PL212125B1 - Biopolymer biocomposite of antimicrobial activity - Google Patents

Description

The subject of the invention is a biopolymer biocomposite with antimicrobial activity, containing ingredients used in the production of food and / or drugs and / or cosmetics.

The biocomposite according to the invention can find applications in the food, pharmaceutical or cosmetic industries, especially for encapsulating and producing edible protective coatings.

The purpose of using biologically active substances as food additives or applying them in the form of bioactive protective coatings is to ensure a high standard of quality, freshness and food safety, in conjunction with pro-ecological measures in packaging. The beginnings of using natural protective coatings date back to 3000 BC. and are still invariably popular, e.g. the digestive tract of slaughter animals, waxes, mainly due to the ability to pass water vapor, fragrances and gases, as well as plastic features related to adapting to the shape of the product. However, the susceptibility of food raw materials to, for example, microbiological contamination, mechanical damage, as well as dimensional limitations, are the reasons why casings and packaging made of synthetic materials are currently gaining such wide application. Artificial casings can be made of synthetic and natural raw materials. Traditional packaging materials, such as polypropylene, polystyrene or polyamides, are produced by low-pressure polymerization of propylene, styrene or made of coal, natural gas or crude oil. These substances can be a source of health problems related to the migration of harmful plastic components both to the product and the human body, and additionally increase the pool of hard-to-degradable waste that threatens the natural environment.

Nowadays, the technologies of producing protective coatings, in the form of edible films formed directly on the surface of raw materials or products, based on such natural substrates as proteins, polysaccharides or lipids, with the addition of a number of preservatives, are of great interest (Cutter CN, 2002. Incorporation antimicrobials into packaging materials.

AMSA Rec. Meat Conf. Http://www.meatscience.org/pubs/proceedings.htm; Dawson P., Acton J. Ogale A., 2002. Biopolymer films and potential applications to meat and poultry products. AMSA Reciprocal Meat Conference http://www.meatscience.org/pubs/proceedings.htm).

Among the proteins used to form the coatings, the most commonly used are: collagen, gelatin, wheat gluten, soy proteins, whey proteins and zein. On the other hand, polysaccharide films are obtained from derivatives of cellulose and agar, carrageenan, pectins, alginates, and chitosan. The coatings can also be formed from beeswax, camauba wax and triacylglycerols. The latter are also used as substances increasing the hydrophobicity and reducing the vapor permeability of protective films (Debeaufort F., Quezada-Gallo JA, Voilley A., 1998. Edible films and coatings: tomorrow's packagings: A review. Critical Reviews in Food Science, 38: 299 -313). The design of next-generation edible coatings and films is to increase their functionality by introducing natural or chemical substances with antimicrobial or antioxidant activity, functional ingredients or enzymes that have a probiotic effect, are vitamins and / or minerals (Bifani V., Ramirez C ., Ihl M., Rubilar M., Garcia A., Zaritzky N., 2007. Effects of murta (Ugni molinae Turcz) extract on gas and water vapor permeability of carboxymethylcellulose-based edible films. LWT-Food Science & Technology, 40 : 1473-1481; Vargas M, Pastor C, Chiralt A, McClements DJ, Gonzάlez-Martinez C., 2008. Recent advances in edible coatings for fresh and minimally processed fruits. Critical Reviews in Food Science and Nutrition 48: 496-511) . Bacteriocins are commonly used, e.g. Iaktocin (Scannell AGM, Hill C., Ross RP, Marx S., Hartmeier W., Arendt EK, 2000. Development of bioactive food pack-aging materials using immobilized bacteriocins lacticin 3147 and nisaplin. International Journal. of Food Microbiology, 60: 241 - 249), pediocin (Santiago-Silva P., Soares NFF, Nóbrega JE, Jwiior MAW, Barbosa KBF, Volp ACP, Zerdas ERMA, Wϋrlitzer NJ, 2009. Antimicrobial efficiency of film incorporated with pediocin ( AL-TA®2351) on preservation of sliced ham. Food Control, 20: 85-89), nisin (Sanjurjo K., Flores S., Gerschenson L., Jagus R., 2006. Study of the performance of nisin supported in edible films. Food Research International, 39: 749-754) and enzymes such as: chitinase, glucose oxidase (Suppakul P., Miltz J., Sonneveld K., Bigger SW 2003. Active packaging technologies with an emphasis on antimicrobial packaging and its Applications. Journal of Food Science, 68: 408420), and lactoperox ydaza (Mecitoglu ę „Yemenicioglu A., 2007. Partial purification and preparation

PL 212 125 B1 of bovine Iactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films. Food Chemistry, 104: 726-733).

Lactoperoxidase has both antimicrobial and antioxidant properties and is localized in the nipples, salivary glands, tear glands of mammals and their secretions (Seifu E., Buys EM, Donkin EF, 2005. Significance of the lactoperoxidase system in the dairy industry and its potential applications: a review. Trends in Food Science and Technology, 16: 137-154). This enzyme has a killing effect against Gram (+) and Gram (-) bacteria (Kussendrager KD, van Hooijdonk ACM, 2000. Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications. British Journal of Nutrition, 84: 19-25). Many natural substances, commonly known as flavors, are also bacteriostatic and have been used successfully in protein and polysaccharide coatings. These are essential oils isolated from oregano, rosemary and garlic (Seydim AC, Sarikus G., 2006. Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and essential garlic oils. Food Research International, 39: 639-644) and oils obtained in various concentrations from lemongrass or cinnamon, (Rojas-Graϋ MA, Avena-Bustillos RJ, Friedman M., Henika PR, Martin-Belloso O., McHugh TH, 2006. Mechanical, barrier, and antimicrobial properties of apple puree edible films containing plant essential oils. Journal of Agricultural and Food Chemistry, 54: 9262-9267).

There are several methods of producing films with modified cellulose and chitosan with good physicochemical properties (Sebti I., Chollet E., Degraeve P., Noel C., Peyrol E., 2007. Water sensitivity, antimicrobial, and physiccochemical analyzes of edible films based on HPMC and / or chitosan. Journal of Agricultural and Food Chemistry, 55: 693-699; De Moura MR, AvenaBustillos RJ, Mchugh TH, Krochta JM, .Mattoso LHC, 2008. Properties of novel hydroxypropyl methylcellulose films containing chitosan nanoparticles. Journal of Food Science, 73: N31-N37) and antimicrobial (Molier H., Grelier S., Pardon P., Coma V., 2004. Antimicrobial and physicochemical properties of chitosan-HPMC based. Journal of Agriculture and Food films Chemistry, 52: 6585-6591; Park SI, Stan SD, Daeschel MA, Zhao Y., 2005. Antifungal coatings on fresh strawberries (Fragaria x ananassa) to control mold growth during cold storage Journal of Food Science, 70: M202-M207; Sebti, I., Cho llet E., Degraeve P., Noel C., Peyrol E., 2007. Water sensitivity, antimicrobial, and physicochemical analyzes of edible films based on HPMC and / or chitosan. Journal of Agricultural and Food Chemistry, 55: 693-699).

The use of chitosan and nanosilver was also analyzed in terms of antimicrobial properties (Rhim JW, Hong SI, Park H.-M., Ng PKW, 2006. Preparation and characterization of chitosan-based nanocomposite with antimicrobial activity. Journal of Agricultural and Food films Chemistry, 54: 5814-5822), as well as the synergistic protective effect of chitooligosaccharides and lysozyme on meat spoilage (Rao MS, Chander R., Sharma A., 2008. Synergistic effect of chitooligosaccharides and lysozyme for meat preservation. Food Science and Technology, 41: 1995-2001).

There is known a method (application WO2008036243) relating to the production of lysozyme-chitosan films intended for use in food for the purpose of antimicrobial protection.

Patent description CN101496534 describes a method of inhibiting the deterioration of chilled meat by soaking or spraying it with a mixture of composites with antimicrobial properties, containing from 0.01 to 0.02 parts of lysozyme, from 0.005 to 0.01 parts of chitosan and 0.001 to 0.002 parts of nisin. . The treated meat is then vacuum packed and subjected to a pressure treatment with pressure parameters ranging from 100 MPa to 180 MPa for 5-15 minutes.

Japanese patent JP4267867 describes the composition of a mixture which protects food against deterioration, which is characterized by the content of the following substances: vitamin E, chitin, chitosan and its hydrolysates or modifications, chitosan oligosaccharides, pectin hydrolysates, lysozyme, etc. These substances were added to ethanol and organic acids, such as acetic acid, lactic acid, citric acid, malic acid.

The Czech patent description 293987 presents a method of producing biologically active packaging based on a polymer immobilizing protective substances such as: antibiotics and / or silver compounds and / or phenol derivatives and / or bacteriocins and / or p-hydroxybenzoic acid esters and / or monoglycerides fatty acids and / or benzoic acid and / or sorbic acid and / or chitosan and / or 2,2,4-trichloro-2-hydroxyphenyl ether and / or lysozyme and / or nisin and / or hexamethylenetetramine.

From the application No. WO2006065581, a composition of nanoparticle silver and at least one of the following substances is known: silver salt of zinc oxide, chitosan or chito derivatives4

It can be used as a biocide or in the production of a preparation with a biocidal effect.

Japanese patent specification 63036767 describes the use of aqueous solutions of natural fibers such as cellulose, chitin, chitosan and other and polyhydroxy compounds such as monosaccharides, oligosaccharides, polysaccharides, pullulan, gelatin, pectin for the production of edible coatings.

A significant shortcoming of the solutions described above using biopolymers to protect organic substances and food, nutritional products or drugs consumed by humans against deterioration is the limited spectrum of antimicrobial activity determined by the recipe composition and physicochemical properties that are difficult to modify.

The invention relates to a multi-component biocomposite with antimicrobial activity produced on the basis of natural biopolymers, containing in its composition an aqueous solution in which additional substances used in the production of food and / or drugs and / or cosmetics may be dissolved, complementarily modifying such features, properties and functions of the biocomposite, such as: the ability to inhibit the growth of microorganisms, the rate of release of biologically active substances, viscosity, adhesiveness, structure, strength, flexibility, barrier, color, taste, smell, solubility, storage stability, reducibility, etc.

The essence of the invention is a biopolymer biocomposite that contains hydroxypropyl methylcellulose (HPMC) in an amount of 0.1 to 50% by weight, a plasticizer, especially glycerin, in an amount of 0.025 to 20% by weight, protein substances, especially lysozyme and / or cystatin, in an amount from 0.01 to 20% by weight and nanoparticle non-ionic form of colloidal silver, in an amount from 1 to 50 ppm, and the remainder is an aqueous solution in an amount that varies and depends on the degree of water immobilization and / or dehydration of the biocomposite, respectively the result of its immobilization in the spatial lattice which is formed in the sol or gel by the used biopolymers and / or the degree of drying of the structured form of the biocomposite, which degree depends on the equilibrium humidity and the time of its holding.

Preferably, the aqueous solution contains non-hydrated chitosan forms and / or its salts and / or its modifications and / or its hydrolysates and / or its oligomers or these forms dissolved in organic acids in an amount of 0.1 to 50% by weight, especially in lactic or acetic acid and or their mixtures, at a concentration of 1 to 20% by weight,

It is also preferred that the aqueous solution contains bacteriocins of microbial origin in an amount from 1 ppm to 1000 ppm or phytoncides, oleoresins, extracts, isoIates of plant origin in an amount from 1 ppm to 5000 ppm.

The aqueous solution may also contain cross-linkers, dyes, flavors, including smoke preparations and chemical preservatives, or reducing agents and antioxidants, in amounts ranging from 1 ppm to 5,000 ppm.

The use of hydroxypropyl methylcellulose (HPMC) biocomposites as a base substance for the production of biodegradable, biologically active biocomposites creates the possibility of obtaining completely new and different solutions, in relation to the previously known multicomponent preparations.

This is possible thanks to the specific properties of hydroxypropyl methylcellulose, which include, among others:

• biodegradability and biocompatibility, • transparency and sensory neutrality, • resistance to endogenous enzymes of the human gastrointestinal tract, • shielding to protein substances, • stability of physicochemical properties in a wide gradient of active acidity and heat energy delivered or received, • no toxicity.

Unexpectedly, it turned out that thanks to the use of HPMC as a biocomposite matrix, a number of substances can be incorporated into its composition, complementary modifying, among others: the ability to inhibit the growth of microorganisms, the release rate of biologically active substances, viscosity, adhesiveness, structure, mechanical strength, flexibility, barrier, color, taste, smell, solubility, storage stability, reducibility.

In addition, the use of colloidal silver nanoparticles in the composition of the biocomposite significantly extends its spectrum of antimicrobial activity.

PL 212 125 B1

The invention uses substances and ingredients approved for human consumption, which makes the use of a biocomposite in a form permanently integrated with a food product or drug in the form of a coating or a protective coating, edible and non-toxic.

The following non-limiting examples illustrate the subject of the invention.

Example 1. A 5% aqueous solution of hydroxypropyl methylcellulose (HPMC) is prepared. Then it is diluted to the final value of 0.5% with water, additionally adding to the solution an aqueous solution of lysozyme with an activity of 2000 U / mg of protein, silver nanocolloid, lactic acid and glycerin in such amounts as to the concentration of lysozyme, silver colloid and of lactic acid, in the final form of the biocomposite sol, it was 1%, 1 ppm and 1%, respectively, and glycerol 25%, based on the dry weight of the biopolymers.

The obtained biocomposite can be applied in the form of a sol directly on the surface of the protected product, or poured onto a plate covered with Teflon, where, after drying, it transforms into a structured form of a protective coating in the form of a film. Such coatings or coatings can be applied, with the use of known technological devices and operations, directly to the surface of products of the food, pharmaceutical or cosmetic industry. Such a film has a water vapor permeability of 7.6E-10 g / m * s * Pa and a free radical scavenging capacity of about 82%. Furthermore, it completely inhibits the growth of Bacillus cereus and reduces the growth of Micrococcus Aavus by 3 log orders, and inhibits the growth of Aspergillus niger in 40% of the non-grown Petri dish surface.

Example 2. Aqueous solutions of hydroxypropyl methylcellulose (HPMC) and lysozyme and nisin with concentrations of 5%, 10% and 1%, respectively, and a 4% solution of chitosan in 2% lactic acid are prepared. Additionally, nanosilver colloid (50 ppm concentration), glycerin and water are used to prepare the biocomposite sol. Then, the prepared solutions are mixed together in such proportions and amounts that the sol of the finished biocomposite contains: 0.5% HPMC, 1% chitosan, 1% lysozyme, and 25% glycerol in relation to the dry weight of biopolymers.

A biocomposite of such composition fully inhibits the growth of both Gram (+) bacteria of the genus Bacillus cereus, Micrococcus Aavus and Gram (-): of the genus Escherichia coli, Pseudomonas Auorescens. It can be used to encapsulate the ingredients and to protect the food, pharmaceutical or cosmetic industry products against microbial growth with the use of known technological equipment and operations.

Example 3. A water solution of hydroxypropylmethylcellulose with a starting concentration of 5% and an acidic chitosan solution with a concentration of 4% are prepared. The solutions are mixed in appropriate proportions with the addition of distilled water, a solution of bacteriocins and glycerol, to the final concentrations of: 0.5% hydroxypropyl methylcellulose, 2% chitosan, 1% acetic acid, 20 ppm nisin and 25% glycerol based on the dry weight of biopolymers.

The sol-shaped biocomposite is characterized by the complete reduction of Gram (+) bacteria: Bacillus cereus, Micrococcus fluorescens, Gram (-) bacteria: Escherichia coli, Pseudomonas fluorescens and Candida famata.

Example 4. A 5% aqueous solution of hydroxypropyl methylcellulose, 10% lysozyme with an activity of 2000 U / mg protein is prepared. These solutions are mixed and diluted with water, rosemary extract and glycerol to final concentrations of 0.5% and 0.5%. However, the final concentration of the extract in the sol is diluted to 1000 ppm, and glycerol to 25% based on the dry weight of the polymers.

Such sol applied to meat that has been refrigerated for 4 weeks is characterized by a reduction of microorganisms by 0.2 logarithmic per 1 g of meat. Such a coating, 15 mm long, 7 mm wide and 0.05 mm thick, has a tensile strength of 3.1 MPa.

Example 5. An aqueous solution of hydroxypropyl methylcellulose, a solution of chitosan in lactic acid, is prepared, mixed in appropriate proportions with trasglutaminase and silver nanocollide and filled with water so that the final biocomposite contains the following concentrations of the components mentioned: 0.5% hydroxypropyl methylcellulose, 2 % chitosan, 1% lactic acid, 3000 ppm transglutaminase, 2 ppm silver nanocolloid and 25% glycerol based on the sum of the dry weight of all polymers.

This biocomposite completely inhibits the growth of such bacteria as: Bacillus cereus, Micrococcus fluorescens, Escherichia coli, Pseudomonas fluorescens, as well as Candida famata and Aspergillus niger fungi by 77%.

PL 212 125 B1

Example 6. A solution of hydroxypropylmethylcellulose with a starting concentration of 5% is prepared, diluted with distilled water and the remaining components to a concentration of 0.5%. The remaining components of the system are added in such amounts and concentrations that in the end they constitute lysozyme - 0.5%, ascorbic acid 0.05% and 25% glycerol based on the dry weight of biopolymers.

A biocomposite with such composition inhibits the growth of Aspergillus niger in 86% and has the ability to sweep away

Claims (9)

1. Biopolymer biocomposite with antimicrobial activity, containing biopolymer biocomponents, plasticizer, protein substances and nanoparticle non-ionic form of colloidal silver, characterized in that it consists of hydroxypropyl methylcellulose in an amount from 0.1 to 50% by weight, a plasticizer in an amount from 0.025 to 20% by weight, protein substances in an amount from 0.01 to 20% by weight and a nanoparticle non-ionic form of colloidal silver in an amount from 1 to 50 ppm and the remainder is an aqueous solution in an amount that varies and depends on the degree of water immobilization and / or dehydration of the biocomposite, respectively its immobilization in the lattice formed in the sol or gel by the used biopolymers and / or the degree of drying of the structured form of the biocomposite, which degree is determined by the equilibrium humidity and the time of its maintenance. 2. Biocomposite. according to p. The process of claim 1, wherein the plasticizer is glycerin. 3. The biocomposite according to claim The method of claim 1, characterized in that the protein substance is lysozyme and / or cystatin. 4. The biocomposite according to claim A method according to claim 1, characterized in that the aqueous solution contains non-hydrated chitosan forms and / or its salts and / or its modifications and / or its hydrolysates and / or its oligomers or these forms dissolved in an acid selected from the group consisting of lactic acid or acetic acid and / or mixtures thereof in an amount from 0.1 to 50% by weight. 5. The biocomposite according to claim 1 A process as claimed in claim 1, characterized in that the aqueous solution contains bacteriocins of microbial origin in an amount from 1 to 1000 ppm. 6. A biocomposite according to claim A method according to claim 1, characterized in that the aqueous solution contains biologically active substances from the group of phytoncides, oleoresins, extracts, isolates of plant origin, in an amount from 1 ppm to 5000 ppm. 7. The biocomposite according to claim The process of claim 1, wherein the aqueous solution contains cross-linkers, dyes, flavors, including smoke preparations and chemical preservatives, in an amount from 1 ppm to 5000 ppm. 8. A biocomposite according to claim The process of claim 1, wherein the aqueous solution contains organic acids in an amount of from 1 to 20% by weight. 9. The biocomposite according to claim 1 A process as claimed in claim 1, characterized in that the aqueous solution contains substances with reducing and antioxidant properties.