Use of nutritional composition in promoting bidirectional development of gut and brain via gut-brain axis

Abstract

A use of a nutritional composition in promoting the bidirectional development of the gut and brain via the gut-brain axis. The nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galactooligosaccharide, 1,3-dioleic acid-2-palmitic acid triglyceride, lutein, nucleotide, lactoferrin, casein phosphopeptide, and probiotics. By combining and strengthening multiple functional active components, the gut health and brain cognitive development of the organism can be bidirectionally promoted by means of the microbiota-gut-brain axis. In particular, the present application can promote the regulation of flora in the gut of the organism, increase the abundance of beneficial bacteria, and decrease the abundance of harmful bacteria related to brain development diseases. The content of butyric acid in the gut can be increased, and the contents of acetic acid and propionic acid are decreased to promote brain and cognitive development.

Description

Use of nutritional composition to promote bidirectional development of intestine and brain through gut-brain axis Technical Field

[0001] The present invention belongs to the technical field of functional nutrient research, and specifically relates to the use of a nutrient composition in promoting the bidirectional development of the intestine and brain. Background Art

[0002] Breast milk is the ideal natural food for infants, promoting the development of their physique, gastrointestinal tract, brain, neurocognitive, and immune systems. For infants who do not have enough breast milk or are unable to receive breastfeeding, formula is the best alternative. Infant formula is based on cow's milk, goat's milk, or plant-based milk (such as soy milk), with additional protein, fat, and carbohydrates added to make the formula's nutritional composition more similar to breast milk. With the continuous deepening of nutrient analysis technology for breast milk and cow's and goat's milk, differences have been found in the nutritional profile, molecular structure, and content of individual components. Consequently, various new functional ingredients, such as human milk oligosaccharides (HMOs) and probiotics, are being developed in the hope that infant formula can mimic the nutritional composition and functions of breast milk.

[0003] Numerous studies have reported that the gut-brain axis is a two-way communication pathway, in which intestinal microorganisms and brain health influence each other. After the first few days of life, the microbiome focuses on extracting nutrients to support the rapid development of the host brain and body. A key point of difference in the intestinal flora may depend on whether the infant is breastfed or formula-fed. Despite heterogeneity between population characteristics and research techniques, most studies have shown that the diversity and richness of the intestinal flora of breastfed infants are lower than those of formula-fed infants. Studies have also shown that breastfed infants are superior to formula-fed infants in brain and neuropsychological development.

[0004] Therefore, in the current design of infant formula, on the one hand, the nutritional composition of the formula is continuously approaching the level of breast milk through the addition of new functional ingredients. On the other hand, in terms of clinical function, there is also the expectation that effective ingredients can bring infants' gastrointestinal and brain development closer to the level of breastfed infants. Currently, the gut-brain axis has become a current research hotspot, and many substances that may affect the gut-brain axis have been discovered and studied.

[0005] Human milk oligosaccharides (HMOs) are the third largest solid component of breast milk, after lactose and fat. Over 200 different structures have been identified. Literature reports that subtle structural differences among HMOs confer distinct physiological functions, potentially influencing brain maturation and neurodevelopment. HMOs serve as a food source for developing intestinal microbes, influencing brain development through the gut-brain axis. They are also direct or indirect sources of sialic acid, an essential nutrient for brain tissue. Current methods for synthesizing HMOs primarily include enzymatic and microbial fermentation. However, due to limitations such as genetic modification, only two neutral oligosaccharides synthesized by microbial fermentation—2'-fucoylactose (2'-FL) and lacto-N-neotetraose (LNnT)—are currently permitted in infant and toddler dairy products. Other HMOs, particularly acidic oligosaccharides containing sialic acid groups that support brain development, are not yet approved for use in infant and toddler foods.

[0006] Research on the effects of probiotics on the gut-brain axis has also been reported. For example, the combination of Bifidobacterium lactis subsp. lactis Bb-12 and other strains has shown promise in treating schizophrenia through gut-brain axis effects, promoting increases in the neurotrophic factor BDNF while reducing the incidence of severe dyspnea. However, as one of the few probiotics approved for inclusion in infant formula, little is known about whether Bb-12 can benefit infants through the gut-brain axis. Summary of the Invention

[0007] Problems to be solved by the invention

[0008] Although existing technologies have studied functional substances that may play a role in the gut-brain axis, such research still cannot be said to be sufficient. There are very few studies on various functional substances and their combinations, as well as their addition to infant nutritional formula to mediate intestinal and brain development through the gut-brain axis.

[0009] To this end, the present invention aims to provide a nutritional composition for promoting bidirectional development of the intestine and brain through the gut-brain axis.

[0010] Solutions for solving problems

[0011] [1] A nutritional composition for use in preparing a food for promoting bidirectional development of the intestine and brain through the gut-brain axis; characterized in that the nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptide and probiotics.

[0012] [2] The use according to [1], characterized in that the nutritional composition further comprises choline, inositol, taurine and L-carnitine.

[0013] [3] The use according to [1] or [2], characterized in that the probiotics include Bifidobacterium animalis subsp. lactis.

[0014] [4] The use according to any one of [1] to [3], characterized in that the nucleotides include disodium 5'-cytidylate, disodium 5'-uridylate, adenosine 5'-monophosphate, disodium 5'-guanylate and disodium 5'-inosinate.

[0015] [5] The use according to any one of [1] to [4], characterized in that the promoting bidirectional development of the intestine and brain through the gut-brain axis comprises at least one of increasing the abundance of Lactobacillus in the intestine, increasing the abundance of Eubacterium in the intestine, and reducing the abundance of Sutterellaceae in the intestine.

[0016] [6] The use according to any one of [1] to [5], characterized in that the promoting bidirectional development of the intestine and brain through the gut-brain axis includes at least one of increasing the content of butyric acid in the intestine, reducing the content of acetic acid in the intestine, and reducing the content of propionic acid in the intestine.

[0017] [7]. The use according to any one of [1] to [6], characterized in that the food is infant formula.

[0018] [8] The use according to any one of [1] to [7], characterized in that the nutritional composition further comprises any one or more of the following ingredients: animal milk, protein components, fat components, carbohydrate components, vitamins and minerals.

[0019] [9] The use according to [8], characterized in that the nutritional composition is a powdered solid.

[0020]

[0010] . The use according to any one of [1] to [9], characterized in that the nutritional composition contains human milk oligosaccharides, and the human milk oligosaccharides include at least one of 3'-sialyllactose, 6'-sialyllactose, 4'-galactosyllactose, 3'-galactosyllactose, 6'-galactosyllactose, 2'-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose and N-acetylneuraminic acid.

[0021] Effects of the Invention

[0022] The present invention provides a use of a nutritional composition in preparing a food that promotes the bidirectional development of the intestine and brain through the gut-brain axis. By combining and strengthening multiple functional active components such as docosahexaenoic acid, eicosatetraenoic acid, oligogalactose, 1,3-dioleyl-2-palmityl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptide and probiotics, the body's intestinal health and brain cognitive development can be promoted bidirectionally through the microbial-gut-brain axis. In particular, while ensuring the physical development of the body, the development of organs such as the heart, liver, spleen, and kidneys, and the development of intestinal structure without any difference, the nutritional composition can further promote brain development and improve the brain index; it can promote the regulation of intestinal flora, increase the abundance of psychobiotics Lactobacillus, and increase the abundance of Blautia that promotes brain and cognitive development through immune homeostasis; at the same time, it can reduce the abundance of harmful bacteria Sutterellaceae related to brain development diseases; and it can increase the butyric acid content in the intestine and reduce the acetic acid and propionic acid content to promote brain and cognitive development; it provides assistance for the development of infant formula. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: Comparison of body weight of rats after 28 days of intervention with different formulas and doses of formula powder.

[0024] Figure 2A: Comparison of the abundance of the beneficial bacteria genus Lactobacillus in the intestines of rats after 28 days of intervention with different formulas and doses of formula powder.

[0025] Figure 2B: Comparison of the abundance of the beneficial bacteria genus Blautia in the intestines of rats after 28 days of intervention with formula powders of different formulas and doses.

[0026] Figure 3: Comparison of harmful bacteria at the intestinal level in rats after 28 days of intervention with different formulas and dosages of formula powder.

[0027] Figure 4A: Comparison of the production of short-chain fatty acid butyrate in the intestine of rats after 28 days of intervention with formula powders of different formulas and doses.

[0028] Figure 4B: Comparison of the production of short-chain fatty acid acetic acid in the intestine of rats after 28 days of intervention with formula powders of different formulas and doses.

[0029] Figure 4C: Comparison of the production of short-chain fatty acid propionate in the intestine of rats after 28 days of intervention with different formulas and doses of formula powder. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various modifications can be made within the scope of the present invention. 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.

[0031] <Term Definition>

[0032] In the present invention, the terms "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited components or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, excluding additional, unrecited components or method steps.

[0033] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0034] In the present invention, "infant" is used to refer to a human group aged 0 to 6 months.

[0035] In the present invention, "older infants" are used to refer to a human group of 6 to 12 months of age.

[0036] In the present invention, the term "infant" refers to a human group aged 12 to 36 months.

[0037] In the present invention, "infants" are used to refer to the human group under 3 years old.

[0038] In the present invention, the term "infant formula" encompasses infant formula, follow-on formula, and toddler formula. Generally, infant formula is used as a breast milk substitute from birth, follow-on formula is used as a breast milk substitute from 6 to 12 months after birth, and toddler formula is used as a breast milk substitute from 12 to 36 months after birth.

[0039] In the present invention, "animal milk" is used to refer to the liquid obtained from the mammary glands of mammals during lactation. The term "animal milk" should be interpreted broadly and covers both raw milk (i.e., liquid obtained directly from the mammary gland) and standardized milk products (such as, for example, skim milk or whole milk).

[0040] In the present invention, for the convenience of describing fatty acid glycerides, the following characters are used to refer to different types of fatty acids: P: palmitic acid (C16:0); O: oleic acid (C18:1).

[0041] In the present invention, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0042] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] <Nutritional Composition>

[0044] The present invention has found that by combining and strengthening multiple functional active components such as docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptides and probiotics, the body's intestinal health and brain development can be promoted bidirectionally through the microbial-gut-brain axis.

[0045] In some embodiments, the nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptides, and probiotics.

[0046] In some embodiments, the nutritional composition further comprises choline, inositol, taurine, and L-carnitine.

[0047] In some specific embodiments, the nutritional composition comprises the following essential components: choline, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptides and probiotics.

[0048] In some specific embodiments, the nucleotides include disodium 5'-cytidylate, disodium 5'-uridine, adenosine 5'-monophosphate, disodium 5'-guanylate, and disodium 5'-inosinate.

[0049] In some embodiments, the probiotics include Bifidobacterium animalis subsp. lactis. Exemplarily, the Bifidobacterium animalis subsp. lactis includes Bb-12 strain, HN109 strain, Bi-07 strain, etc. In some preferred embodiments, the probiotics include Bifidobacterium animalis subsp. lactis Bb-12 strain.

[0050] In some specific embodiments, the nutritional composition comprises the following essential components: choline, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoylglycerol, lutein, disodium 5'-cytidylate, disodium 5'-uridylphosphate, adenosine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-inosinate, lactoferrin, casein phosphopeptide and Bifidobacterium animalis subsp. lactis Bb-12.

[0051] In order to meet the basic nutritional needs of consumers, in some embodiments, the nutritional composition further comprises any one or more of the following ingredients: animal milk, protein components, fat components, carbohydrate components, vitamins and minerals.

[0052] By adding the above raw materials, in some embodiments, the nutritional composition contains a variety of human milk oligosaccharides, for example, at least one of 3'-sialyllactose, 6'-sialyllactose, 4'-galactosyl lactose, 3'-galactosyl lactose, 6'-galactosyl lactose and N-acetylneuraminic acid.

[0053] By combining a variety of raw materials and / or human milk oligosaccharides with the necessary components of the above-mentioned nutritional composition, a more effective effect of promoting the body's intestinal health and brain development in both directions through the gut-brain axis can be further achieved.

[0054] In some embodiments, the source of the animal milk includes cows and / or sheep. In some specific embodiments, the animal milk is raw cow milk.

[0055] In some embodiments, the source of the protein component includes at least one of whole milk powder, skim milk powder, concentrated whey protein powder, whey protein powder, hydrolyzed whey protein powder, and demineralized whey powder.

[0056] In some embodiments, the source of the fat component includes at least one of structured mixed esters, sunflower oil, coconut oil, linseed oil, corn oil, rapeseed oil, and soybean oil.

[0057] In some embodiments, the source of the carbohydrate component comprises lactose.

[0058] In some embodiments, the vitamins include at least one of vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, calcium pantothenate, vitamin C, biotin, and niacinamide.

[0059] In some embodiments, the mineral comprises at least one of copper sulfate, magnesium sulfate, ferric pyrophosphate, zinc sulfate, calcium citrate, dibasic calcium phosphate, potassium iodate, sodium selenite, manganese sulfate, potassium chloride, calcium carbonate, tricalcium phosphate, sodium citrate, and ferrous sulfate.

[0060] For ease of use and transportation, etc., in some embodiments, the nutritional composition of the present invention is a powdered solid.

[0061] In some embodiments, based on dry weight, in the nutritional composition, the protein content is 8-20 g / 100 g, the fat content is 17-28 g / 100 g, and the carbohydrate content is 30-75 g / 100 g.

[0062] <Food>

[0063] The nutritional composition of the present invention can be used to prepare a variety of foods, including infant formula, etc. In some embodiments, the infant formula includes infant formula milk powder.

[0064] In addition, in some embodiments, the food further comprises any acceptable excipients, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0065] The present invention does not impose any particular limitation on the amount of the nutritional composition and its components used in food.

[0066] In some embodiments, based on dry weight, the content of docosahexaenoic acid in the food is greater than or equal to 40 mg / 100 g, preferably greater than or equal to 100 mg / 100 g, and preferably less than or equal to 190 mg / 100 g.

[0067] In some embodiments, based on dry weight, the content of eicosatetraenoic acid in the food is greater than or equal to 75 mg / 100 g, preferably greater than or equal to 170 mg / 100 g, and preferably less than or equal to 350 mg / 100 g.

[0068] In some embodiments, based on dry weight, the content of the galacto-oligosaccharide in the food is greater than or equal to 1 g / 100 g, preferably greater than or equal to 2 g / 100 g, and preferably less than or equal to 6.2 g / 100 g.

[0069] In some embodiments, based on dry weight, the content of 1,3-dioleoyl-2-palmitoyl triglyceride in the food is greater than or equal to 1 g / 100 g, preferably greater than or equal to 3 g / 100 g, and preferably less than or equal to 7 g / 100 g.

[0070] In some embodiments, based on dry weight, the lutein content in the food is greater than or equal to 200 μg / 100 g, preferably greater than or equal to 205 μg / 100 g, and preferably less than or equal to 220 μg / 100 g.

[0071] In some embodiments, based on dry weight, the content of the nucleotide in the food is greater than or equal to 20 mg / 100 g, preferably greater than or equal to 25 mg / 100 g, and preferably less than or equal to 50 mg / 100 g.

[0072] In some embodiments, based on dry weight, the content of lactoferrin in the food is greater than or equal to 35 mg / 100 g, preferably greater than or equal to 40 mg / 100 g, and preferably less than or equal to 500 mg / 100 g.

[0073] In some embodiments, based on dry weight, the content of the casein phosphopeptide in the food is greater than or equal to 30 mg / 100 g, preferably greater than or equal to 35 mg / 100 g, and preferably less than or equal to 50 mg / 100 g.

[0074] In some embodiments, based on dry weight, the choline content in the food is greater than or equal to 90 mg / 100 g, preferably greater than or equal to 200 mg / 100 g, and preferably less than or equal to 460 mg / 100 g.

[0075] In some embodiments, based on dry weight, the content of inositol in the food is greater than or equal to 20 mg / 100 g, preferably greater than or equal to 35 mg / 100 g, and preferably less than or equal to 200 mg / 100 g.

[0076] In some embodiments, based on dry weight, the taurine content in the food is greater than or equal to 15 mg / 100 g, preferably greater than or equal to 33 mg / 100 g, and preferably less than or equal to 80 mg / 100 g.

[0077] In some embodiments, based on dry weight, the content of L-carnitine in the food is greater than or equal to 5 mg / 100 g, preferably greater than or equal to 10 mg / 100 g, and preferably less than or equal to 50 mg / 100 g.

[0078] <Use of promoting bidirectional development of the gut and brain through the gut-brain axis>

[0079] The present invention strengthens multiple functional active components such as docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptides and probiotics, which can promote the intestinal health and brain cognitive development of the body in both directions through the microbial-intestinal-brain axis. Furthermore, foods containing such nutritional compositions can promote the bidirectional development of the intestine and brain through the gut-brain axis. Moreover, the combination of the above-mentioned multiple functional active components with other ingredients in the nutritional composition can further enhance the effect of promoting the development of the intestine and brain in both directions.

[0080] In some embodiments, the promoting bidirectional development of the intestine and brain through the gut-brain axis includes increasing the abundance of Lactobacillus in the intestine, increasing the abundance of Blautia in the intestine, and reducing the abundance of Sutterellaceae in the intestine.

[0081] In some embodiments, promoting bidirectional development of the gut and brain through the gut-brain axis comprises at least one of increasing the content of butyrate in the gut, decreasing the content of acetate in the gut, and decreasing the content of propionate in the gut.

[0082] Example

[0083] The embodiments of the present invention will be described in detail below with reference to the examples. However, it will be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used were commercially available conventional products.

[0084] 1. Nutritional composition function evaluation method

[0085] 1.1 Experimental Animals

[0086] Fifty SPF male Sprague-Dawley rats, 3 weeks old (21-27 days old), were weaned and, after 7 days of adaptive feeding, randomly divided into six groups based on body weight: a group supplemented with 15% control formula, a group supplemented with 15% experimental formula, a group supplemented with 20% control formula, a group supplemented with 20% experimental formula, a group supplemented with 30% control formula, and a group supplemented with 30% experimental formula. The rats were fed for 4 weeks. Body weight and food intake were measured on days 0, 7, 14, 21, and 28 of group feeding. Experimental animals were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., with experimental unit use license number: SYXK (Beijing) 2022-0049 and experimental animal license number: SCXK (Beijing) 2021-0011. The animal experiments were approved by the Capital Medical University Ethics Committee, with animal ethics review number: AEEI-2023-035.

[0087] 1.2 Experimental animal feed

[0088] Basal feeds were supplemented with 15% control formula, 15% experimental formula, 20% control formula, 20% experimental formula, 30% control formula, and 30% experimental formula. The experimental formulas contained appropriate amounts of DHA, ARA, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoylglycerol, lutein, nucleotides, lactoferrin, and casein phosphopeptides. The control formulas in this experiment did not contain any of these ingredients. All feeds were provided by Keao Xieli (Tianjin) Feed Co., Ltd. The control and experimental formulas replaced corn starch in the feed at three different doses: 15%, 20%, and 30%.

[0089] 1.3 Biological sample collection and testing

[0090] On day 29 after group feeding, five SD rats were randomly selected from each group. The rats were immobilized, their tails lifted, and the lower abdomen gently pressed with fingers. Fresh feces were collected using the stress defecation method and stored in sterile EP tubes at -80°C until further use (n = 5). On day 35 after group feeding, the rats were anesthetized with an intraperitoneal injection of tribromoethanol and euthanized. The brain, heart, liver, spleen, and kidneys were quickly collected. The organs were repeatedly rinsed with 0.9% saline, dried with filter paper, weighed, and stored at -80°C until further use.

[0091] 1) Fecal 16S rRNA fecal intestinal flora sequencing

[0092] Genomic DNA was extracted from SD rat feces; PCR amplification was performed on the 338F_806R region, and the PCR products were quantitatively detected using the QuantiFluorTM-ST blue fluorescence quantitative system. The PCR products were mixed according to the corresponding ratios based on the sequencing requirements of each sample; Miseq library construction was performed; Miseq sequencing results were obtained; PCR synthesis was performed using DNA fragments as templates and primers with fixed base sequences in the chip to obtain target DNA fragments to be detected in the chip; DNA clusters were generated; laser scanning was performed on the surface of the reaction plate to read the nucleotide types polymerized by each template sequence in the first round of reaction; and the collected fluorescence signals were statistically analyzed to obtain the sequence of template DNA fragments.

[0093] 2) Fecal short-chain fatty acid level detection

[0094] Sample processing:

[0095] Weigh 20 mg of fecal sample into a 2 ml grinding tube and add 800 μL of 0.5% phosphoric acid (containing 10 μg / mL of 2-ethylbutyric acid as internal standard). Cryo-grind the sample for 3 minutes (50 Hz), then sonicate for 10 minutes and centrifuge at 13,000 g at 4°C for 15 minutes. Remove 200 μL of the supernatant and transfer it to a 1.5 mL centrifuge tube, then add 200 μL of n-butanol solvent for extraction. Vortex for 10 seconds, sonicate for 10 minutes, and centrifuge at 13,000 g at 4°C for 5 minutes. Transfer the supernatant to a vial and load it onto the instrument.

[0096] GC-MS detection:

[0097] The analytical instrument used in this experiment was an Agilent Technologies Inc. (CA, UAS) 8890B-7000D GC / MSD gas spectrometer. Chromatographic conditions: HP FFAP capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas: high-purity helium (≥99.999%), flow rate: 1.0 mL / min, inlet temperature: 180°C. Injection volume: 1 μL, split injection, split ratio: 10:1, solvent delay: 2.5 min. Temperature program: The column oven temperature was initially set at 80°C, ramped at 20°C / min to 120°C, then at 5°C / min to 160°C, followed by a 3-min hold at 220°C. Mass spectrometry conditions: electron impact ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, transfer line temperature 230°C, electron energy 70 eV. Scanning mode was selected ion scanning mode (SIM).

[0098] 3) HE staining of ileum and colon tissue

[0099] Paraffin-embedded tissue sections:

[0100] (1) Sampling: Fresh tissue was fixed in 4% paraformaldehyde for at least 24 hours. The tissue was removed from the fixative and trimmed flat with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box.

[0101] (2) Dehydration: Place the dehydration box in the hanging basket and dehydrate in the dehydrator in a gradient of alcohol. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, ethanol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, wax I for 1 hour, wax II for 1 hour, wax III for 1 hour.

[0102] (3) Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block.

[0103] (4) Sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4 μm. Float the slices on a 40°C warm water slide to flatten the tissue. Pick up the tissue with a glass slide and bake it in a 60°C oven. Once the water is dried and the wax is melted, remove the slices and store them at room temperature for later use.

[0104] HE staining:

[0105] (1) Dewaxing of paraffin sections: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and then wash with distilled water.

[0106] (2) Hematoxylin staining of cell nuclei: Stain sections with Harris hematoxylin for 3-8 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia solution, and rinse with running water.

[0107] (3) Eosin staining of cytoplasm: Slice into eosin staining solution and stain for 1-3 minutes.

[0108] (4) Dehydration and sealing: Dehydrate the sections in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to make them transparent. Take the sections out of xylene, let them dry slightly, and seal them with neutral gum.

[0109] (5) Microscopic examination, image acquisition and analysis.

[0110] 4) Statistical analysis

[0111] SPSS 21.0 software was used for statistical analysis of the data to obtain relevant information. Descriptive statistics were used to analyze the experimental data, and quantitative indicators were expressed as mean ± standard deviation (mean ± SD). For comparisons of the five groups of measurement data, one-way analysis of variance was used for data that met normality and had homogeneous variances, while nonparametric tests (Kruskal-Wallis tests) were used for data that did not meet normality or had heterogeneous variances. A P value of < 0.05 indicated statistical significance. Graphs were generated using GraphPad Prism 5.0.

[0112] 2. Preparation of Experimental Formula Milk Powder

[0113] The raw milk and other proteins, fats, carbohydrates, vitamins, minerals and other functional ingredients produced by the applicant's own ranch are prepared into powder through processes such as sterilization, mixing, sterilization, homogenization, concentration and spray drying. Among them, proteins include skim milk powder, concentrated whey protein powder, whey protein powder, and hydrolyzed whey protein powder; lipids are mainly edible vegetable blended oils (1,3-dioleyl 2-palmityl triglyceride, sunflower oil, coconut oil, flaxseed oil); carbohydrates include lactose and oligosaccharides; vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, vitamin C, biotin; minerals include Including copper sulfate, magnesium sulfate, ferric pyrophosphate, zinc sulfate, calcium citrate, calcium hydrogen phosphate, potassium iodate, sodium selenite, manganese sulfate; other functional active ingredients include choline, inositol, taurine, L-carnitine, docosahexaenoic acid (DHA), arachidonic acid (ARA), lutein, nucleotides (disodium 5'-cytidylate, disodium 5'-uridine, adenosine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-inosinate), lactoferrin, casein phosphopeptide, Bifidobacterium animalis (Bb-12).

[0114] The composition of HMOs and lipid nutrients in the final experimental formula product is shown in the following table.

[0115] Table 1 Composition of HMOs and lipid nutrients in the experimental formula

[0116] 3. Preparation of Control Formula Milk Powder

[0117] Raw cow's milk and other proteins, fats, carbohydrates, vitamins, minerals, and other functional ingredients are prepared into powder through processes such as sterilization, mixing, sterilization, homogenization, concentration, and spray drying. Proteins include demineralized whey powder, whole milk powder, skim milk powder, and whey protein powder; lipids are mainly vegetable oils (corn oil, rapeseed oil, coconut oil, sunflower oil, and soybean oil); carbohydrates are mainly lactose; and other food additives include phospholipids, retinyl acetate, cholecalciferol, dl-α-tocopheryl acetate, phytonadione, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, cyanocobalamin, niacinamide, folic acid, D-calcium pantothenate, L-ascorbic acid, D-biotin, inositol, taurine, potassium chloride, calcium carbonate, tricalcium phosphate, sodium citrate, ferrous sulfate, zinc sulfate, magnesium sulfate, copper sulfate, manganese sulfate, potassium iodate, sodium selenite, choline chloride, and L-carnitine.

[0118] 4. Differences in nutritional indicators between the experimental formula and the control formula

[0119] Table 2 Nutritional composition of experimental formula and comparative formula

[0120] 5. Example and Comparative Example Setup

[0121] Example 1: 15% of the experimental formula was mixed into the basic feed of rats.

[0122] Example 2: 20% of the experimental formula was mixed into the basic feed of rats.

[0123] Example 3: 30% of the experimental formula was mixed into the basic feed of rats.

[0124] Comparative Example 1: 15% of the control formula was mixed into the basic feed of rats.

[0125] Comparative Example 2: 20% of the control formula was mixed into the basic feed of rats.

[0126] Comparative Example 3: 30% of the control formula was mixed into the basic feed of rats.

[0127] Finally, the energy supply ratio and energy density of the three major macronutrients in the rat feed of each embodiment and comparative example are shown in the following table:

[0128] Table 3 Basic feed and experimental feed formula

[0129] 6. Body weight test of rats in each group

[0130] After 28 days of group feeding, no significant difference in body weight was found among the rats in each group, as shown in Figure 1. This indicates that the addition of different types of formula milk powder and different doses of formula milk powder had no significant effect on the increase in rat body weight.

[0131] 7. Organ development test of rats in each group

[0132] After 28 days of group feeding, the development of various organs of the rats was measured and characterized by organ index. Organ index = weight of each organ / body weight. The differences in brain index, heart index, liver index, spleen index and kidney index of rats in the experimental formula milk powder group and the control formula milk powder group were compared using t-test. The results are shown in the following table. It was found that except for the brain index of the experimental formula group (Example 1, Example 2 and Example 3), which was significantly higher than that of the control formula group (Comparative Example 1, Comparative Example 2 and Comparative Example 3) (p < 0.01, p < 0.05 and p < 0.001), there was no significant difference in liver index, spleen index and kidney index between the comparative example and the example. The above results prove that the experimental formula milk powder has the effect of promoting brain development.

[0133] Table 4 Organ indexes of rats after 28 days of intervention with different ratios and different milk powders

[0134] 8. Length and structure of the colorectum and ileum of rats in each group

[0135] This study examined the length and structure of the colorectum and ileum of rats in each group. Results showed no significant differences in colorectal and ileal length among the rats after 28 days of intervention feeding. HE staining revealed that the colorectal and ileal structures of all groups were essentially normal, with clear and intact villi, densely packed mucosal epithelial cells, and no necrosis. Crypts were densely packed, with no obvious abscesses or dilation. Edema was absent in the submucosa. No significant inflammatory cell infiltration was observed in any of the tissues. Therefore, neither the experimental nor the control formula affected the normal development of intestinal structure.

[0136] 9. Investigation of intestinal flora of rats in each group

[0137] Many studies have reported that the mammalian intestinal flora coexists with the host and is closely related to the host's digestive, immune and nervous system development. The active metabolites produced by the intestinal flora can act as neurotransmitters. Although the occurrence and growth of nerve cells mainly occur in the fetal period, the formation of glial cells, synapses and myelin sheaths will continue throughout infancy. The continued development of nerve cells throughout infancy will be affected by many factors, one of which is the intestinal flora. Some studies believe that the development of the nervous system is related to the intestinal flora. Intestinal flora imbalance can lead to changes in its metabolites, thereby affecting the gut-brain axis communication system, brain immune function, central nervous system inflammation, and the integrity of the blood-brain barrier.

[0138] In this study, rat fecal samples were subjected to 16S rRNA sequencing analysis to analyze the rat intestinal flora, and the effects of different formula milk powders on intestinal flora were compared. It was found that at the genus level, the abundance of beneficial bacteria was higher in the experimental group formula milk-fed rats. Fig. 2 A shows that the abundance of lactobacillus (Lactobacillus) in the intestinal tract of the experimental group (Example 1, Example 2 and Example 3) formula-fed rats after 28 days was significantly higher than that in the control group (Comparative Example 1, Comparative Example 2, and Comparative Example 3). And along with the continuous increase of the formula dosage, the abundance of lactobacillus (Lactobacillus) continuously increases. As is well known, lactobacillus is an important sign of human intestinal health. It has the important physiological functions of regulating host intestinal flora, promoting nutrient absorption and metabolic regulation, and studies have shown that lactobacillus can play an important role in the prevention and treatment of depression. Animal models and / or human clinical trials show that antidepressant lactobacilli mainly include lactobacillus (Lactobacillus) and bifidobacterium, both of which are referred to as psychobiotics. Figure 2B shows the abundance comparison of the genus Blautia of the Lachnospiraceae family in the experimental group and the control group. It can be seen that the abundance of this bacterium in the intestine of the milk powder-fed rats in the experimental group is higher than that in the control group. After the Mann-Whitney test, it was found that Example 1 and Example 2 were significantly higher than those in Comparative Example 1 (p < 0.05) and Comparative Example 2 (p < 0.01), respectively. Blautia is a core genus of bacteria in the intestine. Studies have shown that it has antibacterial activity against some specific pathogens and has a certain anti-inflammatory effect. The immune system and neuroimmunity are one of the ways for the gut-brain axis to communicate. Studies have also shown that immune homeostasis disorders can affect brain and neural development in early life.

[0139] Figure 3 shows the difference in the abundance of Sutterellaceae in the intestines of rats in the experimental groups (Example 1, Example 2 and Example 3) and the control groups (Comparative Example 1, Comparative Example 2 and Comparative Example 3) after being fed with the formulas for 28 days. The model genus of this family is Sutterella, which is related to human diseases, especially brain development-related diseases such as autism and Down syndrome. It is also related to the occurrence of inflammatory bowel disease (IBD). From the results of this study, it can be seen that the abundance of this family in the intestines of rats in the experimental formula groups is lower than that of the corresponding control groups, and after the Mann-Whitney test, it was found that the abundance of Sutterellacea in the intestines of rats in Example 3 was significantly lower than that in Comparative Example 3 (p < 0.05).

[0140] 10. Investigation of the production of short-chain fatty acids in the intestine of rats in each group

[0141] Short-chain fatty acids (SCFAs) are one of the main metabolites of intestinal flora. Studies have reported that SCFAs are related to cognitive development. SCFAs can stimulate the synthesis and secretion of 5-hydroxytryptamine (5-HT) in the intestine. After 5-HT binds to its receptor, it plays a role in regulating motility, intervening in neuronal development and differentiation, and regulating emotions through neural signals. Many studies have reported that butyrate can regulate mitochondrial function, stimulate oxidative phosphorylation and fatty acid oxidation, and upregulate physiological stress pathways. It has been reported that butyrate can regulate social behavior in autism mouse models. This study found that the experimental group formula can promote the production of butyrate in the rat intestine, as shown in Figure 4A. The butyric acid content in Example 2 and Example 3 groups was significantly higher than that in Comparative Example 2 and Comparative Example 3, respectively (p < 0.01). Studies have reported that the levels of acetic acid and propionic acid in the intestines of autistic patients are higher than those in normal children. Higher concentrations of acetic acid and propionic acid can penetrate the intestinal barrier and the blood-brain barrier and enter the brain, affecting physiological processes related to autism, such as cell signal transduction, neurotransmitter synthesis and release, free radical production, immune function, etc., and have neurotoxicity. This study found that the experimental formula group can reduce the content of acetic acid and butyric acid in the rat intestine after intervention. As shown in Figures 4B and 4C, the content of acetic acid in the rat intestine in the three example groups was significantly lower than that in the respective control groups, while the propionic acid content in Example 1 was significantly lower than that in Control Group 1 (p < 0.01). The propionic acid content in Examples 2 and 3 was lower than that in the corresponding control groups, but the difference was not significant (p > 0.05).

[0142] Industrial applicability

[0143] The nutritional composition provided by the present invention can be widely used in industry for preparing food that promotes bidirectional development of the intestine and brain through the gut-brain axis.

Claims

1. A nutritional composition for preparing a food for promoting bidirectional development of the intestine and brain via the gut-brain axis; characterized in that: The nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharide, 1,3-dioleyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptide and probiotics.

2. The use according to claim 1, characterized in that The nutritional composition also includes choline, inositol, taurine, and L-carnitine.

3. The use according to claim 1 or 2, characterized in that The probiotics include Bifidobacterium animalis subsp. lactis.

4. The use according to any one of claims 1 to 3, characterized in that The nucleotides include 5'-cytidylate disodium, 5'-uridine monophosphate disodium, adenosine 5'-monophosphate, 5'-guanylate disodium and 5'-inosinate disodium.

5. The use according to any one of claims 1 to 4, characterized in that The promoting bidirectional development of the intestine and brain through the gut-brain axis includes at least one of increasing the abundance of Lactobacillus in the intestine, increasing the abundance of Eubacterium in the intestine, and reducing the abundance of Sutterellaceae in the intestine.

6. The use according to any one of claims 1 to 5, characterized in that The promoting bidirectional development of the intestine and brain through the gut-brain axis includes at least one of increasing the content of butyric acid in the intestine, reducing the content of acetic acid in the intestine, and reducing the content of propionic acid in the intestine.

7. The use according to any one of claims 1 to 6, characterized in that The food is infant formula.

8. The use according to any one of claims 1 to 7, characterized in that The nutritional composition further comprises any one or more of the following ingredients: animal milk, protein component, fat component, carbohydrate component, vitamins and minerals.

9. The use according to any one of claims 1 to 8, characterized in that The nutritional composition is a powdered solid.

10. The use according to any one of claims 1 to 9, characterized in that The nutritional composition contains human milk oligosaccharides, and the human milk oligosaccharides include at least one of 3'-sialyllactose, 6'-sialyllactose, 4'-galactosyl lactose, 3'-galactosyl lactose, 2'-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 6'-galactosyl lactose and N-acetylneuraminic acid.

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