WO2007077459A2 - Moss-derived antimicrobial composition - Google Patents

Abstract

The invention provides an antimicrobial polysaccharide mixture derived from moss or peat which is essentially free of 5-keto-D-mannuronic acid residues and, for example, is obtainable by fractionation of the polysaccharides released by polysaccharide cleavage of moss or moss-derived peat and contains at least 20 mole % Rha, at least 35 mole % Ga1A3 less than 4.5 mole % Man and less than 9.5 mole % Gal.

Description

88061P.631

Moss-derived product

This invention relates to a moss-derived antimicrobial agent, to compositions containing it and to its use as an antimicrobial agent, e.g. in treatment of surfaces or of foodstuffs or in medicine.

It has been known for some time that peat which derives from moss has an antimicrobial and preservative effect. This has been attributed to a saccharide composition that has been called sphagnan. Much of the work on this material was carried out by Professor Terence Painter of NTNU in Trondheim, Norway.

Professor Painter identified the active ingredient of sphagnan as polysaccharides containing residues of the unusual monomer D-lyxo-5- hexosulo pyranuronic acid (5-keto-D-mannuronic acid or 5-KMA). See for example: Painter, Carbohydrate Research 124: C22-C26 (1983); Smidsrød and Painter, Carbohydrate Research 127: 267-281 (1984); Andresen, Grasdalen, Holsen and Painter "Structure, properties, and potential applications of sphagnum holocellulose" in "Ind. Polysaccharides. The impact of biotechnology and advanced methodologies", Ed. Stivala et al., 1987; Painter, Carbohydrate Polymers 15: 123-142 (1991); Painter,

Chemistry & Industry, pages 421-424, 17 June 1991; Painter, Carbohydrate Polymers 36: 335-347 (1998); and Børsheim, Christensen and Painter, Innovative Food Science & Emerging Technologies 2: 63-74 (2001).

The use of 5-KMA-containing sphagnum and sphagnum extracts in toothpaste has been proposed in WO 2005/041912 and the use of sphagnum in tampons has been described in Russian Patent Application RU 94002278/14 and by Podterab et al in Pharmaceutical Chemistry Journal 36(4) (2002).

However, we have now surprisingly found that a moss extract which is free of 5-KMA has particularly effective antimicrobial properties.

Thus viewed from one aspect the invention provides an antimicrobial polysaccharide mixture derived from moss or peat which is essentially free of 5-keto-D-mannuronic acid residues and, for example, is obtainable by fractionation of the polysaccharides released by polysaccharide cleavage of moss or moss-derived peat and contains at least 20 mole % Rha, at least 35 mole % GaIA₃ less than 4.5 mole % Man and less than 9.5 mole % Gal.

The polysaccharide mixture of the invention preferably has a weight average molecular weight of at least 5000 Da, more preferably 10 to 200 kDa.

The polysaccharide mixture preferably has less than 1 reactive carbonyl group per 10 monosaccharide units, more preferably less than 5 per 100 monosaccharide units.

The polysaccharide mixture of the invention may be in a salt form (eg with a group 1 metal counterion such as sodium) or, more preferably, wholly or partially (eg at least 80% of relevant functional groups) in its acid form. The acid (H⁺) form is preferred.

The mixture, either with or without formulation with other components, may be used as a surface disinfectant, to promote wound healing, as an internally applied antibiotic, or to treat foodstuffs, e.g. as a preservative or to prolong shelf-life.

By essentially free from 5-KMA it is meant that the polysaccharide contains less than 1 mole % 5-KMA, preferably less than 0.5 mole %, especially less than 0.1 mole %, particularly less than 0.01 mole %. The polysaccharide preferably contains (i) at least 25 mole % Rha and/or (ii) at least 40 mole % GaIA and/or (iii) less than 3 mole % Man and/or (iv) less than 7.5 mole % Gal.

Viewed from a further aspect the invention provides an antimicrobial composition comprising a mixture according to the invention together with a carrier or diluent, and optionally also a further antimicrobial agent.

Viewed from a yet further aspect the invention provides a method of preservative or disinfectant treatment of an inanimate object which method comprises applying to said object a mixture or composition according to the invention. Viewed from a still further aspect the invention provides the use of a mixture according to the invention for the manufacture of a medicament for use in a method of antibiotic or disinfectant treatment of a human or non- human (e.g. mammalian, avian, reptilian or piscine, especially mammalian) subject.

Viewed from a yet still further aspect the invention provides a method of antibiotic or disinfectant treatment of a human or non-human (e.g. mammalian, avian, reptilian or piscine, especially mammalian) subject, which method comprises applying to said subject an effective amount of a mixture or composition according to the invention.

Viewed from a yet further aspect the invention provides a process for the preparation of a mixture according to the invention, said process comprising: cleaving polysaccharides in moss or moss-derived peat; and fractionating polysaccharides released by this cleavage.

The compositions of the invention may of course contain sphagnan, preferably as no more than 50 mole % of the saccharide present, especially no more than 10 mole %, particularly no more than 1 mole %. Mole percentages as referred to herein relate to percentages of the total monosaccharides or monosaccharide residues present.

The moss or peat used according to the invention may be any moss, e.g. Anomodon sp., Atrichum sp., Aulacomium sp., Barbula sp., Brachythecium sp.j Bryum sp., Calliergonella sp., Calypogeia sp., Cephalozieϊla sp., Ceratodon sp., Cinclidotus sp., Climacium sp., Cratoneuron sp., Ctenidium sp., Dicranella sp., Dicranoweisia sp., Dicranum sp., Diplophyllum sp., Distichium sp., Drepanocladus sp., Dryptodon sp., Eurhynchium sp., Fissidens sp., Frullania sp., Funaήa sp., Grimmia, sp., Hedwigia sp., Homalia sp., Homalothecium sp., Hookeria sp., Hygrohypnum sp., Hylocomium sp., Hypnum sp., Isothecium sp., Lejeunea sp., Lepidozia sp., Leptodictyum sp., Leskea sp., Leucobryum sp.,

Leucodon sp., Mnium sp., Myurella sp., Nardia sp., Neckera sp., Orthothecium sp., Orthotrichum sp., Pellia sp., Philonotis sp., Plagiochila sp., Plagiomnium sp., Plagioihecium sp., Pleurozium sp., Polytήchum sp., Porella sp., Ptilidium sp., Racomitrium sp., Rhizomnium sp., Rhodobryum sp., Rhynchostegium sp., Rhytidiadelphus sp., Rhytidium sp., Schistidium sp., Schistostega sp.,

Scleropodium sp., Sphagnum sp., Thamnobryum sp., Thuidium sp., Tortella sp., Tortula sp., and Tήchocolea sp., as well as Bryales\ however Sphagnum and Calliergonella mosses are preferred, in particular C cuspidatum, S. papillosum, 5. palustre, S. quinquefarium, S. imbricatum, S. magellanicum, S. capϊllifolium, S. fimbriatum, S. fuscum, S. girgensohnii, S. molle, S. rubelllum, S. russowii, S. subnitensj S. warnstorfii, S. denticulatum, S. contortum, S. inundatum, S. platyphyllum, S. subsecundum, S. squarrosum, S. teres, S. tenellum, S. angustifolium, S. balticum, S. brevifolium, S. cuspidatum, S. fallax, S. flexuosum, S. lindbergii, S. majus, S. obtusum, S. ήparium, S. compactum, S. auriculatum, and 5. recurvum, particularly C. cuspidatum, S. magellanicum and 5. papillosum, and peats derived therefrom. In general however, Sphagnum mosses, in particular 5. papillosum, and peats derived therefrom are most preferred. The moss or moss-derived peat is preferably cleaned and separated from other materials, and particularly preferably only the leaves or leaf residue is used. Particularly preferably this leaf material is milled, i.e. powdered or otherwise fragmented.

Particularly where the starting material is moss rather than peat, it is preferred before the polysaccharide cleavage reaction to oxidize the organic material. This oxidative treatment is preferably effected in an aqueous environment, especially an acidic aqueous environment, e.g. of pH 1 to 5, particularly preferably using an oxidizing salt, e.g. a peracid or perhalite, especially a chlorite, e.g. at a concentration of 0.05 to 0.15 M, especially about 0.1M. Typically the treatment is effected for 10 minutes to 24 hours, more especially 3 to 7 hours. If desired, further oxidant may be added to replenish the reaction mixture. The reaction is preferably performed at elevated temperature, e.g. 30 to 95°C, preferably 60 to 80°C, especially about 75°C. Before oxidative treatment, the moss or peat is preferably treated with an organic solvent to remove waxes and pigments. This may typically be achieved by boiling in acetone for 1 to 30 minutes, e.g. 2 to 10 minutes.

Following oxidative treatment, which essentially oxidizes aromatic compounds in the moss or peat while leaving the polysaccharide structure minimally modified, the moss or peat is preferably washed and dried.

Washing is preferably effected first with water and subsequently with one or more organic solvents, e.g. acetone and then methanol. The polysaccharides in the moss or peat are then cleaved, e.g. enzymatically (for example using hydrolases, esterases, lyases, and especially pectinaseSj materials which are available commercially) or by hydrolysis, particularly by autohydrolysis (where the protons for the hydrolysis reaction are released from the carboxyl groups of the uronic acids it is only necessary to add water and heat the mixture; the pH then drops to 3-4), preferably in an acidic aqueous medium, e.g. pH 3 to 5.5, preferably pH 4 to 5, and preferably at an elevated temperature, e.g. at least 35°C, more preferably at least 70°C, particularly a few degrees below boiling (e.g. 98°C). This releases water-soluble polysaccharides into the solvent phase which can be separated (e.g. by filtration or centrifugation) from the solid phase. Particularly preferably, the separation is effected 1 to 3 times daily for a period of up to 10 days, more preferably 2 to 4 days. After each separation the solid phase is placed in a fresh aqueous medium to allow the hydrolysis to continue. While the early stage separations are preferably by filtration, separation of solid and liquid phases may also be effected by centrifugation with the supernatent taking the place of the filtrate as the separated liquid phase. In general, the final separation will be effected by centrifugation. The filtrates and/or supernatents are then preferably combined and concentrated down, optionally with further filtration to remove residual solids. The dissolved polysaccharides may then be converted to salt form, e.g. with a group 1 metal counterion such as sodium.

The polysaccharide solution is then fractionated to produce the mixture of the invention. Typically this may be effected using an anion exchange column with a salt (e.g. chloride) gradient. The initial fraction eluting from the column is discarded so as to obtain the desired mixture and material remaining bound to the column may be removed by washing with a reverse flow of an alkali (e.g. sodium hydroxide). This "column wash" is desirably included with the collected eluate. The combined material is then preferably dialyzed against water until the conductivity is below 3 μScnr¹, whereafter the solution may be dried, e.g. by lyophilization.

Where the polysaccharide solution is applied at a concentration of 5 mg/mL in 20 mL of 0.02M sodium phosphate to a column of DEAE- Sepharose GL-6B anion exchange resin (2.7 x 33 cm) at a flow rate of 0.5 mL/min and eluted with 160 mL of 0.02M sodium phosphate buffer pH7 (buffer A)₃ followed by 2000 mL of a mixture of buffer A and 0.5 M NaCl in 0.02 M sodium phosphate buffer, pH 7 (buffer B) where the percentage of buffer B increases linearly from 0-100%, a mixture according to the invention may be produced by discarding the first 500 mL of the eluate, preferably the first 750 mL, more preferably the first 1000 mL. Once again the mixture is preferably produced by including the column wash fraction.

The monomer composition of a polysaccharide mixture may be verified by conventional techniques, e.g. as described in the Examples below. Verification of the 5-KMA-free status of the mixture of the invention may be effected indirectly by assaying for the presence of gluconic acid, the product of reduction of 5-KMA. Again this is described in the Examples below. The antimicrobial compositions of the invention may be produced by formulation of the mixture with appropriate carriers, diluents or other components. Where the compositions are for internal use, e.g. for administration into an externally-voiding body cavity, such as the GI tract, etc., the other components may be physiologically active (e.g. other antibiotic agents) but must be physiologically tolerable and preferably of pharmaceutical standard. Where the composition is to be applied to the surface of a living body, again the other components may be physiologically active (e.g. other antibiotic agents) but must be physiologically tolerable and preferably of pharmaceutical standard. Where the composition is to be applied to an inanimate object which is later to be consumed, the other components should be suitable for consumption. Where the composition is to be applied to an inanimate object which is not to be consumed, the other components need not meet these standards, although the composition will still desirably be sterile. Examples of such further components include: antimicrobials, solvents, bulking agents, binding agents, pH modifiers, viscosity modifiers, aromas, antioxidants, preservatives, carriers (e.g. holocellulose (e.g. sphagnum holocellulose), webs or other porous substrates), etc. The production of the antimicrobial compositions may be effected using conventional techniques and using such other components in conventional quantities. The antimicrobial mixture (in terms of its saccharide content) will typically form 0.01 to 90% wt., more typically 0.05 to 10% Wt₅ especially 0.1 to 2% Wt₃ more especially 0.2 to 0.5% wt, of the antimicrobial compositions. There has been some doubt in the literature as to whether mosses contain lignin as such or simply lignin-like materials such as phenolics. For the avoidance of doubt, by the term moss holocellulose herein is meant moss (eg sphagnum) treated to render it lignin-free, e.g. by an oxidative treatment as described above, and by lignin in this regard is meant lignin and lignin-like materials (eg aromatics, in particular phenolics) that are removed by the oxidative treatment.

Such lignin-free moss holocellulose may itself be used as an antimicrobial agent analogously to the mixtures of the invention and it, compositions containing it (in particular compositions for external topical application or application to inanimate objects) and surface treatments of inanimate objects or of external surfaces of animate objects using it form further aspects of the present invention.

The antimicrobial compositions may take any appropriate administration form, e.g. powders, tablets, coated tablets, sprays, solutions, dispersions, suspensions, emulsions, creams, impregnated wipes, etc. However the use of solutions is preferred.

The compositions of the invention may be applied to a surface to be treated by smearing, wiping, spraying, soaking, dusting, etc. according to the administration form. Administration may be a single event (as for example in the treatment of a foodstuff) or may be a repeated event (as for example in the treatment of an inanimate surface). The dosage of the mixture of the invention will then depend on the nature of the composition and the surface being treated. For foodstuffs, e.g. raw or cooked fish or meat, especially fish, it is preferred to apply a dose of 0.1 to 100 mg saccharide/cm², especially 0.5 to 50 mg/cm², particularly 5 to 20 mg/cm².

For internal treatment of a human or non-human animal it is preferred to apply a dosage of 0.001 to 0.1 g saccharide/kg bodyweight. Previously, moss holocellulose has been known for use as a water absorber, e.g. in tampons, or as a whole fish tanning and preservative agent (see Børsheim et al, Innovative Food Science & Emerging Technologies 2:63- 74(2001)). However it has not previously been recognised that it reduces odour formation or that it can be used with raw food (eg meat) having an exposed muscle or internal organ surface. (Børsheim, supra, specifically mentioned at page 69 that it was ineffective with naked fish muscle). However we have surprisingly found that moss holocellulose, in particular sphagnum holocellulose, has both an odour suppressing effect and that it is effective for use with exposed raw muscle or organ tissue to limit odour and microbial contamination. It thus may be used in objects where this occurs, in particular where such contamination may be transmitted to or back to a human or non-human animal as for example is the case where food, food waste or body exudates may serve as growth media for microbes such as bacteria, yeast or fungi.

Thus viewed from one aspect the invention provides an odour and contamination reducing water-absorbent structure comprising a carrier material and a water-insoluble particulate moss holocellulose.

Viewed from a further aspect the invention also provides the use of water-insoluble particulate moss holocellulose as an odour and microbial contamination reducing agent in a water-absorbent structure.

Viewed from a further aspect the invention also provides the use of water-insoluble particulate moss holocellulose as an odour and microbial contamination reducing agent in the manufacture of a water-absorbent structure.

Examples of structures according to the invention include cleaning webs (eg cloths) and pads, food packaging inserts, sanitary pads, incontinence pads, sweat-absorbers, and insoles.

Such structures may be incorporated within larger objects, e.g. handle- mounted cleaning cloths and pads, food packages, wrapped sanitary or incontinence pads, etc., and such larger objects are deemed also to form further aspects of the invention. Thus viewed from a further aspect the invention provides a cleansing device having a handle with attached thereto a water-absorbent cloth or pad containing a water-insoluble particulate moss holocellulose.

Viewed from a still further aspect the invention provides a food package comprising a sealed container, a foodstuff selected from the group consisting of cut raw plant material and animal material having exposed raw internal organ or muscle tissue, and a water-absorbent structure comprising a water-insoluble particulate moss holocellulose.

By cut plant material is meant herein peeled, diced, sliced or chopped vegetable, fruit or fungi. The term cut does not cover the case where the material is simply plucked or severed from roots, stems, stalks, or leaves. Examples of suitable materials include peeled, sliced or diced potatoes, onions and carrots.

Viewed from another aspect the invention provides an armpit sweat- absorber comprising a water-pervious casing containing a water-insoluble particulate moss holocellulose.

Viewed from another aspect the invention provides an incontinence pad comprising a water-pervious casing containing a water-insoluble particulate moss holocellulose. Viewed from another aspect the invention provides an insole comprising a water-pervious casing containing a water-insoluble particulate moss holocellulose.

By water-insoluble, it is meant that the moss holocellulose retains its particulate nature in water, even though some water-soluble materials in it may go into solution.

By a moss holocellulose is meant herein moss oxidized to substantially eliminate phenolic content, e.g. chlorite-oxidized moss or more preferably moss treated with gaseous chlorine dioxide. The use of gaseous chlorine dioxide in this regard is novel and forms a further aspect of the invention. Viewed from this aspect the invention provides a process for producing moss holocellulose which process comprises contacting moss, preferably damp but preferably not in an aqueous environment (i.e. not in aqueous dispersion or suspension), with gaseous chlorine dioxide. By "damp" in this regard is meant a water content of for example up to 30g/g dry moss, eg at least 5g, preferably at least 1Og.

For holocellulose production, the moss used may be any moss (or moss-derived peat), e.g. those mentioned herein, but is preferably a sphagnum moss. The moss may be divided before treatment, and if desired root material (ie surface or substrate binding material since sphagnum has no roots as such) may be removed before oxidative treatment. Likewise it may be washed in water and/or organic solvents before treatment and it may be broken or cut into smaller particles before treatment. Desirably however, where it is to be incorporated into a cloth or fabric (whether woven or not) which will be crumpled or wrung during use, it is produced with a mean maximum particle size of at least 3 mm, more especially at least 10 mm. The moss in this instance is preferably treated in whole plant or whole leaf branch form, optionally with the removal of roots. Chlorine dioxide may be generated and used analogously to the manner in which it is used to treat wood pulp in paper manufacture. Thus chlorine dioxide may for example be produced by acidification of a buffered alkaline aqueous solution (at pH 9, a 15% concentration may be achieved) or by passing chlorine gas (generally diluted with nitrogen) through pellets of a chlorite, e.g. sodium chlorite. Apparatus for chlorine dioxide generation is available from CDG of Bethlehem, PA, USA. Chlorite treatment may be effected using a chlorite salt, e.g. sodium chlorite, in aqueous acid solution. Treatment may be determined to be complete when the product is bleached and substantially phenolic free. Moss contains lignins or lignin-like phenolics which are removed by this process leaving a product which is both visually acceptable and free of phenolics which would be undesirable for products intended to come into contact with foods or food carriers.

The moss holocellulose, by virtue of the oxidative treatment during its production is advantageously not only essentially colour free but also sterile. The absorbent structures of the invention will generally contain further water-absorbent materials, e.g. cellulose materials such as wood pulp or cotton or plastics sponge or web, and may be carried by a carrier, e.g. a water- pervious container (e.g. a pervious cellulosic or plastic wrapper, envelope or bag) or a water-pervious or impervious substrate layer or layers. Particularly desirably the moss holocellulose is carried within or as an integral component of a woven or non-woven web or pad. Such webs or pads may be produced in conventional fashion, e.g. by draining and compressing a fibrous suspension including or consisting of the holocellulose, by foam/sponge production using a plastics material containing fibrous holocellulose and a blowing agent, or by incorporating fibrous holocellulose in the formation of threads from which a woven fabric is to be produced.

The absorbent structures may of course incorporate other materials suitable for the intended end use, e.g. abrasives (for example stiff fibres), deodorants, absorbents (e.g. activated charcoal), water-impermeable backing sheets, adhesive tags, handles, cords etc. Likewise such structures may conveniently be packaged inside applicators or water-impermeable packaging. In the case of food packaging, the package according to the invention contains a raw foodstuff typically comprising cut plant material or, more preferably, muscle tissue or internal organ from, for example, a mammal, bird or fish, especially fish, e.g. liver or meat, especially skin-free meat, having underneath it a holocellulose or holocellulose containing absorbent structure, e.g. a pad or more preferably a shaped (eg moulded) cup. Desirably a holocellulose pad or lid is also above the raw foodstuff. Where a shaped cup is used, this may typically be of an absorbent card shaped to conform to and hold the foodstuff much as is the case with egg boxes. Such packages preferably have a sealed plastics container optionally with an outer wrapper, e.g. of paper or card. Less preferably the raw foodstuff is placed above the holocellulose in a metal foil container sealed with card or metal or plastics foil. The food package is preferably sealed containing an oxygen-depleted atmosphere as is standard in MAP of foodstuffs or alternatively an oxygen scavenger may be included in the food package. Thus, other conventional barriers or hurdles to oxidation or contamination of the foodstuff may be used in conjunction with the moss holocellulose. The holocellulose insert in the package is preferably slightly moist or damp when the food is added so as to prevent undue drying of the food during storage. The hollocellulose insert (eg the pad, cup or lid) will preferably be 1 to 5mm thick, especially 2 to 4mm. TTie packaging may of course contain other food materials besides the raw plant or animal material;, eg flavoring additives, etc.

Where the absorbent structure is in pad, sheet or moulded cup or lid form, it may consist essentially of the moss holocellulose, eg containing 0 to 20% Wt₃ especially 0 to 5%wt, of other materials such as binders, colorants and other water-absorbers. Such structures form further aspects of the invention and viewed from these aspects the invention provides a moss holocellulose sheet, pad, cup or lid.

The absorbent structure, in certain embodiments, eg where it is to be in sustained contact with animate or inanimate flesh or plant matter, preferably has as a contact surface a layer of material which permits water passage in essentially only one direction, towards the holocellulose. Such materials are known for example in wound dressings, eg as described in WO2005/034797, WO2006/081403 and WO2006/127292, the contents of which are incorporated herein by reference. Wound dressings with such a one-way contact layer and containing moss holocellulose in a backing layer form a further aspect of the invention. Likewise, moss holocellulose may be used according to the invention as a component in toilet paper and other tissues for absorbing moisture, especially biological fluids. Such paper and tissue forms a further aspect of the present invention.

In a yet further aspect, the invention provides a plant container, eg a basket, plant pot or plant pot insert, in the form of a moulded water- absorbent body comprising particulate water-insoluble moss holocellulose. Such containers, typically of material 1 to 5mm thick, especially 2 to 4mm thick, may consist essentially of the moss holocellulose or may contain it as a component as described above for food packaging inserts.

In the absorbent structures of the invention, the holocellulose is preferably present at from 5 to 100% wt, especially 10 to 80% wt, particularly 20 to 75% wt. The invention will now be described further with reference to the following non-limiting Examples and the accompanying drawings, in which: Figure 1 is a photograph of culture plates bearing bacterial colonies; Figure 2 is a schematic cross-sectional drawing of a food package according to the invention;

Figure 3 is a schematic cross-sectional drawing of an absorbent structure according to the invention; and Figure 4 is a schematic drawing of a handled washing pad according to the invention.

Referring to Figure 2, there is shown a food package 1 comprising a plastics tray 1, sealed by plastics foil lid 2 and containing a water-absorbent card cup 3 having a water-absorbent card lid 4. Both the cup 3 and the lid 4 contain moss holocellulose 5. Within the card cup is housed a filet 6 of skin- free fish, eg salmon. The gaseous atmosphere 7 within the package is a modified atmosphere (i.e. an oxygen-depleted atmosphere) such as is standard in sealed packages for raw animal or fish meat.

Referring to Figure 3, there is shown a water-absorbent structure 8 comprising a water-absorbent body 9 surrounded by a water-permeable sleeve 10, for example of perforated paper or plastics film. Body 9 comprises an absorbent cotton or cellulose matrix 11 containing fibrous moss holocellulose 12. Structure 8 may for example be an incontinence pad, a sanitary pad, an armpit sweat shield, or an insole. Referring to Figure 4, there is shown a cleaning pad 13 for washing-up, comprising an absorbent pad 14 containing moss holocellulose 15. The absorbent pad 14 is held by the head 17 of a plastics handle 16.

Example 1 Preparation of chlorite-treated leaves

Sphagnum papillosum plants were harvested in Tømmerdalen, Bymarka, Trondheim, Norway. Immediately after picking, whole fronds of moss were dried over a period of three days in a current of air at 6O⁰C. Dried leaves were stripped manually from the stems and the latter discarded. The leaf material (50 g) was boiled in acetone (2 L, 57⁰C, 3 min) and collected by manual filtration through a nylon filter mesh (pore size 60 μm). This was repeated three times, until the filtrate (containing extracted waxes and pigments) was almost colourless. Finally the leaf residue was extracted once more with dry methanol and air-dried at room temperature.

Once dried, acetone/methanol-treated leaves (47 g) were split into two portions and stirred mechanically in water (3 L) at 75°C. Glacial acetic acid (30 ml) was then added, followed by sodium chlorite (30 g), added in small amounts over 1 hour to generate chlorine dioxide (and other chlorine free radicals). After 3 hours these additions were repeated, and after a further 3 hours the mixture was cooled and filtered as before through a 60 μm nylon filter mesh. The purpose of this process was to selectively oxidise all aromatic compounds with chlorine dioxide and leave behind the pure white intact leaves with minimal modification to their polysaccharide structure. The pure white chlorite-treated leaves were washed well with water followed by ice-cold 0.02 N HCl, and then with distilled water. The residue was finally washed with acetone, then methanol and then air-dried to give chlorite-treated leaves in their H⁺-form.

Sphagnum holocellulose prepared in this way was used in Examples 5, 6 and 7 below.

Moss holocellulose was also prepared from whole Sphagnum magellanicum and, separately, from Calliergonella cuspidatum mosses by an analogous process. For each gram of these mosses, l.Og sodium chlorite and ImL glacial acetic acid in a total volume of 10OmL was used. The treatment was effected for a single period of 4 hours.

Example 2

Preparation of sphagnan

Dry phenol-free leaves (33 g in their H⁺ form, from Example 1) were made into a thick slurry with 2 L degassed distilled water (final pH was 4.5) which was then heated at 98°C. At daily intervals for 3 days, the residual solid was collected by vacuum filtration through a Whatman GF/D glass filter and re- suspended in another 1.5 L of distilled water, which was then heated again. On the fourth day, when the slurry was impossible to filter in a reasonable time, it was settled by centrifugation at 10,000 rpm (16264 g) for 10 minutes at room temperature and the supernatant was decanted off and pooled with the earlier filtrates. This liquid solution (5.5 L) was concentrated in a rotary evaporator at 3O⁰C to 500 ml before progressive filtration through GF/D, GF/C, GF/F (Whatman glass filters), 0.45 and finally 0.22 μm membranes (Millipore nitrocellulose). The filtrate was then first dialysed against 0.5 M NaCl to convert the sphagnan into its Na⁺ form and then repeatedly against distilled water. Finally, the dialysate was sterile filtered through a 0.22 μm membrane and freeze-dried. The yield of light-brown crude solid (Na⁺-form) was 1O g. This was stored in the refrigerator until used.

Example 3

Fractionation of sphagnan by preparative anion-exchange chromatography Sphagnan from Example 2 is dissolved to a concentration of 5mg/ml in 20 ml 0.02 M sodium phosphate, pH 7. This sample is then subjected to anion- exchange chromatography on a column of DEAE-Sepharose CL-6B (2.7 x 33cm) at a flow rate of 0.5 ml/min. Buffer A is 0.02 mM sodium phosphate, pH 7 and buffer B is 0.5 M NaCl in 0.02 M sodium phosphate, pH 7. After elution in 160ml buffer A, a linear gradient of chloride is produced from 0-

100% in 2 L of eluent using a mixture of buffer A and buffer B. The first 500 mL (preferably 750 mL, more preferably 1000 mL) of eluate is discarded. The remaining eluate is collected. Material that remains bound to the anion- exchange resin at the end of the chloride gradient is removed and collected by washing the column with a reverse-flow of 1 M NaOH. The collected eluate is combined together with the material eluted by the NaOH wash, and dialysed against distilled water until the conductivity is below 3 μScπr¹. Material recovered from dialysis is then freeze-dried and stored in the fridge until further use.

Example 4 Analysis 5-KMA Analysis 25 mg of the material from Example 3 is dissolved in 50 ml of 2 % w/v sodium borohydride and incubated at room temperature for 24 hours, prior to the addition of acetic acid to destroy excess borohydride, followed by dialysis against distilled water until the conductivity is below 3 μScnr¹. The material in the dialysate is recovered and evaporated to dryness followed by repeated drying/distillation in portions of 2% acetic acid in methanol to remove boric acid. The material in the dialysis tubes is also recovered for later analysis with GC (see below).

The recovered dried material is dissolved in 50 ml of water and sodium ions are removed by mixing for 2 hours with an H⁺ exchange-capacity excess of AG-50W-X8 resin (60 mesh, H⁺-form, 1.7-1.9 meq/ml wet volume). The sample is filtered to remove the resin and again evaporated to dryness. Finally the sample is dissolved in methanol, evaporated to dryness, dissolved in water and freeze-dried. As a positive control and standard 50 mg of 2-keto gluconic acid (2-KGA) is dissolved in 50 ml of 2% sodium borohydride or sodium borodeuteride and treated in an identical fashion for 24 h and just 1 hour and then further treated as described above.

The recovered standards and sample collected from the dialysate are analysed in water by standard direct injection electrospray-ionisation mass spectroscopy in negative mode. 2-KGA Ca²⁺-form, and gluconic acid Na⁺- form, with and without borohydride treatment, are also used as standards. Secondly, 0.5-5 mg samples are subjected to methanolysis and analysed by gas chromatography as described below. Both 2-KGA and gluconic acid are again used as standards and are prepared for GC-analysis in an identical fashion to the other samples assayed.

Monosaccharide analysis

Samples (1 mg) are dried in vacuum over P2O5 for 24 hours prior to methanolysis for 24 hours at 8O⁰C in 4 M methanolic-HCl spiked with 100 μg of mannitol as internal standard. Samples are then dried with nitrogen gas at 35°C and dried a further three times following repeated additions of anhydrous methanol. Samples are then stored in vacuum over P2O5 for at least 1 hour prior to the addition of 200 μl of a mixture of pyridine- hexamethyldisilazane-chlorotrimethylsilane (5:2:1) followed by incubation for 30 min at room temperature. Quantitative analysis is then carried out by gas chromatography on a DB-5 capillary column calibrated with carbohydrate standards common to the polysaccharides of plants that are treated in the same way as the samples.

Determination of glvcosyl-linkage/methylation analysis

Carboxyl groups are activated with carbodiimide at the polymer level (H⁺- form) and reduced with sodium borodeuteride to yield 6,6' dideuterio neutral sugars. Hydroxyl groups are then de-protonated with a mixture of sodium hydroxide and dimethylsulphoxide followed by methylation with methyl iodide. These methylated polysaccharides are hydrolysed for 2 hours at 110°C in 2.5M TFAj subsequently reduced with sodium borodeuteride to yield partially methylated alditols, and finally acetylated with acetic anhydride to yield partially methylated alditol acetates. These are then dissolved in anhydrous methanol, separated by gas chromatography and analysed online by El-mass spectrometry. Data is processed with Fisons Masslab software. The relative proportion of uronic acid to corresponding neutral sugar and 2- and 4-linked pentopyranosyl units is determined and effective carbon- response factors are applied for quantitation.

NMR

Samples of 60 mg are dissolved in 600 μl of D2O and spiked with 100 μl of an internal reference standard of 1% 3-(trimethylsilyl)propionic-2,2,3,3j-d4 Na salt (TSP) in D2O. ¹H and ¹³C NMR are recorded on a Bruker Avance DPX 300 spectrometer. For ¹H NMR₃ spectra are obtained at 90 ⁰C using a 30° pulse angle, a spectral width of 3591 Hz and a data-block size of 32K. After 4 dummy scans 64 more are accumulated. For ¹³C NMR₅ spectra are recorded at 90 °C with power-gated proton decoupling using a 45° pulse angle, a spectral width of 18832 Hz and a data block size of 64K. After 8 dummy scans 20000 more are accumulated.

Determination of molecular weight and intrinsic viscosity Samples, pullulan standards and alginate (positive control) are dissolved in

0.05 M sodium sulphate, 0.01 M EDTA₃ pH 6 to a concentration of 2 mg/ml prior to size-exclusion chromatography (SEC) on 3 HPLC columns connected in series (TSK 6000, 5000 and 4000 PWXL). These columns are eluted at room temperature at a flow of 0.5 ml/min in the same buffer used to dissolve the samples. Effluent is monitored by multi-angle laser light scattering (MALLS) followed by refractive index (RI) detection on a DAWN DSP instrument. All data is processed with ASTRA software. After RI- detection, on-line intrinsic viscosity is also monitored using a Viscotek 301 Triple array detector. Data is collected and processed on TriSEC GPC Triple detector software. Off-line reduced capillary viscosity is used to determine intrinsic viscosity.

Ion-exchange capacity

Five replicates of 20 mg of the product of Example 3, Sigma P-3850 poly- galacturonic acids, 85% GaIA (control) and alginate from Lamina^ήa hyperborea stipe (control) are dissolved in 5 ml of distilled water and placed into a pre-washed dialysis sack. Repeated dialysis is carried out against daily renewal of 0.2 M magnesium nitrate for 5 days, followed by changes of distilled water until a conductivity below 3 μScπr¹ outside the dialysis bag is achieved. Each individual dialysis tube is then dialysed against 25 ml 0.2 M nitric acid to release bound Mg²⁺. The volume of the dialysate is recorded each day (via weight assuming a density of lg/ml) for a total of 4 days with daily renewal of acid. Each day 10 ml of dialysate is collected and stored until analysis Of Mg²⁺ by flame atomic absorption spectrometry over a linear range of 0-1 ppm Mg²⁺ against matrix matched standards. Polysaccharide dry weight is determined by thermo-gravimetrical analysis on a Netzsch STA 449 instrument at 160 °C over a 30 min period. An aluminium oxide standard is used for calibration. Ion-exchange capacity is expressed as mequiv/g dry weight polysaccharide corrected against replicate water blanks.

Colorimetric assays Total carbohydrate is determined by the phenol-sulphuric acid reaction using D-glucose as the standard. 6-deoxy-hexoses are determined by the cysteine- sulphuric acid reaction using L-rhamnose as the standard. Uronic acids are determined by the carbazole-sulphuric acid reaction (or more preferably the m-hydroxydiphenyl reaction) using D-galacturonic acid as the standard.

Example 5

Absorbent pads for food travs

Absorbent pads were made from chlorite-treated (bleached) Sphagnum papillosium and placed on the top and bottom of Atlantic salmon (Salmo salar) individual fillet slices. Each fillet slice was packed in a high-density polyethylene tray in a modified atmosphere of 60:40 N2/CO2 to a gas:fillet slice volume » 5:1 and stored at +4°C. These trays, and controls (fillet slices with a standard paper absorptive pad or no pad) packed in the same way, were independently removed from cold storage and sampled (n=3) at selected time intervals over a 30 day period. Tray gas composition, water content, water holding capacity, pH, colour, texture, smell, viable bacterial count, amount of soluble protein, amount of acid soluble peptides and free amino acid were assessed for each fillet slice. By the end of the experiment and compared to controls, the smell of the fillet slices stored with Sphagnum pads was deemed to be acceptable to a consumer. Between storage days 7-12 and 19-30 days in the stationary bacterial growth phases these same fillet slices had roughly half the viable bacterial counts per gram slice. No significant differences, between all fillet slices at each sampling interval, were found for any of the other parameters which were assessed, except for free amino acid content. These results show that absorbent pads made from Sphagnum moss may, with further development, have a commercial potential in that they can help extend the shelf-life and the quality of packaged fresh foods such as fish. ZViATERIALS AND METHODS Pad production

66 Sphagnum sheets (12 x 24 x 0.1 cm) were produced to ISO standard 5269-1 using a custom-made sheet former, and a press (PTI₃ model 40140). Each sheet comprised 4 g of ground chlorite-treated whole 5. papillosum plants produced according to Example 1. Finally the sheets were cut down to 9.5 x 11 x 0.1 cm to form the pad.

Salmon packaging, storage and sampling Fifteen salmon (Salmo salar) of 13-15 kg were obtained from a commercial fish farm. These were live-chilled, bled and gutted, and immediately iced prior to transport. All fish were manually filleted 3 days after slaughter to obtain fillet slices of 8 x 9 x 3 cm- One day later (defined as day 0) the fillet slices were individually packed in high-density 750 ml polyethylene trays and sealed with a polyethylene film in a modified atmosphere of 60:40 N2/CO2 to a gas:fillet slice volume » 5:1. Three different sample series (treatments) were prepared: fillet slices packed without a pad, slices packed on their underside with a standard absorbent paper pad currently used in food packaging, and fillet slices packaged with Sphagnum pads on the top and bottom side. In total 65 fillet slices were packed; 21 in each pad treatment and 23 without a pad. These were immediately stored at +4 ⁰C. After 2, 6₅ 9, 13, 16, 20, 27 and 30 days three replicate fillet slices from each treatment were removed from cold storage for analysis. At day 0 only 2 fillet slices packed with no pad were sampled.

Analytical analysis (n=3) of sampled fillet slices

The O2, CO2 and N2 content of the tray were quantified by a gas analyser. Muscle pH was measured in the fillet slices. Water content was determined by drying 2 g samples (n=3) of each fillet slice at 105 °C for 24 h. Water holding capacity was determined on ground muscle as described by Eide et al. in Journal of Food Science. 47, 347-349, 354 (1982). Fillet slice colour was determined with a salmon graduate colour card. Extractable protein, acid soluble peptides and free amino acids were determined as previously described by Hultmann et al. in Journal of Aquatic Food Product Technology 11: 105-123 (2002) and Food Chemistry 87: 31-41(2004).

Microbiological analysis A 50 g piece of fillet slice (n=l) was aseptically cut and homogenized at 200 rpm for 30 second in filter bags with 4 parts sterile peptone saline, pH 7.2, using a stomacher. These homogenates were further diluted (1:5) with peptone saline. Appropriate dilutions (10"² - 10"⁶) were spread on Long and Hammer agar plates (n=3) with 1% (w/v) added NaCl and incubated at 15 ⁰C for 7 days, prior to counting the number of colony forming units (CFU) and expressing them per gram wet weight of fish (N.B. The water content of each fillet slice was relatively constant). Typical isolated colonies were identified by 16S rDNA sequence analysis (NCIMB Ltd, Aberdeen, Scotland).

Textural properties and smell analysis

Textural properties were measured with a TA.XT2 Texture Analyser as previously described (Hultmann et al., supra, 2002). Smell was determined by sensory analysis by a panel of 5-7 scientists each smelling the fillet slice immediately and 10 min after opening the package. Smell was ranked on a integer scale from 1 to 6 (1- fresh seaweed-like smell, acceptable, 2- odourless, acceptable, 3- slight fishy odour, acceptable, 4- significant fishy odour, borderline acceptable, 5- strong fishy odour, not acceptable, 6- totally off, i.e. putrid smell, not acceptable).

RESULTS AND DISCUSSION

The proportion of CO2 in the atmosphere of all the packages (no significant differences between treatments) decreased from 60% at day 0 to a near constant value of around 44-45% after day 6. This is possibly because of diffusion of CO2 into the fish muscle. On the other hand the concentration of O2 in the trays increased slightly from not detectable at the start of the experiment to a maximum of 0.18% by day 6. This is probably because of a slow diffusion of air into the tray. By day 9 no O2 was detected in any tray because all is consumed, primarily by bacterial respiration. The pH and colour of all fillet slices remained constant at around 6 and salmon red respectively throughout the experiment. It is likely that the CO2 content inside the tray had a buffering effect on fillet slice pH and a stabilising effect on the fillet slices pigments.

Not surprisingly the modified atmosphere packaging (MAP) also had a large influence on the microbiology of the fillet slices over the course of the experiment. At the start of the experiment the observed viable bacterial count was about 10² CFU/g which increased at an exponential rate in the log- growth phase to 10⁶ CFU/g after 6 days. Such levels of viable bacteria are typical for MAP packed fish fillet slices. Although there is only one sampling point it is interesting to note that after 3 days storage in the log growth phase, fillet slices in contact with a standard pad used in current food packaging, had about a one and a half magnitude greater viable bacterial count (10⁵) than fillet slices in the other treatments.

Between 6 and 12 days of storage the stationary growth phase became established were the viable bacterial count remained relatively constant. The dominant bacterium in this growth phase was identified as Photobacterium phosphoreum; typical of MAP packaged fish. Between day 12 and 16 there is a death phase followed by a secondary log and stationary phase. P. phosphoreum remains the dominant bacteria in this growth phase, but in addition colonies of Lactobacillus maltaromaticus start to appear. In food microbiology it is usually standard practice to consider significant differences in bacterial growth in terms of orders of magnitude. In this case no marked difference is seen in the stationary growth phases between fillet slices in each of the three treatments. However, examination of revealed that the viable count of bacteria in the fillet slices treated with the Sphagnum pad, in both the first and second stationary growth phases, is consistently about 50% less than the other control treatments. It therefore seems clear that the presence of such a pad has some influence in terms of decreasing the overall maximum viable bacterial count of the fillet slices. In addition to these properties we also found that the smell of the fillet slices stored with the Sphagnum pads were deemed acceptable to a consumer, in contrast to control fillet slices, right up to the end of the experiment. All fillet slices that contained pads had no free liquid floating around in the trays because as expected all was absorbed by the pad. Nevertheless, pad treatment had no significant effect on fillet slice texture, water content, water holding capacity, extractable protein or acid extractable peptides. The content, however, of extractable free amino acids was significantly different.

Example 6

Dishcloth

To get an indication of how well bleached Sphagnum moss holocellulose would work as an antibacterial dishcloth we performed a simple test by comparing it to a viscose cloth, and a woven cotton cloth

This simple test gave a clear indication that titiere is far less growth of bacteria in the water surrounding holocellulose than in the water surrounding a viscose or cotton cloth. The water used as the initial inoculum contained 2.I x IO⁶ cfu/mL as assessed on plate count agar. After 24 hours incubation, all samples from water from viscose and woven cotton contained above 10⁶ cfu/mL on plate count agar, while the samples from water from holocellulose contained less than 10⁵ (see figure 1). After 48 hours incubation, all samples from water from viscose and woven cotton contained above 10¹⁰ cfu/mL on plate count agar, while the samples from water from holocellulose contained less than 10⁶. Sensory analysis of the smell showed that the holocellulose had no undesirable bad smell in contrast to the rotten smell of the other materials.

Method 1. Six food-soiled plates were selected from a canteen and kept overnight at ambient temperature on a laboratory bench. They were then washed in lukewarm tap water in a bucket. 2. 1 gram dry weight of each material (holocellulose, woven cotton cloth and viscose cloth) in three replicates was put into each of nine separate plastic tubes.

3. 20 ml of the washing up water, cooled to ambient temperature was added to each tube, and the tubes were kept overnight on a rotating table with the lid loosely open.

4. After 24 and 48 hours incubation, a 100 μl sample of the dishwater from each tube was diluted in a log series in successive portions of 900 μl 0.9% NaCl and an aliquot of each was spread onto plate count agar (Difco) in standard culture media dishes. The dishes were incubated at 22°C overnight and counts of viable bacteria were made the following day. Counts of 30-300 colonies were accepted, above 300 colonies, the medium was considered to be overgrown.

5. Sensory analysis of smell was performed by a panel of two people at each 24 and 48 hour sampling point.

Example 7 Dishcloth

After each of six successive overnight incubation periods of 18-24 hours, and as an extension to Example 6, the dishwater was renewed. Since the bacterial growth in the cotton cloth and viscose cloth in Example 6 were so similar, only the latter cloth was used as a positive control.

This test shows little or no growth of bacteria in the water surrounding holocellulose at the end of the first five incubation periods. In contrast and during the same period there was rapid growth of bacteria in the water surrounding the viscose cloth. Total cfu/mL in this period were less than 10⁴ in the water surrounding the holocellulose while in the water surrounding the viscose cloth it was >10⁷ cfu/mL. There was ≤ 10² cfu/mL in the new dishwater at the start of each of the first five incubation periods. After the sixth and final incubation period, growth of bacteria was observed in the water surrounding the holocellulose (<10⁷) and although there were more bacteria observed in the water surrounding the viscose cloth (>10⁷) the difference between the two treatments was smaller than observed in previous incubations.

Method

Food soiled dishes were washed-up as described in Example 6. To 3 replicates of 1 g holocellulose and viscose cloth in a 60 ml open plastic syringe, 20 ml of freshly cooled dishwater was added. After 18-24 h incubation under the conditions described in Example 6 the water was pressed out of the material in the syringe by human force with a corresponding plastic plunger designed to fit the syringe. The bacteria in this water and in water at added at the start of the incubation were assayed as described in Example 6. The dishwater in the syringe was then renewed and the process repeated again another six times over an experimental period of one week.

Example 8 Preparation of chlorite/chlorine dioxide-treated leaves

Sphagnum papillosum plants were harvested from Klεebu, Trondheim, Norway. Immediately after picking, whole fronds of moss were dried over a period of three days in a current of air at 60°C. Whole plants of dried moss (25g) were placed inside a plastic bag (with zip-lock). A stock solution containing 37.5 g of added NaOCb in 500 ml distilled water was prepared. Six times 50 ml portions of the stock solution were added to the moss in the bag at intervals over 1.45 h. 3.5 ml of concentrated acetic acid was added immediately after each addition of the stock solution, the bag sealed, and the liquid massaged into the sphagnum moss. The bag containing the sphagnum moss was incubated by floating it on top of a water bath held at 75⁰C inside a fume cupboard and behind a Perspex shield. After a period of 10 minutes incubation a yellow/green gas (presumably mostly chlorine dioxide) developed inside the bag.

At the end of the experiment the remaining gas was released and leaves thoroughly washed with distilled water. The product was off-white but otherwise essentially the same as that of Example 1. To obtain a more pure white product the process may be repeated once again.

Claims

Claims

1. An antimicrobial polysaccharide mixture derived from moss or peat which is essentially free of 5-keto-D-mannuronic acid residues and, for example, is obtainable by fractionation of the polysaccharides released by polysaccharide cleavage of moss or moss-derived peat and contains at least 20 mole % Rha₃ at least 35 mole % GaIA₃ less than 4.5 mole % Man and less than 9.5 mole % Gal.

2. An antimicrobial composition comprising a mixture according to claim 1 together with a carrier or diluent, and optionally also a further antimicrobial agent.

3. A method of preservative or disinfectant treatment of an inanimate object which method comprises applying to said object a mixture or composition according to claim 1 or 2.

4. The use of a mixture according to claim 1 for the manufacture of a medicament for use in a method of antibiotic or disinfectant treatment of a human or non-human subject.

5. A method of antibiotic or disinfectant treatment of a human or non- human subject, which method comprises applying to said subject an effective amount of a mixture or composition according to claim 1 or claim 2.

6. A process for the preparation of a mixture according to claim I₃ said process comprising: cleaving polysaccharides in moss or moss-derived peat; and fractionating polysaccharides released by this cleavage.

7. An antimicrobial composition comprising lignin-free sphagnum leaves together with a carrier or diluent₃ and optionally also a further antimicrobial agent.

8. A method of preservative or disinfectant treatment of an inanimate object which method comprises applying to said object a composition as claimed in claim 7.

9. The use of lignin-free sphagnum leaves for the manufacture of a medicament for use in a method of antibiotic or disinfectant treatment of an external body surface of a human or non-human subject.

10. An odour and contamination reducing water-absorbent structure comprising a carrier material and a water-insoluble particulate moss holocellulose.

11. The use of water-insoluble particulate moss holocellulose as an odour and microbial contamination reducing agent in a water-absorbent structure.

12. The use of water-insoluble particulate moss holocellulose as an odour and microbial contamination reducing agent in the manufacture of a water- absorbent structure.

13. A cleansing device having a handle with attached thereto a water- absorbent cloth or pad containing a water-insoluble particulate moss holocellulose.

14. A food package comprising a sealed container, a foodstuff selected from the group consisting of cut raw plant material and animal material having exposed raw internal organ or muscle tissue, and a water-absorbent structure comprising a water-insoluble particulate moss holocellulose.

15. A package as claimed in claim 14 wherein said animal material comprises raw mammalian, avian or piscine muscle.

16. A package as claimed in either of claims 14 and 15 wherein said structure is in the form of a cup carrying said foodstuff.

17. A package as claimed in claim 16 wherein said cup is provided with a water-absorbent lid comprising water-insoluble moss holocellulose.

18. A water-absorbent food packaging cup comprising water-insoluble moss holocellulose.

19. An armpit sweat-absorber comprising a water-pervious casing containing a water-insoluble particulate moss holocellulose.

20. An incontinence pad comprising a water-pervious casing containing a water-insoluble particulate moss holocellulose.

21. An insole comprising a water-pervious casing containing a water- insoluble particulate moss holocellulose.

22. A water absorbent moss holocellulose sheet.

23. A water absorbent moss holocellulose pad.

24. A water absorbent moss holocellulose cup.

25. A plant container in the form of a moulded water-absorbent body comprising particulate water-insoluble moss holocellulose.

26. A process for producing moss holocellulose which process comprises contacting moss with gaseous chlorine dioxide.