FIELD OF THE INVENTION

The present invention relates to a process for the preparation of bacterial nanocellulose comprising a fermentation step actuated by bacteria and yeasts. The nanocellulose obtained from the process is used in textile production, in the preparation of cosmetics, medical devices, sensors and building products.

PRIOR ART

Bacterial nanocellulose (BNC) is an extracellular biopolymer produced through a microbial fermentation process in which bacteria from vinegar are commonly used. BNC has many excellent properties, such as a high purity (free of lignin and hemicellulose), a high crystallinity, a high degree of polymerisation, a nanostructured network, a high capacity to retain water and good biocompatibility. These features distinguish BNC from plant cellulose. In view of these advantageous features, BNC is used for applications in many fields, such as biomedicine, the food industry, cosmetics, advanced acoustic diaphragms, paper production and the textile industry.

The BNC production process involves culture media mainly consisting of laboratory solutions comprising chemical compounds, such as, for example, solvents. Furthermore, each fermentation process uses microorganisms with different modifications and features depending on the fate of the biomaterial. These culture media lead to having a fairly loosely textured cellulose which allows to incorporate active ingredients therein. However, the BNC thus obtained does not show good tensile strength and is rather delicate. Therefore, it is not currently possible to obtain a BNC which can be used in different sectors, but a different process must be developed for each application. Furthermore, BNC is produced using chemical solutions which show different problems, both related to their environmental impact and their high production cost. Therefore, the need to find alternatives for the production of BNC through the use of substances with a very low environmental impact and with low production costs is greatly felt. Furthermore, a culture medium must be found which allows the creation of a polyvalent neutral fabric for the different areas of application.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a process for the preparation of bacterial nanocellulose comprising a fermentation step actuated by bacteria and yeasts. Preferably, the bacteria belong to the genus Acetobacter and the yeasts to the species Saccharomyces cerevisiae.

A second aspect of the present invention relates to the bacterial nanocellulose obtained from the process described above.

A third aspect of the present invention relates to the use of bacterial nanocellulose in the textile industry, preferably for the production of clothing and clothing accessories.

A fourth aspect of the present invention relates to a medical device comprising the bacterial nanocellulose described above. Preferably, the medical device is selected from: a gauze, a bandage, a surgical binder, a patch and a hydrocolloid device with soothing properties.

A fifth aspect of the present invention relates to a cosmetic composition comprising the bacterial nanocellulose described above. The composition object of the present invention may further comprise at least one pharmacologically acceptable excipient, i.e., a compound, acceptable for cosmetic use.

Another aspect of the present invention relates to a building product comprising the bacterial nanocellulose. Preferably, the building product is selected from: a panel, a wall, a roofing sheet and external insulation for homes.

A further aspect of the present invention relates to an electronic device comprising the bacterial nanocellulose. Preferably, the electronic device is selected from a sensor, a switch, a touch pad and a touch cover.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an image obtained through scanning electron microscope (SEM) of a bacterial nanocellulose obtained with classical techniques (A) and of nanocellulose obtained with the process of the present invention (B); Figure 2 shows the results of tensile tests carried out on bacterial nanocellulose with a thickness 0.8 mm (sample 1 ), 0.6 mm (sample 2) and 0.4 mm (sample 3);

Figure 3 shows the results of tensile tests carried out on bacterial nanocellulose with a thickness 0.3 mm (sample 1 ), 0.8 mm (sample 2) and 0.5 mm (sample 3);

Figure 4 shows the results of tensile tests carried out on bacterial nanocellulose with a thickness 0.3 mm (sample 1 ), 0.6 mm (sample 2), 0.7 mm (sample 3) and 0.8 mm (sample 4);

Figure 5 shows the results of tensile tests carried out on bacterial nanocellulose with a thickness 0.8 mm (sample 1 ), 0.6 mm (sample 2) and 0.5 mm (sample 3);

Figure 6 shows a semi-logarithmic scale graph showing the resistance of the bacterial nanocellulose as a function of the variation in water content;

Figure 7 shows a graph showing the resistivity of the bacterial nanocellulose as a function of the variation in water;

Figure 8 shows the variations in weight of the bacterial nanocellulose with respect to the moisture in the air;

Figure 9 shows the variations of the resistivity of the bacterial nanocellulose with respect to the moisture in the air;

Figure 10 shows the bacterial nanocellulose in the form of a round-shaped film obtained with the process according to the present invention with moisture content;

Figures 1 1 -12 show the bacterial nanocellulose obtained with the process according to the present invention without moisture content. DEFINITIONS

In the context of the present invention, the term "by-product" or "waste product" means a secondary product derived from the processing of other products, in particular fruit and vegetables, normally available at low cost or free of charge. The terms “by-products” or “waste products” are used interchangeably in the text.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a process for the preparation of bacterial nanocellulose comprising a fermentation step actuated by bacteria and/or yeasts. In an embodiment, the process comprises the steps of: a) inoculating bacteria belonging to the genus Acetobacter and/or yeasts belonging to the species Saccharomyces cerevisiae with by-products or waste products of the processing of fruit and vegetable products, thereby forming a fermentation mixture; b) incubating the fermentation mixture, thereby forming the bacterial nanocellulose.

Preferably, the fermentation mixture comprises at least one source of theine, at least one sugar and at least one source of acetic acid. The at least one source of theine is preferably selected from: coffee, cocoa, tea, cola, guarana and mate. In a preferred embodiment, the at least one source of theine is tea.

According to an embodiment, the at least one source of theine is present in a concentration comprised between 20 and 70 g/L, preferably between 30 and 60 g/L.

Preferably, the at least one sugar is selected from: sorbitol, glucose, fructose and combinations thereof. In a preferred embodiment, the at least one sugar is sorbitol. According to an embodiment, the at least one sugar is present in a concentration comprised between 170 and 250 g/L, preferably between 180 and 230 g/L.

Preferably, the at least one source of acetic acid is selected from: vinegar, preferably wine vinegar, alcohol vinegar, wine, wine must, derived from fermentation processes of fruit waste, preferably of apricots, apples, oranges, exhausted fruit juice, peaches, hydrolysate of sulphite fibre sludge and thin stillage.

In a preferred embodiment, the at least one source of acetic acid is vinegar. According to an embodiment, the at least one source of acetic acid is present in a concentration comprised between 150 and 230 ml/L, preferably between 170 and 220 ml/L.

In an embodiment, the by-products or waste products are obtained from fruit selected from among: citrus fruits, preferably oranges, pineapples, clementines, lemons, mandarins, peaches, apricots, plums, carrots, apples, melons, pears and grapes.

In an embodiment, the waste by-products are obtained from vegetables selected from: tomatoes, potatoes, salads, courgettes, pumpkin, carrots, onions, fennel, aubergines and peppers.

In a preferred embodiment of the invention, the by-products or waste products are obtained from the processing of fruit or exhausted juices.

In an embodiment, the preparation of the fermentation mixture provides for the preparation of a homogeneous solution comprising the at least one source of theine and the at least one source of acetic acid. Subsequently, the at least one sugar is added to the solution which is preferably mixed until the latter is completely dissolved, and then the by-products or waste products are added. Lastly, the bacteria and yeasts are added to start the fermentation.

In an embodiment, the bacteria and yeasts used in the process of the present invention are cultured and replicated in vitro before being brought into contact with the by-products or waste products. Preferably, the bacteria and yeasts are replicated separately in vitro at least 72 hours before inoculation with the by-products to obtain the fermentation mixture.

In an embodiment, the bacteria belonging to the genus Acetobacter preferably belong to the species Acetobacter xylinum, more preferably they belong to at least one strain selected from: IFO 13693, AJ 12712, Ku-1 , Acetobacter sp. A9, IFO 1772, ATCC10245, ATCC 53524, NBRC13693 and combinations thereof.

In a preferred embodiment of the invention, the bacteria belonging to the genus Acetobacter are inoculated with the by-products or waste products to obtain a final concentration in the fermentation mixture comprised between 10*10 ³ and 20*10 ³ colony forming units/ml (CFU/ml), preferably between 12*10 ³ and 18*10 ³ CFU/ml.

In a preferred embodiment of the invention, the yeasts belonging to the species Saccharomyces cerevisiae are inoculated with the by-products or waste products to obtain a final concentration in the fermentation mixture comprised between 3*10 ³ and 10*10 ³ CFU/ml, preferably between 5*10 ³ and 9*10 ³ CFU/ml.

The fermentation of the waste or by-products, as defined in step (b) of the present process, occurs at a temperature comprised between 18°C and 30°C, preferably between 20°C and 28°C, at a relative humidity comprised between 25 and 50%, preferably between 30 and 45%, for a time comprised between 72 hours (start of formation of the aggregate biofilm) and 240 hours (thickness comprised between 1.5 cm and 3 cm), preferably comprised between 80 hours and 180 hours.

During the fermentation, the bacterial nanocellulose is produced and covers the container where the fermentation mixture is placed. As shown in the example and figures, the bacterial nanocellulose obtained has the appearance of a film. Preferably, the nanocellulose obtained has a thickness comprised between 1 .0 cm and 3 cm, preferably between 0.8 and 2.2 cm. Following the reduction of the moisture content thereof, the nanocellulose has a thickness comprised between 0.3 and 1.2 mm, preferably between 0.6 and 1 mm. Preferably, the reduction of moisture content is comprised between 75 and 90% water/total weight, preferably between 80 and 90% water/total weight.

The Applicant has demonstrated that the process described above allows to obtain a bacterial nanocellulose with a denser texture with respect to the nanocellulose obtained with classical methods, i.e., by using fermentation mixtures comprising solutions of non-natural origin. In fact, when viewed under an electron microscope, the nanocellulose obtained with the method described shows a dense and compact texture (Figure 1 B), while the nanocellulose obtained with classical techniques shows a loose texture (Figure 1 A).

Therefore, a second aspect of the present invention relates to the bacterial nanocellulose obtained by the method described in detail above. The nanocellulose obtained also shows a greater resistance than the nanocellulose obtained with classical techniques. Preferably, the bacterial nanocellulose of the present invention with a thickness comprised between 0.5 and 0.8 mm shows a tensile strength comprised between 20 and 35 kg, preferably between 22 and 30 kg.

The nanocellulose obtained has a texture with high breathability, is very resistant to tearing and high temperatures, therefore it is partially flame retardant, and is biodegradable. Therefore, the nanocellulose is an excellent material for the textile industry. In fact, the nanocellulose itself can be used for the production of clothing and clothing accessories, for the production of furnishings, for fitting out motor vehicle interiors.

Therefore, a third aspect of the present invention relates to the use of the bacterial nanocellulose in the textile industry, preferably for the production of clothing, clothing and furnishing accessories, for fitting out motor vehicle interiors, for example trimmings, design inserts, finishes, smart pockets and coverings. Thanks to its beneficial properties for the skin, in particular thanks to the ability of the bacterial nanocellulose to promote wound healing, it can also be used for the preparation of medical devices, such as gauzes, bandages, surgical binders, patches and hydrocolloids. Furthermore, the texture of the bacterial nanocellulose allows to fix active ingredients or molecules with pharmacological activity therein. In this case, the nanocellulose acts as a carrier for the topical application of active ingredients.

Therefore, a fourth aspect of the present invention relates to a medical device comprising the bacterial nanocellulose described above. Preferably, the medical device is selected from: a gauze, a bandage, a surgical binder, a patch and a hydrocolloid device with soothing properties.

In an embodiment, the medical device comprises at least one active ingredient, preferably an active ingredient to promote wound mending and healing.

A fifth aspect of the present invention relates to a cosmetic composition comprising the bacterial nanocellulose described above. The composition object of the present invention may further comprise at least one pharmacologically acceptable excipient, i.e., a compound, acceptable for cosmetic use.

In an embodiment, the bacterial nanocellulose is de-structured with techniques known to the person skilled in the art (for example through soaking), and used for the preparation of the cosmetic composition.

Said excipient can be at least one moisturising, occlusive or emollient, soothing, toning and nourishing conditioning agent of the skin or hair/body hair. Furthermore, said conditioning agent can be an oil or a vitamin, or an antioxidant, such as a phenol or resveratrol, preferably obtained from grapes or wine.

Furthermore, the bacterial nanocellulose can be used as a cosmetic facial mask, replacing the fabric masks present on the market.

Thanks to the high strength and malleability of the nanocellulose of the present invention, it can be employed for numerous uses, such as for the production of building products.

Therefore, a further aspect of the present invention relates to a building product comprising bacterial nanocellulose. Preferably, the building product is selected from: a panel, a wall, a roofing sheet and external insulation for homes.

Finally, the Applicant has found that the bacterial nanocellulose is capacitive and able to respond to external stimuli. Therefore, the bacterial nanocellulose can be used in the production of electronic devices.

A further aspect of the present invention relates to an electronic device comprising the bacterial nanocellulose. Preferably, the electronic device is selected from a sensor, a switch, a touch pad, a touch cover.

EXAMPLE

The production of bacterial nanocellulose involves a fermentation process carried out by bacteria and yeasts. The microorganisms coexist symbiotically and the species in question are Acetobacter xylinum and Saccharomyces cerevisiae. The culture medium necessary for the production of the biofilm has the following components: tea: the presence of theine (alkaloid); sorbitol: simple sugar which allows a better structuring of the cellulose fibres of the final biofilm, making it more compact and thicker; apple vinegar: essential to obtain a saturated environment for the bacteria and therefore modify the metabolic output of the acetobacter; pulp fruit (production waste from the agri-food chains): rich in fructose, a simple sugar which acts as a starter in production;

Symbiotic plate comprising Acetobacter xylinum and Saccharomyces cerevisiae.

The fermentation process consists of two steps: 1 ) cleavage of the complex sugars into simple sugars; 2) production of ethanol from the conversion of the simple sugars obtained in the first step. The choice of integrating simple sugars into the culture medium is to ensure that they are immediately available for the microorganisms, thus reducing production times.

The preparation of the culture medium involves the following steps: a homogeneous solution of vinegar and tea is made in a ratio of 1 :5. Then the sugar is added, in a sugar:tea ratio of 1 :5. The amount of sugar used is obtained through a fructose extraction process from the waste (fruit, wine, vegetables as indicated above) completed with the addition of sorbitol and mixed until the latter is completely dissolved. The fructose extraction process is carried out by filtering the pulp left in maceration with a 5% ethanol solution or by filtering the juice obtained through the pressing of the waste substances. The quantification of the extracted fructose takes place with sample analysis with UV spectrophotometry, rotary evaporation or evaluation of the glycaemic index by means of a glucometer.

The pH is monitored and adjusted with solution acidifiers throughout the process. At the end of the preparation, the fruit pulp is added and only at the end of the preparation of the medium the plate with the microorganisms is added. The growth process requires oxygen and is carried out under standard conditions: temperature 25°C and relative humidity 40% for a time comprised between 7 and 15 days. The bacterial nanocellulose grows, completely covering the surface of the container in which the medium was placed. The fermentation process structures a cellulose consisting of pure microfibrils. The approach used is of the “bottom-up” type.

Once the product has been obtained, it can take different forms: nanocellulose sheets of variable thickness - wet with a thickness of 1 cm, dry with a thickness of 0.8 mm - to be directed towards different markets, including that of fashion, textiles, biomedical devices and cosmetics; bioink to be used as coating, to be introduced in the industries for the production of building products (panels, walls, ceramic materials, roofing and insulation sheets) and design products (lamps or capacitive furnishing elements); powder/mixture to be mixed in other materials during their manufacture.

The technical features obtained thanks to specific tests carried out on the samples are shown below.

Structure

Images obtained by means of SEM observation. Two different samples are compared to highlight how with known media the texture is loose (Figure 1A) sample obtained with acidified solution based on sugars and water, while with the proposed medium the texture is much denser and more compact (Figure 1 B) sample obtained with the previously proposed solution (apple vinegar, sorbitol, pulp fruit and tea).

Resistance Tensile tests have been carried out to highlight the resistance of the bacterial nanocellulose. The results are shown in Tables 1 -4 and Figures 2- 5.

Tensile tests carried out on samples of bacterial nanocellulose of different thicknesses. Table 1 : strength tests on nanocellulose of thickness 0.8 mm (sample 1 ), 0.6 mm (sample 2) and 0.4 mm (sample 3).

Table 2: strength tests on nanocellulose of thickness 0.8 mm (sample 1 ), 0.6 mm (sample 2) and 0.4 mm (sample 3). Table 3: strength tests on nanocellulose of thickness 0.3 mm (sample 1 ), 0.6 mm (sample 2) 0.7 mm (sample 3) and 0.8 mm (sample 4).

Table 4: strength tests on nanocellulose of thickness 0.8 mm (sample 1 ), 0.6 mm (sample 2) and 0.5 mm (sample 3).

Electronic behaviour of nanocellulose

The electronic behaviour of the nanocellulose was tested under different moisture conditions, highlighting the relationship between variation of water quantity in the nanocellulose and resistivity taking into account the weight variation of some nanocellulose samples. To assess the relationship between the two parameters, the weight variation induced by the increase in moisture in relation to the detected resistance value was studied. As can be seen in Figures 6-9, there is rapid resistivity growth. Consequently, a fit was developed to identify the mathematical trends (Figure 6).

Figure 7 shows the resistivity of the material as a function of the variation in water. For each experimental point, the corresponding resistivity value was calculated. The graph in Figure 7 is shown on a semi-logarithmic scale and the values vary from about 10 ⁸ to about 10 ⁶ , therefore of two orders of magnitude.

To bring the units of measurement back to the conventional units, the variation in weight of the sample was studied, in hydrostatic equilibrium with the environment. The nanocellulose obtained with sorbitol is particularly thick. Figure 8 shows the trend of this relationship, the red line represents the fit in the first order of the data:

Parameters: a = 350 ± 60 b = 60 ± 30

Using this relationship, the graph shown in Figure 9 is obtained, in which, however, the resistance value of the sample was reported for clarity and not the variation with respect to the laboratory value, as in the previous case.

The resulting formula of the tests carried out is therefore: p = 1.72, 1012 ± 0.23 MQ (glass range).