Stable dry probiotic compositions for special dietary uses

Abstract

A stable dry probiotic composition for special dietary uses is provided. The compositions comprise one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides, but do not comprise trehalose. The composition has a viability of at least 1 * 1010 CFU / g and a loss of viability of less than 1 log unit / g after 3 months at a temperature of 40 DEG C and a relative humidity of 33%. A method for preparing the dried stable probiotic composition is also provided.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680051904.4, filed on July 8, 2016, entitled "Stable dried probiotic composition for special dietary purposes".

[0002] This application relates to and claims the benefit of U.S. Provisional Application No. 62 / 198,220, filed July 29, 2015, entitled STABLE DRY PROBIOTIC COMPOSITIONS FOR SPECIAL DIETARY USES, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to stable probiotic compositions for special dietary uses, such as infant formula. Background Technology

[0004] Currently, there are various probiotic microorganisms (also known as probiotics) used to supplement the gastrointestinal tract of animals (including humans). These microorganisms can modulate the natural microflora in the animal's gut to achieve the desired biological effects.

[0005] One of the challenges in delivering an effective amount of probiotic bacteria to a host is maintaining their viability under the harsh conditions of typical industrial manufacturing processes and during long-term storage at high temperatures and humidity. While encapsulation and formulation technologies for delivering biological materials to the digestive systems of animals have been developed, encapsulation or stabilization technologies for protecting the viability of probiotics during manufacturing, dispensing, and storage have seen little development. Stabilization technologies are needed that enable probiotic bacteria to survive exposure to a variety of harsh environments, especially those associated with elevated temperature and humidity.

[0006] Furthermore, the inherent humidity of probiotic products presents another challenge, as probiotics are typically sensitive to water activity, especially when combined with high temperatures. To date, no technology or process has been established to provide significant probiotic protection under moderate humidity conditions (i.e., water activity of about 0.2 and above, or as high as about 0.4 and above) and high temperatures during dispensing and storage (e.g., at least about 30°C, or as high as about 40°C and above). Therefore, there is a need for stable probiotic compositions suitable for dispensing in diverse geographical locations, including those with tropical climates, where probiotic viability can be compromised.

[0007] Other challenges include regulatory restrictions on the use of common food ingredients in special dietary formulations intended for human consumption, such as for infants, young children, and the elderly. Common synthetic encapsulating and stabilizing compounds, as well as even some natural compounds (such as gum arabic, alginate, milk protein, and certain sugars, such as trehalose), are not recommended for use in these special dietary formulations. The FAO / WHO Codex Alimentarius Commission has established a recommended list of nutrient compounds permitted for special dietary uses.

[0008] Therefore, there is a need for stable probiotic compositions containing probiotic microorganisms (e.g., probiotic bacteria) and other ingredients suitable for specific dietary uses, and for stabilization techniques used to prepare such compositions. Summary of the Invention

[0009] This invention provides a stable dried probiotic composition for special dietary uses and a method for preparing the same.

[0010] According to one aspect of the invention, a dried composition is provided. The composition comprises one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides. The composition does not contain trehalose. The viability of the composition is at least 1 × 10⁻⁶. 10 The viability loss after 3 months at 40℃ and 33% relative humidity was less than 1 log unit / g (CFU / g).

[0011] This composition can provide probiotic benefits to the host in a special dietary product. The special dietary product may be selected from infant formula, follow-on formula, processed cereal-based food, canned baby food, animal supplement or treatment, and / or special foods for medical purposes. In some specific embodiments, the special dietary product is infant formula.

[0012] Live probiotic microorganisms can be selected from live probiotic bacteria, fungi, and yeast.

[0013] Based on the total dry weight of the composition, the composition may contain at least 50% of one or more hydrolyzed proteins. The one or more hydrolyzed proteins may be selected from milk proteins, plant proteins, and combinations thereof. The one or more hydrolyzed proteins may be selected from hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof.

[0014] Based on the total dry weight of the composition, the composition may contain less than 20% of one or more disaccharides. The one or more disaccharides may be selected from sucrose, lactose, and combinations thereof.

[0015] Based on the total dry weight of the composition, the composition may contain 5% to 30% of one or more oligosaccharides. The one or more oligosaccharides may be selected from inulin, maltodextrin, dextran, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), mannan-oligosaccharide (MOS), and combinations thereof.

[0016] Based on the total dry weight of the composition, the composition may contain 1% to 10% of one or more polysaccharides. The one or more polysaccharides may be selected from carrageenan, guar gum, gum arabic, locust bean gum, starch, modified starch, and combinations thereof.

[0017] The composition may also contain one or more additional reagents. Based on the total weight of the composition, the composition may contain 0.5% to 10% of one or more additional reagents. The one or more additional reagents may be selected from carboxylates, tocopherols, and combinations thereof. The carboxylates may be selected from ascorbate and citrate. The one or more additional reagents may contain one or more tocopherols and one or more carboxylates in a weight ratio of 1:4 to 4:1. Preferably, the one or more additional reagents contain vitamin E and sodium ascorbate in a weight ratio of 4:1.

[0018] According to another aspect of the present invention, a method for preparing the composition of the invention is provided. The method includes one or more drying processes selected from air drying, vacuum drying, fluidized bed drying, and spray drying.

[0019] According to another aspect of the invention, a method for preparing the composition of the invention is provided. The method comprises: (a) combining one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides in an alkaline aqueous solvent to form a slurry; (b) snap-freezing the slurry in liquid nitrogen to form solid frozen particles in the form of beads, droplets, or strings; (c) performing a primary drying step on the solid frozen particles by evaporation under vacuum while maintaining the temperature of the particles above their freezing temperature, thereby forming a primary-dried formulation; and (d) performing a secondary drying step on the primary-dried formulation under 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. As a result, the composition of the invention is prepared. The method may further include sterilizing the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides, and the one or more polysaccharides prior to step (a). The method may also include cutting, crushing, milling, or pulverizing the composition into a free-flowing powder. The particle size of the powder may be less than about 1000 μm.

[0020] In the method of this invention, the composition may contain an effective amount of one or more live probiotic microorganisms to provide probiotic benefits to the host in a special dietary product. The method may also include preparing a special dietary product using the composition. The special dietary product may be selected from infant formula, subsequent formula, processed cereal-based foods, canned infant foods, animal supplements or therapeutics, and / or special foods for medical purposes. In some particular embodiments, the special dietary product is preferably infant formula. Attached Figure Description

[0021] Figure 1 The storage stability of the sample from Example 2 under accelerated storage conditions of 40°C and 33%RH is shown. Detailed Implementation

[0022] This invention provides novel, stable, dried probiotic compositions (preferably for specific dietary uses) and methods for preparing such compositions. These compositions provide the desired stability and protection for probiotic microorganisms. The probiotic microorganisms can be protected during the manufacturing process for consumer products, through distribution channels, and under extreme storage conditions. Most probiotic formulation manufacturers utilize extremely high numbers of bacterial cells in their products, sometimes up to 10 times and even 100 times the effective dose, where it should be understood that a significant number of cells eventually lose viability and die during the manufacturing process, transportation, and storage.

[0023] As used herein, the term "special dietary use" refers to a special dietary product prepared or applied for a host. Preferably, the Codex Alimentarius Commission of the United Nations, in conjunction with the FAO / WHO, recommends special dietary products in its document entitled "Standard For Infant Formula and Formulas For Special Medical Purposes Intended for Infants, CODEX STAN 72-1981" ("US Standard Codex 72"). Examples of special dietary products include infant formula, follow-up formula, processed cereal-based foods, canned infant foods, and special foods for medical purposes. Preferably, the special dietary product is infant formula.

[0024] The host can be any animal, including fish; birds, such as chickens; or mammals, such as ruminants, pigs, or companion animals (such as horses, dogs, or cats). In some particular embodiments, the mammal is a human. The human host can be an infant, a child, or an elderly person. Preferably, the human host is an infant.

[0025] The term “infant” as used in this article refers to a person from birth to approximately 12 months of age.

[0026] The term “child” as used in this article refers to a person who is approximately 12 months to approximately 12 years old.

[0027] The term “older person” as used in this article refers to a person who is at least about 55, 60, 65 or 70 years old (preferably at least about 65 years old).

[0028] The terms “probiotic microorganism” and “probiotic” are used interchangeably herein and refer to live microorganisms that provide or confer probiotic benefits to the host when administered to the host in an effective amount. The term “effective amount” as used herein refers to the amount of probiotic microorganisms sufficient to achieve the desired probiotic benefits in the host when administered to the host, for example, via a dietary product (e.g., a special dietary product). Probiotic microorganisms may be selected from live probiotic bacteria, fungi, and yeasts. The desired probiotic benefits can be any beneficial health or nutritional effects, such as maintaining a healthy gut microbiota, boosting immunity, preventing allergic reactions and colds, and preventing diarrhea, atopic dermatitis, and urinary tract infections.

[0029] The term “viability” as used herein refers to the ability of probiotic microorganisms in a composition to form colonies on a nutrient medium suitable for the growth of probiotic microorganisms, and may be expressed as colony forming units (CFU) relative to the weight of the composition, such as CFU / g.

[0030] As used in this article, the term "relative humidity (RH)" refers to the amount of water vapor in the air, typically at a given temperature. Relative humidity is usually less than the humidity required to saturate the air and is typically expressed as a percentage of saturated humidity.

[0031] As used herein, the term "dry" refers to the physical state of a substance (e.g., the composition of the present invention) that is dehydrated or anhydrous, for example, substantially free of liquid. For example, the substance of the composition of the present invention can be dried by one or more drying processes (e.g., air drying, vacuum drying, fluidized bed drying, spray drying, and freeze drying).

[0032] As used herein, the term "water activity (Aw)" refers to the availability of water in a substance (e.g., the compositions of the present invention), representing the energy state of water in the substance. It can be defined as the vapor pressure of water above the substance divided by the vapor pressure of pure water at the same temperature. The water activity of pure distilled water is precisely 1, i.e., Aw = 1.0. The Aw of dry matter can be about 0.5 or less, preferably about 0.3 or less, more preferably about 0.2 or less, and most preferably about 0.1 or less.

[0033] A dried composition is provided. The composition comprises one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides. The initial viability of the composition is at least 1 × 10⁻⁶. 9 1×10 10 1×10 11Or 1×10 12 CFU / g, preferably 1×10 10 CFU / g. The composition loses 1 log unit / g of viability after a predetermined period of time under predetermined conditions. Preferably, the composition does not contain trehalose.

[0034] The composition may contain an effective amount of one or more live probiotic microorganisms to provide probiotic benefits to the host in a special dietary product. The special dietary product may be infant formula, follow-up formula, processed cereal-based foods, canned infant foods, or special foods for medical purposes, preferably infant formula.

[0035] The predetermined time period can be approximately 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 12, 18, 24, or 36 months, preferably approximately 1, 2, or 3 months, more preferably 1 or 3 months. The specified time period may include shorter or longer periods within 10% of the specified time period. The term "3 months" as used herein refers to a time period of approximately 84 to 90 days. The term "2 months" as used herein refers to a time period of approximately 56 to 60 days. The term "1 month" as used herein refers to a time period of approximately 28 to 30 days.

[0036] The predetermined conditions may include a predetermined temperature and a predetermined relative humidity (RH). The predetermined temperature may be at least about 25°C, 37°C, 40°C, 45°C, 50°C, or 55°C. The predetermined relative humidity (RH) may be at least about 10%, 20%, 30%, 33%, 35%, 40%, 50%, 60%, 70%, or 80%.

[0037] Predetermined conditions can be accelerated storage conditions. For example, predetermined conditions may include at least about 40°C and at least about 33%RH, or at least about 45°C and at least about 33%RH.

[0038] The viability of the composition may be reduced by less than 1 log unit / g after about 3 months at about 40°C and 33%RH or after 1 month at about 45°C and 33%RH.

[0039] Based on the total dry weight of the composition, the composition may contain about 1% to 30%, 10% to 25%, 10% to 20%, or 15% to 20% of one or more live probiotic microorganisms. Suitable probiotic microorganisms include, but are not limited to: yeasts, such as *Saccharomyces*, *Debaromyces*, *Candida*, *Pichia*, and *Torulopsis*; molds, such as *Aspergillus*, *Rhizopus*, *Mucor*, *Penicillium*, and *Torulopsis*; and bacteria, such as the genera *Bifidobacterium*, *Clostridium*, *Fusobacterium*, *Melissococcus*, etc. Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, and Lactobacillus. Specific examples of suitable probiotic microorganisms can be represented by the following species, including all culture biotypes of these species: *Aspergillus niger*, *Aspergillus oryzae*, *Bacillus coagulans*, *Bacillus lentus*, *Bacillus licheniformis*, *Bacillus mesentericus*, *Bacillus pumilus*, *Bacillus subtilis*, *Bacillus natto*, *Bacteroides amylophilus*, *Bacillus capillosus*, *Bacillus ruminocola*, *Bacillus suis*, *Bifidobacterium adolescentis*, *Bifidobacterium animalis*, and *Bifidobacterium breve*.Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcus cremoris, Enterococcus diacetylactis, Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis, Enterococcus muntdii, Enterococcus thermophilus, Escherichia coli, Kluyveromyces fragilis, Lactobacillus acidophilus Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus cellobiosus, Lactobacillus delbrueckii ss. bulgaricus, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Leuconostoc mesenteroides Pediococcus mesenteroides, Pediococcus cereviseae (harmful Pediococcus (P. damnosus)), Pediococcus acidilactici, Pediococcus pentosaceus, Propionibacterium freudenreichii, Propionibacterium chejeni (Prop.The probiotics include *Saccharomyces cereviseae*, *Staphylococcus carnosus*, *Staphylococcus xylosus*, *Streptococcus infantarius*, *Strep. salivarius thermophilus*, *Strep. thermophilus*, and *Strep. lactis*. Preferably, the probiotics are lactic acid bacteria and bifidobacteria.

[0040] Based on the total dry weight of the composition, the composition may contain at least about 40% or 50%, preferably at least about 50%, of one or more hydrolyzed proteins. For example, the composition may contain about 40% to 80%, 40% to 70%, 50% to 70%, or 50% to 60%, preferably 40% to 80%, of hydrolyzed proteins.

[0041] The terms "hydrolyzed protein" and "protein hydrolysate" are used interchangeably herein and refer to proteins that have been broken down into shorter peptide fragments and / or amino acids through hydrolysis or digestion. Hydrolysis or digestion can be carried out using strong acids, strong bases, enzymes, or combinations thereof. Hydrolyzed proteins can be derived from animals or plants, preferably from mammals, and more preferably from dairy sources. Hydrolyzed proteins can be milk proteins, plant proteins, or mixtures thereof.

[0042] Hydrolyzed proteins can be partially or completely hydrolyzed, preferably completely hydrolyzed. The hydrolyzed protein can be a mixture of peptides and amino acids. In some embodiments, at least about 60%, 70%, 80%, 90%, 95%, or 99%, preferably at least about 70%, of the hydrolyzed protein has a molecular weight of less than about 100,000, 75,000, 50,000, 25,000, 10,000, 5,000, 1,000, or 500 Daltons, preferably about 50,000 Daltons, more preferably about 10,000 Daltons, and even more preferably about 2,000 Daltons. For example, at least about 50%, 60%, 70%, 80%, or 90%, preferably at least about 70%, of the hydrolyzed protein has a molecular weight of less than about 2,000 Daltons.

[0043] Suitable proteins for preparing hydrolyzed proteins used in the compositions of the present invention include ovalbumin, gelatin, milk protein, casein, whey protein, egg white, soy protein, pea protein, rice protein, wheat protein, and other plant proteins. Preferably, the proteins are those recommended for specific dietary uses.

[0044] Examples of hydrolyzed proteins include hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof. In a preferred embodiment, the hydrolyzed protein comprises hydrolyzed casein or hydrolyzed pea protein, wherein at least about 80% has a molecular weight of less than about 2,000 Daltons.

[0045] The composition comprises a mixture of disaccharides, oligosaccharides, and polysaccharides in which probiotic microorganisms are embedded. Without being bound by any theory, it is believed that the matrix formed by combining the carbohydrate mixture and thoroughly hydrolyzed proteins as described herein allows for faster drying and contributes to the desired amorphous and rigid structure of the resulting dried composition.

[0046] Based on the total dry weight of the composition, the composition may contain less than about 30%, 20%, or 10%, preferably less than 20%, of one or more disaccharides. For example, the composition may contain about 1% to 30%, 1% to 20%, 1% to 10%, 5% to 30%, 5% to 20%, 5% to 10%, 10% to 20%, 10% to 15%, or 10% to 20%, preferably about 5% to 30%, of disaccharides.

[0047] The disaccharides are preferably those recommended for specific dietary uses. The disaccharides can be lactose, sucrose, maltose, fructose, or combinations thereof, preferably lactose or sucrose, more preferably lactose. The disaccharide is preferably not trehalose. In some preferred embodiments, the compositions of the present invention do not contain trehalose.

[0048] Based on the total dry weight of the composition, the composition may contain about 1% to 30%, 1% to 20%, 1% to 10%, 5% to 30%, 5% to 20%, 5% to 10%, 10% to 20%, 10% to 15%, or 10% to 20% of one or more oligosaccharides. Preferably, the composition contains 5% to 30% oligosaccharides.

[0049] Oligosaccharides are soluble fibers commonly considered prebiotics in nutritional applications. Advantageously, soluble fiber passes through the stomach undigested and becomes available for digestion by the gut microbiota. The incorporation of soluble fiber also helps protect probiotics from digestive enzymes and the high acidity of the stomach.

[0050] Oligosaccharides are preferably those recommended for specific dietary uses. Oligosaccharides can be inulin, maltodextrin, dextran, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), oligomannans (MOS), or combinations thereof, preferably maltodextrin or inulin, more preferably inulin.

[0051] Based on the total dry weight of the composition, the composition may contain about 0.1% to 40%, 0.5% to 30%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, or 5% to 10% of one or more polysaccharides. Preferably, the composition contains 1% to 10% polysaccharides. The polysaccharides are preferably those recommended for specific dietary uses. The polysaccharides may be carrageenan, guar gum, gum arabic, locust bean gum, starch, modified starch, or combinations thereof, preferably locust bean gum or guar gum, more preferably locust bean gum. Preferably, the polysaccharide is not alginate or chitosan. In some preferred embodiments, the composition does not contain alginate or chitosan. In other preferred embodiments, the composition does not contain trehalose or alginate.

[0052] In some embodiments, the composition comprises 0.1% to 20% polysaccharides, 5% to 30% oligosaccharides, and 1% to 20% disaccharides, based on the total dry weight of the composition. In particular, the composition may comprise 0.1% to 20% locust bean gum, 5% to 30% inulin, and 1% to 20% lactose, based on the total dry weight of the composition.

[0053] The compositions of the present invention may also contain one or more additional agents. These additional agents may provide additional benefits to the probiotic microorganisms, the host, or both. For example, the additional agents may provide a therapeutic or immunogenic effect to the host. The additional agents may be selected from vitamins, antioxidants, trace elements, sterols, magnesium stearate, calcined silica, surfactants, peptides and steroids, and combinations thereof.

[0054] Based on the total weight of the composition, the composition may contain 0.1% to 20%, 0.5% to 20%, 1% to 20%, 0.1% to 10%, 0.5% to 10%, 1% to 10%, or 1% to 5% of additional reagents. The additional reagents are preferably those recommended for specific dietary uses.

[0055] The additional reagent may be selected from carboxylates, tocopherols, and combinations thereof. The carboxylates may be selected from ascorbate and citrate. In some embodiments, the additional reagent comprises one or more tocopherols and one or more carboxylates in a weight ratio of 4:1 to 1:4. For example, the additional reagent comprises vitamin E and sodium ascorbate in a weight ratio of 4:1.

[0056] In some embodiments, the composition comprises, based on its total weight, 40% to 80% hydrolyzed protein, 5% to 30% disaccharides, 5% to 30% oligosaccharides, and 1% to 10% polysaccharides. In a preferred embodiment, the composition comprises, based on its total weight, 54% hydrolyzed pea protein, 8% lactose, 14% inulin, and 3% locust bean gum. The composition may also contain, based on its total weight, 4% of an additional agent comprising vitamin E and sodium ascorbate in a 4:1 weight ratio.

[0057] The compositions of the present invention can be prepared using techniques known in the art. Preparation methods may include, for example, processes such as mixing, freezing, freeze-drying, ambient air drying, vacuum drying, spray drying, or combinations thereof. The resulting probiotic compositions, whether alone or integrated into specific dietary products, exhibit enhanced viability when exposed to a wide range of temperature and humidity conditions.

[0058] The probiotic microorganisms used to prepare the composition are preferably fermented harvests concentrated to a wet paste consistency with a solid content of about 5 to 30% w / v. The probiotic concentrate can be in the form of a wet, frozen, or thawed paste before being combined with other ingredients. Starting with probiotic microorganisms in a dried form is an alternative.

[0059] The preparation of a stable probiotic composition may include concentrating selected probiotics, mixing the ingredients with the concentrated probiotics to form a slurry, flash-freezing the slurry in liquid nitrogen to form particles in the form of droplets, strings, or beads, drying the particles by evaporating the moisture in the particles under reduced pressure while supplying heat to the particles, and subsequently packaging or assembling the resulting stable probiotic composition into a special dietary product, which may be a nutritional product, such as infant formula milk powder.

[0060] A suitable mixing method may involve directly adding a dried mixture of all components of the composition, except for the probiotic microorganisms, to a concentrated culture or culture medium solution containing the probiotic microorganisms to form a slurry. The dried mixture may be pre-dissolved at 20 to 80°C in an aqueous solution adjusted to pH 8 to 9 with a concentrated alkaline solution (e.g., 1 M or 5 M sodium hydroxide (NaOH) solution). In the slurry, the dry weight of the probiotic microorganisms may account for about 5 to 30% w / v, while the dried mixture may account for about 70 to 95% or 80 to 90% w / v. The total solids content in the slurry may be about 20 to 60% or 30 to 50%. The amount of polysaccharides in the dried mixture can be adjusted to achieve a desired slurry viscosity that allows for efficient drying while avoiding rubber formation or over-foaming that may occur during drying. The desired slurry density can be obtained by any means known in the art, such as by degassing under vacuum or injecting a gas (e.g., air, nitrogen, carbon dioxide, or argon).

[0061] The slurry can be rapidly frozen to approximately -30°C to approximately -180°C, or rapidly frozen in liquid nitrogen by atomization, dripping, or injection into a liquid nitrogen bath. The resulting particles, in the form of beads, strings, or droplets, can be collected and dried in a freeze dryer or vacuum dryer, or stored in a deep freezer (e.g., -30°C to -80°C) for subsequent use in frozen form or for subsequent drying, such as by freeze-drying or vacuum drying.

[0062] Generally, available drying processes include freeze drying, or evaporating and drying the thawed slurry in a vacuum oven or centrifugal evaporator while maintaining the temperature of the slurry or dried product above its freezing point (e.g., -20 to -5°C), followed by grinding to the desired particle size. Preferably, the probiotic microorganisms are coated with a non-crystalline amorphous material within the particles. The advantage of coating the probiotic microorganisms with an amorphous material is improved particle physical stability and reduced harmful crystal formation within the particles. It should be noted that obtaining a non-crystalline amorphous structure is not a prerequisite for long-term stability, as some microorganisms progress better in a more crystalline state. In a suitable exemplary embodiment, the quick-frozen slurry can be loaded onto a tray at a loading rate of about 0.1 kg / sqft to about 1.5 kg / sqft and then immediately transferred to a vacuum drying chamber, wherein the drying process can be carried out in three main steps, including: (a) an optional short temperature adaptation and structural stabilization step of freezing the particles under a vacuum pressure of less than <1000 mTORR; (b) primary drying or primary evaporative drying under vacuum and particle temperature above its freezing point; and (c) secondary drying under full-intensity vacuum pressure and elevated heat source temperature for a time sufficient to reduce the water activity of the resulting dried composition to, for example, 0.3 Aw or lower. The resulting dried composition can be glassy amorphous.

[0063] The terms “lyophilization” and “freeze-drying” are used interchangeably herein and refer to the preparation of a composition in a dried form by rapid freezing and dehydration (sometimes referred to as sublimation) in a frozen state. Lyophilization occurs at temperatures that cause the components in the composition to crystallize.

[0064] As used in this article, "primary drying" refers to drying a product at a temperature significantly below that of the heat source (i.e., the heat source temperature or shelf temperature) to prepare a primary-dried product. Generally, most primary drying can be carried out by a large amount of evaporation while maintaining the product temperature significantly below the heat source temperature.

[0065] As used herein, the term "secondary drying" refers to drying a product that has undergone primary drying at a temperature close to that of the heat source (i.e., the heat source temperature or shelf temperature) to prepare a dried product. This process can be carried out under vacuum sufficient to reduce the water activity of the resulting dried product. In a typical drying process, the secondary drying step reduces the water activity of the formulation to, for example, 0.3 or lower Aw.

[0066] In one embodiment, the composition of the present invention is prepared by a method comprising: (a) combining one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides in an alkaline aqueous solvent to form a slurry; (b) rapidly freezing the slurry in liquid nitrogen to form solid frozen particles in the form of beads, droplets, or strings; (c) performing a primary drying step on the solid frozen particles by evaporation under vacuum while maintaining the particle temperature above its freezing temperature, thereby forming a primary dried formulation; and (d) subjecting the primary dried formulation to secondary drying under full 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, thereby preparing the composition.

[0067] The method may further include sterilizing the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides, and the one or more polysaccharides prior to step (a). Sterilization may be performed by any method known in the art. For example, the mixture of hydrolyzed proteins, disaccharides, oligosaccharides, and polysaccharides may be heated under pressure and then cooled prior to step (a).

[0068] The method may further include dissolving the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides, and the one or more polysaccharides prior to step (a).

[0069] The method may further include cutting, crushing, grinding, or pulverizing the composition into a free-flowing powder. The particle size of the powder may be less than about 10,000, 1,000, 500, 250, or 100 µm, preferably less than about 1,000 µm, and more preferably less than about 250 µm.

[0070] The dried composition of the present invention can be used directly as flakes, or pulverized into powder and sieved to an average particle size of about 1 to 10,000 μm, preferably 10 to 1,000 μm.

[0071] The compositions of the present invention can be applied as concentrated powders or reconstituted liquids (e.g., beverages). They can be introduced into existing foods in the form of flakes or powders.

[0072] The method may further include preparing a special dietary product using the composition of the present invention containing an effective amount of one or more live probiotic microorganisms, for providing probiotic benefits to a host in the special dietary product. Examples of special dietary products may include infant formula, follow-up formula, processed cereal-based foods, canned infant foods, and special foods for medical purposes. Preferably, the special dietary product is infant formula.

[0073] The resulting dry, stable powder containing probiotics can be agglomerated with molten fat. The dry powder can be placed in a planetary mixer at 40°C, and molten fats such as cocoa butter, natural waxes or palm oil, stearic acid, stearine, or mixtures thereof are slowly added to the warm powder. The mixture can be cooled below the melting temperature of the fat while continuing to mix until the agglomerated powder achieves a visually uniform size. The weight percentage of molten fat in the resulting composition can be about 20-70%, preferably 30-50%.

[0074] Example 1. Preparation of a dried and stable composition

[0075] Hydrolyzed pea protein (65 g, Marcor, Carlstadt, NJ) was dissolved in 100 ml of warm distilled water (75°C). The pH of the pea solution was adjusted to 8.5 using a 20% NaOH solution. A 4:1 w / w mixture (5 g) of locust bean gum (3 g, Belcamp, MD), lactose (10 g, Foremost Farms, Rothschild, WI), inulin (17 g, Cargill Minneapolis, MN), vitamin E, and sodium ascorbate was dried, blended, and added to the pea solution using an impeller mixer at 500 rpm. The solution was cooled and maintained at 35°C to 40°C while continuously mixing.

[0076] The resulting stabilized composition is translucent, with a syrupy consistency and an amber color. The syrup solution is transferred to a dual planetary mixer (DPM, 1qt, Ross Engineering, Inc. Savannah, GA) equipped with a temperature-controlled jacket. The mixer jacket temperature is 37°C. Frozen bacteria (100 g, *Bifidobacterium* sp.) are added and mixed at 45 rpm for 2 to 3 minutes or until all bacteria are well thawed and evenly distributed. The probiotic mixture is cooled to 4°C and held at this temperature for 30 to 60 minutes. The mixture is then added dropwise and flash-frozen in a liquid nitrogen bath to form frozen beads, which are harvested from the liquid nitrogen and stored at -80°C for subsequent drying.

[0077] For drying, frozen beads were spread at a loading capacity of 800 g / sqft on pre-cooled trays (-20°C) and immediately placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY). The vacuum was then adjusted to 1800 to 2200 mTORR, and the shelf temperature was raised to +20°C. These temperature and vacuum pressure settings were maintained for 12 hours. Prior to primary drying, the frozen beads were optionally acclimatized to approximately -20°C by applying a vacuum pressure of approximately 1000 mTORR for about 10 minutes. The primary drying step was then performed by adjusting the vacuum pressure to 2000 to 2700 mTORR and the shelf temperature to +20°C. These temperature and vacuum pressure settings were maintained for 12 hours. A secondary drying step was then performed under full vacuum (150 to 200 mTORR), with the shelf temperature maintained at 40°C for an additional 12 hours. The formulation was completely dried, and its water activity, measured using a Hygropalm Aw1 instrument (Rotonic Instrument Corp., Huntington, NY), was Aw 0.23. The dried material was then ground and sieved to a particle size ≤250 μm and stored at 4°C.

[0078] Example 2. Storage stability of the dried probiotic composition

[0079] Samples containing a dry-stabilized composition of probiotic bacteria from Example 1 or a conventional freeze-dried bacterial suspension in 10% trehalose were placed in a desiccator under accelerated storage conditions of 40°C and 33% RH. Periodic sampling was performed for microbial CFU assessment using standard microbial dilutions and LMRS agar plate inoculation procedures. Figure 1 Storage stability under accelerated storage conditions of 40°C and 33%RH was demonstrated. Unprotected probiotic bacteria completely lost their viability in the first few weeks under accelerated storage conditions, while the probiotic bacteria dried composition of the present invention lost only 0.70 log units / g after 84 days at 40°C and 33%RH.

[0080] Example 3. Effects of carbohydrates and proteins on storage stability

[0081] The compositions of the present invention eliminate the need for carbohydrate stabilizers that are not included in the recommended list of nutrient compounds permitted for specific dietary uses according to the Joint FAO / WHO Codex Alimentarius Commission.

[0082] a. Composition containing maltodextrin: A composition comprising 50 g of pea protein hydrolysate (Marcor, Carlstadt, NJ), 33 g of maltodextrin (Tate & Lyle, Decatur, IL), 10 g of inulin (Cargill, Minneapolis, MN), 2 g of locust bean gum (Tic gum, Belcamp, MD), and 5 g of a mixture of vitamin E and sodium ascorbate (4:1 w / w). The pea protein hydrolysate was dissolved in 100 g of distilled water, and the pH was adjusted to 7.5. The carbohydrates were dried, blended, and added to the pea protein solution. The mixture was heated to 70°C to dissolve all compounds, and the solution was cooled to 37°C. Frozen beads (100 g) of Lactobacillus rhamnosus sp. were added to the solution as described in Example 1, and the slurry was dried. The initial count of viable bacteria in the dried composition was 9.92 logCFU / g. As described in Example 1, a sample of the product was subjected to an accelerated stability challenge. After one month, the sample showed a loss of 1.19 log units / g and therefore failed the challenge.

[0083] b. Composition containing wheat protein isolate: A composition comprising 25 g pea protein hydrolysate (Marcor, Carlstadt, NJ), 33 g Prolite 200 (wheat protein isolate, Archer Daniels Midland Company Decatur, IL), 25 g maltodextrin (Tate & Lyle, Decatur, IL), 10 g inulin (Cargill, Minneapolis, MN), 2 g locust bean gum (Tic gum, Belcamp, MD), and 5 g a mixture of vitamin E and sodium ascorbate (4:1 w / w) was prepared. The composition containing *Lactobacillus rhamnosus* species was prepared as described in Examples 1 and 2 above and dried. The initial count of viable bacteria in the dried composition was 10.17 log CFU / g. A sample of this product was placed under an accelerated stability challenge as described in Example 1. After two weeks, the sample showed a loss of 1.71 log units / g and therefore failed the challenge test.

[0084] c. Composition containing whey protein isolate: A composition comprising 25 g pea protein hydrolysate (Marcor, Carlstadt, NJ), 25 g whey protein isolate (Davisco, Eden Prairie, MN), 33 g maltodextrin (Tate & Lyle, Decatur, IL), 10 g inulin (Cargill, Minneapolis, MN), 2 g locust bean gum (Ticgum, Belcamp, MD), and 5 g a mixture of vitamin E and sodium ascorbate (4:1 w / w) was prepared. The composition containing *Lactobacillus rhamnosus* species was prepared as described in Examples 1 and 2 above and dried. The initial count of viable bacteria in the dried composition was 10.40 log CFU / g. A sample of this product was placed under an accelerated stability challenge as described in Example 1. After two weeks, the sample showed a loss of 1.30 log units / g and therefore failed the challenge test.

[0085] d. Lactose-containing composition: A composition comprising 50 g of pea protein hydrolysate (Marcor, Carlstadt, NJ), 33 g of lactose (Foremost Farms, Rothschild, WI), 10 g of inulin (Cargill, Minneapolis, MN), 2 g of tic gum (Belcamp, MD), and 5 g of a mixture of vitamin E and sodium ascorbate (4:1 w / w) was prepared. The composition containing *Lactobacillus rhamnosus* species was prepared as described in Examples 1 and 2 above and dried. The initial count of viable bacteria in the dried composition was 9.77 log CFU / g. A sample of this product was placed under an accelerated stability challenge as described in Example 1. The sample passed the first month of stability testing but failed the challenge test after two months due to a loss of 1.89 log units / g.

[0086] e. The composition of the present invention: A composition comprising 65 g of pea protein hydrolysate (Marcor, Carlstadt, NJ), 10 g of lactose (Foremost Farms, Rothschild, WI), 17 g of inulin (Cargill, Minneapolis, MN), 3 g of tic gum (Belcamp, MD), and 5 g of a mixture of vitamin E and sodium ascorbate (4:1 w / w). The composition containing Bifidobacterium was prepared and dried as described in Examples 1 and 2 above, except that the pH of the pea protein hydrolysate solution was adjusted to pH 8.5. The initial count of viable bacteria in the dried composition was 10.87 log CFU / g. A sample of this product was placed under an accelerated stability challenge. After 3 months, the sample lost only 0.70 log units / g, thus passing the challenge test.

[0087] Example 4. Effect of inulin on storage stability

[0088] US Standard Code 72 permits the use of oligosaccharides such as inulin and maltodextrin in infant formula. The effect of inulin levels from 0% to 30% in the compositions was evaluated. Compositions were prepared containing 50 g of pea protein hydrolysate (Marcor, Carlstadt, NJ), 43 g of lactose (Foremost Farms, Rothschild, WI), 2 g of Tic gum (Belcamp, MD), 5 g of a mixture of vitamin E and sodium ascorbate (4:1 w / w), and 0 g, 10 g, 15 g, or 30 g of inulin (subtracting the amount of inulin added from the amount of lactose in the composition). Compositions containing Lactobacillus acidophilus sp. were prepared and dried as described in Examples 1 and 2 above. The initial viable bacterial counts in the dried compositions containing 0 g, 10 g, 15 g, and 30 g of inulin were 9.42, 9.57, 9.59, and 9.80 log CFU / g, respectively. Samples from each composition were placed under an accelerated stability challenge. The sample containing 0 g of inulin (without oligosaccharides) lost 1.14 log units / g after 2 months and 2.25 log units / g after 3 months, while the sample containing 15 g and higher of inulin lost less than 2 log units / g after 3 months. Therefore, it was determined that the minimum amount of inulin in the compositions of the present invention must be greater than 10%.

[0089] Example 5. Effect of disaccharides on storage stability

[0090] U.S. Standards Code 72 restricts the use of trehalose in infant formula but permits the use of sucrose and lactose. The following examples demonstrate that, according to the composition of the invention, an increased amount of hydrolyzed pea protein and only a small amount of the disaccharide lactose successfully replace trehalose.

[0091] a. Composition containing trehalose: A composition comprising 25 g pea protein hydrolysate (Marcor, Carlstadt, NJ), 62 g trehalose (Cargill, Minneapolis, MN), 5 g inulin (Cargill, Minneapolis, MN), 3 g locust bean gum (Tic gum, Belcamp, MD), and 5 g a mixture of vitamin E and sodium ascorbate (4:1 w / w) was prepared. The composition containing *Lactobacillus acidophilus* species was prepared as described in Examples 1 and 2 above and dried. The initial count of viable bacteria in the dried composition was 9.49 log CFU / g. Samples of the composition were subjected to an accelerated stability challenge. After 3 months, the sample lost 0.83 log units / g, exhibiting relatively good stability. However, trehalose is not permitted in infant formula.

[0092] b. Effect of using lactose instead of trehalose: A composition was prepared containing 50 g of pea protein hydrolysate (Marcor, Carlstadt, NJ), 2 g of Tic gum (Belcamp, MD), 5 g of a mixture of vitamin E and sodium ascorbate (4:1 w / w), and 33 g of lactose (Foremost Farms, Rothschild, WI) and 10 g of inulin (Cargill, Minneapolis, MN), or 13 g and 30 g of inulin. Compositions containing *Lactobacillus acidophilus* species were prepared as described in Examples 1 and 2 above and dried. The initial viable bacterial counts in the dried compositions containing 33 g or 13 g of lactose were 9.57 log CFU / g and 9.80 log CFU / g, respectively. Samples of each composition were placed under accelerated stability challenges. The sample containing 33 g of lactose lost 1.25 log units / g after 1 month, while the sample containing 13 g of lactose maintained this loss for 2 months. Therefore, it was determined that 13% lactose in the stabilization composition, along with 50% hydrolyzed pea protein, can effectively replace trehalose.

[0093] c. Stability of compositions without trehalose and alginate: To evaluate the effectiveness of alternatives to undesirable components (e.g., trehalose and alginate) in infant formula milk powder in compositions with more than 50% disaccharides, three compositions were prepared (Table 1) comprising 25 g of pea protein hydrolysate (Marcor, Carlstadt, NJ), 5 g of inulin (Cargill, Minneapolis, MN), 5 g of a mixture of vitamin E and sodium ascorbate (4:1 w / w), 3 g of alginate (ISP Corp., Wayne, NJ) or 3 g of locust bean gum (Tic gum, Belcamp, MD), and 62 g of trehalose (Cargill, Minneapolis, MN) or 62 g of lactose (Foremost Farms, Rothschild, WI). Compositions containing *Lactobacillus rhamnosus* species were prepared and dried as described in Examples 1 and 2 above. The initial count of viable bacteria in the three dried compositions was approximately 10 log CFU / g (Table 1). Accelerated stability challenges were performed on samples from each composition. Compositions containing only alginate and trehalose showed a loss of less than 1 log unit / g after 84 days, while the other two compositions, in which lactose and locust bean gum were substituted for trehalose and alginate respectively, showed a loss greater than 1 log unit / g after only 28 days. Therefore, trehalose and alginate are essential components in compositions typically containing more than 50% disaccharides to achieve the stability requirement of less than 1 log unit / g loss after 3 months at 40°C and 33% relative humidity.

[0094] Table 1. Effects of trehalose and alginate substitutes on probiotic stability

[0095]

[0096] Example 6. Effect of polysaccharides on storage stability

[0097] Polysaccharides provide essential structural support in probiotic compositions. U.S. Standards Code 72 restricts the use of many polysaccharides, such as alginate, in infant formula, but permits the use of guar gum, locust bean gum, and starch. Sodium alginate, guar gum, locust bean gum, and starch are commercially available polysaccharides, and their role in stabilizing the compositions of this invention has been evaluated.

[0098] a. A composition containing guar gum: a composition comprising 36 g of casein hydrolysate (DMV International Nutritionals, Delhi, NY) and 25 g of inulin (Cargill, Minneapolis, MN). 36 g sucroseA composition containing Sigma and 3 g of guar gum (Tic gum, Belcamp, MD) was prepared and dried as described in Examples 1 and 2 above. The initial count of viable bacteria in the dried composition was 9.90 log CFU / g. A sample of this product was placed under an accelerated stability challenge as described in Example 1. The sample passed the stability test for the first month, but failed the challenge test after 2 months due to a loss of 1.10 log units / g.

[0099] b. Composition containing sodium alginate: A composition comprising 17 g casein hydrolysate (DMV International Nutritionals, Delhi, NY), 5 g inulin (Cargill, Minneapolis, MN), 75 g sucrose (Sigma), and 3 g sodium alginate (ISP Corp., Wayne, NJ) was prepared. The composition containing *Lactobacillus rhamnosus* species was prepared as described in Examples 1 and 2 above and dried. The initial count of viable bacteria in the dried composition was 9.90 log CFU / g. A sample of this product was placed under an accelerated stability challenge as described in Example 1. The sample passed the stability test for the first month but failed after 2 months due to a loss of 1.25 log units / g.

[0100] b. Composition containing Tic gum: A composition comprising 25 g pea protein hydrolysate (Marcor, Carlstadt, NJ), 5 g inulin (Cargill, Minneapolis, MN), 62 g trehalose (Cargill, Minneapolis, MN), 3 g Tic gum (Belcamp, MD), and 5 g a mixture of vitamin E and sodium ascorbate (4:1 w / w) was prepared. The composition containing *Lactobacillus rhamnosus* species was prepared as described in Examples 1 and 2 above and dried. The initial count of viable bacteria in the dried composition was 9.93 log CFU / g. A sample of this product was subjected to an accelerated stability challenge as described in Example 1. The sample passed the stability test after 2 months with a loss of 0.81 log units / g. Tic gum was determined to replace the alginate function in the stabilized probiotic composition.

[0101] Example 7. Infant formula milk powder

[0102] A stable, dried composition containing Bifidobacterium was prepared according to Example 1 and subsequently sieved into two particle size groups (above 50 μm and below 250 μm). Infant formula containing probiotic bacteria was prepared by mixing 99.9 g of Gerber Good Start (Nestle Infant Nutrition, Florham Park, NJ.) with 0.1 g of dried composition particles with a particle size of 50 µm to 250 µm. The final product contains approximately 10 μm of probiotic bacteria per 100 g of infant formula. 8 CFU of Lactobacillus GG. Probiotic infant formula powder was packaged in 180 cc HDPE bottles and exposed to controlled temperature / humidity at 40°C / 33%RH. Microbiological stability testing was performed monthly for 12 months or until a value below 5 × 10⁵ was observed. 7 / The number of measurements per unit dose decreased.

[0103] Example 8. Probiotic Supplement

[0104] A stable, dried composition containing *Lactobacillus acidophilus* was prepared according to Example 1 and formulated into oral dosage forms, such as tablets, caplets, or capsules. Orange-flavored tablets containing 99.9 g of compression agent (dextrose) and 0.1 g of dried formulation particles with a particle size of 50 µm to 250 µm were prepared by direct compression on a rotary machine using a 1 / 2" circular standard concave tool. The final product contained approximately 10... 8 CFU / unit dose. The tablet hardness is 8-10 kp, and the disintegration time is approximately 20 seconds. Compressed tablets are packaged in 100-tablet packs into 180 cc HDPE bottles and exposed to controlled temperature / humidity at 40°C / 33%RH. Microbiological stability testing is performed monthly for 12 months or until a disintegration rate below 1×10⁻⁶ is observed. 6 / The number of measurements per unit dose decreased.

[0105] Example 9. Functional Beverages

[0106] A stable, dried composition containing *Lactobacillus acidophilus* was prepared according to Example 1 and formulated into a dried mixture comprising (wt%) 71% sucrose, 14% maltodextrin, 10% inulin, 2% dextrose, 1% anhydrous citric acid, 0.3% gum arabic, 0.3% flavoring, 0.3% tricalcium phosphate, and 0.1% dried probiotic composition particles (*Lactobacillus acidophilus*) with a particle size of 50 µm to 250 µm. The final product contains approximately 10 9CFU / unit dose (30 g dry mixture). This product is packaged in small aluminum foil pouches (30 g unit dose / pouch) for consumption by stirring in 340 mils of water. Microbiological stability testing of the probiotic bacteria in the beverage dry mixture is performed monthly for 12 months or until less than 1 × 10⁻⁶ is observed. 7 / The number of measurements per unit dose decreased.

[0107] Example 10. Multivitamin / Probiotic Tablets

[0108] 10 g of dry powder composition was produced as described in Example 1. To prepare tablets, a dried and stable probiotic composition (100 mg) was mixed with 400 mg of a commercially available multivitamin powder (Centrum®, Pfizer) containing 2% w / w magnesium stearate and 2% w / w hydrophilic fumed silica (AEROSIL® 200, Evonik Industries), and compressed in a hand-held pill press (using a ½" tablet diameter shell). Each tablet contains approximately 10 g of the dry powder composition. 7 (CFU / tablet). Tablets were packaged in 100-tablet packs into 180 cc HDPE bottles and exposed to controlled temperature / humidity at 40°C / 33%RH. Microbiological stability tests were performed on the bottles monthly for 12 months or until a value below 1×10⁻⁶ was observed. 6 The number of samples measured decreased.

[0109] The term “about” as used herein, when referring to measurable values ​​(e.g., quantity, percentage, etc.), means covering a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of a given value, as such variation is appropriate.

[0110] All references, books, manuals, papers, patents, published patent applications, guidelines, abstracts, and other references cited herein are incorporated herein by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. The specification and embodiments are intended to be exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

[0111] The following content corresponds to the original claims in the parent application and is incorporated herein as part of the specification:

[0112] 1. A dried composition comprising one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides, wherein the composition does not contain trehalose, and wherein the viability of the composition is at least 1 × 10⁻⁶. 10 CFU / g, and wherein the viability loss of the composition is less than 1 log unit / g after 3 months at a temperature of 40°C and a relative humidity of 33%.

[0113] 2. The composition of claim 1, wherein the composition provides probiotic benefits to the host in a special dietary product.

[0114] 3. The composition of claim 2, wherein the special dietary product is selected from infant formula milk powder, subsequent formula milk powder, processed cereal-based foods, canned infant foods, and special foods for medical purposes.

[0115] 4. The composition of claim 2, wherein the special dietary product is infant formula milk powder.

[0116] 5. The composition of claim 1, wherein the live probiotic microorganism is selected from live probiotic bacteria, fungi, and yeast.

[0117] 6. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition comprises at least 50% of the one or more hydrolyzed proteins.

[0118] 7. The composition of claim 1, wherein the one or more hydrolyzed proteins are selected from milk proteins, plant proteins, and combinations thereof.

[0119] 8. The composition of claim 1, wherein the one or more hydrolyzed proteins are selected from hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof.

[0120] 9. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition contains less than 20% of the one or more disaccharides.

[0121] 10. The composition of claim 1, wherein one or more disaccharides are selected from sucrose, lactose, and combinations thereof.

[0122] 11. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition comprises 5% to 30% of the one or more oligosaccharides.

[0123] 12. The composition of claim 1, wherein the one or more oligosaccharides are selected from inulin, maltodextrin, dextran, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), oligomannans (MOS), and combinations thereof.

[0124] 13. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition comprises 1% to 10% of the one or more polysaccharides.

[0125] 14. The composition of claim 1, wherein one or more polysaccharides are selected from carrageenan, guar gum, gum arabic, locust bean gum, starch, modified starch, and combinations thereof.

[0126] 15. The composition described in item 1, further comprising one or more other reagents.

[0127] 16. The composition of claim 15, wherein, based on the total weight of the composition, the composition contains 0.5% to 10% of the one or more of the other reagents.

[0128] 17. The composition of claim 15, wherein the one or more additional reagents are selected from carboxylates, tocopherols, and combinations thereof.

[0129] 18. The composition of claim 17, wherein the carboxylate is selected from ascorbate and citrate.

[0130] 19. The composition of claim 15, wherein the one or more additional reagents comprise one or more tocopherols and one or more carboxylates in a weight ratio of 1:4 to 4:1.

[0131] 20. The composition of claim 15, wherein the one or more additional agents comprise vitamin E and sodium ascorbate in a weight ratio of 4:1.

[0132] 21. A method for preparing the composition described in item 1, comprising one or more drying processes selected from air drying, vacuum drying, fluidized bed drying and spray drying.

[0133] 22. A method for preparing the composition described in item 1, comprising:

[0134] (a) Combining the one or more live probiotic microorganisms, the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides and the one or more polysaccharides in an alkaline aqueous solvent to form a slurry;

[0135] (b) The slurry is rapidly frozen in liquid nitrogen to form solid frozen particles in the form of beads, droplets or strings;

[0136] (c) A primary drying step is performed on the solid frozen particles under vacuum by evaporation while maintaining the temperature of the particles above their freezing temperature, thereby forming a primary dried formulation; and

[0137] (d) The primary-dried formulation is subjected to secondary drying under full-intensity 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, thereby preparing the composition described in item 1.

[0138] 23. The method of item 22, further comprising: sterilizing the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides and the one or more polysaccharides prior to step (a).

[0139] 24. The method of claim 22, further comprising cutting, crushing, grinding or pulverizing the composition into a free-flowing powder.

[0140] 25. The method of item 24, wherein the particle size of the powder is less than about 1000 μm.

[0141] 26. The method of claim 22, wherein the composition comprises an effective amount of the one or more live probiotic microorganisms to provide probiotic benefits to a host in a particular dietary product.

[0142] 27. The method of item 26, further comprising preparing the special dietary product using the composition.

[0143] 28. The method of claim 26, wherein the special dietary product is selected from infant formula milk powder, subsequent formula milk powder, processed cereal-based foods, canned infant foods, and special foods for medical purposes.

[0144] 29. The method of item 26, wherein the special dietary product is infant formula milk powder.

Claims

1. A dried composition comprising one or more live probiotic microorganisms, one or more hydrolyzed proteins, one or more disaccharides, one or more oligosaccharides, and one or more polysaccharides. Based on the total dry weight of the composition, the composition comprises at least 40% of the one or more hydrolyzed proteins, and the one or more hydrolyzed proteins are selected from hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and combinations thereof. Based on the total dry weight of the composition, the composition contains less than 30% of the one or more disaccharides, and the one or more disaccharides are selected from sucrose, lactose, and combinations thereof. Based on the total dry weight of the composition, the composition contains 1% to 30% of one or more oligosaccharides, and the one or more oligosaccharides are selected from inulin, maltodextrin, dextran, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), oligomannans (MOS), and combinations thereof. Based on the total dry weight of the composition, the composition contains 0.1% to 40% of one or more of the polysaccharides, and the one or more polysaccharides are selected from carrageenan, guar gum, gum arabic, locust bean gum, starch, modified starch, and combinations thereof. The viability of the composition is at least 1 × 10⁻⁶. 10 The composition exhibits a viability loss of less than 1 log unit / g after 3 months at 40°C and 33% relative humidity.

2. The composition of claim 1, wherein the composition comprises an effective amount of the one or more live probiotic microorganisms to provide probiotic benefits to a host in a particular dietary product.

3. The composition of claim 1, wherein the live probiotic microorganism is selected from live probiotic bacteria, fungi and yeast.

4. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition comprises at least 50% of the one or more hydrolyzed proteins.

5. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition contains less than 20% of the one or more disaccharides.

6. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition comprises 5% to 30% of the one or more oligosaccharides.

7. The composition of claim 1, wherein, based on the total dry weight of the composition, the composition comprises 1% to 10% of the one or more polysaccharides.

8. The composition of claim 1, further comprising one or more other reagents.

9. The composition of claim 8, wherein, based on the total weight of the composition, the composition comprises 0.5% to 10% of one or more of the other reagents.

10. The composition of claim 8, wherein the one or more additional reagents are selected from carboxylates, tocopherols, and combinations thereof.

11. The composition of claim 10, wherein the carboxylate is selected from ascorbate and citrate.

12. The composition of claim 8, wherein the one or more additional reagents comprise one or more tocopherols and one or more carboxylates in a weight ratio of 1:4 to 4:

1.

13. The composition of claim 8, wherein the one or more additional agents comprise vitamin E and sodium ascorbate in a weight ratio of 4:

1.

14. A method for preparing the composition of claim 1, comprising: (a) Combining the one or more live probiotic microorganisms, the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides and the one or more polysaccharides in an alkaline aqueous solvent to form a slurry; (b) The slurry is rapidly frozen in liquid nitrogen to form solid frozen particles in the form of beads, droplets or strings; (c) A primary drying step is performed on the solid frozen particles under vacuum by evaporation while maintaining the temperature of the particles above their freezing temperature, thereby forming a primary dried formulation; and (d) The primary-dried formulation is subjected to secondary drying under full 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, thereby preparing the composition of claim 1.

15. The method of claim 14, further comprising: Prior to step (a), the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides, and the one or more polysaccharides are sterilized.

16. The method of claim 9, further comprising cutting, crushing, grinding or pulverizing the composition into a free-flowing powder.

17. The method of claim 16, wherein the particle size of the powder is less than about 1000 μm.

18. The method of claim 14, wherein the composition comprises an effective amount of the one or more live probiotic microorganisms to provide probiotic benefits to a host in a particular dietary product.

19. The method of claim 14, further comprising preparing the special dietary product using the composition.

20. The method of claim 14, wherein the special dietary product is selected from infant formula milk powder, subsequent formula milk powder, processed cereal-based foods, canned infant foods, and special foods for medical purposes.

21. The method of claim 19, wherein the special dietary product is infant formula milk powder.

22. A method for preparing infant formula milk powder using the composition of claim 1, comprising: (a) Sterilize the one or more hydrolyzed proteins, the one or more disaccharides, the one or more oligosaccharides and the one or more polysaccharides; (b) Combining the sterilized one or more live probiotic microorganisms, the sterilized one or more hydrolyzed proteins, the sterilized one or more disaccharides, the sterilized one or more oligosaccharides and the sterilized one or more polysaccharides in an alkaline aqueous solvent to form a slurry; (c) The slurry is rapidly frozen in liquid nitrogen to form solid frozen particles in the form of beads, droplets or strings; (d) While maintaining the temperature of the solid frozen particles above their freezing temperature, the particles are subjected to a primary drying step by evaporation under vacuum to form a primary dried formulation; (e) The primary-dried formulation is subjected to secondary drying under full 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, thereby preparing the composition of claim 1; (f) Cutting, crushing, grinding, or pulverizing the composition of claim 1 into a free-flowing powder, wherein the particle size of the powder is less than about 1000 μm, and the powder contains an effective amount of the one or more live probiotic microorganisms to provide probiotic benefits to the host in infant formula milk powder; and (g) Prepare the infant formula milk powder using the powder.

Images