CN118044614A - Use of nutritional composition for promoting intestinal and brain bi-directional development through intestinal brain axis - Google Patents

Abstract

The invention belongs to the technical field of functional nutrient research, and in particular relates to an application of a nutritional composition in promoting intestinal and brain bidirectional development, wherein 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. Experiments show that the combination of the functional active components can promote the intestinal health and the brain cognitive development of the organism through the microorganism-intestinal-brain axis bidirectional. In particular, the microbial inoculum can promote the regulation of flora in intestinal tracts of organisms, promote the abundance of beneficial bacteria and reduce the abundance of harmful bacteria related to brain development diseases; and can promote the content of butyric acid in intestinal canal, reduce the content of acetic acid and propionic acid, and promote brain and cognitive development.

Description

Use of nutritional composition for promoting intestinal and brain bi-directional development through intestinal brain axis

Technical Field

The invention belongs to the technical field of functional nutrient research, and particularly relates to application of a nutritional composition in promoting intestinal tract and brain bidirectional development.

Background

Breast milk is the most ideal natural food for infants, and can promote the development of infant physique, gastrointestinal tract, brain, neuro-cognition and immune system. Formula milk is the best alternative for infants who are not breast-fed or who are not receiving breast-feeding. Infant formula is based on cow milk, sheep milk or plant milk (such as soybean milk), and the nutritional composition of the formula is more similar to that of breast milk by adding protein, fat and carbohydrate. With the continuous and deep research of nutrient component analysis technologies such as breast milk, cow milk and sheep milk, the two are found to have differences in terms of the nutrition composition profile and the molecular structure and content of single components, so that various novel functional raw materials such as breast milk oligosaccharides (HMOs), probiotics and the like are continuously developed, and the infant formula powder can simulate the nutrition composition and the function of breast milk.

Numerous studies report that the intestinal axis is a two-way communication pathway, and that intestinal microorganisms interact with brain health. After the first few days of life, the focus of the microbiota is on the extraction of nutrients in order to support rapid development of the host brain and body. One key point of gut flora differences may depend on whether the infant is breast fed or formula fed. Despite the heterogeneity between population characteristics and study technology, most studies indicate that the diversity and richness of the intestinal flora of breast-fed infants is lower than that of formula-fed infants. And breast-fed infants are considered superior to formula-fed infants in terms of brain and neuropsychological development.

Therefore, in the current design of infant formula, on one hand, the nutritional composition of the formula is continuously close to the breast milk level by adding novel functional raw materials, and on the other hand, effective raw materials are expected to be capable of enabling infants to approach the breast-fed infant level in the aspects of gastrointestinal tract, brain development and the like in clinical function. The intestinal brain axis has become the current research hotspot, and a number of substances which can affect the intestinal brain axis have been discovered and studied.

Breast milk oligosaccharides (HMOs) are the third largest solid component in breast milk after it has been supplemented with lactose and fat, and at present, there are 200 different structures of breast milk oligosaccharides identified. There are literature reports that subtle differences in HMOs structure confer different physiological functions that can affect brain maturation and neural development. HMOs are on the one hand a food source for developing intestinal microorganisms, affecting brain development through the intestinal brain axis, and on the other hand HMOs are also a direct or indirect source of sialic acid, which is a necessary nutrient for brain tissue. The current synthetic methods of breast milk oligosaccharide raw materials mainly comprise an enzyme method and a microbial fermentation method, but are limited by factors such as transgenosis, and only two neutral oligosaccharides, namely 2 '-fucosyllactose (2' -FL) and lactose-N-neotetraose (LNnT), which are synthesized by microbial fermentation are allowed to be applied to infant and children dairy products. While other types of HMOs, in particular acidic oligosaccharides containing sialic acid groups and contributing to brain development have not been allowed to be added to infant foods.

Research on the action of probiotics on the intestinal brain axis has also been reported, for example, the combination of animal bifidobacterium lactis subspecies Bb-12 and other strains plays a certain role in the treatment of schizophrenia through the action of the intestinal brain axis, and can promote the promotion of neurotrophic factor BDNF, reduce the occurrence of serious defecation difficulty and the like. But Bb-12 is one of the few probiotics that can be added to infant formulas and it is very poorly understood whether infants can produce certain benefits through the gut brain axis.

Disclosure of Invention

Problems to be solved by the invention

Although the prior art has been directed to functional substances that may act in the intestinal brain axis, such studies have not been adequate, and there has been very little research directed to various functional substances and combinations thereof and their addition to infant nutritional formulas to mediate intestinal and brain development through the intestinal brain axis.

In this regard, the present invention aims to provide the use of a nutritional composition for promoting the bi-directional development of the intestine and brain through the intestinal-brain axis.

Solution for solving the problem

[1] Use of a nutritional composition for the preparation of a food for promoting intestinal and brain bi-directional development via the intestinal-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 phosphopeptides and probiotics.

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

[3] The use according to [1] or [2], wherein the probiotic comprises bifidobacterium animalis subspecies lactis.

[4] The use according to any one of [1] to [3], wherein the nucleotide comprises disodium 5' -cytidine, disodium 5' -uridine, adenosine 5' -monophosphate, disodium 5' -guanylate, and disodium 5' -inosinate.

[5] The use according to any one of [1] to [4], wherein the promotion of intestinal and brain bi-directional development by the intestinal brain axis comprises at least one of increasing the abundance of lactobacillus in the intestinal tract, increasing the abundance of truffle in the intestinal tract and decreasing the abundance of sarsashimi in the intestinal tract.

[6] The use according to any one of [1] to [5], wherein the promotion of intestinal and brain bi-directional development through the intestinal brain axis comprises at least one of an increase in the content of butyric acid in the intestinal tract, a decrease in the content of acetic acid in the intestinal tract, and a decrease in the content of propionic acid in the intestinal tract.

[7] The use according to any one of [1] to [6], wherein the food is an infant formula.

[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.

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

[10] The use according to any one of [1] to [9], characterized in that the nutritional composition comprises a milk oligosaccharide comprising 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.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention provides application of a nutritional composition in preparing food capable of promoting intestinal and brain bidirectional development through intestinal and brain axes, and can promote intestinal health and brain cognitive development of an organism through microorganism-intestinal and brain axes in a bidirectional way through combining and strengthening various functional active components such as docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharide, 1, 3-dioleate-2-palmitic acid triglyceride, lutein, nucleotide, lactoferrin, casein phosphopeptide and probiotics. In particular, under the condition of ensuring physical development of organisms, visceral development of heart, liver, spleen, kidney and the like and no difference in intestinal canal structural development, the nutritional composition can further promote brain development and promote brain index; it can promote regulation of flora in intestinal tract, promote abundance of probiotic Lactobacillus, promote abundance of truffle (Blauthia) promoting brain and cognitive development by immune homeostasis; simultaneously, the abundance of harmful bacteria Sagnac bacteria (Sutterellaceae) related to brain development diseases can be reduced; and can promote the butyric acid content in intestinal canal, reduce the acetic acid and propionic acid content to promote brain and cognitive development; provides help for the development of infant formulas.

Drawings

Fig. 1: different formulations different dosage formulations rats were compared for body mass after 28 days of intervention.

Fig. 2A: different formulations different doses of formula powder are compared in the abundance of beneficial bacteria Lactobacillus in the intestinal tract of rats after 28 days of intervention.

Fig. 2B: different formulations different doses of formula powder comparison of beneficial bacteria Blauthia abundance in the rat intestinal tract 28 days after intervention.

Fig. 3: different formulations different doses of the formula powder are compared with harmful bacteria at the level of the intestinal internal medicine of the rat after 28 days of intervention.

Fig. 4A: different formulations different doses of the formulations were used to compare the amount of short chain fatty acid butyrate produced in the intestinal tract of rats 28 days after intervention.

Fig. 4B: different formulations different doses of the formulations were used to compare the amount of short chain fatty acid acetate produced in the intestinal tract of rats 28 days after intervention.

Fig. 4C: different formulations different doses of the formulations were used to compare the amount of short chain fatty acid propionic acid produced in the intestinal tract of rats 28 days after intervention.

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the invention as claimed, and embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention.

< Definition of terms >

In the present invention, the terms "comprising," "having," "including," or "containing" may be used to refer to inclusion or patenting, and do not exclude additional, unrecited components or method steps. In the meantime, "comprising," "having," "including," or "containing" may also mean enclosed, excluding additional, unrecited components or method steps.

In the present invention, the terms "a" or "an" or "the" may mean "one" or "one or more", "at least one", and "one or more".

In the present invention, "infant" is used to denote a group of humans of 0 to 6 months of age.

In the present invention, "older infant" is used to mean a group of humans of 6 to 12 months of age.

In the present invention, "young children" means a group of humans of 12 to 36 months of age.

In the present invention, "infant" is used to denote a group of humans under 3 years of age.

In the present invention, the "infant formula" encompasses infant formulas, older infant formulas, and young child formulas. Typically, infant formulas are used as a breast milk substitute from the birth of the infant, and older infant formulas are used as breast milk substitutes from 6 to 12 months after the birth of the infant, and toddler formulas are used as breast milk substitutes from 12 to 36 months after the birth of the infant.

In the present invention, the use of "animal milk" means a liquid obtained from the mammary glands of a mammal during lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e. liquid obtained directly from the mammary gland) and standardized dairy products (such as, for example, skim milk or whole milk).

In the present invention, for convenience of expression for fatty acid glycerides, the following characters are used to refer to different kinds of fatty acids: p: palmitic acid (C16:0); o: oleic acid (C18:1).

Reference in the present disclosure to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," etc., means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

Unless defined otherwise, 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.

< Nutritional composition >

The research of the invention finds that the combination of the components for strengthening various functions, such as docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1, 3-dioleate-2-palmitic acid triglyceride, lutein, nucleotide, lactoferrin, casein phosphopeptide and probiotics can promote the intestinal health and the brain development of organisms in a bidirectional way through a microorganism-intestinal-brain axis.

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.

In some embodiments, the nutritional composition further comprises choline, inositol, taurine, and l-carnitine.

In some specific embodiments, the nutritional composition comprises the following essential components: choline, inositol, taurine, l-carnitine, docosahexaenoic acid, eicosatetraenoic acid, galactooligosaccharides, 1, 3-dioleate-2-palmitoleic acid triglyceride, lutein, nucleotides, lactoferrin, casein phosphopeptides, and probiotics.

In some specific embodiments, the nucleotides include disodium 5' -cytidine, disodium 5' -uridine, adenosine 5' -monophosphate, disodium 5' -guanylate, and disodium 5' -inosinate.

In some embodiments, the probiotic includes bifidobacterium animalis subspecies lactis. Exemplary bifidobacterium animalis subspecies include the Bb-12 strain, the HN109 strain, bi-07, and the like. In some preferred embodiments, the probiotic includes a bifidobacterium animalis subspecies lactis Bb-12 strain.

In some specific embodiments, the nutritional composition comprises the following essential components: choline, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid, galactooligosaccharides, 1, 3-dioleoyl-2-palmitoyl triglyceride, lutein, disodium 5' -cytidine, disodium 5' -uridylate, adenosine 5' -monophosphate, disodium 5' -guanylate, disodium 5' -inosinate, lactoferrin, casein phosphopeptides, and bifidobacterium animalis subspecies of milk Bb-12.

To meet the basic nutritional needs of the consumer, 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.

By the addition of the above-described raw materials, in some embodiments, the nutritional composition comprises a plurality of breast milk oligosaccharides including, for example, at least one of 3' -sialyllactose, 6' -sialyllactose, 4' -galactosyllactose, 3' -galactosyllactose, 6' -galactosyllactose, and N-acetylneuraminic acid.

By combining various raw materials and/or breast milk oligosaccharides with the essential components in the above-described nutritional composition, it is possible to further obtain an effect of more effectively promoting the intestinal health and brain development of the organism in both directions through the intestinal brain axis.

In some embodiments, the source of animal milk comprises cattle and/or sheep. In some specific embodiments, the animal milk is cow milk.

In some embodiments, the source of the protein component comprises at least one of whole milk powder, skim milk powder, concentrated whey protein powder, hydrolyzed whey protein powder, and desalted whey powder.

In some embodiments, the source of the fat component comprises at least one of structural blend ester, sunflower oil, coconut oil, linseed oil, corn oil, rapeseed oil, and soybean oil.

In some embodiments, the source of the carbohydrate ingredient includes lactose.

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.

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

For ease of use and transportation, etc., in some embodiments, the nutritional compositions of the present invention are powdered solids.

In some embodiments, the protein content is 8 to 20g/100g, the fat content is 17 to 28g/100g, and the carbohydrate content is 30 to 75g/100g in the nutritional composition on a dry weight basis.

< Food >

The nutritional composition provided by the invention can be applied to preparing various foods, including infant formulas and the like. In some embodiments, the infant formula comprises an infant formula.

In addition, in some embodiments, the food product further comprises any acceptable adjuvant, including, but not limited to, solvents, antioxidants, antimicrobial agents, thickeners, diluents, co-solvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, flavoring agents, food colors, and the like.

The amount of the nutritional composition and its components in the food is not particularly limited in the present invention.

In some embodiments, the docosahexaenoic acid is present in the food product in an amount of 40mg/100g or more, preferably 100mg/100g or more, preferably 190mg/100g or less on a dry weight basis.

In some embodiments, the eicosatetraenoic acid is present in the food product in an amount of 75mg/100g or more, preferably 170mg/100g or more, preferably 350mg/100g or less on a dry weight basis.

In some embodiments, the galacto-oligosaccharides are present in the food product in an amount of 1g/100g or more, preferably 2g/100g or more, preferably 6.2g/100g or less on a dry weight basis.

In some embodiments, the 1, 3-dioleoyl-2-palmitoleic acid triglyceride is present in the food product in an amount of 1g/100g or more, preferably 3g/100g or more, preferably 7g/100g or less on a dry weight basis.

In some embodiments, the lutein is present in the food at a level of 200 μg/100g on a dry weight basis, preferably 205 μg/100g or more, preferably 220 μg/100g or less.

In some embodiments, the nucleotides are present in the food product in an amount of 20mg/100g or more, preferably 25mg/100g or more, preferably 50mg/100g or less on a dry weight basis.

In some embodiments, the lactoferrin is present in the food product in an amount of 35mg/100g or more, preferably 40mg/100g or more, preferably 500mg/100g or less on a dry weight basis.

In some embodiments, the casein phosphopeptide is present in the food product in an amount of 30mg/100g or more, preferably 35mg/100g or more, preferably 50mg/100g or less on a dry weight basis.

In some embodiments, the choline is present in the food product in an amount of 90mg/100g or more, preferably 200mg/100g or more, preferably 460mg/100g or less on a dry weight basis.

In some embodiments, the inositol is present in the food product in an amount of 20mg/100g or more, preferably 35mg/100g or more, preferably 200mg/100g or less on a dry weight basis.

In some embodiments, the taurine is present in the food product in an amount of 15mg/100g or more, preferably 33mg/100g or more, preferably 80mg/100g or less on a dry weight basis.

In some embodiments, the L-carnitine is present in the food in an amount of 5mg/100g or more, preferably 10mg/100g or more, preferably 50mg/100g or less, on a dry weight basis.

< Use of the intestinal and brain Axis to promote the bidirectional development of the intestinal and brain >

The invention can promote the intestinal health and the brain cognitive development of organisms in a bidirectional way through microorganism-intestinal-brain axis by combining and strengthening various functional active components such as docosahexaenoic acid, eicosatetraenoic acid, galactooligosaccharide, 1, 3-dioleate-2-palmitic acid triglyceride, lutein, nucleotide, lactoferrin, casein phosphopeptide and probiotics. Further, foods containing such nutritional compositions are capable of promoting intestinal and brain bi-directional development through the intestinal-brain axis. In addition, the functional active components can further improve the effect of promoting the development of the intestinal tract and the brain in both directions after being combined with other components in the nutritional composition.

In some embodiments, the promoting intestinal and brain bi-directional development via the intestinal brain axis comprises at least one of increasing Lactobacillus (Lactobacillus) abundance in the intestinal tract, increasing truffle (Blautia) abundance in the intestinal tract, and decreasing sarsashimi (Sutterellaceae) abundance in the intestinal tract.

In some embodiments, the promoting intestinal and brain bi-directional development via the intestinal brain axis comprises at least one of increasing the content of butyric acid in the intestinal tract, decreasing the content of acetic acid in the intestinal tract, and decreasing the content of propionic acid in the intestinal tract.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The materials used, or the instruments, unless otherwise specified, are conventional products available commercially.

1. Method for evaluating functions of nutritional composition

1.1 Laboratory animals

50 SPF-grade male SD rats with 3 weeks of weaning age (21-27 days) were fed adaptively for 7 days, and then were randomly divided into a 15% control formula group, a 15% experimental formula group, a 20% control formula group, a 20% experimental formula group, a 30% control formula group and a 30% experimental formula group 6 according to body weight, and fed for 4 weeks. And the body mass and food intake of rats were determined on days 0,7, 14, 21, 28 of group feeding. Experimental animals were purchased from beijing vernalia experimental animal technologies limited, the experimental units used license numbers: SYXK (Beijing) 2022-0049, laboratory animal license number SCXK (Beijing) 2021-0011. Animal experiments were approved by the ethical committee of the university of capital medical science, animal ethical review number: AEEI-2023-035.

1.2 Experimental animal feed

Basic feed, 15% of control formula feed, 15% of experimental formula feed, 20% of control formula feed, 20% of experimental formula feed, 30% of control formula feed and 30% of experimental formula feed. The experimental formula refers to milk powder added with a proper amount of DHA, ARA, galacto-oligosaccharide, 1, 3-dioleate-2-palmitic acid triglyceride, lutein, nucleotide, lactoferrin and casein phosphopeptide, and the control formula in the experiment is not added with the special components. The feed is provided by Tianjin feed limited company, australian, and the corn starch component in the feed is replaced by the control formula milk powder and the experimental formula milk powder according to three different dosages of 15%, 20% and 30%.

1.3 Biological sample collection and detection

On day 29 after group feeding, 5 SD rats per group were randomly selected, the tail of the rat was fixed, the lower abdomen of the rat was gently pressed with a finger, fresh feces were collected by a stress defecation method, and stored in a number of sterile EP tubes and stored in a-80 ℃ low temperature refrigerator for later use (n=5). On day 35 of group feeding, rats were anesthetized by intraperitoneal injection of tribromoethanol, and after euthanasia of the rats, brains, hearts, livers, spleens, kidneys were collected rapidly. Repeatedly rinsing the viscera with 0.9% physiological saline, drying with filter paper, weighing, and storing in a refrigerator at-80deg.C.

1) Fecal 16S rRNA fecal intestinal flora sequencing

Extracting genome DNA from SD rat feces; then PCR amplification is carried out according to a 338F_806R region, PCR products are quantitatively detected through a QuantiFluorTM-ST blue fluorescent quantitative system, and are mixed according to the sequencing quantity requirement of each sample and the corresponding proportion; miseq construction of a library; miseq sequencing results; the DNA fragment is used as a template, a primer is used for fixing a base sequence in a chip, and a target DNA fragment to be detected in the chip is obtained through PCR synthesis; generating a DNA cluster; carrying out laser scanning on the surface of the reaction plate to read the nucleotide types polymerized by the template sequences in the first round of reaction; and carrying out result statistics on the collected fluorescent signals to obtain a template DNA fragment sequence.

2) Fecal short chain fatty acid level detection

Sample treatment:

20mg of fecal sample was weighed into a 2mL grind tube and 800. Mu.L of 0.5% phosphoric acid water (10. Mu.g/mL with internal standard 2-ethylbutyric acid) was added. The sample was freeze-milled for 3min (50 HZ) and then sonicated for 10min,4℃and centrifuged at 13000g for 15min. The supernatant was removed from the tube to a 1.5mL centrifuge tube, and then extracted with 200. Mu.L of n-butanol solvent. Vortex for 10s, ultrasonic at low temperature for 10min, centrifuge at 4 ℃ and 13000g for 5min, take supernatant solution to sample injection vial.

GC-MS detection:

The analytical instrument for this experiment was an 8890B-7000DGC/MSD gas chromatograph-mass spectrometer (Agilent Technologies Inc. CA, UAS). Chromatographic conditions: HP FFAP capillary column (30 m×0.25mm×0.25 μm, agilent J & WSCIENTIFIC, folsom, calif., USA), carrier gas is high purity helium (purity not less than 99.999%), flow rate 1.0mL/min, and sample inlet temperature 180 ℃. The sample feeding amount is 1 mu L, the split sample feeding is carried out, the split ratio is 10:1, and the solvent is prolonged for 2.5min. Programming temperature: the initial temperature of the column temperature box is 80 ℃, the temperature is programmed to 120 ℃ at 20 ℃/min, the temperature is programmed to 160 ℃ at 5 ℃/min, and the column temperature box is operated at 220 ℃ for 3min. Mass spectrometry conditions: the electrons bombard an ion source (EI), the ion source temperature is 230 ℃, the quaternary rod temperature is 150 ℃, the transmission line temperature is 230 ℃, and the electron energy is 70eV. The scanning mode is selected from an ion scanning mode (SIM).

3) Ileum and colon tissue HE staining

Tissue paraffin embedded sections:

(1) Drawing materials: fresh tissue was fixed to 4% paraformaldehyde for more than 24 h. And taking out the tissue from the fixing solution, trimming the tissue of the target part in a fume hood by using a surgical knife, and placing the trimmed tissue and a corresponding label in a dehydration box.

(2) Dehydrating: and placing the dehydration box into a basket, and sequentially carrying out gradient alcohol dehydration in a dehydrator. 75% alcohol 4h, 85% alcohol 2h, 90% alcohol 2h, 95% alcohol 1h, absolute alcohol I30 min, absolute alcohol II 30min, alcohol benzene 5-10min, xylene I5-10 min, xylene II 5-10min, wax I1h, wax II 1h, wax III 1h.

(3) Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20deg.C, solidifying, removing the wax block from the embedding frame, and trimming.

(4) Slicing: the trimmed wax block was placed on a paraffin microtome for slicing to a thickness of 4 μm. The slices float on warm water at 40 ℃ of a slice spreading machine to flatten the tissues, the tissues are fished up by using glass slides, and the slices are put into a 60 ℃ oven to be baked. And taking out the water after the water is baked to dry the wax and bake the wax, and preserving the wax at normal temperature for standby.

HE staining:

(1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min, xylene II 20min, absolute ethanol I10 min, absolute ethanol II 10min, 95% ethanol 5min, 90% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min, and distilled water.

(2) Hematoxylin-stained nuclei: the slices are stained with Harris hematoxylin for 3-8min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, washed with tap water, and returned to blue with 0.6% ammonia water, and washed with running water.

(3) Eosin-stained cytoplasm: the slices are dyed in eosin dye solution for 1-3min.

(4) And (3) removing the water sealing piece: sequentially placing the slices into 95% alcohol I5 min, 95% alcohol II 5min, absolute alcohol I5 min, absolute alcohol II 5min, xylene I5 min and xylene II 5min for dewatering and transparency, taking out the slices from the xylene, slightly airing, and sealing the slices with neutral resin.

(5) Microscopic examination, image acquisition and analysis.

4) Statistical analysis

Statistical analysis of the data was performed using SPSS21.0 software to obtain relevant information. For analysis of experimental data, the quantitative index was expressed using mean ± standard deviation (mean ± SD) using descriptive statistical methods. For comparison of five sets of metrology data, one-way anova was used for data that were normalized and plausible, whereas non-parametric test (Kruskal-Wallis test) was used for data that were not normalized or plausible, where P <0.05 indicated that the significance level of the difference was statistically significant. Mapping was performed using GRAPHPAD PRISM 5.0.0.

2. Preparation of experimental formula milk powder

Raw milk produced by applicant in pasture and other proteins, fats, carbohydrates, vitamins, minerals and other functional components are prepared into powder through the processes of sterilization, mixing and stirring, sterilization, homogenization, concentration, spray drying and the like. Wherein the proteins comprise skimmed milk powder, concentrated whey protein powder, whey protein powder and hydrolyzed whey protein powder; the lipid is mainly edible vegetable blend oil (1, 3-dioleate 2-palmitic acid triglyceride, sunflower seed oil, coconut oil, linseed oil); carbohydrates include lactose and galactooligosaccharides; vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinic acid, folic acid, pantothenic acid, vitamin C, and biotin; the minerals include copper sulfate, magnesium sulfate, ferric pyrophosphate, zinc sulfate, calcium citrate, calcium hydrogen phosphate, potassium iodate, sodium selenite, and manganese sulfate; other functional active ingredients also 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 phosphopeptides, bifidobacterium animalis (Bb-12).

The composition of HMOs and lipid nutrients in the final experimental formulation obtained is shown in the table below.

Table 1 composition of HMOs and lipid nutritional substances in the Experimental formulation

3. Preparation of control formula milk powder

Raw milk and other proteins, fats, carbohydrates, vitamins, minerals and other functional components are prepared into powder through the processes of sterilization, mixing and stirring, sterilization, homogenization, concentration, spray drying and the like. Wherein the proteins comprise desalted whey powder, whole milk powder, skimmed milk powder and whey protein powder; the lipid is mainly vegetable oil (corn oil, rapeseed oil, coconut oil, sunflower seed oil, soybean oil); the carbohydrate is mainly lactose; other food additives include phospholipids, retinyl acetate, cholecalciferol, dl-alpha-tocopherol acetate, phytomenaquinone, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, cyanocobalamin, nicotinamide, folic acid, calcium D-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, L-carnitine.

4. Nutrition index difference between experimental formula and control formula

Table 2 experimental and comparative formulation nutritional composition

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5. Examples and comparative example setup

Example 1: 15% of the experimental formulation was mixed into the basal feed for rats.

Example 2: to the rat basal feed, 20% of the experimental formulation was mixed.

Example 3: to the rat basal feed, 30% of the experimental formulation was mixed.

Comparative example 1: in the rat basal feed, 15% of the control formulation was mixed.

Comparative example 2: in the rat basal feed, 20% of the control formula was mixed.

Comparative example 3: in the basal feed for rats, 30% of the control formulation was mixed.

The energy ratios and energy densities of the three macronutrients in the final and comparative rat feeds are shown in the following table:

table 3 basal feed and experimental feed formulation

Species of type	Comparative example 1	Example 1	Comparative example 2	Example 2	Comparative example 3	Example 3
							Proteins	17.3	17.3	17.3	17.3	17.3	17.3
Fat	21.8	20.9	20.4	21.1	19.5	19.8
							Carbohydrates	61.5	62.2	62.3	61.6	63.2	62.9
kcal/g	3.97	3.95	3.92	3.93	3.89	3.90

6. Body weight test of rats in each group

No significant difference in body weight was found for each group of rats after 28 days of group feeding, and the results are shown in fig. 1. The addition of different types of formula milk powder and different doses of formula milk powder has no obvious effect on the weight increase of rats.

7. Test of organ development of rats in each group

The development of each organ of the rats was measured 28 days after group feeding and characterized by the 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 were compared with the experimental formula group and the control formula group using t-test, and as a result, as shown in the following table, it was found that liver index, spleen index and kidney index were not significantly different in the comparative examples and examples except that the brain index experimental formula group (example 1, example 2 and example 3) was significantly higher than the control formula group (comparative example 1, comparative example 2 and comparative example 3) (p <0.01, p <0.05 and p < 0.001), respectively. The results prove that the experimental formula milk powder has the effect of promoting brain development.

TABLE 4 organ index of rats 28 days after intervention with different ratios and different milk powders

Comparative example 1

Example 1

Comparative example 2

Example 2

Comparative example 3

Example 3

Brain index

0.45±0.019

0.49±0.039

0.45±0.033

0.48±0.015

0.45±0.017

0.53±0.035

Heart index

0.38±0.050

0.40±0.057

0.37±0.062

0.37±0.041

0.38±0.044

0.36±0.030

Liver index

3.73±0.44

3.77±0.24

3.52±0.40

3.48±0.39

3.39±0.16

3.62±0.37

Spleen index

0.19±0.025

0.19±0.024

0.23±0.099

0.20±0.024

0.18±0.014

0.21±0.033

Kidney index

0.85±0.10

0.84±0.062

0.82±0.12

0.79±0.044

0.80±0.060

0.85±0.060

8. Length and Structure investigation of colorectal and ileum of groups of rats

The study examined the length and structure of colorectal and ileum of each group of rats, and found that the length of colorectal and ileum of each group of rats was not significantly different after 28 days of feeding by intervention, and that the structure of colorectal and ileum of each group was substantially normal after HE staining, the villi in the visual field was clear and complete, the mucosal epithelial cells were closely arranged, and necrosis was not seen. The crypts were closely packed and no apparent abscess or distension was seen. Edema was not seen in submucosa. No obvious inflammatory cell infiltration was seen in the tissues. Therefore, the experimental formula and the control formula do not influence the normal development of the intestinal structure from the aspect of the intestinal structure development.

9. Examination of intestinal flora of rats in each group

Many studies report that mammalian intestinal flora is symbiotic to the host and is closely related to host digestion, immune and nervous system development. Active metabolites produced by the intestinal flora may act as neurotransmitters. Although the development and growth of nerve cells occurs mainly in fetal phase, glial cells, synapses and myelination can continue throughout infancy. And the continued development of nerve cells throughout infancy will be affected by a number of factors, one of which is the intestinal flora. It is considered that the development of nervous system is related to intestinal flora, and the disorder of intestinal flora can cause the change of metabolites thereof, thereby affecting the intestinal-brain axis communication system, brain immune function, central nervous system inflammation, blood-brain barrier integrity and the like.

In the study, the 16S rRNA sequencing analysis of the rat fecal samples is carried out to analyze the composition of the intestinal flora of rats, and the influence of different formulas on the intestinal flora is compared. The results found that the abundance of beneficial flora was higher in rats fed with the experimental group formula at the belonging level. Fig. 2A shows that the abundance of Lactobacillus in the intestinal tract is significantly higher in the experimental group (example 1, example 2 and example 3) than in the control group (comparative example 1, comparative example 2 and comparative example 3) after 28 days of formula feeding of rats. And with increasing formulation doses, the abundance of Lactobacillus (Lactobacillus) is increasing. As is well known, lactobacillus is an important sign of human intestinal health, has important physiological functions of regulating host intestinal flora, promoting nutrient absorption and metabolic regulation, and the like, and has been shown to play an important role in the prevention and treatment of depression. Animal models and/or human clinical trials have shown that antidepressant lactic acid bacteria mainly include Lactobacillus and bifidobacterium, both of which are known as mental probiotics (Psychobiotics). FIG. 2B shows comparison of the abundance of Eubacterium mucilaginosa (Blauthia) in the experimental and control groups, and it can be seen that the abundance of this bacterium in the intestinal tract of milk-fed rats in the experimental group is higher than that in the control group, and that example 1 and example 2 were found to be significantly higher than comparative example 1 (p < 0.05) and comparative example 2 (p < 0.01), respectively, after Mann-WHITNEY TEST test. Blauthia is a core fungus in the intestinal tract, and researches show that Blauthia has antibacterial activity on part of specific pathogenic bacteria and has a certain anti-inflammatory effect. Immune system and nerve immunity are one of the ways of intestinal brain axis communication, and studies have also demonstrated that dysregulation of immune homeostasis affects brain and nerve development early in life.

Fig. 3 shows the difference in abundance of sarsashimi (Sutterellaceae) in the gut after 28 days of feeding rats with the experimental (example 1, example 2 and example 3) and control (comparative example 1, comparative example 2 and comparative example 3) formulas, respectively. The genus Sagnac of this family is Sagnac (Sutterella), sutterella is associated with human diseases, in particular brain development related diseases such as autism, down syndrome, and in addition to the occurrence of Inflammatory Bowel Disease (IBD). From the results of this study, the abundance of this bacterial family in the intestinal tract of the rats of the experimental formulation group was respectively lower than that of the corresponding control group, and the abundance of Sutterellacea bacterial families in the intestinal tract of the rats of example 3 was found to be significantly lower than that of comparative example 3 (p < 0.05) after Mann-WHITNEY TEST examination.

10. Investigation of the production of short-chain fatty acids in the intestinal tract of rats of each group

Short Chain Fatty Acids (SCFAs) are one of the major metabolites of the intestinal flora. SCFAs are reported to be related to cognitive development, and can stimulate synthesis and secretion of 5-hydroxytryptamine (5-HT) in intestines, and after the 5-HT binds to a receptor of the SCFAs, the SCFAs play roles in regulating motility, intervening in neuron development and differentiation, regulating emotion through nerve signals and the like. Various studies report that butyrate can regulate mitochondrial function, stimulate oxidative phosphorylation and fatty acid oxidation, up regulate physiological stress pathways, and that butyrate can regulate social behavior in mouse models of autism, and the experimental formulation can promote the production of butyrate in the intestinal tracts of rats, as shown in fig. 4A. Wherein the butyric acid content in the groups of example 2 and example 3 is significantly higher than that of comparative example 2 and comparative example 3 (p < 0.01), respectively. It has been reported that acetic acid and propionic acid are higher in the intestinal tract of autistic patient than normal children, and that higher concentration of acetic acid and propionic acid can penetrate the intestinal barrier and blood brain barrier to enter the brain, affect physiological processes related to autism such as cell signal transduction, neurotransmitter synthesis and release, free radical generation, immune function, etc., and have neurotoxicity. This study found that the dry prognosis of the experimental formulation group reduced the levels of acetic acid and butyric acid in the rat intestinal tract, as shown in FIGS. 4B and 4C, with the levels of acetic acid in the rat intestinal tract being significantly lower than the respective comparative examples in the 3 example groups, whereas the propionic acid levels in example 1 were significantly lower than comparative example 1 (p < 0.01), with the propionic acid levels in example 2 and example 3 being lower than the respective comparative examples, but the differences were insignificant (p > 0.05).

Industrial applicability

The application of the nutritional composition provided by the invention in preparing food for promoting the intestinal and brain bidirectional development through the intestinal and brain axes can be widely used in industry.

Claims (10)

1. Use of a nutritional composition for the preparation of a food for promoting intestinal and brain bi-directional development via the intestinal-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 phosphopeptides and probiotics.

2. The use according to claim 1, characterized in that the nutritional composition further comprises choline, inositol, taurine and l-carnitine.

3. Use according to claim 1 or 2, characterized in that the probiotics comprise bifidobacterium animalis subspecies lactis.

4. The use according to any one of claims 1 to 3, wherein the nucleotides comprise disodium 5' -cytidine, disodium 5' -uridylate, adenosine 5' -monophosphate, disodium 5' -guanylate and disodium 5' -inosinate.

5. The use according to any one of claims 1 to 4, wherein said promoting intestinal and brain bi-directional development via the intestinal brain axis comprises at least one of increasing the abundance of lactobacillus in the intestinal tract, increasing the abundance of truffle in the intestinal tract and decreasing the abundance of sarsashimi in the intestinal tract.

6. The use according to any one of claims 1 to 5, wherein said promotion of intestinal and brain bi-directional development via the intestinal brain axis comprises at least one of increasing the content of butyric acid in the intestinal tract, decreasing the content of acetic acid in the intestinal tract and decreasing the content of propionic acid in the intestinal tract.

7. Use according to any one of claims 1 to 6, characterized in that the food is an 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 components, fat components, carbohydrate components, vitamins and minerals.

9. 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 comprises a milk oligosaccharide comprising at least one of 3 '-sialyllactose, 6' -sialyllactose, 4 '-galactosyllactose, 3' -galactosyllactose, 2 '-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 6' -galactosyllactose and N-acetylneuraminic acid.