CROSS REFERENCE TO RELATED APPLICATIONS

[001 ] This application claims the benefit of U.S. Provisional Application No. 62/485,855, filed April 14, 2017, which is herein incorporated by reference in its entirety.

SUMMARY

[002] Disclosed are processes for the production of pullulan by microorganisms in a fermentation medium containing calcium phosphate as a principal phosphate source.

[003] Pullulan is a polymer of glucose, more particularly, a polysaccharide consisting of a-1 ,4-linked maltotriose units which are connected by a-1 ,6-linkages between the terminal glucosidic residues of the trisaccharide.

[004] Pullulan is obtained from a strain of Aureobasidium pullulans extracellularly when the strain is cultured aerobically in a fermentation medium. The degrees of polymerization of the thus obtained pullulan are reported as falling in the range of a few hundreds to a several thousand kilodaltons (kDa). High product quality consistency can be obtained by controlling the fermentation process.

[005] The films formed with pullulan have a variety of properties that make such films suitable for forming products such as capsules. Such resulting films have excellent homogeneity and transparency. In addition, such films have very low oxygen permeability. Accordingly, capsules made from pullulan are particularly useful for the filling of oxygen sensitive products such as fish and vegetable oils. Such films and resulting capsules made from pullulan also have relatively low water content, and exhibit high stability over storage, such as with respect to mechanical and dissolution properties.

[006] The natural origin of pullulan and its superior properties as a film forming material has made pullulan a desired polymer for the manufacture of capsules having ingredients of all natural origin. This would enable capsules made with pullulan to meet the requirements for "organic" labeling. Notwithstanding the commercial need for a capsule meeting the organic labeling requirements, no satisfactory pullulan capsules have to date been developed that meet the organic labeling requirement. This is primarily due to the absence of pullulan as a raw material that has been made so as to satisfy the organic labeling requirements.

[007] In one embodiment, a process for manufacturing pullulan is provided, comprising: a. preparing a fermentation medium comprising water, a nitrogen source, a carbon source, a phosphate source, and a magnesium source; b. inoculating said fermentation medium with a pullulan producing microorganism; c. culturing said microorganism in said fermentation medium so as to produce pullulan in said fermentation medium; wherein said phosphate source comprises calcium phosphate.

[008] In one embodiment, the phosphate source comprises, consists essentially of or consists of calcium phosphate.

[009] In one embodiment, the fermentation medium has an initial calcium phosphate concentration of from about 0.25 g/L to about 4 g/L.

[010] In one embodiment, the magnesium source is magnesium chloride.

[01 1 ] In one embodiment, the fermentation medium has an initial concentration of magnesium chloride of from 0.01 g/L to 1 g/L. [012] In one embodiment, the nitrogen source is yeast extract, NhUOH, L-glutamine, NaNC , NhUCI, arginine, or a mixture thereof.

[013] In one embodiment, the nitrogen source is yeast extract.

[014] In one embodiment, the carbon source is glucose, maltose, lactose, sucrose, fruit and vegetable syrups, fruit and vegetable molasses, or a mixture thereof.

[015] In one embodiment, the carbon source is sucrose.

[016] In one embodiment, the microorganism is Aureobasidium pullulans.

[017] In one embodiment, the microorganism is Aureobasidium pullulans ATCC No. 42023.

[018] In one embodiment, the culturing of the microorganism is stopped by pasteurization.

[019] In one embodiment, the molecular weight of the pullulan is reduced by pullulanase produced by the microorganism, and the process is free from a step of adding an acid or heat to reduce the molecular weight of the pullulan.

[020] In one embodiment, the fermentation medium has an initial pH of from 2.5 to 8.

[021 ] In one embodiment, the microorganism is cultured for at least 3 days.

[022] In one embodiment, pullulan is produced from the foregoing processes.

[023] In one embodiment, the pullulan has a molecular weight of from 200 to 500 kDa.

[024] In one embodiment, the pullulan has a Ca ²⁺ content of at least 50mg/kg, or at least 1 00 mg/kg, or at least 200mg/kg, or at least 300mg/kg, or even at least 500 mg/kg.

[025] In one embodiment, the pullulan has a Mg ²⁺ content of at least 1 0 mg/kg, or 20mg/kg, or 30mg/kg, or at least 40 mg/kg, or even at least 50mg/kg.

[026] In one embodiment, the pullulan has a ratio of (Mg ²⁺ + Ca ²⁺ )/K ⁺ that is greater than 3, or greater than 5, or greater than 1 0, or even greater than 15.

[027] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWING

[028] FIG. 1 is a time course plot of pullulan concentration, molecular weight, and biomass concentration measured in Example 1 .

[029] FIG. 2 shows pullulan concentration, molecular weight, and biomass concentration measured in Example 2.

DETAILED DESCRIPTION

[030] Definitions

[031 ] As used herein, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one." The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. The term "about" as used in the disclosure of numerical ranges indicates that deviation from the stated value is acceptable to the extent that the deviation is the result of measurement variability and/or yields a product of the same or similar properties.

[032] As used herein, "w/w %" and "wt%" means by weight as a percentage of the total weight. [033] Process for Making Pullulan

[034] A process for manufacturing pullulan comprises the steps of

a. preparing a fermentation medium comprising water, a nitrogen source, a carbon source, a phosphate source, and a magnesium source;

b. inoculating said fermentation medium with a pullulan producing microorganism;

c. culturing said microorganism in said fermentation medium so as to produce pullulan in said fermentation medium.

[035] The fermentation medium may contain any carbon source suitable for the production of pullulan. Suitable known carbon sources include saccharides such as sucrose, fructose, and glucose; modified saccharides such as isomerized sugar, and invert sugar; carbohydrates such as starch; and natural products such as tapioca and dates. In a preferred embodiment, the carbon source is certified as meeting organic labeling requirements. In one embodiment, the carbon source is sucrose. The carbon source, for example, sucrose, is initially present in the fermentation medium at a concentration of from 1 0g/L to 200 g/L, more preferably from 60g/L to 1 1 0g/L.

[036] The fermentation medium may contain any nitrogen source suitable for the production of pullulan. Generally, ammonium salts, nitrates, peptone and yeast extract may be used as nitrogen sources. In a preferred embodiment, the nitrogen source is certified as meeting organic labeling requirements. In one embodiment, the nitrogen source is yeast extract. The nitrogen source, for example, yeast extract, is present in the fermentation medium initially at a concentration of from 1 g/L to 6g/L (0.08 g/L to 0.4 g/L nitrogen equivalent)..

[037] The fermentation medium also contains a phosphate source comprising calcium phosphate. In one embodiment, the phosphate source consists of calcium phosphate. Calcium phosphate is a preferred source of phosphate as it is capable of being certified as meeting organic labeling requirements. The calcium phosphate is present in the fermentation medium initially at a concentration of from 0.25 g/L to 4 g/L.

[038] The fermentation medium also contains a magnesium source suitable for the manufacture of pullulan. In one embodiment, the magnesium source consists of magnesium chloride. Magnesium chloride is a preferred source of magnesium as it is capable of being certified as meeting organic labeling requirements. The magnesium source is present in the fermentation medium initially at a concentration of from 0.01 g/L to 1 g/L. (0.1 mM to 1 0.5mM Mg or 0.002 g/L to 0.26 g/L Mg)

[039] The fermentation medium may contain other optional materials. In one embodiment, the fermentation medium may contain ascorbic acid. The ascorbic acid may be present in an amount of from 0.2 mg/ml to 3 mg/ml of the initial fermentation medium.

[040] Other optional materials that may be included in the fermentation medium include salts such as sodium chloride.

[041 ] The pH of the fermentation medium may range from 2.5 to 8. The pH of the fermentation medium may be adjusted by addition of acids, bases, or buffers, such as sodium hydroxide, and hydrochloric acid. [042] The fermentation medium may be prepared in any vessel suitable for culturing microorganisms, such as shake flasks or bioreactors. The fermentation medium ingredients and water are added to the vessel and stirred or agitated to dissolve or uniformly disperse the ingredients. The fermentation medium is then sterilized, for example, by heating to a temperature of at least 60 C for at least 30 min time prior to introduction of the microorganism.

[043] The pullulan producing microorganisms employable in the present invention include Aureobasidium pullulans, Pullularia fermentans var fermentans IFO 6401 , Pullularia fermentans var fusca IFO 6402, Pullularia pullulans AHU 9553, Pullularia pullulans IFO 6353, and Dematium pullulans IFO 4464. A preferred pullulan producing microorganism is Aureobasidium pullulans. Aureobasidium pullulans may be obtained from ATCC. A preferred clone is Aureobasidium pullulans ATCC No. 42023.

[044] After heat sterilizing of the fermentation medium and pH adjustment, the microorganism is cultured in the medium with aeration and/or agitation, at 25 °-30°C, preferably at 27 °C, for about 3-7 days. Aeration may be performed by sparging. Agitation may be performed by impellor. About 3 days after the start of the fermentation, accumulation of a considerable amount of pullulan is observed, and the viscosity of the culture mixture increases.

[045] The pullulan concentration and molecular weight in the medium may be determined at certain intervals, and cultivation may be discontinued when the amount of pullulan concentration approaches a concentration greater than 20g/L and has a molecular weight between 200kDa and 500kDa.

[046] Fermentation may be stopped when the pullulan molecular weight is between 200kDa and 500kDa and the viscosity of a 1 0wt% solution of the pullulan is between 10OcP and 190cP. Fermentation may be stopped by pasteurization.

[047] The molecular weight of the pullulan produced during fermentation changes over time during the fermentation process. This is due in part to the production of pullulanase by the microorganism. In one embodiment, the molecular weight of the pullulan produced during the process is controlled principally by the pullulanase produced by the microorganism. This eliminates a further step of addition of a material, such as an acid or an elevated temperature, to the bioreactor to reduce the molecular weight of the pullulan.

[048] The microbial cells may be removed from the liquid medium by centrifuging, and the cell-free liquid medium may be decolorized with activated carbon, if desired.

[049] The pullulan may be purified by precipitation in an organic solvent. Preferably, the supernatant following centrifugation is mixed with a hydrophilic organic solvent, such as methanol or ethanol, to precipitate the accumulated pullulan. If desired, the recovered pullulan is dissolved in warm water and again precipitated by solvent.

[050] The pullulan obtained after drying is a whitish powder which very readily dissolves in water to form a viscous solution. The molecular weight of the pullulan obtained varies depending on the culture conditions between 50,000 and 4,500,000 Da. The yield of pullulan also varies from 20 to 75% depending on the culture conditions.

[051 ] In one embodiment, the resulting pullulan has a relatively high concentration of calcium (Ca ²⁺ ) and magnesium (Mg ²⁺ ) ions. The fermentation media contains calcium phosphate and magnesium chloride. This results in a final pullulan product containing relatively high amounts calcium and magnesium. In addition, the use of a precipitation process, and the absence of an ion exchange process to remove ions, retains a significant fraction of the starting calcium and magnesium. In one embodiment, the pullulan has a Ca ²⁺ content of at least 50mg/kg, or at least 1 00 mg/kg, or at least 200mg/kg, or at least 300mg/kg, or even at least 500 mg/kg. In another embodiment, the pullulan has a Mg ²⁺ content of at least 1 0 mg/kg, or 20mg/kg, or 30mg/kg, or at least 40 mg/kg, or even at least 50mg/kg. In one embodiment, the ratio of (Mg ²⁺ + Ca ²⁺ )/K ⁺ is greater than 3, or greater than 5, or greater than 10, or even greater than 15.

[052] In another embodiment of the invention, a dry fermentation media is provided that is suitable for use for making a fermentation medium for making organic pullulan. In one embodiment, the dry fermentation media comprises

(a) a carbon source in an amount of from 89wt% to 99wt%

(b) a nitrogen source in an amount of from 0.9wt% to 9wt%; and

(c) a phosphate source consisting of calcium phosphate in an amount of from 0.2wt% to 0.7wt%.

[053] In one embodiment, the dry fermentation media comprises magnesium chloride in an amount of from 0.009wt% to 1 .6wt%.

[054] It should be understood that the embodiments described herein are not limited thereto. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosed embodiments. The following examples should be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims.

[055] Example 1

[056] An organic pullulan fermentation medium was prepared as follows. 2 g/L of monocalcium phosphate (Ca(H2P04)2), 0.2 g/L of magnesium sulfate (MgSC ), and 1 g/L sodium chloride (NaCI) were added to water and allowed to evenly disperse under vigorous agitation for at least 15 minutes. The monocalcium phosphate did not completely dissolve, but evenly dispersed into a suspension. 3 g/L of organic yeast extract was then added to the mixture and allowed to dissolve. Finally, 1 00 g/L of organic sucrose was added to the mixture and allowed to dissolve. After adjusting to the final volume with additional water, the solution pH was measured at 4.85. The media was steam sterilized at 121 C for 40 minutes.

[057] An inoculum culture was prepared by inoculating a sterilized 125 mL shake flask containing 30 mL of the organic pullulan fermentation medium with a scraping from a plate culture of A. pullulans. The plate culture was grown on potato dextrose agar. Shake flasks were then passaged at a dilution rate of 1 :60 at least every 4 days until enough biomass was achieved to obtain 0.32 mg/mL initial biomass concentration in bioreactors.

[058] The bioreactors were pre-filled with sterile organic pullulan fermentation media and controlled to a temperature of 28C and agitated at a power input of 700 W/m ³ with a single Rushton impeller prior to inoculation and for the duration of the fermentation. Inoculum was then added to the reactor at a volume sufficient to yield a reactor biomass concentration of 0.32 mg/mL. Dissolved oxygen was controlled to >50% air saturation by sparging pure oxygen through a 15μιη stone sparger. The reactor gassing system was designed such that an oxygen transfer rate of at least 1 1 0 mM/hr was achieved in order to maintain non- hypoxic conditions. The exhaust gas was routed through a condenser in which the condensate was returned directly to the fermentation media. The working volume of the reactors was 1 .25 L, and the total reactor volume was 2L. The fermentation was then allowed to progress for 93 hours. The pH was uncontrolled. The fermentation was halted by heating the culture to >60C at 93 hrs. [059] The culture was sampled daily to measure biomass concentration, pullulan concentration, and pullulan molecular weight.

[060] Biomass concentration was determined as follows. A 30 ml_ sample was removed from the bioreactor and placed in a pre-weighed 50 ml_ centrifuge tube. The sample was then centrifuged at a force of 10OOOg for 20 minutes. The supernatant was retained to determine pullulan concentration and molecular weight. The pellet was rinsed with 10 mL water, re-suspended, and centrifuged for an additional 20 minutes at a force of 1 0OOOg. The pellet was then flash frozen and lyophilized to dryness. The dried pellet was then weighed and the biomass concentration calculated based on the volume of the bioreactor sample and weight of the pellet.

[061 ] Pullulan concentration was determined by precipitating 20mL of the first supernatant from the biomass analysis in 90% cold ethanol at a ratio of 1 :1 .4. The precipitated pullulan was then dissolved in water at 1 0 wt%. This solution was then precipitated again in fresh cold ethanol at the same ratio. The resulting pellet was then dried and weighed. The pullulan concentration was then determined based on the volume of supernatant sample and weight of dried pullulan pellet.

[062] The pullulan molecular weight was determined by dissolving the dried pullulan pellet in water to a concentration of 1 0 mg/mL. The resulting solution was then filtered through a 0.45um filter and then injected onto a High Pressure Liquid Chromatography (HPLC) system. The H PLC system was outfitted with a size exclusion column. The pullulan polymer was retained on the column for a time directly related to the polymer size and conformation. The retention time was determined by detecting the pullulan elution via a refractive index detector. The polymer size was determined by the response of the polymer in a multi-angle laser light scattering detector. The response in the multi-angle laser light scattering detector was normalized and verified by the measurement of other known molecular weight standards.

[063] Example 2

[064] In this Example cell cultures were passaged and maintained as described in Example 1 , including the passaging of the inoculation train and the organic pullulan media recipe. In order to evaluate a wholly certified organic pullulan media, we evaluated substituting MgS04 with a certified organic MgCl2 (Mitoku; Tokyo, Japan). In this experiment, 3 separate 125mL PETG shake flasks were inoculated with 0.5mLs of inoculum into a volume of 30mLs organic pullulan media (1 :60). One of these shake flasks contained media with the same composition as described in Example 1 . For the other two shake flasks, MgCl2 was substituted for the MgSC at concentrations of 0.1 6g/L and 0.32g/L (the rest of the components were the same as outlined in Example 1 ). These shake flask cultures were grown for 4 days in an orbital shaker incubator at 28 ^' C at 200 RPM shaking. On the fourth day, these shake flask cultures were analyzed for pullulan yield and biomass using the same procedures as described in Example 1 . The results from this study are shown in FIG 2.

[065] Example 3

[066] In this example, pullulan was produced following the process of Example 2. This pullulan (termed pullulan A) and a representative sample of pullulan produced by Hayashibara (termed pullulan B) was analyzed for Mg2+ and Ca2+ content following the standard protocols SM31 1 1 B and D (Flame Atomic Absorption Spectroscopy) respectively. Each pullulan sample was analyzed in triplicate. From this analysis it was determined that the concentration of both Mg2+ and Ca2+ is higher in pullulan A than pullulan B.

Table 1. Results of Metal Analysis of Pullulan

Pullulan A Pullulan B

Mg ²⁺ Ca ²⁺ K ⁺ (Mq ²⁺ +Ca ²⁺ Mg ²⁺ Ca ²⁺ K ⁺ (Mq ²⁺ +Ca ²⁺

(mg/kg) (mg/kg) (mg/kg) ) (mg/kg (mg/kg) (mg/kg) )

K ⁺ ) K ⁺

Replicat 66.8 746 48.1 1 6.9 2.57 20.5 1 1 .4 2.0 e 1 I

Replicat 66.0 696 44.2 1 7.2 2.73 21 .9 1 1 .4 2.2 e 2

Replicat 66.0 688 43.6 1 7.3 2.52 39.7 1 1 .1 3.8 e 3

Mean ± 66.3±0. 710±31. 45.3±2. 17.1 ±0.2 2.6±0.1 27.4±10. 11.3±0. 2.6±1.0

St. Dev 5 4 3 7 2

Values expressed in mg metal per kilogram pullulan