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    dry mixes, composition, method for preparing a dry mix and method for increasing the germination, growth, metabolism and/or enzymatic activity of bacterial spores in one aspect, the present invention relates to a dry mix that comprises a bacterial spore and a germinal compound, as well as methods of preparing the intimate mixture. in another aspect, the present invention relates to a composition comprising such an intimate mixture. the present invention also relates to methods of increasing the germination, growth, metabolism and/or enzymatic activity of bacterial spores, which comprise preparing an intimate mixture of a bacterial spore and a germinal compound.
    公开号:BR112015018783B1
    申请号:R112015018783-8
    申请日:2014-02-06
    公开日:2022-01-04
    发明作者:Tommie Eugene Hashman；Michael Matheny
    申请人:Envera Lic, Llc；
    IPC主号:
    专利说明:

    CROSS REFERENCE TO RELATED ORDERS
    [001] This application claims the priority benefit of US Interim Application Serial No. 61/849,973, filed February 6, 2013, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION
    [002] In one aspect, the present invention relates to a dry intimate mixture comprising a bacterial spore and a germinal compound, as well as methods of preparing the mixture. In another aspect, the present invention relates to a composition comprising such a mixture. The present invention also generally relates to methods of increasing the germination, growth, metabolism and/or enzymatic activity of bacterial spores, which comprise the preparation of a dry intimate mixture comprising a bacterial spore and a germinal compound. BACKGROUND OF THE INVENTION
    [003] The use of spore-forming bacteria, including certain strains of Bacillus as probiotics for humans and animals, has become frequent in recent years. As indicated in Knap et al (WO 2010/070005), species such as Bacillus subtilis and Bacillus licheniformis are used as supplements in animal feed in order to promote growth through increased digestion and availability of nutrients in animal feed. Bacillus coagulans is the active ingredient of commercial probiotic products for human consumption, which aid in the digestion of proteins, lactose and fructose.
    [004] As indicated in Maathuis et al (2010, Beneficial Microbes 1(1): 31-36), these bacteria must be present in the small intestine in their germinated or vegetative form in order to function as probiotics. Although these microbes are resistant to stomach acid and bile salts in their spore form, they are susceptible to these environments in their vegetative states. Therefore, if employed in their vegetative state, Bacillus strains must be contained in a pharmaceutically acceptable “enteric” or acid-resistant carrier. See Paragraph 7 of Farmer (US Patent Application No. 2003/0124104).
    [005] Unfortunately, it is difficult to formulate Bacillus species in their vegetative form so that they have adequate shelf life. As indicated in the Ganeden BC product literature, traditional vegetative probiotics do not survive high heat and pressure in the manufacturing process, die quickly in storage, and are sensitive to stomach acids and bile enzymes in the intestine. On the other hand, formulations of these species in their spore form are much more suitable for practical and commercial use. Therefore, as indicated by Cartman et al (2008), Applied and Environmental Microbiology, August, pp. 5254-5258), “bacterial spores are particularly suitable for use as live microbial products as they are metabolically dormant and highly resilient to environmental stress. These intrinsic properties are highly desirable from a commercial point of view and indicate that spore-based products have a long shelf life and retain their viability during distribution and storage.”
    [006] The use of certain compounds, particularly certain L-amino acids, to stimulate the germination of Bacillus spores, has been reported in the literature. Therefore, for example, Foerster et al (1966, Journal of Bacteriology 91 (3): 1168-1177) describe that the addition of L-alanine to spore suspensions in aqueous solutions will cause a number of Bacillus species to germinate. Furthermore, Maathius et al, op. cit., suggests that germination of Bacillus coagulans spores in Ganeden BC could be triggered early in the small intestine by ingesting them in conjunction with a diet containing L-alanine or by including L-alanine with these spores in a formulation in dust. The approaches suggested by Maathius, for example, present several important challenges to establishing a probiotically effective bacterial culture: 1. Although the Bacillus coagulans spores used in Ganeden BC themselves are very resistant to the low pH of the stomach, exposure to these acids can generate delay in germination when these spores enter a more neutral pH. For example, Blocher et al (1985, Applied and Environmental Microbiology 50 (2): 274-279) demonstrated that germination of B. cereus spores was inhibited at pH 4.5, even in the presence of the germinal compounds L-alanine or L-cysteine. Spores exposed sequentially to buffer at pH 4.5, followed by buffer at pH 7.0, were able to germinate upon exposure to these L-amino acids, but exhibited delayed germination effort. Any substantial delay in germination is highly undesirable considering the relatively short period of time that spores can be present in the small intestine before being excreted. This is particularly true in smaller animals such as chicks, which have feeding transit times of about one and a half hours at one day of age and transit times of less than two hours at seven days of age (see BC Watson et al. (2006, Poultry Science 85: 493-497)) and shrimp, which have transit times of less than ninety minutes (see Beseres et al, 2005, Journal of Shellfish Research 24 (1): 301308). There remains, therefore, the need to accelerate and increase the germination of bacterial spores under low pH exposure conditions, such as those found in the stomach. 2. Diets high in L-alanine may also be high in D-alanine. As indicated by Atluri et al (2006, Journal of Bacteriology 188 (1): 28-36) and Blocher et al (noted above), D-alanine is a potent inhibitor of Bacillus germination. In addition, there may be large amounts of other germination inhibitors (e.g., other D-amino acids, organic and inorganic acids, fatty acids, and bile salts) present in the small intestine that would compete with L-alanine if mixed in powdered form with Bacillus spores. There is, therefore, a need to develop a method of enhancing spore germination in the presence of germination inhibitors that can compete with germinal compounds.
    [007] Accordingly, it is an object of the present invention to provide a bacterial spore formulation that is capable of providing these benefits. BRIEF DESCRIPTION OF THE INVENTION
    [008] It has been surprisingly found that the germination, growth, metabolism and enzymatic activity of bacterial spores are increased through the formation of an intimate mixture of the bacterial spore and a germinal compound. Unexpectedly, intimate mixing also enhances germination and bacterial spore growth in the presence of germination inhibitors.
    [009] In one aspect, the present invention relates to a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the bacterial spore and the germinal compound are held in close proximity until they reach an environment conducive to germination. .
    [0010] In another aspect, the present invention relates to a composition comprising a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the bacterial spore and the germinal compound are held in close proximity until they reach an environment leading to germination.
    [0011] In a further aspect, the present invention relates to a method of preparing a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the method comprises: a. preparing a solution comprising a bacterial spore and a germ compound; and b. drying the solution to obtain a dry intimate mixture comprising a bacterial spore and a germinal compound; wherein the bacterial spore and germinal compound are held in close proximity until they reach an environment conducive to germination.
    [0012] In yet another aspect, the present invention relates to a method of increasing the germination, growth, metabolism and/or enzymatic activity of bacterial spores, comprising: a. preparing a solution comprising a bacterial spore and a germ compound; B. drying the solution to obtain a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the bacterial spore and the germinal compound are held in close proximity until they reach an environment conducive to the germination of the bacterial spores; and c. exposing the intimate mixture to an environment conducive to bacterial spore germination, wherein the germination, growth, metabolism and/or enzymatic activity of the bacterial spores in the intimate mixture is increased relative to a corresponding bacterial spore formulation that does not contain a germinal compound. DETAILED DESCRIPTION OF THE INVENTION
    [0013] The mixtures according to the present invention are composed of a bacterial spore and a germinal compound. The bacterial species employed can be any species that forms spores and typically exhibits desirable probiotic activity in humans or animals. Bacterial species that have other industrial applications, including those useful in agriculture, environmental remediation, composting or methane production, however, cleaning materials and the like may also be employed. These species include spore-forming members of the phylum Firmicutes and spore-forming members of the phylum Actinobacteria, as listed in Bergey's Manual of Systematic Bacteriology, second edition (2009), fully incorporated herein by reference. Members of the phylum Firmicutes include aerobic spore-forming species (generally formerly defined as Bacillus species) and anaerobic spore-forming species (generally formerly defined as Clostridium species). Illustrative species of the phylum Firmicutes include B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B. centroporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentimorbus, B. lentus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C. ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuria penetrans, Pasteuria thornei and Pasteuria nishizawa, as well as genetically modified variants of these species. Preferred species include B. amyloliquefaciens, B. brevis, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. laterosporus, B. lentus, B. licheniformis, B. megaterium, B. polymyxa, B. B. pumilus, B. simplex, B. sphaericus, B. stearothermophilus, B. subtilis, B. thuringiensis and C. butyricum. Members of the phylum Actinobacteria include species of Streptomyces as well as genetically modified variants of these species. Preferred species include Streptomyces viridochromogenes, Streptomyces griseoviridis, Streptomyces lydicus, Streptomyces plicatus, Streptomyces sindeneusis, Streptomyces rochei, Streptomyces alni, Streptomyces viridis, Streptomyces thermovulgaris, Streptomyces griseus, Streptomyces acidiscabies, Streptomyces aureofaciens, Streptomyces galbus, Streptomyces microflavus, and Streptomyces aureofaciens.
    [0014] The germinal compound may comprise any compound which is effective in causing germination of the specific spore-forming bacteria species with which it is intimately mixed and which is suitable for useful processes of forming such intimate mixture, such as spray drying, drying by freezing, air drying or drum drying. Typically, such germ compounds are L-amino acids, including L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine, L-phenylalanine and analogues thereof. Such analogs can be created by those of ordinary skill in the art by making substitutions on or within base chemistry groups. Therefore, L-alanine analogues include, for example: L-Leu-L-Ala, L-Ala-L-Leu, L-Pro-L-Ala, L-Ala-L-Pro, aL-Glu-L- Ala, L-Ala-L-Glu, L-His-L-Ala, L-Ala-L-His, L-Ala-L-Ala, Gly-L-Ala, L-Ala-Gly, dicyclo- N42-(Methylsulfonyl)ethyloxycarbonyl-L-alanine (N-MSOC-L-Ala) hexylammonium, Nt-butoxycarbonyl-L-alanine (Nt-BOC-L-Ala), N-acetyl-L-alanine (N-Ac -L-Ala), N-2,4-dinitrophenyl-L-alanine (N-DNP-L-Ala), N-carbobenzoxy-L-alanine (N-CBZ-L-Ala), cyclohexylamine salt of 5-Dimethylamino-1-naphthalenesulfonyl-L-alanine (N-dansyl-L-Ala), N-benzoyl-L-alanine (N-Bz-L-Ala), L-alanine methyl ester hydrochloride, ethyl ester hydrochloride L-alanine, t-butyl ester hydrochloride of L-alanine, benzyl ester hydrochloride of L-alanine, L-alaninamide, L-alanine hydrochloride p-nitroanilide and L-alaninol. In one embodiment, the germ compound is an amino acid selected from the group consisting of L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine and L-phenylalanine. Preferably, that germ compound is L-alanine.
    [0015] These amino acids can be used as individual compounds or in the form of polypeptides. In one embodiment, the polypeptides are protein hydrolysates, such as casein hydrolyzate. Useful polypeptides will typically comprise at least 50% amino acids that will function as seedlings, such as L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine and L - phenylalanine, where these percentages are based on the amount of amino acids in the polypeptide.
    [0016] In a preferred embodiment, the germ compound is selected from the group consisting of L-amino acids, proteins, sugars and salts. Particularly preferred combinations of germ compounds and bacterial spores include: - L-alanine + Bacillus subtilis, L-alanine + Bacillus licheniformis, L-alanine + Bacillus pumilus, L-alanine + Bacillus amyloliquefaciens, L-alanine + Bacillus coagulans, L-alanine + Bacillus cereus, L-alanine + Bacillus clausii, L-alanine + Clostridium butyricum - L-valine + Bacillus subtilis, L-valine + Bacillus licheniformis, L-valine + Bacillus pumilus, L-valine + Bacillus amyloliquefaciens, L-valine + Bacillus coagulans, L-valine + Bacillus cereus, L-valine + Bacillus clausii, L-valine + Clostridium butyricum - L-alanine + glucose+fructose+potassium ions (GFK) + Bacillus subtilis, L-alanine + glucose+fructose+ potassium ions (GFK) + Bacillus licheniformis, L-alanine + glucose+fructose+potassium ions (GFK) + Bacillus pumilus, L-alanine + glucose+fructose+potassium ions (GFK) + Bacillus amyloliquefaciens, L-alanine + glucose+fructose+potassium ions (GFK) + Bacillus coagulans, L-alani na + glucose+fructose+potassium ions (GFK) + Bacillus cereus, L-alanine + glucose+fructose+potassium ions (GFK) + Bacillus clausii, L-alanine + glucose+fructose+potassium ions (GFK) + Clostridium butyricum - L-asparagine + glucose+fructose+potassium ions (GFK) + Bacillus subtilis, L-asparagine + glucose+fructose+potassium ions (GFK) + Bacillus licheniformis, L-asparagine + glucose+fructose+potassium ions ( GFK) + Bacillus pumilus, L-asparagine + glucose+fructose+potassium ions (GFK) + Bacillus amyloliquefaciens, L-asparagine + glucose+fructose+potassium ions (GFK) + Bacillus coagulans, L-asparagine + glucose+fructose+ potassium ions (GFK) + Bacillus cereus, L-asparagine + glucose+fructose+potassium ions (GFK) + Bacillus clausii, L-asparagine + glucose+fructose+potassium ions (GFK) + Clostridium butyricum - L-alanine + inosine + Bacillus subtilis, L-alanine + inosine + Bacillus licheniformis, L-alanine + inosine + Bacillus pumilus, L-alanine + inosine + Bacillus amyloliquefacie ns, L-alanine + inosine + Bacillus coagulans, L-alanine + inosine + Bacillus cereus, L-alanine + inosine + Bacillus clausii, L-alanine + inosine + Clostridium butyricum - L-proline + glucose + Bacillus megaterium, L-proline + Bacillus megaterium and L-lactate + Clostridium butyricum.
    [0017] In certain embodiments, preferred mixtures according to the present invention include those wherein the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus; and the germ compound is selected from the group consisting of L-alanine, L-valine and L-asparagine.
    [0018] The germinal compound is present in sufficient quantity to cause the bacterial spore employed to germinate. While this can be readily determined for any specific mixture of bacterial spores and germ compound through routine experimentation, these germ compounds are typically formulated, prior to drying, at concentrations from 0.0001 mg/ml to 170 mg/ml. In some embodiments, the germinal compounds are formulated, prior to drying, at concentrations of 0.0003 mg/ml to 170 mg/ml, 0.0003 mg/ml to 30 mg/ml, 0.001 mg/ml to 100 mg/ml or 0.001 mg/ml to 10 mg/ml. In a preferred embodiment, the germinal compound is formulated, prior to drying, in concentrations from 0.001 mg/ml to 1 mg/ml.
    [0019] As used in the present, the expression "intimate mixture" designates a mixture in which the spores and germinative compounds are kept in a close position until they reach an environment that leads to germination.
    [0020] This intimate mixing can be achieved employing processes such as spray drying, freeze drying, air drying or drum drying. In a preferred embodiment, the intimate mixture is produced by spray drying or freeze drying. When forming such intimate mixtures, it is important that the spore and germ compost are not mixed together under conditions that would allow the germ compost to cause the spore to germinate, as this would cause premature germination with detrimental effect on the life of the mixture in storage. This can be avoided by employing separate streams in a spray dryer, either using two nozzles or a single nozzle that allows simultaneous spraying of two separate streams; or by freeze-drying under conditions (such as temperatures) that are not conducive to germination. Premature germination can also be prevented by introducing the spore mass into a solution containing the germinal compound immediately before drying.
    [0021] In a preferred embodiment, the germ compound is adsorbed or absorbed by the bacterial spore in the intimate mixture. In a further embodiment, the bacterial spore and germ compound are finely dispersed throughout the intimate mixture. In yet another embodiment, the bacterial spore and germ compound are microscopically dispersed throughout the intimate mixture such that individual particles consisting essentially of bacterial spores and individual particles consisting essentially of germ compounds are not visible to the eye. naked.
    [0022] In one embodiment, the intimate mixture is prepared by combining the bacterial spore and the germ compound in a solution prior to drying. Preferably, the bacterial spore and germ compound are combined in a solution just before drying.
    [0023] While not wishing to be bound by any theory, it is believed that the formation of an intimate mixture places the germinal compound in a proximate position, in which it can more preferably bind to the spore germination initiator sites when the mixture to reach a suitable environment for germination. This close position allows the germinal compound to win the competition with compounds that interfere with germination (such as D-amino acids) that may be present and, as a result, will germinate a higher percentage of spores. Due to the logarithmic growth of bacteria after entering the vegetative state, this higher percentage can quickly result in a large logarithmic increase in crop formation.
    [0024] The present invention also relates to compositions comprising an intimate mixture comprising (a) a bacterial spore and (b) a germinal compound, wherein the spore and the germinal compound are maintained in an inactive form such that the germinal compound does not induce spore germination until this composition is subjected to an activation environment.
    [0025] As used in the present, the expression “activation environment” designates an environment that allows the interaction between the germinal compound and the spore, in such a way that the spore is induced to enter a vegetative state. This activation environment may involve a combination of factors such as temperature, humidity, pH or salinity.
    [0026] The formation of an intimate mixture of a germinal compound and a bacterial spore can increase the usefulness of the bacterial spore by enhancing one or more characteristics of interest of the bacterial spore, including, but not limited to, germination, growth, metabolism, enzymatic activity, and tolerance to environmental stresses such as low pH, high concentration of salts and exposure to toxic metals. In some embodiments, intimate mixing increases the characteristic of interest by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500% relative to a formulation of corresponding bacterial spores that do not contain a germ compound.
    [0027] In order to further increase germination, it is preferred that the spore undergoes shock prior to the formation of the intimate mixture. Spores can be shocked by a number of standard methods, such as osmotic shock, heat shock, pressure shock, nutrient deprivation and/or exposure to certain acids. Heat shock involves heating the spores for a sufficient period of time at a temperature sufficient to induce the production of heat shock proteins. Atluri et al, op. cit., describes one such heat shock treatment for Bacillus subtilis.
    [0028] The compositions according to the present invention comprise an intimate mixture comprising a bacterial spore and a germinal compound. Such compositions may further comprise additional components, including cogerminants, nutrients, and formulation aids (e.g., surfactants and/or enteric coatings), depending on their intended use.
    [0029] Cogerminants that may be employed include purine nucleosides such as inosine or adenosine, salts, sugars (such as glucose and fructose) and the like; all of which are well known to those skilled in the art.
    [0030] Nutrients may also be included, including dextrose, starches and micronutrients that will aid in the multiplication of bacterial colonies after spore germination.
    [0031] When the composition is intended for use as a probiotic, the use of enteric coating is preferably employed, in order to avoid the reduction of spore germination associated with the exposure of spores to low pH environments. This enteric coating is designed to resist solution in the stomach and dissolve in neutral or alkaline intestinal fluid. This coating can be pH sensitive, for example, not dissolving in an acidic environment like that found in the stomach, but dissolving in a neutral environment like that found in the small intestine. Alternatively, the enteric coating may dissolve when exposed to specific metabolic events, such as encountering a digestive enzyme that is found in the small intestine. The coating is digested, for example, by a pancreatic enzyme such as trypsin, chymotrypsin or pancreatic lipase. Digestion or dissolution of the coating allows the entry of the germinal compound and Bacillus spores into an environment conducive to spore germination.
    [0032] Enteric coating materials that may be employed are known in the art and include alginates, malic acid-propane 1,2-diol; cellulose derivatives such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate (HPMCP); cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and anionic polymers of methacrylic acid and methyl methacrylate; and an emulsion in water of a copolymer of ethyl acrylate and methacrylic acid or hydroxypropyl methyl cellulose acetate succinate (HPMAS).
    [0033] When employed in agricultural or industrial uses, these compositions may further comprise standard formulation aids, such as surfactants, emulsifiers, other active ingredients, etc., provided that these other components do not interfere with germination or impair the viability of the germinated spores. . Phenolic compounds that are otherwise not particularly sporocidal, for example, are known to inhibit germination at concentrations up to 0.2% (phenol), 0.08% (cresol), and 0.02% (chlorocresol) ( weight/volume). Other compounds that can inhibit germination are also well known in the art. See, for example, A.D. Russell, Bacterial Spores and Chemical Sporicidal Agents, Clinical Microbiology Reviews, April 1990, pp. 99-119.
    [0034] The present invention also provides a method of preparing a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the method comprises: a. preparing a solution comprising a bacterial spore and a germ compound; and b. drying the solution to obtain a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the bacterial spore and the germinal compound are held in close proximity until they reach an environment conducive to germination.
    [0035] In a preferred embodiment, the drying in the above-mentioned method is spray drying, freeze drying, air drying or drum drying. In another preferred embodiment, the spore of the above-mentioned method is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B. centroporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C. ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae and Streptomyces spp.
    [0036] In a further preferred embodiment, the spore of the above-mentioned method is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus; and the germ compound of the above-mentioned method is selected from the group consisting of L-alanine, L-valine and L-asparagine.
    [0037] The germinal compound of the above-mentioned method can be formulated, before drying, in concentrations from 0.0001 mg/ml to 170 mg/ml. In some embodiments, the germinal compounds are formulated, prior to drying, at concentrations of 0.0003 mg/ml to 170 mg/ml, 0.0003 mg/ml to 30 mg/ml, 0.001 mg/ml to 100 mg/ml or 0.001 mg/ml to 10 mg/ml. In a preferred embodiment of the above-mentioned method, the germinal compound is formulated, prior to drying, in concentrations from 0.001 mg/ml to 1 mg/ml. In certain embodiments, the germ compound of the above-mentioned method is a polypeptide. In a preferred embodiment of the above-mentioned method, the spore has been shocked.
    [0038] The present invention also provides a dry intimate mix produced by means of the above mentioned methods.
    [0039] In another aspect, the present invention provides a method of increasing the germination, growth, metabolism and/or enzymatic activity of bacterial spores, comprising: c. preparing a solution comprising a bacterial spore and a germ compound; d. drying the solution to obtain a dry intimate mixture comprising a bacterial spore and a germinal compound, wherein the bacterial spore and the germinal compound are held in close proximity until they reach an environment conducive to the germination of the bacterial spores; and is. exposing the intimate mixture to an environment conducive to bacterial spore germination, wherein the germination, growth, metabolism and/or enzymatic activity of the bacterial spores in the intimate mixture is increased relative to a corresponding bacterial spore formulation that does not contain a germinal compound.
    [0040] In some embodiments of the above-mentioned method, intimate mixing increases the characteristic of interest by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500% against a corresponding bacterial spore formulation that does not contain a germ compound.
    [0041] In a preferred embodiment of the aforementioned method, the percentage of germination, growth, metabolism and/or enzymatic activity of bacterial spores in the intimate mixture increases by at least 10% with respect to the corresponding bacterial spore formulation that does not contain a compound germinal. In a further preferred embodiment of the above-mentioned method, the drying is spray drying, freeze drying, air drying or drum drying.
    [0042] In certain embodiments, the spore of the above-mentioned method is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B. boronophilus, B. brevis, B. caldolyyicus, B. centrolyyicus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C. ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae and Streptomyces spp. In a preferred embodiment, the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus; and the germ compound is selected from the group consisting of L-alanine, L-valine and L-asparagine.
    [0043] In some embodiments of the above-mentioned method, the germ compound is a polypeptide. In a preferred embodiment, the germinal compound of the above-mentioned method is formulated, prior to drying, in concentrations from 0.0001 mg/ml to 170 mg/ml. In some embodiments of the above-mentioned method, the germinal compounds are formulated, prior to drying, in concentrations of 0.0003 mg/ml to 170 mg/ml, 0.0003 mg/ml to 30 mg/ml, 0.001 mg/ml to 100 mg/ml or 0.001 mg/ml to 10 mg/ml. In a preferred embodiment of the above-mentioned method, the germinal compound is formulated, prior to drying, in concentrations from 0.001 mg/ml to 1 mg/ml. In a further preferred embodiment of the above-mentioned method, the spore has been shocked. EXAMPLES
    [0044] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention in any way.
    [0045] In the Examples below, the terms "GOSD" and "GO+" designate compositions in which a germinal enhancer (L-alanine, unless otherwise specified) has been spray dried with the particular Bacillus species indicated. Spores of Bacillus species were spray dried by introducing L-alanine to the spore mass immediately before spray drying as a solution containing 0.044 grams of alanine per milliliter of distilled water.
    [0046] The term “GO-“ indicates compositions in which the Bacillus species has been spray-dried in a similar manner without the presence of a germinal compound.
    [0047] In addition, the following method was employed to determine spore germination in the Examples below, unless otherwise noted. When the spores are placed in nutrient solutions and begin to germinate, they release dipicolinic acid and ions, which results in browning. This germination indicator results in reduced optical quenching of visible light by a spore suspension. The germination rate was therefore determined by counting the light/dark spore ratio in the phase and monitoring the optical density reduction at 600 nm (OD 600) of germinating spore suspensions under an ultraviolet visible spectrophotometer. This is then converted into percentage of germination. EXAMPLE 1 INCREASED GERMINATION OF B. SUBTILIS ENV 923 IN INTIMATE MIXTURE WITH L-ALANINE
    [0048] In order to compare the germination rate of spores of intimate mixtures according to the present invention with that of spores conventionally mixed with a germinant, the following treatments were carried out: A. Formation of intimate mixture: spores of B. subtilis ENV 923 were spray dried by introducing L-alanine to the spore mass immediately prior to spray drying as a solution containing 0.044 grams of alanine per milliliter of distilled water. The intimate mixture produced germinated by subsequent introduction into a solution consisting of 0.01 M phosphate buffer in resulting distilled water with pH 7 and calibrated to an initial OD 600 of 0.6. B. Conventional mixture of spores with a germinant. B. subtilis ENV 923 spores were spray dried and then introduced into a solution consisting of 0.01 M phosphate buffer in the resulting pH 7 distilled water. Spores were added to the buffer solution for calibration at an initial OD 600 of 0.6. Alanine was added to the solution at a concentration of 0.0001 grams of alanine per milliliter of solution. C. Germination of isolated spores: Spores of B. subtilis ENV 923 were spray dried and then introduced into a solution consisting of 0.01 M phosphate buffer in resulting pH 7 distilled water and calibrated to initial OD 600 of 0.6.
    [0049] Two replicates of each of these treatments were performed. Table 1 below shows the average results of these treatments that affect the germination of B. subtilis ENV 923 as measured by the percentage drop in optical density. The drop in optical density indicates germination progress. Optical density (OD) was measured at a wavelength of 600 nm with a Jenway model 6320D visible range spectrophotometer. The L-alanine used was 99% pure and was obtained from Alfa Aeser, Heysham, Lancashire, UK. TABLE 1 PERCENTAGE OF REDUCTION OF DO (600 NM) OVER THE BASELINE OVER TIME

    [0050] The above results demonstrate that spores in intimate mixtures according to the present invention germinate faster than spores which are not intimately mixed with the same germinant. Spores mixed with germinant exhibited much higher germination than spores alone. EXAMPLE 2 INCREASE GERMINATION OF ENV923 LINEAGE OF BACILLUS SUBTILIS TREATED WITH GOSD
    [0051] Bacillus subtilis spores were treated with GOSD by spray drying the spores in the presence of an L-alanine solution and germination levels were determined by means of Optical Density (OD) readings.
    [0052] 0.01 M potassium phosphate buffer, pH 7.
    [0053] 0.01 M potassium phosphate buffer was prepared using solutions of 1 M K2HPO4 (87.09 g dissolved in 0.5 L of distilled water) and 1 M KH2PO4 (68.045 g dissolved in 0.5 L of distilled water). A combination of 61.5 ml of 1 M K2HPO4 was carried out with 38.5 ml of 1 M KH2PO4 and dilution to 1000 ml with distilled water of 0.1 M potassium phosphate buffer at pH 7.0. After further dilution of 0.1 M potassium phosphate buffer with distilled water at a ratio of 1:10, 0.01 M potassium phosphate buffer, pH 7.0, was obtained. The buffer was sterilized by autoclaving at 121 °C for 60 (sixty) minutes.
    [0054] Bacillus subtilis spore suspensions were prepared at a concentration of 1.7 x 108 cfu/ml in 0.01 M potassium phosphate buffer, pH 7.0, incubated in a previously heated 37 °C water bath and evaluated to determine the germination percentage at five-minute intervals over a 45-minute period. TABLE 2

    [0055] Conclusion: treatment with GOSD significantly increased the percentage of germination and the germination rate of Bacillus subtilis spores. EXAMPLE 3 INCREASE IN GERMINATION OF ENV100 LINEAGE OF BACILLUS LICHENIFORMIS TREATED WITH GOSD
    [0056] Bacillus licheniformis spores were treated with GOSD by spray drying the spores in the presence of 0.044 grams of L-alanine per ml of distilled water as described in Example 1. Germination levels were determined by readings of the Optical Density (OD) as described above using 0.01 M potassium phosphate buffer, pH 7.0, for spore suspension preparation.
    [0057] Bacillus licheniformis spore suspensions were prepared at a concentration of 1.29 x 108 cfu/ml in 0.01 M potassium phosphate buffer, pH 7.0, incubated in a previously heated 37 °C water bath and evaluated to determine the germination percentage at five-minute intervals over a 45-minute period. TABLE 3 PERCENTAGE OF GERMINATION OF TREATED AND UNTREATED SUSPENSIONS OF BACILLUS LICHENIFORMIS SPORES

    [0058] Conclusion: treatment with GOSD significantly increased the percentage of germination and the germination rate of Bacillus licheniformis spores. EXAMPLE 4 INCREASE GERMINATION OF BACILLUS SUBTILIS LINEAGE ENV923 TREATED WITH GOSD AT VARIOUS PH LEVELS
    [0059] Spores GO+ and GO- of Bacillus subtilis strain ENV293 were prepared as described above and resuspended in 0.01 M potassium phosphate buffer at various pH levels. OD600 measurements were performed as described above.
    [0060] 0.01M potassium phosphate buffer under pH 3.0-7.0.
    [0061] 0.01M potassium phosphate buffer, pH 7.0, was prepared as described in Example 1.
    [0062] To prepare 0.01 M of potassium phosphate buffer with a pH range of 3.0 to 6.0 as a base buffer, 0.1 M of potassium phosphate buffer, pH 6.0, was used through mixing 13.2 ml of 1 M K2 HPO4 and 86.8 ml of 1 M KH2 PO4 solutions (described in Example 1), bringing the volume to 1 L with distilled water.
    [0063] To obtain 0.01 M potassium phosphate buffer pH 5.0 to pH 3.0, 0.1 M potassium phosphate buffer pH 6.0 was diluted with distilled water, the pH of the buffer was reduced to pH 5.0, pH 4.0 and pH 3.0 using 1 M of H2PO4 and the final volume was brought up with distilled water, keeping the ratio between 0.1 M of buffer and distilled water of 1: 10.
    [0064] Prepared buffers were stored at 4 °C and, before each experiment, the pH of the buffers was readjusted using 1 M NaOH or 1 M H2PO4. TABLE 4
    [0065] Germination results with GOSD (GO+) and without GOSD (GO-) at various pH levels. The table displays the germination percentage measured at five-minute intervals.

    [0066] Conclusion: GOSD treatment allows Bacillus subtilis spores to germinate faster and overcome the effects of lower pH levels. EXAMPLE 5
    [0067] Increased germination of ENV923 strain of Bacillus subtilis treated with GOSD. Spore germination reaction affected by different temperature levels. The table displays the germination percentage measured at ten-minute intervals.
    [0068] Bacillus subtilis ENV 923 GOSD spores and control spores were prepared as described above. Spore germination was tested using optical density (OD) measurements as described above. Spore suspensions for OD600 measurements were prepared and cooled to 4 °C in 0.01 M potassium phosphate buffer, pH 7.0 (phosphate buffer, pH 7.0 preparation) at a concentration of 1.7 x 108 cfu/ml. For each spore suspension, three culture tubes filled to a volume of 3 ml were prepared. After shaking and measuring the initial OD600, the tubes were incubated in water baths previously heated at 25 °C, 30 °C and 37 °C for 120 minutes. At ten minute intervals, the tubes were shaken and OD600 measurements were taken. TABLE 5 PERCENTAGE OF GERMINATION OF BACILLUS SUBTILIS WITH GO- AND GO+ AT 37 °C, 30 °C and 25 °C

    [0069] Conclusion: GOSD (GO+) treatment allows Bacillus subtilis spores to germinate faster and overcome the effects of lower temperature regimes. EXAMPLE 6 PERCENTAGE OF GERMINATION OF BACILLUS LICHENIFORMIS WITH AND WITHOUT GOSD IN THE PRESENCE OF DIFFERENT MOLAR SOLUTIONS OF NACL QUITE
    [0070] The medium was a diluted tryptic soy broth (mTSB) (BD, 211822). The medium was prepared by suspending 50 mg of tryptic soy broth powder in 1 L of water with heat and stirring until complete dissolution. It was then split into bottles and autoclaved for thirty minutes at 121 °C. Based on the powder content reported by the manufacturer, the mTSB medium contained per liter: Pancreatic casein digestion: 28.3 mg Soy papain digestion: 5.0 mg Dextrose: 4.2 mg Sodium chloride: 8.3 mg Dipotassium Phosphate: 4.2 mg
    [0071] Sodium chloride (Amresco X190) was added to induce osmotic tension where appropriate, such that final concentrations were 0.5 M or 1.5 M (29.22 g/l and 87.66 g /l, respectively) before heating and autoclaving the medium. SPORE SUSPENSIONS
    [0072] Spore powders from strain ENV100 of Bacillus licheniformis treated or not with GOSD were suspended in sterile water with 0.1% Octosol SLS (FT-SLS-246DRUM, Tiarco Chemical, Dalton, GA) in a sterile mixing jar. The spores were suspended by mixing at five second intervals for a total of at least fifteen seconds or until the spores were completely visually suspended. This was done in such a way that the final concentration in the mixing jar was 1x1010 cfu/ml. From this spore suspension, 250 μl were transferred to tubes containing 4.75 ml of mTSB to generate a final concentration of 5x108 cfu/ml. These concentrations are determined by optimizations performed on each batch of spores to achieve an initial OD600 of about 0.6. OD GERMINATION TEST
    [0073] Tubes containing the suspended spores were immediately vortexed, measured at OD600 for time zero and incubated in a 37 °C water bath. At corresponding time intervals, the time was recorded, the tubes were removed, vortexed, measured at OD600 and returned to the water bath. The percent reduction of OD600 was determined by subtracting the measured value from time zero, divided by time zero and multiplied by 100%. Total germination was previously documented, corresponding to the percentage of OD600 reduction of 60%. The percentage of germination was therefore determined by multiplying the percentage reduction of OD600 by 1.67. TABLE 6 PERCENTAGE OF SPORE GERMINATION OF BACILLUS LICHENIFORMIS FOR ONE HOUR WITH (GO+) AND WITHOUT (GO-) GOSD IN THE PRESENCE OF 0.5 MOLLAR AND 1.5 MOLLAR SOLUTIONS OF NACL


    [0074] Conclusion: GOSD treatment allows Bacillus licheniformis spores to germinate faster and overcome the osmotic stress effects of various levels of salt (NaCl). EXAMPLE 7 PERCENTAGE OF GERMINATION OF BACILLUS LICHENIFORMIS WITH AND WITHOUT GOSD IN THE PRESENCE OF DIFFERENT SOLUTIONS OF PARTS PER MILLION COPPER QUITE
    [0075] mTSB medium was prepared as above, but supplemented with NaCl to a final concentration of 50 mM to create an osmotically balanced medium. A standard suspension of 1x105 ppm of copper(II) nitrate semi(pentahydrate) (Alfa Aesar 12523) was prepared by suspending 2 g in 20 ml of water and filtering 0.22 μm. This was added to mTSB plots to achieve final concentrations of 0, 50, 100 and 200 ppm. SPORE SUSPENSIONS
    [0076] Powders of Bacillus licheniformis strain ENV100 spores treated or untreated with GOSD were suspended in sterile water with 0.1% Octosol SLS (FT-SLS-246DRUM, Tiarco Chemical, Dalton, GA) in a sterile mixing jar . The spores were suspended by mixing at five second intervals for a total of at least fifteen seconds or until the spores were completely visually suspended. This was done in such a way that the final concentration in the mixing jar was 2x109 cfu/ml. From this spore suspension, 250 μl were transferred to tubes containing 4.75 ml of mTSB to generate a final concentration of 1x108 cfu/ml. These concentrations are determined through optimizations performed on each batch of spores to achieve an initial OD600 of about 0.6.
    [0077] OD germination test: performed and calculated as indicated in Example 6. TABLE 7 PERCENTAGE OF BACILLUS LICHENIFORMIS SPORE GERMINATION FOR ONE HOUR WITH (GO+) AND WITHOUT (GO-) GOSD IN THE PRESENCE OF 0.50 PPM SOLUTIONS , 100 PPM AND 200 PPM OF COPPER IONS

    [0078] Conclusion: GOSD treatment allows Bacillus licheniformis spores to germinate faster and overcome the stress effects of various levels of copper ions. EXAMPLE 8 PERCENTAGE OF GERMINATION OF BACILLUS LICHENIFORMIS WITH AND WITHOUT GOSD IN THE PRESENCE OF DIFFERENT SOLUTIONS OF PARTS PER MILLION ALUMINUM QUITE
    [0079] mTSB medium was prepared as above, but supplemented with NaCl to a final concentration of 50 mM to create an osmotically balanced medium. A standard solution of 1000 ppm of Al3+ was prepared by suspending 0.62 g of Al2(SO4)3*14 H2O (Alfa Aesar 12362) in 50 ml of water and filtering 0.22 μm. This was added to mTSB plots to achieve final concentrations of 0, 0.25, 0.50 and 1.0 ppm. The pH of the medium was then brought to pH 4.5 by adding HCl to allow complete separation of the Al3+ ion. SPORE SUSPENSIONS
    [0080] Spore powders from strain ENV100 of Bacillus licheniformis treated or untreated with GOSD were suspended in sterile water with 0.1% Octosol SLS (FT-SLS-246DRUM, Tiarco Chemical, Dalton, GA) in a sterile mixing jar . The spores were suspended by mixing at five second intervals for a total of at least fifteen seconds or until the spores were completely visually suspended. This was done in such a way that the final concentration in the mixing jar was 1x1010 cfu/ml. From this spore suspension, 250 μl were transferred to tubes containing 4.75 ml of mTSB to generate a final concentration of 5x108 cfu/ml. These concentrations are determined through optimizations performed on each batch of spores to achieve an initial OD600 of about 0.6.
    [0081] OD germination test: performed and calculated as in Example 6. TABLE 8 PERCENTAGE OF BACILLUS LICHENIFORMIS SPORE GERMINATION FOR ONE HOUR WITH (GO+) AND WITHOUT (GO-) GOSD IN THE PRESENCE OF SOLUTIONS OF 0, 0.25 PPM, 0.50 PPM AND 1.0 PPM ALUMINUM IONS

    [0082] Conclusion: GOSD treatment allows Bacillus licheniformis spores to germinate faster and overcome the stress effects of various levels of aluminum ions. EXAMPLE 9 PERCENTAGE OF GERMINATION OF BACILLUS LICHENIFORMIS WITH AND WITHOUT GOSD IN THE PRESENCE OF DIFFERENT MILLIMOLAR SOLUTIONS OF BILE SALTS QUITE
    [0083] mTSB medium was prepared as above, but supplemented with NaCl to a final concentration of 50 mM to create an osmotically balanced medium. A standard solution of 80 mM of bile salts was prepared by suspending 2.5 g of sodium taurodeoxycholate (Sigma T0875), 1.1 g of sodium glycodeoxycholate (Sigma G9910) and 0.346 g of sodium deoxycholate ( Sigma D5670) in 100 ml of water and 0.22 μm filtration. This was added to mTSB plots to reach final concentrations of 0, 4, 6 and 8 mM. SPORE SUSPENSIONS
    [0084] Powders of Bacillus licheniformis strain ENV100 spores treated or untreated with GOSD were suspended in sterile water with 0.1% Octosol SLS (FT-SLS-246DRUM, Tiarco Chemical, Dalton, GA) in a sterile mixing jar . The spores were suspended by mixing at five second intervals for a total of at least fifteen seconds or until the spores were completely visually suspended. This was done in such a way that the final concentration in the mixing jar was 1x1010 cfu/ml. From this spore suspension, 250 μl were transferred to tubes containing 4.75 ml of mTSB to generate a final concentration of 5x108 cfu/ml. These concentrations are determined by optimizations performed on each batch of spores to achieve an initial OD600 of about 0.6.
    [0085] OD germination test: performed and calculated as above. TABLE 9 PERCENTAGE OF SPORE GERMINATION OF BACILLUS LICHENIFORMIS FOR ONE HOUR WITH (GO+) AND WITHOUT (GO-) GOSD IN THE PRESENCE OF SOLUTIONS OF 0, 4 PPM, 6


    [0086] Conclusion: GOSD treatment allows Bacillus licheniformis spores to germinate more quickly and overcome the stress effects of various levels of bile salts that can be found in the gastrointestinal tract. EXAMPLE 10 AVERAGE GROWTH OF THREE REPLICAS OF BACILLUS LICHENIFORMIS WITH OR WITHOUT GOSD IN DEFINED POTASSIUM PHOSPHATE AND 2% GLUCOSE MEDIUM
    [0087] Bacillus licheniformis strain ENV 431 GO+ spores were treated with GOSD (procedure described above). As a control, GO- spores from the same crop that were spray-dried without the use of GOSD were used. The growth test was performed in minimal salts medium supplemented with 2% glucose. QUITE
    [0088] The medium was prepared by dissolving (NH4)2SO4 (1.26 g/l), MgCl2 (0.81 g/l), CaCl2 (0.15 g/l), NaCl (0.05 g/l) in distilled water and addition of 1 ml/l of 1000* mixture of trace minerals (MnSO4 (0.85 g/50 ml), ZnSO4 (0.15 g/50 ml), FeSO4 *7H2O (0. 15 g/50 ml), thiamine hydrochloride (0.05 g/50 ml). Prepared solution was poured into flasks (90 ml/vial) and autoclaved at 121 °C for forty minutes. Prior to inoculation, the medium was supplemented with 4 ml of sterile filtered solution of 25x potassium phosphate (K2HPO4 (3.44 g/50 ml), KH2PO4 (2.81 g/50 ml)) and 2 ml of 50x (100 g/200 ml) glucose until final concentration of 2%. GROWTH OF BACILLUS LICHENIFORMIS CELLS
    [0089] The amount of spores used for inoculation was determined using GO- and GO+ spore powder counts. Concentrated Bacillus licheniformis strain ENV 431 spore suspensions (1000x) were prepared by mixing spores in sterile mixing jars using sterile water and added to vials with medium to the concentrations indicated in the table below as 0 h. Initial culture counts were obtained by performing dilutions and plate counts of mixed spore suspensions. Three vials were prepared for each spore sample.
    [0090] The flasks were incubated at 30 °C, 150 rpm and cultured for 48 hours. Samples were taken and plate counts were performed after 24 hours and 48 hours. TABLE 10 AVERAGE GROWTH OF THREE REPLICAS OF BACILLUS LICHENIFORMIS WITH GOSD (GO+) OR WITHOUT GOSD (GO-) IN MINIMUM POTASSIUM PHOSPHATE MEDIUM AND 2% GLUCOSE. DATA IS DISPLAYED IN CFU/ML

    [0091] Conclusion: GOSD treatment significantly increased the rate of growth and germination of Bacillus licheniformis. EXAMPLE 11 GROWTH OF BACILLUS LICHENIFORMIS OVER A TWO-DAY PERIOD; COMPARISON OF TREATMENTS WITH OR WITHOUT GOSD IN THE PRESENCE OF DIFFERENT CONCENTRATIONS OF NACL QUITE
    [0092] mTSB was prepared as above, but with addition of sodium chloride (Amresco X190) to induce osmotic strain where appropriate, such that final concentrations were 0, 0.5, 1.0 or 1.5 M (0, 29.22, 58.44 or 87.66 g/l, respectively). The medium was heated, divided into flasks and autoclaved.
    [0093] Plate Agar Counter (PCA) (BD 247910) was prepared according to the manufacturer's instructions: 23.5 g of powder suspended in 1 l of water, bring to the boil with frequent agitation, divide in glass jars and perform autoclave. Media jars were cooled in a 45 °C water bath until needed. The powder manufacturer reports the following PCA levels per liter: Pancreatic casein digestion: 5.0 g Yeast extract: 2.5 g Dextrose: 1.0 g Agar: 15.0 g SPORE SUSPENSIONS
    [0094] Spore powders of strain ENV100 of Bacillus licheniformis treated or untreated with GOSD were suspended in sterile water with 0.1% Octosol SLS (FT-SLS-246DRUM, Tiarco Chemical, Dalton, GA) in a sterile mixing jar . The spores were suspended by mixing at five second intervals for a total of at least fifteen seconds or until the spores were completely visually suspended, followed by serial dilutions in sterile water. This was done in such a way that the final concentration in the culture flasks was 1x102 cfu/ml. QUANTIFICATION
    [0095] The flasks were incubated at 37 °C by shaking at 150 rpm for 28 hours (“one day”) or 50 hours (“two days”). Portions of each flask were serially diluted in Petri dishes with PCA cooled to <45 °C poured over the top, shaken and kept solidifying. The plates were inverted and incubated for approximately 24 hours at 37 °C. Colonies were counted and concentrations calculated based on dilutions. Samples of about 10 µl from each vial were also streaked onto PCA plates for purity testing. TABLE 11 GROWTH OF BACILLUS LICHENIFORMIS TREATED WITH GOSD (GO+) WHEN CHALLENGED BY OSMOTIC TENSION FROM SALT SOLUTION

    [0096] Conclusion: GOSD treatment significantly increased the germination and growth of Bacillus licheniformis under osmotic salt stress. EXAMPLE 12 BACILLUS SUBTILIS LINEAGE ENV 923 PROTEASE ACTIVITY
    [0097] Bacillus subtilis strain ENV 923 spores treated with GOSD (GO+) as described above and untreated (GO-) were used for the protease activity test. QUITE
    [0098] Chemically defined salt medium (CDSM) was used for cell propagation in a protease assay. The medium was prepared by dissolving components of the base solution (g/l): (NH4)SO4, 1.26 g; L-glutamic acid, 1.18 g; MgCl2, 0.81; CaCl2, 0.155 and 85% L-lactic acid (0.530 ml/l) in distilled water and addition of 1 ml/l of 1000* trace mineral mixture (g/50 ml). Vials with base solution (48 ml/vial) were autoclaved for forty minutes. Prior to inoculation, 2 ml of separately prepared filter-sterilized 25x buffer solution with glucose (g/50 ml): MOPS, 11.6; KH2PO4, 0.6; glucose, 4.5. GROWTH OF ENV923 CELLS FROM BACILLUS SUBTILIS
    [0099] The amount of spores used for inoculation was determined using GO- and GO+ spore powder counts. Concentrated (1000x) Bacillus subtilis strain ENV923 spore suspensions were prepared by mixing spores in sterile mixing jars using 0.01 M sterile potassium phosphate buffer, pH 7.0, and added to the vials at a concentration of approx. of 1 x 10 4 cfu/ml. Initial culture counts were confirmed by performing dilutions and plate counts of mixed spore suspensions. Three vials were prepared for each spore sample.
    [00100] Vials were incubated at 30°C, 150 rpm and samples were taken after 48 hours. PROTEASE ACTIVITY TEST
    [00101] Protease activity assay was conducted on cell supernatants using casein as substrate and Folin & Ciocalateu phenol reagent which reacts with tyrosine and facilitates the development of blue staining. The unit of protease activity was defined as the amount of enzyme that releases 1 μg of tyrosine in one minute. The amount of tyrosine in test tubes was determined by measuring OD650 on a Jenway 7305 spectrophotometer and calculating the tyrosine released using a standard curve. REAGENTS
    [00102] Reagent 1: 0.05M potassium phosphate buffer, pH 7.0.
    [00103] 0.1 M Potassium Phosphate Buffer, pH 7.0 (prepared as described in Example 1) was diluted 1:1 with distilled water to obtain 0.05 M Potassium Phosphate Buffer, pH 7 ,0.
    [00104] Reagent 2: 0.65% casein solution.
    [00105] 0.65 g of casein was dissolved in 80 ml of 0.05 M potassium phosphate buffer, pH 7.0, heated to bring the casein into solution, and the final volume was brought to 100 ml with 0. 05M potassium phosphate buffer, pH 7.0.
    [00106] Reagent 3: 15% trichloroacetic acid (TCA).
    [00107] 15 g of TCA was dissolved in distilled water and the final volume was brought to 100 ml.
    [00108] Reagent 4: 20% Na2CO3.
    [00109] 20 g of Na2CO3 was dissolved in distilled water and the final volume was brought to 100 ml. PROTEASE TEST - 10 ml of culture were centrifuged and the supernatant was filtered through a 0.2 μm filter in sterile tubes. - 3 ml of filtered supernatant was mixed with 3 ml of 0.65% casein solution and placed in a water bath at 37 °C for one hour. - The reaction was suspended by adding 6 ml of 15% TCA and the samples were centrifuged for five minutes. - 0.5 ml of each sample was mixed with 1 ml of 20% Na2CO3, followed by the addition of 0.5 ml of Folin &Ciolcallieu's phenol reagent and incubation for twenty minutes at room temperature to allow the development of a blue color. - 3 ml of distilled water was added to each sample and, after mixing, the OD650 was measured. - To calculate the protease activity, the standard curve for tyrosine was prepared by obtaining a serial dilution of tyrosine dissolved in distilled water, treating it under the same conditions as the culture samples and measuring OD650. TABLE 12 PROTEASE PRODUCTION OF BACILLUS SUBTILIS TREATED WITH GOSD (GO+) COMPARED TO CONTROL (GO-)

    [00110] Conclusion: treatment of Bacillus subtilis spores with GOSD allows for increased production of enzymes such as protease. EXAMPLE 13 GERMINATION OF STREPTOMYCES VIRIDOCHROMOGENES IN THE PRESENCE OF GERMINATIVE COMPOUNDS
    [00111] Spores of Streptomyces viridochromogenes were collected from plates by pouring 10 ml of TX buffer (0.05 M Tris-HCl buffer, pH 7.3, with 0.001% Tween 80) and removing the spores with a sterile cotton swab. Plaque spore suspensions were poured into sterile 50 ml tubes. When all spore suspensions from all samples were obtained, heat shock was performed by placing the tubes with spore suspensions in a heat block, allowing the temperature to reach 55 °C and maintaining the temperature at 55 °C. for ten minutes. After the heat shock, spore suspensions were cooled in ice water for five minutes and centrifuged for thirty minutes. The supernatant was discarded, the spores were resuspended in 25 ml of 0.02 M potassium phosphate buffer, pH 7.0 at 4 °C and centrifuged for fifteen minutes. After pouring out the supernatant, the spores were resuspended in 20 ml of 0.02 M potassium phosphate buffer, pH 7.0, and vigorously mixed to obtain the spore suspension that was used in the experiment.
    [00112] Samples were prepared by mixing 1.5 ml of 2x germinant mixture with 1.5 ml of spore suspension. All germinating mixtures were prepared in distilled water in the form of 2x50 ml solutions. Calcium chloride was prepared as a 100X solution (0.4 g/10 ml) and 20 μl was added to 10 ml of 2X germination mixtures. The final concentrations of the germinal compounds were as follows: 0.89 mg/ml of L-alanine, 1.17 mg/ml of L-valine, 13.2 mg/ml of L-asparagine, 2.25 mg/ml of glucose and 2.25 mg/ml fructose. After the initial OD600 measurement, the samples were transferred to a 30°C water bath and OD600 was measured at fifteen minute intervals for ninety minutes to determine germination rates. TABLE 13 ENV 151 (STREPTOMYCES VIRIDOCHROMOGENES): PERCENTAGE OF REDUCTION IN OPTICAL DENSITY.

    [00113] Conclusion: treatment of spores of Streptomyces viridochromogenes with germinal compounds increased germination. EXAMPLE 14 COMPARISON OF GERMINATION RATES BETWEEN INTIMATE MIXTURES AND CONVENTIONAL MIXTURE OF BACTERIAL SPORES AND GERMINATIVE COMPOUNDS
    [00114] In order to compare the germination rate of spores from intimate mixtures with that of spores conventionally mixed with a germinant, the following treatments were performed: A. Formation of intimate mixture: spores of B. subtilis, B. amyloliquefaciens, B. brevis, B. cereus, B. coagulans, B. firmus, B. laterosporus, B. licheniformis, B. megaterium, B. mycoides, B. popilliae, B. polymyxa, B. pumilus, B. thuringiensis, Pasteuria penetrans , Pasteuria thornei, Pasteuria nishizawae, Streptomyces viridochromogenes, Streptomyces griseoviridis, lydicus Streptomyces plicatus Streptomyces, Streptomyces sindeneusis, Streptomyces rochei, Streptomyces alni, viridis Streptomyces, Streptomyces thermovulgaris, Streptomyces griseus, Streptomyces acidiscabies, aureofaciens Streptomyces galbus Streptomyces microflavus Streptomyces and Streptomyces aureofacien are dried with L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L-glutamine, L-asparagine or L-phenylalanine, which are i introduced into the spore mass immediately before drying in the form of a solution containing 0.044 grams of the amino acid per milliliter of distilled water. The intimate mixture produced germinated by means of subsequent introduction into a solution consisting of 0.01 M phosphate buffer in distilled water at pH 7. B. Conventional mixture of spores with a germinant: spores of B. subtilis, B. amyloliquefaciens , B. brevis, B. cereus, B. coagulans, B. firmus, B. laterosporus, B. licheniformis, B. megaterium, B. mycoides, B. popilliae, B. polymyxa, B. pumilus, B. thuringiensis, Pasteuria penetrans Pasteuria thornei, Pasteuria nishizawae, Streptomyces viridochromogenes, Streptomyces griseoviridis, lydicus Streptomyces plicatus Streptomyces sindeneusis Streptomyces, Streptomyces rochei, Streptomyces alni, viridis Streptomyces thermovulgaris Streptomyces, Streptomyces griseus, Streptomyces acidiscabies, aureofaciens Streptomyces galbus Streptomyces microflavus Streptomyces and Streptomyces aureofacien are hydrated and dried. These spores are germinated by introduction into a solution consisting of 0.01 M phosphate buffer in resulting pH 7 distilled water and 0.0001 grams of L-alanine, L-valine, L-proline, L-leucine , L-cysteine, L-threonine, L-glutamine, L-asparagine or L-phenylalanine per milliliter of solution. C. Germination of isolated spores: spores of B. subtilis, B. amyloliquefaciens, B. brevis, B. cereus, B. coagulans, B. firmus, B. laterosporus, B. licheniformis, B. megaterium, B. mycoides, B .popilliae, B. polymyxa, B. pumilus, B. thuringiensis, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, Streptomyces viridochromogenes, Streptomyces griseoviridis, Streptomyces lydicus, Streptomyces plicatus, Streptomyces sindeneusis, Streptomyces rochei, Streptomyces alni, Streptomyces virididicus, Streptomyces viridis viridis , Streptomyces griseus, Streptomyces acidiscabies, Streptomyces aureofaciens, Streptomyces galbus, Streptomyces microflavus and Streptomyces aureofacien are hydrated and dried. The spores are then introduced into a solution consisting of 0.01 M phosphate buffer in the resulting pH 7 distilled water.
    [00115] The resulting spore germination for each of these treatments is measured by the percentage drop in optical density or by counting the amount of germinated spores under a microscope. The intimately blended compositions were found to germinate faster than their corresponding conventionally blended equivalent.
    权利要求:
    Claims (15)
    [0001]
    1. INTIMATE DRY MIXTURE, characterized by comprising a bacterial spore and an L-amino acid, in which the bacterial spore and L-amino acid are dried together, so that the bacterial spore and the L-amino acid are kept in close position until they reach a environment conducive to germination.
    [0002]
    2. MIXTURE according to claim 1, characterized in that the spore is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus , B. boronophilus, B. brevis, B. caldolyyicus, B. centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C. ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, and Streptomyces spp.
    [0003]
    3. MIXTURE according to claim 1, characterized in that the L-amino acid is selected from the group consisting of L-alanine, L-valine, L-proline, L-leucine, L-cysteine, L-threonine, L -glutamine, L-asparagine, L-phenylalanine and their analogues.
    [0004]
    4. MIXTURE, according to claim 1, characterized in that this mixture is produced by means of spray drying, freeze drying, air drying or drum drying.
    [0005]
    MIXTURE according to claim 1, characterized in that the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus; and the L-amino acid is selected from the group consisting of L-alanine, L-valine and L-asparagine.
    [0006]
    6. COMPOSITION, characterized in that it comprises the dry intimate mixture, as defined in claim 1.
    [0007]
    7. COMPOSITION according to claim 6, characterized in that this mixture is produced by means of spray drying, freeze drying, air drying or drum drying.
    [0008]
    COMPOSITION according to claim 6, characterized in that this composition additionally comprises an enteric coating.
    [0009]
    A COMPOSITION according to claim 6, characterized in that the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus; and the L-amino acid is selected from the group consisting of L-alanine, L-valine and L-asparagine.
    [0010]
    10. METHOD FOR PREPARING A DRY INTIMATE MIXTURE, comprising a bacterial spore and an L-amino acid, characterized in that the method comprises: (a) preparing a solution comprising a bacterial spore and an L-amino acid; and (b) drying the solution to obtain a dry intimate mixture comprising a bacterial spore and an L-amino acid; wherein the bacterial spore and the L-amino acid are held in close proximity until they reach an environment conducive to germination.
    [0011]
    11. METHOD according to claim 10, characterized in that the drying is spray drying, freeze drying, air drying or drum drying.
    [0012]
    METHOD according to claim 10, characterized in that the spore is selected from the group consisting of B. alcalophilus, B. alvei, B. amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus , B. boronophilus, B. brevis, B. caldolyyicus, B. centrosporus, B. cereus, B. circulans, B. clausii, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B larvae, B. laterosporus, B. lentus, B. lentimorbus, B. licheniformis, B. megaterium, B. mesentericus, B mucilaginosus, B. mycoides, B. natto, B. pantothenicus, B. popilliae, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. simplex, B. sphaericus, B. sporothermodurans, B. stearothermophilus , B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis, B. weihenstephanensis, C. thermocellum, C. ljungdahlii, C. acetobutylicum, C. beijerinckii, C. butyricum, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, and Streptomyces spp.
    [0013]
    METHOD according to claim 10, characterized in that the spore is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus; and the L-amino acid is selected from the group consisting of L-alanine, L-valine and L-asparagine.
    [0014]
    14. INTIMATE DRY MIXTURE, characterized in that it is produced by means of the method, as defined in claim 10.
    [0015]
    MIXTURE, according to claim 1, characterized in that the L-amino acid is formulated, before drying, in concentrations from 0.0003 mg/ml to 170 mg/ml.
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    JP2016506735A|2016-03-07|
    US20160362654A1|2016-12-15|
    EA201591463A1|2016-04-29|
    KR102302522B1|2021-09-14|
    PL2954041T3|2019-05-31|
    SI2954041T1|2019-01-31|
    CA2899606A1|2014-08-14|
    AU2014214917A1|2015-08-13|
    PT2954041T|2018-10-22|
    KR20150133699A|2015-11-30|
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    US20140220662A1|2014-08-07|
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    MX2015010124A|2016-04-25|
    WO2014124120A1|2014-08-14|
    AU2014214917B2|2019-09-26|
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    法律状态:
    2019-09-17| B25A| Requested transfer of rights approved|Owner name: ENVERA LIC, LLC (US) |
    2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-11-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2022-01-04| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/02/2014, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201361849973P| true| 2013-02-06|2013-02-06|
    US61/849,973|2013-02-06|
    US14/174,099|2014-02-06|
    US14/174,099|US9447376B2|2013-02-06|2014-02-06|Dried spore germinative compound mixtures|
    PCT/US2014/015076|WO2014124120A1|2013-02-06|2014-02-06|Dried spore germinative compound mixtures|
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