Nutritional composition and use thereof for improving sleep

Abstract

The invention belongs to the field of food, and particularly relates to a nutritional composition and application thereof in improving sleep. The invention provides a nutritional composition. The nutritional composition comprises necessary active ingredients shown as (I) and (II) as follows: (I) casein phosphopeptides and (II) neutral non-fucosylated breast milk oligosaccharides, wherein the casein phosphopeptides are casein phosphopeptides, the casein phosphopeptides are casein phosphopeptides, the casein phosphopeptides are casein phosphopeptides, the neutral non-fucosylated breast milk oligosaccharide at least contains lactose-N-neotetraose, and in the nutritional composition, the mass ratio of the casein phosphopeptides to the lactose-N-neotetraose is 1: (0.1-15.0). According to the invention, the combination of the casein phosphopeptides and the lactose-N-neotetraose can effectively improve the sleep problem, even can improve the anxiety state caused by the sleep problem, and the casein phosphopeptides and the lactose-N-neotetraose also have a synergistic interaction effect.

Description

Technical Field

[0001] The invention belongs to the field of food, and in particular relates to a nutritional composition and an application thereof in improving sleep. Background Art

[0002] Sleep is a restorative process that regulates brain states. It is reversible and homeostatic, embedded in circadian rhythms and sociophysiological organization, and involves species-specific resting postures, a degree of perceptual detachment, and an elevated arousal threshold. Sleep is when humans are most vulnerable and vulnerable, yet evolutionarily, humans still spend one-third of their lives sleeping, demonstrating the importance of sleep to human health. Scientific research shows that sleep contributes to the maintenance of the human brain, heart, gastrointestinal tract, systemic immunity, growth and development, and cell repair, making it extremely important.

[0003] Currently, solutions to sleep problems include pharmacotherapy and non-drug interventions. Drug therapy primarily utilizes medications such as benzodiazepines, non-benzodiazepine sedative-hypnotics, and melatonin receptor agonists to control sleep. However, these medications can have significant side effects, including cognitive side effects, withdrawal symptoms, and long-term safety concerns, as well as the risk of dependency. Non-drug interventions include behavioral interventions. For example, Cognitive Behavioral Therapy for Insomnia (CBT-I) is currently recognized internationally as the preferred first-line treatment for chronic insomnia. It is particularly effective for special populations such as pregnant women and the elderly who are concerned about medication dependency or side effects. However, this approach requires professional psychiatric guidance and has issues such as low accessibility, poor compliance, and a long treatment timeframe. Another non-drug intervention involves the use of nutritional supplements to improve sleep. For example, cited document 1 discloses a composition with the functions of improving sleep and anti-oxidation, which found that a specific combination of γ-aminobutyric acid, L-theanine, α-lactalbumin, casein phosphopeptide and lily extract has a strong synergistic antioxidant activity and a significant effect on sleep regulation; cited document 2 discloses a milk-derived polypeptide protein beverage that relieves stress and helps sleep, which contains raw milk, hydrolyzed casein powder, casein phosphopeptide, whey protein hydrolyzate, γ-aminobutyric acid, coconut powder, passion fruit concentrate, pear concentrate, and asparagus powder. Casein and whey protein are hydrolyzed to produce "opioid peptides," which exert anesthetic and analgesic effects, helping to relieve fatigue and induce sleep. Gamma-aminobutyric acid can inhibit or block excessive excitation of nerve cells, relax the nerves, increase the body's inhibitory neurotransmitters, and thoroughly relax the nerves, inducing natural sleep and creating a tranquil state of mind and body, which helps with falling asleep. Coconut powder, passion fruit, pear, asparagus powder, and natural herbal nutritional formulas participate in regulating sleep rhythms and over-arousal, reducing cortisol secretion levels, relieving stress, stabilizing emotions, and promoting pineal gland synthesis. However, currently common functional ingredients that improve sleep, such as melatonin, gamma-aminobutyric acid, L-theanine, magnesium, and some plant extracts (valerian extract, chamomile extract, etc.), are not suitable for people throughout their life cycle. For example, they may not be suitable for infants, children, or pregnant women, and some may even cause gastrointestinal discomfort. In addition, some of these functional ingredients are limited in source and are expensive.

[0004] Therefore, there is an urgent need in this field to develop a nutritional composition that is highly safe, suitable for use by people throughout their life cycle, has no side effects, and has a reliable source to improve sleep.

[0005] Bioactive peptides from bovine milk have antibacterial, antioxidant, antithrombotic, immunomodulatory, and mineral-binding properties in human health and physiology, and are considered important health-promoting ingredients in food and pharmaceutical applications. Casein phosphopeptides (CPPs) are a group of bioactive peptides involved in a variety of functional activities and are used worldwide as nutritional supplements, promoting mineral absorption, influencing bone growth, and enhancing immunity. As mentioned in References 1 and 2, while existing technologies have used casein phosphopeptides to prepare sleep-improving compositions, limited research has examined whether casein phosphopeptides themselves can improve sleep.

[0006] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk, after lactose and fat. Based on their molecular structure, HMOs can be divided into neutral HMOs and acidic HMOs. Neutral HMOs include neutral fucosylated HMOs and neutral non-fucosylated HMOs, while acidic HMOs are primarily acidic sialylated HMOs. Several reported physiological functions of neutral HMOs include regulating the intestinal microbiota, preventing pathogen adhesion, immunomodulating, strengthening the intestinal barrier, and providing antiviral effects. Studies have also shown that neutral HMOs can improve sleep. For example, Reference 3 discloses a composition for improving sleep maturation in non-infants, such as improving sleep difficulties. The composition comprises, consists of, or consists essentially of, an effective amount of one or more fucosylated HMOs, preferably neutral fucosylated HMOs. Neutral fucosylated HMOs may be supplemented with other human milk oligosaccharides, such as LNT and / or LNnT. Structurally, LNnT is an important core oligosaccharide in HMOs. Its specific structure is lactose as the reducing end, with N-acetyllactosamine linked to lactose via a β-1,3 glycosidic bond to form the core structure lactoyl-N-neotetraose. N-acetyllactosamine can be repeatedly extended to up to approximately 15 molecules, and both lactose and N-acetyllactosamine molecules can be modified with fucose or sialic acid groups. However, little research has been conducted on whether LNnT itself has a sleep-enhancing effect.

[0007] References:

[0008] Reference 1: CN114601919B;

[0009] Reference 2: CN111743001A;

[0010] Reference 3: CN116528695A. Summary of the Invention

[0011] Problems to be solved by the invention

[0012] Although there are currently methods of using nutritional supplements to intervene in sleep, common functional ingredients that improve sleep, such as melatonin, γ-aminobutyric acid, L-theanine, magnesium, and some plant extracts, are not suitable for people throughout their life cycle, and some functional ingredients can also cause side effects of gastrointestinal discomfort. Other ways to improve sleep also have their limitations. For example, drug treatments have large side effects, and behavioral interventions have low accessibility, poor compliance, and long time consumption. Therefore, there is still a demand for research and development of nutritional compositions that are safe, suitable for people throughout their life cycle, have no side effects, and are from reliable sources to improve sleep.

[0013] In this regard, the present inventors have conducted extensive research and unexpectedly discovered that the combination of casein phosphopeptide and lactose-N-neotetraose has a good effect on improving sleep, and even improves the anxiety state caused by sleep, and when the mass ratio of the two is within a certain range, the two have a synergistic effect.

[0014] Solutions for solving problems

[0015] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0016] [1] A nutritional composition, wherein the nutritional composition comprises the following essential active ingredients (I) and (II):

[0017] (I) casein phosphopeptides,

[0018] (II) neutral non-fucosylated human milk oligosaccharides;

[0019] Wherein, the neutral non-fucosylated human milk oligosaccharide contains at least lactose-N-neotetraose.

[0020] [2] The nutritional composition according to [1], wherein the neutral non-fucosylated human milk oligosaccharide further comprises lactose-N-tetraose.

[0021] [3] The nutritional composition according to [1] or [2], wherein the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose in the nutritional composition is 1:(0.1-15.0).

[0022] [4] Use of the nutritional composition according to any one of [1] to [3] in the preparation of a food that helps improve sleep and / or helps improve anxiety caused by sleep problems.

[0023] [5] The use according to [4], wherein the aiding in improving sleep includes any one or more of improving the number of awakenings, improving the duration of awakenings, improving the number of sleep bouts, and improving the duration of sleep bouts; and / or, the aiding in improving the anxiety state caused by sleep problems includes improving haptotaxis.

[0024] [6] The use according to [4] or [5], wherein the helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems includes improving the content of melatonin and / or γ-aminobutyric acid in brain tissue.

[0025] [7] The use according to any one of [4] to [6], wherein the method for improving sleep and / or improving anxiety caused by sleep problems comprises improving the content of N-methyl-D-aspartate receptors and / or cyclic adenosine monophosphate in nerve cells.

[0026] [8] The use according to any one of [4] to [7], wherein the food comprises any one or more of infant food, children's food, adolescent food, pregnant and lying-in women food, adult food and food for the middle-aged and elderly.

[0027] [9] The use according to any one of [4] to [8], wherein the food contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any acceptable excipients.

[0028]

[10] . The use according to any one of [4] to [9], wherein, in the food, based on the total dry matter content of the food, the content of casein phosphopeptide is 50-300 mg / 100 g, preferably 80-220 mg / 100 g, more preferably 130-181 mg / 100 g; and the content of lactose-N-neotetraose is 0.05-2 g / 100 g, preferably 0.1-1 g / 100 g, more preferably 0.20-0.5 g / 100 g.

[0029]

[11] Use of the nutritional composition according to any one of [1] to [3] in preparing a food having any one or more of the following effects (a) to (j):

[0030] (a) improving the amount of wakefulness activity, preferably reducing the amount of wakefulness activity;

[0031] (b) improving the number of awakenings, preferably reducing the number of awakenings;

[0032] (c) improving wakefulness time, preferably reducing wakefulness time;

[0033] (d) improving the number of sleep bouts, preferably increasing the number of sleep bouts;

[0034] (e) improving sleep bout duration, preferably increasing sleep bout duration;

[0035] (f) improving thigmotaxis, preferably reducing thigmotaxis;

[0036] (g) improving the content of melatonin in brain tissue, preferably increasing the content of melatonin in brain tissue;

[0037] (h) improving the content of gamma-aminobutyric acid in brain tissue, preferably increasing the content of gamma-aminobutyric acid in brain tissue;

[0038] (i) improving the content of N-methyl-D-aspartate receptors in nerve cells, preferably increasing the content of N-methyl-D-aspartate receptors in nerve cells;

[0039] (j) improving the cyclic adenosine monophosphate content in nerve cells, preferably increasing the cyclic adenosine monophosphate content in nerve cells.

[0040]

[12] The use according to

[11] , wherein the food comprises any one or more of infant food, children's food, adolescent food, maternal and infant food, adult food and food for the middle-aged and elderly.

[0041]

[13] The use according to

[11] or

[12] , wherein the food contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any acceptable excipients.

[0042]

[14] . The use according to any one of

[11] to

[13] , wherein, in the food, based on the total dry matter content of the food, the content of the casein phosphopeptide is 50-300 mg / 100 g, preferably 80-220 mg / 100 g, more preferably 130-181 mg / 100 g; and the content of the lactose-N-neotetraose is 0.05-2 g / 100 g, preferably 0.1-1 g / 100 g, more preferably 0.20-0.5 g / 100 g.

[0043] Effects of the Invention

[0044] The present invention proposes that a combination of casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose can effectively improve sleep problems and even improve the anxiety caused by these sleep problems. In particular, the nutritional composition provided by the present invention comprising casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose and the food added thereto or using the same can improve the number of awakenings, awakening time, number of sleep bouts and sleep bout time, improve sleep structure as a whole from multiple dimensions, and improve sleep quality. At the same time, the nutritional composition provided by the present invention comprising casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose and the food added thereto or using the same can improve the body's tactility and effectively alleviate anxiety caused by sleep problems.

[0045] Furthermore, the present invention studied physiological indicators related to sleep and found that the nutritional composition provided by the present invention comprising casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose and foods containing or using the same can improve the levels of various biochemical indicators related to sleep in the body, specifically including improving the levels of melatonin and γ-aminobutyric acid in brain tissue, and N-methyl-D-aspartate receptors and cyclic adenosine monophosphate in nerve cells.

[0046] Moreover, when the mass ratio of casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose is within a certain range, the two have a synergistic effect, especially in improving physiological indicators related to sleep. The synergistic effect between casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose is more obvious. The present invention unexpectedly discovered that neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose and casein phosphopeptides can significantly amplify each other's improvement effects on sleep problems and / or anxiety caused by sleep problems.

[0047] Furthermore, the casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides, such as lactose-N-neotetraose, in the nutritional composition provided by this invention have good safety and are suitable for use throughout the life cycle. They can be applied to a variety of products, including infant formula, nutritional supplements, health products, and nutritional supplements. Compared to behavioral therapy, improving sleep through dietary supplementation is easier to implement and is applicable to families of all economic levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Comparison of the improvement effects of CPP and LNnT in different ratios on zebrafish thigmotaxis. DETAILED DESCRIPTION

[0049] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0050] I. Definition of Terms

[0051] In the present invention, "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, and exclude additional, unrecited elements or method steps.

[0052] In the present invention, the meaning of "can" includes both performing a certain process and not performing a certain process.

[0053] In the present invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0054] In the present invention, the numerical ranges expressed as “value A to value B,” “value A - value B,” or “value A or more / less than value A” refer to ranges including the endpoints A and B.

[0055] Throughout the present invention, the term "about" is used to delineate numerical ranges and parameters of the present invention. Numerical values ​​in the specific embodiments are presented as accurately as possible. Unless otherwise expressly stated, all ranges, quantities, values, and percentages used herein are modified by the term "about." As used herein, "about" generally refers to the actual value being within ±5%, ±3%, ±1%, or ±0.5% of a particular value or range. Furthermore, the numerical values ​​and numerical ranges presented herein are to be understood as including the inevitable systematic errors encountered in industrial production.

[0056] Throughout the present disclosure, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," and the like mean that the particular elements (e.g., features, structures, properties, and / or characteristics) described in connection with that embodiment are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in the various embodiments.

[0057] In the present invention, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used represents weight or mass percentage.

[0058] In the present invention, "infants and young children" refers to a human group under 3 years old, including infants aged 0-6 months, older infants aged 6-12 months, and young children aged 12-36 months.

[0059] In the present invention, "children" refers to a human group aged 3-6 years.

[0060] In the present invention, "adolescents" refers to the human group aged 7-18 years old.

[0061] In the present invention, "pregnant women" include pregnant women and lactating women.

[0062] In the present invention, "middle-aged and elderly" refers to a human group over 41 years old.

[0063] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] II. Nutritional Composition

[0065] The nutritional composition provided by the present invention comprises the following essential active ingredients (I) and (II):

[0066] (I) casein phosphopeptides,

[0067] (II) neutral non-fucosylated human milk oligosaccharides;

[0068] Wherein, the neutral non-fucosylated human milk oligosaccharide contains at least lactose-N-neotetraose.

[0069] The casein phosphopeptide (CPP) described herein is a casein-derived peptide having at least one phosphoserine (SerP) residue. CPPs preferably contain at least one SerP per 20 amino acids, more preferably at least one SerP per 10 amino acids, or even 1-3 SerPs per 7 amino acids. CPPs preferably have a phosphorus content of 0.6 to 1.5% by mass. CPPs can be prepared by enzymatic hydrolysis of casein or caseinates, particularly whole casein, α-casein, κ-casein, or β-casein, using, for example, trypsin, pepsin, chymotrypsin, pancreatic enzymes, or bacterial (Bacillus), fungal, or plant endogenous and / or exoproteases, or mixtures thereof.

[0070] The lactose-N-neotetraose (LNnT) described in the present invention is a linear tetrasaccharide composed of D-galactose, N-acetyl-D-glucosamine, D-galactose and D-glucose, wherein the terminal D-galactose is connected to N-acetyl-D-glucosamine (GlcNAc) via a β-(1→4) bond, connected to D-galactose via a β-(1→3) bond, and connected to the reducing end D-glucose via a β-(1→4) bond. Its molecular formula is C 26 H 45 NO 21 The present invention does not place any particular limitation on the source of LNnT. Typically, it can be obtained by conventional chemical synthesis methods, microbial fermentation methods, etc. In addition, LNnT can also be derived from animal milk, such as cow's milk.

[0071] In some embodiments, the nutritional composition of the present invention comprises (I) casein phosphopeptide and (II) neutral non-fucosylated human milk oligosaccharide containing at least lactose-N-neotetraose as the main effective ingredients (active ingredients). That is, the nutritional composition of the present invention primarily relies on the (I) casein phosphopeptide and (II) neutral non-fucosylated human milk oligosaccharide containing at least lactose-N-neotetraose to exert specific physiologically active functions, such as helping to improve sleep and / or helping to improve anxiety caused by sleep problems. In other words, in some embodiments, the active ingredient of the nutritional composition (the ingredient used to exert a specific physiologically active function, i.e., the ingredient that helps to improve sleep and / or helps to improve anxiety caused by sleep problems) comprises the following components (I) and (II): (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide contains at least lactose-N-neotetraose.

[0072] The present invention found that compared with casein phosphopeptides and neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose alone, the nutritional composition provided by the present invention can more effectively help improve sleep and / or help improve the anxiety state caused by sleep problems, that is, there is a synergistic effect between the two.

[0073] In some embodiments, the neutral non-fucosylated human milk oligosaccharides included in the nutritional composition further comprise lactose-N-tetraose.

[0074] In other embodiments, the nutritional composition is substantially free of neutral non-fucosylated human milk oligosaccharides other than lacto-N-neotetraose.

[0075] In other embodiments, the nutritional composition is substantially free of human milk oligosaccharides other than lacto-N-neotetraose.

[0076] In some embodiments, in the nutritional composition described in the present invention, the (I) casein phosphopeptide and (II) lactose-N-neotetraose in neutral non-fucosylated human milk oligosaccharides are the main effective ingredients (active ingredients), that is, the nutritional composition described in the present invention mainly relies on the casein phosphopeptide and lactose-N-neotetraose contained therein to exert specific physiological active functions, such as helping to improve sleep and / or helping to improve anxiety caused by sleep problems.

[0077] In some embodiments, the nutritional composition comprises the following essential active ingredients (I) and (II): (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide is lactose-N-neotetraose.

[0078] In some embodiments, the active ingredient of the nutritional composition (an ingredient used to exert a specific physiologically active function, i.e., an ingredient that helps improve sleep and / or helps improve the anxiety state caused by sleep problems) is composed of the following components (I) and (II): (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide is lactose-N-neotetraose.

[0079] In some embodiments, the nutritional composition comprises an active ingredient (an ingredient used to exert a specific physiologically active function, i.e., an ingredient that contributes to improving sleep and / or improving anxiety caused by sleep problems) and an inactive ingredient (a substance that does not contribute to improving sleep and / or improving anxiety caused by sleep problems). Exemplarily, the inactive ingredient may be other nutrients, any food-acceptable excipient, and / or a substance produced during the production or acquisition of the active ingredient that cannot be effectively separated from or does not need to be separated from the active ingredient. In some embodiments, the nutritional composition consists of the active ingredient and the inactive ingredient.

[0080] In some embodiments, the nutritional composition consists of the following components (I) and (II): (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide is lactose-N-neotetraose.

[0081] In some specific embodiments, in the nutritional composition, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.1-15.0); for example, it can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1 , 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5. 2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7.0, 1:7.1, 1:7.2, 1:7 .3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8.0, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9.0, 1:9.1, 1:9.2, 1:9.3, 1: 9.4、1:9.5、1:9.6、1:9.7、1:9.8、1:9.9、1:10.0、1:10.1、1:10.2、1:10.3、1:10.4、1:10.5、1:10.6、1:10.7、1:10.8、1:10.9、1:11.0、1:11.1、1:11.2、 1:11.3, 1:11.4, 1:11.5, 1:11.6, 1:11.7, 1:11.8, 1:11.9, 1:12.0, 1:12.1, 1:12.2, 1:12.3, 1:12.4, 1:12.5, 1:12.6, 1:12.7, 1:12.8, 1:12.9, 1:13.0 , 1:13.1, 1:13.2, 1:13.3, 1:13.4, 1:13.5, 1:13.6, 1:13.7, 1:13.8, 1:13.9, 1:14.0, 1:14.1, 1:14.2, 1:14.3, 1:14.4, 1:14.5, 1:14.6, 1:14.7, 1:14.8, 1:14.9 or 1:15.0, etc.; preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.3-13.0); more preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.6-9.0); even more preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.8-7.0); further preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(1.0-3.0).

[0082] The present invention does not particularly limit the form of the nutritional composition, and typically, it can be liquid or solid, etc. From the perspective of production, transportation, storage and ease of use, the nutritional composition of the present invention is preferably a powdered solid.

[0083] III. Uses of the nutritional composition

[0084] The present invention proposes that compounding casein phosphopeptide with neutral non-fucosylated human milk oligosaccharides such as lactose-N-neotetraose in a certain ratio can help improve sleep and anxiety caused by sleep problems, and the two substances have a synergistic effect.

[0085] In some embodiments, the sleep problems include increased amount of awakening activity during sleep, more awakenings, longer awakening times, fewer sleep bouts, and / or shorter sleep bouts.

[0086] The method of helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems described in the present invention is not intended to treat or prevent diseases. At the same time, the sleep problems and the anxiety state caused by sleep problems described in the present invention have not reached the level that can be identified as diseases.

[0087] Based on this, the present invention provides use of the nutritional composition in preparing a food that helps improve sleep and / or helps improve anxiety caused by sleep problems.

[0088] In some embodiments, the helping to improve sleep includes any one or more of improving the amount of wakefulness, improving the number of wakefulnesses, improving the duration of wakefulness, improving the number of sleep bouts, and improving the duration of sleep bouts.

[0089] In some embodiments, the improving the amount of wakefulness activity is improving an increase in the amount of wakefulness activity.

[0090] In some embodiments, the improving the number of awakenings is improving an increase in the number of awakenings.

[0091] In some embodiments, the improving wakefulness time is improving an increase in wakefulness time.

[0092] In some embodiments, the improvement in the number of sleep bouts is an improvement in a decrease in the number of sleep bouts.

[0093] In some embodiments, the improvement in sleep bout time is an improvement in a decrease in sleep bout time.

[0094] In some embodiments, the helping to improve sleep includes improving the amount of wakefulness, improving the number of awakenings, improving the duration of awakenings, improving the number of sleep bouts, and improving the duration of sleep bouts.

[0095] In some embodiments, said helping to improve an anxious state caused by a sleep problem comprises improving haptotaxis.

[0096] In some embodiments, the helping to improve sleep and / or helping to improve anxiety caused by sleep problems comprises improving the content of melatonin and / or gamma-aminobutyric acid in brain tissue.

[0097] In some embodiments, the helping to improve sleep and / or helping to improve anxiety caused by sleep problems comprises improving the levels of melatonin and gamma-aminobutyric acid in brain tissue.

[0098] In some embodiments, the helping to improve sleep and / or helping to improve anxiety caused by sleep problems comprises improving the content of N-methyl-D-aspartate receptors and / or cyclic adenosine monophosphate in nerve cells.

[0099] In some embodiments, the helping to improve sleep and / or helping to improve anxiety caused by sleep problems comprises improving the levels of N-methyl-D-aspartate receptors and cyclic adenosine monophosphate in nerve cells.

[0100] In some embodiments, improving the melatonin content in brain tissue is improving the decrease in the melatonin content in brain tissue.

[0101] In some embodiments, improving the content of γ-aminobutyric acid in brain tissue is improving the decrease of the content of γ-aminobutyric acid in brain tissue.

[0102] In some embodiments, improving the content of N-methyl-D-aspartate receptors in nerve cells is improving the decrease of N-methyl-D-aspartate receptors in nerve cells.

[0103] In some embodiments, improving the content of cyclic AMP in nerve cells is improving the decrease of the content of cyclic AMP in nerve cells.

[0104] In some embodiments, the helping to improve sleep and / or helping to improve anxiety caused by sleep problems comprises improving the levels of melatonin and gamma-aminobutyric acid in brain tissue, and N-methyl-D-aspartate receptors and cyclic adenosine monophosphate in nerve cells.

[0105] The present invention does not particularly limit the specific food containing the above nutritional composition or the food that can be prepared using the above nutritional composition.

[0106] In some embodiments, under normal temperature conditions, the food of the present invention is in the form of liquid or solid.

[0107] In some embodiments, the food is infant food, children's food, adolescent food, pregnant and lying-in women food, adult food or food for the middle-aged and elderly.

[0108] In some embodiments, the food of the present invention is candy, such as hard candy, jelly candy, crisp candy, compressed candy, and aerated candy.

[0109] In some embodiments, the food of the present invention is a beverage, such as a carbonated beverage, a tea beverage, a coffee beverage, a fruit and vegetable juice beverage, and a lactic acid bacteria beverage.

[0110] In some embodiments, the food of the present invention is a dairy product, such as milk powder, cheese, yogurt, liquid milk, etc.

[0111] In some embodiments, the food of the present invention is a baked food, such as bread, cakes, and biscuits.

[0112] In some embodiments, the food of the present invention is a dietary supplement, such as a hard capsule, a soft capsule, a tablet, an oral solution, a pill, a granule, a powder, and the like.

[0113] In some embodiments, in the food described in the present invention, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.1-15.0); preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.3-13.0); more preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.6-9.0); even more preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.8-7.0); further preferably, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(1.0-3.0).

[0114] The present invention does not particularly limit the specific absolute content of the lactose-N-neotetraose and the casein phosphopeptide in the food, as long as the requirements of local food-related laws and regulations are met.

[0115] In some embodiments, in the food, based on the total dry matter content of the food, the content of the casein phosphopeptide is 50-300 mg / 100 g, preferably 80-220 mg / 100 g, and more preferably 130-181 mg / 100 g; the content of the lactose-N-neotetraose is 0.05-2 g / 100 g, preferably 0.1-1 g / 100 g, and more preferably 0.20-0.5 g / 100 g.

[0116] In addition to the components described above for the nutritional composition, the food may also contain other ingredients, such as proteins / amino acids, carbohydrates, fats, vitamins, minerals and other common food ingredients.

[0117] In addition, depending on the type of food and the final needs of the applicable object, in some embodiments, the food contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable excipients.

[0118] For plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica chinensis and bilberries or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts; and some botanical Chinese medicinal materials or their extracts that are both medicinal and edible.

[0119] Examples of animal dairy ingredients include fresh milk from cows, sheep, etc., as well as reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder.

[0120] Examples of animal meat product ingredients include meat product ingredients from pigs, cattle, sheep, aquatic products or poultry.

[0121] Functional added ingredients include vitamin supplements (such as vitamin A, β-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, biotin, etc.), mineral supplements (such as iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.), nucleotide supplements (such as), dietary fiber (such as inulin, konjac flour, galacto-oligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.), functional polyunsaturated fatty acid supplements (such as arachidonic acid oil powder, docosahexaenoic acid oil powder, etc.), etc.

[0122] As for any acceptable excipients, examples include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0123] The embodiments of the present invention will be described in detail below with reference to the examples and experimental examples. However, it will be understood by those skilled in the art that the following examples and experimental examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific conditions are not specified in the examples and experimental examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used were commercially available conventional products.

[0124] The experimental instruments used in the experimental examples of the present invention include: a zebrafish recirculating aquaculture system (ESEN-AW-S1, Beijing Aisheng Technology Development Co., Ltd.); an electronic balance (AR-2140, Shanghai Ohaus Instrument Co., Ltd.); an ultrapure water system (TC-RO-100, Shanghai Likang Instrument Co., Ltd.); a pH meter (PH9500, Shanghai Peirui Instrument Co., Ltd.); a conductivity meter (HM-100TDS, South Korea HM Digital Co., Ltd.); a thermometer (high precision, Odashi Technology Co., Ltd.); an ultra-low temperature refrigerator (HYCD-205, Qingdao Haier Co., Ltd.); a mini centrifuge (LX-500, Anhui Zhongke Co., Ltd.); and a zebrafish behavioral analysis system (DanioVision, Noldus, the Netherlands).

[0125] The experimental animals used in the experimental examples of the present invention include wild-type AB zebrafish, which were raised and maintained by the Center for Precision Nutrition and Active Health of Yantai University:

[0126] (1) Rearing environment: Adult zebrafish were housed in an Aisheng zebrafish recirculating aquaculture system with a 14 h / 10 h light / dark cycle. The water temperature was maintained at 28 ± 0.5°C, pH 7.0-8.0, and conductivity around 500 μs. Fresh brine shrimp eggs were fed twice daily.

[0127] (2) Zebrafish egg mating: After the adult zebrafish finish eating at night, they are placed on both sides of the mating box isolation plate in a ratio of 1:2 between male and female fish. The isolation plate is removed the next morning. After the male and female fish chase each other, they lay eggs and fertilize in vitro. After 0.5-1 hour, the adult fish are collected in the circulating water system and the date is marked. The embryos are collected using a filter and placed in a glass culture dish filled with E3 water. They are cultured at 28±0.5℃. Normal embryos aged 6 hours are selected for subsequent research on the mechanism of the nutritional composition regulating sleep and brain development.

[0128] Experimental Example 1: Study on the effect of nutritional composition on improving sleep disorders in zebrafish

[0129] Zebrafish developed insomnia symptoms after being induced by p-chlorophenylalanine. In this experiment, the improvement effects of casein phosphopeptide (CPP) (purchased from Guangzhou Green Extract Biotechnology Co., Ltd.) and lactose-N-neotetraose (LNnT) (purchased from DSM-Firmenich) at different doses alone or in combination on such insomnia symptoms were investigated.

[0130] 1. Experimental Methods

[0131] 1.1 Construction of sleep deprivation model and experimental grouping

[0132] Wild-type AB zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 per well. A blank control group (Comparative Example 1), a model group (Comparative Example 2), a positive control group (Comparative Example 3), and sample-treated groups (Comparative Examples 4-9 and Examples 1-9) were established. The blank control group was cultured normally with E3 water (no other treatments). The remaining groups were treated with 2 mM p-chlorophenylalanine and subsequently remained untreated, serving as the model group. The positive control group was treated with 150 μg / mL melatonin. Comparative Examples 4-9 were treated with low, medium, and high doses of CPP or LNnT, respectively. Examples 1-9 were treated with CPP and LNnT mixed in varying ratios. The specific experimental groupings are shown in Table 1.

[0133] Table 1 Design schemes for improving sleep with different ratios of nutrients

[0134]

[0135] 1.2 Evaluation of zebrafish locomotor behavior

[0136] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in a 6-well plate, with 30 zebrafish treated per well. Samples were dissolved in water and treated for 24 hours at 28°C. The treated juveniles were removed and placed one per well in a 96-well plate. The bottom light in the monitoring area was turned on, and the 96-well plate was placed in the behavioral monitoring area for 10 minutes to allow acclimatization. The camera was adjusted to capture the movement of each well. Once the movement of the zebrafish in each well was successfully captured, the behavioral trajectory of the zebrafish juveniles was monitored. Statistical results are expressed as mean ± SE.

[0137] (1) Autonomous activity: The activity level (total movement distance) of zebrafish in the awake state was measured using a behavioral analyzer within 60 min.

[0138] (2) Sleep structure: In zebrafish larvae, if the total activity time per minute is less than 0.1 seconds, they are considered to be in a sleep state; a continuous period of sleep is defined as a sleep bout. By analyzing the number of sleep bouts of zebrafish in each treatment group under light and dark conditions within 5 hours, we can understand the characteristics of their sleep structure and then evaluate their sleep quality.

[0139] 2. Experimental Results

[0140] (1) Results of zebrafish awakening activity (activity distance)

[0141] The experimental results are shown in Table 2. As can be seen from the table, the average awakening activity distance of normal zebrafish with normal sleep behavior without any treatment (Comparison Example 1) is about 2840.84 mm, while the awakening activity distance after modeling treatment (Comparison Example 2) is as high as 4694.89 mm. After one-way ANOVA difference significance analysis, it was found that the difference was extremely significant (p < 0.0001), which indicates that the modeling is successful. At the same time, we gave melatonin treatment (Comparison Example 3) as a positive control group and found that after melatonin intervention, the awakening activity distance of zebrafish decreased to about 2445.74 mm, which was significantly lower than that of the model group (Comparison Example 2) (p < 0.0001), and there was no significant difference with the normal control group (Comparison Example 1) (p > 0.05); Comparison Examples 4-6 are the awakening activity distances of zebrafish after CPP intervention at low, medium and high doses. The results showed that the values ​​were all reduced to about 3000 mm. After the difference significance analysis, it was found that the three comparison examples All were significantly lower than those in the model group (Comparison 2) (p < 0.0001). Comparisons 7-9 showed the awakening distances of zebrafish after low, medium, and high doses of LNnT. The results showed that the values ​​of the three comparisons ranged from approximately 4200 to 4400 mm, which were higher than those of CPP alone, but still lower than those of the model group. Analysis of significance revealed that comparison 7 was not significantly different from the model group (p > 0.05), while comparisons 8 (p < 0.05) and 9 (p < 0.05) were significantly lower than those of the model group. These results indicate that CPP alone can reduce the nocturnal distance of zebrafish, while LNnT at low doses had no significant effect, but at medium and high doses it did. When CPP and LNnT were combined in varying ratios, the combined treatment reduced the zebrafish's wake-activity distance to a range of 2400-2700 mm. Analysis of significance revealed that the treatment distances for all nine combined treatment groups were significantly lower than those in the model group (p < 0.0001) and showed no significant differences from the melatonin-treated positive control group (Comparative Example 3) (p > 0.05). This indicates that the combined treatments of CPP and LNnT in varying ratios (Examples 1-9) significantly improved the zebrafish's wake-activity distance compared to either substance alone (Comparative Examples 4-9), as confirmed by the results of the significance analysis, as shown in Table 3. This demonstrates that, at appropriate doses, CPP and LNnT alone can improve the wake-activity distance of sleep-disordered zebrafish, and their combination significantly amplifies the improvement achieved by each substance alone, demonstrating a synergistic effect. Furthermore, no significant differences were observed among the nine examples.

[0142] Table 2 Effects of different nutrients or combinations on the wakefulness activity of sleep-disordered zebrafish

[0143]

[0144] Table 3 Significance analysis of the effects of single nutrient and combined nutrient intervention on the arousal activity distance of zebrafish

[0145]

[0146]

[0147] (2) Results of zebrafish sleep structure investigation

[0148] The improvement of sleep structure by CPP and LNnT monomers or complexes of the two at varying ratios was evaluated using the number of nocturnal sleep bouts. The experimental results are shown in Table 4. The table shows that the average number of nocturnal sleep bouts in normal zebrafish is approximately 776 (Comparative Example 1). However, after modeling (Comparative Example 2), this number of nocturnal sleep bouts was significantly reduced to approximately 249 (p < 0.0001), indicating successful modeling. Melatonin intervention (Comparative Example 3) further significantly increased the number of nocturnal sleep bouts to approximately 829. Comparative Examples 4-6 show the number of sleep bouts in zebrafish after intervention with low, medium and high doses of CPP. It can be seen that the number of sleep bouts in zebrafish increased significantly with the increase of CPP concentration, and the number of sleep bouts in the three doses was significantly higher than that in the model group (Comparative Example 2) (p < 0.0001). Comparative Examples 7-9 show the number of sleep bouts in zebrafish after intervention with low, medium and high doses of LNnT. The mean values ​​were 371, 424 and 345, respectively, which were higher than those in the model group, but slightly worse than those in the neutral and high doses of CPP. Effects of high-dose intervention: Examples 1-9 show the number of sleep bouts in zebrafish treated with sleep-disordered zebrafish after treatment with CPP and LNnT in different ratios. Table 4 shows that all nine groups of zebrafish achieved over 800 sleep bouts, significantly higher than those in Comparative Examples 4-9. Specific significance analysis results are shown in Table 5. The combination of CPP and LNnT significantly amplifies the improvement in sleep bouts observed in sleep-disordered zebrafish treated with either CPP or LNnT alone, demonstrating a synergistic effect. Furthermore, a significance analysis revealed that Example 8 significantly improved sleep bouts compared to the melatonin-treated positive control group (Comparative Example 3). The sleep bout numbers in the remaining eight groups showed no significant differences (p>0.05). This suggests that the combination of CPP and LNnT significantly improves the number of sleep bouts in sleep-disordered zebrafish, with the most effective complex formed when CPP and LNnT were mixed at a ratio of 1:0.91.

[0149] Table 4 Effects of different nutrients or combinations on sleep structure in sleep-disordered zebrafish

[0150]

[0151] Table 5 Significance analysis of the improvement of zebrafish sleep bouts by intervention with single nutrients and combined nutrients

[0152]

[0153]

[0154] The above results show that CPP and LNnT have a significant effect on improving sleep disorders in the ratio range of 1:0.41-11, specifically by reducing the amount of wakeful activity in zebrafish and increasing the number of sleep bouts in zebrafish. In particular, Example 8, when CPP and LNnT are combined in a ratio of 1:0.91, has the best intervention effect.

[0155] Experimental Example 2: Study on the effect of the nutritional composition on improving the anxiety state caused by sleep disorders in zebrafish

[0156] 1. Experimental Methods

[0157] 1.1 Construction of sleep deprivation model and experimental grouping

[0158] Same as Experimental Example 1.

[0159] 1.2 Evaluation of zebrafish locomotor behavior

[0160] 4 dpf wild-type AB strain zebrafish were randomly selected and placed in a 6-well plate, with 30 zebrafish treated in each well. The samples were dissolved in water and treated at 28°C for 24 h. The treated juveniles were then removed.

[0161] Haptotaxis: A 12-well plate with a diameter of 24 mm was divided into a central zone (12 mm) and a total zone. One test fish was placed in each well and allowed to move freely within the central zone. Zebrafish larvae were first acclimated to darkness for 5 minutes in a behavioral observation system. The experiment was then conducted under 15-minute alternating light-dark cycles (5 minutes of darkness, 5 minutes of light, and 5 minutes of darkness). Haptotaxis was measured continuously for 15 minutes. The distance and time spent swimming between the two zones were calculated. Haptotaxis was measured as the percentage of the total distance covered in the central zone. Statistical results are expressed as mean ± SE.

[0162] 2. Experimental Results

[0163] Previous studies have shown that after experiencing sleep deprivation, people's perception and evaluation of their own and others' emotions often become negative, leading to anxiety-like behaviors. This experiment used haptotaxis to assess anxiety-like behaviors in zebrafish after sleep deprivation. Specifically, this was done by examining whether zebrafish spontaneously approached the edges of a container in a novel environment, reflecting their anxiety level.

[0164] In the experiment, each well of a 12-well plate was divided into two observation zones: a central zone and a peripheral zone. The central zone was half the area of ​​the entire well. In this experiment, the proportion of zebrafish's movement in the peripheral zone was used to reflect anxiety levels. The experimental results are shown in Table 6. As can be seen from the table, the proportion of zebrafish's movement in the peripheral zone in the normal control group (Comparative Example 1) was 61.70%, while the proportion of zebrafish's movement in the peripheral zone in the model group (Comparative Example 2) increased significantly to 92.05%, with a highly significant difference compared to the control group (p < 0.0001), indicating that the zebrafish in the model group developed severe haptotaxis. The haptotaxis of the positive control group (Comparative Example 3) treated with melatonin was significantly reduced to 64.95%, and there was a very significant difference with the model group (Comparative Example 2) (p < 0.0001), but there was no significant difference with the blank control group (Comparative Example 3) (p > 0.05); Comparative Examples 4-6 show the effects of low, medium and high doses of CPP on the haptotaxis of sleep-disordered zebrafish. From Table 6, it can be seen that the proportion of activity distance in the edge area of ​​the three groups after intervention was lower than that of the model group, and the proportion of activity distance in the edge area was reduced to between 75% and 90%. After the significant difference analysis, it was found that Except for comparison 4, which had no significant difference with the model group (p>0.05), comparisons 5 and 6 were significantly lower than the model group; comparisons 7-9 were the effects of LNnT on haptotaxis after intervention at low, medium and high doses. The results showed that the distance drop of zebrafish in the low-dose group (comparison 7) during edge activity was slightly lower than that of the model group, but after significant difference analysis, it was found that there was no significant difference with the model group (p>0.05), while after medium and high dose intervention, the mean distribution decreased to 79.01% and 72.48%, and after significant difference analysis, it was found that there were significant differences with the model group (p<0.0001). This shows that when CPP and LNnT intervened alone at low doses, there was no significant effect on improving the tactile behavior of zebrafish, but both medium and high doses produced a significant reducing effect; Examples 1-9 show the effects of LNnT combined with different ratios on the tactile behavior of sleep-disordered zebrafish. From Table 6, it can be seen that the intervention of the composition reduced the proportion of zebrafish's activity distance in the marginal area to varying degrees, and after a significant difference analysis with the model group, it was found that they were significantly lower than the model group (p < 0.0001); and after comparison, it was found that the composition was more effective than CPP and LNnT intervention alone. After a significant difference analysis, it was found that Examples 1-9 were significantly lower than Comparative Examples 4-9 (the results of the significant difference analysis are shown in Table 7), that is, the combination of CPP and LNnT can significantly amplify the improvement effect of a single CPP or LNnT on the tactile behavior of sleep-disordered zebrafish, and there is a synergistic effect between the two. The proportion of zebrafish's activity distance in the marginal area in the 9 examples was further compared, and the results are as follows: Figure 1As shown in the figure, Example 8 is significantly lower than the other 9 groups (p < 0.0001). This shows that the combination of CPP and LNnT at a ratio of 1:0.91 has the best effect in improving the haptotaxis of sleep-disordered zebrafish and suppressing the occurrence of anxiety and other problems.

[0165] Table 6 Effects of different nutrients or combinations on haptotaxis in sleep-disordered zebrafish

[0166]

[0167] Table 7 Significance analysis of the effects of single nutrients and combined nutrients on zebrafish haptotaxis

[0168]

[0169]

[0170] The above results show that CPP and LNnT have a significant effect on improving the anxiety state caused by sleep disorders in the ratio range of 1:0.41-11, which is specifically manifested by reducing the activity distance (histotaxis) of zebrafish in the peripheral area. In particular, Example 8, that is, when CPP and LNnT form a combination at a ratio of 1:0.91, the intervention effect is the best.

[0171] Experimental Example 3: Study on the mechanism of improvement of zebrafish sleep disorder by nutritional composition

[0172] 1. Experimental Methods

[0173] 1.1 Construction of sleep deprivation model and grouping

[0174] Same as Experimental Example 1.

[0175] 1.2 Biochemical index detection

[0176] Wild-type AB zebrafish (4 dpf) were randomly selected and plated in 6-well plates, with 30 zebrafish treated per well. The samples were dissolved in water and treated at 28°C for 24 hours. The zebrafish were then lysed in 0.01 M 1× PBS. The lysate containing the zebrafish tissue homogenate was collected and analyzed using ELISA kits for γ-aminobutyric acid (GABA), melatonin (MT), cyclic adenosine monophosphate (cAMP), and glutamate receptor (NMDAR) levels. The specific assay methods are as follows:

[0177] 1) Kit Name: Fish γ-Aminobutyric Acid (GABA) ELISA Detection Kit, Fish Melatonin (MT) ELISA Detection Kit, Fish Cyclic Adenosine Monophosphate (cAMP) ELISA Detection Kit, and Fish N-Methyl-D-Aspartate Receptor (NMDAR) ELISA Detection Kit.

[0178] 2) Sample Collection and Processing: Thirty zebrafish larvae were collected from each group and placed in a centrifuge tube. Residual liquid was aspirated and an appropriate amount of phosphate-buffered saline (PBS) was added at a ratio of 1:9 (w / v), i.e., 1 g of tissue + 9 mL of PBS, to obtain a 10% homogenate lysate. The lysate was then frozen at -20°C until further use.

[0179] 3) Experimental steps:

[0180] ① Take out the required microwell enzyme labeling strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, seal the remaining strips in a ziplock bag and return them to 4°C.

[0181] ② Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well;

[0182] ③ First add 10μL of the sample to be tested to the sample well, and then add 40μL of sample diluent; do not add anything to the blank well.

[0183] ④Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard and sample wells, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60 min.

[0184] ⑤ Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times (you can also use a plate washer to wash the plate).

[0185] ⑥ Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0186] ⑦ Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0187] 4) Result evaluation: Use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve for the standard. Calculate the concentration of each sample according to the curve equation.

[0188] 2. Experimental Results

[0189] (1) Changes in MT and GABA content in zebrafish tissues

[0190] Melatonin (MT) is an endogenous sleep regulator that has been shown to regulate sleep-wake rhythms and significantly improve sleep quality. Mechanistically, melatonin can regulate the circadian clock, provide a calming effect, and induce sleep by activating MT1 and MT2 receptors. Furthermore, melatonin levels during the day and night affect brain levels of gamma-aminobutyric acid (GABA), thereby influencing sleep regulation. Therefore, this experiment examined changes in MT and GABA levels in zebrafish tissues across various groups. The results are shown in Table 8.

[0191] As can be seen from Table 8, the average MT content in the brain tissue of normal zebrafish is approximately 168.86 pg / mL, but the MT content in the brain tissue of zebrafish with sleep disorders is significantly reduced to 101.14 pg / mL, which is significantly different from the blank control group (p < 0.001). This shows that MT is a susceptible indicator for evaluating sleep. Comparative Examples 4-6 show the MT content in zebrafish after intervention with low, medium, and high doses of CPP. As can be seen from Table 8, the MT content increases with the increase in CPP concentration, reaching 168.69 pg / mL at the high dose intervention. After analysis of significance, it was found that all of them were significantly higher than the model group (p < 0.0001). Comparative Examples 7-9 show the MT content in zebrafish after intervention with low, medium, and high doses of LNnT. As can be seen from the table, the MT content in the three groups ranged from 125-138 pg / mL, and after analysis of significance, it was found that all three groups were significantly higher than the model group (p < 0.005, p < 0.01, p < 0.0001); Examples 1-9 show the MT content in zebrafish after combined intervention with compositions formed by combining CPP and LNnT at different ratios. As shown in Table 8, the MT content in all samples was higher than 190 pg / mL, showing extremely significant differences compared to the model group (p < 0.0001). Compared with the control group, the effects of the compositions were significantly greater than those of the two monomers. That is, the combination of CPP and LNnT significantly amplified the improvement effect of either CPP or LNnT alone on the reduced MT content in sleep-disordered zebrafish. The specific results are shown in Table 9, indicating that CPP and LNnT have a synergistic effect. Further comparison of the MT content in each example revealed that the MT content in Example 8 reached a maximum of 211.14 pg / mL, indicating that the complex formed by combining CPP and LNnT at a ratio of 1:0.91 had the best effect in increasing MT content in zebrafish.

[0192] In the mammalian central nervous system, GABA's sleep-inducing mechanism is primarily achieved by regulating neuronal excitability. After binding to receptors on neurons, GABA can inhibit the firing rate of neurons, thereby producing a calming and soothing effect. Table 8 shows the differences in GABA content in the brain tissue of zebrafish in each group. As can be seen from Table 8, the average GABA content in the brain tissue of normal zebrafish is 21.04 μmol / L, but the GABA content in zebrafish with sleep disorders drops rapidly to 13.71 μmol / L, and this difference is significant (p < 0.0001), indicating that the model was successfully established and that GABA content in those with sleep disorders was significantly reduced. Comparative Examples 4-6 represent the GABA content in zebrafish after CPP intervention at low, medium and high doses. As can be seen from Table 8, with the increase of CPP intervention dose, GABA can reach nearly 20 μmol / L, and is significantly higher than that of the model group (p < 0.05, p < 0.001, p < 0.001); Comparative Examples 7-9 are the GABA levels in zebrafish after LNnT intervention at low, medium and high doses. As can be seen from the table, the values ​​are slightly lower than when CPP is intervened alone, but still higher than the model group, and the difference is significant (p < 0.001); Examples 1-9 are the effects of CPP and LNnT combined in different proportions to form a composition on the GABA content in zebrafish. The results show that the GABA content is increased to within the range of 24.09-27.02 μmol / L. After significance analysis, it was found that each group was significantly higher than the model group. The specific significance results are shown in Table 10. As can be seen from Table 10, the combination of CPP and LNnT significantly amplifies the improvement effect of either CPP or LNnT alone on the reduced GABA content in sleep-disordered zebrafish, demonstrating a synergistic effect. In particular, in Example 8, GABA content in zebrafish was significantly increased to 27.02 μmol / L.

[0193] Table 8 Comparison of MT and GABA contents in zebrafish nerve tissues of each group

[0194]

[0195] Table 9 Analysis of the significance of the difference in MT content in zebrafish between the comparative example and the example

[0196]

[0197]

[0198] Table 10 Analysis of the significance of the difference in GABA content in zebrafish between the comparative example and the example

[0199]

[0200]

[0201] (2) Changes in NMDAR and cAMP levels in zebrafish tissues

[0202] NMDA receptors (NMDARs) are known to be receptors for the excitatory neurotransmitter glutamate, and D-serine assists glutamate in activating NMDA receptors. A common assumption is that excitatory neurons should cause wakefulness, while inhibitory neurons should cause sleep. However, a 2019 postdoctoral research paper by Rao Yi's laboratory at the Beijing Center for Brain Science and Brain-Inspired Research and Peking University's McGovern Institute for Brain Research, titled "D-Serine made by serine racemase in Drosophila intestine plays a physiological role in sleep," demonstrated that D-serine promotes sleep through NMDA receptors, clearly demonstrating that the molecular and cellular mechanisms of sleep contradict this common belief. This study also found that the level of NMDAR in the nerve cells of normal zebrafish (Comparative Example 1) was approximately 1397.87 μg / mL (the experimental results are shown in Table 11). However, after modeling (Comparative Example 2), the level of NMDAR in the nerve cells of zebrafish with sleep disorders dropped directly to 854.59 μg / mL, which was significantly different from the normal control group (Comparative Example 1) (p < 0.001), indicating that the modeling was successful and that the NMDAR level in zebrafish decreased significantly with the disturbance of sleep disorders. Comparative Examples 4-6 show the NMDAR levels in zebrafish neurons after CPP intervention at low, medium and high doses. As can be seen from the table, they are all improved compared with the model group. The NMDAR levels are 1096-1249 μg / mL, and after significant difference analysis, it is found that they are significantly improved compared with the model group (p < 0.05, p < 0.001, p < 0.0001); Comparative Examples 7-9 are the NMDAR levels in zebrafish neurons after LNnT intervention at low, medium and high doses. As can be seen from Table 11, the values ​​are all lower than when CPP is used alone, but higher than the model group. After significant difference analysis, there is no significant difference between LNnT low and medium doses and the model group (p > 0.05), and the high dose intervention is significantly higher than the model group (p < 0.001). This shows that CPP intervention alone has the effect of increasing NMDAR levels in zebrafish neurons, and LNnT has this effect at high doses. Examples 1-9 show the effects of combined intervention with CPP and LNnT in different proportions to form a composition on NMDAR levels in zebrafish neurons. The results show that the combined intervention is significantly superior to either single intervention (Examples 1-9 are significantly higher than Comparative Examples 4-9). The specific significance analysis of the differences is shown in Table 12. That is, the combination of CPP and LNnT can significantly amplify the improvement effect of either CPP or LNnT alone on NMDAR levels in zebrafish neurons with sleep disorders, and the two have a synergistic effect.Further comparison of the nine examples revealed that the NMDAR level in Example 8 could reach 1536.5 μg / mL, which had the best effect of increasing the NMDAR level. Furthermore, after significance analysis, Example 8 was found to be significantly higher than Example 2 (p < 0.001), Example 3 (p < 0.01), Example 4 (p < 0.01), Example 5 (p < 0.001) and Example 6 (p < 0.05).

[0203] Cyclic adenosine monophosphate (cAMP, or cyclic AMP, 3'-5'-cyclic adenosine monophosphate) regulates BDNF-induced TrkB phosphorylation, promoting anxiolysis and enhancing sleep through the cAMP / PKA-CREB-BDNF signaling pathway. This experiment found that the cAMP level in neurons of normal zebrafish (Comparative Example 1) was 21.83 nmol / L. However, the cAMP level in neurons of zebrafish modeled with sleep disorders (Comparative Example 2) rapidly decreased to 14.14 nmol / L, a highly significant difference from the control group (Comparative Example 1) (p < 0.0001). This indicates that cAMP content in neurons decreases significantly with the onset of sleep disorders. Comparative Examples 4-6 are the levels of cAMP in zebrafish nerve cells after intervention with CPP at low, medium and high doses (the results are shown in Table 11). The cAMP content was significantly increased, and there was a trend of increasing with the increase of CPP intervention dose. After significant difference analysis, it was found that only the medium and high doses were significantly higher than the model group (p < 0.0001), which shows that CPP intervention alone is effective in increasing cAMP in zebrafish nerve cells with sleep disorders only at medium and high doses, and there is no significant increase at low doses; Comparative Examples 7-9 are the cAMP content in zebrafish nerve cells after intervention with LNnT at low, medium and high doses. The results showed that the cAMP content was higher than the model group, but after significant difference analysis, it was also found that only the medium and high doses had significant differences with the model group (p < 0.05; p < 0.0001), and the cAMP value at low dose was not significantly different from that of the model group (p > 0.05). Examples 1-9 demonstrate the effects of CPP and LNnT combined in varying proportions on cAMP levels in zebrafish with sleep disorders. Table 11 shows that cAMP levels were all above 24 nmol / L after intervention with the combination. Analysis of significance revealed that all nine examples were significantly higher than the model group (p < 0.0001) and significantly higher than comparative examples 4-9. This suggests that the combination of CPP and LNnT significantly amplifies the improvement in cAMP levels in zebrafish neurons caused by either CPP or LNnT alone, demonstrating a synergistic effect. The specific significance of the differences is shown in Table 13. Further comparative analysis of the nine examples revealed that Example 8 achieved the best cAMP level, reaching 28.44 nmol / L.

[0204] Table 11 Comparison of NMDAR and cAMP levels in zebrafish neurons of each group

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[0206] Table 12 Analysis of the significance of differences in NMDAR levels in zebrafish neurons between the comparative examples and the examples

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[0209] Table 13 Analysis of the significance of the difference in cAMP levels in zebrafish nerve cells between the comparative examples and the examples

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[0211]

[0212] The above results show that the composition formed by the combination of CPP and LNnT can improve sleep. After intervention with the composition formed by the combination of CPP and LNnT, the content levels of MT and GABA, key neural factors mediating sleep, in brain tissue were significantly increased. From a mechanistic perspective, it was found that the composition can improve sleep by activating the excitatory neurotransmitter receptor NMDAR in the nervous system and regulating the cAMP / PKA-CREB-BDNF signaling pathway. In particular, in Example 8, the composition formed when CPP and LNnT were combined at a ratio of 1:0.91 had the best effect.

Claims

1. Use of a nutritional composition in preparing a food that helps improve sleep and / or helps improve anxiety caused by sleep problems; the nutritional composition comprises the following essential active ingredients (I) and (II): (I) casein phosphopeptides, (II) neutral non-fucosylated human milk oligosaccharides; in, The neutral non-fucosylated human milk oligosaccharide contains at least lactose-N-neotetraose; Furthermore, in the nutritional composition, the mass ratio of the casein phosphopeptide to the lactose-N-neotetraose is 1:(0.1-15.0).

2. The use according to claim 1, characterized in that The neutral non-fucosylated human milk oligosaccharides also contain lactose-N-tetraose.

3. The use according to claim 1, characterized in that The helping to improve sleep includes any one or more of improving the amount of wakefulness activity, improving the number of wakefulnesses, improving the wakefulness time, improving the number of sleep bouts, and improving the sleep bout time.

4. The use according to claim 1, characterized in that The helping to improve the anxiety state caused by sleep problems includes improving haptotaxis.

5. The use according to claim 1, characterized in that The helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems includes improving the content of melatonin and / or gamma-aminobutyric acid in brain tissue.

6. The use according to claim 1, characterized in that The method of helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems includes improving the content of N-methyl-D-aspartate receptors and / or cyclic adenosine monophosphate in nerve cells.

7. The use according to any one of claims 1 to 6, characterized in that The food includes any one or more of infant food, children's food, adolescent food, pregnant and lying-in women food, adult food and middle-aged and elderly food.

8. The use according to any one of claims 1 to 6, characterized in that The food contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable auxiliary materials.

9. The use according to any one of claims 1 to 6, characterized in that In the food, based on the total dry matter content of the food, the content of the casein phosphopeptide is 50-300 mg / 100 g, and the content of the lactose-N-neotetraose is 0.05-2 g / 100 g.

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