EP4041905A2 - Substance nanocellulosique bactérienne et utilisations associées - Google Patents

Substance nanocellulosique bactérienne et utilisations associées

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Publication number
EP4041905A2
EP4041905A2 EP20792284.0A EP20792284A EP4041905A2 EP 4041905 A2 EP4041905 A2 EP 4041905A2 EP 20792284 A EP20792284 A EP 20792284A EP 4041905 A2 EP4041905 A2 EP 4041905A2
Authority
EP
European Patent Office
Prior art keywords
bnc
process according
range
culturing
medium
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP20792284.0A
Other languages
German (de)
English (en)
Inventor
Isabel ALVAREZ-MARTOS
Deby FAPYANE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellugy Aps
Original Assignee
Cellugy Aps
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 Cellugy Aps filed Critical Cellugy Aps
Publication of EP4041905A2 publication Critical patent/EP4041905A2/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • 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
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H11/00—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
    • D21H11/16—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only modified by a particular after-treatment
    • D21H11/18—Highly hydrated, swollen or fibrillatable fibres
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2301/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives
    • C08J2301/02—Cellulose; Modified cellulose

Definitions

  • the present invention relates to a packaging material based on bacterial nanocellulose (BNC).
  • BNC bacterial nanocellulose
  • the present invention relates to a packaging material, which is compostable.
  • Fossil-based/Biodegradable Fossil fuel based but designed to be biodegradable. Primarily used as additives to improve performance of other bioplastics: a comparatively small group.
  • Bio-based/non-biodegradable Entirely or partially produced from bio-based renewable resources yet technically equivalent to fossil-based counterparts so they are not biodegradable. Around 56.8% of global bioplastics production capacity in 2018.
  • FCMs food contact materials
  • Starch is a polysaccharide produced by green plants for energy storage. Native starch cannot be applied to thermoplastic processing and thus, is usually a complex blend with compostable polymers and additives like plasticizers. This is necessary to improve water resistance, processing properties and mechanical properties. Commonly the starch content in this blends is lower than 50% and not all are allowed for food contact applications.
  • PLAs are amorphous or semi-crystalline polyesters that can be produced from the fermentation of lactic acid. Specific benefits of PLA in packaging applications are its transparency, gloss, stiffness, printability, processability and excellent aroma barrier.
  • PLA is a rigid material with (mechanical) properties that are comparable with Polystyrene (PS), Polypropylene (PP), and Polyethylene Terephthalate (PET).
  • PS Polystyrene
  • PP Polypropylene
  • PET Polyethylene Terephthalate
  • the world of bioplastics is increasingly becoming one of biosciences.
  • the fastest growing bio-based & biodegradable bioplastic is PH A, which is synthesized using "white biotechnology" (/.e. produced by microorganisms).
  • PHAs are generally extracted with chlorinated solvents from heterotrophic bacteria grown on refined sugars or oils.
  • Cellulose long been used as a packaging material, has recently attracted renewed attention because of its biobased character. Besides being the chief component of paper, cellulose is also used to make cellophane and cellulose acetate. Cellophane is a transparent material widely used in confectionery and bakery items. It cannot be thermoformed (i.e. via melting) and a separate sealing layer is required to make the material sealable. Cellophane offers extremely good biodegradability in diverse environments. Cellulose acetate is extremely suitable for food serviceware such as cups for hot drinks and cutlery.
  • nanocellulose can be obtained using "white biotechnology", where it is biosynthesized from glucose using specific bacterial strains. These nanofibers are devoid of other contaminating polysaccharides (lignin, hemicelluloses and pectin) and have greater optical transparency.
  • the mechanism of bacterial nanocellulose (BNC) production is the construction of an interface air/culture medium film. Therefore, isolation and purification is relatively simple, requiring no energy or chemical intensive processes.
  • First commercially available products of BNC was nata de coco, originated in the Philippines during the 1990s. Commercialization is still incipient and focused on high-value niche markets (eg. tissue engineering, wound dressing, artificial skin and blood vessels and carriers for drug delivery).
  • biobased bioplastics are mostly made from food crops (so-called “first generation feedstocks”) such as sugarcane, sugar beet, corn, potato, and wheat. While first generation feedstocks have reached an advanced stage and are widely used in many countries, the European Parliament recently supported the need to set limits to their use, raising concerns about food price, land use impact, and food shortage. Therefore, the bioplastics industry is currently searching for second and third generation feedstocks (e.g. waste, residues, or algae), which don't create additional demand for land for non-food production. Although, these new technologies are still on experimentation and demonstration stage, they leave significant potential for using biotechnology to create new materials for industrial purposes - among them bioplastics
  • an improved bio-based & biodegradable material would be advantageous, and in particular a more efficient and/or reliable process for producing such materials would be advantageous.
  • the present invention relates to a nanocellulose-based film produced by using fruits as raw material.
  • the nanocellulose-based flexible films protects both the food it packs and the environment. Mimicking nature, the material has been designed to either break down into healthy nutrients for soil, or be recycled in a closed-loop process with minimum loss in material performance.
  • the films can be disposed and recycled in the existing system for paper and cardboard and is converted into compost at room temperature in the compost heap or together with organic waste, where it may decompose in 3-16 weeks (see e.g. example 18).
  • the raw materials may be renewable and non-edible or produced by using fruit waste streams, avoiding the competition with crops that might be used to feed the exponentially growing population.
  • the films are created to address different food packaging requirements: (1) Barrier properties, some food products require breathable films, while others require strong barrier against oxygen and water, (2) Sealability, by integrating a sealing layer to close the films using standard heat sealing equipment and (3) Dimensions to obtain films with the required size for different packaging applications (e.g. bags, lidding, or wrapping films).
  • the film can be easily modified by coating with other polymers, generating layers with different properties. All the polymers used in this invention are cellulose-based, with each contributing to provide different properties to the final films. The concentration and thickness of each layer have been optimized to provide the films with the required oxygen and water barrier properties.
  • the film of the present invention utilizes cellulose-based polymers such as CMC (carboxymethylcellulose) or MC (methylcellulose), usually used as thickeners in food or cosmetics. Both polymers are cheap, abundant and most importantly biodegradable, making the perfect match for a 100% biodegradable packaging.
  • CMC carboxymethylcellulose
  • MC methylcellulose
  • the film of the present invention is entirely built from cellulose-based polymers, it behaves like a thin layer of plant cells where nature can degrade it as it degrades plants or wood.
  • the material will disappear in nature and bring benefits as additional soil nutrition.
  • the material is recyclable with cardboard and thus can be disposed in this existing waste collection system.
  • the material is expected not to affect the cardboard quality, as it is pure cellulose (no lignin nor hemicellulose), or even can be used as an additive due to its long fiber nature.
  • an object of the present invention relates to provision of process for producing a bio-based & compostable film, preferably consisting entirely of cellulose-based materials.
  • one aspect of the invention relates a process of producing bacterial nanocellulose (BNC), the process comprising a) culturing in a liquid medium o Komagataeibacter rhaeticus; o Gluconobacter oxydans; and o at least one of Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and Kloeckera lindneri ; and b) removing a hydrogel comprising BNC from the liquid medium, thereby providing BNC.
  • BNC bacterial nanocellulose
  • Another aspect of the present invention relates to a BNC material obtained/obtainable from a process according to the invention.
  • Yet another aspect of the present invention is to provide a process for sealing/gluing one or more BNC materials according to the invention, the process comprising using methylcellulose (MC) and/or Carboxymethylcellulose (CMC) as a sealant/glue to adhere two parts of the BNC material together.
  • Still another aspect of the present invention is to provide a film comprising
  • a base layer comprising bacterial nanocellulose (BNC), wherein 10-20% of the hydroxyl groups are replaced by nitro groups;
  • a sealing layer comprising MC or CMC on at least one side of the film.
  • Yet another aspect of the invention relates to the use of a BNC material according to the invention as a packaging material, a lidding, a sheet, a bag, a lamination material, a wound dressing or a bandage.
  • a further use relates to the use of a composition
  • a composition comprising o Komagataeibacter rhaeticus; o Gluconobacter oxydans; and o at least one of Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and Kloeckera lindneri, ⁇ for production of BNC.
  • a further aspect relates to the provided BNC material according to the invention for use as a medicament.
  • Yet an aspect relates to a product, packed in a material/film according to the invention.
  • Figure 1 shows a schematic overview of the production process, showing different steps from end to end production covering: raw material preparation, cultures treatment and reuse, fermentation conditions and process, BNC treatment and layer structure.
  • A Whole fruit is used as a raw material and feedstock for culture medium during the fermentation.
  • B Fermentation with a symbiotic culture. The arrow indicates "re-use" of the rest of the medium and the culture from for a new fermentation process.
  • Figure 2 shows (A) the bacterial composition of symbiotic cultures and (B) the fungal composition of symbiotic cultures.
  • Figure 3 shows that (A) a strict aerobic fermentation process begins when fermentation medium is mixed with culture. (B) The BNC is synthesized on the surface/interphase of water and air which (C) is easy to be harvested. (D) Silent aeration aerobic fermentation. The method to aerate using secondary aeration tank in order to increase O2 concentration without disturbing the surface static condition.
  • Figure 4 shows a schematic diagram for processing whole vegetable to fruit juice.
  • Figure 5 shows the transparency of the produced films (A) before and (B) after overnight treatment with 1% HNO3.
  • Figure 6 shows heavy metals and fluorine content (ppm on total solids) for the tested material (final dried film). * Microwave digestion was executed on the sample according to DIN EN 13657, before the analysis of the heavy metals; ** Maximum levels for USA (according to ASTM D6868 (2012) heavy metals content must be less than 50% of those prescribed for sludges or composts in the country where the product is sold).
  • Figure 7 shows SEM imaging of (A) dry BNC film and (B) with a spray-coated layer of nitrocellulose. Scalebar 1 pm.
  • FIG 8 shows several applications using heatsealed BNC bags.
  • Figure 9 shows evolution of the disintregration of EcoFLEXY by showing visual presentation of the evolution of the disintegration of dry BNC film coated with 0.25% nitrocellulose at ambient temperature.
  • Figure 10 shows visual presentation of the content of a composting reactor with dry BNC film coated with 0.25% nitrocellulose after 4 weeks of composting at ambient temperature.
  • an aspect of the invention relates to a process for producing bacterial nanocellulose (BNC), the process comprising a) culturing in a liquid culturing medium o Komagataeibacter rhaeticus; o Gluconobacter oxydans; and o at least one of Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and Kloeckera lindneri ; and b) removing a hydrogel comprising BNC from the liquid medium, thereby providing bacterial nanocellulose (BNC).
  • a process has been developed for producing BNC comprising materials.
  • the dominating strains producing the BNC have been identified.
  • example 3 it is also described how the specific bacterial strains are considered to improve the production of BNC.
  • at least two of Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and Kloeckera Hndne such as at least three or such as at least all four strains.
  • one or both of Hanseniaspora uvarum and Brettanomyces bruxellensis present in the culturing medium.
  • the nutritional source can have different origins.
  • the culturing medium in step a) comprises fruit juice and/or vegetable juice, such as filtered or unfiltered fruit or vegetable juice
  • the culturing medium in step a) comprises tea, such as a tea buffered medium.
  • the fruit juice is selected from the group consisting of apple, strawberry, blackberry, aronia (chokeberry), rovada, grape, plum, cranberry, pea, orange, banana and combinations thereof.
  • the vegetable juice is selected from the group consisting of potato, carrot, beet, sugar beet and combination thereof.
  • the culturing medium comprises leftover materials from production of other food products.
  • the culturing medium in step a) comprises fruit and/or vegetable parts such as peels, pulp and juices (or water residue).
  • culturing medium is not sterilized before the bacteria and yeasts are applied to the medium. It has been found that the symbiotic culture of fungi and bacteria will predominate the culturing medium thereby making sterilization unnecessary. Sterilized material may of course also be used.
  • the culturing medium in step a) comprises
  • a sugar content in the range 5 - 15 degrees brix preferably such as 7-13 degrees brix, preferably 8-12 degrees brix;
  • “Brix” or “degrees Brix” is the sugar content of an aqueous solution.
  • One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by mass. If the solution contains dissolved solids other than pure sucrose, then the °Bx only approximates the dissolved solid content.
  • the °Bx is traditionally used in the wine, sugar, carbonated beverage, fruit juice, maple syrup and honey industries.
  • the culturing medium in step a) has a pH value in the range 2.5 - 4, preferably such as in the range 2.8 - 3.8, more preferably in the range 3- 3.5.
  • a pH around 3 is optimal for the culturing medium comprising the symbiotic culture. This is much lower than other BNC producing cultures, which is usually fixed at more neutral pH, 6-7.
  • step a) is performed at a temperature in the range 15-30°C, such as 15-27°C, preferably such as 18-23°C, more preferably such as 20-23°C.
  • a temperature around room temperature is optimal for the culturing medium comprising the symbiotic culture. This was lower than expected.
  • the fermentation conditions should be controlled.
  • the culturing in step a) is aerobic fermentation, preferably static aerobic fermentation, such as without shaking, stirring or any process causing fluctuative medium height level.
  • air is supplied to the medium, by aeration outside the culturing container, such as by aerating a flow tube outside the culturing container.
  • Example 4 and figure 3D describe such an aeration system.
  • the ratio between the bacterial strains can also be optimized.
  • the numerical ratio between Komagataeibacter rhaeticus and Gluconobacter oxydans is in the range 10:1 to 1:10, such as 10:1 to 1:3, such as 10:1 to 1:1, preferably 7:1 to 2:1, more preferably 5:1 to 2:1, such as around 3:1.
  • Example 3 + figure 2A shows that these two strains are dominating bacteria, preferably with a higher number of Komagataeibacter rhaeticus.
  • the ratio by weight between culturing medium and culture is in the range 5:1 to 1:2, such as 5:1 to 1:1, preferably 5:1 to 2:1, more preferably 4:1 to 2:1, such as around 3:1.
  • Example 8 shows data on such ratios.
  • the culturing (fermentation) period in step a) before collecting the produced BNC hydrogel may vary.
  • the culturing in step a) takes place for a period of 2-10 days at room temperature, preferably 3-7 days, even more preferably 4-6 days. The longer the incubation the thicker the hydrogel will be. A thickness around 0.6-0.7 cm is preferred when collecting the hydrogel.
  • the BNC hydrogel in step b) is removed from the surface of the culturing medium or from the interphase between surface and culturing medium. In yet a related embodiment, the BNC hydrogel in step b) is removed, when it has a thickness in the range 0.2 cm to 2 cm, such as in the range 0.3 to 1 cm, preferably in the range 0.5 to 1 cm, more preferably 0.6-0.8 cm, such as around 0.7 cm. A thickness of 0.6-0.7 cm may be obtained after 5 days.
  • the culturing medium After collecting the hydrogel, the culturing medium may be reused. Thus, in an embodiment, the remaining culturing medium after removal of the BNC hydrogel in step b) is used as an inoculum in a new culturing step a).
  • the process further comprises a step c) of sterilizing the provided BNC hydrogel.
  • the sterilization in step c) is performed by incubation in a base such as NAOH and/or NaCICM.
  • NAOH is preferred.
  • NaCICM is preferred.
  • sterilization using NaOH may result in undesired colouring of the sterilized material at higher concentration.
  • the sterilization in step c) is performed in
  • NaOH within this range does not result in undesired colouring of the material, instead a white material is provided.
  • the process further comprises a step d) of treating the sterilized BNC hydrogel with acid, to provide a transparent BNC gel.
  • the acid treatment is performed in HNO3, more preferably at a concentration of 0.3 to 2% (w/w), even more preferably at a concentration of 0.5 to 1% (w/w).
  • Example 10 shows that a transparent material is provided when 0.5 to 1% (w/w) HNO3 is used.
  • the process further comprises a step e) of drying the provided BNC hydrogel, to provide a dried BNC material.
  • the drying step e) is performed at a temperature in the range 30- 40°C such as for 10-20 hours, preferably at 33-39°C for 12-18 hours.
  • the drying is performed on a non-stick surface such as on silicone, preferably an oil-coated non-stick surface such as silicone surface, such as coated with rapseed oil, sunflower or any vegetable-based oil.
  • Example 11 shows optimization of the drying step.
  • the dried BNC material may have different thicknesses, depending on the start material and drying conditions.
  • the provided dried BNC material has a thickness in the range 10-100 pm, preferably 10-60 pm, more preferably 20-60 pm. Additional features of the dried material may be introduced by modifying the material.
  • the process further comprises a step f) of coating the dried BNC material with nitrocellulose, such as coating with a 0.1 to 5% (w/w) solution of nitrocellulose, such as a 0.1 to 3%, such as 0.1 to 2%, preferably 0.5-1.5%, and more preferably 0.8-1.2%.
  • nitrocellulose treatment enhances puncture resistance, water resistance, O2 resistance and flexibility.
  • a nitrocellulose layer is coated on each side of the dried BNC material.
  • the process comprises steps a) to d) as outlined above.
  • the process comprises steps a) to e) as outlined above.
  • the process comprises steps a) to f) as outlined above.
  • antioxidants and polyphenols can improve the output of the process (see example 19).
  • Polyphenols, such as Theaflavins are also present in tea (black tea).
  • the liquid culturing medium comprises tea, preferably black tea.
  • the liquid culturing medium comprises one or more antioxidants and/or polyphenols.
  • the antioxidant is ascorbic acid.
  • the polyphenol is selected from the group consisting of Theaflavin and Epicatechin.
  • the polyphenol is an antioxidant polyphenol, such as Theaflavin or derivatives thereof, wherein the derivative thereof comprises a tropolone moiety.
  • the one or more Theaflavins or derivatives thereof are selected from the group consisting of Theaflavin, Theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3-3'-digallate.
  • the liquid culturing medium comprises one or more antioxidant polyphenols, such as Theaflavin, at a concentration in the range 5-100 g/l, such as in the range 5-80 g/l, preferably in the range 20-50 g/l, and more preferably 30-40 g/l.
  • one or more antioxidant polyphenols such as Theaflavin
  • Another aspect of the invention relates to a BNC material obtained/obtainable from a process according to the invention.
  • he BNC material is transparent (to the naked eye).
  • the material has a HAZE level above 85 % measured according to ASTMD 1003:2013.
  • the BNC material has
  • TS total solid content
  • a moisture content below 40% such as below 30%, such as below 20%, preferably below 15%, and more preferably below 10%;
  • VS,% on TS a total volatile solids content (VS,% on TS) above 60%, such as above 70% such as above 80% preferably above 85%, and more preferably above 87%;
  • an ash content (% on TS) below 40% such as below 30%, such as below 20%, preferably below 15%, and more preferably below 13%.
  • the BNC material is in compliance with EN 13432 (2000), NF T51-800 (2015), ASTM D6868 (2017) and/or CAN/BNQ 0017-088 (2010) in relation to heavy metal content and fluorine content.
  • the BNC material is in compliance with EN 13432, the French standard NF T51-800, the Canadian standard CAN/BNQ 0017-088 and/or the international standard ISO 18606 (2013) in relation to minimum volatile solids content of 50% on total solids (TS).
  • the BNC material is in compliance with biodegradability according to the French standard NF T 51-800 (Plastics - Specifications for plastics suitable for home composting (2015)). In a related embodiment, the BNC material is biodegradable.
  • biodegradable refers to the ability of materials to break down and return to nature. In order for packaging products or materials to qualify as biodegradable, they must completely break down and decompose into natural elements within a short time after disposal - typically a year or less. The ability to biodegrade within landfills helps to reduce the buildup of waste, contributing to a safer, cleaner and healthier environment.
  • Materials that are biodegradable include corrugated cardboard and even some plastics. Most plastics, however, are not biodegradable - meaning they cannot break down easily after disposal and can remain on the planet as waste for decades.
  • the BNC material is compostable.
  • the term “compostable material” is similar to “biodegradable material", as they are both intended to return to the earth safely. However, “compostable materials” go one step further by providing the earth with nutrients once the material has completely broken down. These materials may be added to compost piles, which are designated sites with specific conditions dependent on wind, sunlight, drainage and other factors. While “biodegradable materials” are designed to break down within landfills, “compostable materials” require special composting conditions.
  • Compostable packaging materials include starch-based packing peanuts - an alternative to Styrofoam loose fill packaging that can be dissolved in water and added to composts for safe disposal. See also examples 12, 14 and 18.
  • a BNC material according to the invention is (with thickness 40-50 um) (dried BNC films after basic/acid treatment) is disintegrable in soil after 4 weeks. See example 18.
  • the BNC material is in compliance with 1935/2004/EC, 2023/2006/EC, and/or the Danish food contact regulation Bekendtgorelse nr. 822 of 26.06.2013.
  • the BNC material may also have other properties.
  • the BNC material has a water vapour permeability in the range of 20-150 [g/(m 2 .day)] depending on the material layering procedures.
  • the water vapour permeability was determined using Water Permeance determined in accordance with ASTM E96/E96M-16.
  • the BNC material has a:
  • the BNC material has a puncture resistance of at least 10N.
  • the BNC material has an oxygen permeability of 5-162 [cm 3 /(m 2 x day x bar)] depending on the material layering procedures.
  • the BNC material is burn resistant up to 300C.
  • the BNC material comprises Theaflavin or a derivative thereof, such as selected from the group consisting of Theaflavin, Theaflavin-3-gallate, Theaflavin-3'-gallate, and Theaflavin-3-3'-digallate.
  • the materials comprises theaflavin.
  • the theaflavins or derivatives thereof are present at a concentration in the range 5-100 g/l, such as in the range 5-80 g/l, preferably in the range 20-50 g/l, and more preferably 30-40 g/l.
  • the Theaflavins or derivatives thereof are present in the hydrogel. As shown in example 19, the addition of Theaflavin improves the process substantially. Wihtout being bound by theory, it is believed that the Theaflavin will also be present in the obtained material, such as in the hydrogel.
  • an aspect of the invention relates to a process for sealing/gluing one or more BNC materials according to the invention together, the process comprising using methylcellulose (MC) and/or Carboxymethylcellulose (CMC) as a sealant/glue to adhere two parts of the BNC material together.
  • the sealing can be comprises with heat.
  • a product is sealed inside the one or more BNC materials.
  • the product is selected from the group consisting of solid products such as a food product, a cosmetic product, and tableware. It is of course to be understand that other solid product groups could also be packed inside the material.
  • Figure 8 shows different materials packed inside a sealed material according to the invention.
  • an aspect of the invention relates to a film comprising - a base layer comprising bacterial nanocellulose (BNC), wherein 1-50%, such as 5-40%, preferably 5-30% or more preferably 10-20% of the hydroxyl groups are replaced with nitro groups;
  • BNC bacterial nanocellulose
  • a sealing layer comprising MC or CMC on at least part of the film.
  • the invention relates to a film comprising
  • BNC bacterial nanocellulose
  • a sealing layer comprising MC or CMC on at least part of the film.
  • hydroxyl groups are replaced with nitro groups or more preferably 10-20% of the hydroxyl groups are replaced with nitro groups (in the bacterial nanocellulose (BNC).
  • treatment with HNCb introduces nitro groups in the BNC (see example 10), whereas the nitrocellulose layer introduces further functionalities.
  • the methyl/carboxymethylcellulose may function as a sealant.
  • a nitrocellulose layer is present on both sides of the base layer.
  • the base layer has a thickness in the range 10-100 pm, preferably 10-60 pm, more preferably 20-60 pm;
  • the nitrocellulose layer has a thickness in the range of 1-10 pm.
  • the film is transparent.
  • the material has a HAZE level above 85 % measured according to ASTMD 1003:2013.
  • the film has - a total solid content (TS,%) above 60%, such as above 70%, such as above 80% preferably above 85%, and more preferably above 90%; and/or
  • a moisture content below 40% such as below 30%, such as below 20%, preferably below 15%, and more preferably below 10%;
  • VS,% on TS a total volatile solids content (VS,% on TS) above 60%, such as above 70% such as above 80% preferably above 85%, and more preferably above 87%;
  • an ash content (% on TS) below 40% such as below 30%, such as below 20%, preferably below 15%, and more preferably below 13%.
  • the film is in compliance with EN 13432 (2000), NF T51-800 (2015), ASTM D6868 (2017) and/or CAN/BNQ 0017-088 (2010) in relation to heavy metal content and fluorine content.
  • the film is in compliance with EN 13432, the French standard NF T51-800, the Canadian standard CAN/BNQ 0017-088 and/or the international standard ISO 18606 (2013) in relation to minimum volatile solids content of 50% on total solids (TS).
  • the film is biodegradable.
  • the film is in compliance with biodegradability according to the French standard NF T 51-800 (Plastics - Specifications for plastics suitable for home composting (2015)).
  • the film is compostable.
  • the film is in compliance with 1935/2004/EC, 2023/2006/EC, and/or the Danish food contact regulation Bekendtgorelse nr. 822 of 26.06.2013.
  • the film is water resistant.
  • the film has a
  • the film has a puncture resistance of at least 10N.
  • the film has an oxygen permeability of 5-262 ml/(m2 x day x atm (bar)).
  • the film is burn resistant up to 300°C.
  • the materials comprises theaflavin.
  • an alternative aspect of the invention relates to a BNC hydrogel, wherein the hydrogel comprises Theaflavin or derivatives thereof.
  • the hydrogel and/or film comprises traces of Komagataeibacter rhaeticus, Gluconobacter oxydans, Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and/or Kloeckera Hndneri. Since these strains may be part of the culture medium, it is expected that traces may be present in the gel or film. In the present context, traces may be DNA or RNA signatures, or specific protein signatures.
  • the hydrogel or film according to the invention comprises traces of Komagataeibacter rhaeticus, Gluconobacter oxydans and at least one of Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and Kloeckera Hndneri.
  • the traces are of dead microorganisms, as the process preferably comprises a sterilization step.
  • the BNC material according to the invention may have different uses in either dry or in the hydrogel form (with water content).
  • the invention relates to the use of a BNC material according to the invention as a packaging material, a sheet, a bag, a lamination material, a wound dressing or a bandage.
  • the use is for packaging fruit, such as dried or fresh fruit.
  • hydrogel material as a lamination material, a wound dressing, a facial mask, or as a thickener such as for food or cosmetics.
  • a further use relates to the use of a composition
  • a composition comprising o Komagataeibacter rhaeticus; o Gluconobacter oxydans; and o at least one of Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii and Kloeckera lindneri ; for production of BNC.
  • a further aspect relates to the provided BNC material according to the invention for use as a medicament.
  • the BNC material according to the invention for use as a wound dressing with the proviso that the BNC material is a hydrogel.
  • Dried material may function as artificial skin or a biodegradable bandage.
  • the material is a dried material according to the invention.
  • the product is selected from the group consisting of food, such as dried fruit, fresh fruit, prepacked food, cosmetics, such as soap, a packed shampoo, table wear, household vegetable garbage and household fruit garbage (solid goods).
  • Example 2 Isolation of symbiotic culture composition for production
  • the kombucha fungus starter (Wellness-Drinks, Eleonore-Sterling-Str. 2060433 Frankfurt am Main). Black tea extract (Ahmad tea, UK). No-added sugar pasteurized juice (Rynkeby, Denmark). Sucrose (Sigma Aldrich, Denmark).
  • Refractometer to measure sugar brix at the range of 0-20% with sensitivity 0.1% (ESSKA GmBH, Germany).
  • the symbiotic culture starter was treated with medium consisting of 10% sucrose and 10% black tea extract at 25°C with ratio culture: medium of 1:3.
  • the starter is being cultured in batch mode for more than 20 times (1 week per batch).
  • the resulting new culture emerged from batch no 20, and was used for further testing with diverse type of juice as it showed stability in producing stable wet weight of BNC.
  • the symbiotic culture from tea medium treatment was further grown in large variety of fruit and vegetable juices (no further treatment, used as it is) with the same ratio as before.
  • the brix was measured everyday using refracto meter. At the end of day 3 and 5, %-sugar used were calculated.
  • Table 1 shows that the culture consumes sugar the most in the filtered apple and carrot juice with total sugar consumption up to 38-39%. However, the culture in the carrot juice shown crumbly BNC production due to high fibers content in the juice. Therefore, the culture grown in the filtered apple juice is selected for further test and production. Table 1. Starter culture adaptation and selection using vegetables/fruit juices as medium
  • Example 3 Culture composition based on the Next Generation Sequencing (NGS) of 16s RNA and DNA.
  • NGS Next Generation Sequencing
  • the protocol for the NGS analyses according ISI service 25130 has been optimised to analyse bacteria, fungi and higher organisms simultaneously.
  • Total DNA is extracted and amplified using a 2-step polymerase chain reaction (PCR) targeting the V3-V4 16S regions of the 16S rRNA gene from prokaryotes, as well as three primer sets targeting the hypervariable regions V3-V4 of the 18S rRNA gene from eukaryotes.
  • the NGS amplicon sequencing is performed on a desktop sequencer (MiSeq from Illumina, Inc.).
  • the sequence data is compared to the RDP database for bacteria, and the Silva database for fungi, using the BION software.
  • the output is the number of reads of sequences which can be assigned to defined species in the databases; the number of reads to some degree correlates with the amount of DNA originally present in the sample.
  • PCR reactions were carried out in 50 pi total volume including 5 ul 10 x TAQ polymerase reaction buffer, 1 ul 10 mM dNTP, 0.6 ul lOOpmol/ul oligo 1, 0.6 ul lOOpmol/ul oligo 2, and 3 units TAQ polymerase.
  • oligos were used: for specific amplification of the isocitrate dehydrogenase gene from Komagataeibacter rhaeticus:
  • Oligo 1 (SEQ ID NO: 1): 5' C ATG G C A A AG AT C A AG GTC AG 3' and oligo 2 (SEQ ID NO: 2): 5' CTTACGCCTTGGCCAGTGC 3'
  • Oligo 1 (SEQ ID NO: 3): 5' ATTACGCGCGAAACCCTC 3' and oligo 2 (SEQ ID NO:
  • Figure 2A shows the outcome of the analysis in relation to bacterial population
  • figure 2B shows the outcome of the analysis in relation to fungal population
  • Figure 2C shows the outcome of the specific DNA oligos specific for Komagataeibacter rhaeticus and Gluconobacter oxydans.
  • the culture of the invention comprises a combination of known bacterial and fungal strains, which are dominating in the culture.
  • Komagataeibacter rhaeticus is known to use C6-sugar, such as glucose and fructose, as carbon source to produce nanocellulose.
  • Their byproduct consists of gluconic acid, C5-ketoglutarate or lower carbon derivative such as lactic (C3) and acetic acid (C2).
  • Gluconobacter oxydans is mainly used for biorefinery for its ability turning C3-4 carbon to lower-carbon derivative such as lactic (C3) and acetic acid (C2). It was just found that it contributes to nanocellulose production as well.
  • Hanseniaspora uvarum Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces baiiii and Kloeckera lindneri ;
  • strains of fungus in the symbiotic cultures is known to break down higher sugar such as disaccharide (sucrose) to C6 or lower that can be used by K.rhaeticus or G.oxydans to produce nanocellulose.
  • higher sugar such as disaccharide (sucrose)
  • K.rhaeticus or G.oxydans to produce nanocellulose.
  • the two bacteria protect the fungus from other contaminant by making acidic pH environment.
  • the bacterial strains can be obtained individually in German Collection of Microorganisms and Cell Cultures GmbH (dsmz.de), while the fungus strain can be found in CBS-KNAW fungal collection (CBS.nl).
  • the bacterial BNC (BNC) is produced during an aerobic fermentation process ( Figure 3A). Generally, the BNC will be synthesized inside the medium to form sphere-like gel, made from nanocellulose (using G. xylinus as culture reference).
  • the BNC is synthesized on the interphase of water (medium) and air in which the shape will follow the shape of the mold or fermenter, typically the fermentation is undergone for 7 days in the 10% sucrose tea medium.
  • aeration with bubbles should be avoided.
  • bubbles can be avoided by aerating the medium in a separate aeration outside the fermentation tank. The aerated medium can then be circulated to the fermentation tank.
  • Table 2 static culturing results in a high wet weight of BNC and BNC films are formed as desired. Table 2. The effect of aeration or shaking in the fermentation process
  • Hestin-Schramm medium To optimize sugar content during fermentation, different sugar concentrations were evaluated in Hestin-Schramm medium.
  • Hestin-Schramm medium To optimize pH during fermentation, different pH's during fermentation were evaluated in Hestin-Schramm medium.
  • the medium is conditioned with different pH with alternating amount of citric acid and sodium biphosphate with 10% sucrose.
  • Hestin-Schramm medium containing 10% glucose concentration with 1.03 g/L citric acid and 6.03 g/L sodium biphosphate.
  • Room temperature is the best condition for the culture to grow which is quite different from literature (30 C) (Machado et al., Komagataeibacter rhaeticus as an alternative bacteria for cellulose production. Carbohydrates Polymers. 2016. 152. 841-849).
  • Figure 4 shows shows a schematic diagram of producing whole fruit to fruit juices.
  • Hestin-Schramm medium the medium containing 10% sugar (sucrose/glucose/fructose) with 1.03 g/L citric acid and 6.03 g/L sodium biphosphate.
  • BNC can be produced from different fruit sources or tea produced better nanocellulose yield than generally used HS medium with sucrose/glucose/fructose. It is also shown that the higher medium ratio to culture gives higher BNC production.
  • hydrogel may comprise attached and trapped bacteria/fungi. It is important to sterilize the hydrogel from any entrapped/attached microorganisms before further use.
  • Freshly- harvested bacterial cellulose hydrogels were first washed with warm tap water, followed by incubation in different NaOH/NaCICM concentrations overnight.
  • Example 10 improved transparency
  • Transparent materials after treatment are shown in figure 5 after 0.5% and 1% treatment with HNO3. Further, the AT-IR fingerprint indicated a % of nitro-groups of 10-20% (% of substituted OH-groups) measured by the ratio of nitro:OH IR stretch. Nitro groups emerged at band 1250-1750 nm in the AT-IR fingerprint (not shown).
  • Acid treatment with 0.5-1% HNO3 makes the hydrogel transparent and introduced nitro-groups in the material.
  • the wet thickness of the BNC film should be controlled during harvest time.
  • the drying time and condition are also important in achieving transparency of the film.
  • the film should preferably be dried on an oily nonstick surface, such as a silicone mat.
  • Rapeseed and sunflower Type of oil tested: Rapeseed and sunflower.
  • the film should preferably be dried on an oily nonstick surface, such as a silicone mat.
  • Optimal temperature is around 37°C for 15 hours. Without being bound by theory, it is believed that the vegetable oil C-H group is masking the BNC surface thus make it more transparent comparing with air-dried BNC which makes it more opaque.
  • Example 12 Volatile solid content
  • Tested material was dried BNC films with thickness 40-50 urn.
  • the total solids or dry matter content is determined by drying at 105°C for at least 14 hours and weighing. Determination of moisture content'. The total solids content is given in percent on wet weight. The volatile solids and ash content is determined by heating the dried sample at 550°C for at least 4 hours and weighing. Determination of organic matter and carbon content'. The results are given in percent on total solids.
  • the total solids content (TS), the moisture content, the volatile solids content (VS) on total solids and the ash content on total solids of the test item are shown in Table 11 below. Table 11. Total solids content, moisture content, volatile solids content and ash content of the material of the invention.
  • Tested materials were dried BNC films with thickness 40-50 um.
  • Figure 6 shows heavy metals and fluorine content (ppm on total solids) for the material of the invention (here denoted EcoFLEXY) together with the required values for the above listed regulations.
  • Tested material was dried BNC films with thickness 40-50 urn.
  • the controlled composting biodegradation test is an optimized simulation of an intensive aerobic composting process where the biodegradability of a test item under dry, aerobic conditions is determined.
  • the inoculum normally consists of stabilized and mature compost derived from the organic fraction of municipal solid waste (MS ).
  • MS municipal solid waste
  • the test item is mixed with the inoculum and introduced into a static reactor vessel where it is intensively composted under optimal oxygen, temperature and moisture conditions.
  • a mixture of gases, principally carbon dioxide and water are the final decomposition products while part of the organic material will be assimilated for cell growth.
  • the carbon dioxide production is continuously monitored and integrated to determine the carbon dioxide production rate and the cumulative carbon dioxide production.
  • the percentage of biodegradation can be calculated as the percentage of solid carbon of the test compound, which has been converted to gaseous, mineral C under the form of CO2. Also the kinetics of the biodegradation can be established.
  • the test is considered as valid if the percentage of biodegradation for the reference item is more than 70% after 90 days; The deviation of the percentage of biodegradation for the reference item in the different vessels is less than 20% at the end of the test;
  • the compost inoculum in the blank control has produced more than 10 and less than 50 mg of CO2 per g of volatile solids (mean values) after 10 days of incubation.
  • Results Results are shown in table 12 below.
  • Tested material was dried BNC films with thickness 40-50 um with additional 1 layer (5 pm) of 0.25% nitrocellulose on one side.
  • Nitrocellulose is a cellulose derivative, which can be degraded by cellulase enzyme (secreted by soil bacteria or fungus) at room temperature. It has property of being transparent and water resistance. Thus, nitrocellulose was used for layering the material (EcoFLEXY) to add water resistance. However, there is limit in its application due to several chemical impurities from its production. Based on the food safety assessment, the limit of nitrocellulose layering is 20 mg/dm3 (equal to 1% nitrocellulose layer to cover A4 paper with thickness 10 micron).
  • nitrocellulose coated materials comply with the requirements of 1935/2004/EC, 2023/2006/EC, and the Danish food contact regulation Bekendtgorelse nr. 822 of 26.06.2013.
  • the dried BNC films were sprayed with 5 ml_ 0.25% nitrocellulose in ethanol using tatoo-spraying equipped with air compressor. The coating was done until the nitrocellulose layer was formed homogenously on the dried BNC surface.
  • SEM imaging ( Figure 7) shows that ecoFLEXY has 10 nm wide BNC fibril with length between 25-100 mhi. Addition of nitrocellulose formed layer on the top of the fibril, covering the available hydrophilic OH group with hydrophobic nitro group.
  • Penetration testing is very similar to compression testing with one key difference, the penetrometer probe is typically much smaller than the sample being tested and passes completely through a sample or an element of the product. Puncture is similar, meaning the probe passes into the sample, though not necessarily exiting.
  • This test method can be performed on a wide variety of food products and is a very useful test in simulating a bite or in comparative analysis.
  • the thickness of the samples are converted to 40 pm thick samples. To make this conversion we assume that the structure of the material is the same throughout the sample. Therefore, these numbers are only indications of the barrier properties for a 40 pm thick sample.
  • the water vapor transmission rate (WTR) measured three different places on one sheet is measured according to the ASTM standard F 1249. The method measures how much water is transferred from the air above the film. Water method
  • the water vapor transmission rates measured on both the three different places on the same sheet and on the 10 different sheets were performed with an Electrometer 5100 Payne Permeability Cup.
  • the device was weighed initially and left in a temperature and humidity controlled room for at least 24 hours before it was finally weighed again. At each weight measurements, temperature and humidity were noted. WTR can be calculated by the differences in weight over time.
  • the water vapor transmission rate was measured three different places using both the desiccant method, where calcium chloride is used and the water method where demineralized water is used. The results show that the difference between the rates for evaporating water through the cellulose films are >500 times higher compared to letting water from the air into the films.
  • Haze is cloudiness of a product that is caused by scattering of light. Light may be scattered by particles suspended in the substance, such as pigment particles or contaminants, or by an imperfect surface caused by dirt, oil, or a fine texture. Haze is an important appearance attribute which can be quantified and then used to assess the quality of objects such as liquids, glass, plastics, painted panels, and even metals.
  • Transmission haze is defined as the forward scattering of light from the surface of a nearly clear specimen viewed in transmission. Normally, light scattered back through the sample is not included. Also, only light scattered more than 2.5° from the incident light is considered to contribute to haze. When measuring haze, the percentage of light diffusely scattered compared to the total light transmitted is reported
  • TGA Thermogravimetric analysis
  • ecoFLEXY and its derivatives is stable up to 300°C before it burns.
  • Table 20 The TGA analysis of ecoFLEXY and its derivatives
  • nitrocellulose forms layer on the top of the fibril, covering the available hydrophilic OH group with hydrophobic nitro group.
  • Example 17 Sealing layer/sealant
  • Methyl cellulose (MC), carboxymethyl cellulose (CMC) and ethanol were from Sigma Aldrich, Denmark. Methods:
  • the MC or CMC (1 g) was dispersed in 5% ethanol, then 2 ml_ boiled water was added. Final gel was formed by addition 8 ml_ of cold water. Stirring and vigorous mixing was necessary to avoid clumps.
  • Figure 8 shows different materials sealed inside a package consisting of the material of the invention.
  • the adhesive may be sodium Carboxymethylcellulose (CMC) and/or methylcellulose (MC), depending on the desire strength of the adhesion.
  • CMC Carboxymethylcellulose
  • MC methylcellulose
  • the material was dried BNC films with thickness 40-50 um.
  • Test item EcoFLEXY was put into slide frames and mixed with compost inoculum. The obtained mixture is incubated in the dark at ambient temperatures (28°C ⁇ 2°C). The test is performed in 2 replicates (qualitative evaluation).
  • Figure 9 gives a visual presentation of the evolution of the disintegration of test material EcoFLEXY during 4 weeks of composting at ambient temperature.
  • the disintegration of EcoFLEXY has started and has proceeded very swiftly. Already after 1 week of composting small holes started to appear in the test material. One week later by far the major part of the test material had disappeared. Only some small test item pieces remained present at the borders of the slide frames. The disintegration proceeded and after 4 weeks of composting all slide frames were completely empty and no test item pieces remained present in the test reactors ( Figure 10).
  • the material according to the invention shows superior disintegration abilities in a composting test.
  • a material with thickness 40-50 pm dried BNC films after basic/acid treatment
  • the nanocellulose production was evaluated using Hestin-Schramm medium: the medium containing 10% sucrose with 1.03 g/L citric acid and 6.03 g/L sodium biphosphate with varied antioxidant/phoplyphenols concentration.
  • Results Results are shown in table 21 below.
  • BNC production can be enhanced using addition of antioxidant/polyphenols in certain concentrations.
  • Theaflavins enhanced BNC production up to 100% at the concentration approximately 30 g/L.
  • Lower or higher concentrations of theaflavins in the medium yielded less BNC production.
  • Epicathecin or ascorbic acid at certain concentration only enhances 10-20% BNC production.

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Abstract

La présente invention concerne un procédé de production de nanocellulose bactérienne (BNC), le procédé consistant a) à cultiver dans un milieu de culture liquide Komagataeibacter rhaeticus, Gluconobacter oxydans et au moins l'une parmi Hanseniaspora uvarum, Brettanomyces bruxellensis, Zygosaccharomyces bailii et Kloeckera lindneri ; et b) à éliminer un hydrogel comprenant du BNC du milieu liquide, ce qui permet d'obtenir de la nanocellulose bactérienne (BNC). L'invention concerne également les hydrogels obtenus, des films séchés, des conceptions de couches et des utilisations associées.
EP20792284.0A 2019-10-01 2020-09-30 Substance nanocellulosique bactérienne et utilisations associées Pending EP4041905A2 (fr)

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