Patent Description:
It is known in the literature that the resinous exudate of black poplar (Populus nigra) constitutes the base element of European propolis, and in general of bee-produced propolis in temperate zones.

Propolis, known for its many pharmacological activities is a substance produced by bees following collecting, by the latter, of resinous exudates (mainly of black poplar). On average, it is comprised of <NUM>-<NUM>% beeswax, <NUM>% pollen, <NUM>% of various substances present on bee legs and about <NUM>% of plant resins.

Throat lozenges such as Apopros for the protection of throat in the form of tablets comprising sugar, glucose, propolis wax, honey, a propolis-xantan gum-hydrolising glycosaminoglycans complex, propolis extract, menthol, Eugenia caryophyllus bud oil and other ingredients are known in the art.

Virey's trattato compiuto di farmacia teorica e pratica, dated <NUM> discloses an ointment made of poppy, henbane, dark solano prepared with liquefied pork fat for use in the treatment for use as soother, for calming pains, inflammations, burns, haemorrhoids, for cicatrisation of various skin regions, for dissipating milk clogs, and as a component of soothing enemas for the low bowel inflammations.

Various uses of poplar buds-based drugs are also known in the literature.

Given the constant and increasing interest for the use of substances of natural origin for the treatment of numerous affections, and given the current dramatic extinction hazard to which bees are exposed, it is important to find products of natural origin which may comparably replace in a comparable, or even more effective manner, bee-produced substances.

The Authors of the present invention have analysed extracts of poplar buds in order to verify the possible use thereof for therapeutic purposes as an alternative completely of plant origin to propolis.

Unlike propolis, the extracts of poplar buds also entail the advantage of not containing substance of animal origin and allergenic substances such as pollens.

The Authors of the present invention have surprisingly verified that, with respect to European propolis, poplar buds extracts exhibit comparable or even greater mucoadhesive properties.

The Authors of the invention have also demonstrated for the first time an effective antibacterial activity, in particular against Streptococcus pyogenes, Pseudomonas aeruginosa and Staphylococcus aureus, of extracts of poplar buds, optionally lyophilised, optionally co-lyophilised with natural gums (gum-supported).

Therefore, object of the present invention is a composition as defined in the claims for use in the treatment of oropharyngeal cavity affections.

Therefore, object of the present invention is a composition comprising
Extract of poplar buds to a percentage in weight from <NUM> al <NUM>% and one or more of pharmaceutically acceptable carriers, excipients, flavours, preservatives for use in the treatment of oropharyngeal cavity affections. wherein the composition is in a fluid form or a solid form, wherein, when the composition is in the fluid form, the composition consists of:.

By "extract of poplar buds", for the purposes of the present invention, it is meant an extract prepared from leaf buds, or mainly leaf buds, of black poplar (Populus nigra).

In particular, the extract according to the invention is an extract of unopened leaf buds. Buds are found unopened mainly in springtime, which in Europe is usually from March to May.

According to one embodiment of the invention, the sole active principle of the composition is represented by the extract of poplar buds.

In the composition according to any one of the above-reported embodiments, the extract of poplar buds may be associated with a natural gum in an extract:gum ratio comprised in the range of from <NUM>:<NUM> - <NUM>-<NUM>.

The composition, as defined above and as further exemplified in the examples below, can be used, as mentioned hereto, for the treatment of oropharyngeal cavity affections. Such affections can be selected, e.g., from sore throat, pharyngitis, aphtha, oral cavity inflammation or infection.

For the carrying out of the composition of the invention, a hydro-alcoholic extract of poplar buds could preferably be used with a gradation of from <NUM> to <NUM> alcohol grades. In a particularly preferred embodiment, the extract could be lyophilised. Optionally, the extract could be co-lyophilised with natural gum in order to increase extract solubility in water and to make the dry extract more easily processable.

However, the data obtained, and that can be seen in the experimental section, show that co-lyophilisation (support) with natural gum increases the mucoadhesive and protective characteristics of the extract, making particularly effective the compositions of the invention comprising the gum-supported extract.

In particular, the extract of poplar buds could be an extract obtainable by a process comprising the following steps:.

In particular, the extracts in a. can be prepared both by extraction by percolate digestion with motion of the sole extraction solvent, and by extraction with an extractor provided with bladed stirrer, and moving both the solvent and the buds. Extraction can be carried out at a temperature comprised between <NUM> and <NUM>, e.g. at a temperature comprised in the range of <NUM> - <NUM> (extremes included), in particular at a temperature of about <NUM> or <NUM>.

The extraction can be carried out for a time length of from <NUM> to <NUM> hours, e.g. for about <NUM> hours.

The extracts, prepared in a. , are mixed as in point c. , preferably in a <NUM>:<NUM> ratio, but may be mixed also in a <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM> ratio. The mixing is carried out by gradually adding (addition rate: <NUM> to <NUM> I/min) the <NUM>° alcoholic extract in the <NUM>° alcoholic extract, but also the reverse addition is possible. The mixing is carried out at a temperature of <NUM>°± <NUM>.

The multi-fraction extract prepared at point c. is subjected to clarification by decanting on containers provided with a conical bottom for a minimum of <NUM> hours. Once said hours have elapsed, the extract is spilled and is filtered on a cellulose filter. Such filtering can be carried out, e.g. on a panel filter with a cut-off of from <NUM> to <NUM> microns (extremes included) in a single step or in plural consecutive steps, such a, e.g., a cut-off of <NUM> microns, <NUM> microns, or a cut-off of <NUM> microns or even higher.

At the end of the filtering, the alcoholic gradation is controlled and adjusted to <NUM>°± <NUM>° alcoholic grades.

Alternatively to decanting, the extract can be separated by centrifugation on a vertical centrifuge fed at a flow rate of <NUM> to <NUM> I/min.

The extract clarified as in d. could be used tel quel as a multi-fraction alcoholic extract.

The extract clarified as in d. is subjected to a concentration and lyophilisation process as in e. to provide a lyophilised extract that can be used for solid formulations.

The extract clarified as in d. , additioned with arabic gum or natural gum is subjected to a concentration and lyophilisation as in f. to provide a lyophilised extract supported on arabic gum, that can be used for solid formulations. Arabic gum, or one or more natural gums, are used in an amount such as to yield a percentage of <NUM>-<NUM>% on the solid.

The extract as obtained according to the above-described process is characterised by the following composition:.

The values are given by the mean of analyses of different extracts according to the above-reported methodology.

According to one embodiment, the extract of poplar buds in any form defined above, could be at a weight percentage of from <NUM> to 45Wt%.

For instance, the composition in fluid form could be according to the following examples.

The composition of the invention could be, e.g., in the form of powder, tablet, capsule, hard or soft gelatine, syrup, spray, suspension.

Therefore, one or more among pharmaceutically acceptable carriers, excipients, flavours, preservatives known to a technician in the field could be used.

In a broad sense, when made in a fluid form the composition will comprise as active principle the extract of poplar buds in any embodiment described herein and one or more of excipients, solvents, flavours, preservatives, etc., known to a technician in the field. By way of a non-limiting example, as excipients one or more of natural gums could be used, such as, e.g.,, arabic gum, xanthan gum, guar gum, tara gum or mixtures thereof; natural and synthetic polyalcohols, such as, e.g., sucrose, mannitol, sorbitol, xylitol or mixtures thereof; cellulose and cellulose derivatives, such as, e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose or mixtures thereof; synthetic polymers, such as, e.g., polyvinylpyrrolidone, polymethacrylates or mixtures thereof; maltodextrins, inulin and/or cyclodextrins.

As solvents, always by way of a non-limiting example, there could be used ethanol, isopropanol, glycerine, propylene glycol or mixtures thereof.

Natural or lyophilised fruit juices could also be used, like, e.g., apple, pear, orange, lemon, raspberry, blueberry juice or mixtures thereof; natural or artificial flavours, such as, e.g., strawberry, lemon, orange, raspberry, peach, cherry, mixed berries, mandarin orange flavour or mixtures thereof; essential oils (E. ) such as, e.g., lemon, orange, mint, eucalyptus essential oil or mixtures thereof.

Broadly speaking, when made in a solid form, the composition will comprise as active principle the poplar buds extract in any embodiment described herein and one or more of excipients, solvents, flavours, preservatives, etc. known to a technician in the field. By way of a non-limiting example, there could be used as excipients one or more of sugars, such as, e.g., sucrose, mannitol, sorbitol, xylitol or mixtures thereof; cellulose and cellulose derivatives such as, e.g., microcrystalline cellulose, sodium carboxymethyl cellulose, or mixtures thereof; maltodextrins, cyclodextrins and/or inulin.

There could be used one or more of starches and starch derivatives, such as, e.g., rice starch, potato starch, corn starch, pregelatinized starch or mixtures thereof; lubricants such as, e.g., stearic acid, magnesium stearate, glyceryl behenate or mixtures thereof; natural and synthetic flavours such as, e.g., strawberry, lemon, orange, raspberry, peach, cherry, mixed berries flavour or mixtures thereof; essential oils, such as, e.g., lemon, orange, mint, eucalyptus essential oil or mixtures thereof; lyophilised fruit juices such as, e.g., apple, pear, orange, lemon, raspberry, blueberry, strawberry, elder juice or mixtures thereof; powder or lyophilised natural extracts of lime (tree), mallow, aloe, althea or mixtures thereof; sweeteners such as, e.g., honey, sugars, aspartame, cyclamates, acesulfame K, stevia extracts, sucralose or mixtures thereof.

Anywhere in the description and in the claims, the term "comprising" could be replaced by the term "consisting in" or "made of".

The following examples are provided in order to further clarify the invention and not for limitative purposes.

A good mucoadhesive effect is a requirement of paramount importance so that the product may remain in the intended site of action (pharyngeal mucosa) and mechanically carry out thereat its protective and curative actions, as already explained in the description above. The main aim of this assay is to assess the in vitro adhesion of the composition of the invention in different formulations on a suitable cell model envisaging the use of buccal cells. A second aim of the assay is to determine bioadhesion resistance over time to a flow of artificial salivary solution. Mucoadhesion of the composition of the invention in different formulations was determined by assessment of the product ability to adhere to cells by inhibiting lectin (a protein having a high affinity for glucoside and mannoside residues) bonding with membrane glycoproteins. The extent of mucoadhesion is measured by a colorimetric reaction enabling to quantify the sites on glycoproteins not engaged by the lectin, as engaged by the mucoadhesive product. The colorimetric reaction is possible thanks to a peculiar lectin labelling system, illustrated in the following diagram:
<CHM>.

The decrease in the absorbency value is proportional to the ability of the product to adhere ("mucoadhere") to cells. The mucoadhesive ability is expressed as a percentage of inhibition of glycoprotein/lectin bonding, and represents the percentage of mucosal sites occupied by the product according to the equation:<MAT>.

The product is applied after dilution, taking into account the fact that in real use, right after delivery, a mixing with the saliva present in the oropharyngeal cavity occurs. The following graphs depict the results obtained with different samples of composition according to the invention in different formulations.

In the second phase of the experimentation, the composition ability to remain adhered to the mucous membrane over time was assessed, by subjecting the system to a <NUM>/min flow of artificial saliva, made of an isotonic aqueous solution containing a pH <NUM> phosphate buffer and <NUM>% mucin. The results obtained highlighted an interesting product ability to remain bioadhered to mucous membranes during the first hour of application.

The resistance of the mucoadhesive layer obtained with the composition Example solid composition <NUM>, diluted <NUM>:<NUM> at different times, <NUM> <NUM> and <NUM> towards a simulated salivary solution (<NUM>% NaCl physiological solution) is reported in Table <NUM>, below.

The results obtained in this experimentation demonstrate that the different formulations of the composition of the invention possess a high mucoadhesive ability towards buccal cells. Moreover, the assessment of the resistance of the mucoadhesive-protective layer to flow with an artificial salivary solution allowed to highlight a good mucoadhesion retentive ability during the first hour of application.

In light of the results obtained, it is possible to state that the composition of the invention, demonstrating to possess good, resistant mucoadhesivity, can play a real protective role on oropharyngeal mucous membrane.

Object of the assay is to demonstrate the action mechanism of the composition of the invention, by assaying its filmogenic and protective ability compared to a known irritating agent: the agent used is lipopolysaccharide (LPS) membrane, a classic model of inflammatory induction. Said assay aims at highlighting the effective ability of the product to limit contact between the mucous membrane and external irritating agents.

The selected irritating agent was isolated from Escherichia coli cell membrane. LPS dimensions are such as to allow to consider the barrier effective in protecting the mucous membrane from dust, smog, pollens and other agents concurring to irritation, and therefore to the onset of different pathologies burdening this particular and delicate environment.

Analyzing the experimental protocol in more detail, the sample ability to act as barrier is assessed by measuring IL6 production and which consequence of the contact between a layer of cells (fibroblasts) and the LPS. The experimental system provides the use of special Transwell wells, equipped with a collagen-coated semipermeable membrane that prevents direct contact between cells, deposited on the bottom, and sample, stratified on the overhanging semipermeable membrane, which represents the sole communication route between the <NUM> portions of the well. LPS is inoculated in the space above the sample, whereby membrane crossing will be all the more difficult the greater the barrier effect exerted by the sample itself. Therefore, the quantification of interleukins produced at +<NUM> hours from LPS addition yields a direct evidence of the barrier effect exerted by the assayed sample. IL6 inhibition is a direct measurement of the barrier effect.

The barrier effect (BE) is expressed as a percentage of the reduction of the release of IL-<NUM> and is obtained through comparison with the value obtained from the Positive Control (C+), i.e. from cells treated with the sole LPS in the absence of the sample.

The results obtained in the experiment conducted in triplicate with different embodiments of the composition of the invention are reported in the following tables. Due to the presence of resins in high concentrations, the barrier assay was carried out both on the <NUM>:<NUM> diluted product and on the <NUM>:<NUM> diluted product. First the cell-secreted IL6 picograms after LPS insult, and, in the next Table, the % inhibition compared to the positive control are reported.

From the results, a remarkable reduction of the concentration of IL-<NUM> produced in the experiments in which the assayed product is present is evident, at both dilutions, exhibiting a barrier protracted in time, effective also in case of partial loss of product due to inability to slowly dissolve the tablet in the mouth.

The IL-<NUM> value equal to <NUM>,<NUM> pg/µL, detected for the negative control, is assimilable to a normal basal value, i.e. to a non-inflammatory condition. The graph in <FIG> clearly shows the protection exerted by the product on the cells: the data are expressed in terms of fold over (F. ) compared to the control C-.

From the mean IL-<NUM> values measured in the barrier assay and in the positive control, the percentage of IL-<NUM> release reduction is calculated: <MAT>.

Therefore, by applying the formula it is obtained a value of reduction of IL-<NUM> production, in the presence of the assayed sample, which is of <NUM>% at <NUM>:<NUM> dilutions and of <NUM>% at <NUM>:<NUM> dilutions, compared to the positive control reported in Table <NUM> for the composition of the invention made in the form of adult tablets (Example solid composition <NUM>) as described in the detailed section above.

Table <NUM> IL6 production in the barrier assay with formulation of the composition of the invention in children tablets (example solid composition <NUM>) according to the examples reported in the detailed section of the invention.

From the mean IL-<NUM> values measured in the barrier assay and in the positive control, the percentage of IL-<NUM> release reduction is calculated: <NUM>-[(IL-SAMPLE)/ IL-6C+) X <NUM>].

Therefore, by applying the formula it is obtained a value of reduction of IL-<NUM> production, in the presence of the assayed sample, which is of <NUM> % at <NUM>:<NUM> dilutions and of <NUM>% at <NUM>:<NUM> dilutions, compared to the positive control reported in Table <NUM> for the composition of the invention made in the form of children tablets (Example solid composition <NUM>) as described in the detailed section above.

From the mean IL-<NUM> values measured in the barrier assay and in the positive control, the percentage of IL-<NUM> release reduction is calculated: <NUM>-[(IL-SAMPLE)/ IL-6C+) X <NUM>] Therefore, by applying the formula it is obtained a value of reduction of IL-<NUM> production, in the presence of the assayed sample, which is of <NUM>% compared to the positive control reported in Table <NUM> for the composition of the invention made in the form of strong spray (Example fluid composition <NUM>) as described in the detailed section above.

From the mean IL-<NUM> values measured in the barrier assay and in the positive control, the percentage of IL-<NUM> release reduction is calculated: <NUM>-[(IL-6SAMPLE)/ IL-6C+) X <NUM>] Therefore, by applying the formula it is obtained a value of reduction of IL-<NUM> production, in the presence of the assayed sample, which is of <NUM> % compared to the positive control reported in Table <NUM> for the composition of the invention made in the form of no alcohol spray (Example fluid composition <NUM>) as described in the detailed section above.

To validate the method used, and to verify that the results depend on the sole barrier effect of the assayed samples and exclude any pharmacological, immunological or metabolic effects (e.g., cytokine synthesis modulation), the barrier assay was assisted by an INTERNAL CONTROL (IC) concomitantly carried out on each sample.

In the IC assay, first the cells are stimulated with the LPS, whereas the sample is added to the Transwell only subsequently: in this case, a nil % of IL6 inhibition would clearly indicate that the presence of the assayed composition affords a mechanical protection, in no way interfering with cellular cytokine synthesis.

IL-<NUM> concentration values measured in internal control experiments, wherein said treatment with the composition in the form of adult tablets (Example solid composition <NUM>) is carried out after LPS insult, are reported in Table <NUM> below:
<IMG>.

IL-<NUM> concentration values measured in internal control experiments, wherein said treatment with the composition in the form of children tablets (Example solid composition <NUM>) is carried out after LPS insult, are reported in Table <NUM> below:
<IMG>.

IL-<NUM> concentration values measured in internal control experiments, wherein said treatment with the composition in the form of strong spray (Example solid composition <NUM>) is carried out after LPS insult, are reported in Table <NUM> below:
<IMG>.

IL-<NUM> concentration values measured in internal control experiments, wherein said treatment with the composition in the form of no alcohol spray (Example fluid composition <NUM>) is carried out after LPS insult, are reported in Table <NUM> below:
<IMG>.

The values obtained in these experiments are found to be homogeneous with those of the positive control for the barrier assay (sample absence), and confirm how the sample applied after LPS insult does not influence the production of inflammation mediators, and therefore has no direct anti-inflammatory effect. This is better represented in the graphs reported in <FIG>, expressing the values of cell-produced IL-<NUM> in terms of Fold Over C.

The scavenger anti-oxidising activity directed by the extract of poplar buds according to the present description (obtained with the above-described method and having the above-described characteristics) was assessed by DPPH (<NUM>,<NUM>-diphenyl-<NUM>-picrylhydrazyl) assay; DPPH is a coloured stable radical: it changes colour when chemically reacting with an anti-oxidising compound, and can be detected by spectrophotometric reading. Therefore, object of this assay is to demonstrate that the analysed samples possess a good anti-oxidising activity which is carried out in a purely chemical way (hydrogen transfer to a free radical) without involving pharmacological, immunological or metabolic mechanisms when applied on biological substrates. The analysed sample is the extract of poplar buds according to the present description, at <NUM>, <NUM> and <NUM>µg/mL concentrations. The activity was compared to that of vitamin C, used as positive control. As is visible from <FIG>, the extract of poplar buds according to the invention exhibits excellent anti-oxidising activity at the higher concentrations of <NUM> and <NUM>µg/ml and good activity at the lower concentration of <NUM>µg/ml.

The methodology used in this assay exploits microorganism ability to adhere and form biofilms on polystyrene plate wells: the biofilm mass will then be determined by Crystal-violet staining. The bacterium used in the assay is the one most frequently involving infections of the pharyngeal mucous membrane, i.e., Streptococcus pyogenes; the analysed sample is the extract of poplar buds according to the present description (obtainable with the above-described method and having the above-described characteristics). The first phase of the assay consists in the assessment of the minimum inhibitory concentration (MIC), i.e., the lowest sample concentration inhibiting microorganism growth. The antibiotic gentamycin was selected as positive control. In the second phase, the ability to inhibit biofilm formation is measured; the assayed samples are added to the medium in concentrations equal to or lower than the MIC, to rule out that the antibiofilm activity be ascribable to a microbial growth inhibition. After an adequate incubation period, the biofilm formed was stained with Crystal-violet; following staining, excess dye was removed with water, and that adhered to biofilm was dissolved in ethanol. The resulting solution was analysed by spectrophotometer at <NUM>: the intensity of absorbance from this analysis will be all the greater the greater the mass of biofilm formed; accordingly, anti-biofilm activity and recorded absorbance are inversely proportional to each other. For each sample, the concentrations used were 1x MIC, <NUM> xMIC and <NUM> MIC. The reported data are the mean of <NUM> measurements carried out in triplicate. Statistical significance calculated with the t test, * p<<NUM> and **p<<NUM> (treated bacteria versus untreated bacteria). The assay shows how the extract of the invention is more effective compared to a standard <NUM>% hydro-alcoholic extract.

The assays were carried out as described above for example <NUM>, assaying the compositions of the invention and a propolis spray at different dilutions.

The data reported in the tables show how, above all at higher dilutions, the compositions of the invention have greater mucoadhesive effect compared to propolis.

As is apparent from the above-reported assays, the assayed compositions show how, in any embodiment assayed (solid, spray with alcohol, spray without alcohol), the extract of poplar buds possesses a mucoadhesive ability greater than that of propolis, appreciable in particular at higher dilutions of the assayed products, as evident from data obtained with <NUM>:<NUM> dilutions.

Black poplar (Populus nigra) buds were fragmented and subjected to two extraction steps, by extractor equipped with mechanical stirrer, in ethanol at decreasing concentrations, performing the first step with <NUM>% ethanol and a second step with <NUM>% ethanol. The D/S ratio used is equal to <NUM>:<NUM>. The extraction temperature is equal to <NUM>° C. The extraction length with <NUM>° ethanol is equal to <NUM> hours, the extraction length with <NUM>° ethanol is equal to <NUM> hours.

The alcoholic extracts obtained as described were reunited in a <NUM>:<NUM> ratio. Mixing envisaged adding the <NUM>° alcoholic extract into the <NUM>° alcoholic extract (with an addition rate equal to <NUM> I/min). The mixing was carried out at <NUM>°± <NUM>. At the end of the addition the mixture was subjected to decantation for <NUM> hours at <NUM>°± <NUM>, then the supernatant was recovered and further filtered on a panel filter equipped with a cellulose filter with a <NUM> cut-off. At the end of the filtering, alcohol gradation is checked and adjusted to <NUM>°±<NUM>° alcohol grades.

The clarified alcoholic extract obtained as described in Example <NUM>, with no correction to the alcohol gradation, is concentrated by ethanol evaporation, by use of a thin film distillation system according to a standard protocol, envisaging the feeding of the extract to be concentrated at a flow rate of about <NUM> I/h; the operation is carried out by setting a residual vacuum of <NUM>-<NUM>. 8bar, and the fluid heating the evaporation walls is set at <NUM>, thereby obtaining, after ethanol elimination, a concentrated aqueous extract. The aqueous extract thus obtained was subjected to lyophilisation.

Claim 1:
A composition for use in the treatment of oropharyngeal cavity affections, wherein the composition is in a fluid form or a solid form, wherein, when the composition is in the fluid form, the composition consists of:
Poplar buds extract at a percentage in weight from <NUM> to <NUM>%,
Excipients at a percentage in weight from <NUM> to <NUM>%
Solvents at a percentage in weight from <NUM> to <NUM>%
Natural or lyophilised fruit juices at a percentage in weight from <NUM> to <NUM>%
Natural or artificial flavours at a percentage in weight from <NUM> to <NUM>%
Essential oils at a percentage in weight from <NUM> to <NUM>%;
and when the composition is in the solid form, the composition consists of:
Dry poplar buds extract at a percentage in weight from <NUM> to <NUM>%,
Excipients at a percentage in weight of <NUM>-<NUM>%
Natural or artificial flavours at a percentage in weight from <NUM> to <NUM>%
Essential oils at a percentage in weight from <NUM> to <NUM>%
Powdered or lyophilised fruit juices and/or natural extracts at a percentage in weight from <NUM> to <NUM>%
Sweeteners at a percentage in weight from <NUM> to <NUM>%,
wherein said extract of poplar buds is an extract obtainable by a process comprising the following steps:
a. preparing a hydro-alcoholic extract of poplar buds by extraction with <NUM>° ethanol
b. preparing a hydro-alcoholic extract of poplar buds by extraction with <NUM>° ethanol
c. obtaining a multi-fraction alcoholic extract, by mixing the extracts prepared in a. and b.
d. decanting and/or centrifuging and filtering said alcoholic extract (c.) collecting the supernatant
e. subjecting the filtered supernatant obtained in d. to concentration and freeze-drying and, optionally,
f. adding to the filtered supernatant obtained in d. arabic gum and then subjecting it to concentration and lyophilisation.