WO2015088310A1 - Plant-based disinfectants for apples (malus domestica) - Google Patents

Abstract

The invention relates to compositions for effectively disinfecting and/or preserving fruit and vegetables contaminated by pathogenic and destructive micro-organisms. The aforementioned compositions contain plant-derived extracts or compounds with antimicrobial activity, which can act either alone or combined with other disinfectant agents, such as, for example, organic acids and chlorine compounds, and surfactants, such as polysorbate 80. The compositions of the invention can eliminate or inactivate microbial contamination of apples, including by pathogenic micro-organisms, without altering the nutritional and/or dietary and/or sensory properties thereof.

Description

 DISINFECTANTS FOR APPLE and Malus domestica) BASED ON PLANTS

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the development of formulations containing compounds with antimicrobial activity present in plants and which are used as disinfectants and preservatives for food, for example of plant and animal origin; more particularly to aqueous formulations based on extracts of Jamaican chalices {Hibiscus sabdariffa), the method by which it is obtained and its uses as an effective formulation to eliminate pathogenic bacteria from plant-based foods, such as fruits and vegetables, but with the most High effectiveness for Apple (Malus domestica).

STATE OF THE TECHNIQUE

 Apple {Malus domestica) is an agricultural product widely consumed throughout the world. In Mexico it is one of the main agricultural products, with a little more than 60 thousand hectares dedicated to its sowing. The most important types of Apple that are sown in Mexico, both in the open field and in protected agriculture, are: Golden Delicious (the one that is most produced), Red Delicious, Creole, Red Chief, Rome Beauty, Starking, Starking Delicious and Top Red (Sagarpa, 2013).

However, it has been reported that there are at least 5,000 to 20,000 varieties / cultivars of apples that are grown worldwide (Pijpers et al., 1986), and from among these the main ones are: Akane, Ambrosia Arkansas Black, Blackjohn Braeburn, Bramley, Carneo, Cortland, Cox's Orange, Pippin, Crabapple, Criterion, Egremont Russet, Empire, Esperiega, Fuji, Gala, Ginger Gold, Granny Smith, Gravenstein, Honeycrisp, Idared, Jazz, Jonagold, Jonathan , Lodi, Mclntosh, Newtown / Pippin, Oid Apple, Pacific Rose, Pink lady, Pinova, Red El, Reineta, Rome Beauty, Royal Gala, Splendor, Spur, Starkrimson, Starking, Maiden Green, Willie Sharp, Winesap, Winter Banana, Worcester, Permain, among many others (Pijpers et al., 1986). However, along with the increase in Apple consumption worldwide, there have been outbreaks of diseases caused by bacteria associated with the consumption of apples or their raw products (CDC, 2005). For example, in 1999, unpasteurized apple juice was the vehicle in a multi-state outbreak, that is to say that it affected several states in the American union, the microorganism involved in this outbreak was Escheríchia coli 0157: H7 (CDC, 2005) . A little more 50 outbreaks of disease of microbial ethology have been reported in the United States of North America (USA) by the consumption of raw apple juices.

This type of sprout has caused strict regulation inside and outside the USA for all apple and unpasteurized juice producers, and of course, also for all apple producers that export to the USA or other parts of the world such as Mexico. All this has meant that apple shipments are frequently retained at the borders, partial or total closure of the export of this product to the countries and economic losses by the producers when they do not comply with microbiological standards.

It should be noted that although in Mexico there are no reports of disease outbreaks of microbial etiology associated with the consumption of whole raw apples or their raw products such as juices, due to poor hygiene practices that generally occur during cultivation, harvest, transport and Marketing of Apples, is to despair the participation of these raw products in disease outbreaks. A fact that supports this observation is the frequency with which Salmonella strains, pathogenic groups of Escheríchia coli, and other pathogenic bacteria have recently been isolated from different raw products in Mexico (Castañeda-Ramírez et al., 2011 ; Castro-Rosas et al., 2012). Recent outbreaks of food diseases associated with apple and by-products created the need to determine the sources of apple contamination and understand the survival and / or growth of pathogenic microorganisms in it; These have led to the development of innovative control technologies. In general, the pathogens in apples could be controlled by preventing contamination during the cultivation and harvest of the products, also by using disinfectants with antimicrobial power in the harvested product, and by storing the apple at low temperature. However, disinfection has been identified as the most important stage for the microbial safety of raw apples.

Usually apples are not consumed directly as they are harvested. After harvesting either in the field or in the industry (and even at home) they receive various treatments that tend to favor their conservation and / or safety. The application of washing and disinfection of apples improves its microbial image. However, it is difficult to safely achieve the inactivation or removal of pathogenic microorganisms even under extreme conditions of treatments that do not sensually damage apples. The prevention of apple contamination is also a control strategy because the growth of pathogens is not required to cause disease. Therefore, additional control measures may be of value. It should be noted that the behavior of pathogenic microorganisms in apples is affected by the location of the pathogen in the product, product quality, storage temperature, type of packaging, and relative humidity. The surface of apples, usually has little nutrients which limits the growth of pathogens during storage at room temperature or refrigeration.

However, it should be noted that pathogenic microorganisms such as Salmonella or E. coli 0157: 1-17 are able to survive for a long time on the surface of apples both in refrigeration and at room temperature (Fisher et al. 1998). Also once on the apple, pathogens such as Salmonella could produce extracellular polymers on the apple which leads to the formation of a biofilm that can protect them against disinfectants (Kroupitski et al., 2009); This behavior of pathogenic microorganisms has been observed in different vegetables such as tomatoes (Iturriaga et al., 2007).

A noteworthy fact is that pathogenic microorganisms such as Salmonella or E. coli 0157: H7, for example, are capable of multiplying in chopped apple or sliced oe apple juice significantly increasing its concentration and making food much more dangerous (Changa and Fang, 2007).

Pathogenic microorganisms on the surface of apples can contaminate internal tissues and infiltrate and subsequently during fruit cutting they can contaminate the chopped product and grow in it (Changa and Fang, 2007). Several research findings indicate that bacterial pathogens can infiltrate plant products, such as apples (Kroupitski et al., 2009; Bartz, 1982; Guo et al., 2002; Ibarra-Sanchez et al., 2004; Zhuang and Beuchat 1995) when there is a temperature differential between the vegetable product and the washing water and the hydrostatic pressure when the vegetables are submerged in the receiving tank (Bartz, 1982; Bartz and Showalter, 1981).

Bacterial infiltration increases in raw vegetables in the presence of wounds and punctures. Infiltrated pathogens are not eliminated by normal washing practices. The main benefit of the addition of antimicrobial chemicals (such as chemical disinfectants based on hypochlorite or organic acids) to the washing water of apples is to control the spread of pathogens, their inactivation and / or prevent their infiltration to apples. However, currently available chemical disinfectants have limited benefits over plant products, such as apples. The antimicrobial effect of solutions of hypochlorite, hydrogen peroxide, peracetic acid and electrolyzed water in its ability to reduce pathogens in plant products during the washing process has been studied. However, it has been concluded that these treatments have a limited effect on the pathogenic microorganisms, presumably because the active agents do not have sufficient contact with the pathogenic microorganisms on the raw plant products.

The disinfection process refers to the physical destruction of microorganisms whose activity compromises the safety or sensory characteristics of a food. The effect can be achieved through physical or chemical means, its effectiveness depending on the microorganisms (type and number), the substrate on which they are (presence of organic matter), the structure of the material (which allows direct access from germicide to microorganisms) and germicide (concentration, temperature and contact time) (Fernández, 2000). In the disinfection process, the germicidal substance participates in chemical reactions, so that the greater the number of microorganisms, the greater the demand of the agent to achieve a total inactivation of the population. The susceptibility to a specific germicide varies among microorganisms; some are inactivated from the first moment of contact, while at the other end there may be survivors. Finally, keep in mind that among the microorganisms it is possible to select strains with increasing resistance to the effect of a specific germicidal agent. Consequently, over time, much higher concentrations of the disinfectant than the initial ones are required to reach the same level of inactivation (Fernández, 2000).

Different studies show that disinfection treatments of raw agricultural products often have a weak or limited effect. For example, washing and disinfection with 200 mg / L of active chlorine (hypochlorite), iodine (iodophore), chlorine dioxide or 100 mg / L of a commercial product based on grapefruit seed extract (citricidal) reduced alfalfa sprout content in only 1-2 logio; the decrease in S. typhi or V. cholerae OI inoculated in the laboratory was not greater than 1.5 logio CFU / g (Castro-Rosas and Escartín, 1999).

The food industry has a variety of germicidal agents. Its virtues and limitations require carefully selecting those that best fit each particular need (Fernández, 2000). The inactivation of pathogenic bacteria in food processing plants is a basic requirement to control them and prevent their access to the finished product (Álvarez, 1998).

It is common for a germicide to be considered effective when it demonstrates the ability to inactivate at least 3 Logos of a microorganism suspension in 30s (Fernández, 2000).

Chlorine-based solutions are a cheap and available disinfectant as hypochlorite or in its slow-release forms (chloramines, for example) (Lelieveld et al., 2013). Hypochlorites have a broad spectrum of antibacterial activity, although they are less effective against spores than against non-forming bacteria. spores and have low effect against mycobactenas (Russell et al., 2004). Chlorine solutions, such as sodium hypochlorite or chlorine dioxide, are widely used by the food industry as a disinfectant. Both are strong oxidants that act at the level of membranes and other cellular constituents (Harmon et al., 1987). However, the former has the disadvantage of reacting easily with organic matter, so it is inactivated faster. In the second, the interference is minimal (Castro-Rosas and Escartin, 1999). The main disadvantage of sodium hypochlorite is that humidity, heat, light and especially the presence of organic matter increase the loss rate of free chlorine. The germicidal activity of chlorine has generally been attributed to hypochlorous acid (HOCI), which is generated in aqueous solutions of sodium hypochlorite and other chlorine-containing compounds.

Disinfectants can be incorporated into the wash water and thus contribute to the reduction of the microbial load. The effectiveness of hypochlorite is not only affected by the exposure time and concentration of free chlorine, but also by other factors such as temperature, pH, type of strain, as well as presence and type of organic matter (Álvarez, 1998). However, some authors point out that the efficiency of hypochlorite in reducing pathogenic microorganisms present in vegetables is limited (Adams et al., 1997).

Chemical compounds derived from chlorine, iodine and silver have typically been used as vegetable disinfectants, such as apples. However, recently several studies show that disinfection treatments with these compounds are inefficient in eliminating or reducing levels of microbial pathogens. For this reason, many countries have abandoned the use of hypochlorite or iodine solutions for the disinfection of raw vegetables.

Organic acids have traditionally been used as food preservatives or in solutions to disinfect raw vegetables. Its antimicrobial effect is exerted through the undissociated form causing a decrease in pH.

Acetic acid is a harmless substance; There are no official limits for daily intake in man. When acetic acid is incorporated into a food, two effects are expressed, one acidifying and the other preservative. At 1-2% concentration It inhibits almost all of the total flora within reasonably high initial load limits. At 0.1% it acts on the majority of pathogens and sporulates; 0.5% has an effect on toxigenic fungi. The efficacy of acetic acid against some specific pathogens has been evaluated using some foods as a medium. Published reports are often difficult to compare because acid concentrations have been variables expressed as percentage, molarity or final pH of the acidified test medium. The antimicrobial activity depends on the exposure time, temperature, type of acid, acid concentration, dissociation level and pH (Harmon et al., 1987). However, the general results show that the effectiveness of acetic acid increases as the concentration increases, the pH decreases, the temperature increases and the microbial load decreases (Harmon et ai., 1987). Among bacteria, Gram positive are usually more resistant than Gram negative bacteria (Rameshkumar et al., 2007). Bacterial spores and viruses are more resistant than vegetative cells. However, organic acids have also shown little effectiveness in disinfecting raw vegetables (Fernández, 2000).

Recent research indicates that antimicrobial chemicals in the vapor phase can significantly reduce plant surface pathogen populations. The use of 5 mg / liter of chlorine dioxide gas for 1 h was significantly more effective against Salmonella in the scar of the peduncle of tomatoes that were aqueous solutions of 200 ppm of sodium hypochlorite (2 min of exposure) and 1200 ppm of acidified sodium hypochlorite (2 min exposure) (Yuk et al., 2005). The use of 10 mg / liter of ozone completely inactivates 7 log CFU of Salmonella enterítidis from the surface of cherry tomatoes after 15 min, however, the color of tomatoes is affected (Das et al., 2003).

Because the vapor phase antimicrobial agents can be effective against bacteria adhering to locations of raw agricultural products not reached by active agents in aqueous solution, their use in packaged products (in plastic bags) or during product processing (in the company) could provide an extra benefit in pathogen control. However, this type of steam treatment would not be an optional or practical treatment for primary apple growers in the field since they are usually Producers sell their product packed in cardboard boxes or wood among other things for ease and to avoid accumulation and humidity what would happen if plastic bags were used. In addition, this would not be a practical treatment to apply in restaurants or homes.

The use of chemical substances as raw vegetable disinfectants to improve or preserve their safety is a procedure universally used by producers. However, some of these antimicrobials may be toxic to consumers; This is the case of hypochlorite solutions. Recent reports indicate that hypochlorite in solution can form cancer precursors. In addition, many of the chemical disinfectants, such as solutions based on iodine or colloidal silver, show limited or varied antimicrobial effects in products such as raw vegetables; A similar situation occurs with food preservatives. Due to this, disinfectants and preservatives obtained from plants have recently emerged as a viable alternative, since these could have equal or greater antimicrobial potential and with minimal risk to consumers.

The application of garlic extracts in fresh fruit against postharvest diseases has obtained complete control of the brown rot of peaches caused by Monilinia fructicola (Roller, 2003). Yucel and Karapinar (2005) evaluated the reduction of S. typhimurium in onions by applying lemon juice, vinegar and their mixtures, observing a respective reduction of 0.87-293, 0.66-2.92 and 0.86-3.24 Log CFU / g.

Essential oils from plants are capable of inactivating the pathogens of interest in fresh products. Of 96 different types of essential oils examined, only 3 were effective against £. co // ^' 0157: H7 and enteric Salmonella which were oregano, thyme, and cinnamon. In another study, 16 individual compounds of the most effective oils against E. coli 0157: H7 and Salmonella were tested and the most effective compounds were found to be thymol, cinnamaldehyde, and carvacrol (Friedman et al., 2002). This information was obtained using the oil in the liquid phase. There is limited information available on the effectiveness of essential oils in the form of steam. In another study, Muñoz (2003) evaluated the effect of two concentrations of carvacrol and the commercial disinfectant Boradantix © (EVESA, Extracts Vegetables SA) on the survival of L. monocytogenes, P. fíuorescens, E. coli, Erwinia caratovora and S. typhimurium in Apple and carrot juice. All study microorganisms were inhibited at both concentrations of carvacrol. The bacteria studied showed greater sensitivity towards carvacrol than Boradantix ©. Lin et al., (2000) evaluated the effect of allyl and methyl isocyanate (AITC / MITC) (key components of green mustard) on L. monocytogenes, E. coli 0157: H7 and S. montevideo, inoculated on tomato surface . AITC was more effective against Salmonella and £. coli, achieving 8 Log reduction with a generated steam treatment of 400 μΙ of AITC after 4 and 2 days, respectively on apple. 8 Logs of reduction of S. Montevideo on tomato cuticle with 500 μΙ of AITC were also reached.

There have been relatively few studies of the antimicrobial action of essential oils in model food systems and in real foods. However, the effectiveness of essential oils in vitro is often much better than in vivo or in situ, that is in food. Generally, when applying a plant antimicrobial to a food or in vitro, 10 to 100 times the concentration of antimicrobial is needed than what is observed in vivo. For example, the essential oil of peppermint (Mentha piperita) has been shown to inhibit the growth of Salmonella enteritidis and L. monocytogenes in culture media for 2 days at 30 ° C. However, the effect of peppermint essential oil on Greek snacks tzatziki (pH 4.5) and taramasalata (pH 5.0) and in pate (pH 6.8) at 4 ° C and 10 ° C was variable (Roller, 2003). Salmonella enteritidis was eliminated in snacks under all conditions examined but not when it was inoculated in pate and kept at 10 ° C. In this same study, L. monocytogenes behaved similarly, since the microbial count decreased in snacks but increased in pate (Roller, 2003). The growth of £. coli, Salmonella sp., L. monocytogenes and Staphylococcus aureus were inhibited by the essential oil of oregano in culture broths. However, when these oils were tested in foods such as eggplant, taramasalata or mayonnaise, reactions such as pH increase, temperature increase were observed, and in the case of emulsions, separation of used oil (Roller, 2003). In another study L monocytogenes and S. typhimurium were inhibited in meat treated with essential oil of clove and oregano, respectively. A marked reduction in Aeromonas hydrophila has also been reported in cooked pork that was treated with clove or coriander oils, vacuum packed or air-packed and stored at 2 ° C and 10 ° C. (Roller, 2003). The differences observed between studies of antimicrobial effect when directly applied oils extracted from plants on microorganisms (microorganisms in aqueous suspension) and those in which there is a food or organic matter in between, it is possible that it occurs by interference with the components of food or organic matter (proteins, fats, sugars, salts). Therefore, it is very possible that only a proportion of the essential oil added to the food has antibacterial activity. On the other hand, the spatial distribution of the different phases (solid / liquid) in a food and the lack of homogeneity of factors such as pH, at _w among others, can play a role in efficacy. Due to all of the above, studies aimed at the search for alternative antimicrobials are under way in various parts of the world (Jongen, 2005). Among the new disinfectant alternatives, natural compounds with a broad antimicrobial capacity have been chosen.

It should be noted that the extracts obtained from some plants have shown antimicrobial effect against strains of antibiotic multiresistant pathogens, which opens up a whole new field for the development of new antimicrobials for use in humans and animals.

As an antecedent to the present application, the antimicrobial effect of about 60 different plants used in herbalism has been evaluated (Cruz-Gálvez et al., 2013); where some of these have shown a high antimicrobial power against different pathogenic microorganisms, such as Salmonella or Escheríchia coli 0157: H7, among others, as well as against food spoilage microorganisms (Pseudomonas aeruginosas, for example), and the plant that has the greatest antimicrobial effect It has been shown that the chalices of the Jamaican flower have been, in some cases the antimicrobial effect being greater than that of commercial disinfectants based on hypochlorite, iodine, colloidal silver or that of antibiotics such as penicillin. The extracts of the chalices of Jamaica have been separated by column chromatography to obtain fractions with greater antimicrobial power; With selected fractions, solutions have been developed that have been evaluated to determine their antimicrobial potential. In fact, different researchers have also reported that the chalices of the Jamaica flower (Hibiscus sabdariffa L) have substances with high antimicrobial power (Aziz et al., 1998; Fernández et al., 1996; Kang er a /., 2007) .

Jamaica is one of the plants in which the presence of antimicrobial compounds in dehydrated chalices has recently been reported (Aziz et al., 1998; Fernández et al., 1996; Kang et al., 2007). A range of phytochemical compounds that could be responsible for the observed antimicrobial effect, such as polyphenols (Tajkarimi et al., 2010), including some phenolic acids (Tajkarimi ef al. ., 2010), as well as flavonoids, catechins and epicatechin (Friedman et al. 2002). However, there are no specific studies that show which are the chemical molecules or compounds responsible for the antimicrobial effect observed in the chalices of Jamaica. Different researchers agree that further studies are necessary to identify the specific molecules responsible for the antimicrobial effect caused by the calyxes of Jamaica in solution.

There are few patent documents that describe extracts of the chalices of the flower of Jamaica (Hibiscus sabdariffa) and its use as a material with antimicrobial properties.

For example, patent application JP2002128602 describes its use in an agrochemical composition to protect plants in fields of crops, while application US20100323046 describes the use of a crude extract of the calyces of Jamaica to produce a drug to treat urinary infections caused by Escherichia coli and Candida albicans.

Patent application KR20080092186 describes an extract from Jamaica that is used to improve the quality of beef, pork and chicken and to increase its storage stability. The extract is prepared by extraction with ethanol and subjected to a cold drying process. The concentration of the extract in the composition is 500 mg / ml and the meat is treated with a preparation of 0.5 to 3.0% (by weight). On the other hand, the application US20120015062 describes compositions comprising extract of the Agapanthus afrícanus plant and compositions comprising this extract plus other extracts of other different plants, such as for example plants of the Rosa or alfalfa family for use as agents in the Biological protection of other plants including their seeds. Although this patent application document refers to the article published by Leksomboon et al. (Kasetsart, Journal Natural Science 35: 392-396, 2001.) where it is mentioned that extracts obtained from various plants (Hibiscus sabdariffa, Psidium guctjava, Púnica granatum, Spondias pinnata and Tamarindus indica) have an antimicrobial function, this document does not provide no experimental evidence involving the extracts of Hibiscus sabdariffa for the same use that is given to the extracts of Agapanthus afrícanus.

Therefore, it is necessary to have effective protective antimicrobial compositions to prevent and / or combat microbial contamination of food, especially those that are eaten raw, such as apples, for the purpose of preserving and consuming them without the risk of acquire diseases caused by their contamination with pathogenic microorganisms.

Until before the present invention, it had not been possible to develop effective compositions to disinfect efficiently and without damage to the product as described herein, and which at the same time allowed to preserve the nutritional properties of fruits and vegetables and not affect, for example the quality of apples, whereby it is possible with the present invention to obtain innocuous raw apples. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The nuclear magnetic resonance (NMR) spectrum of

PROTON ( ¹ H) of the dry acetonic extract obtained from the Jamaican calyces that was used in the present invention. Figure 2. The nuclear magnetic resonance (NMR) spectrum of PROTÓN ( ¹ H) of the fraction collection called III is shown, which was obtained from an acetonic extract of the chalices of Jamaica and which was the collection that was used in the present invention.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the aforementioned problems, there is a need to provide a more effective formulation to inactivate or remove pathogenic microorganisms from the apple (Malus domestica) even under extreme conditions of treatment, but which do not sensorially damage the food.

The present invention relates to compositions containing phytochemicals present in plant extracts that are used as disinfectants of plant and animal foods, for example directed to the disinfection and preservation of fruits and vegetables, particularly the disinfection and / or preservation of apple (Malus domestica). One embodiment of the present invention relates to obtaining a vegetable preparation comprising a methanolic extract of the chalices of the flower of Jamaica (Hibiscus sabdariffa) or specific chromatographic fractions obtained from the acetonic extract of the calyces of Jamaica, which are useful to eliminate pathogens present in food (disinfectant effect) and to delay the deterioration of food or preserve its safety (conservative effect).

Another embodiment of the present invention relates to obtaining plant-derived extracts that are used as disinfectants against pathogenic microorganisms present in food and to delay the deterioration of food and / or preserve its safety, that is, as food preservatives , those that constitute an alternative to the use of traditional disinfectants that can become toxic to humans, animals or the environment. Another embodiment of the present invention relates to the preparation of compositions containing the extract of Jamaican calyces (Hibiscus sabdariffa L.) that have a food disinfectant and preservative function together with other compounds that have disinfectant properties, for example acetic acid, hypochlorite, etc.

Another embodiment of the present invention relates to obtaining extracts obtained from Jamaican chalices that have a disinfectant or preservative effect when applied to food. One aspect of this modality refers to the application of extracts obtained from chalices of the Jamaican plant (Hibiscus sabdariffa L.) that have a disinfectant or preservative effect when applied to foods of plant origin, preferably apple.

Another embodiment of the present invention is the development of a method for obtaining acetonic extract from Jamaican calyces, an extract that proves to be useful as a food disinfectant and preservative.

Another embodiment of the present invention relates to the method for obtaining specific chromatographic fractions with antimicrobial effect obtained from the acetonic extract of the chalices of Jamaica, which are useful for eliminating pathogens present in food (disinfectant effect) and for delaying deterioration. of food or preserve its safety (conservative effect).

Another embodiment of the present invention is a method of treatment and / or preservation of foods of animal and / or vegetable origin by applying compositions containing extracts of Jamaican chalices that allow disinfection and preservation thereof.

The use of Jamaican calyx extracts as a disinfectant and / or food preservative is another modality described in the present invention.

Compounds from the calyces of Jamaica can be useful in the development of an efficient disinfectant to eliminate the pathogenic bacteria present in raw vegetables, such as apples. In the present invention, It describes an extract of Jamaican chalices and a specific fraction obtained by column chromatography from an acetonic extract of Jamaican chalices, which includes phytochemicals, which can be used as a disinfectant and / or food preservative due to its efficiency in the elimination of pathogenic bacteria from raw vegetables such as apples.

Unlike other compositions known so far for the same purpose, the compositions of the present invention are capable of eliminating the pathogenic bacteria present in raw vegetables, such as apples per se, without altering their nutritional properties as well as quality characteristics. of the product. Consequently, the application of the compositions of the present invention in raw vegetables allows their preservation, as well as their effective disinfection, which makes them safe food for consumption. DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention comprise plant extracts with known antimicrobial activity, such as Jamaican acetonic extracts and specific chromatographic fractions obtained from the acetonic extract of Jamaican calyces, either alone or in combination with other components with proven disinfectant activity, such as for example organic acids that include acetic acid and chlorine compounds that include sodium hypochlorite. In the case of the disinfection of raw vegetables such as apples, the compositions of the invention that include a mixture of acetonic extracts of plants with antimicrobial activity as well as acetic acid and sodium hypochlorite and polysorbate, and a mixture of specific chromatographic fractions obtained from the acetonic extract of the chalices of Jamaica as well as acetic acid and sodium hypochlorite and polysorbate, are usually very effective in eliminating the microorganisms resident in the plant, achieving at the same time that their organoleptic and / or nutritional properties are not affected and without altering, for example the commercial quality of apples.

For purposes of the present invention, the compositions described herein comprise: a) Extracts derived from plants, which exhibit antimicrobial properties, such as extracts derived from Jamaican chalices (Hibiscus sabdariffa),

 b) Chromatographic fractions obtained from acetonic extracts derived from plants, which exhibit antimicrobial properties, such as chromatographic fractions obtained from calyxes of Jamaica (Hibiscus sabdariffa),

 c) An organic acid with disinfectant activity, such as acetic acid, lactic acid, citric acid, peracetic acid, octanoic acid, peroxyethanoic acid and 1-hydroxyethylidene-1,1-diphosphonic acid, and mixtures thereof, in a concentration p / p from 0.01% to 10%, preferably from 0.1% to 1%,

d) A chlorine compound with disinfectant activity, such as sodium hypochlorite, calcium hypochlorite, chlorine dioxide and mixtures thereof in a w / w concentration of 0.001% to 10%, preferably 0.001% to 0.1%, and e) A surfactant with emulsifying activity of natural fats or waxes found on the surface of apples such as, for example, polysorbates, Polysorbate 80, Polysorbate 20, C12-C18 alkyl dimethyl betaine (cocobetaine, C10-C16 alkyl dimethyl betaine (lauryl betaine) ), Sulfobetaine acyl (C10-C14 fatty) amidopropylene (hydroxypropylene), sulfobetaine, Cyclodextrins, B-cyclodextrins and β-Cyclodextrin and mixtures thereof in a w / w concentration of 0.1% to 5%, preferably 0.5% to 1 For the purposes of the invention, the compositions are added to the food to be disinfected and / or preserved through methods known in the art, such as direct application, through aerosols, the complete immersion of fruits and vegetables. ures in disinfectant solutions or through devices that allow their adequate dispersion in the food to be treated. The compositions of the invention can be added or contacted with food in an amount of 0.1 mL per 1000g of food, preferably 0.1 to 1mL per 100g of food, or added in larger volumes according to the needs of disinfection. of food. After applied, the compositions can remain the necessary time until the disinfectant and / or Desired preservation in fruits and vegetables. Prior to consumption, the fruits and vegetables treated with the compositions described herein are simply washed with drinking water to remove said compositions. The compositions described herein can be obtained by mixing their components in the desired concentrations, to then store them at room temperature, so they are ready to be applied to food when deemed necessary. For purposes of the invention, the compositions described herein may contain only plant extracts with antimicrobial activity, such as extracts derived from Jamaican chalices, or chromatographic fractions obtained from acetonic extracts of Jamaican calyces, which are placed in contact with food, for example raw plant-based foods such as apples, in order to disinfect and / or preserve them. In the present invention, the disinfectant activity of extracts derived from Jamaica and chromatographic fractions obtained from the extracts of the chalices of Jamaica, in the disinfection and / or preservation of foods, for example raw fruits and vegetables, is described. which can be used directly or as part of compositions containing them. In this sense, chromatographic extracts or fractions derived from the chalices of Jamaica can be added or contacted with the food to be disinfected and / or preserved in a w / w concentration of 0.001% to 10%, preferably 0.1% to 1 %. The disinfectant and / or preservation effectiveness of the compositions described herein is such that it inactivates or eliminates human pathogenic bacteria or food spoilage that may be present in them, while at the same time not affecting the organoleptic properties and / or nutritious food. In the case of fresh foods such as apples, the compositions of the invention adequately disinfect the food without affecting its nutritional properties, while at the same time not affecting the organoleptic or quality properties. The plant extracts of the present invention and the chromatographic fractions can be obtained by the following method: a) Place the dried plant in a container under aseptic conditions, add acetone in a 1: 9 ratio; preferably 100 g of the dried plant are placed in a container (flask) under aseptic conditions, 900 ml of methanol are added and allowed to stand for 7 days;

b) Remove the chalices and recover the acetonic extract; preferably the resulting extract is recovered after pressure on the flask walls to remove excess liquid;

c) Pass the extract through a sieve and recover the filtered extract; preferably the extract is passed through a No. 200 sieve;

d) Remove the acetone from the extract by rotary evaporation at a temperature of 40 ° C, a rotation of 80 rpm and a vacuum pressure of 72 mbar;

e) Recover the dry extract; preferably in a previously sterile contain; f) Obtain fractions of acetonic extracts by column chromatography using solvents of different polarity;

g) Remove the solvent from the fractions obtained by rotary evaporation at a temperature of 40 ° C, a rotation of 80 rpm and a vacuum pressure of 72 mbar; and h) Recover the dry fractions; preferably in a container.

Obtained extracts and fractions, these are stored at room temperature until use. Once the extracts and chromatographic fractions are obtained, they can be used alone, or in combination with other disinfectants to obtain the compositions of the invention, which can be obtained by methods known in the art where it involves the combination of the various elements that they are formed to form solutions and / or suspensions capable of being subsequently applied to food to be disinfected and / or preserved, by methods known in the art.

The present invention constitutes the first report of the use and effectiveness of compositions containing plant extracts with microbial activity, either alone or in combination with other disinfectants, for disinfection and / or preservation of food, particularly fruits and vegetables, such as apples. As can be seen later, the compositions of the invention are capable of disinfecting and / or eliminating microorganisms present in apples in a very efficient manner, so that it is possible to have innocuous apples ractoctobiologically and safe for consumption.

The following examples are included below for the sole purpose of illustrating the present invention, without implying any limitation on its scope. Example 1. Materials and methods.

 1.1. Vegetal material.

 Dry Jamaican calyces (Hibiscus sabdariffa) of the Creole variety of Oaxaca were used, while in the case of the apple {Malus domestica) the Golden Delicious variety was used. The apples had a uftiform size.

1.2. Bacterial strains

Strains of E. coli 0157 were used: H7 (P1C6, isolated from an outbreak of disease), enteroinvasive coli (4VC81-5, isolated from clinical case) E. Enteri toxogenic coli (1620 TL, isolated from clinical case), E. Enteropathogenic coli (52 GM 291, isolated from clinical case), Salmonella typhimurium (ATCC 14028), Salmonella choleraesuis (ATCC 10708), Listeria monocytogenes (ATCC 19115), Listeria monocytogenes Scott A, Staphylococcus epidermis (ATCC 12228), Staphylococcus Staphyus (ATCC 12228) 25923), Pseudomonas aeruginosa (ATCC 27853), Bordetella (ATCC 12741) Shigella sonnei (ATCC 25931) and Shigella flexneri (ATCC 12022), V. cholerae (87151, Inaba serotype isolated from the environment) and Pseudomonas aeruginosa (ATCC 27853). Strains of E. coli 0157: H7 and that of V. cholerae OI were donated by Dr. Fernández Escartin of the Autonomous University of Querétaro. All strains were marked with resistance to the antibiotic rifampicin (R +) to eliminate interference from the native microbial flora of the extract (Castro-Rosas and Escartín, 2000). This resistance to the antibiotic was maintained throughout the study. The strains were maintained at 4-7 ° C in blood-based agar (ABS, Merck®, Germany) with biweekly transfers, activating in tryptosesein soy broth (CST, Bioxon®, Mexico) with incubation at 35 ° C / 24h. 1.3. Obtaining aqueous extract from the chalices of Jamaica.

 Under aseptic conditions 100 g of Jamaican chalices were placed in an Erlenmeyer flask, to which 900 ml of distilled water was added, boiling the mixture for 20 minutes. Once the treatment was finished, it was allowed to cool to room temperature. The calyces were removed from the extract (after pressure on the flask walls to remove excess liquid from it) and subsequently the extract was passed through a No. 200 sieve (MONTIMAX) to remove particles. Finally, all the water was removed from the extract by broken evaporation using a broken evaporator (Buchi R-205) using the following conditions: temperature of 40 ° C of the tub, rotation of 80 rpm and a vacuum pressure of 72 mbar. The dried extract was recovered in a sterile bottle and stored at room temperature until use.

1.4. Obtaining methanolic and acetonic extract from the chalices of Jamaica.

 Under aseptic conditions 100 g of Jamaican chalices were placed in an Erlenmeyer flask, to which 900 mL of methanol or acetone was added and stored for 7 days at room temperature. Once the treatment was finished, the calyces were removed from the extract (after pressure on the flask walls to remove excess liquid from it) and subsequently the extract was passed through a No. 200 sieve (MONTIMAX) to remove particles. Finally, all methanol or acetone was removed from the extract by broken evaporation using a broken evaporator (Buchi R-205) using the following conditions: temperature of 40 ° C of the tub, rotation of 80 rpm and a vacuum pressure of 72 mbar. The dried extracts (methanolic or acetonic) were recovered separately in a sterile bottle and stored at room temperature until use.

1.5. Obtaining fractions from the methanolic extract by column chromatography

Once the dry extract (solvent-free) was obtained, it was mixed with silica (in order to make the extract manageable, since it still had moisture), it was added to the packed column. Cotton was placed at the bottom of the column with the help of a rod to prevent the silica gel from detaching when the key was opened, the column was held with two tweezers and made sure it was straight. The silica gel was mixed with hexane approximately 8: 1g (silica gel: extract), this amount was mixed with hexane until a fluid paste was obtained, the paste was poured into the column, the amount of hexane added should be sufficient to prevent the silica from drying out or air entering the paste, subsequently the extract was added little by little, a small layer of sodium sulfate (this serves as a drying agent) was added, a cotton layer was placed on top of it to cushion the solvent drop when added and thus avoid dispersion of the calcium sulfate and the extract, after this procedure the column was filled with the solvent (hexane) and the key was opened to begin lowering the fractions with the different solvent mixtures, recovering them in amounts of 50 ml each, which subsequently evaporated with the help of the rotary evaporator, and these were placed in vials, considering each of these as a fraction. To change the solvent mixture, thin plate chromatography was performed, and finding clearly visible differences (due to the appearance of different bands in size and shape) between fractions the mixture was changed from lower to higher polarity (hexane, ethyl acetate and methanol ) Table 1 shows the solvent mixtures used and with which it was eluted.

Table 1. Solvents and mixtures used to obtain fractions from methanolic extract

From a total of 28.5 g of methanolic extract, 193 fractions were obtained, after determining the similarity of the fractions by thin layer chromatography, 7 fraction collections were obtained (Table 2). Table 2. Number of fractions collected in each collection obtained from the methanolic extract

1.6. Obtaining fractions from the acetonic extract by column chromatography.

Once the dry extract (solvent-free) was obtained, it was mixed with silica (in order to make the extract manageable, since it still had moisture), it was added to the packed column. Cotton was placed at the bottom of the column with the help of a rod to prevent the release of the silica gel when the key was opened, the column was fastened with two tweezers and secured in such a way that it was straight. The silica gel was mixed with chloroform approximately 8: 1g. (silica gel: extract), this amount was mixed with chloroform until a fluid paste was obtained, the paste was poured into the column, the amount of chloroform added should be sufficient to prevent the silica from drying out or air entering the paste, subsequently the extract was added little by little, a small layer of sodium sulfate (this serves as a drying agent) was added, a cotton layer was placed on top of it to cushion the solvent drop when added and thus avoid dispersion of the calcium sulfate and the extract, after this procedure the column was filled with the solvent (chloroform) and the key was opened to begin lowering the fractions with the different solvent mixtures, recovering them in amounts of 50 ml each, which later they evaporated with the help of the rotary evaporator, and these were placed in viáles, considering each one of these as a fraction. To change the solvent mixture, thin plate chromatography was performed, and finding differences between fractions the mixture was changed from lower to higher polarity (chloroform-acetone) in table 3 the solvent mixtures used and with which it was eluted are shown. . Table 3. Solvents and mixtures used to obtain fractions from acetonic extract

From a total of 20.5 g of acetonic extract, 117 fractions were obtained, after determining the similarity of the fractions by thin layer chromatography, 7 fraction collections were obtained (Table 4).

Table 4. Number of fractions collected in each collection obtained from the acetonic extract

Finally, all methanol or acetone was removed from each collection by broken evaporation using a broken evaporator (Buchi R-205) using the following conditions: temperature of 40 ° C of the tub, rotation of 80 rpm and a vacuum pressure of 72 mbar . Dry collections (methanolic or acetonic) were recovered separately in clean jars and stored at room temperature until use. 1.7. Determination of the antimicrobial activity of aqueous, methanolic and acetonic extracts, acetic acid, hypochlorite and the corresponding fractions from Jamaican calyces in culture medium (in vitro studies).

1.7.1. Preparation of inoculum of strains.

Test tubes with 24 h cultures in CST of each R + strain were centrifuged at 3500 rpm for 20 min. Subsequently, the supernatant was discarded; The cell packet was resuspended by adding 3 mL of sterile isotonic saline and stirred in vortex for 10 s. The above procedure was repeated two more times. Subsequently, the concentration of each strain was approximately 1x10 ⁹ CFU / mL. Finally, each strain was diluted decimally in isotonic saline solution only once. 1.7.2. Preparation of solutions of extracts or fractions.

 From the dry extracts or dry fractions (collections), aqueous solutions were prepared using sterile distilled water or a solution of Polysorbate 80: water in a 20:80 ratio. The aqueous and methanolic extracts and acetonic fractions (fraction III) and methanolic fractions (fraction IV) and were solubilized in distilled water while acetonic extracts and non-polar or low polarity fractions were solubilized in the solution of Polysorbate 80: water. Water or polysorbate 80: water were added with dry extracts or fractions in a 1: 10 and 1: 100 ratio (water: extract or water: fraction) separately and deposited in sterile bottles.

1.7.3 Antimicrobial effect of extracts and fractions in culture medium.

Separately, 100 iL of the first dilution of the cultures of the pathogens were inoculated on boxes of AST supplemented with 10 mg / L of the antibiotic rifampin, the inoculum was distributed over the entire surface of the agar using the surface extension technique. On the inoculated boxes, separately, 10 μί aliquots of the extract solution (aqueous, methanolic or acetonic), or chromatographic fractions were placed. Four repetitions were performed for each treatment. After the extract or fractions were absorbed by the agar, the culture boxes were incubated at 35 ± 1 ° C, for 24 h. Finally, the diameter of each of the inhibition halos formed on the surface of the inoculated medium was measured.

1.8. Evaluation of the antimicrobial effect of aqueous, methanolic, acetonic, chromatographic fractions and specific formulations in the reduction of Salmonella and E. coli 0157: H7 in the contaminated apple.

1.8.1. Preparation of disinfectant solutions.

 The solutions of Jamaican calyx extracts, mixtures based on extracts and chromatographic fractions as well as mixtures containing acetic acid, hypochlorite and / or polysorbate 80% were prepared at the concentrations, proportions or mixtures described in Table 5. By For example, to prepare 100 ml of a solution containing 1% Jamaican calyces methanolic extract, 0.1% acetic acid and 100 mg / L hypochlorite: to 100 mL of distilled water was added 1 g of dry methanolic extract of calyces of Jamaica, in addition 1 ml of a 10% acetic acid solution and 0.2 ml of a 5% hypochlorite solution.

18.2. Strains

 For these studies we worked with 7 Salmonella serotypes: (3 typhimuríum [ATCC 14028, one isolated from tomato, J1, and another from alfalfa seed, GA1], Salmonella choleraesuis [ATCC 10708], typhi, geminara, and ontevideo) and 3 of E. coli 0157: H7 (two isolated in our laboratory from raw ground beef [P1C6 and M5C8] and another isolated from an outbreak caused by meat consumption in the United States of America [E09]), this strain was donated by Dr. Eduardo Fernández Escartin of the Autonomous University of the State of Hidalgo. From the native strains mutant strains antibiotic resistance rifampin (R +) were obtained, this to be used in the studies. By incorporating the antibiotic into the culture medium to monitor the behavior of the mutant strains, the interference of the native microbial flora of the extracts, fractions and plant material of study would be eliminated (Castro-Rosas and Escartin, 2000).

1.8.3. Preparation of inoculum of strains.

Test tubes with 24 h cultures in CST of each R + strain were centrifuged at 3500 rpm for 20 min. Subsequently, the supernatant was discarded; he Cell pack was resuspended by adding 3 mL of sterile isotonic saline and stirred in vortex for 10 s. The above procedure was repeated two more times. The resulting concentration of each strain was approximately 1x10 ⁹ CFU / mL. One milliliter of each Salmonella strain was mixed in an empty test tube to have a mixture of the 7 Salmonella strains examined. The same was done with strains of E. coli 0157: H7, to have a mixture of the three strains of E. coli 0157.Ή7.

Table 5. Treatments to which apples contaminated with the mixtures of Salmonella or E. coli strains OI 57: H7 were subjected separately

No Treatments

^" 1 No treatment (control) ~ ^"

2 1% aqueous extract

 3 1% acetonic extract

 4 1% methanolic extract

 5 Acetonic fraction 1%

 6 1% methanolic fraction

 7 Sodium hypochlorite 100 ppm

 8 Acetic acid 0.1%

 9 Acetic acid 0.5%

 10 1% aqueous extract + 0.1% acetic acid

 11 1% aqueous extract + 0.5% acetic acid

 12 Acetonic extract 1% + acetic acid 0.1%

 13 Acetonic extract 1% + acetic acid 0.5%

 14 1% methanolic extract + 0.1% acetic acid

 15 1% methanolic extract + 0.5% acetic acid

 16 1% acetonic fraction + 0.1% acetic acid

 7 Acetonic fraction 1% + acetic acid 0.5%

 18 1% methanolic fraction + 0.1% acetic acid

 19 1% methanolic fraction + 0.5% acetic acid

 20 1% aqueous extract + 0.1% acetic acid + sodium hypochlorite 100 ppm

 21 1% aqueous extract + 0.5% acetic acid + sodium hypochlorite 100 ppm

 22 Acetonic extract 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm

23 Acetonic extract 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm

24 1% methanolic extract + 0.1% acetic acid + sodium hypochlorite 100 ppm

25 1% methanolic extract + 0.5% acetic acid + 100 ppm sodium hypochlorite

26 Acetonic fraction 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm

27 Acetonic fraction 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm

28 1% methanolic fraction + 0.1% acetic acid + 100 ppm sodium hypochlorite

29 1% methanolic fraction + 0.5% acetic acid + sodium hypochlorite 100 ppm

30 1% aqueous extract + 0.1% acetic acid + sodium hypochlorite 100 ppm + Polysorbate 80 2%

 31 1% aqueous extract + 0.5% acetic acid + 100 ppm sodium hypochlorite + 2% Polysorbate 80

 32 Acetonic extract 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm + Polysorbate 80 2%

 33 Acetonic extract 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm + Polysorbate 80 2%

 34 Methanocolol extract% + 0.1% acetic acid + sodium hypochlorite 100 ppm + Polysorbate 80 2%

35 Methanocol extract% + 0.5% acetic acid + sodium hypochlorite 100 ppm + 802% Polysorbate 36 Acetonic fraction 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm + Polysorbate 80 2%

 37 1% acetonic fraction + 0.5% acetic acid + 100 ppm sodium hypochlorite + 802% Polysorbate

 38 Methanolic fraction 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm + Polysorbate 80 2%

 39 Methanolic fraction 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm + Polysorbate

 80 2%

 For the elaboration of the solutions the base was used:

 A) the dry extract of the calyces of Jamaica in the previous section, B) Sodium ipochlorite solution with 5% free hypochlorite, C) 10% glacial acetic acid, d) Polyoxyethylene Sorbitan Monooleate, or polysorbate 80 ( Polysorbate 80), d) Sterile distilled water at pH 6

1.8.4. Inoculation of apples

Golden Delicious apples were used; The apples were obtained from a crazy producer! Prior to inoculation, apples were cleaned with a clean cloth to remove dust particles. Apples of a uniform or similar size were used and did not show visible damage. Separately, individual apples were inoculated placing in the central part (not in peduncle) of the fruit and drops or aliquots of 10 μί of a suspension of each type of mixture of pathogenic bacteria (Salmonella or E. coli 0157: H7) containing approximately 1 x 10 ⁷ CFU, the 5 inoculums were close without coming together the inoculated apples were placed in a tray and placed in a bioclimatic hood for two hours at a relative humidity of 90 ± 1% and 26.5 ± 1 ° C. The purpose of this treatment was to cause the adhesion or infiltration of the cells of the pathogenic bacteria under study to simulate the natural conditions, in other words, to have a model that resembles as much as possible what happens when apples They are contaminated by natural or common sources of contamination with pathogenic bacteria. 1.8.5. Apple disinfection treatment.

After two hours in the bioclimatic chamber, each fruit was washed separately to remove microorganisms that did not adhere, the washing consisted of submerging and stirring the inoculated part of the apple in distilled water for 10 s, the part was allowed to drain washed at room temperature until completely dry and subsequently separately the inoculated part of different apples was immersed for 10 min in the different disinfectant solutions indicated in Table 5. A treatment with only distilled water served as a positive control. 1.8.6. Counting of microorganisms surviving the treatments

 After the treatment, the apples were removed from the disinfectant solution and to remove the remaining disinfectant the inoculated part was immersed in distilled water for 10 s, then the inoculated part was cut (a box of approximately 2 x 2 cm and with a depth of approx. 2 cm) with the help of a sterile scalpel, each portion was placed independently in a plastic bag and 10 ml of peptone diluent was added. Subsequently, the materials were shaken manually by pressing and rubbing the inoculated part and the entire apple portion from the outside of the bag for a minute. After this time, each bag was counted using the plate pouring technique using agar for standard methods (Bioxon, Mexico) added 100 mg / L of Rifampicin (Sigma, Mexico), the boxes were incubated at 35 ° C / 24-8 h. This procedure was performed in duplicate for each replica. Each treatment was carried out in quintuplicate.

1.8.7. Statistic analysis

 The results obtained were statistically analyzed with a one-way analysis of variance (ANOVA) comparing the means with the Tukey test, with a significance level of 0.05.

1.9. Nuclear Magnetic Resonance (NMR) of extract and collection IV

The NMR spectrum of the proton ( ¹ H) was determined from both the dry methanolic extract obtained from the chalices of Jamaica and from

of fractions referred to as "IV" which was obtained from the methanolic extract of the calyces of Jamaica and which was the collection that was used in the formulations.

The Carbon NMR spectrum ( ¹³ C) was also obtained only from the collection of fractions referred to as "IV" which was obtained from methanolic extract of the calyxes of Jamaica.

The dried methanolic extract and / or collection IV were solubilized in deuterated water. NMR spectra were obtained using a nuclear magnetic resonance spectrometer (They vary NMR, 400 MHz). NMR spectroscopy studies atomic nuclei. This spectroscopic technique can only be used to study atomic nuclei with an odd number of protons or neutrons (or both), to determine the structures of organic compounds. This situation occurs in the atoms of ¹ H, ¹³ C, ¹⁹ F and ³¹ P. These types of nuclei are magnetically active, that is, they have spin, just like electrons, since the nuclei have a positive charge and have a movement of rotation on an axis that makes them behave as if they were small magnets. The NMR spectrometer detects these signals and records them as a graph of frequencies versus intensity, which is called the NMR spectrum

Example 2. Antimicrobial effect of extracts from Jamaican chalices.

The three types of extracts (aqueous, methanolic and acetonic) showed a marked antimicrobial effect (Table 6). All the microorganisms tested were inhibited from the first moments of contact. The inhibitory effect observed suggests the presence of antimicrobial substances in the extracts. This effect can cause lethal damage to the cell or only cause a sublethal effect or cellular stress (Busta, 1976). Different components of the vegetable could be responsible for this antimicrobial effect. In order to separate, isolate and / or concentrate the antimicrobial substances present in the extracts of the chalices of Jamaica, the extracts were separated into different compounds or groups of compounds based on their polarity; for this, the compounds were separated by column chromatography, in this way different groups of compounds or groups of fractions were obtained (collections of fractions, see methodology). Subsequently, the antimicrobial effect of the collections of fractions obtained was tested. Because the acetonic and methanolic extract had a greater antimicrobial effect than the aqueous extract, the components of the aqueous extract were not chromatographed. Table 6. Antimicrobial effect of the aqueous extract of Jamaica diluted 1:10 and that of a solution of penicillin (control) on different microorganisms Diameter Diameter Diameter Diameter

Type of inhibition inhibition inhibition inhibition microorganism Extract Extract Methanolic acetonic aqueous penicillin extract (control)

E. co // 11 * 13 * 14 25

E. co // O157: H7 11 14 14 22

£. enteroinvasive coli 11 11 13 25

£ enteropathogenic coff 10 12 11 20

E. co /; ^'

 11 12

 enterotoxigenic 12 20

S. aureus 11 13 14 26

V. cholerae OI 11 14 14 22

S. Typhimurium 12 14 14 24

S. Choleraesuis 11 12 12 20

P. aeruginosas 11 13 14 21

S. flexneri 10 12 14 22

S. sonnei 12 14 13 24

L. monocytogenes 10 ¹ 3 14 23

* (mm)

Example 3. Antimicrobial effect of different chromatographic fractions.

Table 7 shows the inhibitory effect expressed in length of the inhibition halo in millimeters (mm), which was observed in petri dishes seeded with different microorganisms, due to the effect of different collections of chromatographic fractions grouped by polarity obtained from acetonic extract of the calyces of Jamaica. It is observed that only collections II and III from acetonic extract show antimicrobial or inhibitory effect. Collection III is the one that showed the greatest antimicrobial effect (Table 7). This fraction III was used to make the mixtures or formulations that were used in the apple disinfection experiments. Table 7. Antimicrobial effect of acetonic collections Type of Collection Collection Collection Collection Collection Collection bacteria nin ll III n IV n V n VI VII

S.

 0 0 14 0 0 0 0

 Choleraesuis

 E. coli

 0 9 * 8 0 0 0 0

 0157: H7

 S. flexneri 0 19 25 0 0 0 0

 S. aureus 0 13 12 0 0 0 0

 S.

 0 22 18 0 0 0 0

 Typhimurium

 L

 monocytogen 0 18 22 0 0 0 0 en

 S. epidermis 0 13 21 0 0 0 0

 P.

 0 14 19 0 0 0 0 aeruginous

 Bordetella 0 0 12 0 0 0 0

 S. sonnei 0 11 18 0 0 0 0 * Halo of inhibition expressed in millimeters (mm); Zero (0) means that no inhibitory effect was observed.

Table 8 shows the inhibitory effect in mm of different collections of chromatographic fractions grouped by polarity obtained from the methanolic extract of the chalices of Jamaica. It is observed that all the collections caused halos of inhibition which is interpreted as the antimicrobial effect of the collections. However, IV is the collection ^WHAT _^ aosfcó- the greatest antimicrobial effect (Table 8). This fraction IV was used to make the mixtures or formulations that were used in the apple disinfection experiments. Example 4. Disinfectant potential of extracts and fractions alone or in mixtures with acetic acid, sodium hypochlorite and / or Polysorbate 80.

It was found that all treatments had antimicrobial effect with respect to the control. The data of this study are reported in Table 9. It is observed that although all treatments show antimicrobial effect only 4 combinations managed to eliminate the concentration of the levels at undetectable levels. mixtures of each pathogen: treatments 32, 33, 36 and 37 reduced 5 log concentration of both pathogens (Table 9).

Table 8. Antimicrobial effect of methanolic collections

Type of Collection Colecció Colecció Collection Colecció Colecció Collection bacteria n i n II n III IV n V n VI VII

L

 monocytogen 6 10 15 24 12 11 15 en

S. sonnei 8 11 10 30 10 12 6

 P.

 6 10 11 22 8 8 7 aeruginous

 s.

 10 12 13 36 9 13 6

 Choleraesuis

 S. fíexnerí 14 29 10 12 8

 S.

 11 32 12 10 10

 Typhimurium

 Bordetella 10 23 9 13 6

E. co // ^'

 17 20 26 14 11 6

 0157.H7

 S. aumus 13 22 17 30 11 10 7

 S.

 6 15 12 28 10 12 6 epidermidis

* Halo of inhibition expressed in millimeters (mm); Zero (0) means that no inhibitory effect was observed

In the present invention, 4 specific combinations (treatment 32, 33, 36 and 37) of three antimicrobials and one surfactant (polysorbate), the total elimination of the pathogenic microorganisms inoculated on apples was achieved; This is an example of what is currently known as multiple barrier treatment. Multiple barriers are the combination of antimicrobial treatments that enhance the overall antimicrobial effect, which results in stable, safe and safe food. For some reason, when applying acetonic extracts or fractions in or ©

 O C C

CD a. (fí (fí Q.

3 "3

 3 (ñ 3 <t> or O — i O

 OR

 3 3

1.90 ± 0.30

 1.50 ± 0.30 8 U s 3r

 or a · <± 0.30 1.50 ± 0.30 or 8

 ± 0.30 1.20 ± 0.30 3 g- (fí CD

 0.0 Q. 3 s 3r Q co

 CD

 1.40 ± 0.20 C or ± 0.30 CD

CD CD CD

It should be noted the potential role of polysorbate 80 in the observed antimicrobial effect, since being a surfactant it is possible that it has favored the emulsification of the natural wax of apples which could increase the effect of the disinfectant solution by eliminating or decreasing the protective effect that the wax would be providing inoculated microorganisms on apples

Therefore, the compositions of the present invention are an excellent alternative for the disinfection and / or preservation of foods, for example fresh foods, without altering their nutritional properties. In this sense, the compositions described here, allow the effective disinfection of pathogenic microorganisms of fruits and vegetables, preferably apples allowing the safe consumption of such products.

Example 5. Nuclear magnetic resonance (NMR) spectrum obtained from dry acetonic extract.

 The NMR spectrum obtained from the dry acetonic extract of the calyxes of Jamaica is presented in Figure 1. In the spectrum there are several characteristic peaks of the extract that we use in the formulations. This spectrum characterizes the acetonic extract used in the antimicrobial formulations in Table 9.

Example 6. Nuclear magnetic resonance (NMR) spectrum obtained from chromatographic collection III from the acetonic extract.

 From the chromatographic collection III obtained from the acetonic extract, the NMR spectrum was obtained (Figure 2). Several and different characteristic peaks of the chromatographic collection that we use in the formulations are observed. This spectrum characterizes the chromatographic collection III used in the antimicrobial formulations in Table 9. References.

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Claims

CLAIMS Having sufficiently described my invention, I consider as a novelty and therefore claim as my exclusive property, what is contained in the following clauses:

1. A solution with antimicrobial activity to disinfect and/or preserve apples (Malus domestica), characterized in that it comprises: a) Acetonic extract of Jamaican calyxes (Hibiscus sabdariffa);

b) Acetic acid;

c) Sodium Hypochlorite;

d) Polyoxyethylene Sorbitan Monooleate, or polysorbate 80 (Polysorbate 80)

2. The solution with antimicrobial activity according to claim 1, characterized in that a) acetone extract of Jamaican calyxes (Hibiscus sabdariffa) is present in a concentration between 0.01% to 10%. ^'

3. The solution with antimicrobial activity according to claim 1, characterized in that b) acetic acid is present in a concentration between

0.01 to 10%.

4. The solution with antimicrobial activity according to claim 1, characterized in that c) Sodium hypochlorite is present in a concentration between 10 to 1000 ppm.

5. The solution with antimicrobial activity according to claim 1, characterized in that d) Polyoxyethylene Sorbitan monooleate, or polysorbate 80 is present in a concentration between 0.1 to 10%.

6. The solution with antimicrobial activity according to claim 1, characterized in that the e) acetone extract of Jamaican calyxes (Hibiscus sabdariffa) presents a nuclear magnetic resonance (NMR) spectrum as seen in Figure 1.

7. The solution of claim 1, characterized in that it is presented as an aqueous formulation.

8. The solution of claim 1 in accordance with the previous claims, characterized in that where one or several parts of Jamaica (Hibiscus sabdariffa) can be used to obtain the extract.

9. The solution of claim 1 in accordance with the previous claims, wherein the part of the Jamaica plant that is used is the calyxes.

10. The solution defined in the preceding claims, characterized by a nuclear magnetic resonance (NMR) spectrum of the acetone extract obtained from the calyxes of Jamaica {Hibiscus sabdariffa) (Figure 1).

11. The solution of claim 1 in accordance with the previous claims, characterized in that it is useful as a disinfectant and preservative for foods of plant and animal origin.

12. The solution of claim 1 according to the preceding claims, characterized in that the aqueous formulation is useful as a disinfectant and preservative for fruits and vegetables, especially apples.

13. The solution of claim 1 in accordance with the previous claims, characterized in that the main varieties of apple (Malus domestica) on which it acts as a disinfectant are selected from Golden Delicious, Red Delicious, Criolla, Red Chief, Rome Beauty, Starking , Starking Delicious, Top Red, Akane, Ambrosia, Arkansas Black, Blackjohn Braeburn, Bramley, Carneo, Cortland, Cox's Orange, Pippin, Crabapple, Criterion, Egremont Russet, Empire, Esperiega, Fuji, Gala, Ginger Gold, Granny Smith, Gravenstein , Honeycrisp, Idared, Jazz, Jonagold, Jonathan, Lodi, Mclntosh, Newtown/Pippin, Old Apple, Pacific Rose, Pink lady, Pinova, Red El, Reineta, Rome Beauty, Royal Gala, Splendor, Spur, Starkrimson, Starking, Maiden Green, Willie Sharp, Winesap, Winter Banana, Worcester, Permain.

14. A method for disinfecting and/or preserving apples (Malus domestica), characterized in that it comprises applying the solution defined in the preceding claims to the apple.

15. A plant extract with antimicrobial activity to disinfect and/or preserve apples (Malus domestica), characterized in that it is obtained through the following steps: a) Place the dry plant in a container under aseptic conditions, add acetone and store at 22° ± 2 ^o C for 7 days,

b) Pass the extract through a sieve and remove the acetone from the extract, and

c) Recover the dry extract.

16. The extract of claim 15, characterized in that it is obtained with acetone.

17. The extract according to claim 15, wherein one or more parts of the plant can be used to obtain the extract.

18. The extract of claim 5, characterized in that the plant is the Jamaican plant (Hibiscus sabdariffa).

19. The extract of claim 15 in accordance with the previous claims, characterized in that the extract is obtained from the calyxes of Jamaica.

20. The extract of claim 15 in accordance with the preceding claims, characterized in that it presents a nuclear magnetic resonance (NMR) spectrum as seen in Figure 1.

21. The extract of claim 15 in accordance with the preceding claims, characterized in that it is useful as a disinfectant and preservative for foods of animal and plant origin.

22. The extract of claim 15 in accordance with the preceding claims, characterized in that it is useful as a disinfectant and preservative for fruits and vegetables, preferably apples.

23. The extract of claim 15 in accordance with the preceding claims, characterized in that the main varieties of apple (Malus domestica) on which it acts as a disinfectant are selected from Golden Delicious, Red Delicious, Criolla, Red Chief, Rome Beauty, Starking , Starking Delicious, Top Red, Akane, Ambrosia, Arkansas Black, Blackjohn Braeburn, Bramley, Carneo, Cortland, Cox's Orange, Pippin, Crabapple, Criterion, Egremont Russet, Empire, Esperiega, Fuji, Gala, Ginger Gold, Granny Smith, Gravenstein , Honeycrisp, Idared, Jazz, Jonagold, Jonathan, Lodi, Mclntosh, Newtown/Pippin, Old Apple, Pacific Rose, Pink lady, Pinova, Red El, Reineta, Rome Beauty, Royal Gala, Splendor, Spur, Starkrimson, Starking, Verde maiden, Willie Sharp, Winesap, Winter Banana, Worcester, Permain.

24. A method for the disinfection and/or preservation of apple (Malus domestica), characterized in that it comprises applying the extract defined in claims 15 to 23 to the apples.

25. A method to obtain a plant extract with antimicrobial activity to disinfect and/or preserve apple (Malus domestica), characterized in that it comprises the following steps: a) Place the dry plant in a container under aseptic conditions, add acetone and store at 22 ° ± 2 ^o C for 7 days,

b) Pass the extract through a sieve and remove the methanol from the extract, and

c) Recover the dry extract.

26. The method for obtaining the plant extract of claim 25, characterized in that the extract can have a solid or liquid presentation.

27. A method for the preparation of a solution with antimicrobial activity to disinfect and/or preserve apples (Malus domestica), characterized by comprising the steps of: a) Place dried Jamaica (Hibiscus sabdariffa) in a container under aseptic conditions, add acetone and store at 22° ± 2 ^o C for 7 days,

b) Pass the extract through a sieve and remove the acetone from the extract and,

c) Recover the dry extract,

d) Prepare the aqueous solution in a container containing: water, dry acetone extract of the plant, acetic acid, sodium hypochlorite and Polyoxyethylene Sorbitan monooleate or polysorbate 80,

28. The method according to claim 27, wherein the part of the Jamaica plant used is the calyxes.

29. A solution with antimicrobial activity to disinfect and/or preserve apples (Malus domestica), characterized in that it comprises: a) A collection of chromatographic fractions obtained from an acetonic extract of Jamaican calyxes (Hibiscus sabdariffa);

b) Acetic acid;

c) Sodium Hypochlorite;

d) Polyoxyethylene Sorbitan Monooleate, or polysorbate 80 (Polysorbate 80).

30. The solution with antimicrobial activity according to claim 29, characterized in that the a) collection of chromatographic fractions obtained from an acetonic extract of Jamaican calyxes (Hibiscus sabdariffa) is present in a concentration between 0.01% to 10%.

31. The solution with antimicrobial activity according to claim 29, characterized in that b) acetic acid is present in a concentration between 0.01 to 10%.

32. The solution with antimicrobial activity according to claim 29, characterized in that c) sodium hypochlorite is present in a concentration between 10 to 1000 ppm.

33. The solution with antimicrobial activity according to claim 29, characterized in that d) Polyoxyethylene Sorbitan monooleate, or polysorbate 80 is present in a concentration between 0.1 to 10%.

34. The solution with antimicrobial activity according to claim 29, characterized in that the e) collection of chromatographic fraction obtained from an acetone extract of Jamaican calyxes (Hibiscus sabdariffa) presents a nuclear magnetic resonance (NMR) spectrum as observed in Figure 2.

35. The solution of claim 29, characterized in that it is presented as an aqueous formulation.

36. The solution of claim 29 in accordance with the previous claims, characterized in that where one or several parts of Jamaica (Hibiscus sabdariffa) can be used to obtain the collections of chromatographic fractions.

37. The solution of claim 29 in accordance with the previous claims, wherein the part of the Jamaica plant that is used is the calyxes.

38. The solution of claim 29 according to the preceding claims, characterized by a nuclear magnetic resonance (NMR) spectrum of the collection of chromatographic fractions obtained from an acetonic extract of Jamaican calyxes (Hibiscus sabdariffa) (Figure 2).

39. The solution of claim 29 in accordance with the previous claims, characterized in that it is useful as a disinfectant and preservative for foods of plant and animal origin.

40. The solution of claim 29 according to the preceding claims, characterized in that the aqueous formulation is useful as a disinfectant and preservative for fruits and vegetables, especially apples.

41. The solution with antimicrobial activity according to claim 29, characterized in that the main varieties of apple (Malus domestica) on which it acts as a disinfectant are selected from Golden Delicious, Red Delicious, Criolla, Red Chief, Rome Beauty, Starking, Starking Delicious, Top Red, Akane, Ambrosia, Arkansas Black, Blackjohn Braeburn, Bramley, Carneo, Cortland, Cox's Orange, Pippin, Crabapple, Criterion, Egremont Russet, Empire, Esperiega, Fuji, Gala, Ginger Gold, Granny Smith, Gravenstein, Honeycrisp, Idared, Jazz, Jonagold, Jonathan, Lodi, Mclntosh, Newtown/Pippin, Old Apple, Pacific Rose, Pink lady, Pinova, Red El, Reineta, Rome Beauty, Royal Gala, Splendor, Spur, Starkrimson, Starking, Green maiden , Willie Sharp, Winesap, Winter Banana, Worcester, Permain.

42. A method for disinfecting and/or preserving apples (Malus domestica), characterized in that it comprises applying the solution defined in claims 29 to 41 to the apple.

43. A collection of chromatographic fractions with antimicrobial activity to disinfect and/or preserve apple (Malus domestica), characterized in that it is obtained through the following steps: a) Place the dry plant in a container under aseptic conditions, add acetone and store at 22 ° ± 2 ^o C for 7 days,

b) Pass the extract through a sieve and remove the acetone from the extract,

c) Recover the dry extract, d) Carry out chromatography to separate the dry acetone extract into chromatographic fractions using solvents and mixtures of solvents of different polarities,

e) Recover chromatographic fractions of different polarities in containers, f) Remove the solvent from the fractions,

g) Group or collect the fractions in containers to form groups (collections) of equal or similar polarities,

h) Carry out microbiological tests with the collections,

i) Recover collections of fractions with antimicrobial activity

44. The collection of chromatographic fractions according to claim 43, wherein one or more parts of the plant can be used to obtain the collection of chromatographic fractions.

45. The collection of chromatographic fractions of claim 43 in accordance with the preceding claims, characterized in that the plant is Jamaica (Hibiscus sabdariffa).

46. The collection of chromatographic fractions of claim 43 in accordance with the previous claims, characterized in that the fractions are obtained from the calyxes of Jamaica.

47. The collection of chromatographic fractions with antimicrobial activity of claim 43 in accordance with the preceding claims, characterized in that the collection presents a nuclear magnetic resonance (NMR) spectrum as seen in Figure 2.

48. The collection of chromatographic fractions of claim 43 in accordance with the preceding claims, characterized in that it is useful as a disinfectant and preservative for foods of animal and plant origin.

49. The collection of chromatographic fractions of claim 43 in accordance with the preceding claims, characterized in that it is useful as a disinfectant and preservative for fruits and vegetables, preferably apples.

50. The collection of chromatographic fractions with antimicrobial activity according to claim 43 in accordance with the previous claims, characterized in that the main varieties of apple (Malus domestica) on which it acts as a disinfectant are selected from Golden Delicious, Red Delicious, Criolla , Red Chief, Rome Beauty, Starking, Starking Delicious, Top Red, Akane, Ambrosia, Arkansas Black, Blackjohn Braeburn, Bramley, Carneo, Cortland, Cox's Orange, Pippin, Crabapple, Criterion, Egremont Russet, Empire, Esperiega, Fuji, Gala , Ginger Gold, Granny Smith, Gravenstein, Honeycrisp, Idared, Jazz, Jonagold, Jonathan, Lodi, Mclntosh, Newtown/Pippin, Oíd Apple, Pacific Rose, Pink lady, Pinova, Red El, Reineta, Rome Beauty, Royal Gala, Splendor , Spur, Starkrimson, Starking, Maiden Green, Willie Sharp, Winesap, Winter Banana, Worcester, Permain.

51. The method to disinfect and/or preserve fruits and vegetables, preferably apples with the collection of chromatographic fractions with antimicrobial activity of the Jamaican calyx extract defined in claims 43 to 50.

52. A method to obtain collections of chromatographic fractions with antimicrobial activity from a plant extract to disinfect and/or preserve apple (Malus domestica), characterized in that it comprises the following steps: a) Placing the dry plant in a container under aseptic conditions , add acetone and store at 22° ± 2 ^o C for 7 days,

b) Pass the extract through a sieve and remove the acetone from the extract,

c) Recover the dry extract,

d) Carry out chromatography to separate the dry acetone extract into chromatographic fractions using solvents and mixtures of solvents of different polarities,

e) Recover chromatographic fractions of different polarities in containers, f) Remove the solvent from the fractions, g) Group or collect the fractions in containers to form groups (collections) of equal or similar polarities,

h) Carry out microbiological tests with the collections,

i) Recover the collections of fractions with antimicrobial activity.

53. The method according to claim 52, characterized in that the collections of fractions can have a solid or liquid presentation.

54. A method for the preparation of a solution with antimicrobial activity to disinfect and/or preserve apple (Malus domestica), characterized by comprising the steps of: a) Placing the dried Jamaica plant (Hibiscus sabdariffa) in a container under aseptic conditions , add acetone and store at 22° ± 2 ^o C for 7 days, b) Pass the extract through a sieve and remove the acetone from the extract,

c) Recover the dry extract,

d) Carry out chromatography to separate the dry acetone extract into chromatographic fractions using solvents and mixtures of solvents of different polarities,

e) Recover chromatographic fractions of different polarities in containers, f) Remove the solvent from the fractions,

g) Group or collect the fractions in containers to form groups (collections) of equal or similar polarities,

h) Carry out microbiological tests with the collections,

i) Recover the collections of fractions with antimicrobial activity, j) Prepare the aqueous solution in a container containing: water, collections of chromatographic fractions with antimicrobial activity, acetic acid, sodium hypochlorite and Polyoxyethylene Sorbitan monooleate or polysorbate 80.

55. The method according to claim 54, wherein the part of the Jamaica plant used is the calyxes.