A nutritional composition for regulating sleep, its preparation method and application

Abstract

This invention relates to the field of nutritional food technology, specifically disclosing a nutritional composition for regulating sleep, its preparation method, and its application. The nutritional composition comprises bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol, and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride. The nutritional composition provided by this invention can promote the expression levels of γ-aminobutyric acid receptors and melatonin, thereby improving sleep, assisting in the regulation of circadian rhythms, and increasing the proportion of deep sleep. This invention provides a new approach for preparing nutritional foods that regulate sleep.

Description

Technical Field

[0001] This invention relates to the field of nutritional food technology, and in particular to a nutritional composition for regulating sleep, its preparation method, and its application. Background Technology

[0002] Sleep plays a vital role in physiological processes, influencing growth, immune function, and cardiovascular health. Quality sleep is considered to have appropriate timing, sufficient duration, and good quality. Sleep is crucial for the growth and development of infants and children, especially during deep sleep when growth hormone secretion peaks. This hormone is essential for the development of bones, muscles, and organs. Regular, good sleep contributes to brain development, including improved memory, attention, and learning abilities; it helps maintain emotional stability and adaptability to environmental changes; and it helps strengthen the body's immune system, reducing the risk of infection. For adults, good sleep allows the brain and body to rest, recuperate, and recover, aiding in daily work and study.

[0003] Currently, in the food sector, such as special medical purpose formula powder, infant formula powder, complementary food and nutritional supplements, sleep is only indirectly improved through basic nutrients such as calcium, vitamin D, prebiotics or probiotics. Moreover, the correlation between the composition design and sleep mechanism is insufficient. This lack of correlation makes it difficult to effectively intervene in sleep health by regulating sleep structure (such as prolonging deep sleep duration and optimizing sleep cycle transitions) or improving sleep quality (such as reducing the number of nighttime awakenings). Summary of the Invention

[0004] In view of this, the present invention provides a nutritional composition for regulating sleep, a method for preparing the same, and its application. The nutritional composition for regulating sleep comprises bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride, and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol triglyceride. The nutritional composition provided by the present invention can increase the expression levels of γ-aminobutyric acid (GABA) receptors and melatonin, thereby improving sleep, regulating circadian rhythms, and increasing the proportion of deep sleep.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] The first aspect of the present invention provides a nutritional composition for regulating sleep, the nutritional composition comprising bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol triglyceride.

[0007] Research has found that GABA is a key inhibitory neurotransmitter that plays a crucial role in the central nervous system of mammals. Its sleep-aiding mechanism primarily works by regulating neuronal excitability. After binding to receptors on neurons, GABA inhibits the firing frequency of neurons, thus producing a calming and sleep-inducing effect. When GABA is insufficient, the brain becomes over-excited, potentially leading to anxiety, restlessness, and fatigue, manifesting as difficulty falling asleep and shallow sleep. Therefore, appropriate GABA supplementation or promoting GABA synthesis in the body can help improve sleep quality. Melatonin is a hormone secreted by the pineal gland in the brain, primarily responsible for regulating the body's circadian rhythm and sleep-wake cycle. When melatonin binds to specific receptors, it initiates cell signal transduction, shortening sleep latency and prolonging sleep duration, thereby overcoming sleep disorders, improving circadian rhythm disturbances, and enhancing sleep quality.

[0008] This invention discloses a sleep-regulating nutritional composition prepared primarily from bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride, and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride. In this nutritional composition, miR-148a, highly expressed in bovine milk exosomes, inhibits DNA methyltransferase 1 (DNMT1), reduces methylation of GABA receptor genes (such as GABRA1 and GABRB2), and promotes their transcription. Furthermore, it indirectly promotes neuronal GABA receptor expression by inhibiting the NF-κB pathway and reducing inflammatory factors (such as IL-6 and TNF-α), and reduces oxidative damage to the pineal gland to maintain melatonin synthesis. 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride optimize fat absorption, promote tryptophan entry into the brain, and subsequently promote serotonin (5-HT) synthesis, which is a precursor to melatonin. More importantly, experiments have shown that the sleep-regulating nutritional composition of the present invention can promote the expression of GABA receptors and melatonin in zebrafish, thereby improving sleep quality and regulating circadian rhythms.

[0009] Preferably, the mass ratio of the bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol triglyceride is (2-20):(40-60):(20-70).

[0010] More preferably, the mass ratio of the bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol triglyceride is (5-10):(40-50):(40-70).

[0011] Preferably, the method for preparing bovine milk exosomes includes the following steps:

[0012] S1. The milk is centrifuged once to separate the solid and liquid, and the supernatant A is obtained; the supernatant A is centrifuged a second time to separate the solid and liquid, and the supernatant B is obtained.

[0013] S2. Centrifuge the supernatant B three times, discard the supernatant, and obtain a solid; add a dispersion to the solid to resuspend the precipitate, and obtain a suspension C;

[0014] S3. The suspension C is finely filtered to obtain the bovine milk exosomes.

[0015] More preferably, in S1, the temperature of the first centrifugation is 3-5℃, the rotation speed is 3000-5000rpm, and the time is 5-15min.

[0016] More preferably, in S1, the temperature of the secondary centrifugation is 3-5℃, the rotation speed is 10000-12000rpm, and the time is 40-60min.

[0017] More preferably, in S2, the temperature of the three centrifugations is 3-5℃, the rotation speed is 10000-12000rpm, and the time is 60-90min.

[0018] More preferably, in S2, the mass-to-volume ratio of the solidified material to the dispersion is 1 g: (5-10) mL.

[0019] More preferably, in S2, the dispersion is a PBS buffer with pH=7.2.

[0020] More preferably, in S3, the specific operation of fine filtration is as follows: the suspension C is filtered sequentially through filter membranes with pore sizes of 0.4-0.6 μm and 0.2-0.3 μm.

[0021] The second aspect of the present invention provides a method for preparing the aforementioned sleep-regulating nutritional composition, comprising the following steps: weighing bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride according to the designed ratio, mixing them evenly to obtain the sleep-regulating nutritional composition.

[0022] In summary, this invention provides a nutritional composition for regulating sleep, its preparation method, and its application. The nutritional composition for regulating sleep comprises bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol, and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol. Through the synergistic effect of its components, the nutritional composition for regulating sleep provided by this invention effectively enhances the expression level of GABA receptors and promotes melatonin secretion, thereby improving sleep, helping to regulate circadian rhythms, and increasing the proportion of deep sleep. Attached Figure Description

[0023] Figure 1 The following graphs show the movement distances of zebrafish in each group, compared with the model control group: *p<0.05, **p<0.01, ***p<0.001;

[0024] Figure 2 The expression levels of GABA receptors in zebrafish in each group are shown in the graphs. Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 3 The graph shows the melatonin expression levels of each group of zebrafish, compared with the model control group: *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0027] To better illustrate the present invention, further examples are provided below; in the present invention, all materials not specifically described are commercially available products.

[0028] Example 1

[0029] This embodiment provides a nutritional composition for regulating sleep, the nutritional composition comprising bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride in a mass ratio of 5:60:24.

[0030] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0031] The method for preparing bovine milk exosomes includes the following steps:

[0032] S1. Centrifuge 50 mL of milk at 4℃ and 4000 rpm for 10 min to separate the solid and liquid, and obtain supernatant A; centrifuge supernatant A at 4℃ and 10000 rpm for 40 min to separate the solid and liquid, and obtain 35 mL of supernatant B.

[0033] S2. Centrifuge the supernatant B at 4°C and 12000 rpm for 60 min, discard the supernatant, and obtain a solid. Add PBS buffer (pH=7.2) at a mass-to-volume ratio of 1 g:5 mL to the solid, resuspend the precipitate, and obtain suspension C.

[0034] S3. The suspension C is filtered sequentially through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the bovine milk exosomes.

[0035] Example 2

[0036] This embodiment provides a nutritional composition for regulating sleep, the nutritional composition comprising bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride in a mass ratio of 5:45:36.

[0037] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0038] The method for preparing bovine milk exosomes includes the following steps:

[0039] S1. Centrifuge 50 mL of milk at 3℃ and 4500 rpm for 10 min to separate the solid and liquid, and obtain supernatant A; centrifuge supernatant A at 5℃ and 10000 rpm for 40 min to separate the solid and liquid, and obtain 35 mL of supernatant B.

[0040] S2. Centrifuge the supernatant B at 4°C and 12000 rpm for 60 min, discard the supernatant, and obtain a solid. Add PBS buffer (pH=7.2) at a mass-to-volume ratio of 1 g:5 mL to the solid, resuspend the precipitate, and obtain suspension C.

[0041] S3. The suspension C is filtered sequentially through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the bovine milk exosomes.

[0042] Example 3

[0043] This embodiment provides a nutritional composition for regulating sleep, the nutritional composition comprising bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol triglyceride and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride in a mass ratio of 5:45:45.

[0044] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0045] The method for preparing bovine milk exosomes includes the following steps:

[0046] S1. Centrifuge 50 mL of milk at 5 °C and 4000 rpm for 10 min to separate the solid and liquid, and obtain supernatant A; centrifuge supernatant A at 4 °C and 10000 rpm for 40 min to separate the solid and liquid, and obtain 35 mL of supernatant B.

[0047] S2. Centrifuge the supernatant B at 5°C and 12000 rpm for 60 min, discard the supernatant, and obtain a solid. Add PBS buffer (pH=7.2) at a mass-to-volume ratio of 1 g:5 mL to the solid, resuspend the precipitate, and obtain suspension C.

[0048] S3. The suspension C is filtered sequentially through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the bovine milk exosomes.

[0049] Example 4

[0050] This embodiment provides a nutritional composition for regulating sleep, the nutritional composition comprising bovine milk exosomes, 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride in a mass ratio of 5:40:48.

[0051] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0052] The method for preparing bovine milk exosomes includes the following steps:

[0053] S1. Centrifuge 50 mL of milk at 5 °C and 4000 rpm for 10 min to separate the solid and liquid, and obtain supernatant A; centrifuge supernatant A at 4 °C and 10000 rpm for 40 min to separate the solid and liquid, and obtain 35 mL of supernatant B.

[0054] S2. Centrifuge the supernatant B at 3°C ​​and 12000 rpm for 60 min, discard the supernatant, and obtain a solid. Add PBS buffer (pH=7.2) at a mass-to-volume ratio of 1 g:5 mL to the solid, resuspend the precipitate, and obtain suspension C.

[0055] S3. The suspension C is filtered sequentially through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the bovine milk exosomes.

[0056] Comparative Example 1

[0057] This comparative example provides a nutritional composition for regulating sleep, the nutritional composition comprising 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol in a mass ratio of 50:50.

[0058] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0059] Comparative Example 2

[0060] This comparative example provides a nutritional composition for regulating sleep. Compared with Example 1, the nutritional composition comprises milk, 1,3-dioleoyl-2-palmitoylglycerol, and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol in a mass ratio of 5:60:24.

[0061] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0062] Comparative Example 3

[0063] This comparative example provides a nutritional composition for regulating sleep, which differs from Example 1 in that the nutritional composition comprises bovine milk exosomes and 1,3-dioleoyl-2-palmitoylglycerol in a mass ratio of 5:60.

[0064] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0065] The method for preparing bovine milk exosomes includes the following steps:

[0066] S1. Centrifuge 50 mL of milk at 4℃ and 4000 rpm for 10 min to separate the solid and liquid, and obtain supernatant A; centrifuge supernatant A at 4℃ and 10000 rpm for 40 min to separate the solid and liquid, and obtain 35 mL of supernatant B.

[0067] S2. Centrifuge the supernatant B at 4°C and 12000 rpm for 60 min, discard the supernatant, and obtain a solid. Add PBS buffer (pH=7.2) at a mass-to-volume ratio of 1 g:5 mL to the solid, resuspend the precipitate, and obtain suspension C.

[0068] S3. The suspension C is filtered sequentially through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the bovine milk exosomes.

[0069] Comparative Example 4

[0070] This comparative example provides a nutritional composition for regulating sleep, which differs from Example 1 in that the nutritional composition comprises bovine milk exosomes and 1-oleic-2-palmitic-3-linoleic acid triglycerides in a mass ratio of 5:24.

[0071] Weigh each raw material component according to the designed ratio, mix them evenly, and obtain a nutritional composition for regulating sleep.

[0072] The method for preparing bovine milk exosomes includes the following steps:

[0073] S1. Centrifuge 50 mL of milk at 4℃ and 4000 rpm for 10 min to separate the solid and liquid, and obtain supernatant A; centrifuge supernatant A at 4℃ and 10000 rpm for 40 min to separate the solid and liquid, and obtain 35 mL of supernatant B.

[0074] S2. Centrifuge the supernatant B at 4°C and 12000 rpm for 60 min, discard the supernatant, and obtain a solid. Add PBS buffer (pH=7.2) at a mass-to-volume ratio of 1 g:5 mL to the solid, resuspend the precipitate, and obtain suspension C.

[0075] S3. The suspension C is filtered sequentially through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the bovine milk exosomes.

[0076] Efficacy test

[0077] 1.1 Laboratory Animals and Grouping

[0078] Zebrafish were cultured in aquarium water at 28℃ (water quality requirements: 200 mg of readily soluble sea salt per 1L of reverse osmosis water; conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3). The husbandry and management met the requirements of the International Committee for Assessment and Certification of Laboratory Animal Husbandry and Management. Wild-type AB strain zebrafish, 5 days post-fertilization (5dpf), were randomly selected and cultured in cell culture plates. Each well contained 30 zebrafish, and the feeding volume was 3 mL per well. Specific grouping and treatment methods are shown in Table 1.

[0079] Table 1 Grouping and Processing Methods

[0080]

[0081] 1.2 Establishment of the Zebrafish Model

[0082] The zebrafish were divided into a blank control group, a positive control group, a model control group, and experimental groups 1-8. The blank control group received no treatment. The model control group, sample treatment group, and positive control group all ingested 140 μg / mL of penetrazol (PTZ). The positive control group ingested penetrazol and 125 μg / mL of oxazepam, while the experimental treatment groups ingested penetrazol and various nutrient combinations.

[0083] 1.3 Effects of different nutrient compositions on the migration distance of zebrafish

[0084] After treatment at 28℃ for 1 day, 10 zebrafish were randomly selected from each group and transferred to a 96-well plate, with one zebrafish per well. The feeding system for each well was 200 μL. The distance of movement of the zebrafish within 10 minutes was then measured using a behavior analyzer to evaluate the effect of different nutritional compositions on improving sleep in model zebrafish.

[0085] 1.4 Effects of different nutrient compositions on the expression levels of GABA receptors and melatonin in zebrafish

[0086] Zebrafish samples were collected from each group 24 hours after feeding. Total RNA was extracted from each group of zebrafish using a kit, and the gene expression levels of GABA receptor and melatonin were detected by q-PCR.

[0087] 1.5 Data Analysis

[0088] Experimental results are expressed as mean ± standard deviation. SPSS software was used to test for normality. If the data conforms to normality, the significance of the data is tested using the T-parameter test or analysis of variance (ANOVA). If the data does not conform to normality, a non-parametric test is used.

[0089] 2. Experimental Results

[0090] 2.1 Results of the effects of different nutrient combinations on the migration distance of zebrafish

[0091] The results of the zebrafish movement distances for each group are as follows: Figure 1 As shown in Table 2.

[0092] Table 2. Results of zebrafish movement distance, GABA receptor gene expression level, and melatonin gene expression in each group.

[0093]

[0094] From Table 2 and Figure 1 It was found that, compared with the blank control group, the zebrafish in the model control group moved a significantly longer distance (P<0.001), indicating successful modeling. Furthermore, compared with the model group, the zebrafish in the positive control group moved a significantly shorter distance (P<0.001), indicating that administering sleep-aiding drugs could significantly reduce the distance and frequency of rapid movement in zebrafish. Compared with the model control group, the zebrafish in each experimental group moved a significantly shorter distance. Notably, the combination of the three components in the examples was more effective than any two components in the comparative examples, indicating a significant synergistic effect between bovine exosomes and 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol.

[0095] Furthermore, compared with Comparative Example 2, Example 1 had a more significant effect on the movement distance of zebrafish, indicating that milk exosomes have a stronger effect on promoting sleep.

[0096] Furthermore, in the examples, when the mass ratio of 1,3-dioleoyl-2-palmitoylglycerol to 1-oleoyl-2-palmitoyl-3-linoleoylglycerol was in the range of 1:1 (Example 3) to 1:1.2 (Example 4), the zebrafish moved a shorter distance, indicating that the synergistic effect was further enhanced.

[0097] 2.2 Effects of different nutrient compositions on the expression levels of GABA receptors and melatonin in zebrafish

[0098] The experimental results of GABA receptor and melatonin expression levels are shown in Table 2. Figure 2 and Figure 3 From Table 2, Figure 2 and Figure 3 As can be seen, compared with the model control group, the experimental groups all showed a significant increase in the expression levels of GABA receptors and melatonin. The effects of the experimental group were superior to those of the control group. With the increase of 1-oleic-2-palmitoyl-3-linoleic acid triglyceride content, the expression levels of GABA receptors and melatonin further increased, indicating that a certain proportion of 1,3-dioleoyl-2-palmitoyl triglyceride and 1-oleic-2-palmitoyl-3-linoleic acid triglyceride have a promoting effect on improving sleep.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nutritional composition for modulating sleep, characterized in that, The nutritional composition comprises bovine milk exosomes, 1,3-dioleic acid 2-palmitic acid triglyceride and 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride, and the mass ratio of the bovine milk exosomes, 1,3-dioleic acid 2-palmitic acid triglyceride and 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride is (2-20):(40-60):(20-70).

2. The sleep-regulating nutritional composition of claim 1, wherein, The mass ratio of the bovine milk exosomes, 1,3-dioleic acid 2-palmitic acid triglyceride and 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride is (5-10):(40-50):(40-70).

3. The sleep-regulating nutritional composition of claim 1, wherein, The preparation method of the bovine milk exosomes comprises the following steps: S1, centrifuging bovine milk once to separate the solid and the liquid to obtain supernatant A; centrifuging the supernatant A twice to separate the solid and the liquid to obtain supernatant B; S2, centrifuging the supernatant B three times to discard the supernatant to obtain solid condensate; adding dispersion liquid to resuspend the precipitate to obtain suspension C; S3, performing precision filtration on the suspension C to obtain the bovine milk exosomes.

4. The sleep-regulating nutritional composition of claim 3, wherein, In S1, the temperature of the first centrifugation is 3-5°C, the rotation speed is 3000-5000 rpm, and the time is 5-15 min; In S1, the temperature of the second centrifugation is 3-5°C, the rotation speed is 10000-12000 rpm, and the time is 40-60 min; In S2, the temperature of the third centrifugation is 3-5°C, the rotation speed is 10000-12000 rpm, and the time is 60-90 min.

5. The sleep-regulating nutritional composition of claim 3, wherein, In S2, the mass-to-volume ratio of the solid condensate to the dispersion liquid is 1g:(5-10)mL; In S3, the specific operation of the precision filtration is that the suspension C is sequentially filtered through filter membranes with pore sizes of 0.4-0.6μm and 0.2-0.3μm.

6. A method for the preparation of a sleep-regulating nutritional composition according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: according to the designed ratio, bovine milk exosomes, 1,3-dioleic acid 2-palmitic acid triglyceride and 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride are weighed and mixed uniformly to obtain a nutritional composition for adjusting sleep.

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