Compositions, products and uses

Abstract

The present application relates to compositions, products and uses. The compositions comprise medium chain fatty acid triglycerides and Bifidobacterium animalis lactis BB-12, wherein the number of viable bacteria of Bifidobacterium animalis lactis BB-12 is from 2.67 x 10 7 CFU to 6 x 10 9 CFU per gram of the medium chain fatty acid triglycerides. The compositions have a synergistic effect in improving gut health, in particular in inhibiting Staphylococcus aureus and / or improving diarrhoea. The present application also relates to the use of the compositions in the manufacture of a product for improving gut health.

Description

Technical Field

[0001] This application relates to compositions, products, and uses. Background Technology

[0002] Diarrhea is a common and prevalent gastrointestinal disease. Infectious diarrhea is an intestinal infectious disease caused by bacteria, fungi, viruses or parasites, with diarrhea as the main clinical manifestation.

[0003] Infectious diarrhea is highly prevalent and has a high incidence rate. Developing countries face a severe medical and economic burden due to diarrhea, making it a significant global public health issue. Furthermore, infectious diarrhea is highly prevalent and widespread among children, seriously endangering their health and being a major cause of childhood malnutrition (Ni Xin et al. Guidelines for the Diagnosis and Treatment of Acute Infectious Diarrhea in Children (2020 Edition) [J]. Chinese Medical Science, 2020, 10 (21): 249-256).

[0004] From an etiological perspective, infectious diarrhea can be caused by a variety of pathogens, including bacteria, viruses, fungi, and parasites, and is a typical intestinal infectious disease. Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus Staphylococcus aureus (SAA) is a zoonotic Gram-positive bacterium with a high carrier rate in both humans and livestock. It can cause chronic infections in cattle, sheep, pigs, chickens, and other animals (Wu Yunpu et al. Comparison of Staphylococcus aureus infection in experimental animals and its molecular typing methods [J]. Laboratory Animal Science, 2024, 41(3): 85-89). The severity of diseases caused by SEA varies, ranging from moderate infections such as skin infections to fatal diseases such as severe pneumonia and sepsis. Treatment can become complicated due to antibiotic resistance, making it one of the most common causes of morbidity and mortality worldwide. The chronic infections and antibiotic resistance induced by SEA pose a serious challenge to public health. Furthermore, SEA is also an important pathogen causing infectious diarrhea.

[0005] Gut health is a crucial foundation for maintaining normal physiological functions. It is not only closely related to nutrient digestion and absorption, but also participates in the body's immune defense through the gut microbiota, intestinal mucosal barrier, and gut-associated immune system. When the gut microbiota is imbalanced or the intestinal barrier is damaged, it can easily induce diarrhea and intestinal inflammation, with the harm being more significant in susceptible populations.

[0006] Currently, Staphylococcus aureus infection and the resulting diarrhea are mainly treated with antibiotics. However, long-term or inappropriate use of antibiotics can easily induce the development of drug-resistant strains and may also cause intestinal flora imbalance and various adverse reactions, limiting their application in long-term conditioning and prevention. In this context, developing safe, effective, and regulatory alternative or adjunctive interventions has become an important research direction in this field.

[0007] Therefore, there remains a need in the field for methods that can improve gut health, particularly by inhibiting Staphylococcus aureus infection and / or improving diarrhea. Summary of the Invention

[0008] To solve the above and other technical problems, the inventors, through in-depth research, discovered that when medium- and long-chain fatty acid triglycerides are combined with Bifidobacterium animalis subsp. lactis BB-12 (… Bifidobacterium animalis subs p. Lactis BB-12 When used in combination at specific dosage ratios, they can synergistically improve gut health, particularly synergistically improve (e.g., relieve or treat) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibit Staphylococcus aureus (in vivo or in vitro).

[0009] In one aspect, this application provides a composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of the medium- and long-chain fatty acid triglycerides, the viable count of Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

[0010] In some embodiments, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 1.33 × 10⁻¹² per gram of the medium- and long-chain fatty acid triglycerides. 8 CFU to 6 × 10 9 CFU.

[0011] In some embodiments, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 1.33 × 10⁻¹² per gram of the medium- and long-chain fatty acid triglycerides. 8 CFU to 2 × 10 9 CFU.

[0012] In some embodiments, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 6.67 × 10⁻¹² per gram of the medium- and long-chain fatty acid triglycerides. 8 CFU to 2 × 10 9 CFU.

[0013] In another aspect, this application also provides a product comprising the composition described in any of the above embodiments.

[0014] In another aspect, this application also provides the use of the composition in the preparation of products for improving gut health, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of said medium- and long-chain fatty acid triglycerides, the viable count of said Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6×10 9 CFU.

[0015] In some implementations, the improvement of gut health includes the improvement of diarrhea.

[0016] In some implementations, the diarrhea is bacterial diarrhea.

[0017] In some embodiments, the bacterial diarrhea is caused by Staphylococcus aureus.

[0018] In another aspect, this application also provides the use of the composition in the preparation of a product for inhibiting Staphylococcus aureus infection, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of said medium- and long-chain fatty acid triglycerides, the viable count of said Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

[0019] In another aspect, this application also provides the use of the composition in the preparation of a product for regulating the intestinal flora of subjects with diarrhea, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of said medium- and long-chain fatty acid triglycerides, the viable count of said Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

[0020] The beneficial effects of this application include: when medium- and long-chain fatty acid triglycerides are combined with Bifidobacterium animalis subsp. lactis BB-12 ( Bifidobacterium animalis subs p. Lactis BB-12 When used in combination at specific dosage ratios, the two have a synergistic effect, which can synergistically improve gut health, particularly synergistically improve (e.g., relieve or treat) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibit Staphylococcus aureus (in vivo or in vitro).

[0021] For example, when each gram of medium- and long-chain fatty acid triglycerides is combined with a live bacteria count of 2.67 × 10⁻⁶... 7CFU to 6 × 10 9 When used in combination with Bifidobacterium animalis subsp. lactis BB-12, the composition synergistically improves gut health, particularly synergistically improves (e.g., alleviates or treats) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibits Staphylococcus aureus (in vivo or in vitro).

[0022] Preferably, when each gram of medium- and long-chain fatty acid triglycerides is combined with a live bacteria count of 1.33 × 10⁻⁶, 8 CFU to 6 × 10 9 When CFU is used in combination with Bifidobacterium animalis subsp. lactis BB-12, the synergistic effect of the composition is more significant and can further synergistically improve gut health, particularly synergistically improve (e.g., alleviate or treat) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibit Staphylococcus aureus (in vivo or in vitro).

[0023] More preferably, when each gram of medium- and long-chain fatty acid triglycerides is combined with a live bacteria count of 1.33 × 10⁻⁶, 8 CFU to 2 × 10 9 When CFU is used in combination with Bifidobacterium animalis subsp. lactis BB-12, the synergistic effect of the composition is more significant and can further synergistically improve gut health, particularly synergistically improve (e.g., alleviate or treat) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibit Staphylococcus aureus (in vivo or in vitro).

[0024] More preferably, when each gram of medium- and long-chain fatty acid triglycerides is combined with a live bacteria count of 6.77 × 10⁻⁶... 8 CFU to 2×10 9 When CFU is used in combination with Bifidobacterium animalis subsp. lactis BB-12, the synergistic effect of the composition is more significant and can further synergistically improve gut health, particularly synergistically improve (e.g., alleviate or treat) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibit Staphylococcus aureus (in vivo or in vitro). Attached Figure Description

[0025] To enable a full understanding of this application and its easy implementation, exemplary embodiments will now be described by way of non-limiting examples, with reference to the accompanying drawings. In the drawings: Figure 1 The diagram shows a statistical graph of intestinal fluorescence intensity in zebrafish after sample treatment according to an embodiment of this application, wherein, compared with the model control group, p < 0.05, p < 0.01, p < 0.001; compared with Comparative Example 1, !p < 0.05, !!p < 0.01; compared with Comparative Example 4, $$p < 0.01, $$$p < 0.001; Figure 2 It shows Figure 1 Typical fluorescence intensity of the zebrafish intestine after sample treatment in the illustrated embodiment; Figure 3 The following is a statistical graph showing the fluorescence intensity of Staphylococcus aureus in the intestine of zebrafish after sample treatment according to an embodiment of this application, wherein, compared with the model control group, p < 0.05, p < 0.01, p < 0.001; compared with Comparative Example 1, !p < 0.05; compared with Comparative Example 4, $$p < 0.01; Figure 4 It shows Figure 3 Typical fluorescence intensity of Staphylococcus aureus in the gut of zebrafish after sample treatment in the illustrated embodiment. Detailed Implementation

[0026] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this application, the technical solution of this application is described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed can and have been performed in a manner commonly known and understood by one of ordinary skill in the art.

[0027] Composition

[0028] In one aspect, this application provides a composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of the medium- and long-chain fatty acid triglycerides, the viable count of Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU. The inventors have surprisingly discovered that when the composition contains the medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12 in the stated proportions, it has a synergistic effect in improving gut health, particularly in improving (e.g., alleviating or treating) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and (in vivo or in vitro) inhibiting Staphylococcus aureus.

[0029] As used herein, the term "medium- and long-chain fatty acid triglycerides" (MLCT) refers to a special type of triglyceride with a molecular structure containing both medium-chain and long-chain fatty acid residues. As used herein, the term "medium-chain fatty acid" refers to fatty acids with 6 to 12 carbon atoms in their carbon chain, such as hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid. As used herein, the term "long-chain fatty acid" refers to fatty acids with 14 or more carbon atoms in their carbon chain, such as 14 to 30 carbon atoms, such as myristic acid, palmitic acid, oleic acid, linoleic acid, stearic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, and nervonic acid. As used herein, the terms "medium-chain fatty acid triglycerides" (MCT) and "long-chain fatty acid triglycerides" (LCT) refer to the esterification products of medium-chain fatty acids and long-chain fatty acids with glycerol, respectively.

[0030] Medium- and long-chain triglycerides, as a star product among novel structured lipids, have attracted widespread attention. Their fatty acid composition is close to that of breast milk, which is beneficial for fat digestion and absorption. They can improve the absorption of lipid nutrients, inhibit the accumulation of body fat, and provide rapid and stable energy (Cheng, X., et al. Medium- and Long-ChainTriacylglycerol: Preparation, Health Benefits, and Food Utilization, Annu. Rev. Food Science and Technology 2024, 15, 381–408). Medium- and long-chain triglycerides have different physicochemical properties, metabolic characteristics, and nutritional value than long-chain or medium-chain triglycerides. Studies have shown that medium- and long-chain triglycerides are not simply equivalent to a physical mixture of long-chain and medium-chain triglycerides (nor are they simply equivalent to a mixture of long-chain and medium-chain fatty acids), and the latter do not possess the physicochemical properties, metabolic characteristics, and nutritional value of the former (Yuan Tinglan. Composition and metabolic characteristics of medium- and long-chain triglycerides in breast milk fat [D]. Wuxi: Jiangnan University, 2021).

[0031] As is known in the art, medium- and long-chain fatty acid triglycerides are produced by using edible vegetable oils and MCTs as raw materials, through transesterification by lipases, followed by processes such as distillation, decolorization, and deodorization.

[0032] This application does not have any particular requirements regarding the medium- and long-chain triglycerides used; medium- and long-chain triglycerides commonly used in the art can be used. Medium- and long-chain triglycerides can be used in pure form or in a non-pure form rich in medium- and long-chain triglycerides. For example, the medium- and long-chain triglycerides used in the compositions of this application can be medium- and long-chain triglycerides with a single composition (i.e., each molecular chain has the same long-chain fatty acid residues and the same medium-chain fatty acid residues), or a mixture of multiple medium- and long-chain triglycerides with two or more different compositions (i.e., each molecular chain has different long-chain fatty acid residues and / or different medium-chain fatty acid residues).

[0033] In some embodiments, the medium- and long-chain fatty acid triglycerides used in this application may contain C6-C6. 12 (e.g., C6, C7, C8, C9, C) 10 C 11 C 12 (or the range defined by either or both) fatty acid residues and C 14 ~C 30 (e.g., C) 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 C 25 C 26 C 27 C 28 C 29 C 30 (or the range defined by either C6-C6) fatty acid residues. In some examples, C6-C6... 12 Fatty acid residues can originate from one or more of the following: hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and lauric acid, etc. C 14 ~C 30 Fatty acid residues can be derived from one or more of the following: myristic acid, palmitic acid, heptadecanic acid, oleic acid, linoleic acid, stearic acid, nonadecanic acid, eicosapentaenoic acid, docosahexaenoic acid, docosapatraenoic acid, docosahexaenoic acid, tricarboxylic acid, tetracarboxyenoic acid, docosapentaenoic acid, docosahexaenoic acid, arachidonic acid, etc. In some examples, C6~C... 12 The fatty acid residues may be derived from one or more of the following: hexanoic acid, caprylic acid, decanoic acid, and lauric acid. In some examples, C... 14 ~C 30Fatty acid residues may be derived from one or more of the following: myristic acid, palmitic acid, oleic acid, linoleic acid, stearic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0034] In some embodiments, the medium- and long-chain fatty acid triglycerides used in this application have C6~C6... 12 Fatty acid residues and C 14 ~C 30 The unit weight ratio of fatty acid residues can be from 0.124 to 2.000. For example, in the medium- and long-chain fatty acid triglycerides used in this application, C6~C 12 Fatty acid residues and C 14 ~C 30 The unit weight ratio of fatty acid residues may be 0.124, 0.125, 0.130, 0.140, 0.150, 0.160, 0.170, 0.180, 0.190, 0.200, 0.300, 0.400, 0.500, 0.600, 0.700, 0.800, 0.900, 1.000, 1.100, 1.200, 1.300, 1.400, 1.500, 1.600, 1.700, 1.800, 1.900, 2.000, or within the range defined by any two of these.

[0035] As used in this article, the term "Bifidobacterium animalis subsp. lactis BB-12" (Latin scientific name: Bifidobacterium animalis subs p. Lactis BB-12 This can be used interchangeably with "Bifidobacterium BB-12" or "BB-12". This strain belongs to the genus Bifidobacterium (Bifidobacterium). Bifidobacterium Bifidobacterium BB-12 is an anaerobic probiotic, isolated from dairy starter cultures by Chr. Hansen in 1983. It has the effects of regulating intestinal flora balance, enhancing immune regulation, and improving diarrhea and constipation, thereby alleviating intestinal discomfort symptoms.

[0036] Currently, whether the combination of medium- and long-chain fatty acid triglycerides and Bifidobacterium BB-12 can synergistically improve gut health, especially synergistically improve (e.g., alleviate or treat) diarrhea (e.g., diarrhea caused by Staphylococcus aureus), or whether it can synergistically inhibit Staphylococcus aureus (in vivo or in vitro) remains to be further studied.

[0037] In some embodiments, preferably, the ratio of Bifidobacterium animalis subsp. lactis BB-12 to medium- and long-chain triglycerides in the composition is such that, based on each gram of medium- and long-chain triglycerides, the viable count of Bifidobacterium animalis subsp. lactis is 1.33 × 10⁻⁶. 8 CFU to 6 × 10 9CFU; or, the ratio of Bifidobacterium animalis subsp. lactis BB-12 to medium- and long-chain triglycerides in the composition can also be expressed as 1.33 × 10⁻⁶ CFU. 8 CFU / g up to 6 × 10 9 CFU / g. When medium- and long-chain fatty acid triglycerides are used in combination with Bifidobacterium BB-12 at the above dosage ratio, the composition can exhibit a more significant synergistic effect, further synergistically improving gut health, particularly synergistically improving (e.g., relieving or treating) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibiting Staphylococcus aureus (in vivo or in vitro).

[0038] More preferably, the ratio of Bifidobacterium lactis subsp. BB-12 to medium- and long-chain triglycerides in the composition can be 1.33 × 10⁻⁶. 8 CFU / g up to 2 × 10 9 CFU / g. When medium- and long-chain fatty acid triglycerides are used in combination with Bifidobacterium BB-12 at the above dosage ratio, the composition can exhibit a more significant synergistic effect, further synergistically improving gut health, particularly synergistically improving (e.g., relieving or treating) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibiting Staphylococcus aureus (in vivo or in vitro).

[0039] More preferably, the ratio of Bifidobacterium lactis subsp. BB-12 to medium- and long-chain triglycerides in the composition can be 6.67 × 10⁻⁶. 8 CFU / g up to 2 × 10 9 CFU / g. When medium- and long-chain fatty acid triglycerides are used in combination with Bifidobacterium BB-12 at the above dosage ratio, the composition can exhibit a more significant synergistic effect, further synergistically improving gut health, particularly synergistically improving (e.g., relieving or treating) diarrhea (e.g., diarrhea caused by Staphylococcus aureus) and synergistically inhibiting Staphylococcus aureus (in vivo or in vitro).

[0040] product

[0041] In another aspect, this application also provides a product comprising the composition as described in any of the above embodiments.

[0042] All descriptions of the composition above apply here and will not be repeated here.

[0043] In some embodiments, the product may include or be a drug.

[0044] In some embodiments, the product may be a pharmaceutical product. The pharmaceutical product may be any dosage form, such as a solid dosage form (granules, powders, tablets, etc.) or a liquid dosage form. Furthermore, those skilled in the art will readily understand that, depending on the dosage form and application scenario, the pharmaceutical product may also include various pharmaceutically permissible excipients.

[0045] The products, such as pharmaceuticals, can be prepared using methods commonly employed in the art, and will not be described in detail here.

[0046] In some embodiments, the content of the medium- and long-chain fatty acid triglycerides is 0.1% to 30% (dry weight) based on the total dry weight of the product. As an example, the content of the medium- and long-chain fatty acid triglycerides may be 0.1%, 0.2%, 0.5%, 0.8%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, or 30.0% (dry weight), or within the range defined by any two of the above, based on the total dry weight of the product.

[0047] use

[0048] In another aspect, this application also provides the use of the composition in the preparation of one or more products for improving gut health, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of said medium- and long-chain fatty acid triglycerides, the viable count of said Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

[0049] In some implementations, the improvement in gut health includes improving (e.g., alleviating or treating) diarrhea. In some examples, the diarrhea is bacterial diarrhea, for example, bacterial diarrhea caused by Gram-positive bacteria, and more specifically, bacterial diarrhea caused by Staphylococcus aureus.

[0050] In another aspect, this application also provides the use of the composition in the preparation of a product for inhibiting Staphylococcus aureus, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of said medium- and long-chain fatty acid triglycerides, the viable count of said Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 109 CFU.

[0051] In another aspect, the present application further provides the use of a composition in preparation of a product for regulating intestinal flora in a subject with diarrhea, wherein the composition comprises medium- and long-chain triglycerides and Bifidobacterium animalis subsp. lactis BB-12, and based on each gram of the medium- and long-chain triglycerides, the viable count of the Bifidobacterium animalis subsp. lactis BB-12 is 2.67 ×10 7 CFU to 6 × 10 9 CFU.

[0052] All the above descriptions of the composition and product of the present application are applicable here, and are not repeated herein.

[0053] In some embodiments, the subject (subject) for improving intestinal health (e.g., improving or relieving diarrhea), regulating intestinal flora, or inhibiting Staphylococcus aureus (in vivo or in vitro) is not particularly limited, and may include, for example, infants, toddlers, children, adolescents, adults, young adults, middle-aged people and / or the elderly. As used herein, the term "infant" refers to a person from birth to 12 months of age; the term "toddler" refers to a person aged 1 to 3 years; the term "child" refers to a person aged 3 to 7 years; the term "adolescent" refers to a person aged 7 to 17 years; the term "adult" refers to a person over 18 years of age; the term "young adult" refers to a person aged 18 to 40 years; the term "middle-aged person" refers to a person aged 41 to 65 years; the term "elderly person" or "the elderly" refers to a person over 65 years of age.

[0054] Examples

[0055] The present application will be more readily understood with reference to the following examples, which are only intended to illustrate certain aspects and embodiments of the present application, and are not intended to limit the present application.

[0056] Unless otherwise stated, the materials used in the examples are all commercially available materials or conventional materials.

[0057] Experimental Animals and Bacteria

[0058] Zebrafish: They are raised in fish culture water at 28°C (water quality: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, conductivity is 450~550 μS / cm; pH is 6.5~8.5; hardness is 50~100 mg / L CaCO3), and are bred and provided by the Fish Culture Center of Genotech Biotechnology Co., Ltd. The laboratory animal use license number is SYXK (Zhejiang) 2022-0004, the breeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number is IACUC-2025-202510290010-01.

[0059] Staphylococcus aureus: Cultured using nutrient broth at 37°C.

[0060] Instruments, consumables and reagents

[0061] Motorized focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan).

[0062] Medium- and long-chain triglycerides (MLCT): Medium- and long-chain fatty acid edible oil, MLCT purity is 61%, sourced from Yihai Kerry Company, production batch number 20250804; Bifidobacterium animalis subsp. lactis BB-12: sourced from Chr. Hansen Ltd., production batch number 3749554; Vancomycin: White powder, sourced from Shanghai Maclean Biochemical Technology Co., Ltd., batch number C12976208, solvent is DMSO; Nutrient broth culture medium: sourced from China Huankai Microbial Technology Co., Ltd., batch number 1094671; Dimethyl sulfoxide (DMSO): sourced from Sinopharm Chemical Reagent Co., Ltd., batch number 20250624; Nile Red: Sourced from Sigma (India), batch number SLBP9326V; CM-DiI: Sourced from Thermo Fisher Scientific (USA), lot number 2335589.

[0063] In the following examples, the amount and ratio of each component are based on the amount of the corresponding active ingredient. For example, Bifidobacterium BB-12 is based on the number of viable BB-12 bacteria contained in the BB-12 raw material; MLCT is based on the content of medium- and long-chain triglycerides in the long-chain fatty acid edible oil in the raw material.

[0064] Example 1: Evaluation of the efficacy of the sample in improving diarrhea

[0065] Wild-type AB strain zebrafish were randomly selected 4 days after fertilization (4 dpf) and placed in 6-well plates with a volume of 3 mL per well. 30 zebrafish were treated in each well.

[0066] Water-soluble samples (concentrations shown in Table 1) were used as examples (MLCT + BB-12) or comparative examples (single-component MLCT or single-component BB-12); the positive control group was given vancomycin at a concentration of 1000 μg / mL; a normal control group (naïve, no treatment) and a model control group (diarrhea model, no administration) were also set up. After treatment at 28℃ for 5 h, Nile red was water-solublely administered to each group as a fluorescent indicator of intestinal contents. After further treatment at 28℃ for 16 h, the samples and Nile red were washed away, and the samples were water-solublely administered again (concentrations shown in Table 1). Except for the normal control group, the other experimental groups were water-solublely administered Staphylococcus aureus to establish a zebrafish diarrhea model. After further treatment at 28℃ for 30 h, 10 zebrafish from each group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of the zebrafish intestine was analyzed, and the statistical analysis results of this index were used to evaluate the efficacy of the samples in improving Staphylococcus aureus-induced diarrhea. Statistical results are expressed as mean ± standard error (mean ± SE). Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0067] For each embodiment and comparative example, the increase in intestinal fluorescence intensity compared to the model control group was calculated (denoted as A); and for each embodiment, the sum of the increases in intestinal fluorescence intensity compared to the model control group for the corresponding comparative example was calculated (denoted as B). For example, for Example 1 (MLCT concentration 30 μg / mL + BB-12 concentration 20000 CFU / mL), the "sum of the increases in intestinal fluorescence intensity compared to the model control group for the corresponding comparative example" is the sum of the "increases in intestinal fluorescence intensity compared to the model control group" for Comparative Example 1 (MLCT concentration 30 μg / mL) and Comparative Example 4 (BB-12 concentration 20000 CFU / mL); and so on. Finally, the synergy coefficient was calculated (denoted as C, C=A / B). When the synergy coefficient > 1, a synergistic effect exists; the larger the value, the stronger the synergistic effect.

[0068] Following the above method, zebrafish were administered MLCT and BB-12 at different dosage ratios to evaluate their efficacy in improving diarrhea. The dosages are shown in Table 1. The results are also shown in Table 1. Figure 1 and Figure 2 As shown.

[0069] Table 1. Experimental results evaluating the efficacy of the samples in improving diarrhea (n = 10)

[0070] Note: Compared with the model control group, p < 0.05, p < 0.01, p < 0.001; Compared with Comparative Example 1, !p < 0.05, !!p < 0.01; Compared with Comparative Example 4, $$p < 0.01, $$p < 0.001.

[0071] From Table 1, Figure 1 and Figure 2 The results showed that, compared with the normal control group, the fluorescence intensity (pixels) of the zebrafish intestines in the model control group was significantly reduced after induction with Staphylococcus aureus via water-soluble administration, indicating that the Staphylococcus aureus-induced diarrhea model was successfully established. The positive control drug (vancomycin, 1000 μg / mL), single-component MLCT (150 μg / mL and 750 μg / mL), single-component BB-12 (60000 CFU / mL and 180000 CFU / mL), and Examples 1-5 all showed significant efficacy in improving diarrhea, specifically manifested as a significant increase in the fluorescence intensity (pixels) of the zebrafish intestines compared with the model control. P <0.05).

[0072] Example 1 is a combination of MLCT (30 μg / mL) + BB-12 (20000 CFU / mL), wherein the ratio of BB-12 to MLCT is 6.67 × 10⁻⁶. 8 CFU / g; Comparative Example 1 was a single-component MLCT (30 µg / mL); Comparative Example 4 was a single-component BB-12 (20000 CFU / mL), i.e., Example 1 is equivalent to a combination of Comparative Example 1 and Comparative Example 4. Compared with the model control group, the increase in intestinal fluorescence intensity of Example 1 was 64848 pixels, while the increases in intestinal fluorescence intensity of Comparative Example 1 and Comparative Example 4 were 1764 pixels and 4596 pixels, respectively. It can be seen that the increase in intestinal fluorescence intensity of Example 1 (64848 pixels) is much greater than the sum of the increases in intestinal fluorescence intensity of Comparative Example 1 and Comparative Example 4 (6360 pixels), indicating that Example 1 has a synergistic effect in improving diarrhea. Similarly, Examples 2 to 5 also showed a synergistic effect in improving diarrhea. These results indicate that when the BB-12 to MLCT ratio is 2.67 × 10⁻⁶, the effect is significant. 7 CFU / g up to 6 × 10 9 When within the CFU / g range, the two have a synergistic effect in improving diarrhea.

[0073] Furthermore, compared to Example 5 (BB-12 to MLCT ratio of 2.67 × 10⁻⁶), 7 CFU / g), Examples 1 to 4 (BB-12 to MLCT ratio of 1.33 × 10⁻⁶) 8CFU / g up to 6 × 10 9 The synergistic coefficient was greater within the CFU / g range, indicating that when BB-12 and MLCT were used in combination at the above dosage ratio range, the synergistic effect in improving diarrhea was more significant.

[0074] Furthermore, compared to Examples 3 and 5 (the ratio of BB-12 to MLCT was 6 × 10⁻⁶), the results showed that... 9 CFU / g and 2.67 × 10 7 CFU / g), Examples 1, 2 and 4 (BB-12 to MLCT ratio at 1.33 × 10⁻⁶). 8 CFU / g up to 2 × 10 9 The synergistic coefficient was greater within the CFU / g range, indicating that when BB-12 and MLCT were used in combination at the above dosage ratio range, the synergistic effect in improving diarrhea was more significant.

[0075] Furthermore, compared to Examples 3 to 5 (where the ratio of BB-12 to MLCT was 6 × 10⁻⁶), the difference was less significant. 9 CFU / g, 1.33 × 10 8 CFU and 2.67 × 10 7 CFU / g), Examples 1 and 2 (BB-12 to MLCT ratio of 6.67 × 10⁻⁶). 8 CFU / g up to 2 × 10 9 The synergistic coefficient (CFU / g) is larger, indicating that when BB-12 and MLCT are used in combination at the above dosage ratio range, the synergistic effect in improving diarrhea is more significant.

[0076] Example 2: Evaluation of the efficacy of the sample in inhibiting Staphylococcus aureus

[0077] 30 zebrafish of wild-type AB strain with 4 dpf were randomly selected and placed in a 6-well plate with a volume of 3 mL per well.

[0078] Water-soluble samples (concentrations shown in Table 2) were used as examples (MLCT + BB-12) or comparative examples (single-component MLCT or single-component BB-12); the positive control group received vancomycin at a concentration of 1000 μg / mL; a model control group (Staphylococcus aureus infection model, no drug administration) was also set up. After treatment at 28℃ for 24 h, each group was given water-soluble Staphylococcus aureus to establish a zebrafish Staphylococcus aureus infection model. After further treatment at 28℃ for 6 h, 10 zebrafish from each group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the fluorescence intensity of Staphylococcus aureus in the zebrafish intestine. The efficacy of the samples in inhibiting Staphylococcus aureus infection was evaluated by statistical analysis of this index. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated statistical significance.

[0079] For each embodiment and comparative example, the reduction in Staphylococcus aureus fluorescence intensity value (pixels) compared to the model control group was calculated; and for each embodiment, the sum of the reduction in Staphylococcus aureus fluorescence intensity values ​​(pixels) compared to the model control group in the corresponding comparative example was calculated, using the same method as described above.

[0080] Following the above method, zebrafish were treated with different ratios of MLCT and BB-12 to evaluate their efficacy in inhibiting Staphylococcus aureus. The dosages are shown in Table 2. The results are also shown in Table 2. Figure 3 and Figure 4 As shown.

[0081] Table 2. Experimental results evaluating the efficacy of the samples in inhibiting Staphylococcus aureus (n = 10)

[0082] Note: Compared with the model control group, p < 0.05, p < 0.01, p < 0.001; Compared with Comparative Example 1, !p < 0.05; Compared with Comparative Example 4, $$p < 0.01.

[0083] From Table 2, Figure 3 and Figure 4The results showed that, compared with the model control group, the positive control group (vancomycin, 1000 µg / mL), single-component MLCT (750 μg / mL), single-component BB-12 (180,000 CFU / mL), and Examples 2, 3, and 5 all significantly inhibited Staphylococcus aureus. Specifically, compared with the model control group, the fluorescence intensity of Staphylococcus aureus in the zebrafish intestine was significantly reduced. P <0.05).

[0084] Example 2 is a combination of MLCT (30 µg / mL) + BB-12 (60000 CFU / mL), wherein the ratio of BB-12 to MLCT is 2 × 10⁻⁶. 9 CFU / g; Comparative Example 1 was a single-component MLCT (30 µg / mL); Comparative Example 5 was a single-component BB-12 (60000 CFU / mL), i.e., Example 2 was the combination of Comparative Examples 1 and 5. Compared with the model control group, the reduction in fluorescence intensity of Staphylococcus aureus in the intestine of Example 2 was 73851 pixels, while the reductions in fluorescence intensity of Staphylococcus aureus in the intestine of Comparative Examples 1 and 5 were -7160 pixels and 28607 pixels, respectively. It is evident that the reduction in fluorescence intensity of Staphylococcus aureus in the intestine of Example 2 (73851 pixels) is much greater than the sum of the reductions in fluorescence intensity of Staphylococcus aureus in the intestine of Comparative Examples 1 and 5 (21447 pixels), indicating that Example 2 has a synergistic effect in inhibiting Staphylococcus aureus. Similarly, Examples 3 and 5 also showed a synergistic effect in inhibiting Staphylococcus aureus. These results indicate that when the ratio of BB-12 to MLCT is 2.67 × 10⁻⁶, the effect is significant. 7 CFU / g up to 6 × 10⁻⁶ 9 Within the CFU / g range, the two exhibit a synergistic effect in inhibiting Staphylococcus aureus.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition, characterized in that, The composition comprises medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, based on each gram of the medium- and long-chain fatty acid triglycerides, the viable count of Bifidobacterium animalis subsp. lactis BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

2. The composition according to claim 1, characterized in that, Based on each gram of the aforementioned medium- and long-chain fatty acid triglycerides, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 1.33 × 10⁻⁶. 8 CFU to 6 × 10 9 CFU.

3. The composition according to claim 1, characterized in that, Based on each gram of the aforementioned medium- and long-chain fatty acid triglycerides, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 1.33 × 10⁻⁶. 8 CFU to 2 × 10 9 CFU.

4. The composition according to claim 1, characterized in that, Based on the amount of the medium- and long-chain fatty acid triglycerides, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 6.67 × 10⁻⁶. 8 CFU to 2 × 10 9 CFU.

5. The product, characterized in that, The product comprises the composition according to any one of claims 1 to 4.

6. Use of the composition in the preparation of a product for improving gut health, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, Based on each gram of the aforementioned medium- and long-chain fatty acid triglycerides, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

7. The use according to claim 6, wherein the improvement of gut health includes improvement of diarrhea.

8. The use according to claim 7, characterized in that, The diarrhea is bacterial diarrhea.

9. The use according to claim 8, characterized in that, The bacterial diarrhea was caused by Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus )cause.

10. Use of the composition in the preparation of a product for inhibiting Staphylococcus aureus infection, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, Based on each gram of the aforementioned medium- and long-chain fatty acid triglycerides, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

11. Use of the composition in the preparation of a product for regulating the gut microbiota of subjects with diarrhea, said composition comprising medium- and long-chain fatty acid triglycerides and Bifidobacterium animalis subsp. lactis BB-12, wherein, Based on each gram of the aforementioned medium- and long-chain fatty acid triglycerides, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BB-12 is 2.67 × 10⁻⁶. 7 CFU to 6 × 10 9 CFU.

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