates (before drying).

(7) TABLE-US-00001 TABLE 1 Dissolution and re-dissolution times (minutes) for various concentrations (wt %) of chitosan (MW 789 kDa) with Aspartic Acid (Asp), Glutamic Acid (Glu), or Acetic Acid (AA) Chitosan Organic acid Re-dissolution concentration concentration (wt %) Dissolution Time (min) for (wt %) Asp Glu AA Time (min) dried powder 1 1 0 0 20 5 1 0.8 0 0 30 15  1 0.7 0 0 >180  N/A 1 0.5 0 0 Still had N/A some particles 1 0 1 0 35 15  1 0.4 0.25 0.25 30 8 1 0.5 0.25 0.25 25 5 3 3 0 0 25 (glass rod N/A stirring) 5 5 0 0 30 (glass rod N/A stirring) 8 8 0 0 40 (glass rod N/A stirring)

(8) TABLE-US-00002 TABLE 2 Dissolution and re-dissolution times (minutes) for various concentrations (wt %) of chitosan (MW 1017 kDa) with Aspartic Acid (Asp), Glutamic Acid (Glu), or Acetic Acid AA) Chitosan Organic Acid Re-dissolution Percentage Percentage (wt %) Dissolution Time (min) for (wt %) Asp Glu AA Time (min) dried powder 1 1 0 0 30 5 1 0.8 0 0 >180  N/A 1 0.7 0 0 Still had N/A some particles 1 0 1 0 40 15  1 0.4 0.25 0.25 35 8 1 0.5 0.25 0.25 30 5 4 4 0 0 30 (glass rod N/A stirring) 1 0 0 1 >300  Only partially re-dissolved

(9) Notes to Tables 1 and 2: N/A: Not available.

(10) All measurements were carried out with 100 mL of solution.

(11) All ratios are given on a mass (or weight) basis

(12) Dissolving was aided with magnetic stirring unless otherwise indicated.

(13) For 789 kDa chitosan at room temperature, the maximum concentration of chitosan in solution was ˜8%.

(14) For 1017 kDa chitosan at room temperature, the maximum concentration of chitosan in solution was ˜4%.

(15) Table 1 indicates that aspartic acid (Asp) was most effective in solubilizing 789 kDa chitosan. The maximum concentration of 789 kDa chitosan in solution was ˜8%, a concentration that could not be achieved with acetic acid. At a chitosan:Asp mass ratio of 1:0.7, it took more than 3 hours for complete dissolution of 1% chitosan. When the chitosan:Asp mass ratio was below 1:0.7, chitosan dissolved only incompletely. However, a slight change in the chitosan:Asp ratio to 1:0.8 or 1:1 led to much faster dissolution times for 1% chitosan—30 and 20 min, respectively. The preferred chitosan:Asp mass ratio is 1:1, or about 1:1. The amount of Asp or Glu can be greater than the amount of chitosan, which could accelerate solubilization; however, for economic reasons it is usually preferred not to use substantially more of the amino acid than would be present in a 1:1 mass ratio with chitosan. Even at the preferred 1:1 ratio, increasing the chitosan and Asp concentrations to 3%, 5%, or 8% required longer dissolution times (25, 30, and 40 minutes, respectively, compared to 20 minutes at 1%). At concentrations of 3% and above, solutions of the 789 kDa chitosan were thick and gel-like.

(16) Glutamic acid (Glu) can be used as an alternative to (or in addition to) Asp. The 789 kDa chitosan took longer to dissolve with Glu than with Asp (35 min v. 20 min.) at a chitosan:amino acid 1:1 mass ratio, and 1% chitosan. Combinations of Asp, Glu and acetic acid (AA) at ratios of 0.4:0.25:0.25 or 0.5:0.25:0.25 dissolved 1% chitosan in 30 or 25 min, respectively.

(17) After drying in air, the chitosan:amino acid powders with higher Asp concentrations had shorter re-dissolution times, ranging from 5-15 min. By contrast, after the chitosan:acetic acid solution was dried, the resulting powder could only be partially re-dissolved in water.

(18) Qualitatively similar results were seen with 1017 kDa chitosan. Because the molecular weight was significantly higher than 789 kDa, dissolution took substantially longer. It took over three hours to completely dissolve 1% chitosan when the Asp mass ratio (e.g., 0.8) was below the preferred chitosan:Asp mass ratio of 1:1. At an Asp mass ratio of 1:0.7 or less, 1% chitosan dissolved only incompletely. At the preferred 1:1 mass ratio, it was possible to prepare an aqueous solution containing up to ˜4% 1017 kDa chitosan, a concentration that was not possible with acetic acid.

Examples 23-29: Measurements of Chitosan Molecular Weight Before and after Drying

(19) The molecular weight of chitosan (having an initial MW of 1017 kDa) after dissolution with various acids or combinations was determined before and after air drying or oven drying. Molecular weight was measured by gel permeation chromatography (GPC), with details as described in Table 3. Briefly, in gel permeation chromatography a polymer is introduced onto a column packed with beads having certain porosity and particle size. Larger molecules are less able to permeate the pores in the beads, and larger molecules therefore elute faster. A shorter retention time indicates a higher molecular weight.

(20) All combinations tested in this set of experiments employed 1% chitosan (1017 kDa). The chitosan was dissolved with one of the following: (a) 1% AA with air drying (25° C.); (b) 1% Asp with air drying (25° C.); (c) 1% Asp with oven drying (80-90° C.); (d) 1% Glu with air drying (25° C.); (e) 1% Glu with oven drying (80-90° C.); (f) a mixture of 0.5% Asp, 0.25% Glu and 0.25% AA with air drying (25° C.); (g) a mixture of 0.5% Asp, 0.25% Glu and 0.25% AA with oven drying (80-90° C.). Because unmodified 1017 kDa chitosan is not water soluble, combination (a), the solution of 1017 kDa chitosan with 1% acetic acid, was used as the control.

(21) The control had a retention time of 9.56 min in the gel permeation column. The other treatments all had retention times between 9.22 and 9.46 min, within 3.6% of the control. These observations suggested that the molecular weight of the 1017 kDa chitosan did not change substantially after dissolution by Asp, Glu, or acetic acid. These observations also showed that neither air drying (25° C.) nor oven drying (80-90° C.) had a substantial effect on the chitosan's molecular weight.

(22) TABLE-US-00003 TABLE 3 Gel permeation chromatography. GPC Equipment Shimadzu Class-VP V6.14 SP2 Column Shodex PLgel column Detector Refractive index detector Mobile phase Water Flow rate 1 mL/min Temperature 40° C. Calibration Pullulan standard

Examples 30-47: Visual Observations of, and pH Values of Freshly-Prepared, Centrifuged, and Filtered Chitosan Solutions

(23) For both the 789 and 1017 kDa MW chitosans, a 1% solution was viscous, and a 3% or greater solution was gel-like. (See Tables 4 and 5). Some visible particles were observed in a 1% chitosan solution when 789 kDa chitosan was dissolved in 0.5% Asp, or when 1017 kDa chitosan was dissolved in 0.7% Asp. Other treatments yielded chitosan solutions that appeared to be homogeneous and viscous, with no precipitant visible following centrifugation. Vacuum filtration was used to evaluate the degree of solubilization; the less residue that could be collected by filtration, the higher the inferred degree of solubilization. Increasing the concentration of Asp or Glu decreased the amount of residue collected on filter paper. The pH values decreased with increased concentrations of Asp.

(24) TABLE-US-00004 TABLE 4 Visual observations, pH values, and residue after filtration for chitosan (MW 789 kDa) in various mixtures Visual observation Residue After Chitosan Organic Acid Filtration Concentration concentration (%) and Drying (%) pH Asp Glu AA Freshly Prepared After Centrifuge (% by mass) 1 3.02 1 0 0 Viscous solution No precipitation <0.2% 1 3.16 0.8 0 0 Viscous solution No precipitation <0.5% 1 3.42 0.7 0 0 Viscous solution No precipitation <2.0% 1 N/A 0.5 0 0 Some undissolved N/A N/A particles visible 1 3.33 0 1 0 Viscous solution No precipitation <0.2% 1 3.40 0.4 0.25 0.25 Viscous solution No precipitation <0.5% 1 3.48 0.5 0.25 0.25 Viscous solution No precipitation <0.2% 3 N/A 3 0 0 Gel N/A N/A 5 N/A 5 0 0 Gel N/A N/A 8 N/A 8 0 0 Gel N/A N/A

(25) TABLE-US-00005 TABLE 5 Visual observations, pH values, and residue after filtration for chitosan (MW 1017 kPa) in various mixtures Visual observation Residue After Chitosan Organic Acid Filtration concentration concentration (%) and Drying (%) pH Asp Glu AA Freshly Prepared After Centrifuge (% by mass) 1 3.06 1 0 0 Viscous solution No precipitation <0.8% 1 3.47 0.8 0 0 Viscous solution No precipitation <2.0% 1 N/A 0.7 0 0 Some undissolved No precipitation N/A particles visible 1 3.38 0 1 0 Viscous solution No precipitation <1.0% 1 3.35 0.4 0.25 0.25 Viscous solution No precipitation <1.0% 1 3.22 0.5 0.25 0.25 Viscous solution No precipitation <0.8% 3 N/A 3 0 0 Gel N/A N/A 4 N/A 4 0 0 Gel N/A N/A Notes to Tables 4 and 5: N/A: not applicable. Asp = aspartic acid, Glu = glutamic acid, AA = acetic acid
Notes to Tables 4 and 5: N/A: not applicable. Asp=aspartic acid, Glu=glutamic acid, AA=acetic acid

Examples 48-58: Viscosity of Chitosan Solutions with Different Percentages of Asp or AA

(26) The viscosity of 1-8% chitosan solutions is shown in Table 6. Increasing the chitosan concentration increased the viscosity of the solution. At a given chitosan concentration, the higher MW chitosan solution was more viscous. The viscosity of a chitosan solution prepared with AA was higher than the viscosity of a solution prepared with Asp in most (but not all) cases. Contrary to a report in the literature that the viscosity of a solution of chitosan with an organic acid is positively correlated with the number of carbons in the organic acid, we found that aspartic acid, with more carbon atoms, produced a lower viscosity chitosan solution than did acetic acid, with fewer carbon atoms. Without wishing to be bound by this hypothesis, we propose that the two carboxyl groups of aspartic acid (or glutamic acid) enhance the solubility of the chitosan polymer more than does the single carboxyl group of acetic acid. Aspartic acid is more effective at dissolving high MW chitosan, and aspartic acid can dissolve higher concentrations of chitosan than acetic acid can.

(27) TABLE-US-00006 TABLE 6 Viscosities of various chitosan solutions Viscosity Speed Sample (cP) Spindle (rpm) 1% chitosan (1017 kDa) & 1% Asp 241 T-F 60 3% chitosan (1017 kDa) & 3% Asp 7820 T-F 60 4% chitosan (1017 kDa) & 4% Asp 14725 T-F 60 1% chitosan (1017 kDa) & 1% AA 492 T-F 60 1% chitosan (789 kDa) & 1% Asp 62 T-F 60 3% chitosan (789 kDa) & 3% Asp 590 T-F 60 5% chitosan (789 kDa) & 5% Asp 8925 T-F 60 8% chitosan (789 kDa) & 8% Asp 20350 T-F 60 1% chitosan (789 kDa) & 1% AA 66 T-F 60 3% chitosan (789 kDa) & 3% AA 770 T-F 60 5% chitosan (789 kDa) & 5% AA 10678 T-F 60 Notes to Table 6: Asp = aspartic acid, AA = acetic acid.

Examples 59-67: Dissolving and Re-Dissolving Chitosan with Various Organic Acids

(28) Various organic acids including acetic, ascorbic, malic, and citric acids were tested for their ability to dissolve high molecular weight (HMW) chitosan (789 kDa or 1017 kDa) in deionized water. The resulting solutions were dried, and the ability of the dried powder to re-dissolve in deionized water was also tested.

(29) We observed: 1. It took more than 5 hours for acetic acid (1%) to dissolve HMW chitosan (1%). After the acetic acid/chitosan solution had been dried, the resulting powder could only be partially re-dissolved. 2. Ascorbic acid (1%) could dissolve 789 kDa chitosan (1%) within 2 hours, and 1017 kDa chitosan within three hours. In both cases, after drying the resulting powder could be re-dissolved within one hour. However, the resulting chitosan solution gradually turned brown with visible precipitates after two days of storage at room temperature, presumably due to oxidation of ascorbic acid. Such oxidation presumably affects the solubility of chitosan and other chemical properties of the chitosan solution, although we have not yet quantitatively tested those effects. Ascorbic acid is therefore not a practical reagent for producing water-soluble HMW chitosan powders. By contrast, there were no observed changes in physical properties (e.g., color) when chitosan/aspartic acid or chitosan/glutamic acid was re-dissolved in water and allowed to stand for five days at room temperature. Even so, the shelf life of the novel powder will be longer than the shelf life of a solution, so it will usually be preferred to store the composition as a powder until it is ready for use. 3. When chitosan was mixed with a malic acid solution or a citric acid solution under constant stirring for over 24 hours, followed by filtration, drying, and weighing, about 10% (for malic acid) and over 80% (for citric acid) of the chitosan remained as undissolved solid particles. In other words, malic acid and citric acid were both poor solubilizers of chitosan. 4. None of the organic acids we tested, other than aspartic acid and glutamic acid, were effective and practical for dissolving and re-dissolving high molecular weight chitosans. Each of the other organic acids tested had one or more drawbacks: viz., the acids were poor solubilizers of HMW chitosan, or the dried powder could not be fully redissolved, or oxidation products appeared when a solution was allowed to stand. Of the organic acids tested, only aspartic acid and glutamic acid overcame all these problems.

(30) Comparison to Huanbutta et al. and to Orienti et al.

(31) Table 7 below compares experimental observations made with our novel compositions to results previously reported by Orienti et al. (2002) or Huanbutta et al. (2013), which represent the closest prior work known to the inventors. The entries in Table 7 corresponding to Orienti et al. (2002) and Huanbutta et al. (2013) reflect what was reported in the respective papers; the present inventors have made no attempts to replicate those experiments. The entries in Table 7 for the “Present Invention” reflect actual experimental observations made by the present inventors. Table 7 shows that the novel compositions possess significant advantages over the earlier compositions.

(32) TABLE-US-00007 Room Spray Drying Temperature Air Spray Drying (Huanbutta et Oven Drying Drying Drying Method; citation (Orienti et al. 2002) al., 2013) (Present Invention) (Present Invention) Molecular weight of chitosan 600 kDa 45 kDa or 789 kDa or 789 kDa or 200 kDa 1017 kDa 1017 kDa Was chitosan found to be soluble soluble soluble soluble soluble in amino acid solution? Ratio of chitosan:amino acid in 1:1 or 1:2 not expressly 1:1 1:1 the solution (molar ratio, based on stated (possibly (mass ratio) (mass ratio) monomer) 3.5% w/w in aqueous solution, based on a reference to Nunthanid et al., 2009, but this is not clear) Temperature for drying Inlet: 105° C. Inlet: 140° C. 80-90° C. 25° C. chitosan/amino acid mixture Outlet: 80-90° C. (chitosan salt) Re-dissolving concentration of 20 mg/10 mL not stated 20 mg/10 mL 20 mg/10 m chitosan salt in aqueous or or solution 200 mg/10 mL (ten 200 mg/10 mL (ten times higher) times higher) Did the dried chitosan salt re- Swelled after 24-48 h; Swelled Soluble within 15 min Soluble within 15 dissolve in acidic buffer (pH did not expressly state (200 kDa) after min 2)? whether the 120 min; did composition was not expressly soluble, only that it state whether swelled the composition was soluble, only that it swelled Did the dried chitosan salt re- not reported not reported Soluble within 15 min Soluble within 15 dissolve in water (pH 7)? min Did the dried chitosan salt re- not soluble; did not Less swelling For 789 kDa For 789 kDa dissolve in Tris-HCl buffer? swell at pH 6.8 (a) Soluble at (a) Soluble at (pH 6.8-7) compared to 200 mg/10 mL after 1 h 200 mg/10 mL after pH 1.2; did not (pH of the buffer 1 h expressly state changed to 6.2 in the (pH of the buffer whether soluble process) changed to 6.2 in the (b) Soluble at process) 20 mg/10 mL after 1 h (b) Soluble at (pH of the buffer 20 mg/10 mL after changed to 6.7 in the 1 h process) (pH of the buffer For 1017 kDa changed to 6.7 in the (a) First swelled and then process) dissolved (soluble) at For 1017 kDa 200 mg/10 mL after 1 h (a) First swelled and (pH of the buffer then dissolved changed to 6.7 in the (soluble) at process) 200 mg/10 mL after (b) First swelled and 1 h then dissolved (soluble) (pH of the buffer at 20 mg/10 mL after 1 h changed to 6.7 in the (pH of the buffer process) changed to 6.7 in the (b) First swelled and process) then dissolved (soluble) at 20 mg/10 mL after 1 h (pH of the buffer changed to 6.7 in the process) Stirred when re-dissolving? no no yes Yes

(33) The composition of the present invention is soluble in both acidic and neutral aqueous solutions; and it is evidently soluble at much higher concentrations than has previously been reported in the literature.

(34) Without wishing to be bound by this hypothesis, one possibility is that the drying method used may have a surprisingly important effect on the solubility of the chitosan salt compositions. We used a lower drying temperature than did Orienti et al. (2002) or Huanbutta et al. (2013). We used moderate-temperature oven drying or room-temperature air drying, while the cited references used spray drying. Although the solutions were exposed to high temperatures for a relatively short time during the spray drying, the surface area exposed to the heat stream during spray-drying is relatively high. The high surface area during exposure to high temperature may promote thermal decomposition, and thus affect a polymer's physicochemical properties, such as the distribution of electrostatic charges, and thus affect its solubility.

(35) Stirring may also affect solubility properties. Our “re-dissolving” mixtures were always stirred, while those of Orienti et al. (2002) and Huanbutta et al. (2013) were evidently not stirred. Note: We have not attempted to replicate the experiments of Orienti et al. (2002) or Huanbutta et al. (2013), either with or without stirring; nor have we carried out “re-dissolving” experiments on our compositions without stirring.

Definitions

(36) The term “solid-state salt” of a chitosan and an acidic amino acid should not be given an overly technical interpretation. Rather, as used in the specification and claims, the term “solid-state salt” of a chitosan and an acidic amino acid (particularly, aspartic acid, glutamic acid, or both) refers to a composition having the following properties: (a) The composition is in the solid state. (b) The composition contains both chitosan and an acidic amino acid, which have at least partially reacted with one another. (c) At least some of the amino groups of the chitosan are protonated. It is not necessary for all amino groups of the chitosan to be protonated. (d) At least some of the carboxyl groups of the acidic amino acid are deprotonated. It is not necessary for all carboxyl groups of the acidic amino acid to be deprotonated. (e) Water molecules may optionally be present in the composition. E.g., waters of hydration may optionally be present. Provided that the composition is in the solid state, the possible presence of water molecules does not remove the composition from the definition of a “solid-state salt.” (g) The amino groups of the chitosan and the carboxyl groups of the acidic amino acid may or may not be in a stoichiometric ratio with one another. An excess of either is permissible, as is a stoichiometric ratio. Also, unless context clearly indicates otherwise, the term “chitosan powder” has the same meaning as “solid-state salt” of a chitosan and an acidic amino acid” as defined above.

(37) As used herein, the term “essentially free of” is the converse of the well-known term “consisting essentially of.” Just as the transitional phrase “consisting essentially of” limits the scope of an invention to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the invention, the transitional phrase “essentially free of” means that the excluded component(s) either are absent from the composition, or if present they are present in such low concentration(s) that they do not materially affect the basic and novel characteristics of the invention as compared to the characteristics of an otherwise identical composition in which the excluded component(s) are entirely absent.

(38) As used herein, the term “mineral acid” means an inorganic compound that releases hydrogen ions when dissolved in water, particularly strong inorganic acids such as sulfuric acid, hydrochloric acid, and nitric acid. By contrast, an acidic compound containing a carboxylic acid group (—COOH) is considered an organic acid, and is not considered a “mineral acid.” Thus, by way of example, amino acids such as glutamic acid and aspartic acid are not considered “mineral acids.” By way of further example, the following would be considered “mineral acids”:phosphoric acid, perchloric acid, hydrofluoric acid, and sulfurous acid.

(39) As used herein, a “surfactant” is an amphiphilic compound containing both hydrophobic groups and hydrophilic groups that can help to disperse otherwise insoluble hydrophobic compounds and hydrophilic compounds with one another, typically by forming micelles. Examples of surfactants include polysorbate 80, sodium stearate, and sodium dodecylbenzenesulfonate. Examples of compounds that are not considered “surfactants” include chitin, chitosan, and amino acids such as aspartic acid and glutamic acid

(40) As used herein, a “plasticizer” is a substance incorporated into a polymer that improves the polymer's flexibility, workability and processability, and decreases brittleness and shrinking during handling and storage. A plasticizer reduces the tension of deformation, hardness, density, viscosity and electrostatic charge of the polymer, while increasing the polymer chain's flexibility, resistance to fracture and dielectric constant. In addition to water, commonly used plasticizers include polyols (e.g., glycerol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol and polyethylene glycol, propylene glycol), sorbitol, mannitol, xylitol, fatty acids, mono-, di- and oligo-saccharides (glucose, mannose, fructose, sucrose), ethanolamine, triethanolamine, vegetable oils, lecithin, and waxes. Compositions of the present invention can optionally include a plasticizer, but in preferred embodiments no plasticizer is used.

(41) As used herein, to “completely dissolve” a solid material, particularly to “completely dissolve” a solid-state salt of a chitosan and an acidic amino acid in water, means that no undissolved solid residue remains at all; or that any undissolved solid residue that remains is less than two percent (<2.0%) of the original solid material by mass, preferably less than one percent (<1.0%), more preferably less than four-fifths of one percent (<0.8%), more preferably less than one-half percent (<0.5%), and more preferably less than one-fifth of one percent (<0.2%)

(42) The complete disclosures of all references cited in this specification are hereby incorporated by reference, as is the complete disclosure of priority application 62/255,697. In the event of an otherwise irreconcilable conflict, however, the present specification shall control.