Use of n-acetylneuraminic acid substance in assisting regulation of gut microbiota of mother and offspring

Abstract

Use of an N-acetylneuraminic acid substance in the preparation of a food product for promoting the regulation of gut microbiota of a mother and offspring thereof via ingestion by the mother. The N-acetylneuraminic acid substance is an N-acetylneuraminic acid anhydrate, an N-acetylneuraminic acid hydrate or an N-acetylneuraminic acid salt. The mother is in a gestational period and / or lactation period, and the offspring is in a fetal stage, infant stage and / or toddler stage. The mother starting to ingest the N-acetylneuraminic acid substance in the gestational period can promote the increase of beneficial bacteria in the gut in the gestational and lactation periods, and can also increase the content of probiotics in the offspring gut, inhibit the proliferation of opportunistic pathogens in the offspring gut and reduce the relative abundance thereof, thereby further contributing to maintaining the health of the nervous system by means of the action of the gut-brain axis.

Description

Uses of N-acetylneuraminic acid compounds in regulating the gut microbiota of mothers and children Technical Field

[0001] This invention belongs to the food field, specifically relating to the use of N-acetylneuraminic acid compounds to help regulate the intestinal flora of mothers and infants. Background Technology

[0002] The gut, a vital part of the human digestive system, is home to a vast and complex array of microorganisms that have co-evolved with the host. Current research has identified key members of the human gut microbiota, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucous Microbes. The gut microbiota is often referred to as the "second brain," and numerous studies have demonstrated its crucial role in human health. The first 1000 days of life are not only a critical period for brain development but also a critical time for the establishment of the gut microbiota. The establishment of a healthy gut microbiota in early life has a significant and far-reaching impact on health outcomes throughout fetal development, childhood, and adulthood.

[0003] Studies have shown that maternal nutrition during pregnancy has a significant impact on the healthy outcomes of the offspring throughout their lifespan. Furthermore, during lactation, breast milk not only provides the fetus with nutrients but also provides immune-active components and beneficial microorganisms crucial for healthy growth and development. Current research suggests that the initial gut microbiota of newborns originates from their mothers. A healthy maternal gut microbiota can pass on a better microbiota to the offspring and influence the establishment of the offspring's gut microbiota, laying a solid foundation for a healthy offspring gut microbiota.

[0004] N-acetylneuraminic acid, also known as neuraminic acid, is a non-carbon monosaccharide with an N-acetylamino group. It is a common form of sialic acid. N-acetylneuraminic acid is typically fused to the non-reducing ends of glycolipids and glycoproteins on cell membranes, participating in cell recognition and the regulation of various biological processes. Studies have found that sialic acid has the function of regulating the anti-inflammatory activity of IgG, enhancing the body's immunity, and affecting the integrity, permeability, and activity of nerve cells. Currently, N-acetylneuraminic acid can be used as a raw material in food.

[0005] Reference 1 (CN102946728B) discloses a method for providing sialic acid to a woman, comprising administering sialic acid in the form of N-acetylneuraminic acid to the woman in at least one of the following stages: preconception, pregnancy, and lactation, wherein the sialic acid provides developmental benefits to the nervous system and / or brain of the woman’s fetus or child.

[0006] Reference 2 (CN119054770A) discloses that sialic acid, as a nutrient for gut microbiota, can promote the growth of microorganisms capable of metabolizing sialic acid, such as Bacteroides fragilis and Myxobacterium ackermannii, thus affecting gut homeostasis. Studies have shown that supplementation with sialic acid-containing glycoproteins or sialylated glycans can alter the composition of the entire gut microbiota, thereby improving health, including reducing the risk of colorectal cancer and atherosclerosis. Dietary intake of sialic acid, especially bound sialylated glycans, is particularly important for human gut health. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] The mother's overall nutritional environment during pregnancy and lactation affects the nutrients she provides to her offspring, but there are currently few studies on improving the mother's nutrition to regulate the establishment of the offspring's gut microbiota.

[0009] Existing research indicates that maternal supplementation with N-acetylneuraminic acid during pregnancy may benefit fetal nervous system development. However, no research has shown that maternal supplementation with N-acetylneuraminic acid during pregnancy can regulate maternal gut microbiota and further regulate offspring gut microbiota. Although, for example, cited reference 2 above discloses the effects of sialic acid on gut microbiota, its research focuses on sialyl polysaccharides. Furthermore, even if nutrients are believed to be directly provided to the body to exert specific health benefits, they may not necessarily promote offspring health when ingested by the mother.

[0010] In the course of in-depth research on the physiological functions of N-acetylneuraminic acid, this invention unexpectedly discovered that maternal intake of N-acetylneuraminic acid during pregnancy can significantly improve their gut microbiota and simultaneously improve the gut microbiota of their offspring. N-acetylneuraminic acid can promote the increase of beneficial bacteria in the gut of pregnant and lactating mothers, while also increasing the content of probiotics in the gut of their offspring, inhibiting the proliferation of opportunistic pathogens in the offspring gut, and reducing their relative abundance.

[0011] Therefore, the object of the present invention is to provide the use of N-acetylneuraminic acid substances for non-therapeutic purposes in simultaneously regulating the gut microbiota of pregnant and / or lactating mothers and their offspring. Furthermore, the object of the present invention is to provide the use of N-acetylneuraminic acid substances in the preparation of foods that, when ingested by the mother, help regulate the gut microbiota of the mother and her offspring.

[0012] Solution for solving the problem

[0013] [1]. Use of N-acetylneuraminic acid derivatives in the preparation of foods that help regulate the gut microbiota of mothers and their offspring when ingested by the mother; wherein the N-acetylneuraminic acid derivative is anhydrous N-acetylneuraminic acid, N-acetylneuraminic acid hydrate or N-acetylneuraminic acid salt, the mother is in the pregnancy and / or lactation period, and the offspring is in the fetal, infancy and / or childhood period.

[0014] [2]. According to the use described in [1], the ability to regulate the gut microbiota of the mother and her offspring includes at least one of increasing the relative abundance of *Muribaculaceae unclassified* in the gut of a pregnant mother, increasing the relative abundance of *Prevotellaceae UCG-001* in the gut of a pregnant mother, and increasing the relative abundance of *Bifidobacterium* in the gut of a pregnant mother.

[0015] [3]. According to the use described in [1] or [2], wherein the aid in regulating the gut microbiota of the mother and her offspring includes at least one of increasing the relative abundance of Muribauculaceae unclassified in the gut of a lactating mother and increasing the relative abundance of Akkermansia in the gut of a lactating mother.

[0016] [4]. The use according to any one of [1] to [3], wherein the aid in regulating the gut microbiota of the mother and her offspring includes at least one of: increasing the relative abundance of Muribauculaceae unclassified in the offspring gut, increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Romboutisa in the offspring gut, and decreasing the relative abundance of Escherichia-Shigella in the offspring gut.

[0017] [5]. The use according to any one of [1] to [4], wherein the food helps maintain the nervous system health of the mother and her offspring by influencing the gut-brain axis function of the mother and her offspring by helping to regulate the gut microbiota of the mother and her offspring.

[0018] [6]. The use according to any one of [1] to [5], wherein the food is infant food, children's food, adolescent food or adult food.

[0019] [7]. Use according to any one of [1] to [6], wherein the food is a beverage, confectionery, dairy product, baked goods or dietary supplement.

[0020] [8]. The use according to any one of [1] to [7], wherein, at room temperature, the food is in liquid form, solid-liquid mixture form, solid block form or solid powder form.

[0021] [9]. The use according to any one of [1] to [8], wherein the food contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any food-acceptable excipients.

[0022]

[0010] . According to any one of [1] to [9], wherein the N-acetylneuraminic acid substance in the food contains 0.03% to 2.0% by mass.

[0023] The effects of the invention

[0024] Based on extensive research, this invention proposes that maternal supplementation with a certain amount of N-acetylneuraminic acid during pregnancy and / or lactation can significantly increase the abundance of beneficial bacteria in their intestines, and simultaneously significantly increase the abundance of beneficial bacteria and reduce the content of harmful bacteria in their offspring's intestines. In particular, it can regulate the abundance of some non-edible beneficial bacteria in the intestines of both mother and offspring. Furthermore, regulating the gut microbiota affects the gut-brain axis function; therefore, this invention proposes that maternal supplementation with a certain amount of N-acetylneuraminic acid during pregnancy and / or lactation is also beneficial for maintaining the nervous system health of both mother and offspring.

[0025] Experimental data show that supplementing the mother's gut with N-acetylneuraminic acid during pregnancy and lactation significantly increased the relative abundance of beneficial bacteria *Muribaculaceae unclassified*, *Prevotellaceae* UCG-001, and *Bifidobacterium* in the mother's gut during pregnancy; and increased the relative abundance of beneficial bacteria *Muribaculaceae unclassified* and *Akkermansia* in the mother's gut during lactation. Simultaneously, it significantly increased the relative abundance of beneficial bacteria *Muribaculaceae unclassified*, *Akkermansia*, and *Romboutisa* in the offspring's gut, while decreasing the relative abundance of opportunistic pathogens *Escherichia-Shigella* in the offspring's gut. Attached Figure Description

[0026] Figure 1: Changes in Muribauculaceae unclassified in the intestine of female mice.

[0027] Figure 2: Changes in the intestinal tract of female mice containing unnamed Prevotellaceae (UCG-001).

[0028] Figure 3: Changes in Bifidobacterium in the intestines of female mice.

[0029] Figure 4: Changes in Akkermansia in the intestines of female mice.

[0030] Figure 5: Changes in Muribauculaceae unclassified in the intestines of baby mice.

[0031] Figure 6: Changes in Akkermansia in the intestines of baby mice.

[0032] Figure 7: Changes in Romboutisa in the intestines of baby mice.

[0033] Figure 8: Changes in Escherichia-Shigella in the intestines of baby mice. Detailed Implementation

[0034] The following describes embodiments of the present invention, but the present invention is not limited thereto. Various modifications can be made within the scope of protection claimed in the present invention, and 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.

[0035] I. Terminology Definition

[0036] In this invention, the terms "a", "an", or "the" may refer to "one", "one or more", "at least one", or "one or more".

[0037] In this invention, the range of values ​​represented by “value A to value B”, “value A to value B”, “value A and above”, “value A and below”, “at least value A” and “at most value A” refers to the range that includes the endpoint values ​​A and B.

[0038] In this invention, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both cases in which the event occurs and cases in which the event does not occur.

[0039] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0040] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0041] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, while specific related values ​​have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range.

[0042] In this invention, "normal temperature" refers to an indoor ambient temperature of 23±2℃.

[0043] In this invention, the term "animal milk" is used to refer to the fluid obtained from the mammary glands of a mammal in the process of lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., the fluid obtained directly from the mammary glands) and standardized dairy products.

[0044] In this invention, "gestation period" and "pregnancy period" can be used interchangeably, referring to the period from fertilization to delivery.

[0045] In this invention, "breastfeeding period" refers to the period from when a mother begins breastfeeding after childbirth until she stops breastfeeding.

[0046] In this invention, "infants and toddlers" refers to the human group under 36 months of age.

[0047] In this invention, "infant" refers to the human group under 12 months of age.

[0048] In this invention, "infant" refers to the human group aged 13 to 36 months.

[0049] In this invention, "children" refers to the group of humans aged 3 years and younger than 12 years.

[0050] In this invention, "adolescent" refers to the group of humans aged 12 years or older and under 18 years old.

[0051] In this invention, "adult" refers to the group of humans aged 18 years or older.

[0052] In this invention, "middle-aged and elderly" refers to the human group aged 45 years and older.

[0053] 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.

[0054] II. N-acetylneuraminic acid derivatives

[0055] N-acetylneuraminic acid (Neu5Ac) is a common form of sialic acid and an acetylated derivative of acidic neuraminic acid. It plays an important role in biorecognition, cellular immunity, and disease, and is one of the most important monomers of sialylated human milk oligosaccharides.

[0056] This invention does not specifically limit the source of N-acetylneuraminic acid-like substances. Typically, they can be extracted from natural resources containing sialic acid, such as bird's nest, eggs, and milk; or they can be synthesized through chemical and enzymatic methods, such as condensing N-acetylglucosamine with the potassium salt of di-tert-butyloxosuccinic acid and decarboxylating it under alkaline catalysis to generate N-acetylneuraminic acid, or producing N-acetylmannosamine by catalyzing N-acetylneuraminic acid aldolase; or they can be obtained through fermentation by suitable microorganisms such as Escherichia coli and Bacillus subtilis; or they can be obtained through whole-cell synthesis.

[0057] In some embodiments, the N-acetylneuraminic acid substances of the present invention are anhydrous N-acetylneuraminic acid (hereinafter referred to as N-acetylneuraminic acid), N-acetylneuraminic acid hydrate, or N-acetylneuraminic acid salts; preferably anhydrous N-acetylneuraminic acid or N-acetylneuraminic acid hydrate; more preferably anhydrous N-acetylneuraminic acid. Exemplary N-acetylneuraminic acid hydrates include N-acetylneuraminic acid dihydrate, which has essentially the same biological efficacy as N-acetylneuraminic acid. The only difference is that in the solid state, there is a slight difference in the presence of water of crystallization, while in the liquid state, the hydrogen bonds connecting the two water molecules of crystallization to N-acetylneuraminic acid are broken, resulting in identical properties. Exemplary N-acetylneuraminic acid salts include sodium N-acetylneuraminic acid, potassium N-acetylneuraminic acid, or ammonium N-acetylneuraminic acid. The present invention does not limit the preparation method of N-acetylneuraminic acid salts; for example, crystals can be precipitated by adding a solvent composed of ketones or alcohols to an aqueous solution of N-acetylneuraminic acid containing sodium, potassium, or ammonium at a pH of 3.0–9.0.

[0058] In some specific embodiments, the N-acetylneuraminic acid substances described in this invention can be prepared in-house or obtained commercially.

[0059] II. Foods containing N-acetylneuraminic acid (including health foods)

[0060] The food containing N-acetylneuraminic acid compounds described in this invention is suitable for people of all ages, especially those who need to regulate their intestinal flora, and is particularly suitable for pregnant and / or lactating mothers. In some specific embodiments, the food described in this invention is infant food, children's food, adolescent food, or adult food; exemplary adult foods include foods for pregnant women, postpartum women, and middle-aged and elderly women.

[0061] The food containing N-acetylneuraminic acid compounds described in this invention can be any type of food. In some specific embodiments, the food described in this invention is a beverage, candy, dairy product, baked goods, or dietary supplement; exemplary beverages include carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages, etc.; exemplary candies include hard candies, gel candies, shortbread candies, compressed candies, and aerated candies, etc.; exemplary dairy products include fermented milk, cheese, and milk powder, etc.; exemplary baked goods include bread, cakes, and biscuits, etc.; exemplary dietary supplements include hard capsules, soft capsules, tablets, oral liquids, granules, and powders, etc.

[0062] The food containing N-acetylneuraminic acid compounds described in this invention can be in any form. In some specific embodiments, at room temperature, the food described in this invention is in liquid form, solid-liquid mixture form, solid block form, or solid powder form.

[0063] In addition to N-acetylneuraminic acid derivatives, the food products described in this invention may also contain any other food-acceptable substances, such as proteins / amino acids, carbohydrates, fats, vitamins, minerals, etc., which can be selected by those skilled in the art according to the actual food type and target population. In some specific embodiments, the food products described in this invention contain any one or more of the following ingredients: plant-based ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives, and any acceptable excipients in the food. Exemplary plant-based ingredients include various vegetables, fruits, grains, nuts, coffee, tea and their extracts, as well as some medicinal and edible herbal plants and their extracts. Exemplary animal dairy product ingredients include fresh milk derived from animals, as well as reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder. Exemplary animal meat product ingredients include meat products from pigs, cattle, sheep, aquatic products, or poultry. Exemplary functional additives include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, and functional polyunsaturated fatty acid supplements. Exemplary excipients acceptable in food products include solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, flavorings, and colorings.

[0064] In some more specific embodiments, the food products described in this invention are infant formula milk powder, baby complementary food, children's formula milk powder, children's snacks, maternal formula milk powder, pregnant women's formula milk powder, middle-aged and elderly milk powder, maternal oral liquid or pregnant women's oral liquid.

[0065] In the food products described in this invention, the content of N-acetylneuraminic acid compounds complies with the requirements of relevant laws and regulations. In some specific embodiments, the mass content of the N-acetylneuraminic acid compounds in the food products described in this invention is 0.03% to 2.0%, for example, it can be 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, preferably 0.08% to 1.3%.

[0066] III. Uses of maternally ingested products that help regulate the gut microbiota of mothers and offspring

[0067] This invention discovers that maternal intake of a certain amount of N-acetylneuraminic acid (NNA) during pregnancy and / or lactation can help regulate the gut microbiota of both the mother and her offspring. Specifically, it can increase the relative abundance of beneficial bacteria in the gut of both the mother and her offspring, while reducing the relative abundance of opportunistic pathogens in the offspring's gut. Furthermore, this invention finds that the effect of NNA, through maternal intake, on regulating the gut microbiota of offspring not only affects fetuses present in the mother's womb and breastfed infants, but also infants who do not consume breast milk but have ample contact with their mothers. Moreover, this regulatory effect can extend into childhood and even have a lasting impact on offspring.

[0068] Based on this, the present invention provides the use of N-acetylneuraminic acid substances and foods containing N-acetylneuraminic acid substances for non-therapeutic purposes in regulating the gut microbiota of mothers and their offspring through maternal ingestion. Simultaneously, the present invention provides the use of N-acetylneuraminic acid substances in the preparation of foods that, through maternal ingestion, help regulate the gut microbiota of mothers and their offspring.

[0069] In some embodiments, the mother is pregnant and / or lactating; preferably, the mother is pregnant and lactating. In some embodiments, the offspring is in the fetal, infancy, and / or childhood stage; preferably, the offspring is in the fetal and / or infancy and optionally childhood stage; more preferably, the offspring is in infancy and optionally childhood stage; even more preferably, the offspring is in infancy. In some embodiments, the offspring in the infancy stage can be breastfed, preferably exclusively breastfed or primarily breastfed. In this invention, "exclusively breastfed" means that the vast majority (at least 90%, preferably at least 95%, more preferably at least 98%) of the nutrients and / or energy ingested by the offspring comes from breast milk. In this invention, "primarily breastfed" means that the nutrients and / or energy ingested by the offspring come primarily (at least 50%, preferably at least 65%, more preferably at least 75%) from breast milk.

[0070] In some embodiments, the “mother” and “offspring” of this invention include mammals, including but not limited to humans, monkeys, orangutans, cattle, sheep, cats, dogs, horses, rabbits, mice, rats, or guinea pigs.

[0071] This invention reveals that although all cases involve the mother ingesting N-acetylneuraminic acid (NNA), the regulatory effects of NNA on the gut microbiota of mothers and offspring at different stages are not entirely the same. In some embodiments, supplementing the mother's gut with NNA during pregnancy and / or lactation can regulate different gut microbiota in pregnant mothers, lactating mothers, and their offspring, respectively.

[0072] In some implementations, the method of helping to regulate the gut microbiota of the mother and her offspring includes at least one of: increasing the relative abundance of beneficial bacteria in the gut of a pregnant mother, increasing the relative abundance of beneficial bacteria in the gut of a lactating mother, increasing the relative abundance of beneficial bacteria in the offspring gut, and decreasing the relative abundance of conditionally pathogenic bacteria in the offspring gut.

[0073] In some implementations, the method of helping to regulate the gut microbiota of mothers and their offspring includes: increasing the relative abundance of beneficial bacteria in the gut of pregnant mothers, increasing the relative abundance of beneficial bacteria in the gut of lactating mothers, increasing the relative abundance of beneficial bacteria in the gut of offspring, and decreasing the relative abundance of opportunistic pathogens in the gut of offspring.

[0074] In some embodiments, the method of helping to regulate the gut microbiota of the mother and her offspring includes: increasing at least one of the following: increasing the relative abundance of *Muribaculaceae unclassified* in the gut of a pregnant mother, increasing the relative abundance of *Prevotellaceae UCG-001* in the gut of a pregnant mother, and increasing the relative abundance of *Bifidobacterium* in the gut of a pregnant mother.

[0075] In some implementations, the method of helping to regulate the gut microbiota of the mother and her offspring includes: increasing the relative abundance of *Muribaculaceae unclassified* in the gut of pregnant mothers, increasing the relative abundance of *Prevotellaceae UCG-001* in the gut of pregnant mothers, and increasing the relative abundance of *Bifidobacterium* in the gut of pregnant mothers.

[0076] In some embodiments, the method of helping to regulate the gut microbiota of mothers and their offspring includes: increasing at least one of the relative abundance of *Muribaculaceae unclassified* in the gut of lactating mothers and increasing the relative abundance of *Akkermansia* in the gut of lactating mothers.

[0077] In some implementations, the method of helping to regulate the gut microbiota of mothers and their offspring includes: increasing the relative abundance of *Muribaculaceae unclassified* and increasing the relative abundance of *Akkermansia* in the gut of lactating mothers.

[0078] In some embodiments, the method of assisting in regulating the gut microbiota of the mother and her offspring includes at least one of: increasing the relative abundance of Muribauculaceae unclassified in the offspring gut, increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Romboutisa in the offspring gut, and decreasing the relative abundance of Escherichia-Shigella in the offspring gut.

[0079] In some implementations, the method of helping to regulate the gut microbiota of the mother and her offspring includes: increasing the relative abundance of Muribauculaceae unclassified in the offspring gut, increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Romboutisa in the offspring gut, and decreasing the relative abundance of Escherichia-Shigella in the offspring gut.

[0080] Muribaculaceae unclassified is an unnamed genus within the family Muribaculaceae. Muribaculaceae is a family of bacteria in the order Bacteroidetes. Muribaculaceae produces short-chain fatty acids through endogenous (mucopolysaccharides) and exogenous polysaccharides (dietary fiber). In recent years, Muribaculaceae has begun to receive attention and research as a potential probiotic family. Studies have found that this family has a cross-feeding relationship with probiotics such as Bifidobacterium and Lactobacillus. Muribaculaceae can effectively break down dietary fiber into monosaccharides, which then serve as a food source for Bifidobacterium and Lactobacillus, promoting the proliferation of Lactobacillus and Bifidobacterium (Zhu Y, Chen B, Zhang X, Akbar MT, Wu T, Zhang Y, Zhi L, Shen Q. Exploration of the Muribaculaceae Family in the Gut Microbiota: Diversity, Metabolism, and Function. Nutrients. 2024 Aug 12;16(16):2660.).

[0081] Prevotellaceae UCG-001 is generally considered a bacterium associated with a healthy plant-based diet. Studies have shown that Prevotellaceae UCG-001 is positively correlated with the AMPK signaling pathway in vivo and negatively correlated with abnormal glucose and lipid metabolism. Other studies have shown that patients with obsessive-compulsive disorder and depression have reduced levels of Prevotellaceae in their gut (Borrego-Ruiz A, Borrego JJ. An updated overview on the relationship between human gut microbiome dysbiosis and psychiatric and psychological disorders. Prog Neuropsychopharmacol Biol Psychiatry. 2024 Jan 10; 128:110861.).

[0082] Bifidobacterium are Gram-negative anaerobic bacteria found in the gut. Studies have shown that Bifidobacterium possesses various probiotic functions, such as improving intestinal diseases caused by immune system disorders; inhibiting the invasion of pathogenic bacteria into the gut; enhancing the activity of host antioxidant enzymes to alleviate oxidative damage; improving diseases caused by gut microbiota imbalance, such as constipation and diarrhea; and secreting β-galactosidase to alleviate lactose intolerance. Studies have found that Bifidobacterium microorganisms have effects on memory and learning behavior, long-term potentiation (LTP), and some biochemical parameters in β-amyloid-induced rat models of Alzheimer's disease (Rezaeiasl Z, Salami M, Sepehri G. The Effects of Probiotic Lactobacillus and Bifidobacterium Strains on Memory and Learning Behavior, Long-Term Potentiation (LTP), and Some Biochemical Parameters in β-Amyloid-Induced Rat's Model of Alzheimer's Disease. Prev Nutr Food Sci. 2019 Sep; 24(3):265-273.).

[0083] Akkermansia microorganisms are Gram-negative anaerobic bacteria in the gut, mainly colonizing the mucus layer in the gastrointestinal tract, protecting the mucus layer and connective tissue (Derrien M, Vaughan EE, Plugge CM, de Vos WM. Akkermansia muciniphila gen.nov., sp.nov., a human intestinal mucin-degrading bacterium. Int J Syst Evol Microbiol. 2004 Sep; 54(Pt 5):1469-1476.). As a new generation of probiotics, they are a biomarker of health (Zhang T, Li Q, Cheng L, Buch H, Zhang F. Akkermansia muciniphila is a promising probiotic. Microb Biotechnol. 2019 Nov; 12(6):1109-1125.). Studies have shown that Akkermansia microorganisms play an important probiotic role in metabolic diseases (such as diabetes and obesity), intestinal diseases (such as inflammatory bowel disease and ulcerative colitis), and the neuropsychiatric system (Huang Qiaoshen, Zhang Yongkang, Wang Shuiping, et al. Research progress on the potential and mechanism of action of Akkermansia myxophilus in the treatment of diseases [J]. Acta Microbiologica Sinica, 2023, 63(09):3360-3373.). With the continuous deepening of research on the gut-brain axis, studies have found that Akkermansia plays an important role in various neuropsychiatric diseases (such as depression and anxiety, autism spectrum disorders (ASDs), Alzheimer's disease, and cognitive impairment).

[0084] Romboutisa is an important genus of commensal bacteria in the human gut. It can utilize various carbohydrates to produce short-chain fatty acids such as formic acid, acetic acid, lactic acid, and butyric acid. It can also ferment monoamino acids and vitamins, influence colonic motility, and possess anti-inflammatory properties. Studies have shown that the relative abundance of Romboutisa in the intestines of depressive rats is significantly reduced (Wang Qiannan, Huang Xinhui, Yang Minxu, et al. Research progress on antidepressant treatment related to gut microbiota [J]. Pharmaceutical Practice and Service, 2022, 40(05):422-426.).

[0085] Escherichia-Shigella is a genus of bacteria in the Enterobacteriaceae family. It is a common pathogenic bacterium in the intestines, a Gram-negative facultative anaerobic bacterium with low nutritional requirements. It produces enterotoxins that are highly pathogenic, and its proliferation can cause intestinal flora imbalance, leading to bacterial dysentery. Studies have shown that Escherichia-Shigella is more abundantly expressed in patients with generalized anxiety disorder (GAD) and is positively correlated with the severity of anxiety (Chen YH, Bai J, Wu D, Yu SF, Qiang XL, Bai H, Wang HN, Peng ZW. Association between fecal microbiota and generalized anxiety disorder: Severity and early treatment response. J Affect Disord. 2019 Dec 1; 259:56-66.).

[0086] Based on the experimental results of this invention and existing literature reports, this invention proposes that supplementing mothers with N-acetylneuraminic acid during pregnancy and / or lactation not only benefits the gut microbiota of both mother and offspring but may also influence the gut-brain axis, thereby potentially contributing to the neurological health of both mother and offspring. This invention's approach to maintaining the neurological health of mothers and offspring is not intended for the prevention or treatment of diseases.

[0087] This invention, through extensive research, has found that supplementing mothers with N-acetylneuraminic acid during pregnancy and / or lactation can simultaneously regulate some of the same gut microbiota in the mother and her offspring during pregnancy and / or lactation, especially some probiotics that are not on the list of edible probiotics in the region and cannot be directly supplemented exogenously for the time being.

[0088] In some implementations, the method of helping to regulate the gut microbiota of mothers and their offspring includes simultaneously increasing the relative abundance of Muribauculaceae unclassified in the gut of mothers and their offspring during pregnancy and lactation.

[0089] In some implementations, the method of helping to regulate the gut microbiota of mothers and their offspring includes simultaneously increasing the relative abundance of Akkermansia in the gut of lactating mothers and their offspring.

[0090] Example

[0091] The implementation scheme of the present invention will be described in detail below in combination with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all conventional products that can be obtained through commercial purchase.

[0092] 1. Detection materials

[0093] 1.1. Experimental animals and grouping

[0094] Forty 8-week-old sexually mature SPF-grade female rats and forty SPF-grade male rats were selected; they were purchased from Hangzhou Medical College, and the animal certificate number was 20240318Aazz0100000836, and the production license number was: SCXK(Zhe)2024-002. Feeding conditions: The animals were raised in a barrier environment, the indoor temperature was 25±1°C, the humidity was 45±5%, the animals had free access to drinking water, and they were raised in a 12h light-dark alternating environment every day. This experiment passed the animal experiment ethics review of Southeast University, and the ethics number was: 20240316003.

[0095] After 3 days of adaptive feeding of the animals, the female and male rats were caged together in a 1:1 ratio, and the start point of gavage intervention was when the female mouse showed a vaginal plug. The female mouse was continuously gavaged until the offspring mice were weaned and then stopped; in the intervention group, the corresponding dose of the sample aqueous solution was given according to the animal grouping, and the control group was given the corresponding dose of normal saline. Gavage was performed once a day for 6 consecutive weeks. Each group was given maintenance feed. Among them, the low and high doses of N-acetylneuraminic acid in the intervention group were 1.5 and 7.5 (mg / d / rat). The specific doses of each group are shown in Table 1 below. The feces of the female mice before gavage, after parturition, and after weaning, as well as the feces of the offspring mice at one week and three weeks (after weaning) were collected respectively for intestinal flora sequencing.

[0096] Table 1 Gavage doses of experimental animals

[0097] 1.2. Instruments, consumables and reagents

[0098] The consumables, reagents and experimental instruments used in the experiment are shown in Tables 2 and 3.

[0099] [[ID=...]]

[0100] Table 3 Main experimental instruments

[0101] 2. Detection methods

[0102] 2.1. Collection of feces from female and offspring mice

[0103] (1) Collection of feces from female mice: Fresh feces from each female mouse were collected in a clean environment before gavage, after parturition, and after weaning. The feces were numbered and stored at -80℃.

[0104] (2) Collection of feces from pups: Fresh feces from each litter of pups were collected at 1 week and 3 weeks after birth (after weaning), numbered, and then frozen at -80℃.

[0105] 2.2. Detection of fecal microbiota in female and offspring mice

[0106] High-throughput sequencing of microorganisms in fecal samples from mother and offspring mice was performed using 16S rDNA.

[0107] (1) Extraction of total microbiome DNA: Total microbiome DNA was extracted from feces using a DNA extraction kit, and the quality of DNA extraction was detected by agarose gel electrophoresis. At the same time, the DNA was quantified using a UV spectrophotometer.

[0108] (2) PCR amplification: The V3-V4 region of bacterial 16S rDNA was selected for gene amplification and sequencing. Primer sequences are shown in the table below:

[0109] Table 4 Primer sequences

[0110] The PCR reaction system is shown in Table 5 below:

[0111] Table 5 Reaction System

[0112] Table 6: PCR reaction conditions

[0113] Table 6 Reaction conditions

[0114] (3) The PCR products were purified by AMPure XT beads (Beckman Coulter Genomice, MA, USA) and quantified by Qubit (Invitrogen, USA).

[0115] (4) The purified PCR products were evaluated using an Agilent 2100 bioanalyzer (Agilent, USA) and Illumina (Kapa Biosciences, Wobum, MA, USA) library quantification kit. Qualified libraries had a concentration above 2 nM. Each qualified sequencing library was serially diluted and mixed according to the required sequencing volume, then denatured into single strands using NaOH before sequencing. 2×250 bp paired-end sequencing was performed using a NovaSeq 6000 sequencer, with the corresponding reagent being the NovaSeq 6000 SP Reagent Kit (500 cycles).

[0116] (5) For the paired-end data obtained from sequencing, the samples were first split according to the barcode information, and adapters and barcode sequences were removed. Then, the sequences were assembled to obtain optimized sequences. After removing chimeric sequences, OUT cluster analysis was performed, and taxonomic analysis was conducted on the representative OUT sequences. Based on the OUT cluster analysis results, various diversity index analyses and sequence depth detection were performed on the OUT sequences; based on taxonomic information, statistical analysis of colony structure was performed at each taxonomic level.

[0117] 3. Test Results

[0118] 3.1 Results of dominant gut microbiota in female mice

[0119] 3.1.1. Changes in Muribauculaceae unclassified in the intestines of female mice

[0120] The changes in Muribaculaceae unclassified in the intestines of female mice are shown in Figure 1. Compared with the control group, the relative abundance of Muribaculaceae unclassified in the intestines of pregnant and lactating female mice in different N-acetylneuraminic acid intervention groups was significantly higher than that in the control group, and the relative abundance in the high-dose N-acetylneuraminic acid intervention group was higher than that in the low-dose intervention group.

[0121] 3.1.2. Changes in the Prevotellaceae genus (UGG-001) in the intestines of female mice

[0122] Figure 2 shows the changes in the Prevotellaceae genus (UCG-001) in the intestines of pregnant mice. Compared with the control, the relative abundance of Prevotellaceae UCG-001 in the intestines of pregnant mice in different N-acetylneuraminic acid intervention groups was higher than that in the control group, and there was no significant difference in relative abundance among the different intervention groups. The relative abundance of Prevotellaceae UCG-001 in the intestines of lactating mice in different N-acetylneuraminic acid intervention groups was lower than that in the control group.

[0123] 3.1.3. Changes in Bifidobacterium in the gut of female mice

[0124] Figure 3 shows the changes in Bifidobacterium in the intestines of pregnant mice. From before gavage to after parturition, the relative abundance of Bifidobacterium in the intestines of pregnant mice in the control group showed a decreasing trend. Compared with the control, the relative abundance of Bifidobacterium in the intestines of pregnant mice in different N-acetylneuraminic acid intervention groups showed an increasing trend, and the high-dose intervention group was higher than the low-dose intervention group. Compared with the control, there was no significant difference in the relative abundance of Bifidobacterium in the intestines of lactating mice in different N-acetylneuraminic acid intervention groups.

[0125] 3.1.4. Changes in Akkermansia in the intestines of female mice

[0126] Figure 4 shows the changes in Akkermansia in the intestines of pregnant mice. Compared with the control group, there was no significant difference in the relative abundance of Akkermansia in the intestines of pregnant mice in different N-acetylneuraminic acid intervention groups. The relative abundance of Akkermansia in the intestines of lactating mice in different N-acetylneuraminic acid intervention groups was significantly increased and higher than that in the control group. The relative abundance in the high-dose intervention group was significantly higher than that in the low-dose intervention group.

[0127] 3.2. Results of dominant gut microbiota in offspring mice

[0128] 3.2.1. Changes in Muribauculaceae unclassified in the intestines of offspring mice

[0129] Figure 5 shows the changes in the intestinal tract of offspring mice. One week after birth, there was no significant difference in the relative abundance of unclassified Muribaculaceae in the intestinal tract of offspring mice from different groups. With the continuation of breastfeeding, the relative abundance of unclassified Muribaculaceae in the intestinal tract of offspring mice in the high-dose N-acetylneuraminic acid intervention group was significantly higher than that in the control group.

[0130] 3.2.2. Changes in Akkermansia in the intestines of offspring mice

[0131] Figure 6 shows the changes in Akkermansia in the intestines of offspring. One week after birth, there was no significant difference in the relative abundance of Akkermansia in the intestines of offspring from different groups. With the continuation of breastfeeding, the relative abundance of Akkermansia in the intestines of offspring in different N-acetylneuraminic acid intervention groups was significantly higher than that in the control group, and the relative abundance in the high-dose N-acetylneuraminic acid intervention group was higher than that in the low-dose intervention group.

[0132] 3.2.3. Changes in Romboutisa in the Intestines of Offspring Rats

[0133] Figure 7 shows the changes in Romboutisa in the intestines of offspring mice. One week after birth, the relative abundance of Romboutisa in the intestines of offspring mice in different N-acetylneuraminic acid intervention groups was higher than that in the control group. With the continuation of breastfeeding, the relative abundance of Romboutisa in the intestines of offspring mice in the high-dose N-acetylneuraminic acid intervention group was significantly higher than that in the control group.

[0134] 3.2.4. Changes in Escherichia-Shigella spp. in the intestines of offspring mice

[0135] Figure 8 shows the changes in Escherichia-Shigella in the intestines of offspring mice. Compared with the control group, the relative abundance of Escherichia-Shigella in the intestines of offspring mice in the high-dose N-acetylneuraminic acid intervention group was lower than that in the control group one week after birth. With the continuation of breastfeeding, the relative abundance of Escherichia-Shigella in the intestines of offspring mice was significantly reduced at three weeks after birth. The relative abundance of Escherichia-Shigella in the intestines of offspring mice in different N-acetylneuraminic acid intervention groups was significantly lower than that in the control group, and the relative abundance in the high-dose intervention group was significantly lower than that in the low-dose intervention group.

Claims

1. The use of N-acetylneuraminic acid derivatives in the preparation of foods that, when ingested by the mother, help regulate the gut microbiota of both the mother and her offspring; among which, The N-acetylneuraminic acid substance is anhydrous N-acetylneuraminic acid, N-acetylneuraminic acid hydrate, or N-acetylneuraminic acid salt. The mother is in the pregnancy and / or lactation period, and the offspring is in the fetal, infancy, and / or childhood period.

2. The use according to claim 1, characterized in that, The benefits of regulating the gut microbiota of mothers and their offspring include: increasing the relative abundance of *Muribaculaceae unclassified* in the gut of pregnant mothers, increasing the relative abundance of *Prevotellaceae UCG-001* in the gut of pregnant mothers, and increasing the relative abundance of *Bifidobacterium* in the gut of pregnant mothers.

3. The use according to claim 1 or 2, characterized in that, The benefits of regulating the gut microbiota of mothers and their offspring include at least one of increasing the relative abundance of *Muribaculaceae unclassified* in the gut of lactating mothers and increasing the relative abundance of *Akkermansia* in the gut of lactating mothers.

4. The use according to any one of claims 1 to 3, characterized in that, The methods for regulating the gut microbiota of the mother and her offspring include at least one of the following: increasing the relative abundance of Muribauculaceae unclassified in the offspring gut, increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Romboutisa in the offspring gut, and decreasing the relative abundance of Escherichia-Shigella in the offspring gut.

5. The use according to any one of claims 1 to 4, characterized in that, The food helps maintain the nervous system health of mothers and their offspring by influencing the gut-brain axis by regulating the gut microbiota of mothers and their offspring.

6. The use according to any one of claims 1 to 5, characterized in that, The food products mentioned are infant food, children's food, adolescent food, or adult food.

7. The use according to any one of claims 1 to 6, characterized in that, The food products mentioned are beverages, candies, dairy products, baked goods, or dietary supplements.

8. The use according to any one of claims 1 to 7, characterized in that, At room temperature, the food is in liquid form, solid-liquid mixture form, solid block form, or solid powder form.

9. The use according to any one of claims 1 to 8, characterized in that, The food contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any food-acceptable excipients.

10. The use according to any one of claims 1 to 9, characterized in that, The N-acetylneuraminic acid derivative in the food contains 0.03% to 2.0% by mass.

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