A composition for promoting growth of akkermansia and use thereof

Abstract

The present application belongs to the technical field of microorganisms and culture medium, and in particular, the present application provides a composition for promoting growth of Akkermansia and application thereof, the composition comprising milk fat globule membrane, alpha-lactalbumin and 3-fucosyllactose, wherein the concentration ratio of the milk fat globule membrane, alpha-lactalbumin and 3-fucosyllactose in the composition is (0.1-3):(0.1-5):(0.1-2). The composition can fully meet the nutritional requirements of Akkermansia bacteria, especially mucinophilic Akkermansia bacteria, can effectively promote the growth of Akkermansia bacteria, and can be used for culturing Akkermansia bacteria in vitro, and can also promote the growth of mucinophilic Akkermansia bacteria in the human intestinal tract, increase the potential probiotic Akkermansia bacteria in the intestinal tract, and improve intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and culture medium technology, and more specifically, relates to a composition for promoting the growth of Akkermansia bacteria and its application. More specifically, it relates to a composition for promoting the growth of Akkermansia myxophilus. Background Technology

[0002] Milk fat globule membrane (MFGM) is a complex biomembrane structure surrounding the surface of milk fat globules, mainly composed of phospholipids (such as phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin), glycolipids (such as gangliosides), membrane proteins (such as mucin, lactoferrin, and immunoglobulins), and cholesterol. MFGM not only has physiological functions such as regulating lipid metabolism, promoting neural development, and enhancing immune responses, but it has also been shown to regulate the gut microbiota through its polar lipid components. Studies have shown that sphingomyelin and its digestive products in MFGM can reach the colon, exerting antibacterial and gut microbiota-regulating effects. Reference 1 reports that in a high-fat diet-induced obese pregnant rat model, MFGM supplementation significantly restored the abundance of Akkermansia bacteria (e.g., Akkermansia muciniphila, or AKK) in the offspring gut (AKK was reduced by 78% in the high-fat diet group).

[0003] Alpha-lactalbumin (α-LA) is the most abundant globular protein in whey protein, rich in essential amino acids such as tryptophan and cysteine, and possesses the ability to bind metal ions (such as zinc and calcium). In addition to its nutritional function, α-lactalbumin releases bioactive peptides after digestion, exhibiting antibacterial, anti-inflammatory, immunomodulatory, and prebiotic properties. Existing research indicates that appropriate concentrations of α-lactalbumin can regulate the diversity and structure of intestinal flora, promoting the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus, while inhibiting the excessive growth of opportunistic pathogens. Reference 2 reports that, confirmed by 16S rRNA high-throughput sequencing, a moderate dose (100 mg / kg) of α-lactalbumin significantly improved the diversity of intestinal flora in rats and promoted the growth of Akkermansia myxophilus, while a high dose (200 mg / kg) showed an inhibitory effect.

[0004] Fucosylated oligosaccharides are a class of human milk oligosaccharides (HMOs) abundant in breast milk, mainly including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), and difucosyllactose (DFL). They possess various physiological functions, such as preventing pathogen adhesion, regulating the immune system, promoting probiotic growth, and promoting nervous system development. Among these, 3-fucosyllactose is one of the most abundant fucosylated oligosaccharides, composed of fucose, galactose, and glucose linked by specific glycosidic bonds. 3-FL resists enzymatic degradation in the upper digestive tract, reaching the colon intact as a prebiotic for selective utilization by gut microbiota. It also inhibits pathogen adhesion by mimicking receptors on the surface of intestinal epithelial cells. In particular, the genomes of Akkermansia bacteria, such as *Ackermania*, encode various glycoside hydrolases (e.g., fucosidase, sialic acidase, sulfatase), enabling them to utilize host mucin oligosaccharides and human milk oligosaccharides as carbon sources. Reference 3 discloses a composition for controlling the proliferation of Akkermansia bacteria in the intestinal flora, comprising any one selected from the group consisting of sialylated human milk oligosaccharides, human milk oligosaccharides with sialic acid as a constituent sugar, lactose, sialic acid, and fucose.

[0005] Akkermania bacteria are a group of anaerobic Gram-negative bacteria belonging to the phylum Verrucomicrobia, widely distributed in the intestines of humans and animals. A representative species, *Ackermania myxotroph*, is a strictly anaerobic Gram-negative bacterium of Verrucomicrobia, colonizing the intestinal mucus layer and surviving by using mucin as a carbon and nitrogen source. *Ackermania myxotroph* is considered a "next-generation probiotic" after Bifidobacteria and Lactobacillus, and its abundance is closely related to improvements in host metabolic health, obesity, type 2 diabetes, and inflammatory bowel disease. *Ackermania myxotroph* stimulates intestinal epithelial cells to secrete more mucus by degrading mucin, thus strengthening the intestinal barrier function; simultaneously, its metabolites, short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid), can provide energy for intestinal epithelial cells and maintain intestinal homeostasis. However, because *Ackermania myxotroph* is not on the list of bacteria permitted for use in food, there is no way to increase its abundance in the human gut through direct exogenous supplementation. Instead, its growth can only be indirectly stimulated by exogenous supplementation with certain substances. Furthermore, *Ackermania myxotroph* is a strict anaerobe with specific nutritional requirements, making in vitro culture difficult, and currently, there are no safe and effective nutritional interventions to promote its colonization and proliferation in vivo.

[0006] In existing technologies, research on the effects of MFGM, α-lactalbumin, and 3-FL on the gut microbiota has largely focused on the regulatory effects of single components on Bifidobacteria, Lactobacilli, or the overall microbiota structure. No studies have disclosed the synergistic proliferative effect of these three specific component combinations on Akkermansia bacteria, particularly Akkermansia myxotropicis, nor have they explored the dose-response effect and optimal ratio of this combination in promoting the growth of Akkermansia bacteria, especially Akkermansia myxotropicis. Reference 4 discloses a nutritional composition containing milk fat globule membrane components, at least one human milk oligosaccharide, and one or more other components, including DHA, ARA, vitamin E, vitamin C, and sphingomyelin, claiming synergistic promotion of nutrient (DHA, etc.) absorption and regulation of general gut microbiota. However, it has not investigated the specific enrichment of Akkermansia bacteria such as Akkermansia myxotropicis, and it does not involve α-lactalbumin. Reference 5 reports an infant formula milk powder rich in milk fat globule membrane protein, phospholipids, and oligosaccharides. This milk powder is made from raw cow's milk and enriched with whey protein powder, α-lactalbumin powder, galactooligosaccharides, and fructooligosaccharides. Through formula adjustments, it enhances the levels of bioactive substances with special functional components, including MFGM-protein, lactoferrin (LF), α-lactalbumin (α-La), total galactooligosaccharides (GOS), total fructooligosaccharides (FOS), sialic acid, total phospholipids, sphingomyelin (SM), lecithin (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), gangliosides (Gang), triglycerides (TAG), and diglycerides (DAG). This promotes the colonization of beneficial bacteria in the infant's gut microbiota, particularly significantly enriching lactic acid bacteria while reducing unclassified bacteria. However, it does not specifically target the proliferation of Akkermansia bacteria such as Akkermansia myxophilus, and the formula does not contain 3-FL.

[0007] References:

[0008] Reference 1: Qichen Yuan et. al, Milk fat globule membranesupplementation to obese rats during pregnancy and lactation promotes neurodevelopment in offspring via modulating gut microbiota, Frontiers inNutrition, 2022;

[0009] Reference 2: Li Menghan et al., Analysis of the effects of α-lactalbumin on rat gut microbiota based on 16S rRNA technology, Food Science, 2020;

[0010] Reference 3: CN118870990A;

[0011] Reference 4: US12342843B2;

[0012] Reference 5: CN112042750B. Summary of the Invention

[0013] The problem the invention aims to solve

[0014] Akkermansia bacteria are strict anaerobes with specific nutritional requirements, making in vitro culture difficult, and currently lacking safe and effective strategies for promoting in vivo colonization. Therefore, nutrients that are readily available and offer broad benefits to overall health have significant application value. However, current research on substances that can promote the growth of Akkermansia bacteria, especially Akkermansia myxotropicum, is still insufficient. Furthermore, existing research on the effects of MFGM, α-lactalbumin, and 3-FL on the gut microbiota focuses primarily on the regulatory effects of single components on Bifidobacteria, Lactobacilli, or the overall microbiota structure. No studies have yet disclosed the synergistic proliferative effect of these three specific component combinations on Akkermansia bacteria, particularly Akkermansia myxotropicum, nor have they explored the dose-response and optimal ratio for promoting the growth of Akkermansia bacteria such as Akkermansia myxotropicum.

[0015] Based on the aforementioned technical problems, this invention studies the utilization capabilities of Akkermansia bacteria, particularly Akkermansia myxophilus, of milk fat globule membrane, α-lactalbumin, 3-fucosylated lactose, and a ternary combination of these three substances. It also explores the dose-effects and optimal ratios of these three substances and the ternary combination, thereby providing a composition that can promote the growth of Akkermansia bacteria, especially Akkermansia myxophilus. Furthermore, a novel application is proposed for a composition containing milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose in promoting the growth of Akkermansia bacteria, particularly in the human intestinal tract.

[0016] Furthermore, the present invention provides a composition and a product that promote the growth of Akkermansia bacteria in the intestine.

[0017] Solution for solving the problem

[0018] To address the problems in the prior art, the present invention provides the following technical solution:

[0019] [1]. The present invention first provides a culture medium composition for promoting the growth of Akkermansia bacteria, wherein the culture medium composition comprises basic nutrient components and additive components,

[0020] The added components include three essential ingredients: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose.

[0021] In the culture medium composition, the concentration ratio of the milk fat globule membrane, α-lactalbumin and 3-fucosylated lactose is (0.1~3):(0.1~5):(0.1~2).

[0022] [2]. According to the culture medium composition of [1], wherein the concentration ratio of the milk fat globule membrane, α-lactalbumin and 3-fucosylated lactose in the culture medium composition is (0.1~2):(0.1~4):(0.25~1.75).

[0023] [3]. The culture medium composition according to [1] or [2], wherein the basic nutrient components include one or more of protein components, lipid components, carbohydrate components, vitamin components, mineral components, and pH adjustment components.

[0024] [4]. The present invention also provides the use of a culture medium composition according to any one of [1] to [3] for promoting the growth of Akkermania bacteria, wherein the culture medium composition is used under anaerobic conditions to promote the growth of Akkermania bacteria.

[0025] [5]. According to the application described in [4], wherein the Akkermansia bacteria include one or more of Akkermansia muciniphila, Akkermansia glycaniphila, and Akkermansia biwaensis.

[0026] [6]. The present invention also provides a composition that promotes the growth of Akkermansia bacteria in the intestine, wherein the composition necessarily comprises three components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose.

[0027] In the composition, the concentration ratio of the milk fat globule membrane, α-lactalbumin and 3-fucosyllactose is (0.1~3):(0.1~5):(0.1~2).

[0028] [7]. The composition according to [6], wherein the Akkermansia bacteria include one or more of Akkermansia muciniphila, Akkermansia glycaniphila, and Akkermansia biwaensis.

[0029] [8]. The present invention also provides the use of a composition in the preparation of a product having the effect of promoting the growth of Akkermansia bacteria in the intestine, wherein the composition necessarily comprises three components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose.

[0030] In the composition, the concentration ratio of the milk fat globule membrane, α-lactalbumin and 3-fucosyllactose is (0.1~3):(0.1~5):(0.1~2).

[0031] [9]. According to the application described in [8], wherein the Akkermansia bacteria include one or more of Akkermansia muciniphila, Akkermansia glycaniphila, and Akkermansia biwaensis.

[0032]

[10] . According to the application described in [8] or [9], wherein the product is a liquid, a semi-solid or a solid.

[0033] The effects of the invention

[0034] Based on the implementation of the above technical solution, the present invention can achieve the following technical effects:

[0035] 1) Unlike existing technologies that only study the regulatory effects of single components or combinations of milk fat globule membrane (MFGM), α-lactalbumin, and 3-fucosylated lactose (3-FL) on general gut microbiota structure or traditional probiotics (such as Bifidobacteria and Lactobacillus), this invention targets Akkermansia bacteria, which are strict anaerobic bacteria with specific nutritional requirements, difficult to culture in vitro, and for which there are currently no safe and effective nutritional interventions to promote their colonization and proliferation in vivo. The invention has found that compositions containing milk fat globule membrane, α-lactalbumin, or 3-fucosylated lactose, as well as ternary combinations containing these three substances, all promote the growth of Akkermansia bacteria, particularly Akkermansia myxophilus. Furthermore, the present invention has found that, compared to the effects of a single composition containing milk fat globule membrane, α-lactalbumin, or 3-fucosylated lactose, a ternary combination of the three substances has a more significant promoting effect on the growth of Akkermania bacteria, particularly Akkermania myxophilus. Milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose have a synergistic effect on the growth of Akkermania bacteria.

[0036] 2) In a further preferred embodiment, the present invention studies the dosage of each component required to promote the growth of Akkermansia bacteria. This is achieved by culturing Akkermansia bacteria, particularly Akkermansia myxophilus, using a composition containing a milk fat globule membrane, α-lactalbumin, or 3-fucosylated lactose, or a ternary combination of these three substances, while ensuring the continued growth of Akkermansia bacteria. The content of the milk fat globule membrane, α-lactalbumin, or 3-fucosylated lactose in the composition used for culturing is adjusted, and a suitable dosage range is screened based on the growth of Akkermansia bacteria after culturing. It has been found that when each component in the composition is within the screened suitable dosage range, the composition provided by the present invention can further effectively promote the growth of Akkermansia bacteria, particularly Akkermansia myxophilus.

[0037] More specifically, the three components of the composition provided by the present invention can promote the growth and / or proliferation of Akkermansia bacteria during the logarithmic growth phase and / or stationary phase, within a wide range of dosage ratios, for example, by increasing the OD of Akkermansia bacteria during the logarithmic growth phase and / or stationary phase. 600 The value is higher. More specifically, the composition provided by this invention can significantly and rapidly increase the OD value of Akkermansia bacteria cultured for 0-10 hours. 600 Value, and / or, significantly improve the OD value of Akkermansia bacteria cultured for 10–36 h. 600 value.

[0038] In summary, the composition provided by this invention can fully meet the nutritional needs of Akkermania bacteria, especially Akkermania myxophilus, effectively promote the growth of Akkermania bacteria, and can be used to culture Akkermania bacteria in vitro. It can promote the growth of Akkermania myxophilus in the human gut, increase the number of potential probiotic Akkermania bacteria (Akkermania myxophilus) in the gut, improve gut health, and provide a new strategy for promoting Akkermania bacterial colonization in vivo. Attached Figure Description

[0039] Figure 1 Growth curves of Akkermansia myxophilus in MRS medium with different glucose concentrations.

[0040] Figure 2 Growth curves of Akkermansia myxophilus in MRS medium supplemented with different doses of MFGM.

[0041] Figure 3 Growth curves of Akkermansia myxophilus in MRS medium supplemented with different doses of α-lactalbumin.

[0042] Figure 4 Growth curves of Akkermansia myxophilus in MRS medium supplemented with different doses of 3-FL.

[0043] Figure 5 Growth curves of Akkermansia myxophilus in MRS medium supplemented with different doses of MFGM, α-lactalbumin and 3-FL ternary combination. Detailed Implementation

[0044] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0045] definition

[0046] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0047] In this specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0048] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0049] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0050] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" indicates weight or mass percentage.

[0051] In this specification, "about" usually means that the standard deviation of the actual value from the theoretical model or theoretical data is within 3%, preferably 2%, and more preferably 1%.

[0052] In this manual, when the term "room temperature" is used, it refers to an ambient temperature between 15°C and 35°C, such as 15°C, 18°C, 20°C, 25°C, 28°C, 30°C, or 35°C. The term room temperature is also called normal temperature or general temperature.

[0053] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0054] In this specification, the terms “comprising” and / or “including” are used to indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0055] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0056] In this specification, the terms “growth” and “proliferation” refer to an increase in the volume and / or number of bacterial cells.

[0057] In this specification, OD 600 and OD 600nm It refers to the optical density value of a certain solution at a wavelength of 600nm.

[0058] In this specification, the term "culture" refers to the growth of a population of cells (e.g., microbial cells) under suitable growth conditions in a liquid, semi-solid, or solid culture medium.

[0059] In this specification, the term "static culture" refers to a technique for culturing cells in a container filled with liquid culture medium without aeration or agitation.

[0060] In this specification, the microorganisms can be cultured according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, and various culture conditions such as temperature, time and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0061] In addition, unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] This invention primarily provides a composition that promotes the growth of Akkermansia bacteria. This composition comprises three essential components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose. This composition can be a culture medium composition or an edible composition. Such a composition fully meets the nutritional requirements of Akkermansia bacteria, particularly Akkermansia myxophilus, thereby effectively promoting the growth of Akkermansia bacteria, especially in the human intestinal tract. This invention is mainly based on the following insights:

[0063] As described above, this invention studies Akkermania bacteria and finds that compositions containing milk fat globule membrane, α-lactalbumin or 3-fucosylated lactose, as well as compositions containing a ternary combination of the three substances, all have the effect of promoting the growth of Akkermania bacteria, especially Akkermania myxophilus. The compositions containing a ternary combination of the three substances show a significant synergistic effect.

[0064] Furthermore, the present invention investigated the dosage of each component required for the composition to promote the growth of Akkermania bacteria, and found that when each component in the composition is within the appropriate dosage range obtained through screening, the composition provided by the present invention can further effectively promote the growth of Akkermania bacteria, especially Akkermania myxophilus.

[0065] In some specific embodiments, the present invention uses a culture that slows down the growth of Akkermansia bacteria, especially Akkermansia myxophila, and allows them to barely maintain growth as a base culture. Different doses of milk fat globule membrane, α-lactalbumin, 3-fucosylated lactose, or a ternary combination containing the three substances are added to obtain the compositions of the present invention. Akkermansia bacteria are inoculated into the obtained compositions for culture, and the growth of Akkermansia bacteria after culture is observed to determine the promoting effect of each composition on the growth of Akkermansia bacteria.

[0066] The technical solution of the present invention will be further described below:

[0067] <First aspect. Culture medium composition for promoting the growth of Akkermansia bacteria>

[0068] In one aspect of the invention, a culture medium composition for promoting the growth of Akkermansia bacteria is provided, the culture medium composition comprising a basic nutrient component and an additive component, the additive component comprising three essential components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose.

[0069] (Basic nutritional components)

[0070] There are no particular limitations on the basic nutrient components that can be used in the culture medium compositions of the present invention, as long as they enable the growth of Akkermania bacteria. In some specific embodiments, such basic nutrient components may include one or more of the following: protein components, lipid components, carbohydrate components, vitamin components, mineral components, and pH adjusting components.

[0071] In some specific embodiments, the basal nutrient component preferably includes components commonly found in culture media used for culturing Akkermania bacteria. Culture media commonly used for culturing Akkermania bacteria include, but are not limited to, MRS medium, brain heart broth (BHI) medium, enhanced Clostridium perfringens (RCM) medium, mMRS medium (modified MRS medium), and other anaerobic culture media. More preferably, the basal nutrient component includes components conventionally used in MRS medium.

[0072] In some preferred embodiments, the culture medium may contain one or more of the following components: tryptone, yeast extract, beef extract, glucose, magnesium sulfate heptahydrate, manganese sulfate monohydrate, sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate trihydrate, and Tween 80, etc.

[0073] In some specific implementation schemes, there are no particular restrictions on the content of each component in the basic nutrient composition. It can be configured in accordance with the usual technical guidelines in the prior art, as long as it can enable the growth of Akkermania bacteria.

[0074] (Added components)

[0075] The culture medium composition of the present invention necessarily includes three components: milk fat globule membrane, α-lactalbumin and 3-fucosylated lactose, wherein the milk fat globule membrane, α-lactalbumin and 3-fucosylated lactose are used in accordance with legal and regulatory standards.

[0076] milk fat globule membrane

[0077] The milk fat globule membrane is a complex biological membrane enclosing milk fat globules. Its main components include proteins, carbohydrates, and lipids primarily derived from mammary gland cell membranes. Current research indicates that the milk fat globule membrane not only possesses excellent emulsifying properties, gastrointestinal digestive stability, and safety, but its specific protein components also exhibit various biological activities such as anti-inflammatory, antiviral, intestinal function regulation, and optimization of motor muscle performance. Furthermore, it can increase the relative abundance of Akkermansia bacteria in the gut.

[0078] The milk fat globule membrane contained in the culture medium composition of the present invention can be a commercially available product or a milk fat globule membrane product obtained through an extraction process. Furthermore, extraction processes for milk fat globule membranes are relatively mature; therefore, the present invention does not impose particular limitations on the extraction process, as long as the milk fat globule membrane can be separated and obtained. For example, the milk fat globule membrane can be extracted by physical methods (such as centrifugation, ultrasound, high-pressure homogenization, etc.), chemical methods (such as solvent extraction, acid-base treatment, etc.), and biological methods (such as enzymatic hydrolysis, fermentation, etc.). In addition, after obtaining the milk fat globule membrane, it can optionally be purified by methods such as gel filtration, ion exchange chromatography, affinity chromatography, etc., to obtain a high-purity milk fat globule membrane.

[0079] Furthermore, there are no particular restrictions on the source of the milk fat globule membrane, which can be derived from various milk sources. In some preferred embodiments, from the perspective of more favorablely promoting the growth of Akkermansia strains, the milk fat globule membrane can be derived from cow's milk, yak milk, buffalo milk, goat milk, camel milk, or horse milk, and more preferably from cow's milk, yak milk, buffalo milk, or goat milk.

[0080] In some other specific embodiments, the milk fat globule membrane can also be used incidentally, that is, by incidentally introducing the milk fat globule membrane using a raw material containing the milk fat globule membrane.

[0081] (α-lactalbumin)

[0082] Alpha-lactalbumin is a protein found in the milk of mammals. It is widely present in the milk of mammals and participates in lactose production as a component of lactose synthase. It also has many biological activities, such as antibacterial, antiviral, immune response enhancement, antioxidant and antitumor activities. Its high tryptophan content can also help improve sleep and relieve stress. Furthermore, moderate doses of alpha-lactalbumin can promote the growth of Akkermansia muciniphila.

[0083] The α-lactalbumin contained in the culture medium composition of the present invention can be a commercially available product or α-lactalbumin obtained through an extraction process. Furthermore, the extraction process for α-lactalbumin is relatively mature, and the present invention does not impose any particular limitations on the extraction process, as long as it can separate and extract α-lactalbumin. For example, it can be obtained by extraction and separation from milk, wherein the extraction method may include steps such as acid precipitation / rennet coagulation, ultrafiltration, ion exchange, adsorption treatment, and chromatography, and the separation method may be achieved through techniques such as preparative chromatography.

[0084] Furthermore, the present invention does not particularly limit the source of α-lactalbumin, and it can be α-lactalbumin derived from the milk of various mammals, such as α-lactalbumin derived from the milk of cows, yaks, buffalo, goats, camels, horses, etc. Moreover, in some preferred embodiments of the present invention, from the perspective of more advantageously promoting the growth of Akkermansia bacteria, the α-lactalbumin can be α-lactalbumin derived from cow's milk.

[0085] In some other specific embodiments, the α-lactalbumin can also be used incidentally, that is, by using raw materials containing α-lactalbumin and incidentally introducing α-lactalbumin.

[0086] 3-Fucose-based lactose

[0087] 3-Fucose-based lactose is a human milk oligosaccharide composed of fucose, galactose, and glucose. It has many health benefits, including inhibiting pathogen infection, regulating gut microbiota, and enhancing immunity. It can also serve as a carbon source for the growth of Akkermania bacteria, especially Akkermania myxophilus strains.

[0088] The 3-fucosyllactose contained in the culture medium composition of the present invention can be a commercially available product or 3-fucosyllactose obtained through a preparation process. Furthermore, the preparation process of the 3-fucosyllactose is not particularly limited, as long as 3-fucosyllactose can be obtained. In some specific embodiments, the preparation process of 3-fucosyllactose includes methods such as breast milk extraction, chemical synthesis, and biosynthesis. Biosynthesis includes, but is not limited to, enzyme-catalyzed synthesis and microbial fermentation synthesis. Specifically, 3-fucosyllactose can be synthesized using Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Yarrowia lipolytica. In addition, after obtaining 3-fucosyllactose, it can be optionally purified by methods such as gel filtration, ion exchange chromatography, affinity chromatography, adsorption treatment (desalting, decolorization, etc.), concentration and crystallization to obtain high-purity 3-fucosyllactose.

[0089] In some other specific embodiments, the 3-fucosyllactose can also be used incidentally, that is, by using raw materials containing 3-fucosyllactose and incidentally introducing 3-fucosyllactose.

[0090] In addition, since 2'-fucosyllactose and difucosyllactose are both fucosylated oligosaccharides with similar structures and functions as 3-fucosyllactose, appropriate amounts of 2'-fucosyllactose and / or difucosyllactose can be used to replace the 3-fucosyllactose of this application in some other specific embodiments.

[0091] (The synergistic effect of milk fat globule membrane, α-lactalbumin and 3-fucosylated lactose)

[0092] Although current research on milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose shows that all three have certain effects on improving the intestinal microecology, and that milk fat globule membrane and 3-fucosyllactose may affect the growth and survival of Akkermansia bacteria, there are no studies or reports on the dose-dependent effects of the individual effects of milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose on the growth of Akkermansia bacteria, or on whether the combination of the three has a synergistic effect on the growth of Akkermansia bacteria.

[0093] The growth-promoting effect of a single substance on Akkermansia bacteria

[0094] This invention first investigated the growth of Akkermania bacteria in a composition containing milk fat globule membrane, α-lactalbumin, or 3-fucosyllactose, and found that the utilization of any one of milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose by Akkermania bacteria was dose-dependent.

[0095] In some specific embodiments, to increase the accuracy of the detection results for Akkermansia spp. growth, this invention performs the detection when Akkermansia spp. can barely maintain growth. Specifically, this invention uses a reduced-sugar medium as the basic nutrient component and forms a culture medium composition for culturing Akkermansia spp. by adding different doses of various additive components. For the reduced-sugar medium, a reduced-sugar MRS medium is preferred, and the amount of glucose added is not particularly limited, and can be 0.01g / 100mL to 0.1g / 100mL, for example 0.01g / 100mL, 0.025g / 100mL, 0.05g / 100mL, 0.075g / 100mL, or 0.1g / 100mL. In some preferred embodiments, the amount of glucose added is 0.025g / 100mL to 0.075g / 100mL. When the glucose content in the reduced-sugar MRS medium is within the above range, the growth rate of Akkermansia spp. is slow, but growth can be maintained, which facilitates subsequent experiments.

[0096] In some specific embodiments, a single milk fat globule membrane can promote the growth of Akkermansia bacteria, wherein the amount of milk fat globule membrane added to the culture medium composition is 0.1 g / L to 3 g / L, for example 0.1 g / L, 0.125 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 2.5 g / L, 3 g / L, preferably 0.1 g / L to 2 g / L, more preferably 0.25 g / L to 2 g / L. When the amount of milk fat globule membrane added is within the above range, the total bacterial count of Akkermansia bacteria is high, the growth rate is fast, and the growth-promoting effect is significant. For example, it increases the OD of Akkermansia bacteria during the logarithmic growth phase and / or stationary phase. 600 The value is higher.

[0097] In some specific embodiments, α-lactalbumin alone can promote the growth of Akkermansia bacteria, wherein the amount of α-lactalbumin added to the culture medium composition is 0.1 g / L to 5 g / L, for example 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.6 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, preferably 0.4 g / L to 1.6 g / L. When the amount of α-lactalbumin added is within the above range, the total bacterial count of Akkermansia bacteria is high, the growth rate is fast, and the growth-promoting effect is significant. For example, it reduces the OD of Akkermansia bacteria during the logarithmic growth phase and / or stationary phase.600 The value is higher.

[0098] In some specific embodiments, 3-fucosyllactose alone can promote the growth of Akkermansia bacteria, wherein the amount of 3-fucosyllactose added to the culture medium composition is 0.1 g / L to 2 g / L, for example 0.1 g / L, 0.25 g / L, 0.438 g / L, 0.656 g / L, 0.875 g / L, 1.313 g / L, 1.75 g / L, 2 g / L, preferably 0.25 g / L to 0.675 g / L. When the amount of 3-fucosyllactose added is within the above range, the total bacterial count of Akkermansia bacteria is high, the growth rate is fast, and the growth-promoting effect is significant. For example, it increases the OD of Akkermansia bacteria during the logarithmic growth phase and / or stationary phase. 600 The value is higher.

[0099] The growth-promoting effect of the ternary combination on Akkermansia bacteria

[0100] Through further investigation, this invention has discovered that when milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose are used in combination, the ternary combination of the three substances can more effectively promote the growth of Akkermansia bacteria compared to a single substance.

[0101] In some specific implementations, when milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose are added to the culture medium composition in specific doses, the growth of Akkermania bacteria, especially Akkermania myxophilus, can be promoted more effectively.

[0102] Based on this, the present invention provides a culture medium or edible nutrient composition that can efficiently promote the growth of Akkermania bacteria, and is superior to the effect of any one of the three single substances. Furthermore, the combination of the three substances has a synergistic effect on the proliferation of Akkermania bacteria, particularly Akkermania myxophilus.

[0103] In some specific embodiments, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose described in this invention in the culture medium or edible nutrient composition is (0.1~3):(0.1~5):(0.1~2), where the unit of concentration is "g / L". The culture medium or edible nutrient composition meeting the above conditions can effectively promote the proliferation of Akkermansia bacteria, and the three components have a synergistic effect.

[0104] In some preferred embodiments, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose can be (0.1~2):(0.1~4):(0.25~1.75), more preferably (0.25~2):(0.4~3.2):(0.25~1.312), and even more preferably (0.25~2):(0.4~1.6):(0.25~1.312). Preferably, the ratio can be (0.25~2):(0.4~1.6):(0.25~0.656); exemplarily, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose can be 2:0.4:0.25, 2:0.4:0.656, 2:1.6:0.25, 2:1.6:0.656, 0.25:0.4:0.25, 0.25:0.4:0.656, 0.2 The following ratios are preferred: 5:1.6:0.25, 0.5:0.8:0.5, 0.5:0.8:1.312, 0.5:3.2:0.5, 0.5:3.2:1.312, 1:0.2:0.328, 1:0.8:0.328, 0.125:0.2:0.328, 0.125:0.8:0.328, etc., with 2:0.4:0.25, 2:0.4:0.656, and 2:1.6:0. 25, 2:1.6:0.656, 0.25:0.4:0.25, 0.25:0.4:0.656, 0.25:1.6:0.25, 0.5:0.8:1.312, 0.5:3.2:0.5, 0.5:3.2:1.312, 1:0.2:0.328, 1:0.8:0.328, 0.125:0.2:0.328, 0.125:0.8:0.328.

[0105] Furthermore, as long as the above concentration ratios are met, and under the premise of ensuring synergistic effects and dosage safety, the present invention does not impose any principle restrictions on the amount of milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose.

[0106] The present invention does not particularly limit the form of the culture medium composition, which can be adjusted according to the culture conditions. In some specific embodiments, the culture medium composition of the present invention may contain a solvent, such as water. Therefore, its form may be, for example, a liquid culture medium, a semi-solid culture medium, or a solid culture medium.

[0107] <Second aspect. Application of culture medium compositions for promoting the growth of Akkermansia bacteria>

[0108] In another aspect of the invention, the use of the above-described culture medium composition for promoting the growth of Akkermania bacteria is also provided, wherein the culture medium composition is used under anaerobic conditions to promote the growth of Akkermania bacteria.

[0109] In some specific embodiments, the Akkermansia genus bacteria are not particularly limited, and may include one or more of Akkermansia muciniphila, Akkermansia glycaniphila, and Akkermansia biwaensis. Preferably, the Akkermansia genus bacteria may be Akkermansia muciniphila.

[0110] Therefore, the culture medium composition of the present invention can effectively promote the growth of Akkermania bacteria under anaerobic conditions, especially Akkermania myxophilus, and the added components therein have a significant synergistic effect.

[0111] In some specific embodiments, the culture medium composition of the present invention can be used to culture Akkermania bacteria under anaerobic conditions. The culture method is not particularly limited as long as it enables the proliferation of Akkermania bacteria; for example, methods commonly used in the culture of Akkermania bacteria can be used directly or modified thereof. The culture temperature is also not particularly limited, for example, it can be 25–50°C, preferably 35–42°C, for example, 25°C, 27°C, 30°C, 33°C, 35°C, 37°C, 40°C, or 42°C. Furthermore, the culture is preferably carried out under strictly anaerobic conditions, for example, under anaerobic conditions, for example, by actively creating an anaerobic environment using anaerobic workstations, anaerobic culture bags, etc. The culture can be carried out, for example, until the Akkermania bacteria have proliferated to the desired extent.

[0112] <Third aspect. Compositions that promote the growth of Akkermansia bacteria in the intestine>

[0113] In another aspect of the invention, a composition is provided that promotes the growth of Akkermansia bacteria in the intestine, wherein the composition necessarily comprises three components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose.

[0114] The contents of milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose are the same as described in the first aspect above; the contents of Akkermania bacteria are the same as described in the second aspect above; and will not be repeated here.

[0115] <Fourth aspect. Application of edible compositions in promoting the growth of Akkermansia bacteria in the intestine>

[0116] In another aspect of the invention, the use of an edible or nutritional composition in promoting the growth of Akkermansia bacteria in the gut is provided; more specifically, the use of a composition in preparing a food having the effect of promoting the growth of Akkermansia bacteria in the gut is provided, wherein the composition comprises the three essential components described above: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose, and optionally comprises other nutritional components.

[0117] The contents of milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose are the same as described in the culture medium composition above. In some preferred embodiments, the concentration ratio of milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose in the composition is (0.1~3):(0.1~5):(0.1~2), where the concentration is expressed in g / L. Culture medium compositions meeting the above conditions can more effectively promote the proliferation of Akkermansia bacteria, as the three components have a synergistic effect. Therefore, they can increase the number of potentially beneficial Akkermansia bacteria in the gut, thus improving gut health.

[0118] In some preferred embodiments, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose can be (0.1~2):(0.1~4):(0.25~1.75), more preferably (0.25~2):(0.4~3.2):(0.25~1.312), and even more preferably (0.25~2):(0.4~1.6):(0.25~1.312). Preferably, the ratio can be (0.25~2):(0.4~1.6):(0.25~0.656); exemplarily, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose can be 2:0.4:0.25, 2:0.4:0.656, 2:1.6:0.25, 2:1.6:0.656, 0.25:0.4:0.25, 0.25:0.4:0.656, 0.2 The following ratios are preferred: 5:1.6:0.25, 0.5:0.8:0.5, 0.5:0.8:1.312, 0.5:3.2:0.5, 0.5:3.2:1.312, 1:0.2:0.328, 1:0.8:0.328, 0.125:0.2:0.328, 0.125:0.8:0.328, etc., with 2:0.4:0.25, 2:0.4:0.656, and 2:1.6:0. 25, 2:1.6:0.656, 0.25:0.4:0.25, 0.25:0.4:0.656, 0.25:1.6:0.25, 0.5:0.8:1.312, 0.5:3.2:0.5, 0.5:3.2:1.312, 1:0.2:0.328, 1:0.8:0.328, 0.125:0.2:0.328, 0.125:0.8:0.328.

[0119] Similarly, provided the above proportions are met, there are no particular restrictions on the mass content of milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose in the composition, and they can be added in accordance with the usual dosage and the requirements of relevant laws and regulations.

[0120] In particular, the composition of this invention can promote the growth and reproduction of Akkermania bacteria in the intestine. Akkermania bacteria, especially *Akkermania mucinosa*, are beneficial intestinal bacteria, but they are not among the species permitted for use in food according to regulations. Therefore, the composition provided by this invention that promotes the growth of Akkermania bacteria in the intestine is of particular significance, as it can promote human health by exerting its effect of promoting the growth of Akkermania bacteria in the intestine.

[0121] The composition of the present invention having the effect of promoting the growth of Akkermansia spp. bacteria in the intestine can be a food composition. Therefore, in addition to the essential milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose, depending on the type of the final product, the composition of the present invention having the effect of promoting the growth of Akkermansia spp. bacteria in the intestine may also optionally contain nutritional components, including but not limited to food ingredients, such as plant or plant extracts thereof, milk-containing components, animal components, functional additives, trace element supplements, and / or any food-acceptable excipients.

[0122] Plants or plant extracts, including fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, rubber, grape, pear, cherry, blueberry, blackberry, raspberry, and bilberry, or their extracts; fruits and vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, foxtail millet, yellow millet, buckwheat, soybean, broad bean, pea, mung bean, red bean, and kidney bean, or their extracts; nuts such as walnut, pistachio, cashew, hazelnut, almond, apricot kernel, pine nut, peanut, sunflower seed, chestnut, macadamia nut, and ginkgo, or their extracts; and coffee or its extracts.

[0123] The product contains milk or protein components. The milk components include dairy products such as fresh milk, milk powder, whey protein, or cheese derived from raw cow (sheep) milk. The protein components may be derived from plant proteins, such as soy protein or peanut protein.

[0124] Animal-derived ingredients, including meat products from cattle, sheep, fish, or poultry.

[0125] The fat component may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, DHA, EPA, ARA, and phospholipids. More specifically, the fat includes safflower seed oil, walnut oil, peanut oil, soybean oil, argan oil, olive oil, tea oil, sacha inchi oil, coconut oil, perilla oil, deep-sea fish oil, cocoa butter, palm oil, butter, cream, lard, medium-chain triglycerides, or lecithin, etc.

[0126] Functional additives include vitamins (one or more of vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, and biotin), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, or L-leucine, etc.), traditional Chinese medicine or its extracts, and dietary fiber (inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, or soybean fiber, etc.).

[0127] Micronutrient supplements may include metal ion salts of organic acids, such as one or more of the following: calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate.

[0128] Any food additives that are acceptable, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0129] Furthermore, the present invention does not particularly limit the form of the composition having the effect of promoting the growth of Akkermansia muciniphila in the intestine; for example, it can be liquid, semi-solid, or solid.

[0130] <Fifth Aspect: Products>

[0131] In another aspect of the invention, a product is provided comprising, or prepared from, the above-described composition having the effect of promoting the growth of Akkermansia spp. bacteria in the intestine. In some specific embodiments, the product of the present invention includes food.

[0132] When the product is a food product, as previously stated, milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose are used in accordance with local laws and regulations.

[0133] In some specific embodiments, the present invention does not particularly limit the form of the product; for example, it may be a liquid, semi-solid, or solid. In some preferred embodiments, the product is a liquid, semi-solid, or solid at room temperature. There are no particular limitations on the food products of the present invention; they can generally be powdered reconstituted foods, baked goods, beverages, confectionery, milk and dairy products, fermented products, pasta products, or oral preparations.

[0134] For powdered reconstituteable foods, typical examples include reconstituteable milk powder products, such as infant formula and adult milk powder, as well as instant coffee, cereal powder, nut powder, or lotus root powder.

[0135] For baked goods, this can be cakes or cookies that are mainly based on butter, eggs, and baking powder.

[0136] Beverages can include carbonated drinks, fruit and vegetable juices, functional drinks, tea drinks, dairy drinks, or alcoholic drinks.

[0137] Candy can include hard candy, hard filled candy, cream candy, gel candy, polished candy, gum-based candy, aerated candy, and compressed candy, etc.

[0138] Milk and dairy products may include fresh milk, milk powder, whey powder, fermented milk, cheese, or condensed milk derived from raw cow (sheep) milk.

[0139] Fermented products typically include fermented milk, fermented soy milk, and may also include fermented sauces.

[0140] For pasta products, this can include staple foods made from flour or other grains, such as steamed buns, pancakes, noodles, and filled pasta products.

[0141] Oral preparations may include tablets, pills, granules, powders, teas, capsules, and oral liquids.

[0142] The food provided by this invention is suitable for all people in principle, especially those who need to improve their gut health. The components of the food can be adjusted accordingly for people with different characteristics. For example, the food provided by this invention can be a functional food, a nutritional food, or a health-promoting food. In addition to the above-mentioned food categories, the food of this invention can also include animal foods such as pet food, livestock feed, and fish feed.

[0143] In some specific embodiments, the content of each component in the food of the present invention is not particularly limited, as long as the concentration ratio of milk fat globule membrane, α-lactalbumin and 3-fucosylated lactose meets the condition of (0.1~3):(0.1~5):(0.1~2), so as to promote the growth of Akkermansia myxophilus in the human intestine, increase the number of potential probiotic Akkermansia bacteria (Ackermansia myxophilus) in the intestine, and improve intestinal health.

[0144] Example

[0145] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0146] The raw materials and equipment involved in the following embodiments are as follows:

[0147] • Milk fat globule membrane (MFGM): Purchased from Friesland, whey protein powder MFGM;

[0148] • Alpha-lactalbumin: Purchased from Arla, catalog number Lacprodan ALPHA-10;

[0149] • 3-Fucose-based lactose (3-FL): Purchased from Huangshan Tongxi Biotechnology Co., Ltd.;

[0150] • Akkermansia bacteria: Akkermansia myxophilus (hereinafter referred to as AKK bacteria), strain number AKK11, isolated from the intestines of healthy infants in Hongyuan Pastoral Area, Sichuan Province, purchased from WeCon Probiotics (Suzhou) Co., Ltd.;

[0151] Materials and equipment: MRS culture medium, purchased from Beijing Aoboxing Biotechnology Co., Ltd.; anaerobic culture bags, purchased from Qingdao Haibo Biotechnology Co., Ltd.; microplate reader, model SpectraMax reg iD3, purchased from Meigu Instruments (Shanghai) Co., Ltd.; fully automated growth curve analyzer, model BK400, purchased from Shandong Boke Biotechnology Co., Ltd.

[0152] The methods for activating and subculturing AKK strains are as follows:

[0153] Take 0.2g of AKK bacteria powder (strain number: AKK11) stored at -28℃ and inoculate it into 20 mL of BHI medium containing 0.4% (w / v) mucin, 0.05% (w / v) L-cysteine ​​hydrochloride and 0.0001% (w / v) resazurin. Shake gently and place in an incubator at 37℃. Activation is completed after 24h of anaerobic culture.

[0154] Example 1: Growth of AKK bacteria in MRS media with different glucose contents

[0155] The above-mentioned AKK bacteria were inoculated at a 1% inoculum into MRS medium with different glucose concentrations (0.01 g / 100 mL, 0.025 g / 100 mL, 0.05 g / 100 mL, 0.075 g / 100 mL, and 0.1 g / 100 mL, respectively). The culture was carried out under anaerobic conditions of 85% N2, 5% H2, and 10% CO2 at 37°C for 20 h. The cultured bacterial solution was then aspirated, centrifuged at 6000 rpm for 5 min (4°C), the supernatant was discarded, and the bacterial cells were resuspended in sterile PBS. The centrifugation and washing were repeated twice. The PBS-resuspended bacterial solution was filtered through a 0.22 μm sterile filter membrane, and the optical density (OD) was measured at a wavelength of 600 nm. 600 This is used to monitor the concentration of the filtered bacterial suspension. For example... Figure 1 As shown, AKK bacteria grow slowly in MRS medium with 0.05 g / 100 mL glucose, barely maintaining growth. Therefore, the glucose concentration in the reduced-sugar MRS medium was set at 0.05 g / 100 mL for subsequent experiments to promote AKK growth using MFGM, α-lactalbumin, and 3-FL.

[0156] Example 2: Growth of AKK bacteria in MRS medium with different doses of MFGM

[0157] Using commercially available infant formula as a reference, a liquid MFGM concentration of 0.5 g / L was set as the baseline dose. Based on this baseline dose, the following concentrations of MFGM were set: 0.1 g / L, 0.125 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, and 2 g / L. Activated second-generation AKK bacterial culture was inoculated at a 1% (v / v) volume fraction into reduced-sugar MRS medium containing the six different doses of MFGM. Reduced-sugar MRS medium without MFGM served as a blank control. The experiment was incubated at 37°C under static conditions, and the optical density (OD) was measured at 600 nm. 600 The concentration of the filtered bacterial suspension was monitored using [method name missing], with a sampling interval of 2 hours, and the operation lasted for 36 hours. The growth curves of AKK bacteria under different doses of MFGM are shown below. Figure 2 As shown.

[0158] Depend on Figure 2 It can be seen that, compared with the control group, AKK bacteria showed a significantly higher OD rate during the logarithmic growth phase in a culture medium containing six different doses of MFGM. 600 The values ​​were higher, and AKK bacteria exhibited larger OD values ​​during the stationary phase in media containing 0.25 g / L, 0.5 g / L, 1 g / L, and 2 g / L MFGM. 600The total bacterial count was high, especially the OD value was highest at an MFGM concentration of 2 g / L. This result indicates that the utilization of MFGM by AKK bacteria is dose-dependent, and MFGM concentrations of 0.25–2 g / L promote the growth of AKK bacteria.

[0159] Example 3: Growth of AKK bacteria in MRS medium with different doses of α-lactalbumin

[0160] Using commercially available infant formula as a reference, a liquid concentration of 0.4 g / L of α-lactalbumin was set as the baseline dose. Based on this baseline dose, the following concentrations of α-lactalbumin were established: 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.6 g / L, and 4 g / L. Activated second-generation AKK bacterial culture was inoculated at a 1% (v / v) volume fraction into reduced-sugar MRS medium containing the six different doses of α-lactalbumin. Reduced-sugar MRS medium without α-lactalbumin served as a blank control. The experiment was incubated at 37°C under static conditions, and the optical density (OD) was measured at 600 nm. 600 The concentration of the filtered bacterial suspension was monitored using a method with a sampling interval of 2 hours, and the operation lasted for 36 hours. The growth curves of AKK bacteria under different doses of α-lactalbumin are shown below. Figure 3 As shown.

[0161] Depend on Figure 3 It was found that, compared with the control group, *AKK* bacteria exhibited a larger OD during the stationary phase in media containing 0.4 g / L, 0.8 g / L, and 1.6 g / L α-lactalbumin. 600 The total bacterial count was high, especially when the α-lactalbumin content was 0.4 g / L, and the OD value was also high. 600 The value was the highest. This result indicates that the utilization of α-lactalbumin by AKK bacteria is dose-dependent, but excessively high doses affect the growth of AKK bacteria. α-lactalbumin at concentrations of 0.4–1.6 g / L promotes the growth of AKK bacteria.

[0162] Example 4: Growth of AKK bacteria in MRS medium with different doses of 3-FL

[0163] Using commercially available infant formula mixing ratios as a baseline, a 3-FL liquid concentration of 0.875 g / L was established as the baseline dose. Based on this baseline dose, the following concentrations of 3-FL were set: 0.25 g / L, 0.438 g / L, 0.656 g / L, 0.875 g / L, 1.313 g / L, and 1.75 g / L (where 0.25 g / L is the minimum legal limit and 1.75 g / L is the maximum legal limit). Activated second-generation AKK bacterial culture was inoculated at a 1% (v / v) volume fraction into reduced-sugar MRS medium containing the six different doses of 3-FL. Reduced-sugar MRS medium without 3-FL served as a blank control. The experiment was incubated at 37°C under static conditions, and the optical density (OD) was measured at 600 nm. 600 The concentration of the filtered bacterial suspension was monitored using a method with a sampling interval of 2 hours, and the operation lasted for 36 hours. The growth curves of AKK bacteria at different doses of 3-FL are shown below. Figure 4 As shown.

[0164] Depend on Figure 4 It can be seen that, compared with the control group, although *AKK* bacteria entered the logarithmic growth phase at approximately the same time in media containing 0.25 g / L to 1.313 g / L of 3-FL and in the control group, *AKK* bacteria exhibited higher absorbance values ​​and higher total bacterial counts during the stationary phase in media containing 0.25 g / L, 0.438 g / L, and 0.656 g / L of 3-FL, especially at a 3-FL concentration of 0.25 g / L. 600 The value was the highest. This result indicates that a moderate dose of 3-FL is needed to promote the growth of AKK bacteria, but excessive doses will affect the growth of AKK bacteria. Concentrations of 3-FL at 0.25~0.656 g / L have a promoting effect on the growth of AKK bacteria.

[0165] Example 5: Growth of AKK bacteria in MRS medium with different dosages of the composition

[0166] According to Example 2, the optimal dosage of MFGM for promoting AKK bacterial proliferation was selected as 0.25~2 g / L. According to Example 3, the optimal dosage of α-lactalbumin was selected as 0.4~1.6 g / L. According to Example 4, the optimal dosage of 3-FL was selected as 0.25~0.656 g / L. MFGM, α-lactalbumin, and 3-FL were combined at the aforementioned optimal dosages for promoting AKK bacterial proliferation, i.e., in a concentration ratio of (0.25~2 g / L):(0.4~1.6 g / L):(0.25~0.656 g / L), resulting in concentration ratios of 2:0.4:0.25, 2:0.4:0.656, 2:1.6:0.25, 2:1.6:0.656, 0.25:0.4:0.25, and 0.25:0.4:0.25. Fifteen compositions of MFGM, α-lactalbumin, and 3-FL were prepared, with ratios of 5:0.4:0.656, 0.25:1.6:0.25, 0.5:0.8:0.5, 0.5:0.8:1.312, 0.5:3.2:0.5, 0.5:3.2:1.312, 1:0.2:0.328, 1:0.8:0.328, 0.125:0.2:0.328, and 0.125:0.8:0.328. Activated second-generation AKK bacterial culture was inoculated at a 1% (v / v) volume fraction into reduced-sugar MRS medium containing different doses of the MFGM, α-lactalbumin, and 3-FL compositions. Reduced-sugar MRS medium without the compositions served as a blank control. The experiment was incubated at 37°C under static conditions, and the optical density (OD) was measured at 600 nm. 600 The concentration of the filtered bacterial suspension was monitored using a method with a sampling interval of 2 hours, and the operation lasted for 36 hours. The growth curves of AKK bacteria under different dosage combinations are shown below. Figure 5 As shown.

[0167] Depend on Figure 5 It was found that AKK entered the logarithmic growth phase at approximately the same time as the control group in all 15 dosage compositions. During the stationary phase, compositions with ratios of 2:0.4:0.25, 2:1.6:0.656, 0.25:1.6:0.25, 0.5:3.2:0.5, and 0.5:3.2:1.312 all promoted AKK growth and increased OD. 600 Values. In particular, the 2:0.4:0.25 composition most significantly promoted AKK growth, approximately equal to the OD value of the control group. 600 1.63 times that.

[0168] Example 6: Growth of AKK bacteria in MRS medium containing appropriate doses of MFGM, α-lactalbumin, 3-FL, and the combination thereof

[0169] According to Example 2, the optimal dose of MFGM for promoting AKK bacterial proliferation was 2 g / L. According to Example 3, the optimal dose of α-lactalbumin was 0.4 g / L. According to Example 4, the optimal dose of 3-FL was 0.25 g / L. According to Example 5, the suitable dose of the composition was a combination of the three components in the following ratios: 2:0.4:0.25, 2:1.6:0.656, 0.25:1.6:0.25, 0.5:3.2:0.5, and 0.5:3.2:1.312, respectively. The activated second-generation AKK bacterial culture was inoculated at a 1% (v / v) volume fraction into reduced-sugar MRS medium supplemented with the three optimal doses of MFGM, α-lactalbumin, 3-FL, and five suitable combinations thereof, respectively. Reduced-sugar MRS medium without MFGM, α-lactalbumin, 3-FL, or any combination thereof served as a blank control. The experiment was incubated at 37°C and statically cultured. The optical density (OD) was measured at 600 nm. 600 The concentration of the filtered bacterial suspension was monitored, with a sampling interval of 2 hours, and the operation lasted for 36 hours. The ΔOD of AKK bacteria was measured under appropriate doses of MFGM, α-lactalbumin, 3-FL, and the combined culture conditions. 600 The changes over time (excluding the blank control) are shown in Tables 1, 2 and 3.

[0170] Table 1: ΔOD of AKK bacteria under different ratios of MFGM, α-lactalbumin, 3-FL combinations and MFGM (2 g / L) culture conditions 600 (Excluding blank control) Changes over time

[0171]

[0172] Note: , as well as The values ​​represent the ΔOD values ​​of the composition and the composition under culture conditions of 2 g / L MFGM, respectively. 600nm Compared to these, P < 0.05, P < 0.01, and P < 0.001.

[0173] Table 2: ΔOD of AKK bacteria under different ratios of MFGM, α-lactalbumin, 3-FL combination and α-lactalbumin (0.4 g / L) culture conditions 600 (Excluding blank control) Changes over time

[0174]

[0175] Note: , as well as The values ​​represent the ΔOD values ​​of the composition and 0.4 g / L α-lactalbumin under culture conditions. 600nm Compared to these, P < 0.05, P < 0.01, and P < 0.001.

[0176] Table 3: ΔOD of AKK bacteria under different ratios of MFGM, α-lactalbumin, 3-FL composition and 3-FL (0.25 g / L) culture conditions 600 (Excluding blank control) Changes over time

[0177]

[0178] Note: , as well as The values ​​represent the ΔOD values ​​of the composition and the composition under 0.25 g / L 3-FL culture conditions, respectively. 600nm Compared to these, P < 0.05, P < 0.01, and P < 0.001.

[0179] Data from Tables 1, 2, and 3 show that individual MFGM (2 g / L), α-lactalbumin (0.4 g / L), and 3-FL (0.25 g / L) all promoted the growth of AKK bacteria (ΔOD). 600nm (Not zero). The five ratio compositions selected in this example (2:0.4:0.25, 2:1.6:0.656, 0.25:1.6:0.25, 0.5:3.2:0.5, 0.5:3.2:1.312) also promoted the growth of AKK bacteria (ΔOD). 600nm (Not zero). ΔOD at corresponding time under the culture conditions of 5 ratio compositions. 600nm All were significantly higher than the ΔOD values ​​under single culture conditions of MFGM (2 g / L), α-lactalbumin (0.4 g / L), and 3-FL (0.25 g / L). 600nm The values ​​(P<0.05, P<0.01, or P<0.001) were significant, and this significant difference persisted for up to 36 hours. Simultaneously, the ΔOD values ​​of all these compositions at the corresponding time points were... 600nm The ΔOD was higher than that of MFGM (2 g / L), α-lactalbumin (0.4 g / L), and 3-FL (0.25 g / L) alone. 600nm The above data shows that the five proportions of the composition selected in this embodiment can all promote the growth of AKK bacteria to varying degrees, and that MFGM, α-lactalbumin and 3-FL have a synergistic effect on the proliferation of AKK bacteria.

[0180] In summary, further evidence demonstrates that the preferred composition ratio is MFGM, α-lactalbumin, and 3-FL concentrations of 0.25~2 g / L, 0.4~3.2 g / L, and 0.25~1.312 g / L, respectively. Compositions meeting these ratios effectively promote the proliferation of AKK bacteria. The most preferred composition ratio is MFGM, α-lactalbumin, and 3-FL concentrations of 0.25~2 g / L, 0.4~1.6 g / L, and 0.25~0.656 g / L, respectively. Compositions meeting these ratios more effectively promote the proliferation of AKK bacteria, exhibiting a synergistic effect among the three components. Therefore, this can increase the number of potentially beneficial AKK bacteria in the gut, improving gut health.

[0181] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0182] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A culture medium composition for promoting the growth of Akkermansia bacteria, characterized in that, The culture medium composition includes basic nutrient components and additive components. The added components include three essential ingredients: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose. In the culture medium composition, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose is (0.25~2):(0.4~1.6):(0.25~0.656). The Akkermansia bacteria in question are Akkermansia muciniphila.

2. The culture medium composition according to claim 1, characterized in that, The basic nutritional components include one or more of the following: protein components, lipid components, carbohydrate components, vitamin components, mineral components, and pH-regulating components.

3. The application of the culture medium composition according to claim 1 or 2 for promoting the growth of Akkermansia bacteria, characterized in that, The culture medium composition is used under anaerobic conditions to promote the growth of the Akkermansia bacteria.

4. A composition having the effect of promoting the growth of Akkermansia bacteria, characterized in that, The composition comprises three components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose. In the composition, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose is (0.25~2):(0.4~1.6):(0.25~0.656). The Akkermansia bacteria in question are Akkermansia muciniphila.

5. The use of a composition in the preparation of a product having the effect of promoting the growth of Akkermansia bacteria, characterized in that, The composition comprises three components: milk fat globule membrane, α-lactalbumin, and 3-fucosylated lactose. In the composition, the concentration ratio of the milk fat globule membrane, α-lactalbumin, and 3-fucosyllactose is (0.25~2):(0.4~1.6):(0.25~0.656). The Akkermansia bacteria in question are Akkermansia muciniphila.

6. The application according to claim 5, characterized in that, The product can be liquid, semi-solid, or solid.

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