An oligosaccharide composition for improving neurodevelopment and dysfunction

Abstract

This invention provides an oligosaccharide composition for improving neurodevelopment and functional disorders, comprising 6'-sialyl lactose and lactose-N-trisaccharide. The oligosaccharide composition provided by this invention can effectively inhibit the accumulation of Aβ protein, which leads to neuronal synaptic disorders, neuronal apoptosis, brain damage, and functional disorders. Therefore, the oligosaccharide composition provided by this invention can protect nerves and improve functional disorders. Furthermore, the oligosaccharide composition provided by this invention can also inhibit acetylcholinesterase activity, protect the normal development of cholinergic neurons, and increase neurotransmitter levels, thus benefiting the improvement of neurodevelopment, learning and memory abilities, and the improvement of behavioral coordination in infants and young children.

Description

Technical Field

[0001] This invention relates to the field of human milk oligosaccharide technology, and more specifically to an oligosaccharide composition for improving neurodevelopment and functional disorders. Background Technology

[0002] Breast milk also contains a variety of bioactive substances, such as human milk oligosaccharides (HMOs), lactoferrin, milk fat globule membrane, and lactoferrin, which play an important role in the health of infants and young children. Among these, human milk oligosaccharides (HMOs), the third largest solid component in human breast milk, are low-polymer sugars composed of multiple monosaccharide molecules linked by glycosidic bonds. These oligosaccharides are abundant in breast milk, possess various biological activities, and have a significant impact on the health and growth of infants. HMOs cannot be digested and absorbed by the upper digestive tract, thus reaching the small intestine and colon intact, where they play functions such as regulating gut microbiota, modulating immunity, supporting the intestinal barrier, and improving cognition. The intestinal epithelial cells of the small intestine and colon form a physical barrier between the intestinal lumen and the circulatory system, serving as the first line of defense for innate immunity. Furthermore, some HMOs have high concentrations of sialic acid adhering to their terminals. Sialic acid participates in the formation of gangliosides and glycoproteins in human brain tissue and is closely related to nerve synapses and nerve conduction; therefore, breastfeeding helps enhance synapse formation and promote the development of the infant's nervous system.

[0003] Human milk oligosaccharides (HMOs) have significant application value, but their composition is highly complex. Current research on the components and functions of each component is insufficient, and whether combining different types of HMOs can achieve better functionality remains unknown. To fully utilize HMOs and realize their biological functions, further research on their different components is necessary. Summary of the Invention

[0004] In view of this, the present invention provides an oligosaccharide composition for improving neurodevelopment and functional disorders, the oligosaccharide composition comprising 6'-sialyl lactose and lactose-N-trisaccharide, the two oligosaccharides having a synergistic effect in improving neurodevelopment and functional disorders caused by neurodegeneration.

[0005] To address the technical problems mentioned in the background section, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides an oligosaccharide composition comprising:

[0007] 6'-Sialolactose and lactose-N-trisaccharide;

[0008] The mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is (1:20) to (20:1).

[0009] Furthermore, the mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is (1:1) to (20:1).

[0010] Secondly, the present invention provides the use of the oligosaccharide composition in the preparation of products for improving neurodevelopment and / or improving functional impairments caused by neurodegeneration.

[0011] Furthermore, the improvements in neurodevelopment include at least one of the following: inhibiting the activity of acetylcholinesterase (AChE), protecting neuronal cells from the toxic effects of Aβ pathogenic protein, protecting the normal development of cholinergic neurons, and increasing the content of neurotransmitters.

[0012] Furthermore, the functional impairments caused by the neurodegeneration include at least one of the following: bradykinesia, incoordination, dysphagia, difficulty in head movement, and paralysis.

[0013] Furthermore, the product is a pharmaceutical product, which also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the application concentration of 6'-sialyl lactose in the product is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL. That is, the product is a powder product, and after reconstitution, the concentration of 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or the concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL.

[0015] Thirdly, the present invention provides the use of the oligosaccharide composition in the preparation of a protein inhibitor that can inhibit the accumulation of Aβ protein.

[0016] Furthermore, in the protein inhibitor, the application concentration of 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL; and / or the mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is 10:1.

[0017] Fourthly, the present invention provides the use of the oligosaccharide composition in the preparation of products for the treatment or adjuvant improvement of diseases associated with Aβ protein accumulation, said diseases including at least one of the neurodegenerative diseases Alzheimer's disease and neurodegenerative functional impairment.

[0018] Furthermore, the present invention provides the use of the oligosaccharide composition in the preparation of a medicament for treating diseases associated with Aβ protein accumulation, said diseases including at least one of the neurodegenerative diseases Alzheimer's disease and neurodegenerative functional impairment.

[0019] Fifthly, the present invention provides the use of the oligosaccharide composition in the preparation of a cholinergic modulator, which can inhibit the activity of acetylcholinesterase, and / or improve and protect the normal development of cholinergic neurons, and / or increase the content of neurotransmitters.

[0020] Furthermore, in the cholinergic regulator, the application concentration of 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL; and / or the mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is 10:1.

[0021] In a sixth aspect, the present invention provides the use of the oligosaccharide composition in the preparation of products for treating or assisting in the improvement of diseases related to acetylcholinesterase, cholinergic neurons or neurotransmitters, characterized in that the diseases include at least one of the neurodegenerative diseases Alzheimer's disease and functional impairment caused by neurodegeneration.

[0022] Furthermore, the present invention provides the use of the oligosaccharide composition in the preparation of a medicament for treating diseases related to acetylcholinesterase, cholinergic neurons, or neurotransmitters, said diseases including at least one of the neurodegenerative diseases Alzheimer's disease and neurodegenerative functional impairment.

[0023] The beneficial effects of the above-mentioned technical solution of the present invention are as follows:

[0024] (1) The oligosaccharide composition provided in this invention can effectively inhibit the accumulation of Aβ protein. Aβ protein accumulation leads to neuronal synaptic disturbances, neuronal apoptosis, and brain damage. Therefore, the oligosaccharide composition provided in this invention can protect the nervous system. Furthermore, the massive accumulation of Aβ protein leads to paralysis in nematodes. Therefore, during this process, the nematode's mobility gradually decreases as the toxicity of the pathogenic protein increases. The oligosaccharide composition provided in this invention can significantly increase the pharyngeal pulsation frequency and head-shaking frequency of nematodes, improve the overall vitality of nematodes, and resist the paralytic behavior of nematodes induced by Aβ protein aggregation.

[0025] (2) The oligosaccharide composition provided in this invention can improve the chemotactic dysfunction of CL2355 nematodes and protect neuronal cells from the toxic effects of Aβ pathogenic protein.

[0026] (3) The oligosaccharide composition provided in this invention can inhibit the activity of acetylcholinesterase (AChE). Abnormal accumulation of AChE can accelerate the hydrolysis of the neurotransmitter ACh, ultimately affecting signal transduction between neurons and further leading to learning and memory impairments. In addition, ACh, as an inflammation regulator, regulates cellular function both inside and outside the nervous system, and its functional changes can have a negative impact on the pathogenesis of neurodegenerative diseases (such as Alzheimer's disease). For cholinergic disorders in the nervous system, inhibiting AChE activity can reduce the hydrolysis of the neurotransmitter ACh and improve cholinergic signal transduction.

[0027] (4) The oligosaccharide composition provided in this invention can protect the normal development of cholinergic neurons.

[0028] (5) The oligosaccharide composition provided in this invention can increase the content of neurotransmitters and is beneficial to the healthy development of the nervous system. Attached Figure Description

[0029] Figure 1 Figure showing the effect of different concentrations of 6'-SL on the paralytic lifespan of CL4176 nematodes;

[0030] Figure 2 Figure showing the effect of different concentrations of LNT2 on the paralyzed lifespan of CL4176 nematodes;

[0031] Figure 3 Figure showing the effect of different concentrations of 2'-FL on the paralyzed lifespan of CL4176 nematodes;

[0032] Figure 4 The effect of different concentrations of the combination (6'-SL and LNT2) on the cholinergic activity of CL4176 nematode;

[0033] Figure 5 Figure showing the effect of 6'-SL on the cholinergic activity of CL4176 nematode;

[0034] Figure 6 Figure showing the effect of LNT2 on the cholinergic activity of CL4176 nematodes;

[0035] Figure 7 Figure showing the effect of 2'-FL on the cholinergic activity of CL4176 nematode;

[0036] Figure 8 The effect of different concentrations of the compositions (2'-FL and 6'-SL) on the cholinergic activity of CL4176 nematodes is shown in the figure.

[0037] Figure 9 The effect of different concentrations of the combination (2-FL and LNT2) on the cholinergic activity of CL4176 nematode is shown in the figure.

[0038] Figure 10 The effect of the composition (6'-SL and LNT2) and its monomers on the swallowing frequency of CL4176 nematodes is shown in the figure.

[0039] Figure 11 The effect of the composition (6'-SL and LNT2) and its monomers on the head-swing frequency of CL4176 nematodes is shown in the figure.

[0040] Figure 12 The effect of the composition (6'-SL and LNT2) and its monomers on the swallowing frequency of N2 nematodes is shown in the figure.

[0041] Figure 13 The effect of the composition (6'-SL and LNT2) and its monomers on the head-swing frequency of N2 nematodes is shown in the figure.

[0042] Figure 14 The effect of the composition (6'-SL and LNT2) and its monomers on the average motility rate of N2 nematodes is shown in the figure.

[0043] Figure 15 The effect of the composition (6'-SL and LNT2) and its monomers on the tactic behavior of CL2355 nematodes is shown in the figure.

[0044] Figure 16 The effect of the composition (6'-SL and LNT2) and its monomers on the cholinergic neurons of LX929 nematode is shown in the figure.

[0045] Figure 17 The graph shows the effect of the composition (6'-SL and LNT2) and its monomers on the neurotransmitter content of N2 nematodes. Detailed Implementation

[0046] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the present invention.

[0047] In a first aspect, the present invention provides an oligosaccharide composition comprising:

[0048] 6'-Sialolactose and lactose-N-trisaccharide;

[0049] The mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is (1:20) to (20:1).

[0050] Secondly, the present invention provides the use of the oligosaccharide composition in the preparation of products for improving neurodevelopment and / or improving functional impairments caused by neurodegeneration. Further, the improvement in neurodevelopment includes at least one of: inhibiting acetylcholinesterase (AChE) activity, protecting neurons from the toxic effects of Aβ pathogenic protein, protecting the normal development of cholinergic neurons, and increasing neurotransmitter levels. Further, the improvement in functional impairments caused by neurodegeneration includes at least one of: bradykinesia, incoordination, dysphagia, difficulty head shaking, and paralysis.

[0051] According to some embodiments of the present invention, the product is a pharmaceutical product, and the pharmaceutical product further includes pharmaceutically acceptable excipients.

[0052] According to some embodiments of the present invention, the application concentration of 6'-sialyl lactose in the product is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL.

[0053] Thirdly, the present invention provides the use of the oligosaccharide composition in the preparation of a protein inhibitor for inhibiting the accumulation of Aβ protein.

[0054] According to some embodiments of the present invention, in the protein inhibitor, the application concentration of 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL; and / or the mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is 10:1.

[0055] Fourthly, the present invention provides the use of the oligosaccharide composition in the preparation of products for the treatment or adjuvant improvement of diseases associated with Aβ protein accumulation, said diseases including at least one of the neurodegenerative diseases Alzheimer's disease and neurodegenerative functional impairment.

[0056] This invention discovers that the oligosaccharides 6'-sialyllactose and lactose-N-trisaccharide in human milk oligosaccharides can act as protein inhibitors to suppress Aβ protein accumulation. Currently, the mainstream view is that Aβ protein accumulation leads to neuronal synaptic dysfunction, neuronal apoptosis, and brain damage, and is a significant cause of Alzheimer's disease. In this invention, *C. elegans* was used as an experimental model. *C. elegans* exhibits Aβ protein accumulation under high-temperature induction (25°C), and large accumulations result in a "paralysis" phenotype. This invention also found that compared to using 2'-fucosylated lactose alone, using 6'-sialyllactose or lactose-N-trisaccharide is more effective in inhibiting Aβ protein accumulation, indicating that the combination of 6'-sialyllactose and lactose-N-trisaccharide can effectively inhibit Aβ protein accumulation.

[0057] Fifthly, the present invention provides the use of the oligosaccharide composition in the preparation of a cholinergic modulator, which can inhibit the activity of acetylcholinesterase, and / or protect the normal development of cholinergic neurons, and / or increase the content of neurotransmitters.

[0058] According to some embodiments of the present invention, in the cholinergic regulator, the application concentration of 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL; and / or the mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is 10:1.

[0059] In a sixth aspect, the present invention provides the use of the oligosaccharide composition in the preparation of products for treating or assisting in the improvement of diseases related to acetylcholinesterase, cholinergic neurons or neurotransmitters, characterized in that the diseases include at least one of the neurodegenerative diseases Alzheimer's disease and functional impairment caused by neurodegeneration.

[0060] This invention also discovered that 6'-sialyl lactose and lactose-N-trisaccharide in human milk oligosaccharides can act as cholinergic regulators to inhibit the activity of acetylcholinesterase, improve and protect the normal development of cholinergic neurons, and increase the content of neurotransmitters.

[0061] Choline is a neurotransmitter crucial for the normal development and function of the nervous system. During infancy and early childhood, the nervous system undergoes rapid development; therefore, any disruption to the neurotransmitter system can harm an infant's neurodevelopment. Cholinergic disorders can further lead to impaired neurodevelopment, behavioral problems, and functional impairments caused by neurodegeneration. Specifically, cholinergic disorders can cause difficulties in learning new things, developing language and memory, and can also lead to incoordination in motor skills, thus affecting the normal development of their motor skills and physical activities. In this invention, using *C. elegans* as an experimental model, experiments were conducted and found that compared to using 6'-sialotrolose or lactose-N-trisaccharide alone, the combination of 6'-sialotrolose and lactose-N-trisaccharide was more effective in inhibiting AChE elevation, protecting the normal development of cholinergic neurons, and increasing neurotransmitter levels. This indicates that the combination of 6'-sialotrolose and lactose-N-trisaccharide has a synergistic effect as a cholinergic regulator.

[0062] The present invention will be further described below through some specific embodiments.

[0063] *C. elegans* is a classic and widely used model organism with several inherent advantages for scientific research: its small size (adults are approximately 1 mm in length), allowing observation of its entire developmental cycle using a standard optical microscope; its primary food source is bacteria, and under laboratory conditions, it can be maintained simply by inoculating *E. coli* OP50 culture medium on NGM medium; it is easy to preserve; and its transparent body allows for observation of various behavioral and developmental indicators under a standard optical microscope, with fluorescence imaging requiring no sample destaining.

[0064] The nervous system of *C. elegans* is its most complex body tissue. Despite this, the lineage and morphology of each neuron have been described in detail, and the brain function of the nematode has been fully elucidated for the first time through neural network circuitry. A complete connectivity map exists for all connections between neurons in the nematode, and for connections from neurons to the nematode's muscles and other tissues (such as the gut and skin) (Cook et al., 2019). Furthermore, almost all nematode neurons possess fluorescent reporter gene markers. Using high-resolution imaging techniques, researchers can precisely track the effects of gene mutations on neuronal cell development phenotypes in *C. elegans*, making its nervous system a primary model system for elucidating the nature of gene regulation in the nervous system. Therefore, *C. elegans* has unique advantages in the study of behavior and neurodevelopment. Adult hermaphroditic nematodes have 302 neurons belonging to two separate nervous systems: a large somatic nervous system (282 neurons) and a small pharyngeal nervous system (20 neurons). These two systems have different topologies and communicate through a pair of RIP interneurons. In the somatic nervous system, neurons and their processes are typically located between the subcutaneous tissue and the muscles of the body wall, sharing the basal layer with the subcutaneous tissue that isolates them from the muscles. Pharyngeal neurons, however, are located directly between the pharyngeal muscles, without being separated from the muscle tissue that forms connections with them through the basal layer.

[0065] 1.1 Raw materials:

[0066] In the following test cases, examples, and comparative examples, unless otherwise stated, the raw materials and preparation methods used are as follows:

[0067] 2'-Fucose-based lactose (2'-FL): FrieslandCampina Ingredients (Beijing) Co., Ltd.;

[0068] 6'-Sialolose (6'-SL): DSM bright science.brighterliving;

[0069] Lactose-N-trisaccharide (LNT2): Shandong Henglu Biotechnology Co., Ltd.;

[0070] OP50 bacterial culture: Streak the OP50 culture onto an LB agar plate and incubate at 37°C for 48 hours. Then, pick a single colony of OP50 and transfer it to a sterilized LB liquid broth medium. Incubate the medium in a shaker at 37°C and 170 rpm for 12 hours. When the OD600 is 0.4, it can be inoculated into nematode growth medium (NGM).

[0071] Growth medium for Caenorhabditis elegans: Add 6.8g technical agar powder, 1.2g sodium chloride, 1.0g tryptone, and 0.08g streptomycin sulfate to every 390mL of tertiary water. Stir well and sterilize at 121℃ for 20min. Before pouring into plates, add 10mL of 1mol / L potassium phosphate buffer, 400uL of 1mol / L magnesium sulfate solution, 400uL of 1mol / L calcium chloride solution, and 400uL of 5mg / mL cholesterol.

[0072] 1.2 In the following examples and comparative examples, N2, CL4176 and CL2355 nematodes were selected for the experiments.

[0073] Wild-type nematode N2: N2 is usually cultured at 20°C. From egg to adult, it goes through four stages from L1 to L4. This short process lasts about 3 days.

[0074] CL4176 nematode: CL4176 nematode is a type of *C. elegans* obtained by co-transfection with human Aβ1-42 and rol-6 mRNA using the temperature-induced system smg-1. Normally, CL4176 nematodes move by circling their bodies. However, under specific conditions, they produce large amounts of Aβ pathogenic protein. When the Aβ protein accumulates to a certain level, the CL4176 nematode loses its original ability to move, becoming unable to swing its body. Unlike N2, CL4176 is sensitive to environmental temperature and is typically cultured at 16°C to slow its growth and reduce Aβ protein accumulation. Therefore, by appropriately inducing a temperature increase (25°C), the development and growth of CL4176 can be accelerated, rapidly accumulating Aβ protein and leading to the appearance of a "paralyzed" phenotype, thus enabling the determination of the paralysis lifespan curve.

[0075] CL2355 nematode: CL2355 is also a temperature-sensitive nematode. After high-temperature induction (25°C), its neurons express large amounts of the pathogenic Aβ1-42 protein (Kalmankar et al., 2021). As Aβ protein gradually accumulates in neurons, its neurotoxicity causes neuronal damage and even death. Motor and chemoreceptive abilities controlled by these neurons are lost, leading to deficits in sensory chemotaxis and associative learning (Kalmankar et al., 2021). Therefore, CL2355 nematode can mimic the learning and memory impairments mediated by Aβ neurotoxicity in the AD brain.

[0076] LX929 nematode: LX929 is a transgenic nematode in which cholinergic neurons are labeled with green fluorescent protein and can be stably inherited during passage.

[0077] Test Example 1: Effect of oligosaccharide 6'-SL on the paralytic lifespan of CL4176 nematodes

[0078] (1) Experimental grouping: The experiment was divided into 4 groups: control group, fed with OP50 bacterial solution; low concentration 6'-SL group, fed with 0.02 mg / mL 6'-SL; medium concentration 6'-SL group, fed with 0.51 mg / mL 6'-SL; and high concentration 6'-SL group, fed with 1.0 mg / mL 6'-SL. Each group contained 60 CL4176 nematodes. The experiment was repeated 3 times, with a total of 180 individuals.

[0079] (2) Sample preparation: Dissolve 6'-SL in sterile water to prepare a 20 mg / mL stock solution, seal with sealing film, and store at 4°C protected from light. Before use, mix the stock solution with OP50 bacterial solution to dilute to three concentrations (0.02, 0.51, and 1.0 mg / mL, respectively), and spread the 6'-SL bacterial solution mixture evenly on NGM plates. After drying, store at 4°C for later use.

[0080] (3) Method for testing the paralyzed lifespan of CL4176 nematodes: Starting from eggs, CL4176 nematodes were placed on NGM plates with or without 6'-SL treatment and cultured at 16℃ for 48 hours. Before the CL4176 nematodes entered the L4 stage (with a crescent-shaped area on the lower abdomen), they were transferred to a 25℃ incubator for further culture. The high temperature was used to induce a "paralyzed" phenotype in the CL4176 nematodes. Paralysis was defined as when the CL4176 nematodes only slightly swayed their heads, and their bodies no longer made rolling or circling movements, or could not be induced to move even when touched by a nematode picker. Timing began when the first "paralyzed" nematode appeared, and the number of paralyzed nematodes was recorded every 2 hours until all individuals were paralyzed. Timing was then stopped, and a lifespan curve was plotted based on the number of paralyzed nematodes every 2 hours.

[0081] The effect of different concentrations of 6'-SL on the paralytic lifespan of CL4176 nematodes in this test case is described in [reference needed]. Figure 1 The paralysis lifespan of CL4176 nematodes is recorded in Table 2.

[0082] Test Example 2: Effect of oligosaccharide LNT2 on the paralytic lifespan of CL4176 nematodes

[0083] (1) Experimental grouping: The experiment was divided into 4 groups: control group, fed with OP50 bacterial solution; low concentration LNT2 group, fed with 0.02 mg / mL LNT2; medium concentration LNT2 group, fed with 0.51 mg / mL LNT2; and high concentration LNT2 group, fed with 1.0 mg / mL LNT2. Each group had 60 individuals, and the experiment was repeated 3 times, for a total of 180 individuals.

[0084] (2) Sample preparation: LNT2 was dissolved in sterile water to prepare a stock solution of 20 mg / mL. The solution was sealed with sealing film and stored at 4°C in the dark. Before use, the stock solution was mixed with OP50 bacterial solution and diluted to low, medium and high concentrations (0.05, 0.35 and 0.65 mg / mL, respectively). The LNT2 bacterial solution mixture was then evenly spread on NGM plates, dried and stored at 4°C for later use.

[0085] (3) Paralysis lifespan test: The test method is the same as in Example 1.

[0086] The effect of different concentrations of LNT2 on the paralytic lifespan of CL4176 nematodes in this test case is described in [reference needed]. Figure 2 The paralysis lifespan of CL4176 nematodes is recorded in Table 2.

[0087] Test Example 3: Effect of oligosaccharide 2'-FL on paralyzed lifespan of CL4176 nematodes

[0088] (1) Experimental grouping: The experiment was divided into 4 groups: control group, fed with OP50 bacterial solution; low concentration 2'-FL group, fed with 0.4 mg / mL of 2'-FL; medium concentration 2'-FL group, fed with 0.8 mg / mL of 2'-FL; and high concentration 2'-FL group, fed with 2.4 mg / mL of 2'-FL. Each group had 60 individuals, and the experiment was repeated 3 times, for a total of 180 individuals.

[0089] (2) Sample preparation: Dissolve 2'-FL in sterile water to prepare a stock solution of 20 mg / mL, seal with sealing film, and store at 4°C protected from light. Before use, mix the stock solution with OP50 bacterial suspension to dilute to the corresponding concentrations (0.4, 0.8, and 2.4 mg / mL, respectively), and spread the 2'-FL bacterial suspension mixture evenly on NGM plates. After drying, store at 4°C for later use.

[0090] (3) Paralysis lifespan test: The test method is the same as in Example 1.

[0091] The effect of different concentrations of 2'-FL on the paralytic lifespan of CL4176 nematodes in this test case is described in [reference needed]. Figure 3 The paralysis lifespan of CL4176 nematodes is recorded in Table 2.

[0092] The concentrations of the samples in Test Examples 1 to 3 are recorded in Table 1. It should be noted that the inventors of this invention conducted experimental tests on the concentrations of 6'-SL, LNT2, and 2'-FL beyond those described in Test Examples 1 to 3, and selected low, medium, and high concentrations for different types of human lactose oligomers from a large number of experimental data.

[0093] Table 1. Concentrations of samples in 6'-SL, LNT2, and 2'-FL

[0094]

[0095] Table 2 Analysis of the survival time of CL4176 nematodes

[0096]

[0097] The average survival time is calculated as follows in the table: Where j represents age (hours), and dj is the age interval (x... j x j+1 The number of paralyzed nematodes is n, where n is the total number of nematodes; median survival time is the time when the survival rate of CL4176 nematodes is 50%; maximum survival time is the time when the survival rate of CL4176 nematodes is 0%, and different letters represent significant differences.

[0098] One of the diseases most closely associated with Aβ accumulation is Alzheimer's disease, a neurodegenerative disorder. In Alzheimer's, the prevailing view is that the accumulation of the pathogenic Aβ protein further leads to neurological disorders, neuronal death, and cognitive impairment. In *C. elegans*, where the nervous system is underdeveloped or completely absent, excessive accumulation of Aβ protein is observed, leading to synaptic disturbances, neuronal apoptosis, and brain damage. *C. elegans* CL4176 exhibits Aβ protein accumulation under high-temperature induction (25°C), with large accumulation resulting in a "paralyzing" phenotype.

[0099] Depend on Figure 1 It can be seen that, compared with the control group OP50, the paralysis curves were significantly shifted to the right when the 6'-SL concentration was 0.02 mg / mL to 1.0 mg / mL. Therefore, 6'-SL in this concentration range can effectively resist the aggregation of Aβ, and 6'-SL in this concentration range has a certain neuroprotective effect.

[0100] Depend on Figure 2 It can be seen that, compared with the control group OP50, the paralysis curves were significantly shifted to the right when the LNT2 concentration was 0.05 mg / mL to 0.65 mg / mL. Therefore, LNT2 in this concentration range can effectively resist the aggregation of Aβ, and LNT2 in this concentration range has a certain neuroprotective effect.

[0101] Depend on Figure 3 It can be seen that, compared with the control group OP50, the paralysis curves were significantly shifted to the right when the 2'-FL concentration was 0.4 mg / mL to 2.4 mg / mL. Therefore, 2'-FL in this concentration range can effectively resist the aggregation of Aβ, and 2'-FL in this concentration range has a certain neuroprotective effect.

[0102] As shown in Tables 1 and 2, the mean survival time and maximum survival time of CL4176 nematodes were increased when the concentration of 6'-SL was 0.02 mg / mL to 1.0 mg / mL, and the mean survival time, median survival time, and maximum survival time of CL4176 nematodes were increased when the concentration of 6'-SL was 0.051 mg / mL. Similarly, the mean survival time, median survival time, and maximum survival time of CL4176 nematodes were increased when the concentration of LNT2 was 0.05 mg / mL to 0.65 mg / mL, and the mean survival time, median survival time, and maximum survival time of CL4176 nematodes were increased when the concentration of 2'-FL was 0.4 mg / mL to 2.4 mg / mL. In this invention, the effective dosage of a single component was determined through test examples 1 to 3. In subsequent tests, the concentration ranges to be tested will be 0.02 mg / mL to 1.0 mg / mL of 6'-SL, 0.05 mg / mL to 0.65 mg / mL of LNT2, and 0.4 mg / mL to 2.4 mg / mL of 2'-FL.

[0103] Example 1: Effect of different concentrations of the composition (6'-SL+LNT2) on the cholinergic activity of CL4176 nematode

[0104] Cholinergic deficiencies alter brain neurons and signal transmission, including impaired acetylcholine (ACh) release, axonal transport defects, and elevated acetylcholinesterase (AChE) levels. Abnormal accumulation of AChE can accelerate the hydrolysis of the neurotransmitter ACh, ultimately affecting interneuronal signal transduction and further leading to learning and memory impairments. Furthermore, rapidly increasing AChE in the brain interacts with Aβ protein to form a stable and more neurotoxic Aβ-AChE complex. In this embodiment, the cholinergic activity of the CL4176 nematode was determined by testing acetylcholinesterase (AChE) activity.

[0105] (1) Experimental grouping: The experiment was divided into 4 groups: control group, fed with OP50 bacterial solution; group 1 with a ratio of 20:1 for the composition (6'-SL+LNT2); group 2 with a ratio of 10:1 for the composition (6'-SL+LNT2); and group 3 with a ratio of 1:1 for the composition (6'-SL+LNT2).

[0106] (2) Preparation of culture medium containing composition 6'-SL and LNT2: The preparation method is the same as in Example 1. The ratio and specific concentration of 6'-SL and LNT2 in the culture medium are shown in Table 3.

[0107] (3) Acetylcholinesterase (AChE) assay method for CL4176 nematodes: Starting from eggs, CL4176 nematodes were placed on NGM plates with or without the combination (6'-SL+LNT2) and incubated at 16°C for 48 h. Before the CL4176 nematodes entered the L4 stage (crescent-shaped abdomen), they were transferred to a 25°C incubator and incubated for another 48 h. At this time, the nematodes were rinsed from the culture dish into centrifuge tubes with buffer, homogenized using an automated tissue homogenizer, and then ground and broken up. After low-temperature centrifugation (4°C, 12000 rpm / min, 10 min), the supernatant was collected and placed at 4°C for testing. AChE activity was determined according to the instructions provided with the kit. In addition, the protein content of each nematode sample was standardized using a protein quantification (TP) kit. Each experiment was repeated three times, with at least 600 nematodes required each time.

[0108] ①AChE definition: Each mg of nematode tissue protein incubated at 37°C for 6 min, with 1 μmol of matrix in the hydrolysis reaction system, is considered one activity unit.

[0109] ②The formula for calculating AChE activity in nematode samples is:

[0110]

[0111] In the formula, A represents the absorbance OD value of each tube; C 标准 The concentration of AChE standard is 1 μmol / mL; C pr The concentration of protein in nematode tissue homogenate is expressed in mgprot / mL.

[0112] ③ The formula for calculating the percentage decrease in AChE activity in Table 2 is as follows:

[0113]

[0114] The effect of different concentrations of the composition on the cholinergic activity of CL4176 nematodes in this embodiment is described in [reference needed]. Figure 4 The effects of different concentrations of the composition on the cholinergic activity of CL4176 nematodes in this embodiment are recorded in Table 4.

[0115] Comparative Example 1

[0116] (1) Preparation of culture medium containing 6'-SL: The preparation method is the same as in Example 1. The concentration of 6'-SL in the culture medium is shown in Table 3. The test concentration range is 0.02 mg / mL to 1.0 mg / mL. Among them, 0.56 mg / mL is the same as the total concentration of 6'-SL+LNT2 in the composition in Example 3.

[0117] (2) Testing the cholinergic activity of CL4176 nematodes: The testing method is the same as in Example 1.

[0118] The effect of 6'-SL on the cholinergic activity of CL4176 nematode in this comparative example is shown in [reference needed]. Figure 5 The effects of 6'-SL on the cholinergic activity of CL4176 nematodes in this comparative example are recorded in Table 4.

[0119] Comparative Example 2

[0120] (1) Preparation of culture medium containing LNT2: The preparation method is the same as in Example 2. The concentration of LNT2 in the culture medium is shown in Table 3. The test concentration range is 0.05 mg / mL to 0.65 mg / mL. Among them, 0.56 mg / mL is the same as the total concentration of composition 6'-SL+LNT2 in Example 3.

[0121] (2) Testing the cholinergic activity of CL4176 nematodes: The testing method is the same as in Example 1.

[0122] The effect of LNT2 on the cholinergic activity of CL4176 nematode in this comparative example is shown in [reference needed]. Figure 6 The effect of LNT2 on the cholinergic activity of CL4176 nematodes in this comparative example is recorded in Table 4.

[0123] Comparative Example 3

[0124] (1) Preparation of culture medium containing 2'-FL: The preparation method is the same as that of Comparative Example 1. The concentration of 2'-FL in the culture medium is shown in Table 3. The test concentration range is 0.4 mg / mL to 2.4 mg / mL. Among them, 0.56 mg / mL is the same as the total concentration of the composition 6'-SL+LNT2 in Example 3.

[0125] (2) Testing the cholinergic activity of CL4176 nematodes: The testing method is the same as in Example 1.

[0126] The effect of 2'-FL on the cholinergic activity of CL4176 nematode in this comparative example is shown in [reference needed]. Figure 7 The effect of 2'-FL on the cholinergic activity of CL4176 nematode in this comparative example is recorded in Table 4.

[0127] Comparative Example 4

[0128] (1) Preparation of culture medium containing the composition 2'-FL and 6'-SL: The preparation method is the same as in Example 3, and the concentrations of 2'-FL and 6'-SL in the culture medium are shown in Table 3.

[0129] (2) Testing the cholinergic activity of CL4176 nematodes: The testing method is the same as in Example 1.

[0130] The effect of the composition on the cholinergic activity of CL4176 nematode in this comparative example is shown in [reference needed]. Figure 8The effects of different concentrations of the composition on the cholinergic activity of CL4176 nematode in this comparative example are recorded in Table 4.

[0131] Comparative Example 5

[0132] (1) Preparation of culture medium containing the composition 2'-FL and LNT2: The preparation method is the same as in Example 3, and the concentrations of 2'-FL and LNT2 in the culture medium are shown in Table 3.

[0133] (2) Testing the cholinergic activity of CL4176 nematodes: The testing method is the same as in Example 1.

[0134] The effect of the composition on the cholinergic activity of CL4176 nematode in this embodiment is described in [reference]. Figure 9 The effects of different concentrations of the composition on the cholinergic activity of CL4176 nematode in this comparative example are recorded in Table 4.

[0135] Table 3. Specific concentrations of different oligosaccharides and their compositions

[0136]

[0137] Table 4. AChE activity and percentage decrease of different oligosaccharides and their compositions.

[0138]

[0139]

[0140] Cholinergic deficiencies alter brain neurons and signal transmission, including impaired acetylcholine (ACh) release, axonal transport defects, and elevated acetylcholinesterase (AChE) levels. AChE's primary physiological function is the hydrolysis of ACh, playing a crucial role in cholinergic neuronal signal transmission. However, abnormal accumulation of AChE can accelerate the hydrolysis of the neurotransmitter ACh, ultimately affecting interneuronal signal transduction and further leading to learning and memory impairments. Furthermore, ACh acts as an inflammatory regulator, modulating cellular function both within and outside the nervous system; its functional alterations negatively impact the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. To address cholinergic disorders in the nervous system, cholinergic signal transmission can be improved by inhibiting AChE activity to reduce the hydrolysis of the neurotransmitter ACh. In this invention, using *C. elegans* as an experimental model, experiments were conducted, revealing that the composition 6'-SL and LNT2 inhibited acetylcholinesterase (AChE) in *C. elegans*.

[0141] Analysis of the data from Example 1, Comparative Examples 1-5, and Tables 3 and 4 shows that the composition of 6'-SL and LNT2 at a ratio of 10:1 and concentrations of 0.51 mg / mL and 0.051 mg / mL, respectively, exhibited significantly higher inhibitory effects on AChE activity than 0.56 mg / mL of 6'-SL, 0.56 mg / mL of LNT2, and 0.56 mg / mL of 2'-FL, with a 40.37% reduction in AChE activity. Furthermore, compared to other compositions, the composition of 6'-SL and LNT2 at a ratio of 10:1 and concentrations of 0.51 mg / mL and 0.051 mg / mL, respectively, showed significantly higher inhibitory effects on AChE activity than 2'-FL+6'-SL and 2'-FL+LNT2. Moreover, the inhibitory effects of 2'-FL+6'-SL and 2'-FL+LNT2 at certain ratios were even worse than those of the single components, indicating an antagonistic effect between the components. In summary, the composition (6'-SL+LNT2) exhibits a good effect in reducing AChE levels. Furthermore, compared with the effects of single components and other compositions, a synergistic effect between the composition 6'-SL and LNT2 in this invention can be observed.

[0142] Example 2: Effects of composition (6'-SL+LNT2) and its monomers, along with other compositions, on the motility of CL4176 nematodes.

[0143] The massive accumulation of Aβ protein leads to the paralysis process in CL4176 nematodes. Therefore, during this process, the motility of CL4176 nematodes gradually decreases as the toxicity of the pathogenic protein increases. This embodiment evaluates the changes in the overall viability of the composition (6'-SL+LNT2) and its monomers during paralysis by detecting the swallowing rate, head-shaking frequency, and average movement rate of CL4176 nematodes.

[0144] (1) The groups were formed according to the grouping method described in Example 1, wherein the composition (6'-SL+LNT2) was in a ratio of 10:1, and other compositions were set as control groups. The other compositions included: the composition (6'-SL+2'-FL) group in a ratio of 25:1; and the composition (2'-FL+LNT2) group in a ratio of 10:1.

[0145] (2) Prepare NGM plates containing 6'-SL, LNT2 and the composition according to the method described in Example 1.

[0146] (3) Test method for swallowing frequency of CL4176 nematodes: Starting from eggs, CL4176 nematodes were placed on NGM plates with or without sample treatment and cultured at 16℃ for 48h. Before the CL4176 nematodes entered the L4 stage (crescent-shaped abdomen), they were transferred to a 25℃ incubator for another 48h. After the culture, the CL4176 nematodes were picked and placed on new NGM plates, allowed to adapt for 5min, and their pharyngeal movement was observed under a microscope. The number of pharyngeal movements of the CL4176 nematodes within 30s was recorded. Ten nematodes were randomly selected from each group for observation. This test was repeated three times.

[0147] (4) Test method for head oscillation frequency of CL4176 nematodes: Starting from eggs, CL4176 nematodes were placed on NGM plates with or without sample treatment and cultured at 16℃ for 48h. Before the CL4176 nematodes entered the L4 stage (crescent-shaped abdomen), they were transferred to a 25℃ incubator for another 48h. After the culture was completed, the CL4176 nematodes were transferred to a new NGM plate and allowed to move freely for 1 minute before observation. The standard for measuring head oscillation was: the nematode's head oscillated from left to right and then back to left as one count. The number of head oscillations of CL4176 nematodes within 30s was recorded. Ten nematodes were randomly selected from each group for observation. This test was repeated three times.

[0148] In this embodiment, the effects of the composition (6'-SL+LNT2) and its monomers, along with other compositions, on the swallowing frequency of CL4176 nematodes are described in [reference needed]. Figure 10 The effect of the composition (6'-SL+LNT2) and its monomers, along with other compositions, on the head-waving frequency of the CL4176 nematode in this embodiment is described in [reference needed]. Figure 11 .

[0149] Depend on Figure 10 , Figure 11 It can be seen that, compared with the control group OP50, 6'-SL, LNT2, and the composition can significantly increase the pharyngeal pulsation frequency and head-shaking frequency of CL4176 nematodes. Compared with the control group OP50, they increased the swallowing frequency by 4.25%, 2.94%, 10.67%, 4.80%, and 5.89%, respectively; and the head-shaking frequency by 7.39%, 4.84%, 23.44%, 9.04%, and 7.83%, respectively. Furthermore, in the test of improving pharyngeal pulsation frequency and head-shaking frequency, the composition 6'-SL+LNT2 was more effective than its monomer and the other two compositions. These results indicate that the composition of the present invention can, to a certain extent, alleviate the swallowing and head-shaking frequency of CL4176 nematodes caused by the accumulation of Aβ pathogenic protein, and improve the overall viability of the nematodes.

[0150] Furthermore, the above results are consistent with the paralysis lifetime of monomers 6'-SL and LNT2 in Test Examples 1 and 2, and the decrease in AChE activity of the composition in Example 1. This indicates that 6'-SL, LNT2, and their composition 6'-SL+LNT2 all have the potential to resist nematode paralysis behavior induced by the aggregation of related pathogenic proteins and to improve the overall vitality of nematodes. Moreover, the composition 6'-SL+LNT2 is more effective and has a synergistic effect.

[0151] Example 3: Effects of composition (6'-SL+LNT2) and its monomers, along with other compositions, on the motility of N2 nematodes.

[0152] (1) Grouping is performed according to the grouping method described in Example 2.

[0153] (2) Prepare NGM plates containing 6'-SL, LNT2 and the composition according to the method described in Example 2.

[0154] (3) Test method for swallowing frequency of N2 nematodes: N2 nematodes, starting from eggs, were placed on NGM plates with or without sample treatment and cultured in an incubator at 20℃ for 3 days, 7 days, and 11 days. After the culture, the N2 nematodes were transferred to new NGM plates, allowed to adapt for 5 minutes, and their pharyngeal movement was observed under a microscope. The number of pharyngeal movements of the N2 nematodes within 30 seconds was recorded. Ten nematodes were randomly selected from each group for observation. This test was repeated three times.

[0155] (4) Test method for N2 nematode head swing frequency: N2 nematodes, starting from eggs, were placed on NGM plates with or without sample treatment and cultured in an incubator at 20℃ for 3, 7, and 11 days. After culture, the N2 nematodes were transferred to new NGM plates and allowed to move freely for 1 minute before observation. The standard for measuring head swing was: the nematode's head swinging from left to right and then back to left was recorded as one time. The number of N2 nematode head swings within 30 seconds was recorded. Ten nematodes were randomly selected from each group for observation. This test was repeated three times.

[0156] (5) Test method for the movement rate of N2 nematodes: Starting from eggs, N2 nematodes were placed on NGM plates with or without sample treatment and cultured in an incubator at 20℃ for 3 days. After the culture, 10-30 N2 nematodes of the same size were picked and placed on NGM plates. After acclimatization for 5 minutes, the nematodes were recorded for 1 minute at an interval of 1 second / frame using the Wormlab nematode video acquisition system (MBF Bioscience, Williston, VT, USA). Then, the average movement rate of the nematodes was analyzed based on the acquired video using Wormlab behavior analysis software (MBF Bioscience, Williston, VT, USA). This test was repeated three times.

[0157] In this embodiment, the effects of the composition (6'-SL+LNT2) and its monomers, along with other compositions, on the swallowing frequency of N2 nematodes are described in [reference needed]. Figure 12 .

[0158] In this embodiment, the effects of the composition (6'-SL+LNT2) and its monomers, along with other compositions, on the head-shaking frequency of N2 nematodes are described in [reference needed]. Figure 13 .

[0159] In this embodiment, the effects of the composition (6'-SL+LNT2) and its monomers, along with other compositions, on the average motility rate of N2 nematodes are described in [reference needed]. Figure 14 .

[0160] As N2 nematodes age, their motility gradually declines. Figure 12 , Figure 13 and Figure 14It can be seen that, compared with the control group OP50, the composition 6'-SL+LNT2 can increase the swallowing frequency of N2 nematodes at days 3, 7, and 11. On day 3, the increases were 0.37%, 0.63%, 4.07%, 2.61%, and 0.71%, respectively; on day 7, the increases were 1.91%, 1.82%, 6.79%, 4.53%, and 3.59%, respectively; and on day 11, the increases were 0.97%, -0.94%, 5.28%, 3.48%, and 1.22%, respectively. Furthermore, compared with 6'SL, LNT2, and other compositions, the composition 6'-SL+LNT2 showed a better effect on increasing swallowing frequency. Secondly, at days 3, 7, and 11, the composition 6'-SL+LNT2 also significantly increased the head-swing frequency of N2 nematodes, increasing it by -5.61%, -7.16%, 10.09%, -5.07%, and -8.12% at day 3; by 3.85%, 4.03%, 17.23%, 6.08%, and 8.23% at day 7; and by 6.22%, 5.72%, 25.85%, 7.75%, and 12.3% at day 11. Compared with 6'SL, LNT2, and other compositions, the composition 6'-SL+LNT2 was more effective in increasing head-swing frequency. In addition, by testing the average motility rate of N2 nematodes on day 3, 6'SL, LNT2 and their combination 6'-SL+LNT2 can all increase the motility rate of nematodes, increasing it by 16.27%, 13.40%, 47.75%, 17.83% and 21.71% respectively, thereby improving the overall vitality of nematodes. Moreover, the effect of the combination 6'-SL+LNT2 is better than that of other combinations.

[0161] Furthermore, the above results are consistent with the paralysis lifespan of monomers 6'-SL and LNT2 in Test Examples 1 and 2, the decrease in AChE activity of the composition in Example 1, and the improvement in the motility of CL4176 nematodes by 6'-SL, LNT2 and their composition in Example 2. This indicates that 6'-SL, LNT2 and their composition have the potential to reverse the decline in motility caused by aging and to improve the overall vitality of nematodes, and the composition 6'-SL+LNT2 is more effective.

[0162] Example 4: Effects of the composition (6'-SL+LNT2) and its monomers on the learning and memory abilities of CL2355 nematodes

[0163] (1) Grouping is performed according to the grouping method described in Example 2.

[0164] (2) Prepare NGM plates containing 6'-SL, LNT2 and the composition according to the method described in Example 2.

[0165] (3) Test method for chemotaxis ability of CL2355 nematodes: CL2355 nematodes, starting as eggs, were placed on NGM plates with or without sample treatment and cultured at 16℃ for 48 h, followed by a 48 h incubation at 25℃. After culture, the nematodes were washed with M9 buffer and collected. 80-100 nematodes were then placed in the center of a 120 mm culture dish. 10 μL of odor attractant (5 μL 0.1% benzaldehyde + 5 μL 1% sodium azide) was dropped onto a point on the edge of the plate, and 10 μL of solvent control (5 μL 100% sterile water + 5 μL 1% sodium azide) was added at a symmetrical point. After 30 min, the number of nematodes that moved from the center to the sides was recorded. The chemotaxis index (CI) was calculated as follows:

[0166]

[0167] The effects of the composition (6'-SL+LNT2) and its monomers on the directional behavior of CL2355 nematodes in this embodiment are described in [reference needed]. Figure 15 .

[0168] Depend on Figure 15 It can be seen that with the gradual accumulation of Aβ protein at high temperatures, the CI value of CL2355 nematodes fed with OP50 was lower, indicating that a nematode model simulating Aβ-mediated neurotoxicity has been successfully established in CL2355 transgenic nematodes. However, in CL2355 nematodes, the CI value of nematodes fed with 6'SL, LNT2, and the combination significantly increased. This indicates that 6'SL, LNT2, and the combination can improve the chemotactic dysfunction of CL2355 nematodes, protect neurons from the toxic effects of Aβ pathogenic protein, and the combination is more effective.

[0169] Example 5: Effects of the composition (6'-SL+LNT2) and its monomers on cholinergic neurons of LX929 nematode

[0170] (1) Grouping is performed according to the grouping method described in Example 2.

[0171] (2) Prepare NGM plates containing 6'-SL, LNT2 and the composition according to the method described in Example 2.

[0172] (3) Evaluation method of cholinergic neurons in LX929 nematodes: LX929 nematodes were cultured from eggs on NGM plates with or without sample treatment at 20°C, and the cholinergic neuron status in the nematodes was photographed on days 3, 7, and 11. The experiment was performed three biological replicates, with at least 10 nematodes anesthetized with 1% sodium azide solution and fixed on a glass slide for imaging using an upright fluorescence microscope at 20× magnification.

[0173] The effects of the composition (6'-SL+LNT2) and its monomers on the cholinergic neurons of LX929 nematode in this embodiment are described in [reference needed]. Figure 16 .

[0174] Cholinergic neurons are neurons that synthesize acetylcholine and use it as a neurotransmitter. They are mainly distributed on the ventral side of nematodes and are the most numerous neurons in the nematode body. As nematodes age, abnormal damage to cholinergic neurons may occur, manifesting as neuronal atrophy, loss, and dendritic breakage. These abnormalities can also impair the nematode's learning and memory abilities. Therefore, this example statistically analyzed the ratio of normal to abnormal cholinergic neurons. Figure 16 It was found that 6'-SL and LNT2 significantly increased the proportion of normal phenotypes on days 7 and 11, while decreasing the proportion of abnormal phenotypes; while the combination 6'-SL+LNT2 significantly increased the proportion of normal phenotypes on days 3, 7, and 11, while decreasing the proportion of abnormal phenotypes. This indicates that 6'-SL, LNT2, and the combination 6'-SL+LNT2 can effectively protect the normal development of cholinergic neurons and maintain the learning and memory abilities of nematodes, and the combination has a better effect on the recovery of cholinergic neurons.

[0175] Example 6 Effect of composition (6'-SL+LNT2) and its monomer on neurotransmitter content in N2 nematodes

[0176] (1) Grouping is performed according to the grouping method described in Example 2.

[0177] (2) Prepare NGM plates containing 6'-SL, LNT2, and the composition 6'-SL+LNT2 according to the method described in Example 2.

[0178] (3) Method for determining the neurotransmitter content of N2 nematodes: N2 nematodes were cultured from eggs on NGM plates with or without sample treatment at 20°C. After 3 days, the glutamate (GLU), γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and dopamine (DA) in the nematodes were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry.

[0179] In this embodiment, the effect of the composition (6'-SL+LNT2) and its monomers on the neurotransmitter content of N2 nematodes is described in [reference needed]. Figure 17 .

[0180] Neurotransmitters are chemical substances that transmit information between neurons. When the synthesis and transmission of neurotransmitters are hindered, the normal functioning of the nervous system is affected. Dopamine (DA) helps regulate motor coordination and enhances the brain's cognitive and memory abilities. Figure 17 It was found that 6'-SL, LNT2, and the combination 6'-SL+LNT2 significantly increased DA levels in *N. 2* nematodes, by 148.71%, 154.15%, and 268.60%, respectively; 5-HT levels were associated with mood and sleep. Figure 17 It was found that 6'-SL, LNT2, and the combination 6'-SL+LNT2 significantly increased 5-HT levels in *N. 2* nematodes, by 72.17%, 77.44%, and 178.23%, respectively; impaired GABA synthesis and transmission may increase the prevalence of neurodegenerative diseases. Figure 17 It was found that 6'-SL, LNT2, and the combination 6'-SL+LNT2 increased GABA levels in *N. 2* nematodes, but the increase was not statistically significant, at 34.72%, 44.81%, and 110.07%, respectively. GLU is one of the earliest molecules formed during fetal development and plays a crucial role in the development of the brain and other organs. Therefore, abnormal GLU activity can seriously affect neural development, thereby impacting brain neural development and health. Figure 17 It was found that 6'-SL, LNT2, and the combination 6'-SL+LNT2 significantly increased GLU levels in N2 nematodes, by 68.47%, 65.51%, and 152.86%, respectively. Therefore, 6'-SL, LNT2, and the combination 6'-SL+LNT2 can effectively increase the levels of neurotransmitters, including DA, 5-HT, GABA, and GLU, in N2 nematodes, and are beneficial to the healthy development of the nervous system.

[0181] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An oligosaccharide composition, characterized in that, The composition comprises: 6'-Sialolactose and lactose-N-trisaccharide; The mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is (1:1) ~ (20:1).

2. The use of the oligosaccharide composition of claim 1 in the preparation of a pharmaceutical for improving neurodevelopment and / or improving functional impairment caused by neurodegeneration.

3. The application according to claim 2, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

4. The application according to claim 2 or 3, characterized in that, The application concentration of 6'-sialyl lactose in the drug is 0.02 mg / mL to 1.0 mg / mL, and / or the application concentration of lactose-N-trisaccharide is 0.05 mg / mL to 0.65 mg / mL.

5. The use of the oligosaccharide composition of claim 1 in the preparation of a medicament for treating or adjuvantly improving diseases related to Aβ protein accumulation, characterized in that, The disease is at least one of the neurodegenerative diseases Alzheimer's disease and functional impairment caused by neurodegeneration.

6. The application according to claim 5, characterized in that, Of the aforementioned medicine, The application concentration of the 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or The application concentration of the lactose-N-trisaccharide is 0.05 mg / mL ~ 0.65 mg / mL; and / or The mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is 10:

1.

7. The use of the oligosaccharide composition of claim 1 in the preparation of a pharmaceutical remedy for treating or adjuvantly improving diseases related to acetylcholinesterase, cholinergic neurons, or neurotransmitters, characterized in that, The disease is at least one of the neurodegenerative diseases Alzheimer's disease and functional impairment caused by neurodegeneration.

8. The application according to claim 7, characterized in that, Of the aforementioned medicine, The application concentration of the 6'-sialyl lactose is 0.02 mg / mL to 1.0 mg / mL, and / or The application concentration of the lactose-N-trisaccharide is 0.05 mg / mL ~ 0.65 mg / mL; and / or The mass ratio of 6'-sialyl lactose to lactose-N-trisaccharide is 10:1.

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