RO128966B1 - Biofilm with antimicrobial action, process for obtaining the same and uses thereof - Google Patents

Abstract

The invention relates to a film-type bioactive packing, meant for microbial reduction at the surface of the meat products from the "ready to consumption" category, to a process for obtaining the same and the use thereof during the products refrigeration period. According to the invention, the packing comprises, as a main ingredient, 2% chitosan combined with aqueous extracts of green tea and basil. As claimed by the invention, the process consists in mixing chitosan acidulated with glacial acetic acid, with aqueous plant extract, the mixture is sterilized at a temperature of 110°C for 10 min, it is poured into moulds which are dried at a temperature of 37°C for 2 h and stored.

Description

The invention relates to a range of bioactive film-type packaging, with antimicrobial activity, intended for the packing of food products from cams, of the type “ready for consumption, to the process of obtaining them, and to their use during the refrigeration period of the products.

In order to inhibit microbial development in foods ready for consumption, it is known that substances with antimicrobial action are added. The disadvantage of the solution presented is given by the antimicrobial substances, which can be reduced or inactivated due to the interaction of antibacterial compounds with food compounds (Appendix P, Hotchkiss JH., 2002, Review of antimicrobial food packaging. Innov. Food Sci. Emerg. Technol. 3 , 113-126; Neetoo H., Ye M. Chen H. 2007, The effectiveness and shelf-life of plastic films coated with nisin for inhibition of Listeria monocytogenes. J. Food Prot. 70,1267-1271; Takahashi H., Kashimura M., Miya S., KuramotoS., Koiso H., Kuda T., Kimura B. 2011, Effect of paired antimicrobial combinations on Listeria monocytogenes growth inhibition in ready-to-eatseafood products., Food Control 26, 397-400 ).

The antioxidant activity of films made from chitosan and green tea extract, together with the process of obtaining them, are known (Siripatrawan U., Map B. 2010., Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract. Hydrocolloids, 24: 770-775). Thus, it is shown that green tea, in combination with chitosan, has an antioxidant effect. The limitations of the presented solution are given by the impossibility of their use as antimicrobial films, because they are not sterile. Obtaining the film presented above does not consider the sterilization process, which would allow a faster solubilization, and a homogeneous mixing of the film components.

Antimicrobial activity of basil oil films is known (Zivanovic S., Chi S., Draughon AF. 2005., Antimicrobial activity of chitosan films enriched with essential oils. J Food Sci, 70 M45-M51). In this study, basil oil (for antimicrobial activity) is added later to the sterilization process. The technical limitations of the solution come from the fact that the antimicrobial activity is exerted by the aqueous extract of basil, which contains silver nanoparticles, which are easily linked to the chitosan base of the biofilm.

The technical problem proposed to be solved by the present invention consists in the production of bioactive film-type packages, efficient for reducing microbial germs by their action on the surface of meat preparations of the ready-to-eat type.

Bioactive film-type packaging eliminates the disadvantages mentioned above by using chitosan (a natural polymer, obtained by deacetylation of chitin, which is the major component of the crustacean exoskeleton) as a main ingredient, in a concentration of 2%, combined with plant extracts (tea green, basil) in different concentrations, thus avoiding the addition of additives obtained by chemical synthesis, and their interaction with the composition of the product.

A first object of the present invention is a biofilm with antimicrobial action, based on chitosan and green tea, which is obtained by mixing an aqueous plant extract, from green tea and basil, with chitosan acidified with 0.7% glacial acetic acid , the resulting mixture containing 4% green tea, 2% basil and 2% chitosan, this mixture being subsequently sterilized for 10 minutes at 110 ° C, poured into forms, dehydrated at 37 ° C for 2 hours, and then stored at maximum 25 ° C in plastic packages that do not allow their humidification.

RO 128966 Β1

Another object of the present invention is a process for obtaining biofilm 1 with antimicrobial action, consisting in the preparation of a mixture of aqueous plant extract, from green tea and basil, with chitosan acidified with 0.7% acetic acid glacial, mixes - 3 cloves, containing 4% green tea, 2% basil and 2% chitosan, being subsequently sterilized for 10 minutes at 110 ° C, poured into shapes, dehydrated at 37 ° C for 2 hours, and then stored at a maximum of 25 ° C in plastic containers that do not allow their humidification.

A third object of the present invention is the use of the biofilm with antimicrobial action, based on chitosan and green tea, for packaging food or as a constituent part of a packaging. 9

Bioactive film-type packaging comes in the form of a plastic mass that, applied to the surface of meat preparations of the ready-to-eat type, does not alter their sensory properties (taste, smell, color, consistency). Due to their consistency, these packages are easy to use. 13

The packages advantageously present the controlled release of bioactive substances extracted from plants with antimicrobial effect, over a longer period of time, and with action on the surface of the food.

The packages presented are made exclusively from nontoxic compounds, bio-degradable and biocompatible, using aqueous extracts from plants.

The innovative idea is highlighted by the range of bioactive film-type packaging, 19 which represents the alternative to the existing packaging, extending the stability of the product under refrigeration conditions, reducing the incidence of food poisoning through the consumption of type 21 "ready-to-eat foods, and using natural compounds. with antimicrobial activity.

In the present invention, the originality is due to the formulation of bioactive packaging of 23 types of chitosan film, containing biomolecules from plant extracts of plants with antimicrobial effects on meat products of the type "ready for consumption", stored in 25 refrigeration conditions.

The process for obtaining the biofilm is shown in FIG. 1. 27

The ground plants are infused in water at 100 ° C for 15 minutes, and the chitosan at 2% is acidified with 0.7% glacial acetic acid. The extract from 29 plants, filtered and centrifuged, is homogenized together with the acidified chitosan. The mixture was sterilized at 110 ° C for 10 minutes, then cooled and poured into forms. The mold-shaped mixture is dehydrated in the oven at 37 ° C for 4 hours. After the dehydration process, the formed biofilm is detached from the surface of the molds. The film is stored at 33 to 25 ° C in tightly sealed plastic packaging, until used.

A brief description of the biofilm production steps is presented in Table 1. 35

Table 1 37

Technological steps in obtaining the biofilm (1-8)

Technological steps in obtaining the biofilm (1-8)

Stage Name of the stage Stage I Selection and grinding of dried plants Stage II Obtaining the clear extract from the plants, through successive settling and filtration Stage III Obtaining the mixture of chitosan acidified with glacial acetic acid Stage IV Homogenization of plant extract with acidified chitosan with glacial acetic acid Stage V Sterilize the mixture at 110 ° C for 10 min Stage VI Thin coating of the mixture into forms Stage VII Dehydration of the mixture Stage VIII Storage of the biofilm until its use for packaging

RO 128966 Β1

Examples of biofilm production are given below.

Example 1. Chitosan biofilm 2%

Prepare 100 ml of wet mixture, of which 10 biofilm disks of size 18 cm ^{2 are} weighed, weighing 2.7 g. Mix 2 g acidified chitosan with 0.7 ml of glacial acetic acid, and make up to sign (100 ml) with distilled water. According to the scheme of FIG. 1, the mixture is sterilized at 110 ° C for 10 minutes, then poured into plates of 9 cm diameter, in the form of a disc. The plates are exposed to the dehydration process in the oven at 37 ° C for 4 hours. From the surface of the plates the formed disks are detached and stored at temperatures below 25 ° C, and humidity of up to 40%, until its use. .

Example 2. Chitosan biofilm with green tea extract 2%

Prepare 100 ml of wet mixture, of which 10 biofilm disks of size 18 cm ² , with a weight of 2,706 g are produced. The green tea extract is obtained by infusing 2 g of dehydrated green tea in 70 ml water at 100 ° C, for 15 min. The extract was filtered and then mixed with 2 g of chitosan acidified with 0.7 ml of glacial acetic acid, and made (100 ml) with distilled water. According to the scheme of FIG. 1, the mixture is sterilized at 110 ° C for 10 minutes, then poured into plates of 9 cm diameter, in the form of a disc. The plates are exposed to the dehydration process in the oven at 37 ° C, for 4 hours. The formed disks are detached from the surface of the plates and stored at temperatures below 25 ° C, and humidity of up to 40%, until their use. .

Example 3. Chitosan biofilm with green tea extract 4%

Prepare 100 ml of wet mixture, of which 10 biofilm discs of size 18 cm ² , with a weight of 2,712 g are made. The green tea extract is obtained by infusing 4 g of dehydrated green tea in 70 ml at 100 ° C, for 15 min. The extract was filtered and then mixed with 2 g of chitosan acidified with 0.7 ml of glacial acetic acid, and made (100 ml) with distilled water. According to the scheme of FIG. 1, the mixture is sterilized at 110 ° C for 10 minutes, then poured into plates of 9 cm diameter, in the form of a disc. The plates are exposed to the dehydration process in the oven at 37 ° C for 4 hours. From the surface of the plates the formed discs are detached and stored at temperatures below 25 ° C and humidity of up to 40%, until the time of their use.

Example 4. Chitosan biofilm with green tea extract 4% and basil 2%

Prepare 100 ml of wet mixture, of which 10 biofilm disks of size 18 cm ² , weighing 2.88 g are produced. The extract of green tea and basil is obtained by infusing 4 g of dehydrated green tea and 2 g of dehydrated basil in 70 ml of water at 100 ° C for 15 min. The extract was filtered and then mixed with 2 g of chitosan powder acidified with 0.7 ml of glacial acetic acid, and made (100 ml) with distilled water. According to the scheme of FIG. 1, the mixture is sterilized at 110 ° C for 10 minutes, then poured into plates of 19 mm diameter, in the form of a disc. The plates are exposed to the dehydration process in the oven at 37 ° C, for 4 hours. From the surface of the plates the formed disks are detached and stored at temperatures below 25 ° C, and humidity of up to 40%, until their use. .

The explanation given below is mentioned below. 1 ... 5.

FIG. 1, the technological scheme for obtaining the biofilm;

FIG. 2, spectrophotometric fingerprints specific to plant extracts used in the production of biofilms from examples 2 ... 4;

FIG. 3, chromatographic imprints specific to plant extracts used in the production of biofilms from examples 2 ... 4;

FIG. 4, FTIR fingerprints specific to biofilms presented in examples 1 ... 4;

FIG. 5, the inhibitory effect of the biofilms presented in Examples 1 ... 4, on the growth of L. monocytogenes during the refrigeration period.

RO 128966 Β1

Characterization of the extracts used to make the biofilm 1

In order to recognize the biochemical profile of the extracts used, the UV-Vis molecular spectra were recorded, highlighting the maxima specific to the phenolic compounds (Ă = 280 nm) presented in fig. 2.

High performance liquid chromatography (HPLC) highlighted the specific footprints 5 of the extracts used to make the biofilms presented in examples 2 ... 4 (fig. 3), with the highlighting of phenolic compounds (Ă = 280 nm) such as the catechins presented in the table. 2. 7

The concentration of catechins identified increases progressively in the extracts used in Examples 2 ... 4, as the content of plant extracts increases. 9

The phenolic compounds identified in the extracts used to make the biofilms in examples 2 ... 4 are presented in the following table. 11

Table 2 13

Nr. signal Phenol compound name ic Retention time (t _R ) Mg content Example 2 Example 3 Example 4 1. Galocatechina 5.76 1.8 3.2 3.4 2. catechins 11.150 4.4 8.2 9.02 3. epigallocatechin 12.12 4.3 8.6 8.89 4. Galocatechingalat 17.660 0.35 0.71 0.72 5. Epicatechingalat 20.75 1.87 3.52 3.64 6. Catechingalat 21.6 0.1 0.3 0.4

By IR spectroscopy, the following specific bands for O-H groups were identified in the biofilms presented in Examples 1 ... 4: 2800 ... 2900 cm ^-1 ; the presence of polysaccharides is identified by the bands from 1155 ... 1032 cm ' ¹ . Another absorption band was identified at 25 1598 cm ¹ , characteristic of the presence of the amino group (-NH ₂ ). The bands from 1654 cm ' ¹ and 1317 cm' ¹ are characteristic of amide I and amide II, specific for the complete non-acetylation of chitosan. 27

Antimicrobial capacity of biofilms

Experimental studies 29 In order to demonstrate the antimicrobial capacity of the biofilms presented above, experimental studies were performed in triplicate for each type of biofilm presented in 31 examples 1 ... 4. The antimicrobial capacity exerted by biofilms was determined on the “ready-to-eat” type product, that is, on the roast pork in vacuum packaged for 33 weeks. Initially, 12 cm ² pieces of steak were inoculated with 3 * 10 ³ CFU / cm ² of L. monocytogenes. The samples were packed in biofilms and then subjected to packaging in 35 nylon / polyethylene using the vacuum packing apparatus. The products were stored at 4 ° C for 8 weeks, taking samples each week, to quantify the density of L. monocytogenes.

Results 39

According to FIG. 5, the control sample (packaging without biofilm) within 8 weeks of inoculation reaches double its microbial load. In the case of the biofilm weighed in Example 1 (packaged with chitosan biofilm), the efficiency is higher in the first 6 weeks, followed by an increase in bacterial density in weeks 7 and 8. In the case of biofilms 43 presented in Examples 2, 3, 4, which use plant extracts as microbial inhibitors, the best antilisteric action is presented, leading to half the initial load. 45

RO 128966 Β1

By applying the invention, the following advantages are obtained:

- microbial reduction in "ready for consumption" type cam products;

- avoiding the use of chemical synthesis substances as antimicrobial additives;

- biodegradability of packaging;

- antimicrobial action on the surface of the products;

- the incidence of food poisoning.

Claims (3)

1. Biofilm with antimicrobial action, based on chitosan and green tea, characterized in that it is obtained by mixing an aqueous plant extract, from green tea and basil, with chitosan acidified with 0.7% glacial acetic acid, the resulting mixture containing 4% 5 green tea, 2% basil and 2% chitosan, being subsequently sterilized for 10 minutes at 110 ° C, poured into forms, dehydrated at 37 ° C, for 2 hours, and then stored at a maximum of 25 ° C , in plastic packages 7 that do not allow their humidification. 2. Process for obtaining the biofilm with antimicrobial action, based on chitosan 9 and green tea, characterized in that a mixture of aqueous plant extract, green tea and basil, is prepared, with 0.7% acidified chitosan glacial acetic, the resulting mixture 11 containing 4% green tea, 2% basil and 2% chitosan, being subsequently sterilized for 10 minutes at 110 ° C, poured into forms, dehydrated at 37 ° C, for 2 hours, and then stored maximum 13 25 ° C in plastic containers that do not allow their humidification. The use of the biofilm defined in claim 1, for packing food or as a constituent part of a package.