WO2019090093A1

WO2019090093A1 - Composition containing viable microorganisms and methods of making

Info

Publication number: WO2019090093A1
Application number: PCT/US2018/058981
Authority: WO
Inventors: Mordechai Harel; Trisha D. RICE; Majid Keshtmand; Erin DICKEY; James J. Reap; Stephen J. GIARRATANO
Original assignee: Advanced Bionutrition Corp.
Priority date: 2017-11-03
Filing date: 2018-11-02
Publication date: 2019-05-09

Abstract

A composition comprising one or more viable microorganisms, one or more metal oxides and/or metalloid oxides and one or more hydrolyzed proteins, wherein the one or more metal oxides and/or metalloid oxides in total constitute at least 1% by weight of the composition. Plant foliage, fruit, p!ant roots, seeds, or soil, a food or feed container or packaging, a medical device, a bandage, human or animal skin, and wounds may be treated or coated by applying the composition thereto. Methods for producing the composition in a dry form are provided.

Description

COMPOSmON CONTAINING VIABLE

MICROORGANISMS AND METHODS OF MAKING

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

62/581,300, filed 3 November 2017, and U.S. Provisional Application No. 62/584,386, Hied 10 November 2017, the contents of each of which are incorporated herein by reference in their entireties for aii purposes.

FIELD OF THE INVENTION

The invention relates to stable compositions containing viable microorganisms and methods of making them. The compositions comprise one or more metal oxides and/or metalloid oxides thereof.

BACKGROUND OF THE INVENTION

One of the challenges to providing an effective amount of live microorganisms to a host is the preservation of their viability under the harsh conditions of typical industrial manufacturing processes and long-term storage at high temperature and humidity. Although there have been developments concerning encapsulation and formulation techniques for protecting biological materials in acidic environments such as the digestive systems of an animal, there has been little development in

encapsulation or stabilization techniques that protect the viability of microorganisms during manufacturing processes, distribution and storage. There is a need for a composition and stabilization technique that enables live microorganisms to survive upon exposure to various harsh environments, especially high temperature and humidity.

The inherent moisture of a consumable product or of a plant seed when coated with live microorganisms poses another challenge in that live microorganisms generally are sensitive to water activity, especially in combination with high temperature. To date, no technology or technique has been identified to provide significant protection of live microorganisms under intermediate moisture conditions (i.e., water activity (Aw) of about 0.4 or higher) and high temperatures during distribution and storage (e.g., temperatures of at least about 25°C, or up to about 40°C or higher) when incorporated into products such as nutritional products, animal feeds and agricultural products. As such, there is a need for stable live microorganism compositions suitable for distribution in various geographic locations, including those in tropical climates, where the viability of the microorganisms couid be compromised.

Additional challenges include nutritional, environmental and regulatory limitations on the use of conventional or non-conventional or non-biodegradable synthetic ingredients suitable for consumption by animals or for various agricultural applications. A recommended list of ingredients allowed for animal feed uses is presented in CFR-21 part 573 and for agricultural and soil uses in CFR-40 parts 112 and 180.

What is desired therefore are stable compositions comprising live

microorganisms such as probiotic bacteria or soil microorganisms, or live attenuated vaccines including fungus, bacteria and viruses, and stabilization techniques for making such compositions.

SUMMARY OF THE INVENTION

The invention provides a composition comprising one or more viable

microorganisms, one or more metal oxides and/or metalloid oxides and one or more hydroiyzed proteins, wherein the one or more metal oxides and/or metalloid oxides in total constitute at least 1% by weight of the composition.

The one or more viable microorganisms may be selected from the group consisting of live or attenuated bacteria, fungi, yeast, unicellular algae, viruses, and bacteriophages.

The one or more hydroiyzed proteins may be selected from the group consisting of hydroiyzed milk proteins, hydroiyzed plant proteins, and combinations thereof.

The one or more hydroiyzed proteins may be selected from the group consisting of hydroiyzed casein, hydroiyzed whey protein, hydroiyzed pea protein, hydroiyzed soy protein, and combinations thereof.

The composition may further comprise one or more carbohydrates that in total constitute 10-70% by weight of the composition.

The one or more carbohydrates may be selected from the group consisting of disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

The one or more carbohydrates may include one or more polysaccharides selected from the group consisting of alginate, gum acacia, locust bean gum, carrageenan, starches, modified starches, and combinations thereof.

The one or more metal oxides and/or metalloid oxides may be selected from the group consisting of calcium oxide, lithium oxide, aluminum oxide, magnesium oxide, silicon dioxide, zinc oxide, titanium dioxide, and combinations thereof.

The one or more metal oxides and/or metalloid oxides may be hydrophilic.

The composition may be a dry composition comprising particles, 90% or more of which may have a diameter having a particle size in the range smaller than about 10,000 pm (in another embodiment, 90% or more of the particles have a particle size in the range smaller than 1000 pm). The composition may comprise at most 75% in total of the one or more metal oxides and/or metalloid oxides. The composition may comprise 1-50%, 5-35 or 10- 25% in total of the one or more metal oxides and/or metalloid oxides.

The composition may further comprise 1*10% in total of one or more carboxyiic acids and/or carboxyiic acid salts by weight.

The one or more carboxyiic adds and/or carboxyiic acid salts may be selected from the group consisting of lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, citric acid, gluconic acid, glutamic add, their salts, and combinations thereof.

The invention also provides a pharmaceutical, nutraceutical, food or feed product or a botanical or agricultural product comprising any of the above-mentioned compositions.

The invention also provides a food or feed container or packaging, a medical device, or a bandage for use on wounds in human or animal skin, wholly or partially coated with or incorporating any of the above-mentioned compositions.

The invention also provides a method for preparing any of the above-mentioned compositions in a dry particulate form, comprising:

(a) combining the one or more viable microorganisms, the one or more hydrolyzed proteins, the one or more metal oxides and/or metalloid oxides, optionally the one or more carbohydrates, and optionally the one or more carboxyiic acids and/or carboxyiic acid salts in an aqueous solvent to form a paste;

(b) freezing or snap-freezing the paste in liquid nitrogen to form a solid frozen cake or particles in the form of beads, droplets or strings;

(c) primary drying of the solid frozen cake or particles by evaporation, under vacuum, while maintaining the temperature of the particles above their freezing temperature, whereby a primary dried formulation is formed; and

(d) secondary drying of the primary dried formulation at full strength vacuum and a heat source temperature of 20°C or higher for a time sufficient to reduce the water activity of the primary dried formulation to 0.3 Aw or lower.

(a) combining the one or more viable microorganisms, the one or more hydrolyzed proteins, the one or more metal oxides, optionally the one or more carbohydrates, and optionally the one or more carboxyiic adds and/or carboxyiic acid salts in an aqueous solvent to form a paste;

(b) extruding or pelleting the paste of step (a) to form semi-dry particles, beads or strings; and (c) drying the product of step (b) to reduce the water activity to 0.3 Aw or lower.

Either of the above-mentioned methods may further comprising cutting, crushing, milling or pulverizing the dry product into a free-flowing powder. The particle size of the free-flowing powder may be less than about 10,000 pm (in another embodiment, less than about 1000 pm).

The invention also provides a method for treating plant foliage, fruit, plant roots, seeds, or soil, or coating a food or feed container or packaging, or a medical device or bandage, for use on wounds in human or animal skin, comprising applying thereto or incorporating therein any of the above-mentioned compositions.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows viability loss over time of samples prepared as described in

Comparative Example 1 and inventive Example 2, evaluated as described in Example 3 under accelerated storage conditions of 25°C and 53%RH.

FIG. 2 shows viability loss of the same compositions evaluated in FIG. 1 under accelerated storage conditions of 25°C and 65%RH.

FIG. 3 shows viability loss over time of a commercial instant dry baker's yeast and the stable particulate composition prepared as described in Inventive Example 16, evaluated under accelerated storage conditions of 30°C and 53%RH.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions for preserving live microorganisms, and methods of preparing the compositions in a dry particulate form. Traditional preserving formulations contain large amounts of small molecules such as sugars, free amino acids and salts that act at the microorganism intracellular and extracellular levels to cryopreserve and protect the live microorganisms from extreme freezing, thawing and drying conditions. Surprisingly, the inventors have found that including metal oxides and/or metalloid oxides according to the invention provides superior protecting and stabilizing effects for a variety of live microorganisms under high moisture and temperature conditions.

Moreover, compositions comprising a hydrophiHc metal oxide and/or metalloid oxide appear to protect the embedded microorganisms much better than those using a hydrophobic metal oxide and/or metalloid oxide. This is a surprising effect, as common knowledge indicates that permeability of water vapor is hard to control, so that the skilled person would expect a rapid and complete loss of the microorganism viability in a high humidity environment due to faster water vapor permeation through the hydrophiHc and porous structure of hydrophiHc metal oxide and/or metalloid oxide as compared with hydrophobic. The composition comprises one or more viable microorganisms, one or more hydroiyzed proteins, and one or more metal oxides and/or metalloid oxides, and optionally one or more carbohydrates and/or one or more carboxyiic acids and/or salts thereof. The viable microorganisms are embedded in the matrix of the composition, providing desirable stability and protection during manufacturing processes for making consumable products, during transport through distribution channels, and during storage under extreme conditions. For example, most traditional probiotlc

microorganism formulations employ an extremely high bacterial cell count, sometimes as high as 10 or even 100 times that required for an effective dose, because a significant number of the ceils ultimately lose viability and die during manufacturing, transportation, and storage. Such high loadings of viable microorganisms are typically not needed for the compositions of this invention.

The composition may provide a biological benefit to a host, which may be any animal, including a mammal, a human, a terrestrial, avian or an aquatic animal. The host may be a plant, plant foliage, plant seed or plant root. The composition may also provide antibacterial or protective effect against harmful microorganisms when coated as a film on a surface of food or feed, packaging materials, devices and skin.

Unless otherwise made dear by the context or explicit statement, all percentages herein are on a weight basis. Unless otherwise made clear by the context or explicit statement, the weight basts is a dry solids basis.

The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and die like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

The term "composition" as used herein refers to a liquid or dry material containing a viable microorganism. The viable microorganism is embedded in a wet or dry matrix comprising hydroiyzed proteins and metal oxide. The composition may be a dry composition comprising particles. Such a composition may be called a dry particulate composition. The particles may be spherical or irregular in shape. About 90% or more of the particles may have a diameter smaller than about 10,000 pm. According to another embodiment, about 90% or more of the particles may have a diameter smaller than about 1000 pm.

The term "microorganism" refers to a live microorganism that provides or confers a biological benefit to a host when administered to the host in an effective amount.

The term "effective amount" as used herein refers to an amount of a viable microorganism that is sufficient to achieve a desirable biological benefit when administered to a host via, for example, a pharmaceutical, nutraceutical supplement or a dietary product, a feed product or immunogenic product or a product for botanical or agricultural applications. The viable microorganism may be selected from the group consisting of live or live attenuated bacteria, fungi, yeast, microalgae, viruses and bacteriophages. The desirable biological benefit may be any beneficial health, prophylactic, immunogenic or nutritional effect or crop or soil enhancement effect- Examples include maintaining healthy gastrointestinal flora; enhancing growth, reproduction, miik yield, or immunity; preventing diseases or allergies; and enhancing agricultural yield and/or disease protection.

The term "viability" as used herein refers to the ability of a microorganism in a composition to form colonies or viral plaques on a nutrient media appropriate for the growth of the microorganism, and may be expressed as colony forming units (CPU) or plaque forming units (PFU) over the weight of the composition, e.g., CFU/g.

The term "relative humidity (RH)" as used herein refers to the amount of water vapor in the air, often at a given temperature. Relative humidity is usually less than that required to saturate the air, and is often expressed in percentage of saturation humidity.

The term "dry" as used herein refers to a physical state of a substance, for example, the particulate composition of the invention, that is dehydrated or anhydrous, e.g., substantially lacking liquid. The substance, for example, the particulates composition of the invention, may be dried by one or more drying process, for example, air drying, vacuum drying, fluidized bed drying, spray drying, and

lyophilization.

The term "water activity (Aw)"as used herein refers to the availability of water in a substance, for example, the composition of the invention, which represents the energy status of water in the substance. It may be defined as the vapor pressure of water above a substance divided by that of pure water at the same temperature. Pure distilled water has a water activity of exactly one, i.e., A«=1.0. A dry substance may have an Aw of about 0.5 or lower, preferably about 0.3 or lower, more preferably about 0.2 or lower, most preferably about 0.1 or lower.

Viable Microoroanisms

The composition comprises an effective amount of the one or more viable microorganisms for providing a biological benefit to a host via a pharmaceutical, a nutraceutical supplement, a dietary product, a feed product, an immunogenic product, a product for botanical, agricultural or soil applications, and/or antibacterial or protective effect against pathogens when coated on or incorporated in food or feed surface, food or feed container or packaging, a medical device or bandage, human or animal skin and wounds therein.

The viable microorganism may be selected from the group consisting of live or attenuated bacteria, plant, soil, or aquatic microorganisms, fungi, microalgae, yeasts, viruses, live attenuated vaccines and phages. For example, one or more viable microorganisms may be live probiotic bacteria for human or animal application.

The one or more viable microorganisms in total may typically constitute at least 1% of the composition, or at least 5% or 10%. They may typically constitute at most 30%, or at most 25% or 20%. Suitable microorganisms include, but are not limited to, micro algae including any marine or fresh water species, yeasts such as

Saccharomyces, Debaromyces, Candida, Pichia and Toruiopsis, moulds such as

Aspergillus, PJhizopus, Mucor, Penicillium and Toruiopsis; bacteria such as the genera Bifidobacterium, Clostridium, Fusobacterium, Melissococcus, Propion/bacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus,

Bacillus, Pediococcus, Micrococcus, Leuconostoc, Welssella, Aerococcus, Oenococcusand Lactobacillus,' and viruses, including any virus that attacks bacteria (Phaginae), plants (Phytophaginae) or animals (Zoophaginae).

Specific examples of suitable probiotic microorganisms may be represented by the following species, including ail culture biotypes within those species: Aspergillus niger, A. oryzae. Bacillus coagulans, S. lentus, B. licheniformis, B. mesentericus, B. pumilus, 8. subtilis, B. natto, Bacteroidesamyiophilus, Bac. capillosus, Bac. ruminocola, Bac. suis, Bifidobacteriumadolescentis, B. animalis, B. breve, B. bifidum, B. infantis, B. lactis, B. longum, B. pseudolongum, 8. thermopMum, Candida pintolepesii, Clostridium butyricum, Enterococcus cremoris, E. diacetylactis, E. faedum, E. intermedius, E. lactis, E. muntdii, E. thermophitus, Escherichia coli, Kiuyveromycesfragitis, Lactobacillus acidophilus, L. alimentarius, L amylovorus, L crispatus, L, brevis, L case L. curvatus, L cellobiosus, L delbrveckii ss< bulgaricus, L fardminis, L. fermentum, L gasseri, L. helveticus, L. lactis, L plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, Leuconostocmesenteroides, P. cereviseae (damnosus), Pediococcus acidilacbci, P. pentosaceus, Propionibacterium freudenreichii, Prop, shermanii,

Saccharomyces cerevisiae, Staphylococcus carnosus, Staph, xylosus, Streptococcus infantarius, Strep. Salivarius, Strep. Thermophilus and Strep. Lactis, and viruses or phages.

Metal Oxide and/or Metalloid Oxide

The composition comprises one or more metal oxides and/or metalloid oxides.

Preferably, the metal oxide or metalloid oxide used to make the composition is a hydrophiiic metal oxide or metalloid oxide. Generally, the preferred metal oxide or metalloid oxide can adsorb moisture up to 0.5 of its weight, or up to 1, 2, or 3 times its weight. In one preferred embodiment the one or more metal oxides and/or metalloid oxides are selected from the group consisting of aluminum oxide, calcium oxide, iron oxide, magnesium oxide, silicon dioxide, titanium dioxide and zinc oxide.

The one or more metal oxides and/or metalloid oxides in total may typically constitute at least about 1%, 10%, 20%, or 25% of the composition. They may constitute at most about 75%, 70%, 60%, or 50% of the composition. The range of the one or more metal oxides and/or metalloid oxides in the composition may be from about 1% to about 50%, preferably from about 5% to about 35%, more preferably from about 10% to 25%.

The terms "metal oxide" or "metalloid oxide" refer to compounds which are formed with a metal or metalloid element cation and oxygen anion in the form of oxide ion (O² ), or in some cases the bonding of the metal or metalloid oxides may be more covaient in nature, for example, that of silicon dioxide. Metal or metalloid oxides are basic in nature and typically exist as solids or dry powders at room temperature.

Generally, a metal oxide or metalloid oxide is insoluble in water and produces a salt or acid. Exemplary metal oxides and/or metalloid oxides include aluminum oxide, calcium oxide, iron oxide, lithium oxide, magnesium oxide, silicon dioxide, titanium dioxide and zinc oxide.

The metal oxide or metalloid oxide used to make the composition of this invention may be in the form of particles. The metal oxide or metalloid oxide particles may have an average particle size in the range from about 10 to about 1000 pm, preferably from about 20 to about 500 um, and most preferably from about 20 to about 200 um. The metal oxide or metalloid oxide particles may be milled by various known methods to reduce their particle sizes into a desirable size range.

The term ""hydrophilic metal oxide or metalloid oxide" refers to a metal oxide or metalloid oxide having both an internal surface area and an external surface area each of which captures, complexes with, or immobilizes at least about 5%, preferably at least about 10% and more preferably at least about 20% moisture.

Hydrpjyzed Proteins

The one or more hydrolyzed proteins may be selected from the group consisting of hydrolyzed milk proteins, hydrolyzed plant proteins, and combinations thereof. In one preferred embodiment, the one or more hydrolyzed proteins may be selected from the group consisting of hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof. The one or more hydrolyzed proteins in total may typically constitute at least about 10% of the composition, or at least about 15% or 20%. They may typically constitute at most about 60%, or at most about 50% or 40%.

The terms "hydrolyzed protein" and "protein hydrolysate" are used herein interchangeably, and refer to proteins broken down by hydrolysis or digestion into shorter peptide fragments and/or amino acids. The hydrolysis or digestion may be carried out by a strong acid, a strong base, an enzyme or a combination thereof. The hydrolyzed protein may be from an animal or a plant. The hydrolyzed proteins may be milk proteins, plant proteins, or a mixture thereof.

The hydrolyzed protein may be partially or extensively hydrolyzed. The hydrolyzed protein may be a mixture of peptides and amino acids. In some

embodiments, at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, preferably at least about 50%, of the hydrolyzed protein has a molecular weight lower than about 100,000, 75,000, 50,000, 25,000, 10,000, 5,000, 1,000 or 500 Daltons, preferably lower than about 50,000 Dalton, more preferably lower than about 10,000 Daltons. For example, at least about 40%, 50%, 60%, 70%, 80% or 90%, preferably at least about 50%, of the hydrolyzed protein has a molecular weight lower than about 20,000 Daltons.

Proteins suitable for making hydrolyzed proteins for the dry particulate composition of the invention include egg proteins, gelatin, milk proteins, casein, whey protein, albumen, soy protein, pea protein, rice protein, wheat protein, and other plant proteins.

Examples of the hydrolyzed proteins include hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof. In one preferred embodiment, the hydrolyzed protein comprises hydrolyzed casein or pea proteins, at least about 50% of which has a molecular weight of less than about 20,000 Daltons.

Carbohydrates

The composition may optionally also comprise one or more carbohydrates selected from disaccharides, oligosaccharides and polysaccharides and combination thereof, in which the viable microorganism is embedded. Without being bound by theory, it is believed that by combining a carbohydrate mixture and hydrolyzed proteins as described herein allows faster drying and contributes to a desirable amorphous and rigid structure of the resulting composition.

If present, the one or more carbohydrates in total may typically constitute at least about 10% of the composition, or at least about 15% or 20%. They may typically constitute at most about 70%, or at most about 60%, 50%, 40%, or 30%. Preferably, the carbohydrates comprise a mixture of disaccharide sugars, oligosaccharides and polysaccharides. The carbohydrate may typically comprise about 5-90% of one or more disaccharides, based on the total dry weight of the

carbohydrate. Preferably, the carbohydrate comprises about 50-80% of disaccharides, based on the total dry weight of the carbohydrate. Exemplary disaccharides include sucrose, lactose, maltose, and trehalose.

The one or more carbohydrates may typically comprise about 1-50% of one or more oligosaccharide, based on the total dry weight of the carbohydrate. Preferably, the carbohydrate comprises about 10-30% of oligosaccharides, based on the total dry weight of the carbohydrate.

The term "oligosaccharides" refers to saccharide polymers containing a small number of simple sugars (monosaccharides), typically from about 3 to about 60 units. Oligosaccharides are soluble fibers often considered as preblotics in nutritional applications. Advantageously, soluble fibers pass through the animal stomach undigested and become available for digestion by the gut microflora. The incorporation of soluble fibers may also help to protect the viable microorganisms from digestive enzymes and high acidity in the stomach.

The oligosaccharides are preferably readily soluble fibers. The oligosaccharide may be inulin, alpha, beta and gamma cydodextrin, maltodextrin, dextran, fructo- oligosaccharide (FOS), ga!acto-oHgosaccharide (GOS), manrtan-oligosaccharide (MOS), or a combination thereof, preferably cydodextrin, maltodextrin or inulin, more preferably inulin or cydodextrin.

The carbohydrate may typically comprise about 0.1-40%, 0.5-30%, 1-30%, 1- 20%, 1-10%, 1-5% or 5-10% of the one or more polysaccharide, based on the total dry weight of the carbohydrate. Preferably, the carbohydrate comprises about 0.1-10% of the polysaccharide, based on the total dry weight of the carbohydrate.

Exemplary polysaccharides may be selected from the group consisting of alginate, carrageenan, guar gum, gum acacia, pullulan, agar, xanthan gum, gum ghatti, gum tragacanth, karaya gum, guaran gum, locust bean gum, starches, modified starches, and modified celluloses (e.g., methyl ethyl cellulose, hydroxypropyi cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose), dextran, and combinations thereof. Exemplary oligosaccharides may be selected from the group consisting of inulin, maltodextrins, dextrans, fructo-oligosaccharides (FOS), galacto- oiigosaccharides (GOS), mannan-oligosaccharides (MOS), and combinations thereof. Exemplary disaccharides may be selected from the group consisting of trehalose, sucrose, fructose, maltose and lactose, and combinations thereof. Preferably, the carbohydrate mixture comprises about 50-80% of disaccharides, about 10-30% of oligosaccharides and about 0.1-10% of the polysaccharide, based on the total weight of the carbohydrates. In a preferred embodiment the carbohydrate is selected from trehalose, sucrose or lactose, inulin or cyclodextrin and alginate, gum Arabic or carrageenan or any combination thereof.

CarbQxvlic Acids

The composition of the invention may further comprise one or more carboxylic acids or salts thereof. The carboxylic add or carboxylic add salt may provide enhanced stability to the composition, as well as an additional benefit to the viable

microorganism, the host or both. For example, the additional carboxylic acid or salt may provide a therapeutic or immunogenic effect to the host. Non-limiting examples of suitable carboxylic acids/salts may be selected from the group consisting of lactic acid, ascorbic acid, maleic acid, oxalic acid, maionic add, malic acid, succinic add, citric add, gluconic add, and glutamic acid. Salts may include cations such as sodium, potassium, calcium, and magnesium. Suitable specific examples include sodium citrate, sodium lactate, sodium maleate, magnesium gluconate, and sodium ascorbate. Preferred carboxylic acids/salts include citric add, ascorbic add, and their salts, for example sodium or potassium ascorbate or citrate and trisodium citrate dehydrate.

If present, the carboxylic acids and/or salts may typically constitute at least about 0.1% of the composition, or at least about 0.5% or 1%. They may typically constitute at most about 20%, or at most 10% or 5%.

Unless the context otherwise makes dear, references herein to "carboxylic acid", "carboxylic salt", and "carboxylate" are understood to include both organic compounds containing a carboxyl group (-COOH) and those containing a carboxylate group, such as a metal salt, for example a sodium or potassium salt.

viability

The microorganism in the composition may have an initial viability of at least about 1x10», IxlO¹⁰, 1x10" or lxlO¹² CFU/g, preferably at least about lxlO¹⁰ CFU/g. The viability loss may be less than 1 log unit/g after a predetermined period of time under predetermined conditions. For example, the composition may have a viability loss of less than about 1 log unit/g after about 1, 2, 3 or 4 weeks, or 1, 2, 3, 4, 5, 6, 12, 18, 24 or 36 months, preferably about 1, 2 or 3 months. A specified time period may include a shorter or longer time period that is within about 10% of the specified time period. The term "3 months" as used herein refers to a time period of about 84-90 days. The term "two months" as used herein refers to a time period of about 56-60 days. The term "one month" as used herein refers to a time period of about 28-30 days. The predetermined conditions may include a predetermined temperature and a predetermined relative humidity (RH). The predetermined temperature may be at feast about 25, 37, 40, 45, 50 or 55°C The predetermined relative humidity (RH) may be at least about 10%, 20%, 30%, 33%, 35%, 40%, 50%, 60%, 70% or 80%.

The predetermined conditions may be accelerated storage conditions. For example, the predetermined conditions may include at least about 40 °C and at least about 43%RH, or at least about 25 °C and at least about 53%RH.

The microorganism in the composition may have a viability loss of less than about 1 log unit/g after about 3 months at about 40°C and 43%RH, or after about one month at about 25°C and 53%RK.

Methods of preparing the composition In a dry particulate form may include processes such as mixing, freezing, freeze-drying, ambient air drying, vacuum drying, spray drying, vacuum spray drying or a combination thereof. The resulting live microorganism particulates, whether alone or integrated into another consumable product, possesses enhanced viability when exposed to a wide range of temperatures and humidity conditions.

The microorganism in the composition may be a fermentation harvest that is concentrated to a cream or paste-like consistency having a solid bacterial content of about 5-30% w/v. The concentrate can be in a form of wet, frozen or thawed cream or paste before being combined with other ingredients. Starting with a microorganism in a dry form is an alternative.

The preparation of a stable dry particulate composition containing viable microorganisms may include concentrating a ferment yield of a selected

microorganism, mixing ingredients or a premix of ingredients with the concentrated microorganism to form a slurry or paste, extruding the slurry or paste in an extruder or a pelleting equipment or freezing the slurry or paste in a freezer at a temperature between about -20°C and about -80°C or snap-freezing in liquid nitrogen to form a cake or particles in the form of droplets, strings or beads, drying the cake or particles by sublimating or evaporating the moisture in the particles under hot air or under a regimen of reduced pressure while supplying heat to the particles, and then crushing or milling to a specific size range and packaging or combining the resulting stable dry particulate composition into a consumable product for animal or agricultural

applications.

The mixing process may involve adding a dry or liquid mixture of all ingredients other than the microorganism directly into a concentrated culture or media dispersion comprising the microorganism to form a slurry or paste. The dry ingredient mixture may be pre-dispersed in heated water or buffer adjusted to pH of 8-9 with a

concentrated alkali solution (e.g., 1, 5 or 10 M sodium hydroxide (NaOH) solution) at about 20-80°C. The total solid content in the slurry may be about 25-90% or 30-80% (w/w).

The slurry or paste may be frozen to about -20°C, -40°C, -60°C or to about -

80°C, or snap-frozen in liquid nitrogen by atomizing, dripping or injecting into a liquid nitrogen bath. The resulting cake or particles in the form of beads, strings or droplets may be collected and dried in a freeze drier or vacuum drier, or alternatively stored in a deep freezer (e.g., between -30°C and -80°C) for later use in a frozen form or for later drying, e.g., by freeze drying or vacuum drying.

In general, useful drying process techniques include air drying, freeze drying, or evaporative drying of a thawed or partially thawed slurry or paste in a vacuum oven or centrifugal evaporator while the temperature of the thawed or partially frozen slurry is maintained above its freezing temperature (e.g., -20 to -5°C), followed by milling to desirable particle size. Preferably, the viable microorganism is coated by or embedded in non-crystallized amorphous materials of the composition. The advantage of coating or embedding the microorganism with materials in an amorphous state is to increase physical stability of the particles and reduce deleterious crystalline formation within the composition matrix of the particles. It should be noted that achieving a non-crystallized amorphous matrix structure is not a prerequisite for long term stability for all microorganisms, as some may fare better in a more crystalline state.

In a suitable exemplary embodiment, the slurry or paste containing live microorganisms may be snap-frozen and loaded onto trays at a loading capacity from about 0.1 kg/sq. ft. to about 1.5 kg/sq. ft. and then immediately transferred to a vacuum drying chamber where the drying process may proceed in two major steps including: (a) primary drying, or primary evaporative drying, under vacuum and at a temperature of the particles above their freezing point, and (b) secondary drying under full strength vacuum pressure and an elevated heat source temperature for a time sufficient to reduce the water activity of the resulting dry composition to, for example, 0.3 Aw or less. The resulting dry composition may be glassy and/or amorphous.

The terms "iyophilization" and "freeze drying^* are used herein interchangeably and refer to rapid freezing and dehydration in the frozen state (sometimes referred to as sublimation). Lyophilization takes place at a temperature significantly lower than the freezing temperature of the product and may results in the crystallization of ingredients in the product composition.

The term "primary drying" as used herein refers to drying a product at a temperature of the product substantially lower than the temperature of a heat source, i.e., heat source temperature or shelf temperature, to make a primary dried product. Typically, the bulk of primary drying may be carried out by extensive evaporation, while the product temperature maintains above its freezing temperature but significantly lower than the temperature of the heat source.

The term ^secondary drying" as used herein refers to drying a primary dried product at a temperature of the product near the temperature of a heat source, i.e., heat source temperature or shelf temperature, to make a dry product. This process may take place under vacuum sufficient to reduce the water activity of the resulting dry product. In a typical drying process, a secondary drying step reduces the water activity of the formulation to, for example, an Aw of 0.3 or less.

According to yet another aspect of die invention, a method for preparing the dry particulate composition of the invention is provided. The method comprises:

(a) combining one or more viable microorganisms, one or more hydrolyzed proteins, one or more metal oxides and/or metalloid oxides, and optionally one or more carbohydrates and/or one or more carboxyiic acid in an aqueous solvent to form a slurry or paste;

(b) freezing the slurry or paste in liquid nitrogen or a freezer to form a solid frozen cake or particles in the form of beads, droplets or strings;

(c) primary drying step of the solid frozen cake or particles by evaporation, under vacuum, while maintaining the temperature of the cake or particles above their freezing temperature, whereby a primary dried formulation is formed;

(d) secondary drying of the primary dried formulation at full strength vacuum and a heat source temperature of 20°C or higher for a time sufficient to reduce the water activity of the primary dried formulation to 0.3 Aw or lower; and

(e) cutting, crushing, milling or pulverizing the dried formulation to produce a free-flowing powder with particle size less than about 10,000 pm (or, in another embodiment, less than about 1000 um). As a result, the particulate composition of the invention is prepared.

According to yet another aspect of the invention, a method for preparing the dry particulate composition of the invention is provided. The method comprises:

(a) combining one or more viable microorganisms, one or more hydrolyzed proteins, one or more metal oxides and/or metalloid oxides, and optionally one or more carbohydrates and/or one or more carboxyiic add in an aqueous solvent to form a paste;

(b) extruding or pelleting the paste of step (a) to form semi-dry particles, beads, or strings; (c) drying the product of step (b) to reduce the water activity to 0.3 Aw or lower; and

(d) optionally cutting, crushing, miiiing or pulverizing the dried formulation to produce a free-Mowing powder with particle size iess than about 10,000 μrn (or, in another embodiment, less than about 1000 pm). As a result, the particulate composition of the invention is prepared.

The drying in step (c) may be performed by applying air or vacuum using a heat source temperature of about 20°C or higher, for example in a convection oven or vacuum oven. The partide size may be iess than about 10,000, 1,000, 500, 250, 100 um or 50 um, preferably less than about 1,000 pm, more preferably less than about 500 pm. Typically, the particle size will be at least about 500 nm or 1 pm.

The method may further comprise sterilizing the one or more metal oxides and/or metalloid oxides, the one or more hydrolyzed proteins, and the optional one or more carbohydrates and one or more carboxylic acid salts before step (a). The sterilization may be achieved by any method known in the art. For example, heating under pressure a dry mixture or an aqueous mixture of the metal oxides and/or metalloid oxides, hydrolyzed proteins and the carbohydrate, and followed by cooling before step (a).

The method may further comprise dissolving or dispersing in water the one or more hydrolyzed proteins, and the optional one or more carbohydrates and one or more carboxylic acids or carboxylic add salts before step (a).

The resulting dry particulate composition comprising viable microorganisms may be agglomerated with molten fats. The dry powder may be placed in a planetary mixer at 40-50°C and molten fats such as cocoa butter, natural waxes or palm oil, stearic acid, stearin or mixtures thereof may be added slowly to the warm powder. The mixture may be cooled down to below the melting temperature of the fats while mixing continues until the molten fat is solidified and a visually uniform size of agglomerated or coated powder is achieved. The weight mass of the molten fats in the resulting composition may be about 20-70%, preferably about 30-50% (w/w).

Applications

The composition of the invention may be used in a variety of applications, induding pharmaceutical, nutraceutical, food or animal feed products. The product may be a consumable (i.e., edible) one. The product may be an immunogenic one, for example a live attenuated vaccine or immune enhandng product. The product may be an agricultural product, for example a botanical, ornamental or crop protection product; a foliage enhancement product; or a seed or root protection and/or enhancement product. The product may be a soil enhancement product, for example a nitrogen, phosphate or micronutrient management product for use in, or in conjunction with, a fertilizer. The product may also be a coated food or feed container or packaging, a medical device, or a bandage for use on human or animai skin.

Applications intended for animal consumption should generally utilize materials that are generally recognized as safe ("GRAS"). If the intended application is for agricultural or soil enhancement, other non-food grade but biodegradable materials may be suitable.

The composition of the invention may be administrated as a concentrated or diluted dry powder to achieve a specific desired potency. It may also be incorporated in or coated on a consumable product, for example a food or feed material. It may also be sprayed on plant foliage, coated on plant foliage or seeds, or applied to soil. Thus, the invention provides methods of providing a benefit to a plant, plant foliage, flowers, plant roots, seeds, fruits or vegetables, comprising applying thereto an effective amount of the viable microorganism-containing composition of the invention.

The composition of the invention may be applied in the form of a liquid or dry particulate protective film or top coat to provide antimicrobial protection or reduce or eliminate bacterial pathogens on surfaces. The surface can be a food or feed surface, a food or feed container or packaging, a medical device or bandage and human or animal skin and a wounds therein. The protecting composition may comprise an effective amount of viral particles (e.g., bacteriophages).

EXAMPLES

COMPARATIVE EXAMPLE 1. Stability of a live microorganism in a dry

comparative particulate composition containing 10% sugar and 3%

hydrolyzed proteins as cryoprotectants

A 100-gram portion of concentrated harvest of rhizobium sp. containing 10% w/w bacterial solids was mixed with log of trehalose (Cargiil, Minneapolis, MN.) and 3g of hydrolyzed pea proteins (Friesland Capina Doma, Paramus, N3) and snap frozen in liquid nitrogen. The frozen beads were freeze dried using a standard drying protocol (full vacuum for 48 h with no heat source). The initial count of live bacteria in the dry form was 10.87 log CFU/g. A sample of this product was placed under accelerated stability challenge at 25"C and 53% RH. The product showed 2 log unit/g loss after 14 days and 2.4 log unit/g loss after 28 days. This demonstrates the inherent instability of a typical dry sample with respect to microorganism survival during storage.

EXAMPLE 2. Preparation of a dry and stable particulate composition of lite invention containing a live microorganism

Hydrolyzed pea protein (12.5g, Friesland Capina Doma, Paramus, NJ) was dissolved in 40 mL warm distilled water (75°C). The pH of the pea solution was adjusted to 8.5 using a 20% concentrated NaOH solution. Optionally, alginate (1.5g, Tic gum, Belcamp, MD), sodium ascorbate (5g, Spectrum Chemical MFG. Corp, New Brunswick, NJ) and cyclodextrin-7 (7.5g, Wacker, MUnchen, Germany) were dry blended and added to the pea solution with continuous hand mixing using a stainless steel spatula. The solution was cooled down and maintained at a temperature between 35°C and 40°C.

Liquid concentrate rhizobium sp., (50g, containing 10% w/w bacterial solids) was added and hand mixed for 2-3 minutes, until ail the bacteria were homogenously distributed. Silicon dioxide (25g, silicagelpackets.com, Harrisburg NC.) was then slowly added under continues mixing until a uniform viscous paste formed. The paste was cooled down to 4°C and kept at this temperature for 30-60 minutes. The paste was then snap-frozen in a liquid nitrogen bath to form frozen beads, where they were harvested from the liquid nitrogen and stored at -80°C for later drying.

For drying, the frozen beads were spread on pre-cooled trays (-20°C) at a loading capacity equivalent to 800 g/ft² and then immediately placed on shelves in a freeze drier (Virtis advantage, Gardiner, NY). Vacuum was then adjusted to between 900-1500 mTORR and the shelf temperature was set to (+)20°C. These temperature and vacuum pressure settings were maintained for 16 hours. A secondary drying step was then performed at full strength vacuum (150-200 mTORR) and a shelf temperature of (+)30°C for an additional 8 hours. The material was completely dried to a water activity 0.2 or lower when measured by a Hygropalm Awl instrument (Rotonic

Instrument Corp., Huntington, NY). The dry material was then milled and sieved to particle size≤ 250 pm and the particulate composition stored at 4°C.

EXAMPLE 3. Storage stability of the stable dry particulate composition of the invention

Samples of the comparative particulate composition from Example 1 and the dry stable particulate composition of the current invention from Example 2 were mixed with equal amount of maitodextrin (1: 1 ratio) and placed in a desiccator under accelerated storage conditions of 25°C and 53%RH. Samples were taken periodically for microbial CFU assessment using standard microbiological dilutions and agar plating procedures. FIG. 1 shows the storage stability results under accelerated storage conditions of 25°C and 53%RH. The unprotected freeze dried microorganism from Example 1 lost 2 logs unit/g viability within the first week under the accelerated storage conditions, while the dry particulate composition of the invention containing the live microorganism lost only 0.94 log unit/g after 28 days at 25°C and 53% RH.

FIG. 2 shows the storage stability results under accelerated storage conditions of 25°C and 65%RH. The unprotected freeze dried probiotic bacteria from Example 1 completely lost its viability within the first week under the accelerated storage conditions, while the dry particulate composition of the invention lost 0.74 log unit/g after 14 days at 25*0 and 65%RH.

EXAMPLE 4. Addition of a metal oxide or metalloid oxide

A. Addition of various amounts of a metal oxide or metalloid oxide, effect on the stability of the dry particulate composition containing a live

microorganism

Dry compositions comprising increasing contents of silicon dioxide and constant amounts of hydroiyzed pea proteins and carbohydrates as shown in Table 1 will be prepared and evaluated. The dry ingredients, hydroiyzed pea protein, trehalose, cydodextrin-7, alginate, ascorbate, and silicon dioxide, will be mixed with a Liquid concentrate, rhizobium sp. (50g, containing 10% w/w bacterial solids) and dried to prepare Compositions 1-6 as described in Example 2. Compositions 1-6 will then be tested for stability as described in Example 3. It is expected that Compositions 2-6, not Composition 1, will show less than one (l) log unit of CFU/g loss after 28 days at 25°C and 53% RH.

Table 1: Variation of metalloid oxide content while maintaining constant amounts of hydroiyzed proteins and carbohydrates

B. Addition of various metal oxides, effect on the stability of the dry

particulate composition containing a live microorganism

Dry compositions comprising increasing contents of a metal oxide or a metalloid oxide and constant amounts of hydroiyzed pea proteins and carbohydrates as shown in Table 2 will be prepared and evaluated. The dry ingredients, hydroiyzed pea protein, trehalose, cydodextrin-7, alginate, ascorbate, and silicon dioxide, will be mixed with a Liquid concentrate, rhizobium sp. (50g, containing 10% w/w bacterial solids), and dried to prepare Compositions 7-11 as described in Example 2. Compositions 7-11 will then be tested for stability as described in Example 3. It Is expected that Compositions 7-11 will show less than one (1) log unit of CFU/g loss after 28 days at 25°C and 53% RH. Table 2: Variation of metal.oxides content_while maintaining constant amounts of hydrolyzed proteins and carbohydrates

C. Addition of silicon dioxide, effect of particle size on the stability of the dry particulate composition containing a live microorganism

Dry compositions comprising a constant amount of silicon dioxide and constant amounts of hydrolyzed pea proteins and carbohydrates as shown in Table 3 will be prepared and evaluated. The dry ingredients, hydrolyzed pea protein, trehalose, cyclodextrin-7, alginate, ascorbate, and silicon dioxide, will be mixed with a Liquid concentrate, rhizobium sp. (50g, containing 10% w/w bacterial solids) and dried to prepare Compositions 12-14 as described in Example 2. Compositions 12-14 will then be tested for stability as described in Example 3. It is expected Compositions 12-14 will show less than one (1) log unit of CFU/g loss after 28 days at 25°C and 53% RH.

Table 3: Variation of silicon dioxide particle size content while maintaining constant anwunts of hvdrvlyised proteins antf carbohydrates

EXAMPLE 5. Effect of a metal oxide or metalloid oxide as an integral part of the particulate composition or as a dry mix with a dry particulate composition

A comparative dry particulate composition containing 12.5g casein hydrolysate (DMV international Amersfoort, the Netherlands} and optional sodium ascorbate (5g, Spectrum Chemical MFG. Corp, New Brunswick, NJ), cyclodextrin-7 (7.5g Wacker, Miinchen, Germany) trehalose (23.5g Cargill Minneapolis, MN) and alginate (l.Sg Tic gum, Belcamp, MD) with no addition of a metal oxide or metalloid oxide is prepared. The protein hydrolysate is dissolved in 40 grams of distilled water and pH adjusted to 8.5. The optional carbohydrate and carboxylic acid salt compounds are dry blended and added to the protein hydrolysate solution. The mixture is heated to 70°C to dissolve all the compounds and the solution cooled down to ambient temperature. Liquid concentrate containing rhizobium sp. {50g, containing 10% w/w solids) is added to the mixture and the slurry snap frozen in liquid nitrogen and dried as described in Example 2. The dry comparative particulate composition is then dry mixed with 50g silicon dioxide. A particulate composition of the invention containing silicon dioxide as an integral part of the particles is prepared and dried as described in Example 2. A sample of each product is placed in open container under accelerated stability challenge as described in Example 3. The comparative sample fails to meet the target stability requirement of < 1 log loss in one month at 25°C and 53% RH storage condition, confirming that the metal oxide must be included as an integral part of the particulate composition.

EXAMPLE 6. Effect of different metal oxide or metalloid oxide (silicon, titanium, calcium, magnesium, zinc and aluminum)

Compositions containing 12.5g casein hydrolysate (DMV international

Amersfoort, the Netherlands), 25g silicon dioxide or titanium dioxide or calcium oxide or magnesium dioxide or zinc oxide or aluminum oxide, and optionally 7.5g

cyclodextrin-7 (Wacker, Miinchen, Germany), 23.5g trehalose (Cargill Minneapolis, MN), l.Sg alginate (Tic gum, Belcamp, MD) and sodium ascorbate (5g, Spectrum Chemical MFG. Corp, New Brunswick, NJ) are prepared. The protein hydrolysate is dissolved in 40 grams of distilled water and pH adjusted to 8.5. The optional carbohydrate and ascorbic acid salt compounds are dry blended and added to the protein hydrolysate solution. The mixture is heated to 70°C to dissolve ali the compounds and the solution cooled down to ambient temperature. Liquid concentrate or frozen beads of a live microorganism (50g, containing 10% w/w solids) is added to the mixture and mixed-in followed by the addition of the metal oxide. The paste is snap frozen in liquid nitrogen and dried as described in Example 2. The initial CFU count of live bacteria in ail dry composition is above 11 log CFU/g. A sample of each product is placed in open container under accelerated stability challenge as described in Example 3. All samples pass the target stability requirement of < 1 log loss in one month at 25°C and 53% RH storage condition thus, either silicon, calcium, magnesium, titanium, zinc or aluminum oxides can be used in the particulate composition of the invention. EXAMPLE 7. Effect of hydrophilic or hydrophobic metal oxide or metalloid oxide

A particulate composition of the invention containing hydrophilic silicon dioxide (available from siiicagelpackets.com, Harrisburg NC) is prepared and dried as described in Example 2. A comparative particulate composition containing hydrophobic silica (Sipernat Dll, Degussa) is prepared and dried as described in Example 2. A sample of each product is placed in open container under accelerated stability challenge as described in Example 3. The comparative sample fails the target stability requirement of < 1 log loss in one month at 25°C and 53% RH storage condition thus, confirming that the preferred metal oxide or metalloid oxide is a hydrophilic metal oxide or metalloid oxide

EXAMPLE 8. Stability of various microorganisms in the particulate composition of the invention (L> acidophilus. Bifidobacterium so_*, rhizoblum sp«, and bacteriophage virus)

Compositions containing I2.5g pea protein hydrolysate (Friedsland Campina Doma, Paramus, N3), optionally 23.5g trehalose (Cargiil, Minneapolis, MN), 7.5g inulin (Cargiii Minneapolis, MN) and 1.5g alginate (Tic gum, Belcamp, MD) and 25g silicon dioxide are prepared. The protein hydrolysate is dissolved in 40 grams of distilled water and pH adjusted to 8.5. The optional carbohydrate compounds are dry blended and added to the protein hydrolysate solution. The mixtures are heated to 70°C to dissolve all the compounds and the solutions cooled down to ambient temperature.

Concentrated ferment liquids or frozen beads of L acidophilus, Bifidobacterium sp._t rhizobium sp., orT2 E.coli bacteriophage virus (50g each) are added to the mixtures followed by the addition of 50g silicon dioxide and the pastes snap freezes in liquid nitrogen and dried as described in Example 2. The initial CFU count of the live bacteria in all dry compositions is at least 10 log units CFU/g. A sample of each product is placed in open container under accelerated stability challenge as described in Example 3. All different microorganism samples pass the target stability requirement of < 1 log unit loss in three month at 40°C and 33% RH storage condition, thus, demonstrating the ability of the particles to stabilize a variety of microorganisms.

EXAMPLE 9. Preparation of a stable seed inoculant microbes

The commercially available biological control agent Rhizobacteria (Advanced Biological Marketing, Van Wert, OH) was incorporated into a dry composition according to the method of Example 2. The effectiveness of the dry Rhfzobacteria composition was evaluated on soybean seed coated with the dry composition powder above 1E+6 CFU/g seed. Commercially available soybean seeds were coated with the dry

Rhfzobacteria composition as follows. About 1 mL of a 0.4% commercially available polymer coating solution (Flo Rite 1706, BASF, Research Triangle Park, NC. USA) was sprayed onto lOOg of soybean seeds in a drum tumbler (Gufstafson Batch Lab Treater, Gufstafson LLC, Skopee, MN) to moisten the surface of the seeds. About 0.5g of a dry powder was siowiy added to the moistened seeds with tumbling. The coated seeds were allowed to tumble and mix for about 4 minutes and were then transferred to a clean dry pan to air dry for about 30 minutes. The dry coated seeds were packaged in paper bags and maintained in a 53% relative humidity chamber at 25°C. The seeds were subjected to weekly microbiological stability testing over a period of one month, during which less than one log loss of viability was observed and a targeted assay count above 1E+5 CPU/seed was maintained.

EXAMPLE 10. Preparation of stable probiotic pet food

A commercially available pelleted pet food for dogs is dried in a convection oven to a water activity lower than 0.3, and then coated with stable L acidophilus dry composition prepared as described in Example 2. The dry pellets are sprayed with about 5% of fat-based moisture barrier (a mixture of 40% chicken fat, 40% cocoa butter and 20% beeswax), mixed in a drum tumbler with the dry powder formulation (usually 0.1-0.5% of the total pet food that provides a dosage of 1E+8 CFU/g), and finally sprayed with an additional coat of the fat-based moisture barrier. The total amount of coating is about 15% relative to the uncoated dry weight of the pet food. Coating time is about 30 min.

EXAMPLE 11. Preparation of stable fish feed containing several probiotic microorganisms

Pelleted probiotic feed for fish culture according to the invention are prepared with a mixture of several probiotics. A stable dry probiotic composition containing a mixture of L Acidophilus and Bifidobacterium iactis is prepared as described in Example 2. A commercially available starter feed for salmon (Zeigler Bros., Gardners, PA) is first dried in a convection oven to a water activity lower than 0.3, and then coated with the probiotics composition in a drum tumbler. The feed pellets (lOOOg) are first sprayed with about 5% by weight of fat-based moisture barrier (a mixture of 40% fish oil, 40% cocoa butter and 20% beeswax), then mixed with lg of the stable dry probiotic composition (to attain a dosage of 1E+7 CFU/g feed), and finally sprayed with additional coat of the fat-based moisture barrier. The total amount of coating is about 10% (of the fish feed}. EXAMPLE 12. Production of animal feed containing stable dry composition containing probiotic bacteria against pathogenic microorganisms

About 500g of commercially available animal feed for either steers or chickens is top coated in a drum tumbler with 3% oil mixture containing one portion of dry stable L acidophilus composition prepared as described in Example 2, and two (2) portions of plant oil such as com oil. The CFU count of the probiotic bacteria is about lE+7/g feed. The coated feed is placed in a 43% relative humidity chamber at 40°C, and after 14 days storage in these extreme conditions the viability loss of the probiotic bacteria is less than one (1) log unit of the initial CFU counts. Another probiotic coated feed is placed in a 33% relative humidity chamber at 30°C and after six (6) month storage in these conditions, during which the viability loss of the probiotic bacteria is less than one (1) log unit of the initial CFU.

EXAMPLE 13. Production of Stable dry particulate composition containing live phages against Vibrio angulllarum

Ten (10) grams of stable composition containing live phages is prepared as described in Example 2. The dry live phage composition is mixed with 20g of canoia oil and the suspension coated on 1 kg tilapia feed pellets. The coated feed is stored under typical warehouse storage conditions (25°C and 43%RH). The viability of the phages in the fish feed is preserved after 14 days exposure to the non-refrigerated warehouse storage conditions when using the compositions and methods of the invention as described in Example 2.

EXAMPLE 14. Coating butcher paper with stable composition containing live phages against Listeria monocytogenes.

Ten (10) grams of stable composition containing live broad-host-range phages AS 11 and P100 for control of L. monocytogenes is prepared in a liquid form according to the composition described in Example 2 (excluding the drying step). The liquid live phage composition suspension is spray coated as a thin film on butcher paper followed by air drying at 45°C. Another coated paper sample is prepared by spraying liquid PBS buffer pH-7.4 containing the phages. The coated papers are stored under typical warehouse storage conditions (25°C and 43%RH). When the paper samples are subsequently tested, the paper coated with phages in the PBS buffer lose their bacteria-killing effect within 7 days while the paper coated with the phages that are embedded in the composition of the present invention, maintain their killing ability against Lysteria monocytogenes up to up to 90 days exposure to the non-refrigerated warehouse storage conditions.

EXAMPLE 15. Coating meat packaging trays with stable dry particulate composition containing live phages against Listeria monocytogenes. Ten (10) grams of stable composition containing live broad- host-range phages A511 and P100 for control of L. monocytogenes is prepared as described in Example 2. The dry live phage composition Is mixed with a warm mixture (45°C) of lOg of molten bees wax and lOg of palm oil and the suspension is coated as a thin film on the bottom of meat packaging trays. The coated trays are stored under typical warehouse storage conditions (25°C and 43%RH). The viability of the phages is preserved up to 90 days exposure to the non-refrigerated warehouse storage conditions when using the compositions and methods of the invention as described in Example 2.

EXAMPLE 16. Production of Stable dry particulate composition containing Baker's yeast ( Saccharomyces cerevislae)

A liquid concentrate of baker's yeast (Saccharomyces cerevislae, 100.2g, containing 20% w/w yeast solids) was mixed on a magnetic stirrer for 10 minutes at a speed of 180 RPM to homogenously disperse the cells in the water. Soy lecithin (i.Og, Soiae LLC, St. Louis, MO) was then added to the cell dispersion, followed by mixing on the magnetic stirrer for an additional 10 minutes at 180 RPM. While mixing, a dry blend containing sodium ascorbate (lOg, Aland Nutraceutical Co., Ltd., JingJiang, Jiangsu P.R. China), trehalose (15g, Hayashibara Co., Ltd., Okayama, Japan), and inulin (15g, Cargill Incorporated, Wayzata, MN) was slowly added to the continuously stirred dispersion and the mixture mixed for an additional 30 minutes on the magnetic stirrer at 180 RPM.

After mixing, silica oxide fumed (Aerosi! 200, lOg, Evonik Industries,

Parsippany, NJ) was slowly incorporated into the mixture In 2g Increments by hand mixing. Then, sodium carboxymethylcellulose (17.5g, Hercules, Inc., Wilmington, DE) and Thermflo Food Starch-Modified (17.5g, Ingredion Inc., Bridgewater, NJ) were kneaded into the formulation. (An alternative method is to incorporate a dry blend, in which a dry mixture containing Aerosil 200, sodium carboxymethy!cei!ulose, and Thermflo Food Starch-Modified are incorporated into the formulation using a standard Kitchen-aid Professional 600 series house-hold mixer (Kitchenaid, Benton Harbor, MI) at a speed of 60 RPM for two minutes, or until all components are fully homogenized within the formulation.)

The formulation was then hand extruded (Clay Extruder, Walnut Hollow,

Dodgeville, WI) through a 1mm die onto a mesh tray (Hubbard Scientific Co.,

Northbrook, III), and the formulation was then dried in a convection oven (IOD, Helena Laboratories, Beaumont, Texas) between 38°C to 40°C for four hours.

After four hours of convection oven drying, the water activity (Aw) was 0.470 and the moisture content was 6%. Water activity was measured using a Hygropalm Awl instrument (Rotonic Instrument Corp., Huntington, NY), while percent moisture was determined with an Ohaus MB45 moisture balance (Parsippany, N3). The formulation was further dried in a freeze dryer (Virtis, Gardiner, NY) at a shelf temperature of 38°C and vacuum pressures of 75 to 230 mTorr for an additional 4 hours.

After drying, the formulation was milled and sieved to particle sizes ranging from 150 microns to 500 microns, and the sieved materials were stored in a

refrigerator within sealed Mylar bags prior to viability and stability assessments.

Stability of the baker's yeast formulation of the present invention was compared against a commercially obtained instant dried yeast (Lesaffre, Milwaukee, WI) under accelerated testing conditions consisting of 30°C/53% relative humidity. Viable ceil counts were evaluated at time points 0, 14, 28, 42, 56, and 84 days, and the corresponding viability loss data plotted in FIG. 3. By 84 days into stability testing, the stabilized yeast had lost 1.32 log units CFU/g compared with a log loss of 2.36 log units CFU/g for the un-stabilized instant dried yeast

EXAMPLE 17. Preparation of a dry and stable particulate composition of the Invention containing Rhlzobacteria

A commercially available biological control agent, Rhizobacteria (Advanced Biological Marketing, Van Wert, OH), is incorporated into a dry composition and extruded as described in Example 16. The effectiveness of the dry Rhizobacteria composition is evaluated on soybean seed coated with the dry composition powder above 1E+6 CFU/g seed. Commercially available soybean seeds are coated with the dry Rhizobacteria composition as follows. About 1 mL of a 0.4% commercially available polymer coating solution is sprayed onto lOOg of soybean seeds in a drum tumbler (Gufstafson Batch Lab Treater, Gufstafson LLC, Skopee, MN) to moisten the surface of the seeds. About 0.5g of a dry powder is slowly added to the moistened seeds with tumbling. The coated seeds are allowed to tumble and mix for about 4 minutes and then transferred to a clean dry pan to air dry for about 30 minutes. The dry coated seeds are packaged in paper bags and maintained in a 53% relative humidity chamber at 25°C. The seeds are subjected to weekly microbiological stability testing over a period of one month. A targeted assay count above 1E+5 CFU/seed is achieved, corresponding to a viability loss of less than one log.

These examples demonstrate that various microorganisms, such as probiotic bacteria, fungi and viruses, used for treating various animals including fish, chickens, swine, and companion animals and for agricultural applications, can be preserved using the compositions and drying methods of the invention and then coated onto or mixed into foods or feeds or agricultural products for long term storage on shelf or for at least two (2) weeks under high humidity and temperature storage conditions typically experienced in handling uncoated seeds or feed.

AM documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only.

Claims

WHAT IS CLAIMED:

1. A composition comprising one or more viable microorganisms, one or more metal oxides and/or metalloid oxides, and one or more hydrolyzed proteins, wherein the one or more metai oxides and/or metalloid oxides in total constitute at least 1% by weight of the composition.

2. The composition of claim 1, wherein the one or more viable

microorganisms are selected from the group consisting of live or attenuated bacteria, fungi, yeast, unicellular aigae, viruses, and phages.

3. The composition of claim 1 or 2, wherein the one or more hydrolyzed proteins are selected from the group consisting of hydrolyzed milk proteins, hydrolyzed plant proteins, and combinations thereof.

4. The composition of claim 1 or 2, wherein the one or more hydrolyzed proteins are selected from the group consisting of hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof.

5. The composition of any preceding daim, wherein the composition is dry composition comprising particles, wherein 90% or more of the particles have a diameter smaller than about 10,000 um.

6. The composition of any preceding claim, further comprising one or more carbohydrates that in total constitute 10-70% by weight of the composition.

7. The composition of claim 6, wherein the one or more carbohydrates are selected from the group consisting of disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

8. The composition of claim 6, wherein the one or more carbohydrates comprise one or more polysaccharides selected from the group consisting of alginate, gum acacia, locust bean gum, carrageenan, starches, modified starches, and combinations thereof.

9. The composition of any preceding daim, wherein the one or more metal oxides and/or metalloid oxides are selected from the group consisting of calcium oxides, aluminum oxides, magnesium oxides, silicon dioxides, zinc oxides, titanium dioxides, and combinations thereof.

10. The composition of any preceding claim, wherein the one or more metai oxides and/or metalloid oxides are hydrophilic.

11. The composition of any preceding daim, wherein the composition comprises at most 75% in total of the one or more metai oxides and/or metalloid oxides by weight.

12. The composition of any preceding claim, wherein the composition comprises 1-50% in total of the one or more metal oxides and/or metalloid oxides by weight.

13. The composition of any preceding claim, wherein the composition comprises 5-35% in total of the one or more metal oxides and/or metalloid oxides by weight.

14. The composition of any preceding claim, wherein the composition comprises 10-25% in total of the one or more metal oxides and/or metalloid oxides by weight.

15. The composition of any preceding claim, further comprising 1-10% in total of one or more carboxylic acids and/or carboxylic acid salts by weight.

16. The composition of claim 15, wherein the one or more carboxylic acids and/or carboxylic acid salts are selected from the group consisting of lactic acid, ascorbic acid, maleic acid, oxalic acid, ma!onic acid, malic acid, succinic add, citric acid, gluconic add, glutamic acid, their salts, and combinations thereof.

17. A pharmaceutical, nutraceuticai, food or feed product or a botanical or agricultural product comprising the composition of any preceding daim.

18. A food or feed container or packaging, a medical device, or a bandage for use on wounds in human or animal skin, wholly or partially coated with or incorporating tiie composition of any one of daims 1-16.

19. A method for preparing a dry composition of any one of claims 1-16, comprising:

(a) combining the one or more viable microorganisms, the one or more hydroiyzed proteins, the one or more metal oxides and/or metalloid oxides, optionally the one or more carbohydrates, and optionally the one or more carboxylic adds and/or carboxylic acid salts in an aqueous solvent to form a paste;

(c) primary drying of the solid frozen cake or particles by evaporation, under vacuum, while maintaining the temperature of the partides above their freezing temperature, whereby a primary dried formulation is formed; and

(d) secondary drying of the primary dried formulation at full strength vacuum and a heat source temperature of 20°C or higher for a time sufftdent to reduce the water activity of the primary dried formulation to 0.3 A* or lower.

20. A method for preparing a dry particulate composition of any one of claims 1-16, comprising: (a) combining the one or more viable microorganisms, the one or more hydrolyzed proteins, the one or more metal oxides and/or metalloid oxides, optionally the one or more carbohydrates, and optionally the one or more carboxylic acids and/or carboxylic acid salts in an aqueous solvent to form a paste;

(b) extruding or pelleting the paste of step (a) to form semi-dry particles, beads or strings; and

(c) drying the product of step (b) to reduce the water activity to 0.3

Aw or lower.

21. The method of claim 19 or daim 20, further comprising cutting, crushing, milling or pulverizing the dry product into a free-flowing powder.

22. The method of claim 21, wherein the free-flowing powder has a particle size less than 10,000 pm.

23. A method of treating plant foliage, fruit, plant roots, seeds, or soil, comprising applying thereto the composition of any one of claims 1-16.