WO2015088309A1 - Phytochemical disinfectants for lettuce (lactuca sativa) - Google Patents

Abstract

The invention relates to compositions for effectively disinfecting and/or preserving food of plant origin contaminated by pathogenic and destructive micro-organisms. The aforementioned compositions contain plant-derived extracts with antimicrobial activity, which can act either alone or in combination 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 lettuce, including by pathogenic micro-organisms, without altering the nutritional and/or dietary and/or sensory properties thereof.

Description

 FITOQUÍ ICOS DISINFECTANTS FOR LECHUGA (Lactuca sativa)

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the development of formulations containing phytochemicals 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 fruits and vegetables, but with the most effective effectiveness for lettuces (Lactuca sativa)

STATE OF THE TECHNIQUE

Lettuce is an agricultural product widely consumed throughout the world. In Mexico it is one of the main agricultural products, with a little more than 17 thousand hectares dedicated to its sowing. The most important types of lettuce that are sown, both in the open field and in protected agriculture, are: Orejona (the one that is most produced), Romana, Iceberg, Boston, Italian, Cos, Baby, Butterhead, Cutting, Stalk, Latin among others (Sagarpa, 2013).

However, along with the increase in the consumption of lettuce, there have been outbreaks of diseases caused by bacteria associated with the consumption of lettuce (CDC, 2012). For example, in 2012 lettuce 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 Escherichia coli 0157: H7 (CDC, 2012).

This type of outbreaks has caused strict regulation for producers that export lettuce to the United States (USA); and has often meant retention of lettuce shipments at the border, partial or total closure of the export of this product to the USA and lost economic by producers to 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 lettuce, due to the poor hygiene practices that generally occur during the cultivation, harvest, transport and marketing of lettuces, participation is expected of these raw products in disease outbreaks. A fact that supports this observation is the frequency with which Salmonella strains and Escherichia coli pathogenic groups have recently been isolated from lettuce salads ready for consumption in Mexico (Castañeda-Ramírez et al., 2011; Castro- Rosas et al., 2012).

Recent outbreaks of food diseases associated with lettuce created the need to determine sources of lettuce contamination and understand the survival and / or growth of pathogenic microorganisms in lettuce; These have led to the development of innovative control technologies. In general, the pathogens in lettuce 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 lettuce to low temperature. However, lettuce disinfection has been identified as the most important stage for the microbial safety of raw lettuce.

Usually lettuces 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 lettuce 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 lettuce. The prevention of lettuce 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 lettuce is affected by the location of the pathogen in the product, the quality of the lettuce, storage temperature, type of packaging, and relative humidity. Lettuce leaves usually have scarce nutrients on their surface 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: H7 are able to survive for a long time on the surface of lettuce leaves both in refrigeration and at room temperature (Changa and Fang, 2007; Kroupitski et al ., 2009). Also, once on the lettuce, pathogenic bacteria such as Salmonella could produce extracellular polymers in the leaves of the lettuce, 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 able to multiply in chopped or cut lettuce by significantly increasing their concentration and making the food much more dangerous (Changa and Fang, 2007).

Pathogenic microorganisms on the surface of lettuce leaves can contaminate the internal tissues and infiltrate and later during the cutting of the leaves they could 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 lettuce leaves (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 product Vegetable and washing water and 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 wash water of lettuces is the control of the spread of pathogens, their inactivation and / or prevent their infiltration to lettuce leaves. However, currently available chemical disinfectants have limited benefits over plant products, such as lettuce. 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, it should be borne in mind that among the microorganisms it is possible to select strains with increasing resistance to the effect of an agent specific germicide 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 log ₁₀ ; the decrease of S. typhio from V. cholerae 01 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 Log ₁₀ 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-spore-forming bacteria and have a low effect against mycobacteria (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 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 lettuce. 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 a concentration of 1-2%, 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. The 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 al., 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 lettuce growers in the field, as producers generally sell their product packed in cardboard or wood boxes, among other things for ease and to avoid accumulation. and humidity what would happen if it They will use plastic bags. 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 0.87-2.93, 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 E. coli 0157: H7 and enteric Salmonella which were of 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, Extractos Vegetales SA) on the survival of L. monocytogenes, P. fluorescens, E. coli, Erwinia caratovora and S. Typhimurium in lettuce 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 the surface of lettuce and tomato. AITC was more effective against Salmonella and E. coli, achieving 8 Log reduction with a generated steam treatment of 400 μΙ of AITC after 4 and 2 days, respectively on lettuce. 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 plant antimicrobial urv to a food or in vitro, 10 to 100 times the concentration of antimicrobial is needed than that observed in vivo. For example, the essential oil of peppermint (Mentha piperita) has demonstrated the growth of Salmonella enteritidis and L monocytogenes in amphitic 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 ° β ίιίβ variable (Roller, 2003). Salmonella enterítidis was eliminated in a ^ esÉstoS, under all the conditions examined but not when it was inoculated in-peté 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 E. coli, Salmonella sp., L. monocytogenes and Staphylococcus aureus were inhibited by the essential oil of oregano in culture broths. However, when these oils: s¾ tested in foods such as eggplant, taramasalata or mayonnaise, reactions such as increased pH, increased temperature and in the case of emulsions separated from the oil were observed used (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 Escherichia coli 0157.Ή7, 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 antibiotics such as penicillin.

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 et al., 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 extract 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 ethanol extraction 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 africanas 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 (Hi Bscus ^ sÉsúariffa, Psidium guctjava, Púnica granatum, Spondias pinnata and Tamarie0 Í d¡ca) have an antimicrobial function This document does not provide any experimental evidence that involves extracts of eus sabdariffa for the same use that is given to extracts of Agapanthus africanus.

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 lettuce, for the purpose of preserving them without taking 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 ρ¾¾ efficiently disinfect and without damage to the product as described here, and that at the same time allowed to preserve the nutritional properties of fruits and vegetables and not affect, for example the quality of lettuce, whereby it is possible with the present invention to obtain microbiologically harmless raw lettuce. BRIEF DESCRIPTION OF THE FIGURES

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

PROTON ( ¹ H) of the dry Aqueous extract obtained from the Jamaican calyces 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 of lettuce (Lactuca sativa) even under extreme conditions of treatment, but which do not sensorially damage the food.

The present invention relates to solutions 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 plant foods, particularly disinfection and / or preservation of lettuce (Lactuca sativa).

One embodiment of the present invention relates to obtaining a vegetable preparation comprising aqueous extracts of the chalices of the flower of Jamaica (Hibiscus sabdariffa) which are useful for eliminating pathogens present in food (disinfectant effect) and for delaying food spoilage 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) that have a food disinfectant and preservative function together with other compounds that have disinfectant properties such as 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) that have a disinfectant or conservative effect when applied to foods of plant origin, preferably lettuce.

Another embodiment of the present invention is the development of a method for obtaining the aqueous extract from calyxes of Jamaica, an extract that turns out to be useful as a disinfectant and food preservative.

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 may be useful in the preparation of an efficient disinfectant to eliminate pathogenic bacteria present in raw plant-based foods, such as lettuce. In the present invention although three types of extracts of Jamaican chalices, one aqueous, one methanolic and the other acetonic, are described, specifically the ACUOSO can be used as a disinfectant and / or food preservative due to its efficiency in the elimination of pathogenic bacteria from raw plant-based foods such as lettuce.

Unlike other compositions known so far for the same purpose, the compositions of the present invention are capable of eliminating pathogenic bacteria present in raw plant-based foods, such as lettuce per se, without altering their nutritional properties as well as Product quality features. Consequently, the application of the compositions of the present invention in raw plant-based foods 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 aqueous and acetonic extracts of Jamaica flower calyces, either alone or in combination with other components with proven disinfectant activity, such as for example organic acids which include acetic acid and chlorine compounds that include sodium hypochlorite. In the case of the disinfection of raw plant-based foods such as lettuce, the compositions of the invention that include a mixture of calyx extracts of the Jamaican flower as well as acetic acid and sodium hypochlorite and polysorbate, which usually be very effective to eliminate microorganisms resident in the plant, while achieving that their organoleptic and / or nutritional properties are not affected and without altering, for example the commercial quality of lettuce.

For the purposes of the present invention, the compositions described herein comprise: a) A plant-derived methanolic extract, which exhibit antimicrobial properties, such as extracts derived from chalices of the Jamaican flower (Hibiscus sabdariff), b) 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%,

 c) 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%, Y

 d) A surfactant with emulsifying activity of natural fats or waxes found in tomatoes, such as 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 purposes of the invention, the compositions are added to 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 in disinfectant solutions. or by means of 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 mL 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 obtaining the desired disinfectant and / or preservation effect on 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, for later store them at room temperature, so they are ready to apply to food when deemed necessary.

For the purposes of the invention, the compositions described must be in admixture with plant extracts with antimicrobial activity, such as extracts derived from Jamaican chalices, which are brought into contact with food, for example, raw plant-based foods such as the lettuce, in order to disinfect and / or preserve them. In the present invention, the disinfectant activity of extracts derived from Jamaican chalices is described, in the disinfection and / or preservation of foods, for example to raw plant-based foods, so they can be used directly or as part of compositions that contain them. In this sense, extracts derived from the chalices of Jamaica can be added or contacted with 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 lettuce, 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 can be obtained by the following method: a) Place the dried plant in a container under aseptic conditions, add water, in a 1: 9 ratio; preferably 100 g of the dried plant are placed in a container (flask) under aseptic conditions, boil at 90-100 ° C for 15-30 minutes and let cool to 20-30 ° C; b) Remove the calyces and recover the aqueous 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 water 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.

Obtained extracts these are stored at room temperature until use.

Once the extracts have been 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 form them to form solutions. and / or suspensions to be applied subsequently 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 the disinfection and / or preservation of food, particularly fruits and vegetables, as per example lettuce. As can be seen later, the compositions of the invention are capable of disinfecting and / or eliminating microorganisms present in lettuces in a very efficient manner, so that it is possible to have microbiologically safe and safe lettuces 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 lettuces (Lactuca sativa) they were used of the Iceberg variety. Lettuces of a physiological size and condition or uniform age were obtained.

1.2. Bacterial strains

 Strains of E. coli 0157.Ή7 (P1C6, isolated from an outbreak of disease), enteroinvasive E. coli (4VC81-5, isolated from clinical case) E. enterotoxigenic coli (1620 TL, isolated from clinical case), E were used 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 (ATCC 12228), Staphylococcus (ATCC 12228) ATCC 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 (+) to eliminate interference from the native microbial flora of the extract (Castro-Rosas and Escartin, 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 calyces 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 chalices 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 were 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. Determination of the antimicrobial activity of aqueous, methanolic and acetonic extracts of Jamaican calyces in culture medium (in vitro studies).

1.5.1. 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.

1.5.2. Preparation of the solutions of the extracts. From the dried extracts, 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 were solubilized in distilled water while the acetonic extracts were solubilized in the solution of Polysorbate 80: water. Water or polysorbate 80: water were added dry extracts in a ratio 1:10 (water: extract) separately and deposited in sterile bottles. 5.3 Antimicrobial effect of extracts in culture medium.

 Separately, 100 pL 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 al aliquots of the extract solution (aqueous, methanolic or acetonic) 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.

1.6. Evaluation of the antimicrobial effect of the aqueous, methanolic, acetonic, acetic acid, hypochlorite and specific formulations in the reduction of Salmonella and E. co /; ^" 0157: H7 in contaminated lettuce.

1.6.1. Preparation of disinfectant solutions.

Solutions of Jamaican calyx extracts as well as mixtures containing extracts, acetic acid and hypochlorite were prepared at the concentrations, proportions or mixtures described in Table 1. For example, to prepare 100 ml of a solution containing methanolic extract of 1% Jamaica calyces, 0.1% acetic acid and 100 mg / L hypochlorite: 100 g of dried methanolic extract of Jamaican calyces was added to 100 mL of distilled water, in addition to 1 ml of a 10% acetic acid solution % and 0.2 ml of a 5% hypochlorite solution. To these solutions were added or not the surfactant polysorbate 80 (also known as tween 80) at the rate described in table 1.

1.6.2. Strains

 For these studies, we worked with 7 Salmonella serotypes: (3 typhimurium [ATCC 14028, one isolated from tomato, J1, and another from alfalfa seed, GA1], Salmonella choleraesuis [ATCC 10708], typhi, gaminara, and montevideo) and 3 of E. coli 0157: H7 (two isolated in our laboratory from raw meat [P1C6 and M5C8] and another isolated from an outbreak caused by meat consumption in the United States of America [E09]). All strains were marked with resistance to the antibiotic nfampicin (R +) to eliminate interference from the native microbial flora of the extract (Castro and Escartín, 2000).

Table 1. Treatments to which the lettuce leaves contaminated with the mixtures of Salmonella strains or E coli strains 0157: H7 were subjected separately

 No Treatments

 1 No treatment (control)

 2 1% aqueous extract

 3 1% acetonic extract

 4 1% methanolic extract

 5 Sodium hypochlorite 50 ppm

 6 Sodium hypochlorite 100 ppm

 7 Acetic acid 0.1%

 8 Acetic acid 0.5%

 9 1% aqueous extract + 0.1% acetic acid

 10 1% aqueous extract + 0.5% acetic acid

 11 Acetonic extract 1% + acetic acid 0.1%

 12 Acetonic extract 1% + acetic acid 0.5%

 13 1% methanolic extract + 0.1% acetic acid

 14 1% methanolic extract + 0.5% acetic acid

 15 1% aqueous extract + 0.1% acetic acid + sodium hypochlorite 50 ppm

 16 1% aqueous extract + 0.5% acetic acid + 50 ppm sodium hypochlorite

 17 1% aqueous extract + 0.1% acetic acid + 100 ppm sodium hypochlorite

 18 1% aqueous extract + 0.5% acetic acid + 100 ppm sodium hypochlorite

 19 Acetonic extract 1% + acetic acid 0.1% + sodium hypochlorite 50 ppm

 20 Acetonic extract 1% + acetic acid 0.5% + sodium hypochlorite 50 ppm

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

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

 23 Methanol extract 1% + acetic acid 0.1% + sodium hypochlorite 50 ppm

 24 1% methanolic extract + 0.5% acetic acid + sodium hypochlorite 50 ppm

25 1% methanolic extract + 0.1% acetic acid + sodium hypochlorite 100 ppm 26 1% methanolic extract + 0.5% acetic acid + 100 ppm sodium hypochlorite

 27 Aqueous extract 1% + acetic acid 0.1% + sodium hypochlorite 50 ppm + Polysorbate 80 2%

 28 Aqueous extract 1% + acetic acid 0.5% + sodium hypochlorite 50 ppm + Polysorbate 80 2%

 29 1% aqueous extract + 0.1% acetic acid + 100 ppm sodium hypochlorite + 802% Polysorbate

 30 Aqueous extract 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm + Polysorbate 80 2%

 31 Acetonic extract 1% + acetic acid 0.1% + sodium hypochlorite 50 ppm + Polysorbate 80 2%

 32 Acetonic extract 1% + acetic acid 0.5% + sodium hypochlorite 50 ppm + Polysorbate 80 2%

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

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

 35 Methanol extract 1% + acetic acid 0.1% + sodium hypochlorite 50 ppm + Polysorbate 80 2%

 36 Methanol extract 1% + acetic acid 0.5% + sodium hypochlorite 50 ppm + Polysorbate 80 2%

 37 1% methanolic extract + 0.1% acetic acid + 100 sodium hypochlorite

 ppm + Polysorbate 80 2%

38 1% methanolic extract + 0.5% acetic acid + 100 sodium hypochlorite

 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 hypochlorite 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

1.6.3. 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; 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. 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. cotí 0 57: H7, to have a mixture of the three strains of E coli 0157: H7.

1.6.4. Lettuce Inoculation Lettuces of the Iceberg varieties were used; Lettuces were obtained from a local producer. Prior to inoculation, the leaves of the lettuce were separated and gently cleaned with a clean cloth to remove dust particles. Whole lettuce leaves of a uniform or similar size were used and did not show visible damage. Separately, the lettuce leaves were inoculated individually by placing in 10 different central parts of each leaf 10 pL of a suspension of each type of pathogenic bacterium mixture {Salmonella or E. coli 0157: H7) containing approximately 1 x 10 ⁷ UFC, the inoculated leaves 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 or common conditions of contamination.

16.5. Disinfection treatment of lettuce leaves.

 After two hours in the bioclimatic chamber, each leaf was washed separately to remove microorganisms that did not adhere, the washing consisted of submerging and shaking the inoculated parts of the lettuce in distilled water for 10 s, the part was allowed to drain washed at room temperature until complete dryness and subsequently, separately, the inoculated part of the lettuces was immersed for 10 min in the different disinfectant solutions indicated in Table 1. A treatment with distilled water alone served as a positive control.

1.6.6. Counting of microorganisms surviving the treatments

After treatment, the lettuce leaves 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 parts were cut with the help of a sterile scalpel, the portions of each lettuce they were placed together 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 portion of inoculated lettuce from the outside of the bag for 2 minutes. After this time, each bag was counted using the plate pouring technique using agar for methods Standard (Bioxon, Mexico) added 100 mg / L of Rifampicin (Sigma, Mexico), the boxes were incubated at 35 ° C / 24-48 h. This procedure was performed in duplicate for each replica. Each treatment was carried out in quintuplicate.

1.6.7. 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.

1.7. Nuclear Magnetic Resonance (NMR) of extracts

The proton NMR spectrum ( ¹ H) of both the dry aqueous extract obtained from the calyxes of Jamaica was determined. The extracts were solubilized in deuterated water. NMR spectra were obtained using a nuclear magnetic resonance spectrometer (Varian 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 the excreta of the chalices of Jamaica.

The three types of extracts (aqueous methanolic and acetonic) showed a marked antimicrobial effect (Table 2). All the microorganisms tested were inhibited from the first moments of contact. However, it was the methanolic extract that showed the highest antimicrobial activity (Table 2). And the one that showed the least effect was the aqueous extract. 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. It is possible that the chalices of Jamaica contain different antimicrobials; that is, the antimicrobial effect may be due to the effect of different molecules with antimicrobial activity, this observation is based on the fact that the extracts were obtained with three solvents of different polarity, and the three types of extracts showed antimicrobial activity.

Table 2. Antimicrobial effect of the aqueous extract of Jamaica diluted 1:10 and that of a solution of penicillin (control) on different microorganisms

 * (mm)

 Example 3. Disinfectant potential of the extracts alone or in mixtures with acetic acid, sodium hypochlorite and / or Polysorbate 80.

It was found that all treatments had an antimicrobial effect with respect to the control (Tables 3). However, only 3 combinations (28, 29 and 30) were 100 percent effective (Tables 3) to disinfect the lettuce. It was with the combination that the aqueous extract of the chalices of Jamaica was included. For some reason, when applying the aqueous extracts in combination with the other components of treatments 28, 29 and 30, directly on the lettuce, these Table 3. Concentration of £ coli 0157: H7 and S. Typhimurium in lettuce at the beginning and after different treatment

 £ coli 0157: H7 Salmonella

 Treatment

 Number Number Number Number

Initial Final Initial Final

1 No treatment (control) 4.80 ± 0.20 "4.60 ± 0.30 5.0010.30 4.8010.20

2 Aqueous extract 1% 4.80 ± 0.20 3.20 ± 0.30 5.0010.30 3.00 ± 0.20

3 Acetonic extract 1% 4.80 ± 0.20 3.00 ± 0.30 5.00 ± 0.30 3.00 ± 0.20

4 Methanol extract 1% 4.80 ± 0.20 3.10 ± 0.30 5.00 ± 0.30 3.10 ± 0.20

5 Sodium hypochlorite 50 ppm 4.80 ± 0.20 3.20 ± 0.30 5.00 ± 0.30 3.60 ± 0.20

6 Sodium hypochlorite 100 ppm 4.80 ± 0.20 2.80 ± 0.40 5.00 ± 0.30 3.30 ± 0.20

7 Acetic acid 0.1% 4.80 ± 0.20 3.20 ± 0.30 5.00 ± 0.30 3.20 ± 0.30

8 Acetic acid 0.5% 4.80 ± 0.20 3.00 ± 0.20 5.0010.30 3.0010.30

9 1% aqueous extract + 0.% acetic acid 4.8010.20 2.80 ± 0.30 5.0010.30 2.9010.40

10 1% aqueous extract + 0.5% acetic acid 4.80 ± 0.20 2.3010.20 5.0010.30 2.20 0.40

11 Acetonic extract 1% + acetic acid 0.1% 4.80 ± 0.20 2.60 ± 0.20 5.0010.30 2.6010.20

12 Acetonic extract 1% + acetic acid 0.5% 4.80 ± 0.20 2.20 ± 0.30 5.0010.30 2.2010.20

13 1% methanolic extract + 0.1% acetic acid 4.80 ± 0.20 2.5010.30 5.0010.30 2.5010.30

14 1% methanolic extract + 0.5% acetic acid 4.80 ± 0.20 2.20 ± 0.20 5.0010.30 2.20 ± 0.30

15 1% aqueous extract + 0.1% acetic acid + sodium hypochlorite 50 ppm 4.80 ± 0.20 2.20 ± 0.30 5.00 ± 0.30 2.00 ± 0.20

16 1% aqueous extract + 0.5% acetic acid + sodium hypochlorite 50 ppm 4.80 ± 0.20 2.10 ± 0.30 5.00 ± 0.30 1.80 ± 0.20

17 1% aqueous extract + 0.1% acetic acid + sodium hypochlorite 100 ppm 4.80 ± 0.20 2.00 ± 0.20 5.00 ± 0.30 1.90 ± 0.30

18 1% aqueous extract + 0.5% acetic acid + sodium hypochlorite 100 ppm 4.80 ± 0.20 1.90 ± 0.30 5.0010.30 2.00 ± 0.20

19 1% acetonic extract + 0.1% acetic acid + sodium hypochlorite 50 ppm 4.80 ± 0.20 1.80 ± 0.30 5.0010.30 2.0010.30

20 1% acetonic extract + 0.5% acetic acid + sodium hypochlorite 50 ppm 4.80 ± 0.20 1.90 ± 0.30 5.0010.30 1.9010.30

21 1% acetonic extract + 0.1% acetic acid + sodium hypochlorite 100 ppm 4.80 ± 0.20 1.40 ± 0.30 5.0010.30 1.6010.30

22 1% acetonic extract + 0.5% acetic acid + sodium hypochlorite 100 ppm 4.80 ± 0.20 1.20 ± 0.30 5.0010.30 2.2010.30

23 1% methanolic extract + 0.1% acetic acid + sodium hypochlorite 50 ppm 4.80 ± 0.20 1.80 ± 0.30 5.0010.30 2.0010.30

24 1% methanolic extract + 0.5% acetic acid + sodium hypochlorite 50 ppm 4.80 ± 0.20 1.9010.30 5.0010.30 1.80 ± 0.20

25 1% methanolic extract + 0.1% acetic acid + sodium hypochlorite 100 ppm 4.80 ± 0.20 1.2010.30 5.0010.30 1.4010.20

26 1% methanolic extract + 0.5% acetic acid + sodium hypochlorite 100 ppm 4.80 ± 0.20 1.4010.40 5.0010.30 1.2010.20

27 1% aqueous extract + 0.1% acetic acid + 50 ppm sodium hypochlorite + 802% Polysorbate 4.80 ± 0.20 1.2010.40 5.00 ± 0.30 1.1010.30

28 1% aqueous extract + 0.5% acetic acid + 50 ppm sodium hypochlorite + 802% Pollsorbate 4.80 ± 0.20 0.00 5.0010.30 0.00

29 1% aqueous extract + 0.1% acetic acid + 100 ppm sodium hypochlorite + 802% Polysorbate 4.8010.20 0.00 5.0010.30 0.00

30 1% aqueous extract + 0.5% acetic acid + 100 ppm sodium hypochlorite + 802% polysorbate 4.8010.20 0.00 S.0010.30 0.00

31 Acetonic extract 1% + acetic acid 0.1% + sodium hypochlorite 50 ppm + Polysorbate 802% 4.80 ± 0.20 1.7010.20 5.00 ± 0.30 1.7010.30

32 Acetonic extract 1% + acetic acid 0.5% + sodium hypochlorite 50 ppm + Polysorbate 802% 4.80 ± 0.20 1.5010.20 5.00 ± 0.30 1.1010.30

33 Acetonic extract 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm + Polysorbate 802% 4.80 ± 0.20 1.7010.20 5.00 ± 0.30 1.8010.20

34 Acetonic extract 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm + Polysorbate 802% 4.80 ± 0.20 1.6010.20 5.00 ± 0.30 1.7010.20

35 Methanolic extract 1% + acetic acid 0.1% + sodium hypochlorite SO ppm + Polysorbate 802% 4.80 ± 0.20 1.5010.20 4.0010.30 1.8010.30

36 Methanolic extract 1% + acetic acid 0.5% + sodium hypochlorite 50 ppm + Polysorbate 802% 4.8010.20 1.4010.20 4.0010.30 1.20 ± 0.20

37 Methanolic extract 1% + acetic acid 0.1% + sodium hypochlorite 100 ppm + Polysorbate 802% 4.80 ± 0.20 1.30 ± 0.20 4.0010.30 1.40 ± 0.20

38 Methanolic extract 1% + acetic acid 0.5% + sodium hypochlorite 100 ppm + Poorsorbate 802% 4.80 ± 0.20 1.20 ± 0.20 4.0010.30 1.10 ± 0.20

Colony Formation Units / inoculated portion, * Average of studies per quintuple, i: standard deviation

In Table 3, it is observed that only 3 combinations managed to eliminate the concentration of the mixtures of each pathogen at undetectable levels: treatments 28, 29, 30 reduced the concentration of both types of pathogens by 5 log (Table 3)

In the present invention, 3 specific combinations (treatment 28, 29, 30) of three antimicrobials and one surfactant (polysorbate), the total elimination of the pathogenic microorganisms inoculated on the leaves of the whole lettuce 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.

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 the lettuce leaves which could increase the effect of the disinfectant solution by eliminating or decrease the protective effect that the wax would be providing the inoculated microorganisms on lettuce leaves

Therefore, the compositions of the present invention are an excellent alternative for the disinfection and / or preservation of foods, for example raw plant-based foods, without altering their nutritional properties. In this sense, the compositions described herein allow the effective disinfection or inactivation of pathogenic microorganisms of raw plant-based foods, preferably lettuce, allowing the safe consumption of such products.

Example 4. Nuclear magnetic resonance (NMR) spectrum obtained from the aqueous extract.

The NMR spectrum obtained from the dry aqueous extract of the chalices 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 aqueous extract used in the antimicrobial formulations in Table 3. 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 lettuce (Lactuca sativa), characterized in that it comprises: a) Aqueous extract of Jamaican calyces (Hibiscus sabdariffa);  b) Acetic acid;  c) Sodium hypochlorite;  d) Sorbitan Polyoxyethylene Monooleate, or Polysorbate 80 (Polysorbate 80) 2. The solution with antimicrobial activity according to claim 1, characterized in that the a) aqueous extract of Jamaican chalices (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 the 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 the 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 the 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) aqueous extract of Jamaican chalices (Hibiscus sabdanffa) presents a nuclear magnetic resonance (NMR) spectrum as seen in Figure 1. 7. The solution of claim 1, characterized in that it has a presentation as an aqueous formulation. 8. The solution of claim 1 according to the preceding claims, characterized in that where one or more parts of Jamaica (Hibiscus sabdanffa) can be used to obtain the extract. 9. The solution of claim 1 according to the preceding claims, wherein the part of the Jamaica plant that is employed is the calyces. 10. The solution defined in the preceding claims, characterized by a nuclear magnetic resonance (NMR) spectrum of the aqueous extract obtained from the chalices of Jamaica (Hibiscus sabdanffa) (Figure 1). 11. The solution of claim 1 according to the preceding claims, characterized in that it is useful as a disinfectant and preservative of 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 of fruits and vegetables, especially lettuce. 13. The solution of claim 1 according to the preceding claims, characterized in that the main varieties of lettuce {Lactuca sativa) on which it acts as a disinfectant are selected from Orejona, Romana, Iceberg, Boston, Italian, Cos, Baby, Butterhead , Cutting, Stalk, Latin. 14. A method for the disinfection and / or preservation of lettuce (Lactuca sativa), characterized in that it comprises applying to the lettuce the solution defined in the preceding claims. 15. A plant extract with antimicrobial activity to disinfect and / or preserve lettuce (Lactuca sativa), characterized in that it is obtained through the following stages: a) Place the dried plant in a container under aseptic conditions, add water, boil for 20 minutes and allow to cool to 22 ° ± 2 ^o C,  b) Pass the extract through a sieve and remove the water from the extract, and  c) Recover the dry extract. 16. The extract of claim 15, characterized in that it is obtained with water. 17. The extract according to claim 15, wherein where one or more parts of the plant can be used to obtain the extract. 18. The extract of claim 15, characterized in that the plant is the Jamaica plant (Hibiscus sabdariffa). 19. The extract of claim 15 according to the preceding claims, characterized in that the extract is obtained from the calyces of Jamaica. 20. The extract of claim 15 according to the preceding claims, characterized in that it has a nuclear magnetic resonance (NMR) spectrum as seen in Figure 1. 21. The extract of claim 15 according to the preceding claims, characterized in that it is useful as a disinfectant and preservative of foods of animal and plant origin. 22. The extract of claim 15 according to the preceding claims, characterized in that it is useful as a disinfectant and preservative of fruits and vegetables, preferably lettuce. 23. The extract of claim 15 according to the preceding claims, characterized in that the main varieties of lettuce (Lactuca sativa) on which it acts as a disinfectant are selected from Orejona, Romana, Iceberg, Boston, Italian, Cos, Baby, Butterhead , Cutting, Stalk, Latin. 24. A method for the disinfection and / or preservation of lettuce (Lactuca sativa), characterized in that it comprises applying the extract defined in claims 15 to 23 to lettuce. 25. A method to obtain a plant extract with antimicrobial activity to disinfect and / or preserve lettuce (Lactuca sativa), characterized in that it comprises the following stages: a) Place the dried plant in a container under aseptic conditions, add water, boil for 20 minutes and let cool to 22 ° ± 2 ° C,  b) Pass the extract through a sieve and remove the water from the extract, and  c) Recover the dry extract. 26. The method of 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 lettuce (Lactuca sativa), characterized by comprising the steps of: a) Place dried Jamaica (Hibiscus sabdariffa) in a container under aseptic conditions, add water , boil for 20 minutes and let cool to 22 ° ± 2 ^o C, b) Pass the extract through a sieve and remove the water from the extract and, c) Recover the dry extract,  d) Prepare the aqueous solution in a container containing: water, dry aqueous 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 calyces.