A composition containing sheep milk globule membrane capable of assisting in improving memory and use thereof

Abstract

The application belongs to the technical field of pharmaceutical compositions, and particularly relates to a composition containing goat milk fat globule membrane and capable of assisting in improving memory and application thereof. The composition comprises goat milk fat globule membrane, L-alpha-glycerophosphocholine, 2'-fucosyllactose, 6'-sialyllactose sodium salt and resveratrol. The composition can reduce the inflammation and oxidative damage level of the body and improve the morphological structure of hippocampus, and has a broad application prospect in the prevention and treatment of memory decline in the elderly.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical composition technology, and specifically relates to a composition containing sheep milk fat globule membrane that can help improve memory and its application. Background Technology

[0002] As my country's population ages rapidly and the proportion of elderly continues to rise, age-related cognitive impairment is becoming increasingly prominent. Aging is often accompanied by neurodegenerative changes such as memory loss and declining learning ability, which seriously affect the quality of life of the elderly and place a heavy burden on families and society. Therefore, finding effective nutritional strategies to intervene in cognitive decline is of great social significance.

[0003] Goat milk, as a traditional nutritional food, is highly nutritious, easily digestible and absorbed, and has low allergenicity. It has a significant effect on improving nutritional health problems in the elderly, such as poor digestion and absorption, and muscle loss. Research has found that the goat milk fat globule membrane (GMFGM) has a unique composition and is rich in gangliosides, sphingomyelin, sialic acid, and other bioactive components. Currently, there are few reports on the effects of goat milk fat globule membrane in improving memory decline and reducing neuroinflammation. Therefore, developing functional foods containing goat milk fat globule membrane and exploring its efficacy when combined with other components is of significant practical importance for improving memory and cognitive function in the elderly. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to propose a composition containing goat milk fat globule membrane that can help improve memory and its applications. This is achieved through the rational compounding of goat milk fat globule membrane (GMFGM), L-α-glycylphosphocholine (GPC), 2'-fucosylated lactose (2'-FL), 6'-sialylated lactose (6'-SL), and resveratrol (RES). This composition balances functionality and nutritional value, demonstrating broad application prospects in improving memory and enhancing cognitive function.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a composition capable of assisting in improving memory, the composition comprising goat milk fat globule membrane, L-α-glucosylcholine, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol, wherein the mass ratio of goat milk fat globule membrane, L-α-glucosylcholine, 2'-FL, 6'-SL, and resveratrol is 30-50:5-20:6-20:5-20:1-6, for example, the mass ratio of goat milk fat globule membrane, L-α-glucosylcholine, 2'-FL, 6'-SL, and resveratrol is 50:10:6:8:2 or 40:5:20:10:1 or 30:15:10:15:6 or 40:10:15:5:6 or 40:20:10:5:1 or 40:5:10:20:1.

[0006] Furthermore, the mass ratio of the goat milk fat globule membrane, L-α-glucosylcholine, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol is 30-40:5-15:10-20:5-10:1-6; Furthermore, the mass ratio of the goat milk fat globule membrane, L-α-glucosylcholine, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol is 30-40:5-10:15-20:5-10:1-6.

[0007] In a second aspect, the present invention provides a product comprising the composition described in the first aspect.

[0008] Furthermore, the product is a health food, feed, or medicine.

[0009] Furthermore, the product also includes excipients acceptable for use in health foods, animal feed, or pharmaceuticals.

[0010] Thirdly, the present invention provides the use of the composition described in the first aspect in the preparation of a product that helps improve memory.

[0011] Furthermore, the product is a health food, feed, or medicine.

[0012] Fourthly, the present invention provides the use of the composition described in the first aspect in the preparation of products capable of assisting in improving cognitive function.

[0013] Furthermore, the product is either animal feed or medicine.

[0014] Fifthly, the invention provides the use of the composition described in the first aspect in the preparation of products capable of reducing the level of inflammation in the body.

[0015] Furthermore, the product is either animal feed or medicine.

[0016] Furthermore, the reduction of the body's inflammation level refers to reducing the level of serum inflammatory factors or increasing the level of serum anti-inflammatory factors.

[0017] In a sixth aspect, the present invention provides the use of the composition described in the first aspect in the preparation of a product capable of alleviating hippocampal neuronal lesions.

[0018] Furthermore, the product is either animal feed or medicine.

[0019] Compared with the prior art, the present invention has the following advantages: This invention demonstrates that combined intervention with goat milk fat globule membrane, L-α-glucosinolate, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol can help improve memory, reduce the body's inflammation level, improve and alleviate hippocampal neuronal lesions, and that the above five components have a synergistic effect in improving age-related memory decline and cognitive function loss. Attached Figure Description

[0020] Figure 1 The results of differential lipid analysis between goat milk MFGM and cow milk MFGM are shown. A is a Venn diagram, B is a principal component analysis (PCA) diagram, C is a partial least squares analysis score (OPLS-DA) diagram, D is a cluster analysis heatmap, and E is a volcano diagram.

[0021] Figure 2 Analysis of the enrichment of the KEGG pathway in goat milk for upregulating MFGM lipids.

[0022] Figure 3 The effects of the composition on novel object recognition, Y-maze, and water maze tests in D-gal-induced aging mice are shown. Where A is the novel object recognition index, B is the percentage of spontaneous alternation in the Y-maze, C is the escape latency, D is the number of times the target area is traversed, E is the time spent in the target quadrant, and F is the movement trajectory.

[0023] Figure 4 The effects of the composition on body weight and organ indices in D-gal-induced aging mice are shown. In this diagram, A represents body weight, B represents brain index, C represents heart index, D represents liver index, and E represents kidney index.

[0024] Figure 5 The effect of the composition on the morphology of hippocampal tissue in D-gal-induced aging mice.

[0025] Figure 6 The effects of the composition on serum inflammatory factor levels and oxidative stress levels in D-gal-induced aging mice are shown. Wherein, A represents LPS content, B represents TNF-α content, C represents IL-1β content, D represents IL-10 content, E represents MDA content, F represents SOD content, and G represents GSH-Px content. Detailed Implementation

[0026] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0027] The sources of each raw material in the examples are as follows: Fresh goat milk was collected from Lianlong Livestock and Poultry Breeding Co., Ltd. in Anqiu City, Shandong Province. Fresh milk was collected from Beijing Sanshi Dairy Farm Co., Ltd. L-α-glycine choline was purchased from Shandong Aobo Biotechnology Co., Ltd. 2'-FL, lactose-N-neotetrasaccharide (LNnT), and 6'-SL were purchased from Suzhou Yixi Biotechnology Co., Ltd. Resveratrol and epigallocatechin gallic acid (EGCG) were purchased from Hebei Weidakang Biotechnology Co., Ltd. Fructooligosaccharides, galactooligosaccharides, vitamin E, and docosahexaenoic acid (DHA) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0028] Example 1: Preparation of goat milk fat globule membrane Fresh goat milk (from healthy animals approximately 3 months after their first calving) was centrifuged at 3000 g for 15 min at 4°C using an ultracentrifuge. After centrifugation, the upper fat layer was collected and washed with an equal volume of pre-cooled ultrapure water to remove contaminating casein and whey protein. The mixture was then centrifuged again under the same conditions (4°C, 3000 g, 15 min), discarding the lower aqueous phase and precipitate, and collecting the washed butter layer. This process was repeated 2-3 times until the lower aqueous phase became clear. The milk fat was then sonicated (200 W, 2 s sonication, 5 s interval, 5-20 min). The milk fat was refrigerated at 4°C for 20-24 h. 5% (v / v) ultrapure water (pre-cooled at 4°C) was added to the refrigerated milk fat, and the mixture was rapidly churned. Churning was stopped when a phase transition occurred. The mixture was filtered through three layers of gauze, and the filtrate was collected as buttermilk. The remaining butter is placed in centrifuge tubes and heated in a 40-45°C water bath for 30-60 minutes until completely melted and separated into layers. After cooling to 4°C, it is centrifuged at 5000 g for 15-20 minutes, and the lower aqueous phase is collected as butter whey. The prepared buttermilk is mixed with the butter whey, and then freeze-dried into powder to obtain goat milk MFGM (GMFGM).

[0029] Bovine milk MFGM (BMFGM) was prepared using fresh cow's milk (from healthy animals approximately 3 months after their first calving) and the same method used to prepare goat milk MFGM.

[0030] Example 2: Difference analysis of MFGM lipids in cow's milk and goat's milk Bovine milk MFGM and goat milk MFGM were prepared according to the method in Example 1. The lipidome of bovine milk MFGM and goat milk MFGM was analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Qualitative analysis was performed based on lipid databases, retention times, and characteristic ion fragments in secondary mass spectrometry. Quantitative analysis was performed using triple quadrupole multiple reaction monitoring (MRM) mode, with peak area integration and internal standard method. Finally, thresholds were set for fold difference (FC) > 2, p-value < 0.05, and variable importance projection (VIP) ≥ 1 to screen for significantly differentially expressed lipids.

[0031] like Figure 1 As shown in Figure A, 993 and 1062 lipids were identified in the MFGM of cow's milk and goat's milk, respectively. Of these, 974 MFGM lipids were common to both cow's and goat's milk, while 19 and 88 MFGM lipids were unique to cow's milk and goat's milk, respectively. Principal component analysis (PCA) was performed on the samples to preliminarily understand the overall metabolic differences between the two groups and the magnitude of variability within each group. Figure 1 As shown in Figure B, significant clustering exists within the lipidomes of goat milk MFGM and bovine milk MFGM, and they can be clearly separated along the first principal component (PC1) axis, indicating a significant difference in lipid composition between the two groups. Lipidome data were analyzed using the OPLS-DA model, and score plots were generated for each group. Figure 1 (C) further demonstrates the differences between goat milk MFGM and cow milk MFGM.

[0032] The original contents of differentially expressed lipids identified by the screening criteria were normalized row-wise, and a cluster heatmap of the differentially expressed lipids was plotted as follows: Figure 1 As shown in D, the lipid profile is clearly divided into two different clusters, indicating that the lipid composition of goat milk MFGM and cow milk MFGM is significantly different between the two samples, while the lipid composition of samples within the goat milk MFGM and cow milk MFGM groups is similar.

[0033] like Figure 1 E shows a volcano plot, which is mainly used to show the differences in lipid content between the two groups of samples and the statistical significance of the differences. It can be seen that among the differential lipids in goat milk MFGM and cow milk MFGM, a total of 568 lipids were upregulated in goat milk and 214 lipids were downregulated in goat milk.

[0034] Table 1. Lipid molecules uniquely expressed in goat milk MFGM Table 1 shows that among the MFGM lipid molecules uniquely expressed in goat milk, lipid molecules belonging to PS have the highest abundance, including PS(21:0_22:6), PS(19:0_17:1), PS(15:0_18:1), etc., followed by PE subclasses, including PE(24:0_20:4), PE(15:1_22:3), PE(20:2_20:5), etc. Lipid molecules belonging to PI include PI(19:0_18:1), PI(19:1_18:2), PI(18:0_19:1), etc., and lipid molecules belonging to PC include PC(18:1_24:1), PC(12:0_16:1), PC(18:1_20:5).

[0035] Example 3: KEGG pathway enrichment analysis of MFGM lipids in goat milk KEGG pathway enrichment analysis was performed on MFGM lipids in goat milk, and the results are as follows: Figure 2 As shown in Figure A, lipids unique to goat milk MFGM are involved in neural regulation, lipid metabolism, and inflammatory signaling pathways. Figure 2 As shown in B, in neural regulation, goat milk MFGM lipids are more enriched in pathways such as the nerve growth factor signaling pathway, reverse endocannabinoid signaling, cholinergic synapse, and glutamatergic synapse than cow milk.

[0036] Example 4: Analysis of MFGM lipids involved in neural regulation and their content The lipids involved in neural regulation, MFGM, and their contents were analyzed, and the results are shown in Table 2. Table 2. MFGM lipids involved in neural regulation and their content. As shown in Table 2, four differentially expressed MFGM lipids from cow's milk and goat's milk are involved in neural regulation. Among them, Cer is involved in the nerve growth factor signaling pathway; PE and PC are involved in reverse endocannabinoid signaling; and FFA is involved in serotonergic synapses. The content of MFGM lipids involved in the nerve growth factor signaling pathway, reverse endocannabinoid signaling, and serotonergic synapses in goat's milk is significantly higher than that in cow's milk.

[0037] Example 5: Effects of goat milk MFGM on the levels of inflammatory factors in BV2 cells Each test substance was dissolved in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and filtered through a 0.22 µm sterile syringe to prepare solutions with concentrations ranging from 1 to 200 mg / mL. BV2 cells were then incubated at 1×10⁻⁶ cells / mL. 5Cells were seeded at a density of 100 cells / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator until cell adhesion or overnight. Bovine milk MFGM and goat milk MFGM were added to the intervention wells, while complete culture medium was added to the control and model groups, and pre-incubated for 4 h. Except for the control group, 1.0 μg / mL LPS was added to each well and cultured for 12–24 h. The cell culture supernatant was collected using sterile tubes. The cells were centrifuged at 1000 g for 20 min at 4°C, and the supernatant was used for analysis, or stored at -20°C or -80°C, but repeated freeze-thaw cycles should be avoided. The levels of IL-6, IL-1β, and TNF-α in the cell culture supernatant were measured according to the kit instructions.

[0038] As shown in Table 3, MFGM intervention can significantly reduce the levels of inflammatory factors IL-6, IL-1β, and TNF-α in BV2 cell supernatant, alleviate LPS-induced neuronal inflammation, and the improvement effect of goat milk MFGM is better than that of cow milk MFGM.

[0039] Table 3. Effects of bovine milk MFGM and goat milk MFGM on the levels of cellular inflammatory factors. Example 6: Raw material screening for the composition Following the method described in Example 5, the levels of IL-6, IL-1β, and TNF-α in the cell culture supernatant were measured, and various raw materials were screened. As shown in Table 4, GMFGM, GPC, 2'-FL, LNnT, 6'-SL, resveratrol, EGCG, vitamin E, and DHA all exhibited activity in reducing the IL-1β level in the LPS-induced BV2 cell supernatant culture medium.

[0040] Taking into account factors such as the bioactivity, economic cost, sensory characteristics, safety, sustainability, recommended intake, and processing methods of the raw materials, goat milk fat globule membrane, L-α-glucosinolate, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol were selected as raw materials for use as components in subsequent compositions.

[0041] Table 4. Effects of the formulation ingredients on cellular IL-1β levels. Example 7 Effects of different compositions on IL-1β levels in BV2 cells Weigh each ingredient according to the following formula, and mix the ingredients in a double helix conical mixer for 20-40 minutes to obtain the composition.

[0042] Composition I: 50 g of goat milk fat globule membrane, 10 g of L-α-glycine choline, 6 g of 2'-fucosylated lactose, 8 g of 6'-sialic acid lactose, and 2 g of resveratrol; Composition II ingredients: 40 g goat milk fat globule membrane, 5 g L-α-glycine choline, 20 g 2'-fucosylated lactose, 10 g 6'-sialic acid lactose, 1 g resveratrol; Composition III ingredients: 30 g goat milk fat globule membrane, 15 g L-α-glycine choline, 10 g 2'-fucosylated lactose, 15 g 6'-sialic acid lactose, 6 g resveratrol; Composition IV Ingredients: 40 g goat milk fat globule membrane, 10 g L-α-glycine choline, 15 g 2'-fucosylated lactose, 5 g 6'-sialic acid lactose, 6 g resveratrol; Composition V Ingredients: 40 g goat milk fat globule membrane, 20 g L-α-glycine choline, 10 g 2'-fucosylated lactose, 5 g 6'-sialic acid lactose, 1 g resveratrol; Composition VI Ingredients: 40 g goat milk fat globule membrane, 5 g L-α-glycine choline, 10 g 2'-fucosylated lactose, 20 g 6'-sialic acid lactose, 1 g resveratrol; Comparative Example 1 Raw material: 76 g of goat milk fat globule membrane; Comparative Example 2 Raw material: L-α-glycine phosphocholine 76 g; Comparative Example 3 Ingredients: 76 g of 2'-fucosylated lactose; Comparative Example 4 Ingredients: 76 g of 6'-sialyl lactose; Comparative Example 5 Raw Material: Resveratrol 76 g.

[0043] The effects of compositions I-VI and comparative examples 1-5 on IL-1β levels in BV2 cell supernatant were determined using the method described in Example 5 to characterize the potential memory-improving effects of each composition.

[0044] The results are shown in Table 5. It can be seen that, with the same total amount of intervention components, composition I-VI has a significantly enhanced activity in reducing the level of IL-1β in the supernatant culture medium of LPS-induced BV2 cells compared with comparative examples 1-5 (single-component intervention group), and can more significantly alleviate LPS-induced neuronal inflammation. Therefore, goat milk fat globule membrane, L-α-glucosinolate, 2'-fucosylated lactose, 6'-sialylated lactose and resveratrol have a synergistic effect in improving memory when used in specific ratios.

[0045] Table 5. Effects of the composition on cellular IL-1β levels Example 8 Effects of different compositions on D-gal-induced aging mice Eight-week-old SPF-grade male BALB / c mice (weighing 18-22g) were selected and housed in an environment with a temperature of 20±2℃, relative humidity of 65±5%, a light-dark cycle of 12 h, and good ventilation. The mice were fed commercial pellets and had free access to water. The animal experiments were reviewed and approved by the Animal Experiment Committee of China Agricultural University.

[0046] Mice were randomly divided into four groups (n=10 per group): normal control (NC), model group (D-gal), goat milk fat globule membrane group (GMFGM), L-α-glycine choline group (GPC), 2'-fucosylated lactose group (2'-FL), 6'-sialylated lactose group (6'-SL), resveratrol group (RES), formula group, and positive control group (Done). Before the experiment, the mice were housed for one week to acclimatize. The intervention details for each group are as follows: NC group (blank control group): The same volume of normal saline was administered by gavage once daily. Four hours after gavage, 0.1 mL / 10 g BW sterile normal saline was injected intraperitoneally.

[0047] D-gal group (model group): Administered an equal volume of physiological saline by gavage once daily. Four hours after gavage, injected intraperitoneally with 300 mg / kg BW D-gal solution.

[0048] GMFGM group: Mice were administered 152 mg / kg BW of goat milk fat globule membrane by gavage once daily. Four hours after gavage, mice were injected intraperitoneally with 300 mg / kg BW of D-gal solution.

[0049] GPC group: Mice were administered L-α-glycine choline 152 mg / kg BW by gavage once daily. Four hours after gavage, they were injected intraperitoneally with 300 mg / kg BW D-gal solution.

[0050] 2'-FL group: Mice were administered 152 mg / kg BW of 2'-fucosylated lactose by gavage once daily. Four hours after gavage, mice were injected intraperitoneally with 300 mg / kg BW of D-gal solution.

[0051] 6'-SL group: Mice were administered 152 mg / kg BW of 6'-sialic acid lactose by gavage once daily. Four hours after gavage, mice were injected intraperitoneally with 300 mg / kg BW of D-gal solution.

[0052] RES group: Mice were administered resveratrol 152 mg / kg BW by gavage once daily. Four hours after gavage, they were injected intraperitoneally with 300 mg / kg BW D-gal solution.

[0053] Formula group (composition group): Composition II from Example 7 was administered by gavage at 152 mg / kg BW once daily. Four hours after gavage, 300 mg / kg BW D-gal solution was injected intraperitoneally.

[0054] Done group (positive control group): 1 mg / kg BW donepezil was administered by gavage once daily. Four hours after gavage, 300 mg / kg BW D-gal solution was injected intraperitoneally.

[0055] The intervention lasted for 8 weeks, with weekly records kept of the mice's physiological status, weight, and food intake. In week 8, the mice underwent new object recognition, Y-maze, and water maze tests. Afterwards, all mice were anesthetized, euthanized by cervical dislocation, and organ and blood samples were collected.

[0056] 1. Effects of the composition on memory in D-gal-induced aging mice 1.1 New Object Recognition Experiment 1.1.1 Experimental Methods The novel object recognition experiment assesses a mouse's ability to explore novel objects in a free state, testing object recognition ability in short-term memory (1-2 hours) and long-term memory (24 hours). The experimental method is as follows: On the first day, during the adaptation phase, mice are allowed to freely explore a box for 5 minutes. 24 hours after adaptation, training begins. On the second day, two identical objects are placed at one-quarter of the height of the box. The mouse is placed into the box from the center of the side wall, equidistant from the two identical objects, and allowed to freely explore for 7 minutes. 24 hours after training, testing is conducted on the third day. A new object of different color and shape replaces one of the old objects. The positions of the two objects are the same as in the second day's training phase. The mouse is placed into the box from the center of the side wall, equidistant from the two identical objects, and allowed to freely explore for 7 minutes. After each experiment, the box and the recognized object are thoroughly disinfected with 75% ethanol. The exploration time for the new and old objects is recorded, and the novel object recognition index is calculated using a formula.

[0057] New object recognition index (%) = (Time spent exploring new objects / (Time spent exploring new objects + Time spent exploring old objects)) × 100% 1.1.2 Experimental Results like Figure 3As shown in Figure A, compared with the NC group, the novel object recognition index of the D-gal group was significantly reduced, indicating that the short-term / long-term memory capacity of the D-gal group mice was reduced, and the modeling was successful. Compared with the D-gal group, the novel object recognition index of mice in all intervention groups was significantly increased, with the Formula and Done groups showing significantly higher novel object recognition indices, and no significant difference compared with the NC group. Compared with the Formula group, the novel object recognition index of mice in the GMFGM, GPC, 2'-FL, 6'-SL, and RES groups was reduced, indicating that the intervention effect of the composition was better than that of the single-component substance at the same dose. These results indicate that the composition can improve the novel object recognition index of mice and alleviate cognitive decline in aging mice, and that GMFGM, GPC, 2'-FL, 6'-SL, and RES have a synergistic effect in maintaining exploratory ability and alleviating cognitive decline.

[0058] 1.2 Y-maze Experiment 1.2.1 Experimental Methods The Y maze test consists of three identical arms, each measuring 34 cm × 5 cm × 15 cm (length × width × height), with an angle of 120° between the arms. The procedure for the Y maze test is as follows: a mouse is placed in the center of the Y maze and allowed to explore freely for 8 minutes. An analysis system is used to record the total number of times the mouse enters an arm and the number of consecutive entries into three different arms, and the percentage of alternation is calculated using a formula.

[0059] The percentage of spontaneous alternations in the Y-maze = (Number of consecutive entries into three different arms) / (Total number of arm entries - 2) × 100% 1.2.2 Experimental Results like Figure 3 As shown in Figure B, the percentage of spontaneous alternation in the Y-maze in the D-gal group mice was 46.89% ± 1.06%, significantly lower than that in the NC group (p < 0.05). Compared with the D-gal group, the percentage of spontaneous alternation in the Y-maze in each intervention group was significantly increased, with no significant difference between the GMFGM group, Formula group, and Done group and the NC group (p > 0.05). Compared with the Formula group, the percentage of spontaneous alternation in the GPC group, 2'-FL group, 6'-SL group, and RES group mice was significantly decreased, while the percentage of spontaneous alternation in the GMFGM group mice was slightly decreased, indicating that the intervention effect of the composition was better than that of the single-component substance at the same dose. These results indicate that the composition can significantly increase the percentage of Y-maze alternation in aging mice.

[0060] 1.3 Water Maze Experiment 1.3.1 Experimental Methods The Morris Water Maze (MWM) experiment was conducted to assess the spatial learning and memory abilities of mice, particularly those dependent on hippocampal function. The MWM consisted of a circular pool (120 cm in diameter and 45 cm in height) and a hidden platform (10 cm in diameter and 20 cm in height), with the water temperature maintained at 20 ± 2°C. Black pigment was added to the water, and software was used to track and identify the mice. The MWM consisted of four quadrants. The hidden platform was placed 1 cm below the water surface in the third quadrant as the mouse's escape target, and this quadrant was recorded as the target quadrant.

[0061] The first four days of the water maze were the training phase. Mice were allowed to freely explore the water maze with escape platforms for 90 seconds each time. Each time, the mice were placed into the water pool facing the wall from different quadrants. The time it took for the mice to find the platform—the latency period—was tracked and recorded using an image acquisition system. For mice that did not find the platform within 90 seconds, the latency period was recorded as 90 seconds, and the mice were guided to the platform and stayed there for 15 seconds.

[0062] Day 5 was the navigation experiment: Mice freely explored a water maze with escape platforms for 60 seconds. An image acquisition system was used to track and record the time it took for the mice to reach the platforms, recording the escape latency. For mice that did not find the platforms, the escape latency was recorded as 60 seconds, and the mice were guided to the platforms, where they stayed for 15 seconds. Day 6 was the spatial exploration experiment: The platforms were removed, and mice freely explored a water maze without escape platforms for 60 seconds. An image acquisition system was used to track and record the mice's swimming trajectory and time the activity.

[0063] 1.3.2 Experimental Results In the positioning and navigation experiment 5 days before the water maze experiment, the escape latency of mice in finding the platform was as follows: Figure 3 As shown in Figure C, the escape latency of mice in the D-gal group was significantly higher than that in the NC group (p<0.05). On day 3, the escape latency of mice in the GMFGM, Formula, and Done groups was significantly lower than that in the D-gal group. On day 5, compared with the D-gal group, the escape latency of mice in the GMFGM, Formula, and Done groups was significantly reduced (p<0.05). These results indicate that the latency of mice to find the platform gradually shortens with increasing training time.

[0064] In the spatial exploration experiment on day 6 of the water maze, the number of times mice traversed the target area and the time spent in the target quadrant were measured. Figure 3As shown in DE, the time spent in the target quadrant by mice in the D-gal group was 12.03 ± 0.17 s, significantly shorter than that in the NC group (p < 0.05). Compared with the D-gal group, the time spent in the target quadrant by mice in the GMFGM and Formula groups was significantly longer (p < 0.05). The time spent in the target quadrant by mice in the Formula group was not significantly different from that in the NC and Done groups (p < 0.05). The number of times mice in the D-gal group crossed the target area was 1.45 ± 0.37, significantly shorter than that in the NC group (p < 0.05). Compared with the D-gal group, the number of times mice in the GMFGM and Formula groups crossed the target area was significantly longer (p < 0.05), and there was no significant difference compared to the NC and Done groups (p > 0.05). Representative mouse movement trajectories are shown below. Figure 3 As shown in F. All the above results indicate that the composition can significantly increase the time spent in the target quadrant and the number of times it crosses the target quadrant in aging mice.

[0065] 2. Effects of the composition on body weight and organ indices in D-gal-induced aging mice like Figure 4 As shown in Figure A, during the experiment, the mouse body weight steadily increased with the extension of experimental time. There were no significant differences in body weight among the NC group, D-gal group, GMFGM group, Formula group, and Done group (p<0.05). The organ indices of the mice were measured, and the results are as follows: Figure 4 As shown in the BE results, the brain index of mice in the D-gal group was significantly lower than that in the NC group (p<0.05). Compared with the D-gal group, the brain index of mice in the GMFGM group and Formula group was significantly increased (p<0.05). There were no significant differences in the heart, liver, and kidney indices of mice in the NC group, D-gal group, GMFGM group, Formula group, and Done group (p>0.05). These results indicate that GMFGM and the combination can increase the brain index of aging mice, without significantly affecting the heart, liver, and kidney indices.

[0066] 3. Effects of the composition on hippocampal morphology in D-gal-induced aging mice like Figure 5As shown, the NC group mice had a large number of hippocampal neurons that were neatly and densely arranged, with clear neuronal structures. Compared with the NC group mice, the D-gal group mice had fewer neurons in the CA1 and CA3 regions of the hippocampus, with looser arrangement, shrunken neuronal cell bodies, irregular morphology, and deep staining (indicated by black arrows). Compared with the D-gal group mice, the GMFGM, Formula, and Done groups mice had significantly increased neuronal cell numbers, significantly improved arrangement, regular morphology, and reduced deep staining. Furthermore, in the DG region, compared with the CON group mice, the D-gal group mice had neurons with shrunken, deeply stained nuclei in the hippocampus (indicated by black arrows), while the neuronal state was significantly improved after Formula composition intervention. These results indicate that the composition can effectively alleviate the severity of hippocampal neuronal lesions.

[0067] 4. Effects of the composition on D-gal-induced inflammation in aging mice Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria and can excessively activate the immune system. TNF-α and IL-1β are core mediators driving neuroinflammation; they can directly damage neurons, disrupt synaptic function, and further amplify the inflammatory response. Excessive neuroinflammation can lead to irreversible nerve damage. IL-10, as a key anti-inflammatory cytokine, can effectively inhibit the production of pro-inflammatory factors, thereby limiting the spread and destruction of inflammation. Figure 6 As shown in the AD diagram, the serum LPS, TNF-α, and IL-1β levels in the D-gal group were significantly higher than those in the NC group, while the IL-10 level was significantly lower (p<0.05). Compared with the D-gal group, the serum LPS, TNF-α, and IL-1β levels in the GMFGM, GPC, 2'-FL, 6'-SL, RES, and Formula groups were significantly lower (p<0.05), while the IL-10 level was significantly higher. Furthermore, compared with the GMFGM, GPC, 2'-FL, 6'-SL, and RES groups, the Formula group showed significantly lower LPS and TNF-α levels and significantly higher IL-10 levels, indicating that the anti-inflammatory effect of the composition was superior to that of the single-component substances at the same dosage. These results suggest that GMFGM, GPC, 2'-FL, 6'-SL, and RES have a synergistic effect in alleviating inflammation in aging mice, and the composition of these five components can effectively reduce serum inflammatory factor levels and increase anti-inflammatory factor levels in aging mice.

[0068] 5. Effects of the composition on D-gal-induced oxidative damage in aging mice Reactive oxygen species (ROS) can damage both enzymatic and non-enzymatic antioxidant systems; their excessive accumulation leads to oxidative damage and accelerates cellular aging. Superoxide dismutase (SOD) and GSH-Px are key antioxidant enzymes in the body that can effectively scavenge ROS and alleviate oxidative damage. Metabolic acid (MDA) is the end product of ROS-induced lipid peroxidation and is often used as an indicator to assess the degree of oxidative damage. Figure 6 As shown in the EG, compared with the NC group, the D-gal group mice showed significantly decreased serum SOD and GSH-Px enzyme activities and significantly increased MDA levels (p<0.05). Compared with the D-gal group, the GMFGM, GPC, 2'-FL, 6'-SL, RES, and Formula groups mice showed significantly increased serum SOD and GSH-Px enzyme activities and significantly decreased MDA levels (p<0.05). Compared with the GMFGM, GPC, 2'-FL, 6'-SL, and RES groups mice, the Formula group showed significantly decreased MDA levels and significantly increased SOD and GSH-Px levels, indicating that the combined intervention was more effective than the intervention of single-component substances at the same dosage. These results indicate that GMFGM, GPC, 2'-FL, 6'-SL, and RES have a synergistic effect in alleviating oxidative damage in aging mice. The combination of these five components can effectively improve antioxidant enzyme activity and lipid peroxidation, and improve the serum antioxidant capacity of aging mice.

[0069] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A composition capable of assisting in improving memory, characterized in that, The composition comprises goat milk fat globule membrane, L-α-glucosylcholine, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol, wherein the mass ratio of goat milk fat globule membrane, L-α-glucosylcholine, 2'-FL, 6'-SL, and resveratrol is 30-50:5-20:6-20:5-20:1-6.

2. The composition according to claim 1, characterized in that, The mass ratio of goat milk fat globule membrane, L-α-glucosinolate, 2'-fucosylated lactose, 6'-sialylated lactose, and resveratrol is 30-40:5-15:10-20:5-10:1-6.

3. A product characterized in that, The product includes the composition according to claim 1 or 2.

4. The product according to claim 3, characterized in that, The product is a health food, feed, or medicine.

5. The product according to claim 4, characterized in that, The product also includes excipients acceptable for use in health foods, feed, or pharmaceuticals.

6. The use of the composition according to claim 1 or 2 in the preparation of a product that helps improve memory, characterized in that, The product is a health food, feed, or medicine.

7. The use of the composition according to claim 1 or 2 in the preparation of a product capable of assisting in improving cognitive function, characterized in that, The product is either animal feed or medicine.

8. The use of the composition according to claim 1 or 2 in the preparation of a product capable of reducing the level of inflammation in the body, characterized in that, The product is either animal feed or medicine.

9. The application according to claim 8, characterized in that, The reduction of the body's inflammation level refers to either reducing the level of serum inflammatory factors or increasing the level of serum anti-inflammatory factors.

10. The use of the composition according to claim 1 or 2 in the preparation of a product capable of alleviating hippocampal neuronal lesions, characterized in that, The product is either animal feed or medicine.

Images