Phytochemical compositions used as disinfectants and food preservatives

Abstract

The present invention describes compositions for disinfection and/or effective preservation of food, for example fresh food, which allows the effective removal of microorganisms from these. The disclosed compositions contain derived extracts from plants with antimicrobial activity, which can act on their own or in combination with other disinfecting agents such as organic acids and chlorine compounds. The compositions of the invention are able to remove microbial contamination, including pathogenic microorganisms in seeds and sprouts obtained therefrom without changing the germination properties and nutritional and/or alimentary properties of the sprouts.

Claims

1. A method for disinfecting contaminated seeds comprising applying an effective amount of a composition to seeds in need thereof, wherein said composition comprises: (a) 1% hibiscus sabdariffa L. calyxes extract; (b) 0.1% acetic acid (c) 0.1 to 1.0% sodium hypochlorite, wherein the seeds are contaminated with foodborne bacteria selected from the group consisting of salmonella and E. coli 0157:H7, wherein the seeds are 70 to 90% germinated, and wherein the composition is administered to the seeds to effectively eliminate the contamination and obtain sprouts without the foodborne bacteria.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Water-treated soybean seeds are shown.

(2) FIG. 2. Soybean seeds treated with the vegetable extract from the invention are shown.

(3) FIG. 3. Soybean seeds treated with a 100 ppm sodium hypochlorite solution are shown.

(4) FIG. 4. Soybean seeds treated with a 10,000 ppm sodium hypochlorite solution are shown.

(5) FIG. 5. Soybean seeds treated with a 0.1% w/w acetic acid solution are shown.

(6) FIG. 6. Soybean seeds treated with a 100 ppm sodium hypochlorite solution and 0.1% w/w acetic acid are shown.

DETAILED SPECIFICATION OF THE INVENTION

(7) This invention describes compositions containing phytochemicals from plant extracts used as disinfectants and/or preservatives for food of vegetable and animal origin. For instance, they are aimed to be used for fresh food disinfection and preservation, particularly for the disinfection and/or preservation of sprouts derived from edible seeds.

(8) One modality of this invention refers to obtaining a vegetable preparation containing a plant extract useful for eliminating pathogenic agents found on foods (disinfectant effect) and for delaying food deterioration or preserving their innocuousness (preservative effect).

(9) Another modality of this invention refers to obtaining plant-derived extracts used as disinfectants against pathogenic agents found on foods, and to delay food deterioration and/or preserve their innocuousness, i.e. as food preservatives, offering an alternative to traditional disinfectants that might be toxic to humans, animals or the environment.

(10) Another modality of this invention refers to the elaboration of compositions containing plant extracts exhibiting disinfectant and food preservative functions, along with other compounds with disinfectant properties, e,g. acetic acid, hypochlorite, etc.

(11) Another modality of this invention refers to obtaining extracts from the calyxes of the hibiscus plant (Hibiscus sabdariffa L.), displaying a disinfectant or preservative effects when applied to food. One aspect of this modality refers to the application of extracts obtained from calyxes of the hibiscus plant (Hibiscus sabdariffa L.), possessing a disinfectant or preservative effect when applied to plants and/or seeds.

(12) Another modality if this invention is the development of a method for obtaining extracts from hibiscus calyxes, which are useful as a disinfectant and food preservative.

(13) Another modality of this invention is a method for treating and/or preserving food of animal and/or vegetable origin through the application of compositions containing extracts from hibiscus calyxes, which allow their disinfection and/or preservation.

(14) The use of extracts from hibiscus calyxes as a disinfectant and/or food preservative is another modality described in this invention.

(15) The compounds from hibiscus calyxes might be useful for the elaboration of an efficient disinfectant in order to eliminate pathogenic bacteria contained in seeds, such as alfalfa seeds, and the mung bean, among others. In this invention an extract from hibiscus calyxes is described. It contains phytochemicals that can be used as a disinfectant and/or food preservative due to its efficiency in eliminating pathogenic bacteria in foods such as seeds or their sprouts.

(16) Unlike other known compositions that have been used to this date for the same purposes, the compositions in this invention are able to eliminate microbial contamination in fresh food considered difficult to preserve, such as seed sprouts and seeds themselves, without altering their alimentary properties or their germination ability. Consequently, the application of the compositions described in this invention to fresh food allows its preservation and effective disinfection, rendering it safe for consumption.

(17) The compositions in this invention are comprised of plant extracts with known antimicrobial activity, as is the case of aqueous hibiscus extracts, either alone or in combination with other components with previously tested disinfectant activity, such as organic acids including acetic acid, and chlorine compounds including sodium hypochlorite. When disinfecting fresh food such as sprouts derived from seeds, the compositions in this invention that include a mixture of aqueous plant extract with antimicrobial activity, as well as acetic acid and hypochlorite, are often effective for eliminating all microorganisms residing in food. At the same time, their organoleptic and/or nutritional properties are not affected and the seed's germination ability is not altered.

(18) For the purposes of this invention, the compositions described herein comprise: a) Extracts derived from plants displaying antimicrobial properties, for instance the extracts derived from hibiscus (Hibiscus sabdariffa L) calyxes, a mixture of extracts from stem, leaves and flowers of the plant known as “Mexican merigold” (Tagetes lucida), an extract from stem and leaves of eucalyptus (Eucalyptus app), an extract from Flourensia resinosa S. F. Blake leaves, and a mixture of extracts from stem, leaves and flowers of absinth (Artemisia absinthium) ______, and a mixture of these at a ______1-20______% (w/w) concentration, preferably at ______1-10______%. b) An organic acid with disinfectant activity, for instance acetic acid, lactic acid, citric acid, peracetic acid, octanoic acid, peroxiethanoic acid and 1-hydroxyethyllindiene-1,1-diphosphonic acid, and mixtures of these at a ______0.1-10______% (w/w) concentration, preferably ______0.1-1______. c) A chlorine compound with disinfectant activity such as sodium hypochlorite, calcium hypochlorite, chlorine dioxide, and mixtures of these at a 0.001-10______% (w/w) concentration, preferably ______0.001-0.1______%.

(19) For the purposes of this invention, these compositions are added in order to disinfect and/or preserve food through the known methods in the art, such as direct application, spraying, or through devices that alllow their dispersion on the food that is being treated. The amount of the compositions in this invention may be added at 0.1______mL per ______1000______g of food, and preferably at ______1______mL per ______100______g of food. They can be added in larger volumes depending on the food disinfection needs. After application, the compositions may remain for the necessary period of time until the desired disinfectant and/or preservative effect is reached in food. Before consumption, food treated with the compositions described herein is simply washed in order for the compositions to be eliminated.

(20) The compositions described herein may be obtained through the mixing of their components at the desired concentrations to be stored later at room temperature, ready to be applied to food when required.

(21) For the purposes of this invention, the compositions described herein may only contain vegetable extracts with antimicrobial activity, such as the extracts derived from hibiscus calyxes, which are added to fresh food, like seed sprouts, in order to disinfect and/or preserve them. In this invention the disinfectant activity of extracts derived from hibiscus for food disinfection and/or preservation is described, such as in the case of fresh food. Thus they can be either applied directly or as part of compositions. In this sense, the extracts derived from hibiscus may be added to food to be disinfected and/or preserved at ______0.001-1______% (w/w) concentrations, preferably ______0.1-1______%. Disinfectant and/or preservative effectiveness on food by the compositions described herein eliminates all microorganisms that the food might contain, whereas its organoleptic and/or nutritional properties are not affected. In the case of fresh foods such as seed sprouts, the compositions described in this invention appropriately disinfect them without affecting their alimentary properties, and their seed germination properties remain unaffected when germination is desired after sprout elaboration. This allows their appropriate preservation, avoiding eventual microbial contaminations.

(22) The vegetable extract of this invention may be obtained by the following method: a) Place the dry plant in a vessel under aseptic conditions, add water and heat until boiling. Preferably, 100 g of dry plant are placed in a container (flask) under aseptic conditions. 900 mL of distilled water are added and boiled for 20 minutes. b) Allow to cool at room temperature, remove the plant debris and recover the aqueous extract. Preferably, the resulting extract is recovered after pressing against the flask wall in order to remove excess liquid. c) Pass the extract through a sieve and eliminate the water from the extract. Preferably, the extract is passed through a No. 200 sieve and water is removed from the extract using a rotatory evaporator at a 40° C. temperature, 80 rpm rotation and a vacuum pressure of 72 mbar. d) Recover the dry extract. Preferably in a sterile container.

(23) When the extract is obtained, it is stored at room temperature until use.

(24) Once the extract is obtained, it can be used alone or in combination with other disinfectants in order to obtain the compositions described in this invention. They may be obtained through the known methods in the art, implying the combination of the different constituting elements in order to form solutions and/or suspensions to be subsequently applied to the food with the purpose of disinfecting and/or preserving it using the methods known in the art. This invention is the first document reporting the use and effectiveness of compositions containing vegetable extracts with antimicrobial activity, either alone or in combination with other disinfectants, for disinfecting and/or preserving food, particularly fresh food such as edible seed sprouts. It is later shown that compositions in this invention are able to efficiently disinfect and/or eliminate microorganisms residing in seeds. It is thus possible to prevent microorganism growth in sprouts from seeds, rendering them safe for consumption. Before this invention, it had not been possible to develop effective compositions for disinfecting fresh food, like those which are described herein, which preserve the food's nutritional properties without affecting the seed's ability to germinate. Thus, through the application of this invention, it is possible to obtain sprouts from disinfected seeds, free from microorganism contamination.

(25) Next, some examples are included with the sole purpose of illustrating this invention, without implying any limitation of its reach

EXAMPLE 1

Materials and Methods

(26) Vegetable Material

(27) Dry hibiscus (Hibiscus sabdariffa L) calyxes were used from the creole variety obtained in Oaxaca, whereas soybean seeds (Vigna radiata L) and alfalfa seeds (Medicago sativa L) were provided by a manufacturer of sprouted seeds. Seeds were acquired from Australia.

(28) Bacterial Strains

(29) The following strains were employed: E. coli O157:H7 (P1C6, isolated from a disease outbreak), enteroinvasive E. coli (4VC81-5, isolated from a clinical case), enterotoxigenic E. coli (1620 TL, isolated from a clinical case), enteropathogenic E. coli (52 GM 291, isolated from a clinical case), Salmonella typhimurium (ATCC 14028), Salmonella choleraesuis (ATCC 10708), Listeria monocytogenes (ATCC 19115), Listeria monocytogenes Scott A, Staphylococcus epidermis (ATCC 12228), Staphylococcus aureus (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). E. coli O157:H7 and V. cholerae O1 strains were donated by Dr. Fernández Escartin at Universidad Autónoma de Querétaro. All strains were marked with resistance to the antibiotic rifampicin (R+) in order to eliminate interference from the local flora of the extract (Castro y Escartín, 2000; Rojas, 2005; Moreno, 2006). This antibiotic resistance was conserved throughout the whole study. Strains were kept at 4-7° C. in blood-based agar (ABS, Merck®, Germany), transference being performed every two weeks, and being activated in typticasein soy broth (CST, Bioxon®, Mexico) with incubation at 35° C./24 h.

(30) Obtaining Aqueous Extract from Hibiscus Calyxes

(31) Under aseptic conditions, 100 g of hibiscus calyxes were placed in an Erlenmeyer flask and 900 mL of distilled water was added. The mixture was boiled for 20 minutes. Once this treatment was finished, the mixture was allowed to cool at room temperature. Calyxes were removed from the extract (after pressing them against the flask walls in order to remove excess liquid) and it was passed through a No. 200 sieve (MONTIMAX) in order to eliminate particles. Finally, all water was separated from the extract using a rotatory evaporator (Buchi R-205) using the following conditions: heating bath temperature of 40° C., 80 rpm rotation and vacuum pressure of 72 mbar. The dry extract was recovered in a sterile flask and stored at room temperature until its use.

(32) Antimicrobial Activity Assessment of Hibiscus Aqueous Extracts

(33) 1. Strain Inoculum Preparation

(34) Test tubes containing each R(+) strain grown for 24 h in STB (soy trypticasein broth) medium were centrifuged at 3500 rpm for 20 min. Supernatant was discarded afterwards and cell pellet was resuspended by adding sterile ISS (isotonic saline solution) and vortexing for 10 s. This procedure was repeated twice. The concentration of each strain was approximately 1×10.sup.9 CFU /mL. Finally, each strain was 10-fold diluted with ISS.

(35) 2. Diffusion Technique

(36) 100 μL from the first dilution of the respective pathogen culture were inoculated in AST plates. Inoculum was completely distributed on agar surface by the surface extension technique. From the dry extract, a 10% solution of hibiscus calyxes was prepared with distilled water. 10 μL aliquots of this extract were placed on each inoculated plate. Four repetitions were carried out. After the extract was absorbed by agar the plates were incubated at 35±1° C. for 24 h. Finally, the diameter of each inhibition zone was measured on the inoculated surface.

(37) Evaluation of Vegetable Extract on Salmonella and E. coli O157:H7 Decrease in Soya Seeds

(38) 1. Strains

(39) 7 Salmonella serotypes were used in this experiment (3 typhimurium [ATCC 14028, one isolated from tomato, J1, and another from alfalfa seeds, GA1], Salmonella choleraesuis [ATCC 10708], typhi, gaminara and montevideo) and 3 E. coli O157:H7 (two of them were isolated in our laboratory from raw meat [P1C6 and M5C8] and the other was isolated from an outbreak caused by meat consumption in the United States [E09]). All strains were marked with resistance to the antibiotic rifampicin (R+) in order to eliminate interference from the extract's microbial flora (Castro and Escartin, 2000; Rojas, 2005; Moreno, 2006).

(40) 2. Strain Inoculum Preparation

(41) Test tubes containing each R(+) strain grown for 24 h in STB medium were centrifuged at 3500 rpm for 20 min. Supernatant was discarded afterwards and cell pellet was resuspended by adding 3 mL of sterile ISS and vortexing for 10 s. This procedure was repeated twice. The resulting concentration of each strain was approximately 1×10.sup.9 CFU /mL. 1 mL of each Salmonella strain was mixed in an empty test tube in order to have a mixture of all 7 Salmonella strains tested. This same procedure was carried out with E. coli O157:H7 in order to have a mixture of all three strains. 1 mL of the washed Salmonella or E. coli O157:H7 was added to 200 mL of sterile water in a beaker. The suspension was homogenized by rotating for 1 min. 100 g of alfalfa or soya seeds were immersed for 10 min in the 200 mL suspensions. After this time, the suspension was poured, the seeds were recovered in a strainer and allowed to drain over a vessel for 20 minutes inside a class II biosafety hood. Seeds were arranged in a monolayer on a plastic tray and allowed to dry for 2 hours at room temperature inside a class II biosafety hood. Afterwards, the tray was covered with a plastic film and stored at room temperature for 15 days. After this period, 10 g of alfalfa or soya seeds were randomly chosen separately and submitted to the treatments mentioned in Table 5.

(42) TABLE-US-00005 TABLE 5 Treatments that were applied separately to alfalfa and soybean seeds previously contaminated with Salmonella sp or E. coli O157:H7 strain mixtures Treatment number Treatment features* 1 Sterile water (control) 2 1% dry hibiscus extract solution.sup.1 3 0.1% acetic acid solution 4 0.5% acetic acid solution 5 1% acetic acid solution 6 100 mg/L sodium hypochlorite solution 7 1,000 mg/L sodium hypochlorite solution 8 10,000 mg/L sodium hypochlorite solution 9 1% dry hibiscus extract and 0.1% acetic acid solution 10 1% dry hibiscus extract and 0.5% acetic acid solution 11 1% dry hibiscus extract and 1% acetic acid solution 12 1% dry hibiscus extract, 0.1% acetic acid, 100 mg/L hypochlorite solution 13 1% dry hibiscus extract, 0.1% acetic acid, 1000 mg/L hypochlorite solution 14 1% dry hibiscus extract, 0.1% acetic acid, 10,000 mg/L hypochlorite solution 15 1% dry hibiscus extract, 0.5% acetic acid, 100 mg/L hypochlorite solution 16 1% dry hibiscus extract, 0.5% acetic acid, 1000 mg/L hypochlorite solution 17 1% dry hibiscus extract, 0.5% acetic acid, 10,000 mg/L hypochlorite solution 18 1% dry hibiscus extract, 1% acetic acid, 100 mg/L hypochlorite solution 19 1% dry hibiscus extract, 1% acetic acid, 1000 mg/L hypochlorite solution 20 1% dry hibiscus extract, 1% acetic acid, 10,000 mg/L hypochlorite solution *In all cases the seeds were completely immersed in 50 mL for 30 minutes

(43) After treatments, solutions were decanted separately and excess water was removed by filtering through a metal strainer, letting it rest for 2 min over a container. Subsequently, the seeds of each treatment were placed in separate bags containing 90 mL of peptone diluent and the counting of surviving bacteria was performed using the pour plate technique using trypticasein soy agar supplemented with 100 mg/L rifampicin. Finally, culture plates were incubated at 35° C. for 48 h.

(44) Similarly, 10 g portions of soybean and alfalfa seeds also subjected to the same treatments described in table 5, were used to continue the germination process. In this case, after treatment, the solutions under study and their excess were removed as described above. Each 10 g portion of treated seeds was placed in 50 mL of sterile water for 4 hours in order to hydrate the seeds. Then, seeds were removed from the water and placed on a plastic tray surface in a monolayer. The tray was covered with plastic film and stored at room temperature for 48 hours. Seeds were irrigated every 24 h using sterile water. Finally, the percentage of non-germinated seeds in each treatment was assessed at 48 h, and subsequently, the count of Salmonella and/or E. coli O157:H7 was carried out using the pour plate technique using trypticasein soy agar supplemented with 100 mg/L rifampicin, as previously described.

(45) Germination Procedure.

(46) 50 g of seeds (alfalfa or soybean) were placed in an Erlenmeyer flask, 200 mL of sterile water was added and the flask was shaken vigorously for 20 s. After this treatment, the water was discarded. Afterwards, 200 mL of sterile water was added to the flask and the seeds were kept submerged in potable water for 4 h at room temperature (hydration). After this period, the water was removed and the seeds were spread on a plastic tray surface in a monolayer. The tray was covered with plastic film to prevent dust pollution. Trays were stored at room temperature for 5 days. Every 24 h, seeds were hydrated by spraying sterile water.

EXAMPLE 2

Determination of the Extract's Antimicrobial Effect

(47) The aqueous plant extract exhibited a marked antimicrobial effect (Table 6). All tested microorganisms were inhibited from the first moments of contact. The observed inhibitory effect suggests the presence of antimicrobial substances in the extract. This effect causes lethal damage to the cell or at least either a sub-lethal effect or cellular stress (Busta, 1976). Several vegetal components could be responsible for this antimicrobial effect, such as anthocyanins (Mazza, 2000). In addition to the potential antimicrobial components, it is assumed that antimicrobial activity of the vegetal extract may be due to its pH, since it has a value between 2 and 4. To explore this, the antimicrobial effect of the vegetal extract was studied at different pH values (data not shown), finding that the antimicrobial effect of the extract decreases by increasing pH. However, a pronounced effect at pH 5 was observed in comparison with normal pH. Regardless of the effect observed with pH increase, our results show that the major antimicrobial effect of the vegetal extract is related to other components and not to pH.

(48) TABLE-US-00006 TABLE 6 Inhibitory effect of 1:10 dilution of aqueous extract of hibiscus and penicillin on different microorganisms Microorganism Inhibition diameter Inhibition diameter Type with hibiscus extract with penicillin E. coli  11* 13 E. coli O157:H7 11 15 Enteroinvasive E. coli   10.5 12 Enterapathogenic E. coli 10 11 Enterotoxigenic E. coli   10.5 12 S. aureus   10.5 13 V. cholerae O1   10.5 13 S. typhimurium 12 15 S. choleraesuis 11 12 P. aeruginosa 11 13 S. flexneri 10 12 S. sonnei 12 14 L. monocytogenes 10 12 *(mm)

EXAMPLE 3

Total Amount of Microorganisms in Seed and Sprout

(49) Table 7 shows the total microorganism count in seeds and in both soybean and alfalfa sprouts before being submitted to pathogen inoculation or to a disinfection method. Because moisture content increases throughout germination, microbial contaminants residing at low levels in seeds can rapidly reach higher levels. In our results an increase up to 6 log CFU/g is observed in sprouts.

(50) TABLE-US-00007 TABLE 7 Aerobic mesophilic bacteria in seed and sprout CFU/g* Plant Soybean Alfalfa Untreated seed 2.30 ± 1.22 3.10 ± 1.0  Untreated sprout 8.30 ± 0.45 8.00 ± 0.32 *Log.sub.10

(51) Raw sprouts consumption has become an important risk factor because pathogenic microorganisms have been found, such as Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila (NACMCF, 1999), Bacillus cereus (Portnoy et al., 1973), Klebsiella pneumoniae, Escherichia coli O157:H7, Shigella and Yersinia enterocolitica (Park et al., 2001). These bacteria may be present in seeds or they contaminate the product during the germination of several seeds such as alfalfa, kidney bean, watercress, mustard and soybean. Some studies show that sprouts support the growth of the aforementioned pathogenic bacteria (Harmon et al., 1987; Beuchat 1996; Hera Kudo et al., 1997).

(52) To prepare solutions, the following were used as a base: A) The extract from dry hibiscus calyxes from the previous section B) Sodium hypochlorite solution with 4% free hypochlorite C) 100% glacial acetic acid D) Sterile distilled water at pH 6

(53) Preparation of solutions was performed as follows:

(54) TABLE-US-00008 Treatment features* 1 Sterile water (control): 100 mL sterile distilled water 2 1% dry hibiscus extract solution: 1 g of dry extract was added to 100 mL sterile distilled water. 3 0.1% acetic acid solution: 0.1 mL of glacial acetic acid was added to 100 mL sterile distilled water. 4 0.5% acetic acid solution: 0.5 mL of glacial acetic acid was added to 100 mL sterile distilled water. 5 1% acetic acid solution: 1 mL of glacial acetic acid was added to 100 mL sterile distilled water. 6 100 mg/L sodium hypochlorite solution: 0.25 mL at sodium hypochlorite solution was added to 100 mL sterile distilled water. 7 1,000 mg/L sodium hypochlorite solution: 2.5 mL of sodium hypochlorite solution was added to 100 mL sterile distilled water. 8 10,000 mg/L sodium hypochlorite solution: 25 mL of sodium hypochlorite solution was added to 75 mL sterile distilled water. 9 1% dry hibiscus extract and 0.1% acetic acid solution: 1 g of dry extract and 0.1 mL of acetic acid were added to 100 mL sterile distilled water. 10 1% dry hibiscus extract and 0.5% acetic acid solution: 1 g of dry extract and 0.5 mL of acetic acid were added to 100 mL sterile distilled water. 11 1% dry hibiscus extract, 1% acetic acid solution: 1 g of dry extract and 1 mL of acetic acid were added to 100 mL sterile distilled water. 12 1% dry hibiscus extract, 0.1% acetic acid, 100 mg/L hypochlorite solution: 1 g of dry extract, 0.1 mL of acetic acid and 0.25 mL of sodium hypochlorite were added to 100 mL sterile distilled water. 13 1% dry hibiscus extract, 0.1% acetic acid, 1000 mg/L hypochlorite solution: 1 g of dry extract, 0.1 mL of acetic acid and 2.5 mL of sodium hypochlorite were added to 100 mL sterile distilled water. 14 1% dry hibiscus extract, 0.1% acetic acid, 10,000 mg/L hypochlorite solution: 1 g of dry extract, 0.1 mL of acetic acid and 25 mL of sodium hypochlorite were added to 75 mL sterile distilled water. 15 1% dry hibiscus extract, 0.5% acetic acid, 100 mg/L hypochlorite solution: 1 g of dry extract, 0.5 mL of acetic acid and 0.25 mL of sodium hypochlorite were added to 100 mL sterile distilled water. 16 1% dry hibiscus extract, 0.5% acetic acid, 1000 mg/L hypochlorite solution: 1 g of dry extract, 0.5 mL of acetic acid and 2.5 mL of sodium hypochlorite were added to 100 mL sterile distilled water. 17 1% dry hibiscus extract, 0.5% acetic acid, 10,000 mg/L hypochlorite solution: 1 g of dry extract, 0.5 mL of acetic acid and 25 mL of sodium hypochlorite were added to 75 mL sterile distilled water. 18 1% dry hibiscus extract, 1% acetic acid, 100 mg/L hypochlorite solution: 1 g of dry extract, 1 mL of acetic acid and 0.25 mL of sodium hypochlorite were added to 100 mL sterile distilled water. 19 1% dry hibiscus extract, 1% acetic acid, 1000 mg/L hypochlorite solution: 1 g of dry extract, 1 mL of acetic acid and 2.5 mL of sodium hypochlorite were added to 100 mL sterile distilled water. 20 1% dry hibiscus extract, 1% acetic acid, 10,000 mg/L hypochlorite solution: 1 g of dry extract, 1 mL of acetic acid and 25 mL of sodium hypochlorite were added to 75 mL sterile distilled water.

(55) Calculations to adjust concentrations and mixtures are described in Table 5.

(56) For example, in order to prepare 100 mL of 1% dry hibiscus extract, 0.1% acetic acid, 100 mg/L hypochlorite solution: 1 g of dry extract, 0.1 mL glacial acetic acid and 2.5 mL hypochlorite solution were added to 100 mL distilled water.

EXAMPLE 4

Comparison of the Antimicrobial Activity of Hypochlorite and Vegetal Extract

(57) A comparison between the activity of aqueous extract and that of hypochlorite (1,000 and 10,000 ppm) was performed in non-inoculated seed. A total count was carried out in all cases. The results obtained are shown in Tables 9 to 11.

(58) TABLE-US-00009 TABLE 8 Antimicrobial effect of aqueous extract and 1,000 ppm hypochlorite on soybean seed Initial Final % of Disinfectant inoculum* concentration* Decrease* decrease Extract 2.59 ± 1.19** 2.23 ± 1.09 0.36 15.05 Hypochlorite 2.59 ± 1.19  2.41 ± 1.08 0.18 6.95 (1,000 ppm) *Log.sub.10 CFU/g, **= ± standard deviation, N = 3.

(59) TABLE-US-00010 TABLE 9 Antimicrobial effect of aqueous extract and 1,000 ppm hypochlorite on alfalfa seed Initial Final % of Disinfectant inoculum* concentration* Decrease* decrease Extract 3.59 ± 0.39** 3.20 ± 0.56 0.36 15.05 Hypochlorite 3.59 ± 0.59  3.41 ± 0.38 0.18 6.95 (1,000 ppm) *Log.sub.10 CFU/g, **= ± standard deviation, N = 3.

(60) TABLE-US-00011 TABLE 10 Antimicrobial effect of aqueous extract and 10,000 ppm hypochlorite on soybean seed Initial Final % of Solution inoculum* concentration* Decrease* decrease Extract 2.31 ± 0.89** 2.01 ± 1.06 0.30 12.99 Hypochlorite 2.31 ± 0.89  1.91 ± 1.23 0.40 17.31 (10,000 ppm) *Log.sub.10 CFU/g, **= ± standard deviation, N = 3.

(61) TABLE-US-00012 TABLE 11 Antimicrobial effect of aqueous extract and 10,000 ppm hypochlorite on alfalfa seed Initial Final % of Solution inoculum* concentration* Decrease* decrease Extract 3.30 ± 0.45** 3.01 ± 0.46 0.30 12.99 Hypochlorite 3.31 ± 0.40   2.9 ± 0.56 0.40 17.31 (10,000 ppm) *Log.sub.10 CFU/g, **= ± standard deviation, N = 3.

(62) It can be observed that the antimicrobial effect of the extract is similar to that of hypochlorite at a 1,000 ppm concentration and it is smaller in comparison to 10,000 ppm hypochlorite. Nevertheless, in neither case is native flora of soybeans and alfalfa reduced to undetectable levels. In order to find an effective medium to ensure microbial safety of sprouts, a great diversity of methods have been investigated (Fett, 2006), including chemical, physical and natural antimicrobial treatments or a combination of these (Fett and Cooke, 2003). Among the solutions studied are disinfectant agents such as sodium and calcium hypochlorite, hydrogen peroxide, ethanol, carbon dioxide, potassium sorbate, calcium propionate, benzoic acid, salicylic acid, and riboflavin, among others (NACMCF, 1999).

EXAMPLE 5

Comparison of Hypochlorite's Toxic Effect and Hibiscus Aqueous Extract on Seed Germination

(63) American literature recommends the use of 10,000 ppm hypochlorite concentration to disinfect sprouts for up to 15 min to decrease the risk of pathogenic agents. This is the only chemical treatment approved by the US Environmental Protection Agency to be used on sprouts, which decreases, but does not eliminate, the number of pathogens. Thus, the potential persistent presence of microorganisms remains a hazard to consumers. A significant drawback of this treatment is that seeds undergo tissue damage, which prevents germination and, because of this, many sprout producers do not follow these recommendations.

(64) Therefore, a study comparing the toxic effect of hypochlorite (1,000 and 10,000 ppm), hibiscus vegetal extract and acetic acid was performed, yielding the results shown in FIGS. 1 to 6. As can be observed, seeds underwent structural damage caused by hypochlorite since they acquired a brown coloring (FIG. 4), which subsequently prevented their germination and decreased the sprout yield by 87.63%. Seeds disinfected with vegetal extract (FIG. 2) displayed no structural damage and consequently a higher germination yield was observed, similar to water-treated sprouts.

(65) The US Environmental Protection Agency, the Food and Drug Administration (FDA) and producers of sprouts meant for human consumption all agree that treatments intended to disinfect seed must eliminate the pathogenic bacteria they contain and should not affect the viability of the treated seed by more than 20%. Additionally pathogenic bacteria should not be detected in sprouts.

(66) Because of this, the hibiscus extract treatment for disinfecting seed in the present invention seems to be an excellent alternative that complies with the requirements laid out by sprout producers and the aforementioned agencies.

EXAMPLE 6

Evaluation of the Antimicrobial Activity of the Hibiscus Extract in Seeds, Used Alone or in Combination With Acetic Acid and Sodium Hypochlorite.

(67) For these studies, only Escherichia coli O157:H7 and S. typhimurium were used because they are two worldwide pathogens of major interest. Antimicrobial activity of the invention's hibiscus aqueous extract was assessed in seeds inoculated with S. typhimurium and E. coli O157: H7. Results are shown in Tables 12 to 15. It is observed that the extract by itself decreases, but does not eliminate, these two pathogens in the seed. A similar effect occurred with organic acid- or hypochlorite-based treatments, which proved to be inadequate for eliminating pathogenic bacteria in seeds, except with the 10,000 ppm treatment. In the case of the 10,000 ppm treatment, it is observed that pathogenic agents were not detected in the samples analyzed from treated batches. However, they were detected (in a large amount) in sprouts, indicating that pathogenic bacteria were not actually eliminated in the seeds. This indicates that, if at least one bacterium survives the treatment, it may grow throughout seed germination, eventually reaching the detected bacterial concentrations (Tables 12 to 15). In fact, in previous studies conducted in our laboratory we observed that when seeds were inoculated with pathogenic bacteria, the bacteria were able to grow during seed germination and consequently remain in concentrations greater than 5 log.sub.10.

(68) Thus, treatment with 10,000 ppm hypochlorite decreases E. coli O157:H7 and Salmonella to undetectable levels in alfalfa and soy seeds. Nevertheless, if treated seeds are allowed to germinate, significant pathogen concentrations are detected in sprouts. This actually indicates that these treatments are not effective for removing pathogens from seeds.

(69) In general, it was also observed that the hibiscus extract of the invention has a greater antimicrobial effect than hypochlorite or organic acid (tables 12 to 15). The combination of either antimicrobial agent (acid or hypochlorite) with the vegetal extract, does not completely eliminate the microorganism (Tables 12 to 15), except in some cases in which pathogens were not detected in sections of seed batches treated with the extract/organic acid mixture. But, as observed in the case of 10,000 ppm hypochlorite treatment, even if they were not detected in seed, they were detected afterwards in sprouts.

(70) However, it was observed that the combination of the three antimicrobial agents (extract, organic acids and hypochlorite) successfully eliminates the presence of Salmonella sp and E. coli O157:H7 in alfalfa and soybean seeds (Tables 12 to 15). Moreover, in agreement with the recommendations issued by the US Environmental Protection Agency, the FDA and producers of sprouts for human consumption, the mixture fulfilling all requests mentioned by these entities is treatment no. 12 (1% dry hibiscus extract, 0.1% acetic acid and 100 mg/L hypochlorite solution). Although, preparation no.13 (1% dry hibiscus extract, 0.1% acetic acid and 1,000 mg/L hypochlorite solution) could also be considered adequate for treating seeds (Tables 12 to 15).

(71) TABLE-US-00013 TABLE 12 Presence of Salmonella sp and germination percentage after application of different treatments to soybean seed Initial Final Concen- concentration concentration tration in (without (after germinated Germinated Treatment treatment) treatment) seed seed (%) 1 4.5.sup.1 ± 0.5.sup.2 4.4 ± 0.2 5.2 ± 0.3 100  2 4.5 ± 0.5 1.6 ± 03  4.1 ± 0.3 100  3 4.5 ± 0.5 3.8 ± 0.3 4.3 ± 0.2 95 4 4.5 ± 0.5 3.2 ± 0.4 4.6 ± 0.1 85 5 4.5 ± 0.5 2.0 ± 0.3 4.6 ± 0.2 80 6 4.5 ± 0.5 4.2 ± 0.1 4.8 ± 0.3 100  7 4,5 ± 0.5 3.0 ± 0.3 4.6 ± 0.3 90 8 4.5 ± 0.5 ND 4,5 ± 0,3 50 9 4.5 ± 0.5 1.2 ± 0.2 4.6 ± 0.2 90 10 4.5 ± 0.5 1.0 ± 0.2 4.7 ± 0.3 85 11 4.5 ± 0.5 0.6 ± 0.2 4.6 ± 0.3 80 12 4.5 ± 0.5 ND ND  87* 13 4.5 ± 0.5 ND ND 75 14 4.5 ± 0.5 ND ND 40 15 4.5 ± 0.5 ND ND 65 16 4.5 ± 0.5 ND ND 60 17 4.5 ± 0.5 ND ND 40 18 4.5 ± 0.5 ND ND 50 19 4.5 ± 0.5 ND ND 40 20 4.5 ± 0.5 ND ND 30 .sup.1log.sub.10 CFU/g (mean value from 3 repetitions); .sup.2standard deviation; ND: not detected; *the least amount of seeds that should germinate after disinfection treatment is 85%.

(72) TABLE-US-00014 TABLE 13 Presence of E. coli O157:H7 and germination percentage after application of different treatments to soybean seed Initial Final Concen- concentration concentration tration in (without (after germinated Germinated Treatment treatment) treatment) seed seed (%) 1 4.2.sup.1 ± 0.3.sup.2 4.3 ± 03  5.2 ± 0.3 100  2 4.2 ± 0.3 1.1 ± 0.2 5.1 ± 0.3 100  3 4.2 ± 0.3 3.2 ± 0.3 5.3 ± 0.3 95 4 4.2 ± 0,3 3.0 ± 0.4 5.6 ± 0.2 85 5 4.2 ± 0.3 2.0 ± 0.2 5.6 ± 0.2 80 6 4.2 ± 0.3 3.8 ± 0.3 5.8 ± 0,3 100  7 4.2 ± 0.3 2.8 ± 0.3 5.6 ± 0.2 90 8 4.2 ± 0.3 ND 5.5 ± 0.3 50 9 4.2 ± 0.3 1.0 ± 0.1 5.6 ± 0.3 90 10 4.2 ± 0.3 0.8 ± 0.2 5.7 ± 0.2 85 11 4.2 ± 0.3 0.6 ± 0.2 5.6 ± 0.3 80 12 4.2 ± 0.3 ND ND  87* 13 4.2 ± 0.3 ND ND 75 14 4.2 ± 0.3 ND ND 40 15 4.2 ± 0.3 ND ND 65 16 4.2 ± 0.3 ND ND 60 17 4.2 ± 0.3 ND ND 40 18 4.2 ± 0.3 ND ND 50 19 4.2 ± 0.3 ND ND 40 20 4.2 ± 0.3 ND ND 30 .sup.1log.sub.10 CFU/g (mean value from 3 repetitions); .sup.2standard deviation; ND: not detected; *the least amount of seeds that should germinate after disinfection treatment is 85%.

(73) TABLE-US-00015 TABLE 14 Presence of Salmonella sp and germination percentage after application of different treatments to alfalfa seed Initial Final Concen- concentration concentration tration in (without (after germinated Germinated Treatment treatment) treatment) seed seed (%) 1 3.5.sup.1 ± 0.3.sup.2 3.4 ± 0.2 5.2 ± 0.2 100  2 3.5 ± 0.3 1.4 ± 0.3 5.1 ± 0.3 100  3 3.5 ± 0.3 2.8 ± 0.3 5.3 ± 0.3 100  4 3.5 ± 0.3 2.2 ± 0.4 5.6 ± 0.3 90 5 3.5 ± 0.3 1.0 ± 0.2 5.6 ± 0.4 80 6 3.5 ± 0,3 2.2 ± 0.3 5.8 ± 0.2 100  7 3.5 ± 0.3 1.0 ± 0.3 5.6 ± 0.2 90 8 3.5 ± 0.3 ND 5.5 ± 0,3 60 9 3.5 ± 0.3 1.0 ± 0.3 5.6 ± 0.2 90 10 3.5 ± 0.3 0.8 ± 0.2 5.7 ± 0.3 80 11 3.5 ± 0.3 ND 5.6 ± 0.2 70 12 3.5 ± 0.3 ND ND  90* 13 3.5 ± 0.3 ND ND 80 14 3.5 ± 0.3 ND ND 50 15 3.5 ± 0.3 ND ND 70 16 3.5 ± 0.3 ND ND 60 17 3.5 ± 0.3 ND ND 40 18 3.5 ± 0.3 ND ND 60 19 3.5 ± 0.3 ND ND 40 20 3.5 ± 0.3 ND ND 30 .sup.1log.sub.10 CFU/g (mean of 3 repetitions); .sup.2standard deviation; ND: not detected; *the least amount of seeds that should germinate after disinfection treatment is 85%.

(74) TABLE-US-00016 TABLE 15 Presence of E. coli O157:H7 and germination percentage after application of different treatments to alfalfa seed Initial concentration Final Concen- (without concentration tration in treatment) (after germinated Germinated Treatment treatment) treatment) seed seed (%) 1 3.2.sup.1 ± 0.4.sup.2 3.0 ± 0.3 5.3 ± 0.4 100  2 3.2 ± 0.4 1.1 ± 0.2 5.2 ± 0.3 100  3 3.2 ± 0.4 2.2 ± 0.4 5.1 ± 0.2 100  4 3.2 ± 0.4 2.0 ± 0.3 5.4 ± 0.3 90 5 3.2 ± 0.4 1.0 ± 0.3 5.5 ± 0.4 80 6 3.2 ± 0.4 2.8 ± 0.3 5.5 ± 0.3 100  7 3.2 ± 0.4 1.8 ± 0.4 5,5 ± 0,3 90 8 3.2 ± 0.4 ND 5.4 ± 0.3 60 9 3.2 ± 0.4 0.8 ± 0.3 5.5 ± 0.4 90 10 3.2 ± 0.4 0.6 ± 0.3 5.2 ± 0.3 80 11 3.2 ± 0.4 0.6 ± 0.3 5.4 ± 0.3 70 12 3.2 ± 0.4 ND ND  90* 13 3.2 ± 0.4 ND ND 80 14 3.2 ± 0.4 ND ND 50 15 3.2 ± 0.4 ND ND 70 16 3.2 ± 0.4 ND ND 60 17 3.2 ± 0.4 ND ND 40 18 3.2 ± 0.4 ND ND 60 19 3.2 ± 0.4 ND ND 40 20 3.2 ± 0.4 ND ND 30 .sup.1log.sub.10 CFU/g (mean value from 3 repetitions); .sup.2standard deviation; ND: not detected; *the least amount of seeds that should germinate after disinfection treatment is 85%.

(75) It should be noted, the importance of removing pathogens from seeds without detecting them in sprouts. In 1999, 157 Salmonella spp. outbreaks linked to consumption of raw alfalfa sprouts were detected in the USA, even though these seeds were disinfected using 10,000 ppm calcium hypochlorite before germination (Proctor et at, 2001). In 2001, another Salmonella sp outbreak was linked to the consumption of the same type of sprouts, resulting in 22 detected cases (Outbreak Alert Database, 2005). Later, in 2004, there was a Salmonella bovismorbificans outbreak in alfalfa sprouts; 35 cases were reported. In the same year, an E. coli O157:H7 outbreak linked to the consumption of alfalfa sprouts was reported in a food establishment, in which 2 cases were detected (Outbreak Alert Database, 2005).

(76) In the present invention, only the combination of three antimicrobials results in complete removal of pathogens from seed, which is an example of what is currently known as a multiple barrier approach (Knochel and Gould, 1995). Multiple barriers are a combination of less severe treatments, resulting in stable, secure and safe food.

(77) Based on this, the compositions of the present invention are an excellent alternative for disinfection and/or food preservation (e.g. fresh food) without altering the food's nutritional properties, In this regard, the compositions described herein allow for effective pathogen disinfection in seeds and sprouts, allowing the safe consumption of such products.

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