WO2025141602A1 - Procédé de production d'acide hyaluronique de faible poids moléculaire ou d'un sel de celui-ci - Google Patents

Procédé de production d'acide hyaluronique de faible poids moléculaire ou d'un sel de celui-ci Download PDF

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Publication number
WO2025141602A1
WO2025141602A1 PCT/IN2024/052402 IN2024052402W WO2025141602A1 WO 2025141602 A1 WO2025141602 A1 WO 2025141602A1 IN 2024052402 W IN2024052402 W IN 2024052402W WO 2025141602 A1 WO2025141602 A1 WO 2025141602A1
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Prior art keywords
salt
molecular weight
low molecular
fermentation
carried out
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English (en)
Inventor
Tushar Ramdas SABALE
Shruti Shrikant Panchwagh
Asma Najirahamed HALAGALE
Babu Mohan
Pramod Shankar Kumbhar
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Praj Industries Ltd
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Praj Industries Ltd
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present subject matter in general, relates to the field of process chemistry. More particularly, the present subject matter relates to process for producing low molecular weight hyaluronic acid or salt thereof, comprising, producing high molecular weight HA or salt thereof by fermentation; and carrying out in-situ fragmentation of said high molecular weight HA or salt thereof; to obtain low molecular weight HA or salt thereof.
  • Hyaluronic acid or hyaluron or hyaluronate is essentially an unsulphated glycosaminoglycan composed of repeating disaccharide units of N- acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) linked together by alternating beta- 1,4 and beta- 1,3 glycosidic bonds.
  • HA is endowed with unique physiological and biological properties such as high water-holding capacity and viscoelasticity and thus occurs widely in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis, and is also suspected of playing a role in numerous physiological functions, such as adhesion, development, cell motility, cancer, angiogenesis, and wound healing. Its biocompatibility accords it with unprecedented applications in the fields of medicine and cosmetics.
  • microbial HA production can be considerably affected by culture conditions, including temperature, pH, aeration rate, agitation speed, dissolved oxygen, shear stress, and type of bioreactor. Therefore, optimization of process parameters through microbial production is invariably advisable.
  • the molecular weight (MW) of HA or salt thereof is the primary quality parameter that determines its suitability for various applications.
  • High molecular weight HA or salt thereof consists of 1,500 kD and larger molecules that cannot feasibly penetrate the skin barrier, and are effortlessly washed off, plummeting their real long-term effect.
  • low molecular weight HA or salt thereof easily absorbs and penetrates the deepest layers of the skin, it is exceedingly recommended for cosmetic applications.
  • low molecular weight HA or salt thereof is commonly carried out by treating the high molecular weight product with inorganic chemicals, and altering physical conditions such as, pH, temperature, agitation, selective filtration, etc.
  • Commercial formulators also achieve low molecular weight HA or salt thereof by applying depolymerization methods involving at least one of ozone radiation, and mechanical fragmentation to the industrially finished HA.
  • aeration is optimized to ⁇ 5 vvm; and preferably, to ⁇ 2.5 vvm.
  • fragmentation or “fragmentized” pertains to a process in which molecular ions are broken into smaller their smaller counterparts such as, ions, radicals, and/or neutral molecules.
  • in-situ fermentation broth is implemented (for fragmentation).
  • ex-situ final product, or HA, as is described in the instant case, is implemented (for fragmentation).
  • the in-situ fragmentation is carried out by ozonation or ozone treatment; and preferably, by ozone gas treatment.
  • ozonation or “ozone gas treatment” or “ozone treatment” refers to an advanced oxidation process (AOP) using ozone (O3).
  • ozonation is carried out at 2 g/h to 20 g/h; and preferably, 5 g/h to 15 g/h.
  • ozonation is carried out at 2 1pm to 15 1pm; and particularly, at 6 1pm to 101pm.
  • ozonation is carried out for 1 to 12 h, preferably for 1 to 10 h; and particularly, at 30°C to 70°C, preferably, at 35°C to 65°C.
  • the in-situ fragmentation is carried out by cavitation; preferably, with or without addition of hydrogen peroxide (H2O2); and particularly, with addition of H2O2.
  • cavitation pertains to a process whereby pressure variations in a liquid can in a short period of time cause countless small cavities to form and then implode.
  • the cavitation is carried out at 2 m 3 /h to 52 m 3 /h; preferably, with 2 kg/m 3 to 12 kg/m 3 pressure; and particularly, for 1 h to 5 h, preferably, for 1. h to 3. h.
  • the cavitation is carried out at 25°C to 50°C; and preferably, at 30°C to 45°C.
  • the in-situ fragmentation reduces the fermentation viscosity to ⁇ 100 cp; and preferably, to ⁇ 50 cp.
  • the in-situ fragmentation reduces microbial contamination; and particularly, deactivates infectious organisms.
  • infectious organisms relate to conventionally known microbial agents associated with human diseases that pose moderate health hazard.
  • cell separation step is carried out after in-situ fragmentation; preferably, using centrifugation; and particularly, without any requirement for addition of water/ fermentation broth dilution.
  • the decolorized supernatant comprising HA or salt thereof is purified; preferably using at least one of cation resin treatment, membrane filtration, electrodialysis, and other conventionally known methods of demineralization/purification; and particularly, using cation resin treatment.
  • cation resin treatment helps in de-mineralizing, and de-alkalizing. They have high capacity strongly acidic cationic exchanger, containing sulphonic acid groups, and are based on crosslinked polystyrene with a gel structure, that have a higher degree of cross linkage to facilitate feasible removal of impurities.
  • pH of the decolorized and purified supernatant comprising HA or salt thereof is adjusted; preferably to > 3 pH.
  • at least one of sodium acetate, or potassium acetate is implemented; and particularly, sodium acetate is implemented.
  • the decolorized and purified supernatant comprising HA or salt thereof is subjected to solvent precipitation.
  • the solvent precipitation is carried out by at least one of acetone, hexane, ethyl acetate, ethanol, n-propanol, isopropanol, or mixtures thereof.
  • Acetone demonstrates better yield and separation of HA or salt thereof from impurities to provide purer final product, as well as lower residual level of about ⁇ 0.1%. Further, in addition to cost-effectiveness, and availability, acetone provides better control and reproducibility in the precipitation process, which facilitates process optimization and ensures consistent product quality. This positively effectuates easier scaling up for reliable industrial HA or salt thereof production.
  • solvent precipitation is carried out by acetone; particularly, in a ratio of 1:1 to 1:5 and following the precipitation, the solvent may be recovered and reused.
  • the precipitate is washed with solvent; preferably to obtain final product or HA.
  • final product (HA or salt thereof) is dried; particularly, to analyze as per industry standards; preferably, as recommended in European Pharmacopoeia - Supplement 2001.
  • ozone gas is introduced in the fermentation broth having viscosity of > 2000 cp at about 5 g/h to 15 g/h (2 1pm to 15 1pm) flow rate for about 60-250 mins (1-5 h), at about 35°C -60°C under mixing conditions (100 rpm - 800 rpm).
  • the fermentation broth is also screened for infectious organisms using microscopy (cell lysis %), and TVC for CFU/ml on BHI media after the ozone gas treatment. Once the fermentation broth viscosity reduces to about ⁇ 50 cp (after about 1-5 h of ozone gas treatment), cell separation is initiated.
  • Separated cell biomass is sent to incineration for disposable purpose, and supernatant is decolorized using granular activated carbon.
  • the decolorized supernatant comprising HA or salt thereof is then passed through cation resin at a flow rate of about 50-180 mL/min to demineralize and de-alkalize, and especially to remove inorganic impurities.
  • pH of the decolorized and purified supernatant comprising HA or salt thereof is adjusted to > 3.
  • the decolorized and purified supernatant comprising HA or salt thereof is subjected to solvent precipitation using acetone in about l-5:5-l ratio. Precipitate is further washed with acetone to remove water from the product and dried overnight at about 30-60 °C overnight to get a final product (HA or salt thereof.
  • strain isolation followed by inoculum preparation, and main fermentation as described in foregoing paragraphs is subjected to cavitation (with or without addition of hydrogen peroxide (H2O2)) of fermentation broth having viscosity of > 2000 cp at about 1 m 3 /h to 7 m 3 /h with about 1 kg/m 3 to 15 kg/m 3 pressure, for about 1-5 h at about 37°C.
  • H2O2 hydrogen peroxide
  • the solvent may be recovered and reused.
  • the final product is low molecular weight HA or salt thereof.
  • the final product is super low molecular weight HA or salt thereof.
  • molecular weight of the low molecular weight HA or salt thereof is ⁇ 1000 kDa; and preferably, is ⁇ 800 kDa. In a preferred embodiment, purity of the low molecular weight HA or salt thereof is > 70%; and particularly, > 80%.
  • loss on drying of the low molecular weight HA or salt thereof is ⁇ 30%; and particularly, ⁇ 20%.
  • recovery of the low molecular weight HA or salt thereof is > 75%; and particularly, > 80%.
  • bacterial count of the low molecular weight HA or salt thereof is ⁇ 50 CFU/g; and particularly, ⁇ 20 CFU/g.
  • the low molecular weight HA or salt thereof is applicable in cosmetics.
  • Soil sample collected from a barn/stable at Hadapsar, Pune was serially diluted using sterile water and then spread on commercially available BHI medium (about 3.7%) comprising about 20% of calf brain infusion, about 25% of beef heart infusion, about 1.0% of protease peptone, about 0.5% of NaCl, about 0.25% of Na2HPO4, and about 0.2% of dextrose.
  • BHI medium about 3.75% comprising about 20% of calf brain infusion, about 25% of beef heart infusion, about 1.0% of protease peptone, about 0.5% of NaCl, about 0.25% of Na2HPO4, and about 0.2% of dextrose.
  • Isolated strain showing positive result was analysed using NCBI Blastn analysis (molecular identification) and showed > 99% homology to Streptococcus zooepidemicus (MTCC 25714.
  • Table 1 Composition of Hi-veg hydrolysate medium (seed ingredients) (for 1000 ml)
  • Color removal or decolorization by granular activated carbon was carried out by passing the supernatant through the charcoal bed at a flow rate of about 120 mL/min. Six bed volumes of the supernatant were passed through the charcoal column. A total of about 52 L of decolorized supernatant comprising HA or salt thereof was collected including about 1 BV (Bed Volume) of water to remove product from the column.
  • BV Bed Volume
  • the decolorized supernatant comprising HA or salt thereof was passed through cation resin at a flow rate of about 120 mL/min (around 1 BV/h) to demineralize and de-alkalize, and especially to remove inorganic impurities such as, iron, zinc, copper, nickel, chromium etc.
  • the cation resin (having a high capacity strongly acidic cation exchanger containing sulphonic acid groups. It is based on crosslinked polystyrene with a gel structure and has a higher degree of cross linkage.) volume was about 5L.
  • the elution water for cation was about 5-7 L.
  • pH of the decolorized and purified supernatant comprising HA or salt thereof was adjusted to about 4.7 by passing sodium acetate through cation resin. A total of about 3 kg of sodium acetate was required to adjust the pH.
  • the decolorized and purified supernatant comprising HA or salt thereof was subjected to solvent precipitation using acetone in about 1:2 ratio. A total of about 120 L of acetone was added at about 40 L/hr flow rate. Precipitate was allowed to settle for about half an hour and separated from the water-acetone mixture. Precipitate was further washed with acetone to remove water from the product and dried overnight at about 35 °C overnight to get the final product (HA), that was analyzed/characterized. The solvent was recovered and reused.
  • Table 11 and Figs. 2, and 3 illustrate the effect of ozonation on molecular weight (kDa) of HA.
  • Table 12 summarizes loss of drying, purity, and intrinsic viscosity for HA or salt thereof production at 100L scale.
  • the purity, intrinsic viscosity, molecular weight, and loss on drying of the final product was found to be about 92.7 %w/w, 1.05 kg/m 3 , 172 kDa and 14 % respectively. Further, the productivity and yield were found to be 0.25 g of HA/L of broth/h, and 0.037 g/g for about 4.5 g/L titre respectively. The % of recovery was > 80%, and the bacterial count was ⁇ 20 CFU/g.
  • Isolation, inoculum preparation, and main fermentation was carried out as described in Examples 1-4. After the fermentation broth achieved viscosity of > 2000 cp after about 18 h of main fermentation, in-situ fragmentation was performed using ozonation (ozone gas treatment) as described in Example 6.
  • Example 10 Comparative study of present process with conventional process for producing HA or salt thereof.
  • Non-requirement of broth dilution (addition of water) step for reducing the viscosity of the fermentation broth) before down streaming results in:
  • the foregoing process can be tailored/regulated/modified according to the consumer and market requirements of HA or salt thereof.
  • the foregoing process can be applied to other HA producing Streptococcus spp. Further, it can be carried out as continuous, semi-continuous, batch, and/or fed batch process. It can also be implemented for small, medium, and/or large-scale production of HA or salt thereof.

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  • Wood Science & Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

La présente divulgation concerne un procédé de production d'acide hyaluronique (HA) de faible poids moléculaire ou d'un sel de celui-ci, comprenant la production d'un HA de poids moléculaire élevé ou d'un sel de celui-ci par fermentation; et la réalisation d'une fragmentation in situ dudit HA de poids moléculaire élevé ou d'un sel de celui-ci, pour obtenir un HA de faible poids moléculaire ou un sel de celui-ci.
PCT/IN2024/052402 2023-12-29 2024-12-20 Procédé de production d'acide hyaluronique de faible poids moléculaire ou d'un sel de celui-ci Pending WO2025141602A1 (fr)

Applications Claiming Priority (2)

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IN202321089681 2023-12-29
IN202321089681 2023-12-29

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WO2025141602A1 true WO2025141602A1 (fr) 2025-07-03

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101294180B (zh) * 2008-05-26 2012-04-18 江南大学 一种在发酵过程中添加过氧化氢和抗坏血酸生产小分子量透明质酸的方法
KR20130128655A (ko) * 2012-05-17 2013-11-27 한국교통대학교산학협력단 저분자량 히알루론산 나트륨의 제조방법
US11155569B2 (en) * 2016-12-30 2021-10-26 Green Valley (Shanghai) Pharmaceuticals Co., Ltd. Method of degrading polysaccharide using ozone

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101294180B (zh) * 2008-05-26 2012-04-18 江南大学 一种在发酵过程中添加过氧化氢和抗坏血酸生产小分子量透明质酸的方法
KR20130128655A (ko) * 2012-05-17 2013-11-27 한국교통대학교산학협력단 저분자량 히알루론산 나트륨의 제조방법
US11155569B2 (en) * 2016-12-30 2021-10-26 Green Valley (Shanghai) Pharmaceuticals Co., Ltd. Method of degrading polysaccharide using ozone

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SERRA MÓNICA, CASAS ANA, TOUBARRO DUARTE, NOVO BARROS ANA, TEIXEIRA JOSÉ ANTÓNIO: "Microbial Hyaluronic Acid Production: A Review", MOLECULES, vol. 28, no. 5, 22 February 2023 (2023-02-22), pages 1 - 16, XP093301838, DOI: 10.3390/molecules28052084 *
YUE WU: "Preparation of low-molecular-weight hyaluronic acid by ozone treatment", CARBOHYDRATE POLYMERS, vol. 89, no. 2, 25 March 2012 (2012-03-25), GB , pages 709 - 712, XP028484877, ISSN: 0144-8617, DOI: 10.1016/j.carbpol.2012.03.081 *

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