Abstract:
Plant essential oils and chitosan salts were tested individually and in combination against a postharvest pathogen  Botrytis cinerea  and two foodborne human pathogens  Escherichia coli  and  Listeria monocytogenes.  Four essential oils (cinnamon, allspice, savory, red thyme) in combination with two chitosan salts (chitosan sorbate and chitosan propionate) demonstrated synergistic antimicrobial activity against all three organisms. The synergistic combinations of essential oils and chitosan salts also demonstrated an eradicant activity against  E. coli  on apple disks previously inoculated with the organism. Synergistic combinations of essential oils and chitosan salts hold promise of giving superior control of both postharvest decay organisms and foodborne human pathogens.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    This invention relates to synergistic combinations of natural antimicrobial compounds that are effective against postharvest and foodborne human pathogens.  
           [0003]    2. Description of the Relevant Art  
           [0004]    Postharvest decay and contamination of fruits and vegetables with foodborne pathogens have been and continue to be of major concern to the fruit and vegetable industry. Conservative estimates place U.S. and Canadian losses of fruits and vegetables from postharvest decay at around 25% of the harvested crops. This problem has been further compounded by the risk of contamination of fresh and processed fruits and vegetables with foodborne pathogens. Several pathogenic bacteria such as Salmonella spp,  Listeria monocytogenes, Clostridium botulinum,  and  Escherichia coli  0157:H7 have been shown to occur at base levels on the outer surfaces of a wide variety of harvested commodities (1988.  Microorganisms in Foods: Application of the Hazard Analysis Critical Control Point  (HACCP)  System to Insure Microbiological Safety and Quality,  Silliker et al., Eds. Blackwell Scientific Publications, Oxford, England). Recent outbreaks of foodborne illness associated with consumption of fresh horticultural products and non-pasteurized fruit juices have weakened consumers confidence in the wholesomeness of fresh produce (Fairchild et al. 1990.  The Packer  33: 1-7; Schwartz et al. 1995.  The Packer  27: 6; Wells et al. 1997.  Plant Dis.  81: 867-872; Parish et al. 1998.  J. Food Protection  61: 280-284).  
           [0005]    Presently, chlorinated washes in conjunction with proper refrigeration, stringent sanitation, and synthetic fungicides are the primary means of controlling foodborne pathogens and postharvest decay. However, the carcinogenicity of trihalomethanes and the possible regulatory restriction of chlorine present major challenges for the fresh produce industry to find safe alternatives. Similar public concern has been raised regarding fungicide safety. As a result, a number of key postharvest fungicides have been recently banned or are undergoing critical re-registration. In addition, some of the fungicides registered for postharvest use, particularly benzimidazole, are becoming ineffective because of the development of fungicide-resistant strains of postharvest pathogens (Spotts et al. 1986.  Plant Dis.  70: 106-108; Eckert, J. W. 1991. In:  Role of Chemical Fungicides and Biological Agents in Postharvest Disease Control. Proceedings of the Workshop on Biological Control of Postharvest Diseases of Fruits and Vegetables,  Shepherdstown, W. Va., USA, Sep. 12-14, 1990, U.S.D.A. and A.R.S. Publication Vol. 92, page 310.). Thus, it has become apparent that new, safe methodologies are needed to reduce both decay and contamination of our food supply by foodborne human pathogens.  
           [0006]    The use of natural plant- and animal-derived antimicrobials, i.e., compounds that are antibacterial and antifungal, as alternatives for the control of foodborne human and plant pathogens provides an attractive means of attacking problems resulting from the contamination of our food with microorganisms. A variety of natural plant compounds including spices, herbs, essential oils, and volatile substances have been shown to suppress the growth of food-poisoning bacteria (Bowles et al. 1993.  J. Food Protection  56: 795-800; Deans et al. 1987.  Int. J. Food Microbiol.  5: 165-180; Aktug et al. 1986.  Int. J. Food Microbiol.  3: 349-353).  
           [0007]    In vitro inhibition of the growth of major postharvest pathogens and the reduction of fruit decay was also observed with several essential oils, volatile substances, and plant extracts (Wilson et al. 1987.  Plant Dis.  71: 316-319; Wilson et al. 1997.  Plant Dis.  81: 204-210; Pesis et al. 1993.  J. Plant Physiol.  142: 717-721; Sholberg et al. 1991.  J. Canad. Inst. Food Sci. Tech.  2: 273-276; Mattheis et al. 1993.  Plant Dis.  77: 810-814; Vaugh et al. 1993.  J. Food Sci.  58: 793-796). Also, the inhibition of the growth of foodborne pathogens has been reported with bacteriocins (Fowler et al. 1990. Antibiotics-nisin. In:  Food Preservatives,  Russel and Gould, Eds. AVI Publishing, New York; Motlagh, A. 1991. Ph.D. Thesis, Univ. Wyoming, Laramie, Wyo.; 1992.  Food Biopreservatives of Microbial Origin,  Ray and Daeschel, Eds. CRC Press, New York), with organic acids (Ray, B. 1992. Diacetyl of Lactic Bacteria as a Food Biopreservative. In:  Food Biopreservatives of Microbial Origin,  supra; Arora et al. 1991.  Handbook of Applied Mycology Vol.  3. Marcel Dekker, Inc., New York. 621 pages; Al Zaemey et al. 1993.  Mycolog. Res.  97: 1463-1468; Sholberg et al. 1995. Hort. Sci. 30: 1271-1275), and with chitosan (Hadwiger et al. 1980.  Plant Physiol.  66: 205-211; El Ghaouth et al. 1992.  Phytopath.  82: 398-402). Some of these compounds (bacteriocins and organic acids) are also used commercially to control food spoilage. Most current available data provide only fragmented information on the effectiveness of combinations of naturally-occurring antimicrobial compounds and on their effect on both postharvest and foodborne pathogens. Development of synergistic combinations of natural compounds can add a new dimension to their use as food preservatives, enhancing their effectiveness for stability, low toxicity, availability, and broad utility.  
         SUMMARY OF THE INVENTION  
         [0008]    We have discovered naturally-occurring compounds that are both antifungal and bactericidal and combinations of particular natural compounds that can be used synergistically to control both major postharvest pathogens and foodborne pathogens.  
           [0009]    In accordance with this discovery, it is an object of the invention to provide a composition of natural compounds that act synergistically and are effective against postharvest pathogens and foodborne pathogens found on fruits and vegetables.  
           [0010]    It is a further object of the present invention to provide a method for protecting fruits and vegetables from postharvest pathogens and foodborne pathogens found on fruit and vegetables by applying to the surface of fruits and vegetables a composition of natural compounds that act synergistically and are effective against bacteria and/or fungi.  
           [0011]    It is a still further object of the present invention to provide a method for reducing the effects of the overall microbial content of a food product by applying to the surface of fruits and vegetables a composition of natural compounds that act synergistically and are effective for eradicating or inhibiting growth and toxin production of bacteria and fungi found on fruits and vegetables.  
           [0012]    An additional object of the present invention is to provide a fruit or vegetable food product having reduced levels of bacterial and/or fungal postharvest pathogens and foodborne pathogens.  
           [0013]    Other objects and advantages of the invention will become readily apparent from the following description.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0014]    The present invention provides combinations of chitosan salts and essential oils that act synergistically both to protect food products from bacterial and fungal contamination and to eradicate or at least inhibit growth and toxin production in foods contaminated with bacteria and fungi. The present invention relates to effective, inexpensive, and environmentally appropriate compositions and methods for controlling postharvest pathogens and foodborne pathogens, as for example, enterotoxigenic bacteria such as  E. coli  and  L. monocytogenes,  on fruits and vegetables.  
           [0015]    Food product here refers to a fruit or a vegetable or part of a fruit or vegetable that can be infected or contaminated by postharvest pathogens and foodborne pathogens. The term “food product” encompasses “exposed fresh fruit” and “exposed fresh vegetable” which in its broadest sense includes the tissue normally covered by the skin of the fruit or vegetable which is exposed when the fruit or vegetable is peeled, cut, segmented or otherwise exposed. The tissue is fresh or raw and is preferably in the form of cut or segmented pieces which have not been heat sterilized or blanched. Generally, one or more of any type of fresh vegetable, fruit or nut, for example, may be treated with the present invention. Suitable examples of fruit include apples, apricots, avocado, bananas, blackberries, blueberries, cherries, cranberries, custard apples, dates, durian, figs, grapefruit, grapes, jack fruit, kiwi fruit, lemons, limes, lychee, mandarins, mangosteen, mangoes, melons, nashi, nectarines, oranges, papaya or paw paw, passionfruit, peaches, pears, pineapple, plums, pomegranates, pomelo, raspberries, rhubarb, star fruit, strawberries, tamarillo, and tangerines of any maturity. Any edible nut is also included. Suitable non-limitative examples of vegetables include: potatoes, corn, tomatoes, onions, herbs, squash, beans, peppers, okra, turnips, broccoli, cauliflower, cabbage, carrots, brussels, sprouts, zucchini, radishes, celery, lettuce, and even prepared mixed vegetable salads. Moreover, any fresh vegetable, fruit or nut may be treated with the present invention, whether grown in the ground or grown hydroponically.  
           [0016]    As used herein, “foodborne pathogen” refers to a bacterium or a fungus capable of contaminating a fruit or a vegetable and causing disease to humans or animals ingesting said fruit or vegetable.  
           [0017]    As used herein, “postharvest pathogen” refers to a bacterium or a fungus capable of infecting a fruit or a vegetable and thereby causing postharvest decay.  
           [0018]    As used herein, the term “synergism” is intended to include both an increased spectrum of activity (i.e., greater activity against a broad spectrum of microorganisms), and/or increased efficacy (i.e., greater activity against specific organisms than that predicted by use of either agent alone). The increased antimicrobial and antifungal activity of the synergistic combination permits the use of smaller amounts of each agent thereby decreasing costs and minimizing other problems, e.g., toxicity, solubility, availability. Effectiveness against a broad spectrum of microorganisms broadens the utility of the synergistic product based on its effectiveness in environments containing many and diverse microorganisms which must be controlled.  
           [0019]    Chitosan is a semisynthetic derivative of chitin produced by the deacetylation of the nitrogen thereof so as to produce the ammonium salt. Chitosan has been shown to have some mild antifungal activity with regard to particular fungal species; see for example, Hadwiger et al., supra; El Ghaouth et al. 1994.  Phytopath.  84: 313-320; El Ghaouth et al., 1992, supra; Allan et al. 1979.  Exp. Mycology  3: 285-287; Stossel et al. 1984.  Phytopath.  11: 82-90; Kendra et al. 1984.  Exp. Mycology  8: 276-281, and Ben-Shalom et al. 1999. U.S. Pat. No. 5,965,545.  
           [0020]    The compositions of the invention comprise combinations of chitosan salts and essential oils that act synergistically both to protect food products from bacteria and fungi and to eradicate or inhibit decay and toxin production in foods contaminated with bacteria and fungi. Particular combinations can be screened in vitro in culture medium prior to testing on food products. Culture medium is inoculated with suspensions of bacteria or fungal spores. Chitosan salts, essential oils, or combinations of chitosan salts and essential oils are (1) added to the culture medium prior to inoculation to determine protective effects or (2) added after inoculation to determine inhibitory or eradicant effects.  
           [0021]    Generally, the compositions according to the invention usually contain in addition to the active material (chitosan salt and essential oil), one or more solid or liquid vehicles and, optionally, one or more surface-active agents. The solid or liquid vehicles and/or surface-active agents utilized in the compositions of the invention must be acceptable in agriculture; inert and conventional vehicles and conventional surface-active agents can be used. The compositions according to the invention are pharmaceutically-acceptable, i.e., the compositions or components are suitable for use in contact with human tissue without undue toxicity, incompatibility, instability, allergic response, and the like. These compositions cover not only compositions that are ready to be applied to the fruits and vegetables, as for example by means of a suitable device, such as a spray device, but also commercial concentrated compositions which have to be diluted before application to the food product.  
           [0022]    In the present account, the term “vehicle” denotes a natural or synthetic, organic or inorganic material with which the active material is combined to facilitate its application on the food product. This vehicle is thus generally inert and it must be agriculturally and pharmaceutically acceptable. The vehicle can be solid as for example, clays, natural or synthetic silicates, resins, and waxes or the vehicle can be liquid, such as water, alcohols, propylene glycol, a vegetable oil or like edible carrier, and the like. An “aqueous solvent” means a water-based solvent, including but not limited to tap water, distilled water, buffers, salt solutions, and the like.  
           [0023]    The surface-active agent can be an emulsifying, dispersing, or wetting agent of ionic or nonionic type or a mixture of such surface-active agents. The presence of at least one surface-active agent is generally indispensable when the active material and/or the inert vehicle is /are not soluble in water and the carrier agent for application is water.  
           [0024]    These compositions can also contain any kind of other ingredients such as, for example, protective colloids, adhesives, binding agents, chelating agents, thickening agents, thixotropic agents, penetrating agents, stabilizing agents, sequestering agents and the like. The compositions used in the method of the present invention may also contain other additives depending on the intended use for the composition. For example, the compositions may contain anti-foam agents, antioxidants, natural or synthetic seasonings and/or flavors, dyes and/or colorants, vitamins, minerals, nutrients, enzymes, insecticides, deodorants, and mixtures thereof. The amount of such optional additives included in the composition of the present invention may vary over a wide range, although amounts of about 0.1 to 10.0 percent of these compositions are generally satisfactory.  
           [0025]    More generally, the chitosan salts and the essential oils can be combined with all the solid or liquid additives corresponding to the conventional formulating techniques.  
           [0026]    As forms of liquid compositions or those intended to constitute liquid compositions at the time of application, solutions, in particular water-soluble concentrates, emulsions, suspension concentrates, aerosols, wettable powders (or powder to be sprayed), pastes or gels are included. The composition of the present invention can be presented to the consumer in dry form to be used after it is wetted with water, i.e., water-activated.  
           [0027]    These compositions can be delivered from for example, bottles, tubes, pumps, squeeze roamers, bags, wipes, and aerosol containers as e.g., volatiles, foams, mousses, lathers, wipes, and dips.  
           [0028]    A composition according to the present invention is most readily used to treat the surface of solid food products. The active materials or combinations may be applied to fruits and vegetables by dipping, spraying, painting, marinating, and/or wiping the surface. In still other embodiments, the composition may be applied as a breading, seasoning rub, glaze, colorant mixture, and the like, the key criteria being that the antimicrobial composition be available to the surface subject to bacterial or fungal degradation and/or contamination. In still other embodiments, the composition may be indirectly placed into contact with the food surface by applying the composition to food packaging and thereafter applying the packaging to the food surface. The optimum amount to be used will depend on the composition of the particular food product to be treated and the method used for applying the composition to the food surface, but can be determined by simple experimentation. It is preferred that the active material or combination be dissolved or dispersed in a vehicle as defined above, at concentrations between 10 and 50% solids. When employing a composition of the invention, the essential ingredients, namely, the essential oils and/or chitosan salts can advantageously be used in amounts ranging from about 3000 ppm to about 10 ppm based on total weight of the food product.  
       
    
    
     EXAMPLES  
       [0029]    The following examples serve as further description of the invention and methods for practicing the invention. They are not intended as being limiting, rather as providing guidelines on how the invention may be practiced.  
       Example 1  
     Bacterial and Fungal Cultures  
       [0030]    [0030] E. coli  (Strain #139 HB101/p5G6) was grown at 24° C. for 48 hr in shake-flask cultures of Lennox broth (LB). Bacterial cells were pelleted by centrifugation in a Sorvall RC-58 centrifuge (Dupont Instruments, Wilmington, Del.) at 3000 g for 20 min, resuspended in sterile distilled water, and centrifuged again. The resulting pellets were dispersed in sterile distilled water and the cell concentration was adjusted to 10 6  CFU per ml using a standard optical density (OD) curve with the OD values of 0.1 and 1 representing viable cell counts of 1×10 6  and 1×10 9 , respectively.  E. coli  015:H7 and  L. monocytogenes  isolates were grown overnight at 37° C. in trypticase soy broth and brain heart infusion, respectively. The concentration of cells was adjusted to 10 6  CFU per ml.  Botrytis cinerea  and  Penicillium expansum  were isolated from infected fruit and maintained on potato dextrose agar (PDA). A spore suspension was obtained by flooding 2 wk cultures of  B. cinerea  with sterile distilled water containing 0.1% (v/v) TWEEN 80. Spore counts were determined with a hemacytometer and spore concentrations were adjusted with sterile distilled water to obtain 10 5  spores per ml.  
       Example 2  
     Inhibitory Effect of Essential Oils and Chitosan Salts  
       [0031]    The object of this experiment was to determine the individual effects of different essential oils and chitosan salts, and the combined effects of chitosan salts with essential oils on the growth of the indicator organism  E. coli  and on spore germination of  B. cinerea.  Autoclaved LB broth was amended with sterile solutions of chitosan salts (chitosan propionate and chitosan sorbate, Sigma, St. Louis, Mo.), essential oils (tarragon, basil, peppermint, wintergreen, savory, thyme red, and allspice; Aroma Vera, Cuber City, Calif.), or combinations of chitosan propionate and chitosan sorbate with individual essential oils to obtain a concentration of 0.1% (v/v) and dispensed into sterile test tubes. Tubes of LB amended with different treatments were inoculated either with 10 6  CFU per ml of  E. coli  cells or 500 spores of  B. cinerea  and incubated on a rotary shaker at 24° C. for 24 hr. For each microorganism, four replicate tubes of each treatment were used; each experiment was repeated twice. Botrytis spore germination was determined microscopically. The viable bacterial cell number was counted by surface plating serially diluted samples in triplicate on LB agar medium. Plates were incubated at 24° C. and colonies were counted at 48 hr.  
         [0032]    Among seven essential oils that were tested for their antimicrobial activity against both  B. cinerea  and  E. coli,  savory, thyme red, and allspice provided the most effective control of both  B. cinerea  and  E. coli.  These three completely inhibited spore germination of  B. cinerea  and substantially reduced the growth of  E. Coli  (Table 1).  
                                                                 TABLE 1                           Effect of essential oils on spore germination of  Botrytis cinerea         and growth of  Escherichia coli  after 48 hr of incubation at 24° C.                % INHIBITION                    Essential Oil     B. cinerea       E. coli                              Control   0   0           Tarragon   0   5           Basil   0   5           Peppermint   100   9           Wintergreen   100   24           Savory   100   80           Thyme Red   100   80           Allspice   100   84                      
 
         [0033]    The effect of time of exposure on the biostatic or biocidal activity of the most effective essential oils and combinations of chitosan salts with essential oils was also assessed. Sterile 0.1 % solutions of chitosan salts (chitosan propionate and chitosan sorbate), essential oils (cinnamon, savory, thyme red, and allspice), or combinations of chitosan salts with individual essential oils were supplemented with 0.1% of autoclaved LB for  E. coli  or 0.1% autoclaved PDB for B. cinerea and dispensed into sterile 10 ml test tubes.  
         [0034]    Test tube cultures were inoculated either with 10 6  CFU per ml of  E. coli  cells or 500 spores of  B. cinerea  and incubated on a rotary shaker at 24° C. An individual test tube served as one replicate and four replicates were sampled after one and four hr of incubation from each treatment for each microorganism. Botrytis spore germination and the viability of bacterial cells were determined as described above.  
         [0035]    In tests of the various essential oil/chitosan salt combinations against spore germination of  B. cinerea  and growth of  E. coli , all four essential oil/chitosan salt combinations completely inhibited spore germination of  B. cinerea  and growth of  E. coli  (Table 2).  
                                                                         TABLE 2                           Biocidal activity of essential oils and different combinations       of natural compounds on spore germination of         B. cinerea  and growth of  E. coli  after 1 and 4 hr.                Inhibition (%)   Cell Counts (CFU) a               B. cinerea       E. coli              Treatments   1 hr   4 hr   1 hr   4 hr                    Control   0   0   TNTC b     TNTC       Chitosan sorbate   0   0   544   181       Chitosan propionate   0   0   527   191       Cinnamon   0   100   &gt;600   &gt;600       Savory   0   0   &gt;600   &gt;600       Allspice   0   100   &gt;600   &gt;600       Chitosan sorbate + Cinnamon   100   100   0   0       Chitosan sorbate + Allspice   100   100   0   0       Chitosan propionate + Savory   100   100   0   0       Chitosan propionate + Red Thyme   100   100   0   0                                  
 
       Example 3  
     Inhibitory Effect of Essential Oils, Chitosan Salts, and Combinations of Essential Oils and Chitosan Salts  
       [0036]    The individual effects of various essential oils and chitosan salts, and the combined effects of essential oils and chitosan salts on the growth of the indicator organism  E. coli  and on the postharvest pathogen  B. cinerea  were determined. To measure the effects of the various treatments on spore germination of  B. cinerea  and growth of the  E. coli , the essential oils: bay, cinnamon, savory, thyme red, allspice, birch, cloves, carvacrol, and hinokitiol (Aroma Vera, Cuber City, Calif.) and MMW chitosan in acetic, propionic, and sorbic acids were combined together with B. cinerea or  E. coli  to yield final concentrations of 0.1 to 0.025% for the essential oils and 0.1 to 0.0016% for the chitosan salts. For the assays, 500 spores of B. cinerea were added to each treatment in microtiter dishes or three ml of a 2X concentration of  E. coli  (i.e., 2×10 6  CFU/ml) were combined with three ml of a 2X concentration of treatment in a 15 ml centrifuge tube, agitated overnight, and plated after 24 hr onto LB agar plates (100 μl suspension/plate). The surfactant (Triton×100) was present at a final concentration of 0.04%. Similarly, for experiments measuring synergy, combinations of individual essential oils (at non-inhibitory concentrations) and chitosan acetate, chitosan propionate, or chitosan sorbate (at non-inhibitory concentrations) were combined with B. cinerea or  E. coli , as described above. Four replicate tubes of each treatment were used; each experiment was repeated twice. The viable bacterial cell number was counted by surface plating serially diluted samples in triplicate on LB agar medium. Plates were incubated at 24° C. and colonies were counted at 48 hr. Botrytis spore germination was determined microscopically.  
         [0037]    Effects on  E. coli  Growth:  
         [0038]    Savory, thyme red, and carvacol, tested individually, were the most effective inhibitors of  E. coli  growth; each, alone, was inhibitory at 0.05% (Table 3). Cinnamon and hinokitiol reduced  E. coli  growth at 0.075%; no effects were seen at 0.05% or lower. Bay, cloves, allspice, and birch oil were the least effective inhibitors; they only inhibited at the final concentration of 0.1%. No effects were observed at 0.075% or lower.  
                                                                                 TABLE 3                           Effect of Concentration of Essential Oil on       Growth of  E. coli  (CFU a )                Concentration (% v/v)                    Essential Oil   0.1   0.075   0.05   0.025                            Bay   13.5   TNTC   TNTC   TNTC           Cinnamon   0   61.5   TNTC   TNTC           Cloves   0   TNTC   TNTC   TNTC           Allspice   0   TNTC   TNTC   TNTC           Thyme Red   0   4.5   0.5   TNTC           Savory   11.5   0   6.0   TNTC           Birch   0   TNTC   TNTC   TNTC           Carvacrol   0   0   0   TNTC           Hinokitiol   0   812   TNTC   TNTC                                              
 
         [0039]    All chitosan salts were effective inhibitors of  E. coli  growth at concentrations of 0.1%-0.0063%, but no effect was seen with concentrations of 0.0032% or lower (Table 4).  
                                                                                             TABLE 4                           Effect of Concentration of Chitosan Salts on       Growth of  E. coli  (CFU a )                Concentration (% v/v)                0.1   0.05   0.025   0.0125   0.0063   0.0032   0.0016                        Chitosan-   0   0   1   0   0   TNTC   TNTC       acetate       Chitosan-   0   0.5   0.5   0   26   TNTC   TNTC       propionate       Chitosan-   0   0   0   0   4.5   TNTC   TNTC       sorbate                                  
 
         [0040]    Essential oils and chitosan salts, each at concentrations shown to be non-inhibitory in Tables 3 and 4, were combined with  E. coli  as described above and their effectiveness at inhibiting the growth of  E. coli  was measured. All combinations inhibited  E. coli  growth (Table 5). The individual essential oils and chitosan salts acted synergistically in combination; each inhibited in combination at concentrations where they were not individually inhibitory. Those essential oils that were found to be the least effective inhibitors of  E. coli  growth, as shown in Table 3, were effective inhibitors when tested together with chitosan salts.  
                                                                             TABLE 5                           Effect of Synergistic Combinations of Essential Oils and       Chitosan Salts on Growth of  E. coli  (CFU a )                Concentration (% v/v)                    Chitosan   Chitosan   Chitosan                   acetate   propionate   sorbate           Conc. (%)   0.0032   0.0032   0.0032   Water                        Water       TNTC   TNTC   TNTC   TNTC       Bay   0.075   75   180   479   TNTC       Cinnamon   0.050   401   582   793   TNTC       Savory   0.025   1803   691   1614   TNTC       Thyme Red   0.025   1044   115   1328   TNTC       Allspice   0.075   0   20   32   TNTC       Birch   0.075   640   1621   2713   TNTC       Cloves   0.075   34   83   193   1212       Carvacrol   0.025   0   0   2   1430       Hinokitiol   0.075   102   56   60   1108                                  
 
         [0041]    Spore germination of  B. cinerea:    
         [0042]    Similar results were observed when spore germination of  B. cinerea  was measured (Tables 6, 7, and 8). The individual essential oils: bay, cinnamon, allspice and cloves were inhibitory at 0.05%; savory and thyme red were inhibitory only at 0.1 % (Table 6). Thus, savory and thyme red, the most effective inhibitors of bacterial ( E. Coli ) growth were not as effective in inhibiting spore germination of the fungus,  B. cinerea;  bay, cinnamon, allspice and cloves were more effective.  
                                                                                                                     TABLE 6                           Effect of Concentration of Essential Oils on Spore Germination of  B. cinerea.                  Percent Inhibition of Spore Germination           Concentration (% v/v)                0.1   0.09   0.08   0.07   0.06   0.05   0.04   0.03   0.02   0.01                        Bay   100   100   100   100   100   100   0   0   0   0       Cinnamon   100   100   100   100   100   100   0   0   0   0       Savory   100   0   0   0   0   0   0   0   0   0       Thyme Red   100   0   0   0   0   0   0   0   0   0       Allspice   100   100   100   100   100   100   0   0   0   0       Cloves   100   100   100   100   100   100   100   0   0   0                  
 
         [0043]    Chitosan-sorbate alone was effective in completely inhibiting B. cinerea spore germination at concentrations of 0.1% to 0.0175% (Table 7). Complete inhibition of spore germination of  B. cinerea  was obtained with chitosan-acetate and chitosan-propionate at 0.1% and 0.08%.  
                                                                                                     TABLE 7                           Effect of Concentrations of Chitosan Salts on       Spore Germination of  B. cinerea.                  Percent Inhibition of Spore Germination           Concentration (% v/v)                0.1   0.08   0.06   0.04   0.02   0.0175   0.015   0.0125                        Chitosan-   100   100   0   0   0   0   0   0       acetate       Chitosan-   100   100   0   0   0   0   0   0       propionate       Chitosan-   100   100   100   100   100   100   0   0       sorbate                  
 
         [0044]    Essential oils and chitosan salts, each at concentrations shown to be non-inhibitory in Tables 6 and 7, were combined with  B. cinerea,  as described above, and their effectiveness at inhibiting spore germination of  B. cinerea  was measured. All combinations of essential oils and chitosan salts, at concentrations where they were not individually inhibitory, showed a synergistic effect and completely inhibited  B. cinerea  spore germination (Table 8). Those essential oils that were found to be the least and the most effective inhibitors of  B. cinerea  spore germination (Table 6) were equally effective when tested together with chitosan salts even though both the essential oil and the chitosan salt were present at concentrations where no inhibition had previously been observed (Tables 6 and 7).  
                                                                             TABLE 8                           Effect of Synergistic Combinations of Essential Oils       and Chitosan Salts on Spore Germination of  B. cinerea.                  Percent Inhibition of Spore Germination           Concentration (% v/v)                    Chitosan   Chitosan   Chitosan               Conc.   acetate   propionate   sorbate           % (v/v)   0.02   0.02   0.006   Water                        Bay   0.013   100   100   100   0       Cinnamon   0.013   100   100   100   0       Savory   0.03   100   100   100   0       Thyme Red   0.03   100   100   100   0       Allspice   0.02   100   100   100   0       Cloves   0.01   100   100   100   0       Hinokitiol   0.067   100   100   100   0                  
 
       Example 4  
     Effect of Combinations of Chitosan Salts and Essential Oils on  E. coli  015:H7 and  L. monocytogenes.    
       [0045]    To determine whether the combinations of essential oils and chitosan salts that were shown in Examples 1-3 to be effective inhibitors of growth of the non-pathogenic indicator strain of  E. coli  would also effectively inhibit pathogenic bacteria, the most promising combinations were tested at the USDA ARS Eastern Regional Research Center (Wyndmoor, Pa.) for their effectiveness in inhibiting the growth of the pathogenic bacteria  L. monocytogenes  and  E. coli  01 5:H7, a strain of  E. coli  pathogenic to humans. Autoclaved LB was amended with a sterile solution of the combination of chitosan-sorbate with cinnamon oil, chitosan-sorbate with allspice, chitosan-propionate with red thyme, or chitosan-sorbate with savory to obtain a final concentration of 0.1 % (v/v). Test tube cultures were inoculated with 10 6  CFU per ml of  E. coli  0157:H7 or L. monocytogenes and incubated on a rotary shaker at 240 C. An individual test tube served as one replicate and four replicates were sampled after 0, 1, 2, and 24 hr of incubation from each treatment for each bacteria. The viable bacterial cell number was counted by surface plating serially diluted samples containing  E. coli  015:H7 and L. monocytogenes in triplicate on LB agar medium and lithium chloride-phenylethanol-moxalactan agar, respectively. Plates were incubated at 24° C. and colonies were counted after 48 hr.  
         [0046]    From these tests, it is apparent that all four combinations of the essential oils and chitosan salts were effective in inhibiting the growth of  E. coli  strain 01 57:H7 and  L. monocytogenes  (Table 9).  
                                                                             TABLE 9                           Biocidal activity of different combinations of natural compounds and essential oils       on growth of  E. coli  0157:H7 and  L. monocytogenes  cells after 0, 1, 4, and 24 hr.                Bacterial Cell Counts (Log CFU) a                    E. coli  0157:H7     L. monocytogenes              TREATMENTS   0 hr   1 hr   4 hr   24 hr   0 hr   1 hr   4 hr   24 hr               Control   6   6   6   6   6   6   6   6       Chitosan sorbate + Cinnamon   6   6   6   0   3   0   0   0       Chitosan sorbate + Allspice   6   6   6   0   3   0   0   0       Chitosan propionate + Red Thyme   6   6   0   0   0   0   0   0       Chitosan propionate + Savory   0   0   0   0   0   0   0   0                          
 
       Example 5  
     Effect of Essential Oils and Chitosan Salts on Contamination of Apple Disk with  E. coli.    
       [0047]    Experiments were conducted to determine whether cinnamon, allspice, savory, chitosan sorbate, and chitosan propionate and/or their combinations could protect fruit surfaces against colonization by  E. coli  and whether  E. coli  could be eradicated once established on fruit surfaces with these treatments. Tree-ripe apples (Malus domestica Borkh) cultivar ‘Red delicious’ were hand-picked at harvest maturity at the Appalachian Fruit Research Station, Kearneysville, W. Va. Fruit were sorted to remove any with apparent injuries or infections and stored at 4° C. under refrigeration before being used in the biocontrol tests. Apple disks (10 mm) were excised from selected Red delicious apples using a cork borer. Apple disks were treated by immersion for 90 min in a 0.1% solution of various essential oils and/or their combinations with 0.1 % chitosan salts. Disks were either treated with the different combinations of natural compounds and then inoculated with  E. coli  by soaking apple disks in a solution of  E. coli  for 90 min or inoculated with  E. coli  and incubated at 24° C. for 24 hr. From each treatment four disks were selected randomly, individually homogenized in 5 ml of sterile water, vortexed, and dilution plated in triplicate on a LB agar medium. Plates were incubated at 24° C. and colonies were counted after 48 hr.  
         [0048]    Chitosan sorbate and chitosan propionate in combination with essential oils of cinnamon, allspice, and savory completely protected apple disks against colonization by  E. coli  and completely eradicated established  E. coli  growth (Table 10).  
                             TABLE 10                           Protectant and Eradicant Effects of Natural Compounds       on Growth of  E. coli  on Apple Disks.                Protectant Activity a     Eradicant Activity b         TREATMENTS e       E. coli  (CFU) c       E. coli  (CFU)               Control      TNTC   d     TNTC       Sorbate   TNTC   TNTC       Propionate   TNTC   TNTC       Chitosan sorbate   133   248       Chitosan propionate   &gt;1800   &gt;1800       Cinnamon   43   450       Allspice   &gt;1800   548       Savory   41   54       Chitosan sorbate + Cinnamon   0   0       Chitosan sorbate + Allspice   0   0       Chitosan propionate + Savory   0   0                                                          
 
         [0049]    All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.  
         [0050]    It is understood that the foregoing detailed description is given merely by way of illustration and that modifications and variations may be made therein without departing from the spirit and scope of the invention.