Patent Application
20180057614
The present invention relates to a method of depolymerization of alginic acid, and the depolymerised alginic acid obtained by the method.
Alginate and alginic acid are the generic terms applied to a natural occurring hydrophilic polysaccharide distributed widely in the cell walls of brown algae and certain microorganisms. In extracted form it absorbs water quickly; it is capable of absorbing 200-300 times its own weight in water.
Alginate is used in various pharmaceutical preparations such as gastric, anti-reflux medication. Alginate is further used as an impression-making material in dentistry, prosthetics, lifecasting and occasionally for creating positives for small-scale casting. It is also used in the food industry, as thickener and gelling agent, and as dietary fibre. Moreover, alginate is used in industrial applications such as textile printing, welding rods and paper coating. Calcium alginate is used in different types of medical products, including burn dressings that promote healing and can be removed with less pain than conventional dressings. Also, due to alginate's biocompatibility and simple gelation with divalent cations such as calcium, it is widely used for cell immobilization and encapsulation.
For many of the above usages such as as gastric, anti-reflux medication and dietary fibre, low molecular weight alginates are needed since a low viscosity of the final product is desired, even though alginates are dosed at high concentrations.
Currently, production methods are based on thermal depolymerisation of intermediates, i.e. alginic acid, or of finished products, i.e. sodium alginate. [H. K. Holmea, K. Lindmoa, A. Kristiansena, O. Smidsrød Thermal depolymerization of alginate in the solid state, Carbohydr. Polym. 2003, 54, 431-438].
KR 20010100250 describes a method comprising the steps of suspending powdered brown seaweed and a powdered tangle in water, leaving it at room temperature for 30 min. and thereafter pre-treating the material with a microwave-treatment.
US 2011/0028708 describes a microwave treatment of an alginic acid material in water.
However, a lean, continuous, and cost-effective method is still desired.
Moreover, seaweeds commonly used for alginate production can under certain storage conditions or due to seasonality occasionally lead to high microbiological count in the final alginate product.
It is an object of embodiments of the invention to provide a lean and cost-effective method that minimizes mechanical or manual handling. It is a further object of embodiments of the invention to provide a method for depolymerisation of alginic acid with a low usage of chemicals for environmental reasons. It is furthermore an object of embodiments of the invention to provide a method which reduces the microbial count in the final alginate product. It is furthermore an object of embodiments of the invention to provide a method which also result in a sterilized depolymerised alginateproduct.
It has been found by the present inventor(s) that using the herein disclosed microwave treatment an effective and fast method for depolymerisation of alginic acid is obtained. In one experiment, the weight average molecular weight of alginic acid is reduced to 43 kDa from 270 KDa after 7 min and 40 sec of microwave treatment. In comparison, treatment in a traditional oven at 105° C. for 2 hours results in a slightly higher weight average molecular weight, i.e. 56 kDa.
Further, no additional processing such as addition of chemicals or storage of intermediates is required for the microwave treatment. Instead, it offers the possibility of setting up a continuous process. Furthermore microwave treatment is an effective method for killing the microorganisms if any in the alginic acid starting material. Results from lab experiments show that 5 minutes of microwave treatment of a sample was sufficient to reduce the total plate count from 104 to <100 colony forming units (cfu)/g, and for mesophilic spores from 104 to <10 cfu/g. Pilot scale results showed that less than 3 minutes of microwave treatment of a sample was sufficient to reduce the total plate count from 104 cfu/g to <100 cfu/g.
So, in a first aspect the present invention relates to method of depolymerisation of alginic acid, said method comprising the steps of:
In a further aspect, the invention relates to a depolymerised alginic acid material obtainable by the method as described herein.
Disclosed herein is a method of depolymerisation of alginic acid, said method comprising the steps of:
Alginate and alginic acids are the linear co-polymers comprised of 1-4 linked 13-D-mannuronic and α-L-guluronic acid as the monomeric building blocks (salts; mannuronate and guluronate). The monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks) or alternating M and G-residues (MG-blocks).
The pKa values of mannuronic and guluronic acid are 3.38 and 3.65 respectively, which limits the solubility of polymannuronic and polyguluronic acid in water at low pH. Alginates with alternating mannuronic and guluronic acid residues (MG) have the highest acid solubility.
In nature alginate is mainly limited to the marine brown algae, Phaeophyta, although extracellular polymeric material resembling alginate from brown algae are also produced by soil bacteria such as Azotobacter vinelandii and several species of Pseudomonas. Alginate exist in the brown algae, as the most abundant polysaccharide comprising up to 40% of the dry matter, and 2-7% of the weight of the wet seaweed. It is located in the intercellular matrix as a gel containing sodium, calcium, magnesium and other multivalent cations. Its main function is structural giving both strength and flexibility to the algal tissue.
The starting material for the present method is preferably based on natural alginate from seaweed. However, it may also be based on alginate arising from Azotobacter vinelandii and other microroganims.
Traditionally alginate processing consists of several steps. The first step after harvesting, and optionally drying of the seaweed, may be milling and washing. Thereafter the alkaline extraction process starts, and after a number of different processing steps pure alginic acid is produced. The alginic acid serves as the raw material for the different alginate salts and ester.
One way to isolate alginate from the biomass is to lower the pH below the pKa of the acid residues. With decreasing pH, calcium and other cations bound by the caboxylic acids become exchanged with protons and the polymer will precipitate as alginic acid coagulate of a gelled or solid stage. The content of cations and calcium has been considerably reduced in the solid/gelled material during this step of purification. The isolated gelled/solid material may be dissolved by increasing pH with alkali to form e.g. sodium alginate, which can then be subjected to further purification steps e.g. filtration. The final isolation step may be carried out in different ways. One of these is the precipitation of the polymer as Ca-alginate followed by an acid wash to convert the water-insoluble Ca-alginate to alginic acid. Another way is to precipitate the liquid sodium alginate directly as alginic acid by addition of e.g. sulphuric acid. In both cases, the resultant alginic acid may be used as the starting material for preparation of the different alginate salts, and also for the covalently modified propylene glycol ester. After the alginic acid has been neutralized, the alginate salt is dried and milled to the desired particle size. Yet another way to isolate the final alginate is to precipitate liquid sodium alginate in alcohol e.g. ethanol or isopropyl alcohol whereby a gelled/solid stade of e.g. sodium alginate is obtained. Salts of sodium, potassium, and ammonium alginate are soluble in water and form viscous solutions. Alginic acid and calcium alginate are insoluble in water.
The starting material used in the herein disclosed method comprises alginic acid.
In the present context, the term “alginic acid” means that the constituting mannuronic and guluronic acid are fully protonated down to the limit where the degree of protonation is 50%, i.e. their pKa-values. Alginate pKa-values have been measured to be up to 4.4 [A. Haug, Dissociation of alginic acid, Acta Chem. Scand. 1961, 15, 950-952].
As described above the alginic acid may be obtained by lowering the pH below the pKa of the acid residues. This enables the removal of, among other cations, calcium, magnesium, iron, potassium and sodium from the polymer.
In one aspect, the alginic acid is in the form of an alginic acid coagulate of a gelled or solid stage. In a further aspect, the alginic acid is insoluble. The insolubility of the alginic acid may be measured by any method known to the person skilled in the art for example as described in “A. Haug, B. Larsen The solubility of alginate at low pH, Acta Chem. Scand. 1963, 17, 1653-1662”.
In one aspect, the alginic acid starting material is in the form of alginic acid fibers.
The alginic acid starting material in the present method should contain enough moisture to prevent browning or burning of the material during microwave treatment.
In one aspect, the dry matter content of the alginic acid starting material is in the range of 5-100% w/w, such as in the range of 5-90% w/w, such as in the range of 15-70% w/w, preferably in the range of 25-45% w/w.
The dry matter content may be adjusted by addition of one or more solvents. In one aspect, the alginic acid starting material comprises alginic acid in a mixture with one or more selected solvents from the group consisting of water, methanol, ethanol, and isopropanol. In a preferred aspect, the alginic acid is mixed with water as the only solvent. In a preferred aspect, the alginic acid starting material comprises alginic acid in water.
The concentration of alginic acid in the alginic acid starting material is preferably in the range of 7.5-100% w/w based on dry matter, such as in the range of 80-100% w/w based on dry matter, preferably in the range of 90-100% w/w based on dry matter. Alginate concentrations can be measured in various ways known to the person skilled in the art, see for example “H. S. Soedjak Colorimetric determination of carrageenans and other anionic hydrocolloids with Methylene Blue, Anal. Chem. 1994, 66, 4514-4518” and references mentioned herein.
In one aspect, the starting alginic acid material has a weight average molecular weight of 1,500,000-50,000 Da. The molecular weight may be measured by the method described herein under “General Procedures”.
In one aspect, the alginic acid starting material has a pH in the range of 0-4.4, such as in the range of 0-3.65, such as in the range of 0-3.5, such as in the range of 0.1-4.2, such as in the range of 0.2-3.65, such as preferably in the range of 0-2.5. In one aspect, the pH is adjusted by addition of acids such as sulphuric acid. By carrying out the microwave treatment of the alginic acid in the pH range of pH 0-4.4 it has been found that a faster cleavage of the glycosidic linkages is obtained, which results in a faster depolymerisation process meaning less energy consumption.
In order to facilitate depolymerization, a suitable agent may be added, i.e. reducing or oxidizing agents. [O. Smidsrød, A. Haug, B. Larsen Degradation of Alginate in the Presence of Reducing Compounds, Acta Chem. Scand. 1963, 17, 2628-2637], [O. Smidsrød, A. Haug, B. Larsen The Influence of Reducing Substances on the Rate of Degradation of Alginates, Acta Chem. Scand. 1963, 17, 1473-1474], [O. Smidsrød, A. Haug, B. Larsen Kinetic Studies on the Degradation of Alginic Acid in the Presence of Iron Salts, Acta Chem. Scand. 1965, 19, 143-152]. Thus, in one aspect, the alginic acid starting material may further comprise a reducing and/or oxidizing agent such as selected from the group of hydroquinone, sodium sulfite, phloroglucinol, sodium hydrogen sulfide, dithionite, cystein, thioglycolic acid, phenylhydrazine, ascorbic acid, reduced diphosphopyridine nucleotide (DPNH) in combination with methylene blue, hydrazine sulfate, dihydroxymaleic acid, hydrogen peroxide with or without the addition of iron(III) chloride.
In the present context, the term “depolymerised alginic acid material” means that the resulting alginic acid material which has been treated has a lower weight average molecular weight than the alginic acid starting material measured for example as described herein.
Preferably the microwave treatment is performed as a continuous process.
In one aspect, the alginic acid starting material may be drained and/or pressed and/or dried before the microwave treatment.
In one aspect, the microwave frequency is between 300 MHz and 300 GHz, preferably 300 MHz to 30 GHz, preferably 300 MHz to 3 GHz. Depending on the desired final viscosity of the depolymerised alginic acid material for the final application, the time or effect of microwave treatment may be adjusted.
In one aspect, the microwave treatment is performed at atmospheric pressure.
In one aspect, the depolymerised alginic acid material has a weight average molecular weight of 300,000-2,000 Da.
In one aspect, the method as described herein provides a depolymerized alginic acid without by-products or a small percentage of by-products, and the depolymerised alginic acid is thus obtained in a high purity.
Optional Treatments after the Microwave Treatment
After the microwave treatment, the depolymerised alginic acid material depending on the further usage may dried and/or milled and/or blended and/or bagged.
In one aspect, the depolymerised alginic acid material is further neutralised after the microwave treatment to obtain various alginates. The neutralisation may be performed by a number of methods known to the skilled person. In one aspect, the neutralisation is performed with one or more selected salts from the group of (NH4)2CO3, Mg(HCO3)2, Ca(HCO3)2, Na2CO3, NaHCO3, K2CO3, KHCO3, NH4HCO3, MgCO2, CaCO3, CaSO4 and Na2SO4, such as from the group of Na2CO3, NaHCO3, K2CO3, KHCO3, NH4HCO3, MgCO2, and CaCO3 to obtain a neutralised, depolymerised alginate. In yet a further aspect, the depolymerised alginic acid is converted into propylene glycol alginate using propylene oxide as reagent.
In yet a further aspect, the neutralised, depolymerised alginic acid material is further dried and/or milled and/or blended and/or bagged.
In yet a further aspect, the neutralised, depolymerised alginic acid material is blended with other salts, such as phosphates, citrates, carbonates and calcium salts, for food or pharma applications.
In the following further embodiments are described:
A method of depolymerisation of alginic acid, said method comprising the steps of:
A method of depolymerisation of alginic acid, said method comprising the steps of:
The method according to any one of embodiments 1-2, wherein the alginic acid is in an insoluble form.
The method according to any one of embodiments 1-3, wherein the microwave frequency is between 300 MHz and 300 GHz, such as 300 MHz to 30 GHz, preferably 300 MHz to 3 GHz.
The method according to any one of the preceeding embodiments, wherein the concentration of alginic acid in the alginic acid starting material is in the range of 7.5-100% w/w based on dry matter, such as in the range of 80-100% w/w based on dry matter, preferably in the range of 90-100% w/w based on dry matter.
The method according to any one of the preceding embodiments, wherein the alginic acid starting material has a pH in the range of 0-4.4, such as in the range of 0-3.5, such as in the range of 0.1-4.2, such as in the range of 0.2-3.65, such as preferably in the range of 0-2.5.
The method according to any one of the preceding embodiments, wherein the dry matter content of the alginic acid starting material is in the range of 5-100% w/w, such as in the range of 5-90% w/w, such as in the range of 15-70% w/w, preferably in the range of 25-45% w/w.
The method according to any one of the preceding embodiments, wherein the starting alginic acid material has a weight average molecular weight of 1,500,000-50,000 Da.
The method according to any one of the preceding embodiments, wherein the alginic acid starting material comprises alginic acid in a mixture with one or more selected solvents from the group consisting of water, methanol, ethanol, and isopropanol.
The method according to any one of the preceding embodiments, wherein the alginic acid starting material comprises alginic acid in water.
The method according to any one of the preceding embodiments, wherein the alginic acid starting material further comprises a reducing and/or oxidizing agent such as selected from the group of hydroquinone, sodium sulfite, phloroglucinol, sodium hydrogen sulfide, dithionite, cystein, thioglycolic acid, phenylhydrazine, ascorbic acid, reduced diphosphopyridine nucleotide (DPNH) in combination with methylene blue, hydrazine sulfate, dihydroxymaleic acid, hydrogen peroxide with or without the addition of iron(III) chloride.
The method according to any one of the preceding embodiments, wherein the depolymerised alginic acid material has a Total Plate Count below 5000 cfu/g, preferably below 1000 cfu/g.
The method according to any one of the preceding embodiments, wherein the alginic acid starting material is drained and/or pressed and/or dried before the microwave treatment.
The method to any one of the preceding embodiments, wherein the depolymerised alginic acid material has a weight average molecular weight of 300,000-2,000 Da.
The method according to any one of the preceding embodiments, wherein the depolymerised alginic acid material is further dried and/or milled and/or blended and/or bagged.
The method according to any one of the preceding embodiments, wherein the depolymerised alginic acid material is further neutralised after the microwave treatment to obtain various alginates.
The method according to any one of the preceding embodiments, wherein the neutralisation is performed with one or more selected salts from the group of (NH4)2CO3, Mg(HCO3)2, Ca(HCO3)2, Na2CO3, NaHCO3, K2CO3, KHCO3, NH4HCO3, MgCO2, CaCO3, CaSO4 and Na2SO4, such from the group of Na2CO3, NaHCO3, K2CO3, KHCO3, NH4HCO3, MgCO2, and CaCO3 to obtain a neutralised, depolymerised alginate.
The method according to any one of the preceding embodiments, wherein the depolymerised alginic acid is converted into propylene glycol alginate using propylene oxide.
The method according to any one of the preceding embodiments, wherein the neutralised, depolymerised alginic acid material is further dried and/or milled and/or blended and/or bagged.
The method according to any one of the preceding embodiments, wherein the neutralised, depolymerised alginic acid material is blended with other salts, such as phosphates, citrates, carbonates and calcium salts, for food or pharma applications.
The method according to any one of the preceding embodiments, wherein the alginic acid starting material is in the form of alginic acid fibers.
A depolymerised alginic acid material obtainable by the method according to any one of embodiments 1-21.
The viscosity of alginic acid is measured by suspending alginic acid in water in a concentric cylinder (cup and bob) using the measuring system CC 27/T200/SS. After adding a few drops of universal indicator the suspension turns dark pink. Sodium carbonate is added slowly until the color turns greenish yellow. As this point the alginic acid is fully dissolved. The viscosity is then measured using a rheometer (Anton Paar, Physica MCR301) at 20° C. with a shear rate of 0.33 s−1.
The M/G ratio of an alginate sample is determined as described in the literature (H. Grasdalen, B. Larsen, O. Smidsrød A Proton Magnetic Resonance Study of the Sequence of Uronate Residues in Alginate Carbohydr. Res. 1979, 68, 23-31) using a Bruker Avance III 600 MHz spectrometer with a 5 mm broad band observe probe.
The weight average molecular weight of alginate may be determined by dissolving the alginate in a running buffer and analysing by Gel Permeation Chromatography equipped with a Multi Angle Light Scattering detector (DAWN EOS, Wyatt Technology Corporation), an RI detector (Optilab rEX, Wyatt Technology Corporation) and GPC columns such as PSS SUPREMA-LUX 1000 Å and PSS SUPREMA-LUX 3000 Å.
A buffer of 0.05 M LiNO3 with 200 ppm N3− is prepared by dissolving 17.23 g LiNO3 p.a. and 1.56 g NaN3 in 5.00 L Millipore Water. The eluent is filtered through a 0.22 μm filter.
1-2 mg/ml samples are dissolved in running buffer under stirring and samples are filtered through a 0.45 μm filter (13 mm GHP 0.45 μm Minispike from Waters). Normal running conditions are: Flow=0.8 mL/min, injection volume=1004, column temperature=40° C., detector temperature=30° C.
The dry matter content of alginate may be determined by weighing the dry residue after evaporation of the water contained in a sample heated at 105° C.
A glass container is dried for 1 hour in an oven at 105° C. and then cooled for 1 hour in a desiccator before using it. Thereafter 2±0.001 g of alginate is placed in the container and the open glass container is placed in an oven at 105° C. for 4 hours. The dry residue is weighed after cooling in a desiccator for 20 minutes.
The dry matter content is calculated as follows:
Samples of AA before and after microwave treatment were analysed according to 3M Petrifilm Aerobic Count Plates, Interpretation Guide, Reminders for Use and Sample Preparation.
Nordval Certificate No. 012. NMKL Method 146.1993 (Ref 3) for total aerobic microbial count. Mesophillic aerobic sporulates were analysed according to American Public Health Association for Food and Drug (APHA), 2001, 4th version, 22.512.
Taken from an industrially produced alginic acid fiber starting material (Dry matter: 32%, pH: 1.7) 160 g alginic acid were placed in a 800 ml glass beaker and microwave treated with 800 watt effect (Electolux) for 7 minutes and 40 seconds.
Alternatively, 130 g alginic acid were placed in an oven at 40° C. for 66 hours, either with or without the addition of 582 μL 35% (w/v) H2O2 solution.
Alternatively, 150 g alginic acid were placed in an oven at 105° C. for 2 hours.
In a different series of trials, 200 g of alginic acid were blended with 17.6 g Na2CO3 in a food processor (KRUPS). The sample was dried at 40° C. for 16 hours in an oven. Hereafter the samples were milled (Retsch ZM 200) and sieved through a 200 μm sieve.
From this sodium alginate, 13 g were placed in an oven at 105° C. for 7 h and 30 minutes. In a final experiment 110 μL 35% (w/v) H2O2 solution were added to 8.75 g sodium alginate. This mixture was placed in an oven at 105° C. for 7 h and 30 minutes.
Weight average molecular weights were determined by Gel Permeation Chromatography equipped with a Multi Angle Light Scattering detector and a RI detector (Wyatt Technology Corporation). 1-2 mg/ml sample were dissolved in a buffer consisting of Millipore water with 0.05 M LiNO3 with 200 ppm NaN3. Prior to measurement the solution was filtered through a 0.45 μm filter.
Normal running conditions were: Flow=0.8 mL/min, Injection volume=1004, column temperature=40° C., detector temperature=30° C., do/dc (mL/g) 0.154. Calculations were done using Astra 5 software from Wyatt Technology Corporation.
Depolymerisation induced by microwave treatment at 850 W (Electrolux), was carried out on two different alginic acid starting materials with characteristics as described in below Table 2.
The duration of the microwave oven treatment was limited since moisture evaporated from the alginic acid. The required treatment seemed to be just short enough, however, to avoid complete dry out and burning of the product. Reducing 1% viscosities of 500-1500 mPa·s to 2000 mPa·s 10% viscosity would require 10-13 minutes of microwave treatment at 850-900 Watt. For calculation of the required depolymerisation time half times were applied. A prerequisite is to measure the 1% sodium alginate viscosity of the starting alginic acid material and convert it to the 10% sodium alginate viscosity. The half-time for the alginic acid was 52 seconds.
A sample of AA based on Laminaria hyperborea a pH of 2.0 and a dry matter content of 24.5% w/w were used in the following. Cultures of Bacillus subtilis and Bacillus licheniformis were produced in the DuPont Food Protection laboratories in Brabrand, Denmark.
From the AA material 3 samples of 150 g were transferred to plastic jugs. 10 ml of Bacillus subtilis and Bacillus licheniformis culture, respectively, were each added to a jug and mixed well by using a spoon for 30 sec. Two of the three samples were treated in a microwave oven, type Electrolux 850W as shown in below table 4.
In addition to the lab bench tests a challenged test was carried out on pilot scale. A batch of microbially contaminated AA was processed in a microwave pipe, type “Thermo-star” from Bertin Technology. Total plate count analysis revealed that the batch contained a level of 3·104 cfu/g wet AA. The objective of the test was to determine the effect of temperature and time on the microbiological quality of the AA.
Samples of AA before and after microwave treatment were analysed according to 3M Petrifilm Aerobic Count Plates, Interpretation Guide, Reminders for Use and Sample Preparation.
Nordval Certificate No. 012. NMKL Method 146.1993 for total aerobic microbial count.
Mesophillic aerobic sporulates were analysed according to American Public Health Association for Food and Drug (APHA), 2001, 4th version, 22.512.
The results presented in Table 6 show the effect of microwave treatment on the total plate count. The results for Jug-1 (reference) indicate high levels above 6·104 cfu/g 12·104 cfu/g for portions 1 and 2. After 5 minutes of microwave treatment at 850 W, the plate count in both portions had decreased to <100 cfu/g, i.e. a decrease of two orders of magnitude. After 10 minutes of microwave treatment no further decrease was observed, suggesting that 5 minutes of microwave treatment is sufficient.
Table 7 presents the corresponding results for the mesophilic spores specifically. Trends similar to those of Table 6 are seen, i.e. after 5 minutes of microwave treatment the mesophilic spores had decreased by some two to three orders of magnitude to <10 cfu/g. Again no further benefit was recorded for 10 minutes of microwave treatment.
The results presented in Table 8 show the effect of microwave treatment on the total plate count on pilot scale. The total plate count was reduced from 104 cfu/g to <102 cfu/g. The specification on microbiological quality of alginate specifies <1,000 cfu/g, hence the microbiologically contaminated batch can be microwave treated to comply with specifications.
All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. The disclosure set forth herein has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope encompassed by the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12199699.5 | Dec 2012 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| 61746853 | Dec 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14649544 | Jun 2015 | US |
| Child | 15590717 | US |
