WO2022136277A1 - Souches bactériennes pour la production de biocellulose - Google Patents

Souches bactériennes pour la production de biocellulose Download PDF

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Publication number
WO2022136277A1
WO2022136277A1 PCT/EP2021/086800 EP2021086800W WO2022136277A1 WO 2022136277 A1 WO2022136277 A1 WO 2022136277A1 EP 2021086800 W EP2021086800 W EP 2021086800W WO 2022136277 A1 WO2022136277 A1 WO 2022136277A1
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WO
WIPO (PCT)
Prior art keywords
biocellulose
rscic
dps
efl
access
Prior art date
Application number
PCT/EP2021/086800
Other languages
English (en)
Inventor
Sandra PUCCIARELLI
Rita GIOVANNETTI
Marco ZANNOTTI
Alessio Mancini
Maria Sindhura JOHN
Joseph Amruthraj NAGOTH
Original Assignee
Irides S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Irides S.R.L. filed Critical Irides S.R.L.
Priority to EP21840612.2A priority Critical patent/EP4267721A1/fr
Priority to KR1020237021058A priority patent/KR20230124919A/ko
Publication of WO2022136277A1 publication Critical patent/WO2022136277A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • 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
    • C12R2001/07—Bacillus

Definitions

  • the present application relates to the field of microbiology and in particular isolated and verified bacterial strains for biocellulose production .
  • a bacterial cultivation system for the production of cellulose gel (HSBC) containing Gluconacetobacter xylinus (G. xylinus) in an interface system between an aqueous and an oily phase in the presence of glucose and mannitol at 30°C is known (Horu Hoshia, Kazuyoshi Yamazakia, Yuki Satob, Takaya Shidaa, Takao Aoyagia, Production of hollow-type spherical bacterial cellulose as a controlled release device by newly designed floating cultivation, Heliyon 4, 2018, e00873) .
  • the inventors of the present invention have isolated new bacterial strains belonging to the genus Bacillus and Brevundimonas capable of producing biocellulose.
  • Said strains have peculiar characteristics compared to strains known in the art. In particular, they are able to synthesise biocellulose under room temperature conditions, i.e. without energy expenditure, compared to known processes which take place at a temperature of around 30°C. In the process, no specific saline solutions are used or prepared, but plain water is used, and the culture medium is not a limiting factor and different types can be used in the bacterial culture without affecting the success of the process for biocellulose production. In contrast to what is known in the art in which the culture media are added with glucose and mannitol, the former at concentrations of at least 3%, in the culture of the present invention glucose or fructose at 1.5% is added.
  • Another object of the present ibnvention is the use of Brevundimonas efl Access no. DPS RE RSCIC 24 for biocellulose production.
  • Still another object of the present invention is a process for biocellulose production including the following steps: a) incubation of Brevundimonas efl Access no. DPS RE RSCIC 24 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth;
  • RECTIFIED SHEET (RULE 91) ISA/EP b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture .
  • a further obj ect of the present invention is biocellulose obtained by means of the above process .
  • Figure 1 shows a representative infrared spectroscopy (FTIR) spectrum of the materials obtained in static or stirring mode with both bacteria : the spectrum indicates that the material in question is cellulose .
  • FTIR infrared spectroscopy
  • Figure 2 shows a reference spectrum from Yim, S . M . ; Song, J . E . , Kim, H . R . Production and characterization of bacterial cellulose fabrics by nitrogen sources of tea and carbon sources of sugar, Process Biochemistry 2017 , 59 , 26-36
  • biocellulose means cellulose of natural , microbial origin characterised by fibres having variable diameter .
  • culture medium suitable for bacterial growth means solid or liquid solutions containing nutrients on which bacterial colonies are possible .
  • the obj ect of the present invention is Brevundimonas efl Access n .
  • DPS RE RSCIC 24 The obj ect of the present invention is Brevundimonas efl Access n .
  • a further obj ect of the present invention is the use of Brevundimonas efl Access n .
  • Another another obj ect of the present invention is a process for biocellulose production including the following steps : a ) incubation of Brevundimonas efl Access n . DPS RE RSCIC 24 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water, addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture .
  • step a Brevundimonas ef l Access no .
  • DPS RE RSCIC 24 is in combination with Bacillus ef l Access no .
  • DPS RE RSCIC 23 is optionally in step a .
  • a further obj ect of the present invention is biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Brevundimonas ef l Access n . DPS RE RSCIC 24 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture .
  • the obj ect of the present invention is also the use of biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Brevundimonas efl Access n . DPS RE RSCIC 24 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture for the absorption of antibiotics and the subsequent slow release thereof into the environment .
  • a further object of the present invention is the use of biocellulose of bacterial origin obtained by a process comprising the following steps : a) incubation of Brevundimonas efl Access n. DPS RE RSCIC 24 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b) dilution of the culture obtained at the end of step a) with water and addition of glucose or fructose in an amount ranging between 1.5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture for the production of biocellulose filters.
  • step a) Brevundimonas efl Access no.
  • DPS RE RSCIC 24 is in combination with Bacillus efl Access no.
  • DPS RE RSCIC 23 is optionally in step a) Brevundimonas efl Access no.
  • the filters can be used in air conditioning or water filtration systems, for example in swimming pools and/or aquariums to block bacterial growth and maintain a healthy environment .
  • the biocellulose spheres contain silver nanoparticles with antimicrobial activity.
  • the bacterial culture medium can be selected from the group consisting of: Hestrin-Schramm, Yamanaka, Zhou, nutrient broth or yeast extract .
  • yeast extract is yeast extract (0.5-1%) .
  • the water is seawater or deionised water.
  • the nutrient broth has the following composition:
  • step a) and step b) the bacteria are incubated at a temperature ranging from 22 and 25 °C.
  • step a) the bacteria are incubated statically or under stirring .
  • the bacteria When in step a) the bacteria are incubated under stirring, they are preferably under stirring by means of a stirrer at a speed of about 150-200 rpm.
  • the adequate bacterial growth is the achievement of an optical density of about 0.6 OD at 550 nanometres.
  • step b) if the water is seawater in an amount equal to
  • step b) if the water is deionised water it is in an amount equal to 90 % of the final volume.
  • step b) glucose or fructose is present in an amount equal to 1.5%.
  • the temperature is 22 °C.
  • the obtained biocellulose is in the form of a sheet when in step a) the incubation is in static mode.
  • the obtained biocellulose is in the form of spheres when in step a) the incubation is under stirring and preferably they have an average diameter ranging between 2 and 10 mm.
  • the first strain Bacillus efl Access no. DPS RE RSCIC 23 was identified as belonging to the genus Bacillus on the basis of the 16S rDNA sequence, it is similar to Bacillus altitudinis, it is a gram-positive rod-shaped bacterium. SEQ ID . NO . 1 PRQKKA_03858 Bacillus 16S ribosomal RNA
  • DPS RE RSCIC 24 was identified as belonging to the genus Brevundimonas on the basis of the 16S rDNA sequence
  • DPS RE RSCIC 24 is similar to Brevundimonas vesicularis adapted to live in Martian conditions .
  • sequences similar to the 16S rRNA gene there is one from the strain Brevundimonas sp . R-37024 isolated from a Lake Lundstrdm in Antarctica .
  • Brevundimonas ef l possesses an enzyme involved in cellulose biosynthesis .
  • the nutrient broth had the following composition:
  • Yeast extract is a source of vitamins, particularly B group vitamins. Sodium chloride maintains the osmotic balance of the medium.
  • the bacteria incubated at 22°C at 150-200 rpm of rotation.
  • the culture is diluted with seawater H 2 0 (50 % of the final volume) or deionised water (90 % of the final volume) . and added with 1.5-2% glucose, and further incubated statically (without stirring) or with stirring at 22°C.
  • biocellulose sheet is produced (static mode)
  • biocellulose spheres are produced (stirring mode) by Bacillus efl Access no. DPS RE RSCIC 23 in nutrient broth diluted with 50% seawater and added with 1.5-2%.
  • biocellulose sheet is produced (static mode)
  • biocellulose spheres are produced (stirring mode) by Brevundimonas efl Access no.
  • DPS RE RSCIC 24 of 25/11/2020 both cultivated in nutrient broth and in yeast extract occurs .
  • biocellulose spheres The ability of the biocellulose spheres to absorb external substances as previously produced was further tested .
  • Methylene blue is partially released into the culture medium: after 18 hours , about l/20th of the initial amount absorbed is released .
  • Table 2 reports the production methods ( rotation speed, medium volume , flask size ) , yield and average size of the biocellulose spheres synthesised by Bacillus ef l and Brevundimonas efl after 5 days of culture .
  • the biocellulose in the form of spheres was also soaked in a solution containing silver nanoparticles ( 20 mg/ml ) having antimicrobial activity, as described in the application incorporated herein for Italian patent reference no . 102019000014121 by the same inventors , 24 hours and subj ected to UV ray sterilisation for 10 minutes . Then the biocellulose spheres were placed in a plate with Muller hinton agar where a strain of Candida albicans was inoculated . The growth inhibitory effect of Candida was visible with a halo of about 4-5 mm even after 72 hours at 22 ° C .

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une souche bactérienne appartenant au genre Brevundimonas et son utilisation pour la production de biocellulose, ainsi qu'un procédé de production de biocellulose et la biocellulose obtenue grâce audit procédé.
PCT/EP2021/086800 2020-12-22 2021-12-20 Souches bactériennes pour la production de biocellulose WO2022136277A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP21840612.2A EP4267721A1 (fr) 2020-12-22 2021-12-20 Souches bactériennes pour la production de biocellulose
KR1020237021058A KR20230124919A (ko) 2020-12-22 2021-12-20 바이오셀룰로오스 생산을 위한 박테리아 균주

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT102020000031769A IT202000031769A1 (it) 2020-12-22 2020-12-22 Ceppi batterici per la produzione di biocellulosa
IT102020000031769 2020-12-22

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WO2022136277A1 true WO2022136277A1 (fr) 2022-06-30

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PCT/EP2021/086800 WO2022136277A1 (fr) 2020-12-22 2021-12-20 Souches bactériennes pour la production de biocellulose

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KR (2) KR20230124918A (fr)
IT (1) IT202000031769A1 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274937A1 (fr) * 2021-07-01 2023-01-05 Irides S.R.L. Production de biomatériaux à partir de déchets alimentaires par des souches bactériennes antarctiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR691410A (fr) 1930-03-03 1930-10-21 Jeu de direction indéréglable pour cycles, motocycles, etc.
IT201900014121A1 (it) 2019-08-06 2021-02-06 Univ Degli Studi Di Camerino Marinomonas ef1 e rhodococcus ef1 per la produzione di metaboliti secondari

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR691410A (fr) 1930-03-03 1930-10-21 Jeu de direction indéréglable pour cycles, motocycles, etc.
IT201900014121A1 (it) 2019-08-06 2021-02-06 Univ Degli Studi Di Camerino Marinomonas ef1 e rhodococcus ef1 per la produzione di metaboliti secondari

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BAGEWADI ZABIN K ET AL.: "CARBOHYDRATE RESEARCH", vol. 491, 2020, PERGAMON, article "Statistical optimization and characterization of bacterial cellulose produced by isolated thermophilic Bacillus licheniformis strain ZBT2"
BAGEWADI ZABIN K ET AL: "Statistical optimization and characterization of bacterial cellulose produced by isolated thermophilic Bacillus licheniformis strain ZBT2", CARBOHYDRATE RESEARCH, PERGAMON, GB, vol. 491, 7 March 2020 (2020-03-07), XP086127970, ISSN: 0008-6215, [retrieved on 20200307], DOI: 10.1016/J.CARRES.2020.107979 *
HOSHI TORU ET AL.: "Production of hollow-type spherical bacterial cellulose as a controlled release device by newly designed floating cultivation", HELIYON, vol. 4, no. 10, 2018, XP055837771, DOI: 10.1016/j.heliyon.2018.e00873
HOSHI TORU ET AL: "Production of hollow-type spherical bacterial cellulose as a controlled release device by newly designed floating cultivation", HELIYON, vol. 4, no. 10, 1 October 2018 (2018-10-01), GB, pages e00873, XP055837771, ISSN: 2405-8440, DOI: 10.1016/j.heliyon.2018.e00873 *
LUCIE BACAKOVA ET AL.: "Versatile Application of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing", NANOMATERIALS, vol. 9, no. 2, 2019, XP055761047, DOI: 10.3390/nano9020164
LUCIE BACAKOVA ET AL.: "VersatileApplication of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing", NANOMATERIALS, vol. 9, no. 2, 2019, XP055761047, DOI: 10.3390/nano9020164
LUCIE BACAKOVA ET AL: "Versatile Application of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing", NANOMATERIALS, vol. 9, no. 2, 29 January 2019 (2019-01-29), pages 164, XP055761047, DOI: 10.3390/nano9020164 *
RICHARD AUTAGRAZYNA ADAMUSMICHAL KWIECIENIZA RADECKAPAUL HOOLEY: "Production and characterization of bacterial cellulose before and after enzymatic hydrolysis", AFRICAN JOURNAL OF BIOTECHNOLOGY, vol. 16, no. 10, 2017, pages 470 - 482
VERHOEF R ET AL: "Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine", CARBOHYDRATE RESEARCH, PERGAMON, GB, vol. 337, no. 20, 5 November 2002 (2002-11-05), pages 1821 - 1831, XP004392178, ISSN: 0008-6215, DOI: 10.1016/S0008-6215(02)00280-X *
YANG HUJEFFREY M. CATCHMARK: "Formation and Characterization of Spherelike Bacterial Cellulose Particles Produced by Acetobacter xylinum JCM 9730 Strain", IOMACROMOLECULES, vol. 11, 2010, pages 1727 - 1734
YIM, S. M.SONG, J. E.KIM, H. R.: "Production and characterization of bacterial cellulose fabrics by nitrogen sources of tea and carbon sources of sugar", PROCESS BIOCHEMISTRY, vol. 59, 2017, pages 26 - 36

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274937A1 (fr) * 2021-07-01 2023-01-05 Irides S.R.L. Production de biomatériaux à partir de déchets alimentaires par des souches bactériennes antarctiques

Also Published As

Publication number Publication date
KR20230124918A (ko) 2023-08-28
EP4267721A1 (fr) 2023-11-01
IT202000031769A1 (it) 2022-06-22
KR20230124919A (ko) 2023-08-28
WO2022136246A1 (fr) 2022-06-30
EP4267720A1 (fr) 2023-11-01

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