Mannooligosaccharide compositions for inhibiting pathogens - Patents.com

Abstract

The present application includes a composition containing mannooligosaccharide (MOS) carbohydrates for use in inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria in a subject, wherein at least 70% by weight of the MOS carbohydrates are mannose subunits. The MOS is derived from mannan material obtained from plant sources. The composition has a low average DP, which improves its solubility. The present application also includes a method for preparing the composition.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 397,993, filed August 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present application relates to the use of mannooligosaccharide compositions for the prevention and treatment of infections. In particular, the present application relates to a highly purified mannooligosaccharide composition for the prevention and treatment of infections, such as infections caused by pathogenic bacteria, and a method for preparing the same. [Background technology]

[0003] Infections in animals and humans are a major cause of illness and death. In production animals such as poultry, livestock, and fish, infections can result in reduced growth rate, reduced feed utilization, and increased mortality. In humans and pet animals, infections, such as gastrointestinal and urinary tract infections, affect health, quality of life, and in severe cases, can cause death.

[0004] Bacterial infections in production animals have historically been treated with antibiotics, often administered prophylactically as growth promoters. While this approach is effective, it has raised concerns about antibiotic overuse, antimicrobial resistance, and antibiotic residues in meat products. The World Health Organization has emphasized that antibiotic overuse is a significant issue with potentially serious consequences for infection control that must be addressed.

[0005] Recurrent bacterial infections in humans, for example, of the gastrointestinal and urinary tract, are also treated with long-term antibiotic administration, resulting in deleterious effects on beneficial bacteria, an increased risk of antibiotic-associated diarrhea, and an increased risk of antibiotic-resistant infections such as Clostridioides difficile (C. difficile).

[0006] There is a clear need to develop alternatives to antibiotics for the prevention and treatment of infections in animals and humans so that antibiotics can be used to treat more serious cases. Various options are being considered, including: (i) compounds such as phages and quorum sensing mechanisms that may target microorganisms; (ii) compounds such as probiotics, prebiotics, polyphenols, and fatty acids that directly or indirectly support the immune system through their metabolites and help resist the harmful effects of pathogens; and (iii) Compounds such as probiotics and prebiotics, which may limit the growth of pathogenic bacteria by increasing the amount of beneficial bacteria and outcompeting pathogenic bacteria (Principi, N. et al., Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections, Front. Pharmacol., 08, May 2019; Jiang, Q. et al., Quorum Sensing: A Prospective Therapeutic Target for Bacterial Diseases, Biomed Res. 2019, April 4).

[0007] Common prebiotics include fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), and xylooligosaccharides (XOS). These prebiotics contain subunits composed of fructose, fructose, galactose, and xylose, respectively. The nature of the bonds between these subunits depends on their source and mode of production. The bond structure, along with the degree of polymerization, can have a substantial impact on interactions with bacteria through binding, transport, and / or metabolism.

[0008] Data from Makelainen et al. (Makelainen H. et al., Xylo-oligosaccharides and lactitol promote the growth of Bifidobacterium lactis and Lactobacillus species in pure cultures, Beneficial Microbes, 2010;1:139-148) show that conventional prebiotics promote the growth of common pathogens, including enterohemorrhagic Escherichia coli (EHEC), Salmonella typhimurium, Clostridium perfringens, and Staphylococcus epidermis (Figure 1). Makelainen showed that in the presence of GOS and FOS, pathogens significantly grew, whereas short-chain xylo-oligosaccharides (scXOS) and XOS with a degree of polymerization (DP) of 2–16 reduced growth. Pathogen growth clearly varies among species and there are significant differences among carbon sources, reflecting differences in the major sugar subunits (fructose, glucose, galactose, xylose), the type of linkage between subunits, and the degree of polymerization. However, of the pathogens studied, very few are inhibited by these prebiotics.

[0009] Mannan-derived oligosaccharides and polysaccharides have also been proposed as prebiotics, but unlike the prebiotics listed above, their use in humans is limited for reasons discussed below. Long-chain mannan oligosaccharides / polysaccharides are derived from yeast cell walls, and these oligosaccharides / polysaccharides are cross-linked to β-glucans and proteins. These yeast-derived long-chain mannan oligosaccharide / polysaccharide fractions contain α, 1-4 linkages and typically have a high degree of polymerization (DP) of 50–100. Although often referred to in the literature as mannan oligosaccharides or mannooligosaccharides (MOS), due to their high DP, they should strictly be defined as polysaccharides. Due to their high degree of polymerization, these mannan fractions are sparingly soluble in water. For example, AgriMOS has a solubility of 8% according to its product insert (Inhibitory effect against unwanted bacteria; Lallemand Animal Nutrition product insert; lallemandanimalnutrition.com). Yeast mannan-derived polysaccharides / oligosaccharides (α-MOS) are typically present in low purity; i.e., 10-30 wt% α-mannan, 10-30 wt% β-glucan, and up to 30% protein (Inhibitory effect against unwanted bacteria; Lallemand Animal Nutrition product insert; lallemandanimalnutrition.com). The low purity and complex bond structures of yeast-derived products make it difficult to assess their mechanism of action; this is because both α-MOS and β-glucan are said to have immune-boosting benefits (inhibitory effects against unwanted bacteria; Lallemand Animal Nutrition product insert; lallemandanimalnutrition.com). These yeast-derived "MOS" products have been used in poultry and livestock, with varying results, likely due to variations in composition and the complex crosslinks between α-mannan / α-MOS, β-glucan, protein, and other components (such as ash and other fiber).

[0010] These low-purity α-mannan / α-MOS products are used in poultry and livestock as natural growth promoters to support the immune system and reduce susceptibility to infections, such as those caused by Salmonella and E. coli. These benefits appear to be indirect; they result from the stimulation of beneficial bacteria by the α-MOS and β-glucan components, which stimulate the production of short-chain fatty acids and other metabolic products. They may also have a direct effect on pathogens by inhibiting type I fimbriae binding, which promotes adhesion to mannose-containing receptors (lectins) lining the gastrointestinal tract, urinary tract, and other tissues in animals and humans. Low-purity products containing β-glucan, α-mannan / α-MOS, and proteins have been suggested to promote microbial aggregation. However, the high degree of polymerization of α-mannan / α-MOS, cross-linking of α-mannan / α-MOS to β-glucan and protein, and low solubility (or insolubility) of α-mannan / α-MOS may affect the efficacy of these yeast-derived products in binding / aggregating pathogens, resulting in variable immune responses and health benefits. Furthermore, it is unclear whether the proposed benefits against specific pathogens are due to the α-mannan, β-glucan, or other components in these mixtures. Variations in yeast source, composition, and processing methods affect the α-mannan, β-glucan, and protein content of the oligosaccharides / polysaccharides, as well as the degree of polymerization (DP), which may contribute to variable results and may also contribute to the lack of efficacy of these products in certain applications. Existing data on MOS in animals is based on these low-purity α-mannan / α-MOS / β-glucan / protein products.

[0011] Ariandi et al. (Ariandi, Y., et al., Enzymatic Hydrolysis of Copra Meal by Mannanase from Streptomyces sp. BF3.1 for the Production of Mannooligosaccharides, HATAYI Journal of Biosciences, Vol. 22(2), pp. 79-86, 2015), Cuong et al. (Cuong, D.B., et al., Bioconversion of Copra Meal into Prebiotic Mannooligosaccharides Using Endo-B-1,4-Mannanase Producing By Aspergillus Niger Bk 01), Science and Technology In several studies, such as those by Rungrassamee et al. (Rungrassamee, W., et al., Mannooligosaccharides from copra meal improve survival of the Pacific white shrimp (Litopenaeus vannamei)), only low-purity β-MOS was prepared in the presence of other compounds. The effect of these other compounds on β-MOS utilization has not yet been evaluated.

[0012] Thus, there is an unmet need for compositions to effectively inhibit the growth of pathogenic bacteria in animals and humans, thereby reducing the use of antibiotics. Summary of the Invention

[0013] This application discloses a highly purified β-MOS composition for use in inhibiting the growth of pathogenic bacteria, improving survival and growth in production animals and aiding in the treatment of infections in humans, thereby reducing the use of antibiotics.

[0014] Accordingly, the present application includes a composition comprising mannooligosaccharide (MOS) carbohydrates for use in inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria in a subject, wherein at least 70% of the MOS carbohydrates are mannose subunits.

[0015] The present application also includes a combination of a mannooligosaccharide (MOS) carbohydrate composition and an antibiotic, wherein the efficacy of the antibiotic is increased by at least 30%.

[0016] The present application also includes a method for producing a MOS carbohydrate composition, comprising subjecting a mannan material to hydrolysis to obtain a crude extract, and purifying the crude extract to obtain a purified extract, wherein at least 70% wt of the MOS carbohydrate is mannose subunits.

[0017] Other features and advantages of the present application will become apparent from the following detailed description, but it should be understood that this detailed description and specific examples are provided by way of illustration and example only, and that the scope of the claims should not be limited to these embodiments, but should be accorded the broadest interpretation consistent with the overall description.

[0018] The embodiments of the present application are described in more detail below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0019] [Figure 1]Aggregate growth of selected pathogens when grown on glucose (control) and on selected prebiotics. Pathogen growth is shown as optical density (OD) and cumulative pathogen growth is shown as area under the curve (AUC) corresponding to OD x time. A large area under the curve indicates a high level of growth; a negative area under the curve suggests potential inhibition. [Figure 2] High-performance liquid chromatogram (HPLC) for the identification and quantification of carbohydrate components in an exemplary purified MOS extract solution from copra meal. [Figure 3] Inhibition of Salmonella enteritidis in the presence of exemplary β-mannooligosaccharides (β-MOS) derived from copra meal. The inhibition is measured by optical density (OD) at 630 nm every 2 hours for 24 hours for equivalent doses in the feed of 0.05 wt% to 0.25 wt%. [Figure 4] Delta optical density (OD600) values ​​versus compound concentration (mg / ml) for six dilutions of exemplary compound solutions of copra-MOS (CMOS), yeast-MOS (YMOS), and YMOS-NaOH measured at a wavelength of 600 nm for minimum inhibitory concentration (MIC) assessment in Vibrio parahaemolyticus. Statistical significance (p<0.05, growth inhibition >20%) is indicated with an asterisk (*). Negative controls were 0 mg / mL of compound in either sterile distilled water (CMOS, YMOS) or 0.4% in 1 M NaOH solution (YMOS-NaOH). Gray dashed lines separate each dilution group. The designations at the top of each section indicate the compound solutions. In each section, the first result represents the CMOS compound solution (C); the second result represents the YMOS compound solution (Y); and the third result represents the YMOS-NaOH (Y-NaOH) compound solution. [Figure 5] Inhibition of Piscirickettsia salmonis in the presence of exemplary β-mannooligosaccharides (C-MOS) derived from copra meal (C-MOS concentrations of 5.5, 11.1, 16.7, or 25 mg / ml). The inhibition was measured after 11 and 16 days of incubation. [Figure 6]Inhibition of P. salmonis in the presence of yeast MOS (Y-MOS) (Y-MOS concentrations of 5.5, 11.1, or 16.7 mg / ml). The inhibition was measured after 11 and 16 days of incubation. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition Unless otherwise indicated, the definitions and embodiments set forth in this and other sections are intended to apply to all embodiments and aspects of the present application as described herein for the purpose of aiding the understanding of those skilled in the art.

[0021] As used herein, the term "comprising" and its derivatives are intended to be open-ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other, unstated features, elements, components, groups, integers, and / or steps. This also applies to words of similar meaning, such as the terms "including," "having," and their derivatives.

[0022] As used herein, terms of degree such as "substantially," "about," and "approximately" refer to a reasonable amount of variation about the modified term without significantly altering the end result. These terms of degree should be considered to include a variation of at least ±5% in the modified term when such a variation does not negate the meaning of the modified term.

[0023] As used herein, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0024] In embodiments that include "additional" or "second" components, the second component herein is chemically distinct from the other components and the first component. A "third" component is distinct from the other components, the first component, and the second component, and further listed or "additional" components are similarly distinct.

[0025] As used herein, the term "and / or" means that the listed items are present or used either individually or in any combination. In practice, the term means the use or presence of "at least one of" or "one or more of" the listed items.

[0026] As used herein, the term "composition of the present application" refers to a composition comprising the mannooligosaccharide (MOS) carbohydrate of the present application.

[0027] As used herein, the term "combination composition of the present application" refers to a composition comprising the mannooligosaccharide (MOS) carbohydrate of the present application and an antibiotic.

[0028] As used herein, the term "the subject process" refers to a method for preparing the subject composition.

[0029] As used herein, the term "monosaccharide" refers to a simple sugar that forms the building block of more complex glycoforms such as oligosaccharides and polysaccharides.

[0030] As used herein, the term "polysaccharide" refers to a carbohydrate that occurs as a long chain made up of repeating monosaccharide units linked together by glycosidic bonds.

[0031] As used herein, the term "oligosaccharide" refers to a polymer of monosaccharides having a degree of polymerization (DP) of 2-10.

[0032] As used herein, the term "mannooligosaccharides (MOS)" refers to polysaccharides containing mannose monosaccharide residues. The mannose residues may be in the form of D-mannose, galactomannan, glucomannan, and mixtures thereof. Mannose-containing polysaccharides may be formed entirely of mannose subunits or may contain a combination of mannose monosaccharide residues and other monosaccharides (e.g., galactose, glucose, and fructose). Mannooligosaccharides may contain multiple oligosaccharides with different degrees of polymerization. The polysaccharides may be α-MOS or β-MOS.

[0033] The term "pathogenic bacteria" as used herein refers to bacteria that can cause disease in a subject. Examples of pathogenic bacteria include, but are not limited to, Vibrio, Tenacibaculum, Clostridia, Salmonella, Escherichia coli, and Piscirickettsia salmonis, or pathogenic bacteria that contain type I fimbriae.

[0034] As used herein, the term "good bacteria" refers to bacteria (such as multiple species of Lactobacillus and multiple species of Bifidobacteria) that are believed to provide health benefits.

[0035] The term "subject" as used herein refers to all members of the animal kingdom, and therefore, for purposes of this application, is applicable to both humans and animals.

[0036] The terms "treating" or "treatment," as used herein and as well understood in the art, refer to an approach to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, attenuation of disease extent, a stabilized (i.e., non-worsening) disease state, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of disease symptoms, reduction in disease recurrence, and remission (partial or complete). "Treating" and "treatment" can also mean prolonging survival as compared to expected survival if not treated. "Treating" and "treatment," as used herein, also include prophylactic treatment. Treatment involves administering a therapeutically effective amount of a composition or combination composition of the present application to a subject, optionally consisting of a single dose or including a series of multiple doses. For example, in some embodiments, the compositions or combination compositions of the present application may be administered at least once a week. In some embodiments, the compositions may be administered to a subject about once every three weeks, or about once a week to about once daily for a given treatment. In another embodiment, the compositions are administered two, three, four, five, or six times daily. The length of treatment depends on various factors, such as the severity of the disease, the disorder or condition, the age of the subject, the concentration and / or activity of the compositions of the present application, and / or combinations thereof. It will also be understood that the effective dosage of the compositions used for treatment may increase or decrease over the course of a particular treatment regimen. Dose modifications may be made or may be revealed by standard diagnostic assays known in the art. In some cases, long-term administration may be necessary. For example, the composition is administered to a subject in an amount for a period of time sufficient to treat the patient.

[0037] As used herein, the terms "prevention" or "prophylaxis," or equivalent terms thereto, refer to a reduction in the risk or probability that a patient will contract a disease, disorder, or condition that is mediated by a pathogenic bacterium, or that is treatable by the inhibition of a pathogenic bacterium, or that presents symptoms associated with a disease, disorder, or condition mediated by a pathogenic bacterium.

[0038] As used herein, the term "disease, disorder, or condition mediated by pathogenic bacteria" refers to a disease, disorder, or condition (such as a bacterial infection) that can be treated by inhibiting the activity of pathogenic bacteria or promoting the growth of beneficial bacteria.

[0039] As used herein, the term "to inhibit the growth of pathogenic bacteria" and variations thereof refers to the detectable inhibition of the growth of, or killing of, the pathogenic bacteria in the presence of a composition or combination composition of the present application compared to otherwise identical conditions except for the absence of the composition of the present application.

[0040] As used herein, the term "to promote the growth of beneficial bacteria" and variations thereof refers to a detectable promotion of beneficial bacterial growth in the presence of a composition or combination composition of the present application compared to otherwise identical conditions except for the absence of the composition of the present application.

[0041] As used herein, the term "effective amount" refers to an amount of a composition or combination of compositions effective to achieve a desired result. For example, in the case of inhibiting the growth of pathogenic bacteria while promoting the growth of beneficial bacteria, an effective amount is, for example, an amount that promotes the growth of the beneficial bacteria while promoting the inhibition of pathogenic bacteria, compared to otherwise identical conditions except for the absence of the composition.

[0042] As used herein, the term "degree of polymerization (DP)" refers to the number of monosaccharides that make up an oligosaccharide or polysaccharide. Thus, for example, a mannooligosaccharide, which is composed of four mannose monosaccharides, has a degree of polymerization of 4, and is therefore described as DP4.

[0043] As used herein, the terms "β-1,4-linked" and "α,1-4-linked" refer to the beta configuration in which oxygen is attached to the C1 carbon of one sugar ring structure and the C4 carbon of another sugar ring structure. The beta configuration differs from the alpha configuration in the position of the attached hydroxyl groups on the two sugar rings. In the beta-linked configuration, the C1 hydroxyl group is above the plane of the sugar ring, whereas in the alpha-linked configuration, the C1 hydroxyl group is below the plane of the sugar ring.

[0044] As used herein, the term "probiotic" refers to live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (internationalprobiotics.org).

[0045] As used herein, the term "prebiotic" refers to a substrate selectively utilized by a host microorganism that confers a health benefit (isappscience.org).

[0046] As used herein, the term "polyphenol" refers to plant-derived organic compounds that contain one or more phenolic groups.

[0047] As used herein, the term "phage" refers to a virus that infects bacteria and replicates within them, destroying them.

[0048] As used herein, the term "clays and minerals" refers to fine-grained earthen materials obtained by hydrolysis, usually from feldspar, to produce kaolinite and smectite.

[0049] As used herein, the term "antibiotic" refers to a drug that inhibits the growth of or destroys bacterial organisms.

[0050] The term "growth promoter" refers to a substance added to feed as a supplement or injection that improves animal feed utilization and animal growth.

[0051] As used herein, the term "short chain fatty acid" refers to a fatty acid having fewer than six carbon atoms. Examples include acetic acid, propionic acid, butyric acid, and the like.

[0052] As used herein, the term "mannan material" refers to a material containing mannans. Examples include, but are not limited to, palm and coconut processing residues, copra meal, coniferous wood (such as pine or spruce), coffee processing residues, and acai seeds and residues.

[0053] As used herein, the term "enzyme" refers to a protein that acts as a biological catalyst for a reaction.

[0054] As used herein, the term "TSB" refers to Tris-buffered saline.

[0055] As used herein, the term "CFS" refers to cell-free supernatant.

[0056] As used herein, the term "OD" refers to optical density.

[0057] As used herein, the term "CFU" refers to colony forming units / ml.

[0058] As used herein, the term "MA / MB medium" refers to marine agar / marine broth.

[0059] As used herein, the term "TSA2 / TSB2 medium" refers to tryptone soy agar / tryptone soy broth.

[0060] As used herein, the term "MIC" refers to minimum inhibitory concentration.

[0061] As used herein, the term "MLC" refers to the minimum lethal concentration.

[0062] As used herein, the term "FCR" refers to feed conversion ratio.

[0063] As used herein, the term "wt" refers to weight.

[0064] Composition of the present application The present application includes compositions comprising mannooligosaccharide (MOS) carbohydrates for use in inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria in a subject, wherein at least 70% by weight of the MOS carbohydrates are mannose subunits.

[0065] In some embodiments, the beneficial bacteria include, for example, multiple species of Lactobacillus and multiple species of Bifidobacterium, and in some embodiments, the composition promotes the growth of beneficial bacteria by at least 10%, at least 30%, at least 50%, or at least 75%, and values ​​therebetween.

[0066] In some embodiments, the MOS is derived from mannan material. In some embodiments, the mannan material is provided from a plant source, including, but not limited to, palm kernel cake, coconut residue, coniferous trees (such as pine or spruce), coffee processing residues, acai seeds and residues, copra meal, etc. Thus, in some embodiments, the MOS is derived from mannan material obtained from a plant source selected from palm kernel cake, coconut residue, coniferous trees (such as pine or spruce), coffee processing residues, acai seeds and residues, and copra meal. In some embodiments, the mannan material is obtained from copra meal.

[0067] In some embodiments, the degree of polymerization (DP) of the MOS is less than 20. In some embodiments, the DP of the MOS is less than 8, or between 1 and 8. In some embodiments, the DP is between 2 and 10. In some embodiments, the DP is between 2 and 6. In one embodiment, the DP is 2, 3, 4, 5, or 6. In one embodiment, the composition comprises mannooligosaccharides having different degrees of polymerization. For example, a portion of the MOS may have a DP of 2, while another portion of the MOS may have a DP of 4.

[0068] In some embodiments, MOS with a DP of 2 is present in the composition at a content of greater than 50 wt%. In some embodiments, MOS with a DP of 2 is present in the composition at a content of about 60 wt%, about 65 wt%, about 70 wt%, or about 80 wt%, and values ​​therebetween. In some embodiments, MOS derived from plant sources provides a high content of MOS with a DP of 2.

[0069] In some embodiments, at least 75% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 80% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 85% by weight, or at least 90% by weight, or at least 95% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 85% by weight of the MOS carbohydrates are mannose subunits.

[0070] In some embodiments, the present compositions have a water solubility of greater than 90% at 25° C. in a 15% to 25% by weight aqueous solution of the composition. In some embodiments, the water solubility of greater than 95% at 25° C. in a 15% to 25% by weight aqueous solution of the composition. In some embodiments, the water solubility of about 95% to about 100% at 25° C. in a 15% to 25% by weight aqueous solution of the composition. In some embodiments, the water solubility of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% at 25° C. in a 15% to 25% by weight aqueous solution of the composition. In some embodiments, the water solubility of about 100% at 25° C. in a 15% to 25% by weight aqueous solution of the composition. Methods for detecting and / or quantifying water solubility are known in the art. In some embodiments, the present compositions, in which MOS with a DP of 2 is present in an amount greater than 50 wt % of the composition, provide improved solubility of the composition.

[0071] In some embodiments, the mannose subunits contain primarily β-1,4 linkages. In some embodiments, the mannose subunits contain α,1-4 linkages. In some embodiments, the β-1,4 linkages of the mannose subunits are highly suitable for the utilization of beneficial bacteria. Thus, in some embodiments, the amount of beneficial bacteria increases, displacing pathogenic bacteria. The beneficial bacteria can be any beneficial bacteria known in the art, such as probiotics, including microorganisms of the genera Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus. In some embodiments, the beneficial bacteria can include microorganisms containing endo, β-1,4 mannanase.

[0072] In some embodiments, the composition further comprises at least one monosaccharide selected from the group consisting of glucose, galactose, xylose, arabinose, and combinations thereof.

[0073] In some embodiments, the glucose content is less than 10% by weight of the MOS. In some embodiments, the glucose content is less than 8% by weight of the MOS. In some embodiments, the glucose content is 3-7% by weight of the MOS. In some embodiments, the glucose content is less than 3% by weight of the MOS.

[0074] In some embodiments, the galactose content is less than 5% by weight of the MOS. In some embodiments, the galactose content is 1-3% by weight of the MOS. In some embodiments, the galactose content is less than 1% by weight of the MOS.

[0075] In some embodiments, the composition further comprises glucose and galactose, hi some embodiments, the total content of glucose and galactose is less than 10% wt. by weight of the MOS.

[0076] In some embodiments, the total monosaccharide content is less than 15% by weight of the MOS. In some embodiments, the total monosaccharide content is less than 13% by weight of the MOS. In some embodiments, the total monosaccharide content is less than 10% by weight of the MOS.

[0077] In some embodiments, the compositions herein are fructose-free.

[0078] In some embodiments, the composition further comprises β-glucan, wherein the β-glucan content is about 0.5% wt to about 5% wt of the MOS. In some embodiments, the composition further comprises β-glucan, wherein the β-glucan content is about 1%, about 2%, about 3%, or about 4%, and values ​​therebetween. In one embodiment, high-purity MOS, even at low levels of β-glucan, about 0.5% wt to about 5% wt, compared to yeast-derived MOS, which typically contains >20% wt of β-glucan (known to support immunity), has been shown to be effective at inhibiting pathogens and to be effective against a broad spectrum of pathogens (including those that do not rely on mannose-containing lectins or agglutination promotion).

[0079] In some embodiments, the composition further comprises a substance selected from the group consisting of probiotics, prebiotics, polyphenols, phages, clays and minerals (such as bentonite and montmorillonite), antibiotics, and short-chain fatty acids.

[0080] The probiotics include microorganisms of the genera Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus.

[0081] Such prebiotics include fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), and xylooligosaccharides (XOS).

[0082] Examples of polyphenols include, but are not limited to, flavonoids, lignans, stilbenes, and phenolic acids.

[0083] Those skilled in the art will understand that phages are specific to particular pathogens, and the selection of a particular phage is within the knowledge of one of ordinary skill in the art.

[0084] The antibiotic may include any antibiotic known in the art, including, but not limited to, penicillin, gentamicin, clindamycin, ceftazidime, and the like.

[0085] The short chain fatty acids include fatty acids having less than six carbon atoms. Examples of short chain fatty acids include, but are not limited to, acetic acid, propionic acid, and butyric acid.

[0086] In some embodiments, the composition is in the form of an aqueous solution or a powder.

[0087] In some embodiments, the aqueous solution or powder is obtained by hydrolysis and purification of α-mannan material.

[0088] In some embodiments, the composition is formulated for oral administration.

[0089] In some embodiments, the composition is formulated in the form of a supplement, food, drink, or feed additive.

[0090] In some embodiments, the composition is formulated in the form of a capsule, tablet, sachet, or liquid.

[0091] In some embodiments, a dose of the composition effective for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria in a subject is: (i) inhibit the growth of the target pathogen; (ii) is lethal to the target pathogen; (iii) promoting the growth of certain production animals in the poultry, livestock, and aquaculture sectors; (iv) increasing the survival rate of infected animals in the poultry, livestock, and aquaculture sectors; (v) alleviating the symptoms of infections in the oral cavity, gastrointestinal tract, and urinary tract in humans; The minimum dosage required; or any such use; or A benefit resulting from improving the immune response or resistance to the presence of or exposure to a pathogen; This is the minimum dose required.

[0092] In some embodiments, the subject is a human or animal.

[0093] In some embodiments, the pathogenic bacteria are selected from the group consisting of Vibrio, Tenacibaculum, Clostridia, Salmonella, Streptococcus, Aeromonas, Campylobacter, Bacillus, Klebsiella, Listeria, Shigella, Escherichia coli, and Piscirickettsia salmonis, or species of pathogenic bacteria containing type I pili.

[0094] In some embodiments, the pathogenic bacterial growth is inhibited by at least 20%, in some embodiments, the pathogenic bacterial growth is inhibited by at least 30%, in some embodiments, the pathogenic bacterial growth is inhibited by at least 40%, at least 50%, at least 75%, or at least 95%, and values ​​therebetween.

[0095] In some embodiments, the composition further comprises an antibiotic. In some embodiments, the MOS enhances the effectiveness of the antibiotic. In some embodiments, the MOS enhances the effectiveness of the antibiotic by at least 30%, at least 40%, at least 60%, and values ​​therebetween. In some embodiments, the antibiotic is selected from penicillins such as amoxicillin, gentamicin, clindamycin, kanamycin, tetracycline, erythromycin, ciprofloxacin, vancomycin, and ceftazidime. In some embodiments, the antibiotic is penicillin.

[0096] Examples of suitable Clostridium species include, but are not limited to, Clostridium perfringens, Clostridium tetani, Clostridium sordellii, and Clostridium botulinum. In some embodiments, the Clostridium species is Clostridium perfringens. In some embodiments, the growth of Clostridium perfringens is inhibited by at least 20%. In some embodiments, the growth of Clostridium perfringens is inhibited by at least 30%. In some embodiments, the growth of Clostridium perfringens is inhibited by at least 40%. In some embodiments, the growth of Clostridium perfringens is inhibited by at least 50%.

[0097] Examples of suitable Salmonella species include Salmonella enterica and Salmonella bongori. In some embodiments, the Salmonella species is Salmonella enteritidis. In some embodiments, the growth of Salmonella enteritidis is inhibited by at least 20%. In some embodiments, the growth of Salmonella enteritidis is inhibited by at least 30%. In some embodiments, the growth of Salmonella enteritidis is inhibited by at least 40%. In some embodiments, the growth of Salmonella enteritidis is inhibited by at least 50%.

[0098] Examples of suitable Tenacibaculum species include, but are not limited to, Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum discolor, Tenacibaculum gallaicum, and Tenacibaculum dicentrarchi. In some embodiments, the Tenacibaculum species is Tenacibaculum maritimum. In some embodiments, growth of Tenacibaculum maritimum is inhibited by at least 20%. In some embodiments, growth of Tenacibaculum maritimum is inhibited by at least 30%. In some embodiments, growth of Tenacibaculum maritimum is inhibited by at least 40%. In some embodiments, growth of Tenacibaculum maritimum is inhibited by at least 50%.

[0099] Examples of suitable Vibrio species include, but are not limited to, Vibrio parahaemolyticus, Vibrio aguillarum, and Vibrio harveyi. In some embodiments, the Vibrio species is Vibrio parahaemolyticus. In some embodiments, the growth of Vibrio parahaemolyticus is inhibited by at least 20%. In some embodiments, the growth of Vibrio parahaemolyticus is inhibited by at least 30%. In some embodiments, the growth of Vibrio parahaemolyticus is inhibited by at least 40%. In some embodiments, the growth of Vibrio parahaemolyticus is inhibited by at least 50%.

[0100] In some embodiments, the Vibrio species is Vibrio aguillarum. In some embodiments, the growth of Vibrio aguillarum is inhibited by at least 20%. In some embodiments, the growth of Vibrio aguillarum is inhibited by at least 30%. In some embodiments, the growth of Vibrio aguillarum is inhibited by at least 40%. In some embodiments, the growth of Vibrio aguillarum is inhibited by at least 50%.

[0101] In some embodiments, the Vibrio species is Vibrio harveyi. In some embodiments, growth of Vibrio harveyi is inhibited by at least 20%. In some embodiments, growth of Vibrio harveyi is inhibited by at least 30%. In some embodiments, growth of Vibrio harveyi is inhibited by at least 40%. In some embodiments, growth of Vibrio harveyi is inhibited by at least 50%.

[0102] In some embodiments, the growth of Piscirickettsia salmonis is inhibited by at least 20%. In some embodiments, the growth of Piscirickettsia salmonis is inhibited by at least 30%. In some embodiments, the growth of Piscirickettsia salmonis is inhibited by at least 40%. In some embodiments, the growth of Piscirickettsia salmonis is inhibited by at least 50%. In some embodiments, the growth of Piscirickettsia salmonis is inhibited by at least 70%, at least 80%, at least 90%, or about 100%, and values ​​therebetween.

[0103] Examples of suitable Streptococcus species include, but are not limited to, mutans, angiosus, pyogenes, agalactiae, and dysgalactieae. In some embodiments, the Streptococcus species is Streptococcus mutans. In some embodiments, the growth of Streptococcus mutans is inhibited by at least 20%. In some embodiments, the growth of Streptococcus mutans is inhibited by at least 30%. In some embodiments, the growth of Streptococcus mutans is inhibited by at least 40%. In some embodiments, the growth of Streptococcus mutans is inhibited by at least 50%.

[0104] In some embodiments, growth of Escherichia coli is inhibited by at least 20%. In some embodiments, growth of Escherichia coli is inhibited by at least 30%. In some embodiments, growth of Escherichia coli is inhibited by at least 40%. In some embodiments, growth of Escherichia coli is inhibited by at least 50%.

[0105] Examples of suitable Listeria species include, but are not limited to, Listeria monocytogenes, Listeria aquatica, and Listeria seeligeri. In some embodiments, the Listeria species is Listeria monocytogenes. In some embodiments, growth of Listeria monocytogenes is inhibited by at least 20%. In some embodiments, growth of Listeria monocytogenes is inhibited by at least 30%. In some embodiments, growth of Listeria monocytogenes is inhibited by at least 40%. In some embodiments, growth of Listeria monocytogenes is inhibited by at least 50%.

[0106] Examples of suitable Staphylococcus species include, but are not limited to, aureus, auricularis, borealis, caprae, cohnii, devriesei, gallinarum, hyicus, lentus, and sciuri.

[0107] Examples of suitable Aeromonas species include, but are not limited to, hydrophila, caviae, salmonocida, and veronii.

[0108] Examples of suitable Campylobacter species include, but are not limited to, Campylobacter jejuni, coli, upsaliensis, fetus venerealis, and lari.

[0109] Examples of suitable Bacillus species include, but are not limited to, cereus, subtills, anthacis, and licheniformis.

[0110] Examples of suitable Klebsiella species include Klebsiella pneumoniae.

[0111] Examples of suitable Shigella species include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella dysenteriae.

[0112] Examples of suitable pathogenic bacteria with type I pili include, but are not limited to, Neisseria and Actinomyces.

[0113] Those skilled in the art will understand that the duration and degree of inhibition of a pathogen will depend on various factors, such as the dose of the composition, the formulation of the composition, the frequency of administration, the characteristics of the pathogen (such as its doubling time), and others.

[0114] In some embodiments, when the composition of the present application is administered to an animal, the composition affects the growth performance of the animal. In some embodiments, the growth performance includes at least one of growth rate and feed conversion ratio (FCR). In some embodiments, the composition increases growth rate. In some embodiments, the composition decreases FCR.

[0115] In some embodiments, the compositions increase the activity of one or more of the IFN-γ, hepcidin, and TLR-9 genes, and thus, by upregulating IFN-γ, the compositions prevent infection, and by upregulating hepcidin and TLR-9, the compositions help support a cellular immune response to reduce the replication of pathogenic bacteria.

[0116] In some embodiments, the compositions of the present application can be used as vaccine adjuvants.

[0117] The present application also includes a combination of a mannooligosaccharide (MOS) carbohydrate composition and an antibiotic, wherein the effectiveness of the antibiotic is increased by at least 30%.

[0118] In some embodiments, the combination provides a synergistic effect in improving the performance of the antibiotic, ie, the antibiotic's effectiveness is increased by at least 40%, at least 50%, at least 60%, at least 70%, and values ​​therebetween.

[0119] The amount of MOS and the ratio of MOS to antibiotic will vary depending on the antibiotic and its molecular weight. In some embodiments, the MOS content in the combination is about 1 wt% to about 25 wt%. In some embodiments, the MOS content in the combination is about 1 wt%, about 2 wt%, about 3 wt%, about 5 wt%, about 10 wt%, or about 25 wt%, and values ​​therebetween. The MOS in the composition improves the efficacy of the antibiotic.

[0120] In some embodiments, the MOS composition comprises mannooligosaccharide (MOS) carbohydrates, wherein at least 70% by weight of the MOS carbohydrates are mannose subunits.

[0121] In some embodiments, at least 75% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 80% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 85% by weight, or at least 90% by weight, or at least 95% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 85% by weight of the MOS carbohydrates are mannose subunits.

[0122] In some embodiments, the MOS is derived from mannan material obtained from a plant source selected from palm kernel cake, coconut residue, coniferous trees (such as pine or spruce), coffee processing residues, acai seeds and residues, and copra meal. In some embodiments, the mannan material is obtained from copra meal.

[0123] The antibiotic may include any antibiotic known in the art. Examples of antibiotics include, but are not limited to, penicillin, gentamicin, clindamycin, ceftazidime, etc. Thus, in some embodiments, the antibiotic is selected from penicillin such as amoxicillin, gentamicin, clindamycin, kanamycin, tetracycline, erythromycin, ciprofloxacin, vancomycin, and ceftazidime. In some embodiments, the antibiotic is penicillin.

[0124] In some embodiments, the degree of polymerization (DP) of the MOS is less than 20. In some embodiments, the DP of the MOS is less than 8 or from 1 to 8. In some embodiments, the DP is from 2 to 10. In some embodiments, the DP is from 2 to 6.

[0125] In some embodiments, the DP 2 MOS is present in the composition in an amount greater than 50 wt%. In some embodiments, the DP 2 MOS is present in the composition in an amount greater than about 60 wt%, about 65 wt%, about 70 wt%, or about 80 wt%, and values ​​therebetween.

[0126] In some embodiments, the combination composition is in the form of an aqueous solution or a powder.

[0127] In some embodiments, the aqueous solution or powder is obtained by hydrolysis and purification of α-mannan material and addition of the antibiotic.

[0128] In some embodiments, the combination composition is formulated for oral administration.

[0129] In some embodiments, the combination composition is formulated in the form of a supplement, food, drink, or feed additive.

[0130] In some embodiments, the combination composition is formulated in the form of a capsule, tablet, sachet, or liquid.

[0131] In some embodiments, the subject is a human or animal.

[0132] In some embodiments, the pathogenic bacterium is selected from the group consisting of Vibrio, Tenacibaculum, Clostridia, Salmonella, Streptococcus, Aeromonas, Campylobacter, Bacillus, Klebsiella, Listeria, Shigella, Escherichia coli, and Piscirickettsia salmonis, or a pathogenic bacterial species containing type I fimbriae. In some embodiments, the pathogenic bacterium is selected from a Streptococcus species. In some embodiments, the pathogenic bacterium is Streptococcus mutans.

[0133] The present application also includes supplements, foods, beverages, or feeds that contain the combination compositions of the present application.

[0134] The present application further includes capsules, tablets, sachets or liquids containing the combination compositions of the present application.

[0135] In some embodiments, the filler of the composition or combination composition is less than 1000 mg per 100 grams of product (such as supplement, food, drink, feed, etc.) in the form of a formulation (such as a tablet, sachet, or liquid).In some embodiments, the filler is less than 800 mg per 100 grams, or less than 600 mg per 100 grams.The filler may vary depending on factors such as the formulation, the subject being treated, the age and sensitivity of the subject, and the optimization of the product; the filler of the composition or combination composition of the present application is within the skill of those skilled in the art.

[0136] Supplements, foods, beverages, feeds, or capsules, tablets, sachets, or liquids containing the compositions or combination compositions of the present application can be administered at least once a week, about once every three weeks, or about once a week to about once daily for a given treatment. In some embodiments, supplements, foods, beverages, feeds, or capsules, tablets, sachets, or liquids containing the compositions or combination compositions of the present application can be administered two, three, four, five, or six times daily.

[0137] The method of the present application The present application also includes a method for producing a MOS carbohydrate composition, the method comprising subjecting a mannan material to hydrolysis to obtain a crude extract, and purifying the crude extract to obtain a purified extract, wherein at least 70% by weight of the MOS carbohydrate is mannose subunits.

[0138] In some embodiments, the mannan material is obtained from a plant source containing β-mannans, including, but not limited to, palm kernel cake, coconut residue, coniferous trees (such as pine or spruce), coffee processing residues, acai seeds and residues, and copra meal. Thus, in some embodiments, the MOS is derived from mannan material obtained from a plant source selected from palm kernel cake, coconut residue, coniferous trees (such as pine or spruce), coffee processing residues, acai seeds and residues, and copra meal. In some embodiments, the mannan material is obtained from copra meal.

[0139] In some embodiments, the mannan material and the enzyme mixture are hydrolyzed at a concentration of about 5 to about 30% w / v. In some embodiments, the mannan material and the enzyme mixture are hydrolyzed at a concentration of about 10% w / v, about 15% w / v, about 20% w / v, about 25% w / v, or about 30% w / v. In some embodiments, the mannan material and the enzyme mixture are hydrolyzed at a concentration of about 15% w / v.

[0140] The mannan material is subjected to hydrolysis using a hydrolysis method such as acid hydrolysis, thermal hydrolysis, enzymatic hydrolysis, microbial fermentation hydrolysis, and combinations of such methods. Thermal hydrolysis may be carried out at a temperature of about 150° C. to about 220° C. In some embodiments, the hydrolysis is enzymatic hydrolysis.

[0141] Hydrolysis of mannan material produces short-chain mannooligosaccharides (which are further broken down into monosaccharides). Therefore, any enzyme capable of hydrolyzing mannans can be used in the enzymatic hydrolysis. Examples of such enzymes include, but are not limited to, β-mannanase, β-mannosidase, β-glucosidase, β-glucanase, β-xylosidase, endo- or exo-xylanase, and combinations thereof. It will be understood that the concentration of the enzyme is affected by its specific activity and purity, with enzymes having higher intrinsic / specific activity and / or purity requiring lower dosages / concentrations, while enzymes with lower specific activity and / or purity require higher dosages / concentrations. Selecting an enzyme concentration based on its specific activity and purity is within the skill of one of ordinary skill in the art. If a low concentration is selected for the hydrolysis, further purification of the MOS may be required.

[0142] In some embodiments, the enzyme is a mixture of β-mannanase enzyme and β-mannosidase enzyme. In some embodiments, the enzyme concentration is about 0.01 to about 0.5% w / v. In some embodiments, the enzyme concentration is about 0.1% w / v.

[0143] In some embodiments, the hydrolysis is carried out at a temperature of about 40°C to about 70°C. In some embodiments, the hydrolysis is carried out at a temperature of about 50°C to about 60°C. In some embodiments, the hydrolysis is carried out at a temperature of about 60°C. As will be appreciated by those skilled in the art, the reaction time depends on the reaction temperature and enzyme concentration / activity, with higher temperatures and higher enzyme concentrations / activities being preferred for more rapid reactions. In some embodiments, the hydrolysis reaction time is about 2 hours to about 12 hours, about 4 hours to about 10 hours, or about 6 hours to about 9 hours.

[0144] In some embodiments, the crude extract obtained contains at least one monosaccharide selected from the group consisting of mannose, glucose, xylose, arabinose, and galactose, as well as oligosaccharides, proteins, polyphenols, and lignin-derived compounds, fats / oils, ash, and extractives. In some embodiments, the composition does not contain fructose.

[0145] In some embodiments, the crude extract is purified by any method known in the art, such as centrifugation, filtration, extraction, absorption, ion exchange, and chromatographic separation. In some embodiments, the crude extract is purified by filtration. In some embodiments, the purified extract is further purified by cooling the crude extract to a temperature below 40°C and decanting off the fat layer to obtain a fat-free extract.

[0146] In some embodiments, the fat-free extract is further purified to remove high molecular weight fractions, low molecular weight fractions and polyphenols.This purification can be carried out by any method known in the art.In some embodiments, the fat-free extract is purified by ultrafiltration, and then a nanofiltration step is carried out in diafiltration mode to obtain a purified extract.Diafiltration mode refers to the addition of water to improve recovery.Various methods for this purification method can be used, and are within the discretion of those skilled in the art.

[0147] In some embodiments, the purified extract is concentrated using any method known in the art for obtaining a purified, concentrated extract (e.g., evaporation and reverse osmosis) to obtain the MOS composition in the form of an aqueous solution or powder. In some embodiments, the extract is concentrated using a multi-effect evaporator.

[0148] In some embodiments, when the MOS composition is in the form of an aqueous solution, the extract may be concentrated to at least 15% wt soluble solids, hi some embodiments, the extract may be concentrated to at least 20% wt, at least 30%, at least 40%, or at least 50% soluble solids.

[0149] In some embodiments, the resulting concentrated purified extract is used as is; alternatively, it is dried into a powder form by any method known in the art, including, for example, refractance window drying, freeze drying, spray drying, fluidized bed drying, and the like.

[0150] In some embodiments, the degree of polymerization (DP) of the MOS is less than 20. In some embodiments, the DP of the MOS is less than 8. In some embodiments, the DP is 2-10. In some embodiments, the DP is 2-6.

[0151] In some embodiments, the MOS with a DP of 2 is present in the composition at a content of greater than 50 wt%. In some embodiments, the MOS with a DP of 2 is present in the composition at a content of about 60 wt%, about 65 wt%, about 70 wt%, or about 80 wt%, and values ​​therebetween. In some embodiments, the methods of the present application provide MOS carbohydrates that contain a high content of MOS with a DP of 2.

[0152] In some embodiments, at least 75% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 80% by weight of the MOS carbohydrates are mannose subunits. In some embodiments, at least 85% by weight of the MOS carbohydrates are mannose subunits.

[0153] In some embodiments, a 15 wt% to 25 wt% aqueous solution of the present composition at 25°C has a water solubility of greater than 90%. In some embodiments, a 15 wt% to 25 wt% aqueous solution of the composition at 25°C has a water solubility of greater than 95%. In some embodiments, a 15 wt% to 25 wt% aqueous solution of the composition at 25°C has a water solubility of about 95% to about 100%. In some embodiments, a 15 wt% to 25 wt% aqueous solution of the composition at 25°C has a water solubility of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, a 15 wt% to 25 wt% aqueous solution of the composition at 25°C has a water solubility of about 100%. Methods for detecting and / or quantifying water solubility are known in the art. In some embodiments, when the MOS having a DP of 2 is present in an amount greater than 50 wt % in the present composition, the solubility of the composition is improved.

[0154] In some embodiments, the mannose subunits mainly contain β-1,4 linkages. In some embodiments, the β-1,4 linkages of the mannose subunits are highly suitable for utilization by beneficial bacteria. Therefore, in some embodiments, the amount of beneficial bacteria increases, and pathogenic bacteria may be eliminated. The beneficial bacteria may be any beneficial bacteria known in the art, such as probiotics, including microorganisms of the genus Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus. In some embodiments, the beneficial bacteria may include microorganisms containing endo-β-1,4 mannanase.

[0155] In some embodiments, the composition further comprises at least one monosaccharide selected from the group consisting of glucose, galactose, xylose, arabinose, and combinations thereof, hi some embodiments, the composition does not comprise fructose.

[0156] In some embodiments, the glucose content is less than 10% by weight of the MOS. In some embodiments, the glucose content is less than 8% by weight of the MOS. In some embodiments, the glucose content is 3-7% by weight of the MOS. In some embodiments, the glucose content is less than 3% by weight of the MOS.

[0157] In some embodiments, the galactose content is less than 5% by weight of the MOS. In some embodiments, the galactose content is 1-3% by weight of the MOS. In some embodiments, the galactose content is less than 1% by weight of the MOS.

[0158] In some embodiments, the composition further comprises glucose and galactose, hi some embodiments, the total content of glucose and galactose is less than 10% by weight of the MOS.

[0159] In some embodiments, the total monosaccharide content is less than 15% by weight of the MOS. In some embodiments, the total monosaccharide content is less than 13% by weight of the MOS. In some embodiments, the total monosaccharide content is less than 10% by weight of the MOS.

[0160] In some embodiments, the composition further comprises β-glucan, wherein the β-glucan content is about 0.5% wt to about 5% wt of the MOS. In some embodiments, the composition further comprises β-glucan, wherein the β-glucan content is about 1%, about 2%, about 3%, or about 4% of the MOS, and values ​​therebetween. The following examples are illustrative of the present invention. These examples should not be construed as limiting the present invention, but rather as illustrative of the present invention, its beneficial properties, and specific embodiments.

[0161] Example General methods and materials Carbohydrates were analyzed using high-performance liquid chromatography (HPLC) with refractive index detection. NREL Laboratory Analytical Procedures (LAPs) were used to measure structural carbohydrates, lignin, ash, and extractives (NREL TPs 510-42622, 510-42625, 510-42619, 510-42618, 510-42623).

[0162] Example 1: Production of high-purity β-MOS Copra meal was hydrolyzed at a concentration of 15% (w / v) in a mixture of β-mannanase and β-mannosidase enzymes (0.1% (w / v)). The slurry was incubated at 60°C for 8 hours to obtain a hydrolysate. The resulting hydrolysate contained unconverted solids, mannose, short- and medium-chain mannooligosaccharides, other carbohydrates (as monomers and oligosaccharides), oils / fats, soluble polyphenols, ash, and other extractives.

[0163] The liquid was recovered from the hydrolysate by a series of solid filtration steps; a clear liquid was obtained. The extract was cooled to 20°C and the fat layer was decanted to remove residual oil.

[0164] The fat-free extract was processed through a series of steps consisting of ultrafiltration (4 kDa molecular weight cut-off (MWCO)) and nanofiltration (450 kDa MWCO) in diafiltration mode to remove high and low molecular weight fractions and polyphenols, and the nanofiltration retentate was concentrated to 18 wt% soluble solids before spray drying. Alternatively, the purified extract may be concentrated to at least 50% by evaporation (e.g., using a multiple-effect evaporator).

[0165] The purified extract was analyzed by HPLC with refractive index detection to determine the DP of the carbohydrates. As can be seen in Figure 2, over 60% of the mannooligosaccharides exhibited a DP of 2 to 6. Mannose elutes at approximately 101 min. The large peak at 63 min and the smaller peak at 68 min represent other oligosaccharides with higher degrees of polymerization; they may be mannooligosaccharides ranging from DP7 to DP10.

[0166] The solubility of the dry powder was at least 20 g in 100 g of water at 25°C.

[0167] Subsequent studies to evaluate the effectiveness of the products used these concentrated liquids or dry powders (less than 5% moisture content).

[0168] Example 2: In vitro studies evaluating the inhibition of Salmonella enteritidis (SE) A 100 μL aliquot of Tris-buffered saline (TSB) containing SE (1.0E+06 CFU (colony forming units) / mL) and a 100 μL aliquot of cell-free supernatant (CFS) were dispensed into individual microtiter plate wells.

[0169] Two types of controls were evaluated: (1) 100 μL of SE (1.0E+06 CFU / mL) and 100 μL of sterile medium (TSB; tryptic soy broth) (positive control); (2) 100 μL of sterile medium and 100 μL of sterile 0.85% saline (medium control).

[0170] High-purity β-MOS dry powder was added to the medium in the treatment plates at doses ranging from 0.05 wt% to 0.25 wt% (doses comparable to those in the diet). Optical density (OD) measurements were obtained by measuring the microtiter plates at 630 nm every 2 hours for 24 hours while maintaining the temperature at 37°C ± 2°C. The results shown in Figure 3 represent the average OD readings of triplicate microtiter wells.

[0171] When the present copra meal-derived, highly purified β-MOS was added to the culture medium, the growth of Salmonella enteritidis was consistently reduced (i.e., inhibited) by 29% at 6 hours, 30% at 8 hours, and 32% at 24 hours. No clear dose-dependence was observed.

[0172] Example 3: In vitro studies evaluating the inhibition of Clostridium perfringens (CP) A 100 μL aliquot of CP in thioglycollate containing beef extract (1.0E+08 CFU (colony forming units) / mL) and a 100 μL aliquot of cell-free supernatant (CFS) were dispensed into individual microtiter plate wells.

[0173] Two types of controls were evaluated: (1) 100 μL of CP (1.0E+08 CFU / mL) and 100 μL of sterile medium (positive control) (2) 100 μL of sterile medium and 100 μL of sterile 0.85% saline (medium control)

[0174] Dry powders of our high-purity β-copra MOS and yeast-"MOS" were added to the medium in the treatment plates at doses ranging from 0.05 wt% to 0.25 wt% (doses comparable to those in feed). The yeast-"MOS" remained in suspension, consistent with the yeast-MOS product specifications (inhibitory effect against undesirable bacteria; Lallemand Animal Nutrition product insert; lallemandanimalnutrition.com). Due to the low solubility of yeast-"MOS," it was difficult to accurately distinguish cells from particles. To obtain optical density (OD) measurements, microtiter plates were measured at 630 nm at 0 and 18 h under anaerobic conditions, maintaining the temperature at 37°C ± 2°C. Results represent the average OD readings of triplicate microtiter wells.

[0175] When our highly purified β-MOS derived from copra meal was added to the medium, CP growth was reduced (i.e., inhibited) by 42% after 18 hours. No clear dose-dependence was observed. Addition of yeast-"MOS" resulted in particulates / precipitation in the medium; OD was lower at 18 hours compared to the positive control, but it is unclear whether this was due to differences in growth or particulates from the additive.

[0176] Example 4: In vitro studies evaluating the inhibition of Vibrio parahaemolyticus and Tenacibaculum maritimum using yeast-"MOS" (YMOS) and highly purified copra MOS (CMOS) Powdered CMOS and YMOS were added to distilled water to prepare a 400 mg / mL stock solution for solubility assessment, including post-treatment by centrifugation at 5,000 g for 5 minutes. Because YMOS was found to be insoluble, an additional test group was added to the study: YMOS partially dissolved in 0.8% 1 M NaOH solution (YMOS NaOH). Test concentrations of each solution were prepared by serial dilution, ranging from 0.21 to 50 mg / mL.

[0177] Tests for Tenacibaculum maritimum were performed over 7–10 days in marine agar / marine broth (MA / MB) at 15°C. Tests for Vibrio parahaemolyticus were performed over 24 hours in tryptone soy agar / tryptone soy broth (TSA2 / TSB2) at 37°C.

[0178] Broth cultures were diluted 1:50 with the respective broth medium, mixed, and dispensed into 96-well plates with a 100 μL pipette. Compound solutions were added in quadruplicate at 2x the final concentration to achieve the desired effective concentration. Each well contained a 1:1 mixture of compound and pathogen and an effective concentration of compound in a total volume of 200 μL of 1:100 diluted pathogen. Negative controls (no product) and blank controls (no pathogen) were prepared. Optical density (OD) at 600 nm was measured, and the OD change was calculated. The initial bacterial concentration was quantified by adding diluted broth onto the agar of the plate.

[0179] The OD data were used to calculate the minimum inhibitory concentration (MIC), which represents the lowest concentration of product that results in a statistically significant decrease in OD compared to the negative control (no product); the MIC also inhibits pathogen growth by at least 20%.

[0180] Growth on agar plates was quantified to assess the minimum lethal concentration (MLC), which represents the lowest concentration of product that stops bacterial growth on agar plates.

[0181] Figure 4 shows the change in optical density (OD) of Vibrio parahaemolyticus for each of the different concentrations of compound solutions. Only the CMOS product produced a statistically significant reduction in Vibrio parahaemolyticus growth at 5.55 mg / mL, 16.67 mg / mL, and 50 mg / mL. Table 1 shows the data on the MIC and MLC of the products.

[0182] [Table 1]

[0183] Evaluation of the optical density (OD) change in Tenacibaculum maritimum for each product revealed that Y-MOS NaOH promoted Tenacibaculum maritimum growth, contrary to the desired / intended effect. This result may be due to impurities in the α-mannan / α-MOS compound "solution." The lower MIC values ​​for the highly purified β-MOS (CMOS) indicate that it is more effective against Tenacibaculum maritimum than the less purified α-mannan / α-MOS product derived from yeast (YMOS). MLC values ​​were comparable. Solubilizing the α-mannan / α-MOS product with NaOH did not improve the MIC and MLC values; in fact, this solubilization resulted in a worsening of the MLC value compared to the MLC values ​​for CMOS and YMOS. Therefore, while solubility may be a contributing factor to the effectiveness of CMOS, enhancing the solubility of YMOS did not improve its performance.

[0184] The lower MIC and MLC values ​​for highly purified β-MOS (CMOS) indicate that CMOS is more effective against Vibrio parahaemolyticus than a low-purity yeast-derived α-mannan / α-MOS product (YMOS). Indeed, even a concentration of 50 mg / ml of YMOS (nearly 10-fold higher than the MIC of CMOS) failed to inhibit the growth of this pathogen. By comparison, the difference in purity and MOS / mannan content between CMOS and YMOS is only 2.5-3-fold. Furthermore, β-glucans have been reported to have biological properties and potential antimicrobial activity, and efficacy has been observed in CMOS products despite the absence of β-glucans [Amer EM, Enhancement of β-Glucan Biological Activity Using a Modified Acid-Base Extraction Method from Saccharomyces cerevisiae, Molecules, 2021, Vol. 26(8), 2113].

[0185] Example 5: In vitro studies evaluating the inhibition of Vibrio anguillarum using yeast-"MOS" (YMOS) and highly purified copra MOS (CMOS) In this example, the method was similar to that of Example 4, using CMOS and YMOS incubated with Vibrio anguillarum at 20° C. for 24 hours in TSA2 / TSB2 medium.

[0186] Both CMOS and YMOS were effective at inhibiting the growth of Vibrio anguillarum at 50 mg / mL; CMOS inhibited growth by 49% and YMOS inhibited growth by 32%.

[0187] Example 6: In vitro studies evaluating the inhibition of Piscirickettsia salmonis Atlantic salmon kidney (ASK) cells were seeded in sterile L15 medium (15% fetal bovine serum, 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)) and grown to confluence. The pH of the medium was adjusted to a range of 7.0–7.6 by adding 1 M sodium hydroxide and 1 M acetic acid. Two products (copra-MOS (CMOS) and yeast-MOS (YMOS)) were added at the indicated amounts. A product-free control was also prepared. ASK cells were monitored daily to assess potential cytotoxicity and measured as the mean cytopathic effect (CPE).

[0188] YMOS was toxic to kidney (ASK) cells at concentrations of 5.55 mg / mL and above, causing cell fragmentation and detachment. CMOS was less toxic at a dose of 50 mg / mL and showed no adverse effects at concentrations of 1.11, 5.55, and 16.67 mg / mL. This indicates a clear difference in safety between high-purity soluble β-MOS preparations and low-purity insoluble α-MOS preparations (containing β-glucan, protein, and other components).

[0189] In parallel experiments, ASK cells were grown as described above. P. salmonis was diluted in L15 medium and added to test wells along with the ASK cells. Wells contained CMOS, YMOS, or a MOS-free control. ASK cells were monitored to assess the cytopathic effect induced by P. salmonis in the presence of YMOS or CMOS, or in the MOS-free control. Because YMOS is toxic to ASK cells as described above, YMOS was tested only at 1.85 mg / mL; CMOS was tested at 0.67, 1.85, 5.55, 16.67, and 50 mg / mL.

[0190] In ASK cells after 19 days, 1.85 mg / mL of YMOS had no appreciable effect on P. salmonis infection.

[0191] After 19 days, 16.67 mg / mL of CMOS reduced the cytopathic effect of P. salmonis by 51%. CMOS concentrations of 0.67 to 5.55 mg / mL had no appreciable effect on P. salmonis progression (Table 2). At 50 mg / mL, CMOS was cytotoxic to ASK cells, so the results for CMOS at this concentration were equivocal.

[0192] [Table 2]

[0193] Example 7: In vivo study to evaluate shrimp survival after exposure to Vibrio parahaemolyticus A concentrated liquid preparation of copra-derived β-MOS (CMOS) was used in a Vibrio challenge study in white-legged shrimp, P. vannamei. The liquid preparation was added to feed pellets as a surface coating at target doses of 0.25 and 0.50 wt% of the diet.

[0194] Four different groups: (i) a negative control group not exposed to Vibrio; (ii) a positive control group exposed to Vibrio but not treated with CMOS; and (iii) two groups treated with CMOS, i.e., nominal doses of "0.25 wt%" and "0.50 wt%"; For each of the five tanks, 12 shrimp were allocated.

[0195] In the other three groups, excluding the negative control group, shrimp were exposed to Vibrio paramemolyticus daily for eight days: a low dose for the first three days, then doubled on days four and five, and increased again by another 2.5-fold on day six. The survival of shrimp in each group was monitored daily.

[0196] Data on the mean survival rate of shrimp receiving CMOS compared to a positive control group exposed to Vibrio but not receiving MOS, and a negative control group not exposed to Vibrio, are shown in Table 3.

[0197] [Table 3]

[0198] The survival data in Table 3 show that survival rates were significantly improved in shrimp fed CMOS after exposure to Vibrio parahaemolyticus, a pathogen of importance to the aquaculture industry.

[0199] In comparison, Rungrassamee et al., "Mannooligosaccharides from copra meal improves survival of the Pacific white shrimp (Litopenaeus vannamei)," observed a dose-dependent modest increase in survival ranging from approximately 40% to 50-60% when vibrio challenge studies were performed using low-purity MOS. Similarly, Cuong et al., "Bioconversion of Copra Meal into Prebiotic Mannooligosaccharides Using Endo-β-1,4-Mannanase Producing By Aspergillus Niger Bk 01," Science and Technology Journal, Vol. 48(3), pp. 43-49, 2010, observed a 70%-97% increase in viability using a weaker vibrio challenge test; their crude MOS preparation required a much higher dose (i.e., 1 wt%) compared to a dose as low as 0.25 wt% in the test shown in Table 3 above.

[0200] Example 8: In vivo test to evaluate shrimp growth A concentrated liquid preparation of copra-derived β-MOS (CMOS) and a powder preparation of yeast-derived impure β-MOS (YMOS) were used for growth studies of vannamei shrimp. The preparations were added to shrimp feed pellets by surface coating. The target doses of CMOS were 0.25 wt% and 0.50 wt% of the diet, and of YMOS was 0.25 wt%.

[0201] Five tanks containing 15 shrimp each were set up for each group. The shrimp were fed either the MOS preparation outlined above or a MOS-free diet. Shrimp weight and growth rate were measured over an 8-week period. Feed conversion ratios were measured at week 4. Growth rates were calculated based on measurements from weeks 0-4, 0-6, and 2-8. The latter calculation excluded the first 2 weeks of growth data to ensure the shrimp were in a linear growth phase.

[0202] All tanks were placed in the same recirculating aquaculture system (RAS) that controlled nitrification, carbon dioxide and oxygen content, and solids removal. Heaters were used to maintain the temperature consistent with commercial operation.

[0203] Summary data on growth rate and feed conversion ratio (FCR) for shrimp receiving different types of MOS are shown in Table 4.

[0204] [Table 4]

[0205] The study data in Table 4 demonstrate consistently superior growth rates for shrimp fed CMOS compared to shrimp fed YMOS, as well as superior growth rates for shrimp fed 0.50% CMOS compared to shrimp fed 0.25% CMOS. While the difference in growth rate for CMOS relative to YMOS is evident throughout the study, the difference between the 0.50 wt% CMOS and 0.25 wt% CMOS groups only becomes apparent after approximately 6 weeks; when the 7- and 8-week data are considered, the difference widens (average growth rates from weeks 2 to 8).

[0206] Feed conversion ratio (FCR) data at week 4 also show that CMOS is superior when comparing shrimp fed CMOS to shrimp fed YMOS (lower FCR is preferred, i.e., less feed consumed per unit weight gain by the animal).

[0207] Example 9: Lactic acid bacteria growth test Growth of L. rhamnosus GG (LGG) was evaluated using various concentrations of highly purified copra MOS (CMOS), yeast MOS (YMOS), FOS, inulin, XOS, and glucose (positive control). A total of 100 μl of broth was added to each well; i.e., 90 μl of 100 mg / ml MRS broth with and without 100 mg / ml prebiotic and 10 μl of pre-cultured bacterial culture, resulting in an approximate OD of 1. Cell density was measured using the OD 600 measurements were taken at regular intervals over a 24 hour period.

[0208] As expected, LGG grew maximally on glucose (OD = 1.6 at 24 h). Growth was similar in the presence of FOS and CMOS (OD approximately 1.0, respectively) and superior to growth in the presence of inulin and XOS (OD approximately 0.6 and 0.7, respectively). Growth in the presence of YMOS was comparable to that of a control grown in MRS medium without prebiotics (OD approximately 0.3).

[0209] Example 10: In vitro studies evaluating MOS immune stimulation and inhibition of Piscirickettsia salmonis in Atlantic salmon head kidney (SHK) cells ECACC-derived SHK-1 cells (ECACC97111106) were grown in sterile L15 medium containing supplements (5% fetal bovine serum, 20 mM HEPES, 40 μM β-mercaptoethanol) and incubated at 20°C in 12-well plates until maximal cell confluency. The pH was adjusted to 7.0–7.6 by adding 1 M sodium hydroxide and 1 M acetic acid. Two products (our copra-MOS (CMOS) and yeast-MOS (YMOS)) were added at the indicated amounts. A product-free control was also prepared. Each treatment and control had a minimum of eight replicates. Initial P. salmonis titers were determined using a modified Spearman-Karber method known in the art, with TCID 50 This was verified by.

[0210] Plates were incubated at 15°C throughout the assay. Observations were made daily for the first 2 days, and then at least every 72 hours for cytopathic effect (CPE) and monolayer detachment, rounded to the nearest 5%, until the negative control reached a mean CPE and / or detachment of 35-55% or the assay continued for a minimum of 20 days. The minimum inhibitory concentration (MIC) was determined by a statistically significant (p<0.05) reduction in CPE (>20%) compared to the negative control.

[0211] Figures 5 and 6 show the inhibition of P. salmonis by copra-MOS and yeast-MOS. A 25 mg / mL dose of yeast-MOS caused cytotoxicity, so full test data could not be obtained at this concentration. High concentrations of YMOS (25 mg / mL) caused chronic cell stress and eventual detachment of the cell layer.

[0212] In particular, at 25 mg / mL, 100% inhibition of P. salmonis was achieved after 11 days and continued until the end of the study. At a dose of 5.5 mg / mL of copraMOS, more than 20% inhibition (the minimum inhibitory concentration criterion) was observed after 11 days.

[0213] Example 11: Biomarker analysis Cells were harvested from well plates during the course of the minimum inhibitory concentration assay and then stored at -80°C before RNA extraction. Cells incubated with 16 and 25 mg / mL CMOS and YMOS were harvested at the beginning and end of the MIC assay, along with positive and negative controls. RNA was isolated and purified from selected cell samples using the Qiagen® RNeasy kit, which also included in-column DNase digestion, and the Zymo® Clean & Concentrator kit according to the manufacturer's instructions. Pure RNA was obtained for all extracted samples. cDNA was synthesized and used to perform a series of SYBR-based qPCR assays to assess gene expression in each sample for genes of interest (campb, cd209, IFN-γ, IL-1β, hepcidin, and TLR-9) and two reference genes (ef1-a and eif-3d). Each sample and gene combination (216 in total) was analyzed in triplicate.

[0214] CMOS altered the expression of IFN-γ, hepcidin, and TLR-9 in cells exposed to the product within 3 hours. On the other hand, YMOS altered the expression of campb, cd209, IFN-γ, IL-1β, hepcidin, and TLR-9 in cells by the end of the cell assay, but in a manner different from the pathogen control. In contrast, CMOS showed differences in the expression of IFN-γ, hepcidin, and TLR-9 compared with the cell and pathogen controls at the end of the study.

[0215] YMOS-induced and ongoing immune responses were observed, consistent with the observed cytotoxicity and YMOS-induced cellular stress. YMOS stimulated inflammatory immune responses via campb, IL-1b, and TLR-9; YMOS also bound to the mannose-binding site of cd209 in SHK cells, which may reduce bacterial replication.

[0216] The near-instant upregulation of IFN-γ by CMOS represents a major pathway for protection against infection. Similarly, upregulation of hepcidin and TLR-9 helps support the cellular immune response and reduces intracellular P. salmonis replication. Long-term immune stimulation by campb was also observed.

[0217] Example 12: Data on MOS in Combination with Antibiotics A pathogen inhibition test was conducted to compare the inhibition of Streptococcus mutans by penicillin with that by the combination of penicillin and the MOS preparation of Example 1. S. mutans was grown in BHI medium. Penicillin alone reduced S. mutans growth by 10%, while penicillin combined with 5 wt% MOS inhibited S. mutans growth by 75%, demonstrating the ability of the MOS composition to enhance antibiotic performance.

[0218] Example 13: Effect of DP of MOS on pathogen inhibition Pathogen inhibition tests were performed using Salmonella enteritidis incubated with (i) medium, (ii) mannose, and (iii) mannooligosaccharides of different degrees of polymerization (including the low DP MOS composition of Example 1 and MOS mixtures prepared with higher average degrees of polymerization). OD 600 Data were obtained and inhibition (or growth promotion) measured relative to growth controls. The relative growth is shown in Table 5 below. A value less than 1 indicates inhibition; a value greater than 1 indicates growth promotion by the substrate.

[0219] [Table 5]

[0220] The data in Table 5 demonstrate the effect of the degree of polymerization of the MOS products, where MOS mixtures with a higher average DP (80% DP4+) promoted the growth of S. enteritidis, while MOS mixtures with a lower average DP (64% DP2) inhibited the growth of S. enteritidis by 37%.

[0221] Example 14: Solubility measurements The solubility of the MOS product was assessed by adding MOS to a specific amount of water to create an "X" wt% solution. Solubility was assessed at ambient temperature (approximately 25°C) and at 40°C. Two different MOS compositions were tested. Composition 1 refers to the composition described in Example 1; Composition 2 has a lower average DP due to the presence of 64% mannobiose (DP2) in the mixture.

[0222] At MOS concentrations up to 15 wt%, composition 1 was completely (100%) soluble at ambient temperature. At MOS concentrations up to 17.5 wt%, composition 1 was completely (100%) soluble at 40°C.

[0223] At concentrations up to 25 wt %, composition 2 was completely (100%) soluble at ambient temperature and 40°C.

Claims

1. A composition comprising manno-oligosaccharide (MOS) carbohydrates, At least 70% by weight (wt%) of the MOS carbohydrate is a β-1,4 linked mannose subunit with a degree of polymerization (DP) of 2 to 10, and MOS with a DP of 2 to 6 is present in greater than 60 wt% of the MOS, and The composition is a composition that has at least 90% water solubility when measured as a 15-25 wt% aqueous solution of the composition at 25°C.

2. At least 85 wt% of the MOS carbohydrate is a β-1,4 linked mannose subunit, and / or The composition according to claim 1, wherein the composition has a water solubility of more than 95% when measured as a 15 wt% to 25 wt% aqueous solution of the composition at 25°C.

3. The composition according to claim 1, wherein a MOS having a DP of 2 is present in an amount of more than 50 wt% to about 65 wt% of the MOS.

4. The composition contains less than 10 wt% of monosaccharides. The composition further contains mannose, and / or The composition according to claim 1, wherein the composition does not contain fructose.

5. The composition according to claim 1, wherein the composition further comprises β-glucan, and the β-glucan is present in an amount of about 0.5 wt% to about 5 wt% of the MOS.

6. The composition according to claim 1, wherein the MOS is derived from mannan material obtained from a plant source selected from palm kernel cake, coconut residue, coniferous trees, coffee processing residue, acai seeds and residues, and copra meal.

7. The composition according to claim 1, wherein the MOS is derived from a mannan material obtained from copramyl.

8. The composition is in the form of an aqueous solution or a powder, and / or The composition according to claim 1, wherein the composition is formulated for oral administration.

9. A supplement, food, beverage, feed additive, or vaccine adjuvant comprising a composition containing a manno-oligosaccharide (MOS) carbohydrate according to any one of claims 1 to 8.

10. A combination comprising a composition containing a manno-oligosaccharide (MOS) carbohydrate according to any one of claims 1 to 8, and an antibiotic.

11. A composition comprising a manno-oligosaccharide (MOS) carbohydrate according to any one of claims 1 to 8, for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria.

12. A composition for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria as described in claim 10, further comprising a substance selected from probiotics, prebiotics, polyphenols, phages, clay minerals, antibiotics and short-chain fatty acids.

13. A composition for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria according to claim 10, wherein the pathogenic bacteria are selected from Vibrio, Tenacibaculum, Clostridia, Salmonella, Streptococcus, Staphylococcus, Aeromonas, Campylobacter, Bacillus, Klebsiella, Listeria, Shigella, Escherichia coli, and Piscirickettsia salmonis, or pathogenic bacteria species containing type I pili.

14. A composition for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria as described in claim 10, wherein the growth of the pathogenic bacteria is inhibited by at least 20%.

15. A composition for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria as described in claim 10, further comprising an antibiotic.

16. A composition for inhibiting the growth of pathogenic bacteria and / or promoting the growth of beneficial bacteria according to claim 10, wherein the growth of Clostridium perfringens is inhibited by at least 20%, the growth of Salmonella enteritidis is inhibited by at least 20%, the growth of Tenacibaculum maritimum is inhibited by at least 20%, the growth of Vibrio parahaemolyticus is inhibited by at least 20%, the growth of Vibrio aguillarum is inhibited by at least 20%, the growth of Vibrio harveyi is inhibited by at least 20%, the growth of Piscirickettsia salmonis is inhibited by at least 20%, and Streptococcus A composition in which the proliferation of mutans is inhibited by at least 20%, the proliferation of Escherichia coli is inhibited by at least 20%, or the proliferation of Listeria monocytogenes is inhibited by at least 20%.

17. A composition comprising a manno-oligosaccharide (MOS) carbohydrate according to any one of claims 1 to 8 and an antibiotic for enhancing the effectiveness of an antibiotic in inhibiting the growth of pathogenic bacteria, wherein the effectiveness of the antibiotic in inhibiting the growth of pathogenic bacteria is enhanced by at least 30%.

18. A composition according to claim 17 for enhancing the effectiveness of an antibiotic that inhibits the growth of pathogenic bacteria, wherein the antibiotic is selected from penicillin, amoxicillin, gentamicin, clindamycin, kanamycin, tetracycline, erythromycin, ciprofloxacin, vancomycin and ceftazidime, and / or A composition in which the pathogenic bacteria are selected from the following species of pathogenic bacteria: Vibrio, Tenacibaculum, Clostridia, Salmonella, Streptococcus, Aeromonas, Campylobacter, Bacillus, Klebsiella, Listeria, Shigella, Escherichia coli, and Piscirickettsia salmonis, or a species of pathogenic bacteria containing type I pili.

19. A method for preparing the composition according to any one of claims 1 to 8, To obtain a crude extract, the mannan material is subjected to enzymatic hydrolysis; the crude extract is purified to obtain the composition containing MOS carbohydrates; and The method includes optionally concentrating the composition containing MOS carbohydrates. Herein, the enzyme is a mixture of β-mannanase enzyme and β-mannosidase enzyme, in this method.

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