Use of active folic acid substance for helping regulate intestinal flora of mother and offspring

Abstract

The use of an active folic acid substance in the preparation of a food product for helping regulate the intestinal flora of a mother and offspring thereof via ingestion by the mother, wherein the active folic acid substance is selected from 6S-5-methyltetrahydrofolic acid and a salt thereof, the mother is in the period of pregnancy and / or lactation, and the offspring is in the fetal period, infant period, and / or childhood.

Description

Uses of active folic acid compounds to help regulate the gut microbiota of mothers and children Technical Field

[0001] This invention belongs to the food field, specifically relating to the use of active folic acid substances to help regulate the intestinal flora of mothers and children. Background Technology

[0002] The gut microbiota is considered a key factor in regulating human health, directly influencing the metabolic, immune, and nervous systems. It is known that the gut microbiota is affected by various factors, including diet, age, lifestyle, medications, antibiotic use, and disease states; different populations and individuals possess different gut microbiota. Studies have shown that maternal gut microbiota can be transported to the placenta via the immune lymphatic system and transmitted to breast milk via the immune mammary system, further influencing the development of the newborn's gut microbiota. Simultaneously, research indicates that regardless of the delivery method or whether breastfeeding is practiced, mothers can influence their infants' gut microbiota through various pathways, such as physical contact.

[0003] Reference 1 (CN117882771A) discloses a nutritional composition, food, and uses containing bovine colostrum and bifidobacteria. Studies have shown that supplementing with bovine colostrum and / or Bifidobacterium longum BB536 during pregnancy and / or lactation can improve the immunity and gut microbiota (e.g., lactobacilli and / or bifidobacteria) of offspring fed by older pregnant women through mother-to-child transmission.

[0004] Folic acid is a B vitamin that the human body cannot synthesize and must be obtained through additional intake. It is known that folic acid supplementation during pregnancy can effectively prevent neural tube defects in the fetus and reduce the incidence of placental malformations, intrauterine growth retardation, premature birth, and low birth weight. Methylenetetrahydrofolate reductase plays a crucial role in the utilization of folic acid in the body, and mutations in the gene determining the synthesis of this enzyme can lead to related diseases. 6S-5-methyltetrahydrofolate, also known as active folic acid, is the most active form of folic acid after it enters the body and can be directly absorbed and utilized by the body. It is not affected by the genotype of tetrahydrofolate reductase in the body and is a better form of folic acid supplementation.

[0005] Reference 2 (CN114340635B) discloses the use of 6S-5-methyltetrahydrofolate (active folic acid) in the preparation of drugs or health foods for the prevention of congenital heart defects in newborns during the perinatal and / or pregnancy period. Studies have shown that folic acid taken by mothers during pregnancy, and the unmetabolized folic acid, can be teratogenic to the embryo. Long-term or excessive supplementation can lead to the occurrence of congenital heart disease. However, 6S-5-methyltetrahydrofolate (active folic acid) is not teratogenic and has the effect of preventing the occurrence of congenital heart disease when taken during pregnancy.

[0006] Reference 3 (CN118252800A) discloses a method for modifying 5-methyltetrahydrofolate liposomes and its use in improving intestinal flora. The method uses soybean lecithin and cholesterol to prepare liposomes to encapsulate 5-methyltetrahydrofolate, and then modifies them stepwise with chitosan and pectin. At the same time, the modified encapsulated material is further coated with a special fermented and enzymatically hydrolyzed plum extract, and then further compounded with dried tangerine peel powder, old tangerine powder and old ginger powder, thereby achieving the effect of regulating intestinal microorganisms. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] Existing research suggests that the initial gut microbiota of newborns originates from their mothers. A healthy maternal microbiota can pass on a better microbiota to offspring and influence the establishment of the offspring's microbiota, laying a good foundation for a healthy offspring microbiota. Therefore, this invention proposes improving the microbiota from the source, i.e., intervening during pregnancy to enhance the health of pregnant and lactating mothers, especially improving their microbiota health, thereby regulating the offspring's microbiota and improving their health. This approach is superior to interventions after birth. However, existing research indicates that even if nutrients are considered to be directly provided to the body to exert specific health benefits, they may not necessarily promote offspring health when ingested by the mother.

[0009] Although cited reference 3 discloses the use of 5-methyltetrahydrofolate liposomes to improve gut microbiota, this effect is not based on 5-methyltetrahydrofolate alone, but rather on the combined use of 5-methyltetrahydrofolate liposomes coated with a specially fermented and enzymatically hydrolyzed plum extract, along with dried tangerine peel powder, aged tangerine peel powder, and aged ginger powder. Currently, no studies have shown the effects of direct maternal supplementation with active folic acid on the gut microbiota of the mother and offspring.

[0010] During the in-depth study of the physiological functions of active folic acid substances, this invention unexpectedly discovered that mothers who take active folic acid substances during pregnancy can significantly improve their gut microbiota and simultaneously improve the gut microbiota of their offspring. Active folic acid substances can promote the increase of beneficial bacteria in the gut of pregnant and lactating mothers and reduce the relative content of opportunistic pathogens, while increasing the content of probiotics in the gut of their offspring and inhibiting the relative abundance of opportunistic pathogens in the gut of their offspring.

[0011] Therefore, the object of the present invention is to provide the use of active folic 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 active folic 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 active folic acid substances in the preparation of foods that help regulate the gut microbiota of mothers and their offspring when ingested by the mother; wherein the active folic acid substance is selected from 6S-5-methyltetrahydrofolate and its salts, 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 active folic acid substance is 6S-5-methyltetrahydrofolate, calcium 6S-5-methyltetrahydrofolate or glucosamine 6S-5-methyltetrahydrofolate.

[0015] [3]. According to the use described in [1] or [2], wherein the assistance in regulating the gut microbiota of the mother and her offspring includes at least one of: increasing the relative abundance of Akkermansia in the gut of a pregnant mother, increasing the relative abundance of Bifidobacterium in the gut of a pregnant mother, decreasing the relative abundance of Escherichia-Shigella in the gut of a pregnant mother, and decreasing the relative abundance of Streptococcus in the gut of a pregnant mother.

[0016] [4]. The use according to any one of [1] to [3], wherein the assistance in regulating the gut microbiota of the mother and her offspring includes: increasing the relative abundance of Akkermansia in the gut of a lactating mother, increasing the relative abundance of Clostridia UCG-014 unclassified in the gut of a lactating mother, increasing the relative abundance of Muribaculaceae unclassified in the gut of a lactating mother, decreasing the relative abundance of Escherichia-Shigella in the gut of a lactating mother, and decreasing the relative abundance of Streptococcus in the gut of a lactating mother.

[0017] [5]. The use according to any one of [1] to [4], wherein the aid in regulating the gut microbiota of the mother and her offspring includes: increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Muribaucaae unclassified in the offspring gut, increasing the relative abundance of Bacteroides in the offspring gut, decreasing the relative abundance of Escherichia-Shigella in the offspring gut, and decreasing the relative abundance of Streptococcus in the offspring gut.

[0018] [6]. The use according to any one of [1] to [5], 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.

[0019] [7]. The use according to any one of [1] to [6], wherein the food is infant food, children's food, adolescent food or adult food.

[0020] [8]. Use according to any one of [1] to [7], wherein the food is a beverage, confectionery, dairy product, baked goods or dietary supplement.

[0021] [9]. The use according to any one of [1] to [8], wherein, at room temperature, the food is in liquid form, solid-liquid mixture form, solid block form or solid powder form.

[0022]

[0010] . According to any one of [1] to [9], 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.

[0023]

[0011] . The use according to any one of [1] to

[0010] , wherein the active folic acid substance in the food has a mass content of 0.0001% to 0.1%.

[0024] The effects of the invention

[0025] Based on extensive research, this invention proposes that maternal supplementation with a certain amount of active folic acid during pregnancy and / or lactation can significantly increase the abundance of beneficial bacteria and reduce the content of harmful bacteria in the mother's gut, while also significantly increasing the abundance of beneficial bacteria and reducing the content of harmful bacteria in the offspring's gut. In particular, it can regulate the abundance of some non-edible beneficial bacteria, such as Akkermansia, in the gut 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 active folic acid during pregnancy and / or lactation is also beneficial for maintaining the nervous system health of both mother and offspring.

[0026] Experimental data show that maternal supplementation with active folic acid during pregnancy and lactation significantly increased the relative abundance of Akkermansia and Bifidobacterium in the maternal gut during pregnancy, while decreasing the relative abundance of Escherichia-Shigella and Streptococcus in the maternal gut during pregnancy. Conversely, during lactation, it increased the relative abundance of Akkermansia, Clostridia UCG-014 unclassified, and Muribaculaceae unclassified in the maternal gut, while decreasing the relative abundance of Escherichia-Shigella and Streptococcus in the maternal gut. Simultaneously, it significantly increased the relative abundance of Akkermansia and Muribaculaceae in the intestines of the offspring of these mothers. The relative abundance of unclassified and Bacteroides was reduced, as was the relative abundance of Escherichia-Shigella and Streptococcus in the intestines of the offspring of the mother. Attached Figure Description

[0027] Figure 1: Changes in Akkermansia in the intestines of female mice.

[0028] Figure 2: Changes in Bifidobacterium in the intestines of female mice.

[0029] Figure 3: Changes in Clostridia UCG-014 unclassified in the intestines of female mice.

[0030] Figure 4: Changes in Muribauculaceae unclassified in the intestines of female mice.

[0031] Figure 5: Changes in Escherichia-Shigella in the intestines of female mice.

[0032] Figure 6: Changes in Streptococcus bacteria in the intestines of female mice.

[0033] Figure 7: Changes in Akkermansia in the intestines of baby mice.

[0034] Figure 8: Changes in Muribauculaceae unclassified in the intestines of baby mice.

[0035] Figure 9: Changes in Bacteroides in the intestines of baby mice.

[0036] Figure 10: Changes in Escherichia-Shigella in the intestines of baby mice.

[0037] Figure 11: Changes in Streptococcus bacteria in the intestines of baby mice. Detailed Implementation

[0038] 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.

[0039] I. Terminology Definition

[0040] In this invention, the terms "a", "an", or "the" may refer to "one", "one or more", "at least one", or "one or more".

[0041] 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.

[0042] 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.

[0043] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] 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.

[0045] 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.

[0046] In this invention, "normal temperature" refers to an indoor ambient temperature of 23±2℃.

[0047] 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.

[0048] In this invention, "gestation period" and "pregnancy period" can be used interchangeably, referring to the period from fertilization to delivery.

[0049] In this invention, "breastfeeding period" refers to the period from when a mother begins breastfeeding after childbirth until she stops breastfeeding.

[0050] In this invention, "infants and toddlers" refers to the human group under 36 months of age.

[0051] In this invention, "infant" refers to the human group under 12 months of age.

[0052] In this invention, "infant" refers to the human group aged 13 to 36 months.

[0053] In this invention, "children" refers to the group of humans aged 3 years and younger than 12 years.

[0054] In this invention, "adolescent" refers to the group of humans aged 12 years or older and under 18 years old.

[0055] In this invention, "adult" refers to the group of humans aged 18 years or older.

[0056] In this invention, "middle-aged and elderly" refers to the human group aged 45 years and older.

[0057] 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.

[0058] II. Active folic acid substances

[0059] In some embodiments, the active folic acid substances described in this invention are selected from 6S-5-methyltetrahydrofolate and its salts.

[0060] In some specific embodiments, the active folic acid substance of the present invention is 6S-5-methyltetrahydrofolate, calcium 6S-5-methyltetrahydrofolate, or glucosamine 6S-5-methyltetrahydrofolate.

[0061] Considering the ease of obtaining raw materials and the widespread use of these materials, in some preferred embodiments, the active folic acid substance described in this invention is 6S-5-methyltetrahydrofolate calcium.

[0062] The present invention does not particularly limit the source of the above-mentioned active folic acid substances. Typically, they can be synthesized by common chemical synthesis methods in the art, such as using folic acid as a raw material, through steps such as reduction, methylation, chiral resolution and optional salt formation.

[0063] II. Foods containing active folic acid (including health foods)

[0064] The food containing active folic 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.

[0065] The food containing active folic 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.

[0066] The food containing active folic acid substances 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.

[0067] In addition to containing active folic acid-like substances, 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, demineralized 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 include solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavorings, and edible colorings.

[0068] 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.

[0069] In the food products described in this invention, the content of active folic acid substances meets the requirements of relevant laws and regulations. In some specific embodiments, the mass content of the active folic acid substances in the food described in this invention is 0.0001% to 0.1%, for example, it can be 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%, preferably 0.0003% to 0.01%.

[0070] III. Uses of maternally ingested products that help regulate the gut microbiota of mothers and offspring

[0071] This invention discovers that maternal intake of a certain amount of active folic acid during pregnancy and / or lactation (through food consumption) can help regulate the gut microbiota of both the mother and her offspring. Specifically, it can increase the relative abundance of beneficial bacteria and decrease the relative abundance of opportunistic pathogens in the gut of both the mother and her offspring. Furthermore, this invention finds that the effect of active folic acid, through maternal intake, in 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.

[0072] Based on this, the present invention provides the use of active folic acid substances and foods containing active folic 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 active folic acid substances in the preparation of foods that, through maternal ingestion, help regulate the gut microbiota of mothers and their offspring.

[0073] 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.

[0074] 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.

[0075] This invention reveals that although both involve maternal intake of active folic acid, the regulatory effects of active folic acid on the gut microbiota of mothers and offspring at different stages are not entirely the same. In some embodiments, maternal supplementation with active folic acid during pregnancy and / or lactation can regulate different gut microbiota in pregnant mothers, lactating mothers, and their offspring, respectively.

[0076] In some embodiments, 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, decreasing the relative abundance of opportunistic pathogens in the gut of a pregnant mother, increasing the relative abundance of beneficial bacteria in the gut of a lactating mother, decreasing the relative abundance of opportunistic pathogens in the gut of a lactating mother, 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.

[0077] In some implementations, the method of helping to regulate the gut microbiota of the mother and her offspring includes: increasing the relative abundance of beneficial bacteria in the gut of a pregnant mother, decreasing the relative abundance of opportunistic pathogens in the gut of a pregnant mother, increasing the relative abundance of beneficial bacteria in the gut of a lactating mother, decreasing the relative abundance of opportunistic pathogens in the gut of a lactating mother, increasing the relative abundance of beneficial bacteria in the gut of the offspring, and decreasing the relative abundance of opportunistic pathogens in the gut of the offspring.

[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 Akkermansia in the gut of a pregnant mother, increasing the relative abundance of Bifidobacterium in the gut of a pregnant mother, decreasing the relative abundance of Escherichia-Shigella in the gut of a pregnant mother, and decreasing the relative abundance of Streptococcus in the gut of a pregnant mother.

[0079] In some embodiments, the method of helping to regulate the gut microbiota of the mother and her offspring includes: increasing the relative abundance of Akkermansia in the gut of pregnant mothers, increasing the relative abundance of Bifidobacterium in the gut of pregnant mothers, decreasing the relative abundance of Escherichia-Shigella in the gut of pregnant mothers, and decreasing the relative abundance of Streptococcus in the gut of pregnant mothers.

[0080] In some embodiments, the method of assisting in regulating the gut microbiota of the mother and her offspring includes at least one of the following: increasing the relative abundance of Akkermansia in the gut of a lactating mother, increasing the relative abundance of Clostridia UCG-014 unclassified in the gut of a lactating mother, increasing the relative abundance of Muribaculaceae unclassified in the gut of a lactating mother, decreasing the relative abundance of Escherichia-Shigella in the gut of a lactating mother, and decreasing the relative abundance of Streptococcus in the gut of a lactating mother.

[0081] In some embodiments, the method of helping to regulate the gut microbiota of mothers and their offspring includes: increasing the relative abundance of Akkermansia in the gut of lactating mothers, increasing the relative abundance of Clostridia UCG-014 unclassified in the gut of lactating mothers, increasing the relative abundance of Muribaculaceae unclassified in the gut of lactating mothers, decreasing the relative abundance of Escherichia-Shigella in the gut of lactating mothers, and decreasing the relative abundance of Streptococcus in the gut of lactating mothers.

[0082] In some embodiments, the method of assisting in regulating the gut microbiota of the mother and her offspring includes at least one of the following: increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Muribaueraceae unclassified in the offspring gut, increasing the relative abundance of Bacteroides in the offspring gut, decreasing the relative abundance of Escherichia-Shigella in the offspring gut, and decreasing the relative abundance of Streptococcus in the offspring gut.

[0083] In some embodiments, the method of assisting in regulating the gut microbiota of the mother and her offspring includes: increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Muribactaceae unclassified in the offspring gut, increasing the relative abundance of Bacteroides in the offspring gut, decreasing the relative abundance of Escherichia-Shigella in the offspring gut, and decreasing the relative abundance of Streptococcus in the offspring gut.

[0084] 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 status (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).

[0085] 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.).

[0086] *Clostridia* UCG-014 unclassified is an unnamed genus within the class Clostridium. It comprises a group of Gram-positive anaerobic or microaerophilic bacteria, with a large number of butyric acid-producing bacteria belonging to this genus. Studies have found a decreased relative abundance of *Clostridia* UCG-014 unclassified in the gut of patients with thyroiditis compared to healthy individuals. Some studies have also found a negative correlation between the relative abundance of *Clostridia* UCG-014 unclassified in the gut during pregnancy and dyslipidemia during pregnancy.

[0087] 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.).

[0088] Bacteroides are Gram-negative anaerobic bacteria found primarily in the gastrointestinal tract of humans and animals. They participate in many important metabolic activities in the human colon, including carbohydrate fermentation, utilization of nitrogenous substances, and biotransformation of bile acids and other steroids. Studies have shown a difference in the amount of Bacteroides in the feces of children with autism compared to healthy controls (Liu J, Gao Z, Liu C, Liu T, Gao J, Cai Y, Fan X. Alteration of Gut Microbiota: New Strategy for Treating Autism Spectrum Disorder. Front Cell Dev Biol. 2022 Mar 3; 10:792490.).

[0089] 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, and this expression 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.).

[0090] Streptococcus is another large group of common Gram-positive cocci among pyogenic cocci. They are widely distributed in nature and in the human nasopharynx and gastrointestinal tract, and include both normal flora and opportunistic pathogens. Pathogenic streptococci can cause purulent inflammation and hypersensitivity reactions in humans.

[0091] Based on the experimental results of this invention and existing literature reports, this invention proposes that supplementing mothers with active folic acid during pregnancy and / or lactation not only benefits the gut microbiota of both mother and offspring but also influences the gut-brain axis, thereby 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 disease prevention or treatment.

[0092] Through extensive research, this invention has discovered that supplementing mothers with active folic acid during pregnancy and / or lactation can simultaneously regulate some of the same gut microbiota in mothers and their offspring during pregnancy and / or lactation.

[0093] In some embodiments, the means for modulating the gut microbiota of the mother and her offspring include at least one of: simultaneously increasing the relative abundance of Akkermansia in the guts of the mother and her offspring during pregnancy and lactation, simultaneously decreasing the relative abundance of Escherichia-Shigella in the guts of the mother and her offspring during pregnancy and lactation, and simultaneously decreasing the relative abundance of Streptococcus in the guts of the mother and her offspring during pregnancy and lactation.

[0094] In some embodiments, the means for modulating the gut microbiota of the mother and her offspring include: simultaneously increasing the relative abundance of Muribaculaceae unclassified in the guts of the mother and her offspring during lactation.

[0095] Examples

[0096] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. Unless otherwise specified, the materials or instruments used are conventional products that can be obtained through commercial purchase.

[0097] 1. Detection materials

[0098] 1.1. Experimental animals and grouping

[0099] 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, with the animal certificate number being 20240318Aazz01000008 and the production license number: SCXK(Zhe)2024-002. Feeding conditions: The animals were raised in a barrier environment, with the indoor temperature at 25±1°C and the humidity at 45±5%. The animals had free access to drinking water and were raised in a 12h light-dark alternating environment daily. This experiment passed the animal experiment ethics review of Southeast University, with the ethics number: 20240316003.

[0100] After 3 days of acclimatization, female and male rats were housed together in a 1:1 ratio. The intervention was initiated by the presence of vaginal plugs in the mother rats, and gavage was continued until the offspring were weaned. The intervention group received a corresponding dose of sample aqueous solution, while the control group received a corresponding dose of physiological saline. Gavage was administered once daily for 6 weeks, with all groups receiving maintenance feed. The intervention group received low and high doses of active folic acid at 5.3 and 10.1 μg / day / rat, respectively. Specific dosages for each group are shown in Table 1 below. Intestinal flora sequencing was performed on feces collected from mother rats before gavage, after parturition, after weaning, and from offspring rats at one and three weeks post-weaning.

[0101] Table 1. Gavage dosage for experimental animals

[0102] 1.2. Instruments, Consumables and Reagents

[0103] The consumables, reagents and experimental instruments used in the experiment are shown in Tables 2 and 3.

[0104] Table 2 Main experimental consumables and reagents

[0105] Table 3 Main Experimental Instruments

[0106] 2. Detection Method

[0107] 2.1. Collection of feces from mother and offspring

[0108] (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℃.

[0109] (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℃.

[0110] 2.2. Detection of fecal microbiota in female and offspring mice

[0111] High-throughput sequencing of microorganisms in fecal samples from mother and offspring mice was performed using 16S rDNA.

[0112] (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.

[0113] (2) PCR amplification: The V3-V4 region of bacterial 16S rDNA was selected for gene amplification and sequencing. Primer sequences are shown in Table 4 below:

[0114] Table 4 Primer sequences

[0115] The PCR reaction system is shown in Table 5 below:

[0116] Table 5 Reaction System

[0117] The PCR reaction conditions are shown in Table 6 below:

[0118] Table 6 Reaction conditions

[0119] (3) The PCR products were purified by AMPure XT beads (Beckman Coulter Genomice, MA, USA) and quantified by Qubit (Invitrogen, USA).

[0120] (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 6000SP Reagent Kit (500 cycles).

[0121] (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.

[0122] 3. Test Results

[0123] 3.1 Results of dominant gut microbiota in female mice

[0124] 3.1.1. Changes in Akkermansia in the intestines of female mice

[0125] Figure 1 shows the changes in Akkermansia in the intestines of female mice. Compared with the control group, different doses of active folic acid substances can increase the relative abundance of Akkermansia in the intestines of pregnant and lactating female mice, and the high dose of active folic acid substances has a significantly higher effect than the low dose of active folic acid substances.

[0126] 3.1.2. Changes in Bifidobacterium in the intestines of female mice

[0127] Figure 2 shows the changes in Bifidobacterium in the intestines of pregnant mice. Compared with the control group, different doses of active folic acid substances can increase the relative abundance of Bifidobacterium in the intestines of pregnant mice. The relative abundance of Bifidobacterium in the intestines of lactating mice in the intervention groups of different doses of active folic acid substances showed a decreasing trend and was lower than that in the control group.

[0128] 3.1.3. Changes in the Clostridia genus (UCG-014 unclassified) in the intestines of female mice

[0129] Figure 3 shows the changes in Clostridia UCG-014 unclassified in the intestines of pregnant mice. Compared with the control group, different doses of active folic acid substances had no significant effect on the relative abundance of Clostridia UCG-014 unclassified in the intestines of pregnant mice. Different doses of active folic acid substances could significantly increase the relative abundance of Clostridia UCG-014 unclassified in the intestines of lactating mice, and there was no significant difference in the increase effect among different dose groups.

[0130] 3.1.4. Changes in Muribauculaceae unclassified in the intestines of female mice

[0131] Figure 4 shows the changes in Muribaculaceae unclassified in the intestines of pregnant mice. Compared with the control group, different doses of active folic acid substances increased the relative abundance of Muribaculaceae unclassified in the intestines of pregnant mice, but the degree of increase was not significantly different from that of the control group. During the lactation period, the relative abundance of Muribaculaceae unclassified in the intestines of control mice showed a decreasing trend, while the relative abundance of Muribaculaceae unclassified in the intestines of mice in the low-dose active folic acid substance intervention group was not significantly different from that after parturition. The relative abundance of Muribaculaceae unclassified in the intestines of mice in the high-dose active folic acid substance intervention group was higher than that after parturition, and also higher than that of the control group and the low-dose active folic acid substance intervention group. The results indicate that active folic acid substance intervention helps to increase the relative abundance of Muribaculaceae unclassified in the intestines of lactating mice.

[0132] 3.1.5. Changes in Escherichia-Shigella spp. in the intestines of female mice

[0133] Figure 5 shows the changes in Escherichia-Shigella in the intestines of female mice. In the control group, the relative abundance of Escherichia-Shigella in the intestines of female mice increased after pregnancy and parturition. However, in the intervention groups of different doses of active folic acid, the relative abundance of Escherichia-Shigella in the intestines of female mice decreased to varying degrees after pregnancy and parturition. Compared with the control group, the relative abundance of Escherichia-Shigella in the intestines of lactating female mice in the intervention groups of different doses of active folic acid was lower than that in the control group, and there was no significant difference in the degree of reduction among the intervention groups of different doses of active folic acid. The results indicate that supplementation with active folic acid during pregnancy can reduce the relative abundance of Escherichia-Shigella in the intestines of pregnant and lactating female mice.

[0134] 3.1.6. Changes in Streptococcus in the Intestine of Female Rats

[0135] Figure 6 shows the changes in Streptococcus spp. in the intestines of pregnant mice. Compared with the control group, the relative abundance of Streptococcus spp. in the intestines of pregnant mice in different doses of active folic acid intervention groups was reduced to varying degrees, and the high-dose active folic acid intervention group was lower than the low-dose active folic acid intervention group. The relative abundance of Streptococcus spp. in the intestines of lactating mice in different doses of active folic acid intervention groups was lower than that in the control group, and there was no significant difference between different doses of active folic acid intervention groups. The results indicate that active folic acid can inhibit the proliferation of Streptococcus spp. in the intestines of pregnant mice and reduce its relative abundance in the lactating intestines.

[0136] 3.2. Results of dominant gut microbiota in offspring mice

[0137] 3.2.1. Changes in Akkermansia in the intestines of offspring mice

[0138] Figure 7 shows the changes in Akkermansia in the intestines of offspring mice. Compared with the control group, there was no significant difference in the relative abundance of Akkermansia in the intestines of offspring in different doses of active folic acid intervention groups one week after birth. With the continuation of breastfeeding, the relative abundance of Akkermansia in the intestines of offspring in different doses of active folic acid intervention groups was significantly higher than that in the control group three weeks after birth, and the high-dose intervention group was higher than the low-dose intervention group.

[0139] 3.2.2. Changes in Muribauculaceae unclassified in the intestines of offspring mice

[0140] Figure 8 shows the changes in Muribaculaceae unclassified in the intestines of offspring. Compared with the control group, there was no significant difference in the relative abundance of Muribaculaceae unclassified in the intestines of offspring treated with different doses of active folic acid at 1 week after birth. With the continuation of breastfeeding, the relative abundance of Muribaculaceae unclassified in the intestines of offspring treated with different doses of active folic acid was significantly higher than that of the control group at 3 weeks after birth, and there was no significant difference in the relative abundance of Muribaculaceae unclassified among the different doses of active folic acid intervention groups.

[0141] 3.2.3. Changes in Bacteroides in the intestines of offspring mice

[0142] Figure 9 shows the changes in Bacteroides in the intestines of offspring mice. Compared with the control group, there was no significant difference in the relative abundance of Bacteroides in the intestines of offspring in different doses of active folic acid intervention groups one week after birth. With the continuation of breastfeeding, the relative abundance of Bacteroides in the intestines of offspring in the high-dose active folic acid intervention group was significantly higher than that in the control group three weeks after birth. The results indicate that administering high doses of active folic acid during the mother's pregnancy and lactation period can increase the relative abundance of Bacteroides in the intestines of offspring.

[0143] 3.2.4. Changes in Escherichia-Shigella spp. in the intestines of offspring mice

[0144] Figure 10 shows the changes in Escherichia-Shigella in the intestines of offspring mice. One week after birth, Escherichia-Shigella was the dominant genus in the intestines of offspring in different doses of active folic acid intervention groups. With the continuation of breastfeeding, the relative abundance of Escherichia-Shigella in the intestines of offspring decreased significantly after three weeks of birth, and the relative abundance of Escherichia-Shigella in the intestines of offspring in the high-dose active folic acid intervention group was significantly lower than that in the control group.

[0145] 3.2.5. Changes in Streptococcus in the Intestines of Offspring Rats

[0146] Figure 11 shows the changes in Streptococcus in the intestines of offspring mice. One week after birth, the relative abundance of Streptococcus in the intestines of offspring in different doses of active folic acid intervention groups was higher than that in the control group. With the continuation of breastfeeding, the relative abundance of Streptococcus in the intestines of offspring decreased significantly after three weeks of birth, and the decrease was greater in different doses of active folic acid intervention groups than in the control group.

Claims

1. Use of an active folate substance in the manufacture of a foodstuff for the modulation of the gut microbiota of a mother and her offspring via maternal ingestion; wherein, The active folic acid substance is selected from 6S-5-methyltetrahydrofolate and its salts, the mother is in the pregnancy and / or lactation period, and the offspring is in the fetal, infancy, and / or childhood period.

2. Use according to claim 1, characterized in that, The active folic acid substance is 6S-5-methyltetrahydrofolate, calcium 6S-5-methyltetrahydrofolate, or glucosamine 6S-5-methyltetrahydrofolate.

3. Use according to claim 1 or 2, characterized in that, The methods for regulating the gut microbiota of mothers and their offspring include at least one of the following: increasing the relative abundance of Akkermansia in the gut of pregnant mothers, increasing the relative abundance of Bifidobacterium in the gut of pregnant mothers, decreasing the relative abundance of Escherichia-Shigella in the gut of pregnant mothers, and decreasing the relative abundance of Streptococcus in the gut of pregnant mothers.

4. Use according to any one of claims 1 to 3, characterized in that, The methods for regulating the gut microbiota of mothers and their offspring include: increasing the relative abundance of Akkermansia in the gut of lactating mothers, increasing the relative abundance of Clostridia UCG-014 unclassified in the gut of lactating mothers, increasing the relative abundance of Muribaculaceae unclassified in the gut of lactating mothers, decreasing the relative abundance of Escherichia-Shigella in the gut of lactating mothers, and decreasing the relative abundance of Streptococcus in the gut of lactating mothers.

5. Use according to any one of claims 1 to 4, characterized in that, The method of helping to regulate the gut microbiota of the mother and her offspring includes at least one of the following: increasing the relative abundance of Akkermansia in the offspring gut, increasing the relative abundance of Muribauculaceae unclassified in the offspring gut, increasing the relative abundance of Bacteroides in the offspring gut, decreasing the relative abundance of Escherichia-Shigella in the offspring gut, and decreasing the relative abundance of Streptococcus in the offspring gut.

6. Use according to any one of claims 1 to 5, 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.

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

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

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

10. Use according to any one of claims 1 to 9, 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.

11. Use according to any one of claims 1 to 10, characterized in that, The active folate substance is present in the foodstuff in an amount of 0.0001% to 0.1%. The active folate substance is present in the foodstuff in an amount of 0.0001% to 0.1%.

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