Abstract:
A bacteriostatic agent and biocidal formulations include a preservative-free bacteriostatic agent in an aqueous solution of a polyol and a dispersing agent. The polyol/dispersing agent ratio is between  30:1  (weight/weight) and  110:1  (weight/weight). The bacteriostatic agent is used as a diluent in the environmentally-friendly biocidal formulations.

Description:
TECHNICAL FIELD OF THE INVENTION 
       [0001]    The present invention relates to a preservative-free bacteriostatic agent that prevents exogenous contamination in biocidal formulations. The invention relates to biocidel formulations with this bacteriostatic agent. The bacteriostatic agent contains an aqueous solution of a polyol and a dispersing agent, wherein the polyol/dispersing agent ratio is comprised between 30:1 (weight/weight) and 110:1 (weight/weight). 
       BACKGROUND OF THE INVENTION 
       [0002]    In recent years there has been interest in developing environmentally-friendly plant protection products that do not contain components that are toxic for humans or for the ecosystem, green biocides being developed. Different families, species and strains of bacteria with biocidel activity, such as  Bacillus  spp.,  Pseudomonas  spp. or yeasts, for example, stand out among green biocides. The biocidel activity of different proteins with insecticidal properties such as cystatins, for example, has also been described. 
         [0003]    Strains of bacteria and yeasts are stored in spores adsorbed in inert substrates, such as the kaolin, silica, zeolites, zinc oxides, clays, diatomaceous earth, polysaccharides or carbonates. Proteins are also immobilized in inert substrates. 
         [0004]    To use the microorganisms in liquid formulations it is necessary to formulate a stable suspension, i.e., a suspension in which separations or precipitations of the inert material will not occur over time. 
         [0005]    It is known that aqueous solutions must contain bacteriostatic agents or preservatives to prevent microbial contamination. The use of parabens, azides or isothiazolines for this purpose is known. However, these products are toxic and environmental pollutants and therefore not suitable for being used in “green plant protection agents/biocides”. 
         [0006]    In turn, it is known that the plant protection agents described in the state of the art are harmful to health due to the presence of toxic preserving agents and biocidal agents. The application of plant protection agents requires taking the necessary precautions to prevent the operator against poisoning: goggles, gloves and applying the product downwind. 
         [0007]    Based on the foregoing, liquid formulations comprising biological material have different drawbacks:
       solutions must be homogenous and must not show phase separation over time,   the formulations must maintain their biocidel activity over time, i.e., the microorganism or protein must be viable for at least two years,   the formulations must not be contaminated with exogenous microorganisms without using bactericidal agents or preservatives.       
 
         [0011]    The person skilled in the art knows of the existence of dispersing agents assuring stability of the suspensions, but the dispersing agents are incompatible with the subsequent viability of the bacteria. In addition, water content is a critical factor; a low water concentration does not assure proper homogenization of the suspensions, precipitation of the excipients or carriers of the bacteria occurring, and in addition, excessive water content will accelerate microbial contamination of the formulations by other species of bacteria or fungi. 
         [0012]    WO200624095, CN101048136, CA110384 and US2006270745 disclose compositions comprising solutions comprising polyols and dispersing agents that are used for formulating oil/water (O/W) emulsions and microemulsions. 
         [0013]    ES 2370561 discloses suspensions with biocidel activity in glycerine with at least 60% by weight being used in the formulations of plant protection products. These suspensions contain dispersing agents, but to prevent exogenous microbiological contamination they use different preservatives such as benzoisothiazolines. The examples detailed in ES2370561 use a polyol/dispersing agent ratio of less than 30:1. 
         [0014]    W02009126647 discloses an aqueous formulation comprising at least one spore at a concentration of between 3-80% (weight/weight), at least one water-miscible solvent and/or a hydrophobic agent. This patent document does not describe any ratio between the polyols which can be water-miscible solvents, and the dispersing agents present in the invention. This patent document also describes the use of preserving agents to prevent exogenous microbiological contamination. 
       OBJECT OF THE INVENTION 
       [0015]    The problem which the invention solves is to find an environmentally-friendly bacteriostatic agent that prevents exogenous microbial contamination in aqueous solutions and assures stability of the suspensions over time. 
         [0016]    The solution found by the inventors is an aqueous preservative-free solution containing a polyol and a dispersing agent, wherein the polyol/dispersing agent ratio is comprised between 30:1 (weight/weight) and 110:1 (weight/weight). 
         [0017]    Another advantage of the invention is that it assures physical stability of the bacterial suspensions inoculated into their inert carrier. Suspensions in which precipitation of suspended inert solids is not observed are obtained by means of selecting sodium polycarboxylate or condensed naphthalene sulfonate as dispersing agents. 
         [0018]    Additionally, the high polyol concentration provides the suspensions with high viscosity, and the use of rheological modifiers, such as xanthan gum, is prevented. 
         [0019]    Another problem solved by the invention is that it assures viability of the microorganisms and proteins in the suspensions and the biocidel activity of the formulations. 
         [0020]    The biocidel formulations with  Bacillus subtilis  showed the same effectiveness as the solid formulations of  Bacillus subtilis  against Sclerotinia in lettuce, Botrytis in tomatoes and Monilia in peaches. The effectiveness was similar to that of chemical fungicides such as thianosan. 
         [0021]    The biocidel formulations with cystatins showed activity against different species of mites, mainly  Tetranychus urticae.    
         [0022]    The formulations obtained with the bacteriostatic agent as a diluent are suitable for the treatment of legumes, olive tree, ornamental plants, grape vines, fruit trees, corn, cotton, citrus fruits, vegetables, strawberries, bulbs, oilseed plants, etc. 
     
    
     DESCRIPTION OF THE INVENTION 
       [0023]    In a first aspect, the invention discloses an aqueous solution of a polyol and a dispersing agent, wherein the polyol/dispersing agent ratio is comprised between 30:1 (weight/weight) and 110:1 (weight/weight). 
         [0024]    The dispersing agents comprise alkyl-sulfonates or salts of sulfuric esters derived from monoalcohols, sulfonates or salts of sulfuric esters of polyalcohols or of amino alcohols or of the derivatives thereof, esters of higher fatty acids with hydroxyalkylsulfonic acids or the salts thereof, products from the sulfation or the sulfonation of fats, oils, waxes or higher fatty acids, or of the esters thereof, with monoalcohols, derivatives of sulfocarboxylic or sulfopolycarboxylic acids of low molecular weights, aryl- or alkylaryl-sulfonates, derivatives of phosphoric acid, amines or polyamines, compounds of quaternary ammonium, compounds of phosphonium and sulfonium, amides, sulfonamides, amino-carboxylic acids, protein hydroxylates, carboxylic acid esters, lignin derivatives, natural or synthetic resins or the salts thereof, glucosides, mucilages or saponins. 
         [0025]    Polyols are compounds having several free or esterified hydroxyl groups, e.g., glycerine, sorbitol, monopropylene glycol, mannitol, PEG-200 or PEG-400. 
         [0026]    The solutions obtained after adding the polyol/dispersing agent mixture to different amounts of water did not show bacterial growth until the amount of water was greater than 50% (weight/weight). The dispersants used were sodium polycarboxylate, condensed sodium naphthalene sulfonate, sodium alkylsulfonate, amphoteric polymeric dispersants, acrylic polymers of modified styrene. 
         [0027]    In a second aspect, the invention provides suspensions with biocidel activity comprising: 
         [0028]    an inert solid with a particle size less than 50 microns,
       buffer media,   a biocidel agent, and   an aqueous diluent containing a polyol and dispersing agent, wherein the polyol/dispersing agent ratio is comprised between 30:1 (weight/weight) and 110:1 (weight/weight).       
 
         [0032]    The best results, without persistent foaming, were obtained when the dispersing agent was sodium polycarboxylate or sodium naphthalene sulfonate, the polyol/dispersing agent ratio was 50:1 (weight/weight) and the water content was less than 50% (weight/weight) of the total suspension. 
         [0033]    Suitable inert solids are kaolin, zeolites or silicates. 
         [0034]    The buffer media assure stability of the biocidel agent at a given pH. Citrates or phosphates can be cited among buffering systems. 
         [0035]    The biocidel agents are selected from active agrochemicals such as herbicides, fungicides, acaricides, antidote or growth regulators. In a preferred embodiment, the biocidel agents are of a natural origin: bacteria, fungi, yeasts or proteins. 
         [0036]    Additionally, the suspensions can contain different wetting agents such as ethoxylated sorbitan esters, ethoxylated alcohols, alkyl polyglucosides and the salts thereof, sorbitol esters, for the purpose of assuring a suitable drop size in the application of the product and the proper adherence thereof on the leaf surface. The concentration of wetting agents is comprised between 0-2% by weight. 
         [0037]    The production of the biocidel suspensions is performed according to the following method. The titrated bactericidal agent is adsorbed in an inert carrier; the aqueous solution is then prepared together with the dispersants and the buffering agent to complete dissolution, the biocidel agent adsorbed in its inert carrier is added under constant stirring between 1000 and 1500 rpm with a Cowles-type disperser until complete incorporation and it is taken to volume with the polyol until reaching the desired concentration of biocidel agent and of the polyol/dispersing agent ratio. 
         [0038]    The preceding method is suitable for any biocidel agent of natural origin:
       Bacteria:  Pseudomonas  spp,  Bacillus  spp. such as:  Bacillus aizawai, Bacillus cereus, Bacillus firmus, Bacillus kurstaki, Bacillus lentimorbus, Bacillus licheniformis, Bacillus megaterium, Bacillus popilliae, Bacillus pumilus, Bacillus sphaericus, Bacillus thuringiensis, Bacillus subtilis, Bacillus polymixa, Azotobacter vinelandii, Rhizobium leguminosarum,      Fungi and yeasts of the class  Basidiomycetes, Chytridiomycetes, Deuteromycetes, Hyphochytridiomycetes, Oomycetes, Plasmodiophoromycetes, Sordariomycetes, Trichomycetes  and  Zygomycetes , specifically the species  Arlhrobolrys superba, Arlhrobolrys irregular, Beauveria bassiana, Fusarium  spp.,  Hirsulella rhossiliensis, Hirsulella Ihompsonii, Lagenidium giganleum, Myrolhecium, Nomuraea rileyi, Paecilomyces lilacinus, Trichoderma  spp.,  Vericillium lecanii , and/or  Verlicillium lecanii, Beauveria bassiana, Paecilomyces lilacinus  and  Saccharomyces spp.      Proteins: cystatins       
 
         [0042]    Accelerated stability studies at 54° C. did not cause a reduction in viability of the microorganisms after 14 days and these results assure that the bacterial suspension at room temperature (20° C.) will be stable for at least 2 years. 
         [0043]    For use in agriculture, the suspensions are diluted in water before application. 
       Example 1. Bacteriostatic Activity of the Polyol/Dispersing Agent Mixtures 
       [0044]    Different samples were prepared by changing the ratio of polyol/dispersing agent in water. The samples were stored in a closed bottle at 20° C. for 6 months. The colony forming units were determined according to the method described in the US Pharmacopoeia. 
         [0000]                                                                                                      Polyol = P   Weight (g)   Dispers. = D   Weight (g)   Water (g)    P/D   Water (%)   D (%)                                1   Glycerine   50.1   Atlox 4915   1.0   30   50.1   37%   1.2       2   Glycerine    50.6   Emulson AGPSBV 4   1.2   30   42.2   37%   1.5       3   Glycerine   52.1   Atlox   1.2   30   43.4   36%   1.4                   Metarsperse 500 L                           4   Glycerine   50.5   Emulson AG/TP1   1   30   50.5   37%   1.2       5   Glycerine   50.6   Emulson AGPSBV 4   1.3   30   38.9   37%   1.1       6   Glycerine   50.9   Emulson AG TRST60   1.3   30   39.2   36%   1.6       7   Glycerine   50.1   Madeol AG W 90   1.3   30   38.5   37%   1.6       8   Glycerine   52   Madeol MW   1.3   30   40.0   36%   1.6       9   Glycerine   50.8   Madeol AG OR 95   0.9   30   56.4   37%   1.1       10           Emulson AGPSBV 4   1.04                       10           Madeol AG W 90   0.03                       10    Sorbitol    84   Total   1.07   44   78.1   34%   0.8       11    Mono-   50.1   Madeol AG W 90   1.2   30   41.8   37%   1.5           propylene                                       glycol                                   12    Glycerine   1148   Emulson AGPSV 4   10.0   80   114.4     6%   0.8       13    PEG-200   50   Atlox 4915   0.9   30   55.6   37%   1.1       14    PEG-200   60   Emulson AGPSBV 4   0.9   30   55.6   37%   0.9       15    PEG-400   70   Atlox   1   40   70.0   36%   1.1                   Metarsperse 500 L                           16    PEG-200   70   Emulson AGPSBV 4   1   40   70.0   36%   1.1       17    Mannitol   50   Emulson AGPSBV 4   0.9   30   55.5   37%   1.1       18    PEG-200   84   Atlox 4915   1.06   44   78.1   34%   0.8                    
where
 
Atlox 4915 is an amphoteric polymeric dispersant,
 
Emulson AG PS BV 4 is a sodium polycarboxylate,
 
Atlox Metarsperse 500 L is a modified acrylic styrene polymer,
 
Emulson AG/TP1 is an acrylic derivative in an aqueous dispersion,
 
Emulson AG TRST60 is an amino salt of a polyaryl phosphate ester,
 
Madeol AG/PWA is a sodium salt of an alkyl naphthalene sulfate,
 
Madeol AG W 90 is a condensed naphthalene sulfonate,
 
Madeol MW is a condensed alkyl naphthalene sulfonate,
 
Madeol AG OR 95 is a condensed naphthalene sulfonate.
 
         [0045]    After incubation in all the samples, the microbial load was less than 1000 colony forming units per gram (&lt;1000 CFU/g). 
       Example 2. Stability of the Suspensions 
       [0046]    Different suspensions in water of an inert material with a particle size of 40 microns were prepared with different polyol/dispersing agent mixtures and with different inert solids. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Weight 
                   
                 Weight 
                 Water  
                   
                 Weight 
               
               
                   
                 Polyol 
                 (g) 
                 Dispers. 
                 (g) 
                 (g) 
                 Solid 
                 (g) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 Glycerine 
                 50.1 
                 Atlox 4915 
                 1 
                 30 
                 Kaolin 
                 1 
               
               
                 2 
                 Glycerine 
                 50.6 
                 Emulson 
                 1.2 
                 30 
                 Zeolite 
                 1.2 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 3 
                 Glycerine 
                 52.1 
                 Atlox 
                 1.2 
                 30 
                 Kaolin 
                 1.2 
               
               
                   
                   
                   
                 Metarsperse 
                   
                   
                   
                   
               
               
                   
                   
                   
                 500 L 
                   
                   
                   
                   
               
               
                 4 
                 Glycerine 
                 50.5 
                 Emulson 
                 1 
                 30 
                 Silica 
                 1.1 
               
               
                   
                   
                   
                 AG/TP1 
                   
                   
                   
                   
               
               
                 5 
                 Glycerine 
                 50.6 
                 Emulson 
                 1.3 
                 30 
                 Kaolin 
                 1.0 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 6 
                 Glycerine 
                 50.9 
                 Emulson AG 
                 1.3 
                 30 
                 Kaolin 
                 1.1 
               
               
                   
                   
                   
                 TRST60 
                   
                   
                   
                   
               
               
                 7 
                 Glycerine 
                 50.1 
                 Madeol AG  
                 1.3 
                 30 
                 Kaolin  
                 1.3 
               
               
                   
                   
                   
                 W 90 
                   
                   
                   
                   
               
               
                 8 
                 Glycerine 
                 52.0 
                 Madeol MW 
                 1.3 
                 30 
                 Kaolin 
                 1.3 
               
               
                 9 
                 Glycerine 
                 50.8 
                 Madeol AG  
                 0.9 
                 30 
                 Kaolin  
                 1.0 
               
               
                   
                   
                   
                 OR 95 
                   
                   
                   
                   
               
               
                 10 
                   
                   
                 Emulson 
                 1.04 
                   
                   
                   
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 10 
                   
                   
                 Madeol AG  
                 0.03 
                   
                   
                   
               
               
                   
                   
                   
                 W 90 
                   
                   
                   
                   
               
               
                 10  
                 Sorbitol  
                 84.0 
                 Total 
                 1.07 
                 44 
                 Kaolin  
                 0.1 
               
               
                 11  
                 Mono- 
                 50.1 
                 Madeol AG  
                 1.2 
                 30 
                 Kaolin  
                 1.2 
               
               
                   
                 propylene 
                   
                 W 90 
                   
                   
                   
                   
               
               
                   
                 glycol 
                   
                   
                   
                   
                   
                   
               
               
                 12  
                 Glycerine 
                 50 
                 — 
                 0 
                 30 
                 Kaolin  
                 1.1 
               
               
                 13  
                 Sorbitol  
                 50.0 
                 Atlox 4915 
                 1 
                 30 
                 Kaolin  
                 1 
               
               
                 14  
                 Sorbitol  
                 50.6 
                 Emulson 
                 1.2 
                 30 
                 Zeolite 
                 1.2 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 15  
                 Sorbitol  
                 52.1 
                 Atlox 
                 1.2 
                 30 
                 Kaolin  
                 1.2 
               
               
                   
                   
                   
                 Metarsperse 
                   
                   
                   
                   
               
               
                   
                   
                   
                 500 L 
                   
                   
                   
                   
               
               
                 16  
                 Sorbitol 
                 50.5 
                 Emulson 
                 1.0 
                 30 
                 Silica  
                 1.1 
               
               
                   
                   
                   
                 AG/TP1 
                   
                   
                   
                   
               
               
                 17  
                 Sorbitol 
                 50.6 
                 Emulson 
                 1.3 
                 30 
                 Kaolin  
                 1.0 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 18  
                 Sorbitol 
                 50.9 
                 Emulson AG 
                 1.3 
                 30 
                 Kaolin  
                 1.1 
               
               
                   
                   
                   
                 TRST60 
                   
                   
                   
                   
               
               
                 19  
                 Sorbitol  
                 50.1 
                 Madeol AG  
                 1.3 
                 30 
                 Kaolin  
                 1.3 
               
               
                   
                   
                   
                 W 90 
                   
                   
                   
                   
               
               
                 20  
                 Sorbitol 
                 52.0 
                 Madeol MW 
                 1.3 
                 30 
                 Kaolin  
                 1.3 
               
               
                 21  
                 Sorbitol 
                 50.8 
                 Madeol AG  
                 0.9 
                 30 
                 Kaolin  
                 1 
               
               
                   
                   
                   
                 OR 95 
                   
                   
                   
                   
               
               
                 22  
                 Sorbitol  
                 49 
                 — 
                 0 
                 30 
                 Kaolin  
                 1.3 
               
               
                 23  
                 Glycerine 
                 1148 
                 Emulson 
                 10.0 
                 80 
                 Kaolin  
                 1 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 24  
                 PEG-200 
                 1148 
                 Madeol AG  
                 10.1 
                 80 
                 Silica  
                 1 
               
               
                   
                   
                   
                 OR 95 
                   
                   
                   
                   
               
               
                 25  
                 Mannitol 
                 1149 
                 Madeol AG  
                 10.0 
                 81 
                 Kaolin  
                 1.2 
               
               
                   
                   
                   
                 OR 95 
               
               
                   
               
             
          
         
       
     
         [0047]    The samples were centrifuged for 15 minutes at 1735 rpm with a Cowles-type agitator. 
         [0048]    The results show that when condensed naphthalene sulfonate or sodium polycarboxylate is used as a dispersant, precipitation of the dispersed solids is not observed after centrifugation. 
         [0049]    Samples 12 and 22, without dispersing agents, showed the precipitation of kaolin. 
         [0050]    The presence of persistent foam or the presence of precipitated solids was observed in the other cases. 
       Example 3. Stability of the Suspensions According to the Solids Content 
       [0051]    Different suspensions were prepared using sodium polycarboxylate (Emulson AG PS BV 4) as a dispersing agent. Different amounts of inert solid were added. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Weight 
                   
                 Weight 
                 Water 
                   
                 Weight 
               
               
                   
                 Polyol 
                 (g) 
                 Dispers. 
                 (g) 
                 (g) 
                 Solid 
                 (g) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 Glycerine 
                 50.6 
                 Emulson 
                 1.2 
                 30 
                 Kaolin 
                 10.1 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 2 
                 Sorbitol  
                 50.4 
                 Emulson 
                 1.2 
                 30 
                 Kaolin 
                 5.5 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 3 
                 Mono- 
                 49.5  
                 Emulson 
                 1.3 
                 30 
                 Kaolin 
                 2.6 
               
               
                   
                 propylene 
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                   
                 glycol 
                   
                   
                   
                   
                   
                   
               
               
                 4 
                 Glycerine  
                 50.1 
                 Emulson 
                 1.3 
                 30 
                 Kaolin 
                 20.0 
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 5 
                   
                   
                 Emulson 
                 10.03 
                   
                   
                   
               
               
                   
                   
                   
                 AGPSBV 4 
                   
                   
                   
                   
               
               
                 5 
                   
                   
                 Tween 85 
                 8.57 
                   
                   
                   
               
               
                 5 
                 Glycerine  
                 1148 
                 Total 
                 18.6 
                 77.9  
                 Kaolin 
                 1.1 
               
               
                 6 
                 PEG-200 
                 1156 
                 Emulson 
                 17.7 
                 80.0  
                 Kaolin 
                 1.1 
               
               
                   
                   
                   
                 AGPSBV 4 
               
               
                   
               
             
          
         
       
     
         [0052]    No precipitates were observed after centrifuging for 15 minutes at 1735 rpm. 
       Example 4. Producing Bacterial Suspensions with Biocidal Activity 
       [0053]    0.10 grams of kaolin containing 10 10  cfu/g of Bacillus subtilis strain IAB, obtained according to ES24002726, was dissolved in distilled water, the dispersing agents were added and the suspension was homogenized. The suspension was then diluted to 100 grams (cps) with polyols. The pH of the suspension was adjusted to pH 7 with citric acid. The concentration of the obtained suspension was 10 7  cfu/g. 
         [0054]    The dispersing agents and polyols used were: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                 Weight of 
                   
               
               
                   
                 Weight of 
                   
                 dispersing 
                 Polyol cps 
               
               
                 Sample 
                 water (g) 
                 Dispersing agent 
                 agent (g) 
                 100 g 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 7.5 
                 Emulson AGPSBV 4 
                 1.20 
                 Glycerine 
               
               
                 2 
                 6.0 
                 Madeol AG W 90 
                 1.50 
                 Sorbitol 70% 
               
               
                 3 
                 7.6 
                 Emulson AGPSBV 4 
                 2.50 
                 Monopropylene 
               
               
                   
                   
                   
                   
                 glycol 
               
               
                 4 
                 8.0 
                 Emulson AGPSBV 4 
                 2.30 
                 Glycerine 
               
               
                 5 
                 12.3 
                 Madeol AG W 90 
                 3.60 
                 Sorbitol 70% 
               
               
                 6 
                 7.5 
                 Madeol AG W 90 
                 1.65 
                 Monopropylene 
               
               
                   
                   
                   
                   
                 glycol 
               
               
                 7 
                 7.6 
                 Emulson AGPSBV 4 
                 1.20 
                 PEG-200 
               
               
                 8 
                 7.5 
                 Emulson AGPSBV 4 
                 1.19 
                 Mannitol 
               
               
                   
               
             
          
         
       
     
         [0055]    The bacterial suspensions were prepared without adding preserving agents, such as azides, isothiazolines or parabens. 
       Example 5. Effectiveness of the Suspensions Against Sclerotinia in Lettuce 
       [0056]    A liquid bacterial suspension of  Bacillus subtilis  strain IAB with a concentration of 10 7  cfu/g was as effective against Sclerotinia in lettuce as a solid formulation of  Bacillus subtilis  strain QST 713 in diatomaceous earth with a concentration of 5.13*10 10  cfu/g. 
         [0057]    The quantitative composition of the suspension was: 
         [0058]    Emulson AGPSBV 4: 1.2 grams 
         [0059]    Water: 7.6 grams 
         [0060]    Kaolin: 1.2 grams 
         [0061]    Glycerine: 91.2 grams 
         [0062]    pH: 7.1 
         [0063]    Polyol/dispersing agent ratio: 75:1 
         [0064]    The bacterial suspension with a concentration of 10 7  cfu/g was diluted to different concentrations in water: 300 mL/100 L; 600 mL/100 L; 1000 mL/100 L, and these dilutions were used as biocidal agent against Sclerotinia in lettuce 
         [0065]    Serenade Max® at 15.65% was used as a control biocidal agent at a dose of 4 kg/Ha, which is almost equivalent to 4 mL/100 L. 
         [0066]    Biocidal activity was observed in all samples. 
       Example 6. Producing Suspensions of Proteins with Biocidal Activity 
       [0067]    100 mg of a cystatin in 1 gram of kaolin was dispersed in 7.7 grams of distilled water. 1.04 grams of sodium polycarboxylate (Emulson AG PS BV 4) and 0.03 of condensed naphthalene sulfonate were added. The mixture was homogenized and 119.34 grams of sorbitol 70 were added. The pH of the suspension was adjusted to pH 7 with citric acid. Concentration of the suspension was 10 7  cfu/g. The suspension that was produced inhibited the activity of cathepsins, so the suspensions of proteins do not lose biocidel activity of the cystatins. 
       Example 7. Stability Study of the Bacterial Suspensions of  Bacillus subtilis    
       [0068]    An accelerated stability study of a bacterial suspension of  Bacillus subtilis  strain IAB with a concentration of 1*10 7  cfu/g obtained according to Example 4, sample 1, was performed. 
         [0069]    The mean parameters were appearance, pH, concentration of the active ingredient, stability of the diluted suspension at 5%. 
         [0070]    The storage conditions were 14 days at 54° C., the samples being packaged into closed plastic bottles. 
         [0071]    The obtained results are detailed below: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
               
                 Assay 
                 Specification 
                 T = 0 
                 T = 54 days 
               
               
                   
               
             
             
               
                 Aspect 
                 Yellowish homogenous 
                 Compliant 
                 Compliant 
               
               
                   
                 dispersion with 
               
               
                   
                 characteristic odor 
               
               
                 pH 
                 7-7.5 
                 7.25 
                 7.22 
               
               
                 Stability of the 
                 Homogenous 
                 Compliant 
                 Compliant 
               
               
                 suspension at 5% 
                 after 18 hours 
               
               
                 
                   B. subtilis 
                 
                 Greater than 
                 1.40*10 7   
                 1.38*10 7   
               
               
                   
                 10 7  cfu/g 
                 cfu/g 
                 cfu/g 
               
               
                   
               
             
          
         
       
     
         [0072]    The obtained results assure stability of the suspension for 2 years at room temperature. 
       Example 8. Effectiveness Against Botrytis in Tomatoes 
       [0073]    A liquid biocidal formulation of  Bacillus subtilis  strain IAB with a concentration of 10 7  cfu/g was as effective against Botrytis in tomatoes as a solid formulation of Bacillus subtilis strain QST 713 in diatomaceous earth with a concentration of 5.13*10 10  cfu/g. 
         [0074]    The quantitative composition was: 
         [0075]    Madeol AG W 90: 2.1 grams 
         [0076]    Water: 8.6 grams 
         [0077]    Kaolin: 1.1 grams 
         [0078]    Polyethylene glycol 200: 83.4 grams 
         [0079]    pH: 7.1 
         [0080]    Polyol/dispersing agent ratio: 39:1 
         [0081]    The bacterial suspension with a concentration of 10 7  cfu/g was diluted to different concentrations in water: 1 L/ha; 2 L/ha I; 3 L/ha, and these dilutions were used as a biocidal agent against Sclerotinia in lettuce. 
         [0082]    Serenade Max® at 15.65% was used as a control biocidal agent with a dose of 4 kg/Ha, which is almost equivalent to 4 mL/100 L. 
         [0083]    Biocidal activity was observed in all samples. 
       Example 9. Effectiveness Against Monilia in Peaches 
       [0084]    A liquid biocidal formulation of  Bacillus subtilis  strain IAB with a concentration of 10 7  cfu/g was as effective against Monilia in peaches as a solid formulation of Bacillus subtilis strain QST 713 in diatomaceous earth with a concentration of 5.13*10 10  cfu/g. 
         [0085]    Madeol AG W 90: 2.0 grams 
         [0086]    Water: 8.6 grams 
         [0087]    Kaolin: 1.5 grams 
         [0088]    Polyethylene glycol 400: 83.8 grams 
         [0089]    pH: 7.5 
         [0090]    Polyol/dispersing agent ratio: 42:1 
         [0091]    Phase separation was not observed in the suspension. 
         [0092]    The bacterial suspension with a concentration of 10 7  cfu/g was diluted to different concentrations in water: 1 L/ha; 2 L/ha I; 3 L/ha, and these dilutions were used as a biocidal agent against Sclerotinia in lettuce 
         [0093]    Serenade Max at 15.65% was used as a control biocidal agent at a dose of 4 kg/Ha, which is almost equivalent to 4 mL/100 L. 
         [0094]    Biocidal activity was observed in all samples. 
       Example 10. Effectiveness of the Formulations of Cystatin 
       [0095]    A liquid biocidal formulation of cystatin with a concentration of 1% w/w was as effective against  Tetranychus urticae  as a formulation of Abamectin with a concentration of 1.8% w/w. 
         [0096]    The cystatin-based biocidal suspension, with a concentration of 1% w/w and with the following excipients: 
         [0097]    Madeol AG W 90: 2.1 grams 
         [0098]    Water: 8.6 grams 
         [0099]    Kaolin: 1.2 grams 
         [0100]    Polyethylene glycol 200: 83.4 grams 
         [0101]    pH: 7.1 
         [0102]    Polyol/dispersing agent ratio: 39:1, 
         [0000]    was diluted in water at a concentration of 20 mL/L, and this dilution was used as a biocidal agent against Tetranychus urticae in strawberry farming. LAOTTA® at 1.8% was used as a control biocidal agent at a dose of 100 mL/100 L. 
         [0103]    Biocidal activity was observed in all samples.