Patent Description:
Laccases are redox enzymes, acting by oxidizing substrates using molecular oxygen. This is different from most other industrial enzymes, which generally belong to the group of hydrolases. While hydrolases are quite specific with respect to substrates, the oxidative nature of laccases results in more unspecific and unpredictable interactions in formulations than hydrolases do, and formulation of laccases may behave differently compared to formulations of hydrolases.

Solid formulations made by granulation are used to stabilize the enzymes and to reduce the amount on enzyme dust released into the environment. <CIT> discloses granulation of hydrolases for detergents, such as proteases and amylases, and shows that an acidic environment in the granules greatly improves the enzyme stability.

<CIT> discloses that the residual activity of a granulated laccase is improved by increasing pH of the laccase concentrate used to prepare the granulate. The granulated laccase of <CIT> does not contain a buffer.

<CIT> discloses granules comprising detergent enzymes and acidic buffer components in the core. The acidic buffer components serve the purpose of stabilizing the detergent enzymes containing granules by neutralizing hostile alkaline materials in the environment.

The present invention provides, in a first aspect, an enzyme granulate, comprising a mixture of a laccase and a buffer, wherein the reserve alkalinity of the granulate is at least <NUM> NaOH per <NUM> granulate;
wherein a <NUM>% w/w aqueous solution/suspension of the granule has a pH in the range from <NUM> to <NUM>; and wherein the laccase is an enzyme according to EC <NUM>.

In an embodiment, the granulate comprises a core and a coating, and the core comprises the laccase and the buffer, or the coating comprises the laccase and the buffer.

Other aspects and embodiments of the invention are apparent from the description and examples.

Unless otherwise indicated, or if it is apparent from the context that something else is meant, all percentages are percentage by weight (% w/w).

The laccase activity of laccase granules is generally improved when the laccase is present in an alkaline environment during production. However, we have found that even though pH is increased to establish an alkaline environment during production of such granules, the residual laccase activity will quickly decrease upon storage; even without mixing the granules with other powder ingredients.

Thus, we have found that it is essential to include an alkaline buffer to maintain the alkaline environment after production of the laccase granules. Not only is it necessary to add an alkaline buffer; the buffer must also be added in a surprisingly high amount to maintain laccase activity.

Without being bound by theory, it is believed that without sufficient amount of alkaline buffer, the pH in the micro-environment of the laccase will slowly decrease during storage, which will eventually reduce the laccase activity.

The granule of the invention comprises a mixture of a laccase and a buffer, wherein the reserve alkalinity of the granulate is at least <NUM> NaOH per <NUM> granulate;
wherein a <NUM>% w/w aqueous solution/suspension of the granule has a pH in the range from <NUM> to <NUM>.

In an embodiment, the buffer has an alkaline pKa; preferably the buffer has a pKa in the range of <NUM> to <NUM>.

In an embodiment, a <NUM>% w/w aqueous solution/suspension of the granule has a pH in the range of <NUM> to <NUM>; and most preferably a pH in the range of <NUM> to <NUM>.

The mixture of the laccase and the buffer is a substantially homogenous mixture. More specifically, the laccase and the buffer are not separated, compartmentalized or arranged in discrete layers.

In an embodiment, the granule comprises a core and at least one coating (outer layer) surrounding the core, wherein the core comprises the mixture of laccase and buffer; or the coating comprises the mixture of laccase and buffer.

In another embodiment, the granule comprises.

Preferably, the first and/or the second coating is a salt, carbohydrate, or polymer coating. Carbohydrates may be sugars, sugar alcohols or starch.

Preferably, the granule is made from ingredients that can be used in foods or food additives.

In an embodiment, the granules has a (weight/volume average) diameter of <NUM>-<NUM>, preferably <NUM>-<NUM>, and more preferably <NUM>-<NUM>.

In another embodiment, the granules have a laccase activity of <NUM>-<NUM> LAMU/g, preferably <NUM>-<NUM> LAMU/g, more preferably <NUM>-<NUM> LAMU/g.

The core may comprise the laccase and the buffer. In an embodiment, the core is essentially free of the laccase.

Suitable cores for use in the present invention include, for example, any material suited for layering in fluid bed processes. The core can be insoluble, dispersible or soluble in water. The core material can preferably either disperse in water (disintegrate when hydrated) or solubilize in water by going into a true aqueous solution. Clays (for example, the phyllosilicates bentonite, kaolin, montmorillonite, hectorite, saponite, beidellite, attapulgite, and stevensite), silicates, such as sand (sodium silicate), nonpareils and agglomerated potato starch or flour, or other starch granule sources such as wheat and corn cobs are considered dispersible. Cores can be produced by various methods known in the art, e.g., by granulation.

The cores can be an organic particulate compound e.g. a natural compound such as microcrystalline cellulose, agglomerated or crystalline carbohydrates, e.g. sugars, sugar alcohols (such as mannitol, xylitol, sorbitol, maltitol, isomalt and erythritol), starch, dextrins, flour (e.g. vegetable flour). The material may have been subjected to a steam treatment.

Nonpareils are spherical particles made of a seed crystal that has been built onto and rounded into a spherical shape by binding layers of powder and solute to the seed crystal in a rotating spherical container. Nonpareils are typically made from a combination of a sugar such as sucrose, and a powder such as cornstarch.

In one embodiment of the present teachings the core is a sodium chloride or sodium sulfate crystal (or agglomerated crystals), also referred to as a seed, or other inorganic salt crystal. In another embodiment of the present teachings, the core is a sucrose crystal. Particles composed of inorganic salts and/or sugars and/or small organic molecules may be used as the cores of the present teachings. Suitable water-soluble ingredients for incorporation into cores include: inorganic salts such as sodium chloride, ammonium sulfate, sodium sulfate, magnesium sulfate, zinc sulfate; or urea, citric acid, sugars such as sucrose, lactose and the like.

Cores of the present teachings may further comprise one or more of the following: active agents, polymers, fillers, plasticizers, fibrous materials, extenders and other compounds known to be used in cores.

Suitable polymers include polyvinyl alcohol (PVA), including partially and fully hydrolyzed PVA, polyethylene glycol, polyethylene oxide, polyvinyl pyrrolidine, and carbohydrate polymers (such as starch, amylose, amylopectin, alpha and beta-glucans, pectin, glycogen), including mixtures and derivatives thereof.

Suitable fillers useful in the cores include inert materials used to add bulk and reduce cost, or used for the purpose of adjusting the intended enzyme activity in the finished granule. Examples of such fillers include, but are not limited to, water soluble agents such as salts, sugars and water dispersible agents such as clays, talc, silicates, cellulose and starches, and cellulose and starch derivatives.

Suitable plasticizers useful in the cores of the present teachings are low molecular weight organic compounds and are highly specific to the polymer being plasticized. Examples include, but are not limited to, sugars (such as, glucose, fructose and sucrose), sugar alcohols (such as, glycerol, lower molecular weight polyethylene glycols, sorbitol, xylitol, mannitol and maltitol and other glycols), polar low molecular weight organic compounds, such as urea, or other known plasticizers such as water.

Suitable fibrous materials useful in the cores of the present teachings include, but are not limited to, cellulose, and cellulose derivatives.

In a particularly preferred embodiment, the core essentially consists of sodium chloride, mannitol and/or microcrystalline cellulose.

The core may have an average diameter of <NUM>-<NUM>, preferably <NUM>-<NUM>, and more preferably <NUM>-<NUM>.

The core can be prepared by crystallization, precipitation, size reduction methods, or by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as pan-coating, fluid bed coating, fluid bed agglomeration or granulation, rotary atomization, extrusion, prilling, spheronization, drum granulation, and/or high shear granulation.

Cores without enzyme (laccase) are prepared by the same techniques, but without enzyme.

Methods for preparing the core can be found in, for example,<NPL>. Other useful references include <NPL>; and <NPL>.

The granule may comprise at least one coating. The coating may comprise the laccase and the buffer. In an embodiment, the coating is essentially free of the laccase.

Coating(s) may also be applied to the cores to improve the laccase storage stability, to reduce enzyme dust formation during handling, to improve adherence of a laccase coating onto the core, or for coloring the granule.

The coating(s) may include a salt coating, a carbohydrate coating, and/or a polymer coating. A polymer coating comprises an organic polymer, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), or polyvinyl alcohol (PVA). A carbohydrate coating comprises a water-soluble carbohydrate, such as a sugar, dextrin, or sugar alcohol.

Examples of enzyme granules with multiple coatings are shown in <CIT> and <CIT>. The coating(s) may also include functional ingredients, such bleach catalysts (e.g. manganese bleach catalysts; MnTACN) and/or bleach activators (e.g. TAED, NOBS).

The coating may be applied in an amount of at least <NUM>% by weight of the core, e.g., at least <NUM>%, <NUM>% or <NUM>%. The amount may be at most <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%.

The coating is preferably at least <NUM> thick, particularly at least <NUM>, at least <NUM> or at least <NUM>. In a particular embodiment the thickness of the coating is below <NUM>. In a more particular embodiment the thickness of the coating is below <NUM>. In an even more particular embodiment the total thickness of the coating is below <NUM>.

The coating should encapsulate the core (and the matrix layer) by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit it is encapsulating/enclosing has few or none uncoated areas. The layer or coating should in particular be homogeneous in thickness.

The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

A salt coating may comprise at least <NUM>% by weight w/w of a salt, e.g., at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or at least <NUM>% by weight w/w.

The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles is less than <NUM>, such as less than <NUM> or less than <NUM>.

The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least <NUM> grams in <NUM> of water at <NUM>, preferably at least <NUM> per <NUM> water, e.g., at least <NUM> per <NUM> water, e.g., at least <NUM> per <NUM> water.

The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than <NUM> carbon atoms, e.g., <NUM> or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

The salt in the coating may have a constant relative humidity at <NUM> (also referred to as 'humidity fixed point') above <NUM>%, particularly above <NUM>%, above <NUM>% or above <NUM>%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in <CIT> or <CIT>.

Specific examples of suitable salts are NaCl (CH<NUM>=<NUM>%), Na<NUM>CO<NUM> (CH<NUM>=<NUM>%), NaNO<NUM> (CH<NUM>=<NUM>%), Na<NUM>HPO<NUM> (CH<NUM>=<NUM>%), Na<NUM>PO<NUM> (CH<NUM>=<NUM>%), NH<NUM>Cl (CH<NUM> = <NUM>%), (NH<NUM>)<NUM>HPO<NUM> (CH<NUM> = <NUM>%), NH<NUM>H<NUM>PO<NUM> (CH<NUM> = <NUM>%), (NH<NUM>)<NUM>SO<NUM> (CH<NUM>=<NUM>%), KCI (CH<NUM>=<NUM>%), K<NUM>HPO<NUM> (CH<NUM>=<NUM>%), KH<NUM>PO<NUM> (CH<NUM>=<NUM>%), KNO<NUM> (CH<NUM>=<NUM>%), Na<NUM>SO<NUM> (CH<NUM>=<NUM>%), K<NUM>SO<NUM> (CH<NUM>=<NUM>%), KHSO<NUM> (CH<NUM>=<NUM>%), MgSO<NUM> (CH<NUM>=<NUM>%), ZnSO<NUM> (CH<NUM>=<NUM>%) and sodium citrate (CH<NUM>=<NUM>%). Other examples include NaH<NUM>PO<NUM>, (NH<NUM>)H<NUM>PO<NUM>, CuSO<NUM>, Mg(NO<NUM>)<NUM> and magnesium acetate.

The suitable salts also include the hydrates of these salts.

Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

A particularly preferred salt is sodium chloride.

The laccase of the invention is any laccase according to enzyme classification EC <NUM>.

The laccases may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts). Preferably, the laccase is a fungal laccase. The laccase may have a pH optimum below pH <NUM>. Fungal laccases generally have an acidic pH optimum.

Suitable examples of fungal laccases include laccases derivable from a strain of Aspergillus, Neurospora (e.g., N. crassa), Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes (e.g., T. villosa, T. versicolor), Rhizoctonia (e.g., R. solani), Coprinopsis (e.g., C. cinerea), Psathyrella, Panaeolus, Myceliophthora (e.g., M. thermophila), Schytalidium (e.g., S. thermophilum), Polyporus (e.g., P. pinsitus), Phlebia (e.g., P. radiata), or Coriolus (e.g., C.

Suitable examples of bacterial laccases include laccases derivable from a strain of Bacillus.

In a preferred embodiment, the laccase is derived from a strain of Coprinopsis or Myceliophthora, such as a laccase derived from Coprinopsis cinerea (<CIT>); or Myceliophthora thermophila (<CIT>).

Laccase activity can be determined from the oxidation of syringaldazin under aerobic conditions, pH <NUM>, <NUM>. The violet color produced is measured at <NUM>. One laccase unit (LAMU) is the amount of enzyme that catalyzes the conversion of <NUM> micromole syringaldazin per minute at these conditions.

As used herein, the term "reserve alkalinity" is a measure of the buffering capacity of the granulate composition (gram NaOH per <NUM> granulate composition) determined by titrating a <NUM>% (w/v) solution of granulate composition with hydrochloric acid to pH <NUM>.

Accordingly, in order to calculate reserve alkalinity as defined herein:<MAT>where.

Dissolve <NUM> granulate product in deionized water in a <NUM> volumetric flask. Fill up with deionized water to the <NUM> mark. Measure and record the pH and temperature of the sample using a pH meter capable of reading to ± <NUM> pH units, with stirring, ensuring temperature is <NUM> +/- <NUM>. Titrate whilst stirring with <NUM> or <NUM> hydrochloric acid until pH measures <NUM>. Note the milliliters of hydrochloric acid used. Carry out the calculation described above to calculate the reserve alkalinity to pH <NUM>.

The granulate of the invention has a reserve alkalinity of at least <NUM> NaOH per <NUM> granulate. Preferably, the reserve alkalinity is at least <NUM> NaOH per <NUM> granulate; more preferably the reserve alkalinity is at least <NUM> NaOH per <NUM> granulate; even more preferably the reserve alkalinity is at least <NUM> NaOH per <NUM> granulate; and most preferably the reserve alkalinity is at least <NUM> NaOH per <NUM> granulate.

One skilled in the art will quickly recognize what type and amount of buffer is needed to achieve the required reserve alkalinity of the granule of the invention.

Many suitable buffers are available to choose from, but the buffer must be capable of maintaining an alkaline pH (an alkaline buffer), as expressed by the reserve alkalinity. Thus, an <NUM>% w/w aqueous solution of the buffer has an alkaline pH; preferably the pH is in the range of <NUM> to <NUM>, more preferably the pH is in the range of <NUM> to <NUM>.

In an embodiment, the buffer has an alkaline pKa, such as a pKa in the range of <NUM> to <NUM>, more preferably a pKa in the range of <NUM> to <NUM>. Examples of suitable buffers include carbonate and many amino acids having alkaline pKa values. Glycine (sodium glycinate) is a preferred buffer.

In a preferred embodiment, the buffer is suitably used in foods and food additives.

The granule of the invention may be used as part of a solid (powder) laccase composition, comprising the granule of the invention, and a laccase mediator.

The laccase mediator acts as an electron donor for the laccase. Thus, the mediator facilitates the electron transfer from the intended substrate to the laccase.

In an embodiment, the laccase mediator can be used in foods or food additives.

In a preferred embodiment, the laccase mediator is chlorogenic acid.

An oral care product of the invention comprises the granule and the mediator as described above. Preferably, the amount of the granule is <NUM> to <NUM>% w/w of the oral care product, more preferably the amount of the granule is <NUM> to <NUM>% w/w of the oral care product, or <NUM> to <NUM>% w/w of the oral care product.

In an embodiment, the oral care product comprises the laccase in an amount of <NUM>-<NUM> LAMU per oral care product (for example, per tablet, per chewing gum, etc.).

In an embodiment, the oral care product comprises the laccase in an amount of <NUM>-<NUM> LAMU/g.

In an embodiment, oral care products are selected from the group consisting of tablets, mints, chewing gums, gels, and toothpastes.

When the granules of the invention are used in oral care products, sugar alcohols and sodium chloride are preferred materials for preparing the granules. They can be used in both the core and coating of the granules. This is because sugar alcohols do not contribute to tooth decay, tastes good and has a low glycemic index. Sodium chloride is extensively used in foods and also tastes good.

The present invention is further described by the following examples.

Chemicals were commercial products of at least reagent grade. The laccase used in the examples below is derived from Myceliophthora thermophila, as disclosed in <CIT>, SEQ ID NO: <NUM>.

A laccase granulate was produced by layered granulation in a Glatt Procell GF3 fluid bed equipped with one <NUM> two fluid nozzle in bottom spray configuration. A liquid laccase concentrate (<NUM>% dry matter, <NUM> LAMU/g) was used in the enzyme feed (Feed <NUM>), which was adjusted to pH <NUM> with 1N NaOH.

The cores for the process was NaCl prepared by sieving Suprasel Fine to <NUM>-<NUM>. <NUM> of the NaCl cores was loaded into the fluid bed. Feeds for the granulation were prepared and applied according to Tables <NUM> and <NUM>. The storage stability of the granulate produced from the granulation is shown in Table <NUM>.

A granulate with a laccase activity of <NUM> LAMU/g was obtained.

Reserve alkalinity was measured on the samples above using a <NUM> HCl. Further the stability of the samples was measured by placing closed glasses of the products at <NUM> and <NUM> for one week. Enzymatic activity was measured, and residual activity calculated relative to samples stored at -<NUM>.

Based on the data in Table <NUM>, it is clear that laccase stability is improved with increasing reserve alkalinity.

Laccase granules were prepared by spraying a laccase concentrate (<NUM> LAMU/g) onto NaCl cores (ESCO <NUM>, <NUM>-<NUM>) in a Glatt Procell GF3 fluid bed with two different buffers, and without a buffer.

A laccase concentrate was adjusted to pH <NUM>, and two different buffers were prepared and also adjusted to pH <NUM>:.

Glycine buffer:     <NUM> glycine + <NUM> deionized water + <NUM> 10N NaOH.

Carbonate buffer:     <NUM> Na<NUM>CO<NUM> + <NUM> NaHCO<NUM> + <NUM> deionized water.

<NUM> NaCl cores were fluidized in a fluid bed, and Granules A, B and C were prepared by spraying the NaCl cores with three different feeds, prepared by mixing the laccase concentrate with the glycine or the carbonate buffer, and without buffer, as shown in Table <NUM>. Each of the feeds were adjusted to pH <NUM> before spraying the NaCl cores.

An accelerated storage stability test was carried out by storing Granules A, B and C in open glasses at <NUM> and <NUM>%RH for <NUM> weeks. The residual laccase activity after storage is shown in Table <NUM>.

Table <NUM> shows that the laccase storage stability is improved by adding an alkaline buffer to the laccase concentrate before preparing the laccase granules.

Claim 1:
An enzyme granule, comprising a mixture of a laccase and a buffer, wherein the reserve alkalinity of the granulate is at least <NUM> NaOH per <NUM> granulate; wherein a <NUM>% w/w aqueous solution/suspension of the granule has a pH in the range from <NUM> to <NUM>; and wherein the laccase is an enzyme according to EC <NUM>.<NUM>.