WO2018018173A1 - Bioactive paper comprising as its base an algae-paper consisting of secondary cellulose fibres in combination with brown algae biomass, and antimicrobial extract obtained from brown algae and added to the algae-based paper - Google Patents

Abstract

The present invention comprises a bioactive paper consisting of secondary cellulose fibres in combination with brown algae biomass, and an antimicrobial extract obtained from brown algae. The method for producing said bioactive paper and the use thereof to protect fruit and vegetables against oxidative stress from the air, bacterial attack and, mainly, phytopathogenic fungi, thereby allowing damage prevention during the storage and transport thereof, is also described.

Description

 BIOACTIVE PAPER THAT UNDERSTANDS AN ALGAL PAPER BASED, WHICH IS COMPOSED BY SECONDARY CELL FABRICS IN COMBINATION WITH ALGAS PARDAS BIOMASS; AND AN ANTIMICROBIAL EXTRACT, OBTAINED FROM SOME BROADS, WHICH IS ADDED TO THE ALGAL PAPER

Technical Sector

 The present invention can be applied in the area of industrial and agricultural, more specifically the product can be used in the protection of fruits and vegetables against oxidative stress of the air, bacterial attack and mainly against phytopathogenic fungi, allowing the prevention of damage during storage and transport stages.

Prior art

 The packages are vain elements that depend on the type of product to be protected, the place of destination and its costs. In this sense, wood packaging, different types of plastics (rigid, flexible and foamed) and cellulose derivatives such as cardboard and paper are manufactured (FAO, 2016). Choosing the right packaging is a key factor, which is why it has motivated efforts to investigate and develop better packaging systems. Thermoplastic materials allow the development of versatile packaging, but these have been affected by the validity of environmental policies in favor of recycling, sustainability and biodegradable elements. This has generated an apparent advantage to the use of packaging from paper and other cellulose derivatives, since they can come from secondary (recycled) sources, but are per se biodegradable and sustainable.

The commercialization of fresh fruits depends on the prevention of damage throughout the entire production chain; harvest, storage, packaging, marketing and transportation (Nunes et al, 2009). The fruit industry makes use of a large number of items for packaging for its fruits, however, regardless of the type of packaging many of the problems are independent of the material or design used in its preparation. The transport of fruits is complex, as they breathe and ripen, for this reason many of the problems are due to changes in environmental conditions inside storage chambers. Mishandling from these factors, it can lead to rot caused by opportunistic phytopathogens, since, in general, packaging offers protection against bumps, scrapes or thermal insulation, but not against microorganisms.

 As an example, the Chilean fruit industry loses between 3 and 5% of total fresh fruit exports annually due exclusively to the attack of post-harvest phytopathogens. If we consider that Chilean exports during the 2014-2015 season were 2,399,976 tons of fruit, the economic losses are millionaire (ODEPA, 2016).

The main phytopathogenic microorganisms are fungi, such as Botrytis cinerea, Penicillium expansum, Alternaría altérnate and Pez / cu / a malicorticis with dire results for the economy (Prusky, 2011; Coulomb 2008; Vargas 2007). Gray mold (B. cinérea) affects more than 200 crop species in the world during the post-harvest period, including; grapes, apples, pears and strawberries, among others (Elad & Shtienberg, 1995). This fungus is complex because it has the ability to grow inside the boxes stored at low temperatures. Synthetic chemical agents are used to control this and other pathogens, such as: benzimidazoles, dithiocarbamates, strobilurines, guanidines, imidazoles, triforine piperazines, pyrimidines, phthalimides, sulfamines and triazoles (Njombolwana et al, 2013; Everett et al, 2005) . However, the use of these compounds in the long term has caused the microorganisms to become resistant. On the other hand, the use of large volumes of these difficult-to-degrade chemical substances has caused damage to the environment. In response to these problems, fruit importing countries have established maximum limits of chemical residues in fruits (Ippolito & Nigro, 2000). The search for new antioxidant and antimicrobial bioactive compounds has motivated the study of macroalgae as a raw material, due to the chemical richness they possess, proving to be an effective natural source of compounds against microorganisms such as bacteria and fungi (Troncoso et al., 2015 ; Ali et al., 2012; Guedes et al., 2012; Stein et al., 2011; Felicio et al., 2010, Lañe et al., 2009). At the same time, there are algae such as those of the Rhizoclonium and Gracilaria genera that have long fibers allowing the manufacture of paper. Therefore, algal fibers can incorporate new structural and functional characteristics based on the bioactive compounds present in them. Considering this background, there have been several technological developments focused on similar problems related to the creation of materials that contemplate the use of algae or derivatives thereof as components capable of increasing or conferring special mechanical or biological presentations. Among the main invention patents related to this subject are:

1. Patent WO2015044821 Al (Kimberly-Clark Worldwide, Inc): "Tree-free fiber compositions and uses in containerboard packaging". The purpose of the patent is to protect a method for the production of corrugated cardboard packaging products that includes at least one layer of cardboard pulp based on tree pulp and at least one corrugated layer in the middle that includes pulp-free material of trees. Tree pulp free material is present in an amount of approximately 5% to 100%. This material can be a wheat straw paste and red algae paste of the genus Gelidium corresponding to the species G. corneum, G. amansii, G. robustum, G. chilensey G. asperum.

2. Patent WO2008131720 (Wendler et al.): "Method for the production of a bioactive cellulose fiber with a high degree of brightness". The invention relates to a method for the production of cellulose molded bodies according to the wet-dry extrusion method with high degree of gloss and bioactive action for use in the textile sector and paper production. In the context of the invention, the term "bioactive" refers to antimicrobial efficacy, based on the antibacterial action of the silver element, which is used as a nanoscalable reagent to increase their efficacy. It is chemically inert and, at the same time, has a bactericidal effect to be used in the production of sportswear and leisure with a high degree of brightness and papers with a prolonged useful life. It is possible to use in the medical sector, for example, for dressing, textiles for hospitals and in the packaging and filter industry.

3. Patent WO2012114045 (Arjo Wiggins Fine Papers Limited): "Methods for preparing paper pulp and for manufacturing paper from seaweed powder". The invention relates to a method for preparing paper pulp from seaweed powder. The method comprises at least the following steps: (a) the powder is prepared by drying seaweed or floating cellulose resulting from seaweed treatment at a temperature below 150 ° C until the seaweed or floating cellulose has a moisture content in the range of 1 to 20% by weight (b) grinding of the seaweed or floating cellulose in order to obtain a powder with a particle size in the range of 5 to 100 μιη (c) mix pre-cooked seaweed powder with water and with wood or plant cellulose fibers, the algae powder is at least 20% of the total mix. Said pulp can be used for the manufacture of paper that has a high opacity and a uniform surface and without inclusions.

Application CN101289821 (Liu Huimin): "Paper pulp and paper sheets made form brown algae and making method thereof". The invention discloses a method for the manufacture of paper pulp by using brown seaweed. The method comprises the following steps: (a) The brown seaweed is treated by acids diluted with sodium acetylide, in addition to other basic extractants (b) the sodium acetylide solution is added with other auxiliary agents which allows solidification of the pulp in gelatinous fibers (c) these fibers are converted into pulp for paper production by conventional methods.

Regarding products available in the market, focused on the established theme or projects that have related objectives, we can mention:

Sulphurous anhydride generator: The product is designed to prevent the appearance of cinematic Botrytis inside boxes for the transport of fresh table grapes. It is a device, which must be arranged between sheets of absorbent paper, composed of sheets of paper and polyethylene, between which sodium matabisulfite (Na ₂ S ₂ 0s) is inserted, which, when coming into contact with moisture, releases sulfurous anhydride (S0 ₂₎ in gas form. (Propel, 2016; Infruta SA, 2016).

FreshPaper: The product is a biodegradable and recyclable paper that absorbs moisture to keep products of vegetable origin, particularly fruits, for longer within domestic refrigeration systems (Fenugreen, 2016) 3. Sentinel: It is an international consortium focused on developing bioactive roles to combat diseases transmitted by bacteria present in foods capable of affecting the health of consumers. The development of paper towels is proposed to clean surfaces that are in contact with potentially contaminated food, which can also detect the presence of pathogenic bacteria (FIBRE, 2016).

 4. Itene Consortium: The Technological Institute of Packaging, Transport and Logistics of the European Union promotes sustainability in the fields of packaging, logistics, transport and mobility, highlighting the following projects: a) Project Plantpack seeks to develop a container to protect High-fat processed foods, such as sausages, cheeses and other pre-cooked foods, from the incorporation of agar, alginate and carrageenan obtained from seaweed and starch fibers to paper and cardboard, and b) Celubioactivepack Project: aims to develop materials Biodegradable multilayer with cellulosic base and biopolymeric coatings with antimicrobial and antioxidant capacity (ITENE, 2016).

Despite the presence of these technologies, there is still a great need for bioactive papers, which allow to protect the products in the transport and distribution stages.

Brief description of the figures Figure 1: Finished product. Figure 2: Comparison of antioxidant capacity. Figure 3: Comparison of antibacterial capacity. Figure 4: Comparison of antifungal capacity.

Figure 5: Comparison over time of the level of protection on apples.

Figure 6: Comparison of the protection level of apples in the laboratory.

Figure 7: Comparison of the level of protection of apples in industrial storage. Disclosure of the Invention

 The present technology corresponds to a paper with biologically active properties, made from renewable sources. The product can be used to protect fruits and vegetables against oxidative stress of the air, bacterial attack and mainly against phytopathogenic fungi, allowing the prevention of damage during storage and transport.

This bioactive paper comprises as a base an algal paper, which is composed of secondary cellulose fibers in combination with brown algae biomass, in addition to an antimicrobial extract also obtained from brown algae, which is added to the algal paper.

As base materials for the preparation of this paper, secondary cellulose fibers are used in combination with brown algae selected from Lessonia spicata, Durvillaea Antarctica, and Macrocystis pyrifera. The ratio used by both parties is from 3: 2 to 9: 1 (p / p). A bioactive mixture from the extracts of the brown algae Macrocystis pyrifera, Lessonia spicata and Dyctiota kunthii is added to the base material. To obtain this mixture of extracts, a pretreatment is carried out that involves cleaning, cold drying and grinding of each of these algae, then they are mixed in varying proportions between 10-20%, 25-40% and 30-60 %, respectively. Then, the compounds are extracted using an apolar solvent in a ratio between 1: 22 - 1:48 of biomass and solvent (w / v), then the extracts are concentrated at a concentration of 5-10 times

The papermaking process, from secondary fibers, comprises at least the following stages in accordance with the standard industrially established process, with some modifications: a. Obtaining reactive pulp: The first stage of production involves the formation of an initial pulp of secondary fiber and algal biomass, at a rate of 3: 2 to 9: 1 (w / w) and a large amount of hot water. This paste is introduced into a tank where it is rotated to release the fibers. b. Washing of the paste (destined): Optionally when the secondary fiber shows the rest of the impression, a destined one must be carried out, which involves multiple washes to extract 99% or more of the ink adhered to the fibers. For this, a fatty acid soap is applied inside the container containing the dirty paste. The soap releases the ink from the fibers, also applying compressed air from the bottom of the container, to generate soap bubbles that attract the released ink particles. These bubbles with adhered ink ascend to the surface of the water to form a dirty foam, which is removed. The procedure is repeated until the paste is completely purified. Some bleaching of the paste may be required to stabilize its whiteness to a uniform and constant level.

Admission box: The paste is 99% water and 1% fiber. Large volume of water is needed to prevent flocculation of the fibers. Otherwise, the sheet of paper will have poor formation. To avoid this, turbulence is generated in the intake box. The intake box distributes a controlled and regular flow of pulp to the next part of the paper machine to start forming the paper sheet.

Sheet formation: The stage takes place in a flat paper or double fabric machine, where the pulp suspension is moved from the intake box to the fabric section by a controlled and constant flow. The fabric is a mesh with fine holes in which the drainage of the suspension begins, which moves at approximately the same speed at which the suspension enters. To prevent the formation of a paper with two different faces, a second fabric processes the upper part of the suspension. The use of turbulence and aspiration favors the drainage of the upper side of the suspension, unifying the distribution of fibers and reducing the difference between the faces. The fabric section increases the degree of dryness from 1 to 16-19%.

Pressed paper sheet: The paper, with high water content, goes through a series of large steel rollers that compress it, expelling the water. The paper sheet is fastened between layers of absorbent felt as it passes between the rollers. The felt acts as a blotting paper in the absorption of water, while some vacuum boxes remove the water from the felt before meeting the paper sheet again. At the end of the pressing section, the degree of dryness is over 40-50%. F. Drying of paper sheet: To set the final degree of moisture in the paper, water is removed by evaporation. The drying stage includes a series of steam-heated cylinders on which the sheet of paper passes. The cylinders are arranged so that they first contact one side of the paper and then the other to ensure their homogeneous dehydration. g. Addition of the antimicrobial extract: To the leaves obtained the extract obtained previously is added through a bath by immersion, or by sprinkling, at low temperature, on both sides. Finally they are dried at low temperature and in darkness for 36-48 hours. The addition of the mixture of extracts in the final stage of the process is due to the fact that if they were incorporated during the initial mixing these extracts would affect the mechanical properties of the paper, and lose their bioactive properties by dilution. Industrially it is unlikely to have a large initial volume of these algae to reach the final concentrations required in the paper obtained with this process.

The papers obtained have physical-mechanical characteristics similar to those of other paper products, when determining their weight values are obtained between 36.7 to 42.67g nr ² , the specific volume has values between 1.81 and 1.86 cm ³ g ^_1 , the explosion index has values between 1.41 and 2.3, the tear index exhibits values between 5.6 and 7.2, finally the tension index showed values between 26.6 and 45.94. All values showed a low standard deviation reflecting that it is a mechanically homogeneous product. The main characteristic from a physical point of view is the homogeneity of the mixture, which allows it to obtain an optimal mechanical property of tension with a value greater than 30 which makes this product technically feasible to produce with the machinery currently available in the industry wastebasket with low modifications and without altering the process itself too much.

The main distinguishing feature on other existing products are their biologically active properties such as antioxidant capacity, activity against the growth of bacteria such as Escherichia coli, Pseudomonas aeruginosa and P. syringae, but above all it stands out in the ability to inhibit growth of the phytopathogenic fungi Botrytis cinerea, Alternaría altérnate and PenicMium genus. All these properties characterized by physical-mechanical, spectrophotometric and biological tests.

The paper used commercially today, sulfite paper, has no antioxidant capacity and has very low antimicrobial activity. The bioactive paper presented in this technology presents a synergy of activities that is not given by the paper alone, nor by the mixture of extracts.

By producing bioactive algal papers to protect fruits and vegetables from the attack of phytopathogenic microorganisms or against oxidative stress present in the air, this product contributes to non-traditional use of macroalgal biomass, diversifying its economic matrix, in addition the product itself promotes recycling when making use of secondary cellulose fibers for its elaboration. Finally, it is a 100% biodegradable material that does not present the restrictions of the plastic products used, for example, to protect export fruits.

Application examples

Example: Manufacture of bioactive algal papers.

The papermaking process comprises at least the following stages according to the standard established by Standards T 412 om-02, T 248 sp-00 and T 205 om-88 (TAPPI, 2008), with some modifications: a. Obtaining reactive pulp: First, the moisture content present in the algal biomass must be determined to be divided into particles with a size of 1 mm in length and then a paste of unrefined secondary fibers mixed with 10% humidity is mixed with pieces, in a ratio between 5: 1 (w / w), to form a mass of 30 g (dry weight) with the addition of water to

300 g, then a refining process begins with 8000 passes in a PFI mill. b. Paste washing (intended): in this case secondary fibers were used without printing, so it was not necessary to use them. C. Admission box: The rehydration stage of the mixture is started inside a helical homogenizer with a volume of 400 ml_ of water for 3 min. The semi-liquid mixture is transferred inside a rectangular container with a capacity for 10 L with constant agitation. Next, 270 mL liquid fractions are taken.

d. Sheet formation: The sheet formation process began with the filling of a metal cylinder with water, coupled to a sifted mold and a drainage system, which makes up the sheet-forming system. The aliquots of 270 are poured into the system in addition to 5 L of water with constant agitation to finally be drained. The newly formed wet leaf was covered by a circular section of virgin pulp and on the latter, 2-4 square sections of extra thick virgin pulp were used, exerting pressure with a metal uslero to remove excess water. and. Pressed paper sheet: The composite sheet is mounted and adjusted to the base of a press by installing a metal disk plus a square section of extra thick virgin pulp on it. The process is repeated achieving a set of 10 sequences of square section of virgin pulp, formed sheet and square section of virgin pulp again. The press is closed and the pressing process started at a pressure of 50 Ib x in ^{~ 2} , gradually reaching the correct pressure within the first 40 seconds, maintained for 5 min. At the end of the period the process was repeated by the reverse of the set of coupled sheets for 2 min at the same pressure with the same precaution. F. Drying of paper sheet: The components of the set from the press are separated and then transferred to the interior of ventilated metal rings, one on top of the other forming a drying tower which was left at room temperature between 24-36 h. Finally the sheets of algal paper are obtained. g. Addition of the antimicrobial extract: The leaves obtained are subjected to a bath by immersion with a 2 mL of macroalgal extract, previously obtained, for 5 min at low temperature, on both sides. Finally they are dried at low temperature and in darkness for 36-48 hours.

For a better understanding of the invention, the Figure is taken as a reference, which corresponds to the finished product and shows a sheet of paper based on a mixture of secondary fibers, algal biomass and algal extract, brown in color with the presence of small spots produced by algal biomass, with a diameter of 16 cm and a thickness of 72 pm.

Example 2: Characterization of bioactive algal papers.

 a) Determination of the physical-mechanical properties: The values were determined using standard equipment under Standards T 402 om-88, T 410 om-02, T 403 om-85, T 414 om-88 and T 494 om-01 ( TAPPI, 2008) inside a room heated to 23 ± 1 ° C with 50 ± 2% relative humidity. Prior to the measurements, the papers were acclimatized for 4 hours. The determined properties were; grammage, specific volume, explosion, tearing and tension, these last 3 were expressed as indexes, which allows comparisons with any other paper products. The importance of the tension value is highlighted as one of the most relevant factors when manufacturing papers, with a low value of 30 being deficient for a standard industrial process.

The values obtained by the paper developed were the following; For the grammage property, values with an average of 39.2 ± 2.1 g nr ^{2 were obtained} , the specific volume had an average value of 1.84 ± 0.01 cm ³ g ^_1 , the explosion index presented an average of 1.75 ± 0.23, the tear index exhibited an average of 6.17 ± 0.54, finally the stress index showed an average value of 39.33 ± 4.71. All values showed a low standard deviation reflecting that it is a mechanically homogeneous product, although visually it looks heterogeneous. The values achieved by the paper developed make its technical feasibility possible, since it has similar values to other products available in the market for similar applications. b) Determination of antioxidant capacity: This property was verified using the ABTS ' ⁺ cation radical method which consists in measuring the discoloration of the radical at a wavelength of 734 nm by means of a spectrophotometer (Dynamica, HALO SB-10 UV-Vis). The discoloration was expressed as the percentage of inhibition as a function of the concentration and the reactivity time of Trolox (6-hydroxy-2,5,7,8-tet-ramet¡chroman-2-carboxylic acid) used as standard . The values were expressed as Trolox Equivalent Antioxidant Capacity (TEAC). The results show that the algal extract has a lower antioxidant capacity than that obtained by the fruit sulphite paper with a TEAC of 0.005 ± 0.0018 and 0.0089 ± 0.0007 pmol of TE g ^_1 , respectively. Furthermore, the bioactive algal paper (paper algal extract algal more developed present a slightly higher value (TEAC of 0.0378 ± 0.002 pmol TE ¹ g paper) to paper without algal extract (TEAC 0.0353 ± 0 0011 pinol of TE g ^_1 of paper) The above demonstrates that algal paper without extract provides 93% of the antioxidant capacity of the final product, Figure 2 shows the values achieved, where the value obtained by the developed paper presents statistically differences significant (HSD Tukey p <0.001) with respect to the algal extract and especially in comparison with the fruit sulphite paper.This positive result demonstrates the potential use as a barrier capable of protecting fruits and vegetables against oxidative stress in the air. c) Determination of the capacity to nti bacteria na: a diffusion disc test was carried out in Petri dishes with Mueller-Hinton Agar culture medium and inoculated with 100 pL of bacterial culture at 10 ⁵ U FC mi ^-1 . Then, 6 mm diameter discs obtained from the developed paper, Gentamicin discs and fruit sulfite paper discs were arranged as control groups. The bacterial strains used were: Escherichia coli (K12), Pseudomonas aeruginosa (PA01) and Pseudomonas psyringae (DC300). Finally, the plates were incubated aerobically at 37 ° C ± 1 ° C for 36 hours.

The results show the presence of bacterial growth inhibition halos of diameter 6 to 10.15 mm. Figure 3 shows the specific results against the bacterial strains tested. The control carried out with fruit sulphite paper discs was able to control the adhesion of bacteria by not allowing its growth on its surface, however, it was not able to inhibit bacterial growth beyond its perimeter. On the other hand, the discs of the developed paper have halos of bacterial growth inhibition of 6.98 ± 1.06 mm with a maximum of 7.98 ± 1.35 mm for £ coli with highly significant differences with respect to sulphite paper ( HSD Tukey, p <0.001) and a minimum of 6.62 ± 0.31 mm against P. aeruginosa. The values observed in the control with the antibiotic Gentamicin had an average of 20.28 ± 0.79 mm in diameter. Notwithstanding the foregoing, the paper developed has an antibacterial capacity, although it is low compared to the control, it is statistically different and superior to that achieved by the fruit sulphite paper. For the Therefore, it promotes its use to protect fruits and vegetables susceptible to attack by these pathogens. d) Determination of antifungal ability: To demonstrate this application, a diffusion disc test was carried out on Petri dishes with Potato Dextrose Agar culture medium inoculated by dispersion. Then, 6 mm diameter discs obtained from the developed paper, Nystatin discs and fruit sulfite paper discs were arranged as control groups. The fungal strains used were: Botrytis cinerea (Bo C12), Penicillium sp. (RGM 902) and Alternaría altérnate (RGM 408). Finally, the plates were incubated aerobically at 25 ° C ± 1 ° C for 6 days. The results show the presence of fungal growth inhibition halos of diameter 7 to 19.3 mm. Figure 4 shows the specific results against the phytopathogenic fungi tested. The control performed with fruit sulphite paper discs did not show fungal growth inhibitory activity, since the discs were covered by fungi. The values obtained show that the paper developed has an antifungal capacity of 11.63 ± 1.39 mm with a maximum of 12.51 ± 1.04 reached against B. cinerea and a minimum of 10.43 ± 0.61 mm achieved against Penicillium sp., Therefore, promotes its use to protect fruits and vegetables susceptible to attack by these phytopathogens.

Example 3: Validation of the use of bioactive algal papers on fruit.

a) In vivo validation of papers developed in the laboratory: To demonstrate this application, an experiment was carried out with 100 export apples of the Royal Gala variety, with an average weight of 174.6 ± 7.5 grams, a pulp firmness of 14 , 6 ± 0.5 Ib and a sweetness of 11.4 ± 0.3 ° Brix. Four standard incisions were made to each block to facilitate the entry of opportunistic pathogens. The treatments consisted of 25 apples each arranged in independent trays, which were protected with fruit sulphite paper, algal paper, bioactive algal paper and unprotected apples as control. They were then stored in a box at room temperature 16 ± 1.6 ° C for 75 days. The results are presented in Figure 5 and show that the growth of pathogens as a function of time was evident after 45 days, indicating a greater infection in the treatment of apples with sulfite paper followed by apples without protection. On the contrary, the lowest infection was obtained in treatments with algal paper and bioactive algal paper.

After 75 days of incubation (Figure 6), the group most affected by the development of phytopathogenic microorganisms was the one protected by fruit sulphite paper showing an infection level of 54%, equivalent to a loss of apples of 92% under this treatment. On the other hand, unprotected apples showed an infection level of 34% equivalent to an apple loss of 80%. However, in apples protected by algal paper the level of infection was 25% equivalent to 60% of the fruits. Finally, apples protected by bioactive algal paper had the lowest level of infection with 13%, which translates into 7 fruits, equivalent to a 28% loss of total apples. The observed values are significantly different (HSD Tukey, p <0.004) and in favor of the use of the developed bioactive algal paper, which reduced the loss of apples by 70% compared to those packed with fruit sulphite paper. b) Validation in vivo under industrial storage conditions: To demonstrate this application, an experiment with 3600 export apples of the Royal Gala variety was mounted, which had an average weight of 197.8 ± 40.5 grams, a firmness of pulp of 14.6 ± 1.6 Ib, sweetness of 11.4 ± 0.8 ° Brix and 58.7 ± 17.7% of Red Color. Apples were grouped into 3 experimental groups: unprotected, protected with fruit sulphite paper and bioactive algal paper, which in turn were divided into 4 subgroups, obtaining 12 treatments with 4 replicates each. The treatments consisted of apples without intervention used to evaluate the microbial load of the garden, in others 2 standard incisions were generated on each of the apples to simulate the deterioration of the fruit by blows, in others the apples were inoculated with spores of Botrytis cinerea to evaluate the viability of the pathogen on the fruit and finally some included incisions and inoculum in order to promote the most favorable condition for the growth of the pathogen B. cinérea becoming the most adverse condition for apples (Table 1). The apples were arranged in trays for 25 units, using 3 trays per box, in a total of 4 boxes, completing 300 apples per treatment. The boxes were grouped together in a pallet and stored at 1.5 ± 1.5 ° C with a relative air humidity of 92.9 ± 1.6% for 90 days. Then the pallet was incubated at 13.2 ± 2.6 ° C with a relative humidity of 91.3 ± 2.7% air for 12 days to favor the growth of pathogens, completing 102 days of experimentation.

The analysis of the results, presented in Figure 7 show that the 12 treatments formed three statistically significant groups (A, B and C) depending on the level of infection observed in apples. The highest level of infection reached corresponds to group A (highly infected), which is composed of treatments 4 and 8 with an infection level of 38.3 ± 3.6% and 44.2 ± 2.5%, respectively . On the other hand, treatment 12 (bioactive algal paper), although it was subjected to the same procedures as those mentioned above (treatments 4 and 8, Table 1), presented a significantly lower infection level of 20.4 ± 2.1% , leaving it out of group A (ANOVA, HSD Tukey p <0.0002). On the other hand, treatments 2, 6 and 10, without inoculum, presented an infection level of 17.6 ± 2%, 22 ± 2.1% and 15.3 ± 1.8%, respectively, which were similar to those obtained by treatment 12 (ANOVA, HSD Tukey p> 0.005) forming group B. With these results, it was possible to affirm that the use of bioactive algal paper was able to successfully inhibit the growth of the phytopathogenic fungus B. cinerea to such an extent to achieve the same level of infection as that observed in those apples that only had incisions without inoculum. On the other hand, group C, composed of treatments 1, 3, 5, 7, 9 and 11 presented an average infection level of 2.9 ± 1.4%, demonstrating the importance of good handling in the fruit, because despite the inoculum applied in 3 of the 6 treatments, the phytopathogen was not able to proliferate because the apples used did not present wounds or blows, maintaining the integrity of the skin which constitutes a difficult barrier to overcome for the pathogens. The above was contrasted with treatments 2, 6 and 10 because they presented an increase in more than 6 times the level of infection observed in group C. Finally, it is concluded that the use of bioactive algal paper reduced by 54 % the level of infection of apples by B. cinérea compared to those apples protected with fruit sulfite paper (treatment 8). Table 1. Experimental design. Description of the treatments.

Claims

1. A CHARACTERIZED bioactive paper because it comprises as a base an algal paper, which is composed of secondary cellulose fibers in combination with brown algae biomass; and an antimicrobial extract, obtained from brown algae, which is added to the algal paper. 2. A bioactive paper according to claim 1, CHARACTERIZED in that the algal paper comprises secondary cellulose fibers in combination with brown seaweed biomass, selected from Lessonia spicata, Durvillaea antarctica, and Macrocystis pyrifera, where the ratio used of both parts is 3 : 2 to 9: 1 (p / p). 3. A bioactive paper according to claim 1, CHARACTERIZED in that the antimicrobial extract comprises extracts of the brown algae Macrocystis pyrifera, Lessonia spicata and Dyctiota kunthii, which are mixed in varying proportions between 10-20%, 25-40% and 30- 60%, respectively. 4. A bioactive paper making process, CHARACTERIZED because it comprises the following stages: to. Obtaining reactive pulp, from secondary fiber and algal biomass, at a rate of 3: 2 to 9: 1 (w / w) and a large amount of water; b. Optionally, when the secondary fiber shows the rest of the impression, a dewatering must be carried out; C. Admission box; d. Formation of sheet of algal paper; and. Pressed sheet of algal paper; F. Drying sheet of algal paper; and g. Addition of the antimicrobial extract. 5. A bioactive papermaking process, according to claim 4, CHARACTERIZED because the addition of the antimicrobial extract is through a dip bath, or by spraying, at low temperature, on both sides. 6. Use of bioactive paper, CHARACTERIZED because it is used to protect fruits and vegetables against oxidative stress of the air, bacterial attack and mainly against phytopathogenic fungi, allowing the prevention of damage during storage and transport.  7. Use of the bioactive paper according to claim 6 CHARACTERIZED because it has activity against the growth of bacteria such as Escherichia coli, Pseudomonas aeruginosa and P. syringae.  8. Use of the bioactive paper, according to claim 6 CHARACTERIZED because it has activity against the growth of the fungi Botrytis cinérea, Alternaría altérnate and of the genus Penicillium.