Nutritional composition and its use for improving sleep

Abstract

The present application belongs to the field of food, and particularly relates to a nutritional composition and its use for improving sleep. The present application provides a nutritional composition comprising essential active ingredients as shown in (I) and (II) below: (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide at least comprises lacto-N-neotetraose, and in the nutritional composition, the mass ratio of the casein phosphopeptide to the lacto-N-neotetraose is 1:(0.1-15.0). The present application proposes that the combination of casein phosphopeptide and lacto-N-neotetraose can effectively improve sleep problems, and even can improve the anxiety state caused by these sleep problems, and the two have a synergistic effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food, in particular to a nutritional composition and its use for improving sleep. BACKGROUND

[0002] Sleep is a restorative process of brain state regulation, reversible and homeostatic, embedded in circadian and socio-physiological organization, involving species-specific postures of quiescence, some degree of perceptual disengagement, and an elevated threshold for arousal. The process of sleep is the most vulnerable time for humans, the moment when they are most susceptible to attack, but from the evolutionary process, humans still spend one-third of their lives sleeping, which shows that sleep is of great significance to human health. Scientific research shows that sleep helps maintain the functions of the human brain, heart, gastrointestinal tract, body immunity, growth and development, and cell repair, so sleep is extremely important.

[0003] Currently, the solutions to sleep problems include drug therapy and non-drug intervention. Drug therapy mainly uses drugs such as benzodiazepines, non-benzodiazepine sedative hypnotics, and melatonin receptor agonists to control sleep, but the side effects of drugs are large, which can cause cognitive side effects, drug withdrawal reactions, and long-term safety problems, and there is a risk of dependence. For non-drug intervention, one is behavioral intervention, for example, cognitive behavioral therapy for insomnia (CBT-I) is currently internationally recognized as the preferred first-line treatment for chronic insomnia, and it is effective for special groups such as pregnant women and the elderly who are concerned about drug dependence or side effects, but this method requires the guidance of professional psychologists, and there are problems such as low accessibility, poor compliance, and long time-consuming; another non-drug intervention is to use sleep-improving nutritional supplements for intervention. For example, reference document 1 discloses a composition for improving sleep and antioxidant, which finds that a specific combination of gamma-aminobutyric acid, L-theanine, alpha-lactalbumin, casein phosphopeptide, and lily extract has strong synergistic antioxidant activity and significant sleep regulation effect; reference document 2 discloses a milk-derived polypeptide protein beverage for relieving stress and helping sleep, which contains raw cow milk, hydrolyzed casein powder, casein phosphopeptide, whey protein hydrolysate, gamma-aminobutyric acid, coconut powder, passion fruit concentrate, pear concentrate, and asparagus powder. The casein and whey protein are hydrolyzed to generate "opioid-like peptides", which play a role in anesthesia and analgesia, helping to relieve fatigue and fall asleep. Gamma-aminobutyric acid can inhibit or block the overexcitation of nerve cells, relax the nerves, increase the inhibitory neurotransmitters in the human body, completely relax the nerves, and induce natural sleep in a state of body and mind. Coconut powder, passion fruit, pear, asparagus powder, and natural herbal nutritional formula participate in the regulation of sleep rhythm and excessive wakefulness, reduce the secretion of cortisol, relieve mental stress, stabilize mood, and promote the synthesis of pineal indoles. However, the functional ingredients commonly used to improve sleep, such as melatonin, gamma-aminobutyric acid, L-theanine, magnesium, and some plant extracts (valerian extract, chamomile extract, etc.), are not suitable for the whole life cycle of the population. For example, they may not be suitable for infants, children, or pregnant women, and some may cause gastrointestinal side effects. In addition, the sources of some of these functional ingredients are limited and expensive.

[0004] Therefore, there is an urgent need in the art to develop a safe, reliable source of nutritional composition that can improve sleep for the whole life cycle of the population without side effects.

[0005] Bioactive peptides in bovine milk can play a role in human health and physiology, such as antibacterial, antioxidant, antithrombotic, immunomodulatory and mineral binding, and are considered important ingredients for promoting health in food or pharmaceutical applications. Casein phosphopeptides (CPP) are a group of bioactive peptides involved in various functional roles, and are used as nutritional supplements worldwide, with effects such as promoting mineral absorption, affecting bone growth, and enhancing immunity. As cited in references 1 and 2 above, although existing technologies use casein phosphopeptides to prepare compositions with sleep improvement effects, there is little research on whether casein phosphopeptides themselves have sleep improvement effects.

[0006] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and fat. According to the molecular structure characteristics, HMOs can be divided into neutral HMOs and acidic HMOs. Neutral HMOs include neutral fucosylated HMOs, neutral non-fucosylated HMOs, and acidic HMOs are mainly acidic sialylated HMOs. As for the physiological functions of several neutral HMOs, the reported functions include regulating intestinal microbiota, preventing pathogen adhesion, immunomodulation, enhancing intestinal barrier and antiviral effects, and some studies have found that neutral HMOs have sleep improvement effects. For example, reference 3 discloses a composition for improving sleep maturation in non-infants, such as improving sleep difficulties, which comprises, consists of or consists essentially of an effective amount of one or more fucosylated HMOs, preferably neutral fucosylated HMOs, which can be supplemented with other human milk oligosaccharides, such as LNT and / or LNnT. Structurally, LNnT is an important core oligosaccharide in HMOs, and its specific structure is: lactose as the reducing end, N-acetyl lactosamine is connected to lactose by a β-1,3 glycosidic bond to obtain the core structure lactosyl-N-neotetraose. The N-acetyl lactosamine can be repeatedly extended to a maximum of about 15 molecules, and the lactose and N-acetyl lactosamine molecules can be modified by fucose or sialic acid groups. However, there is little research on whether LNnT itself has sleep improvement effects.

[0007] Reference:

[0008] Reference 1: CN114601919B;

[0009] Reference 2: CN111743001A;

[0010] Reference 3: CN116528695A. SUMMARY

[0011] Problems to be solved by the invention

[0012] Although there are currently methods of intervening in sleep using nutritional supplements, common functional ingredients that have an improving effect on sleep such as melatonin, gamma-aminobutyric acid, L-theanine, magnesium and some plant extracts are not suitable for the whole life cycle population, and some functional ingredients also have side effects of gastrointestinal discomfort. Other ways to improve sleep also have limitations, for example, drug therapy has the problem of large side effects, and behavioral intervention has the problems of low accessibility, poor compliance, and long time-consuming. Therefore, there is still a need for research and development of nutritional compositions that can improve sleep with high safety, suitable for the whole life cycle population, no side effects, and reliable sources in the current market.

[0013] To this end, the present application has conducted a large number of studies and unexpectedly found that the combination of casein phosphopeptide and lacto-N-neotetraose has a good improving effect on sleep, and even an improving effect on 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] Solution to the problem

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

[0016] [1]. A nutritional composition, wherein the nutritional composition comprises the necessary active ingredients shown as (I) and (II) below:

[0017] (I) casein phosphopeptide,

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

[0019] Wherein the neutral non-fucosylated human milk oligosaccharide at least comprises lacto-N-neotetraose.

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

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

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

[0023] [5]. The use according to [4], wherein the improvement of sleep includes any one or more of improvement of number of awakenings, improvement of time of awakenings, improvement of number of sleep bouts, and improvement of time of sleep bouts; and / or, the improvement of anxiety state caused by sleep problems includes improvement of tactile approach.

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

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

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

[0027] [9]. The use according to any one of [4] to [8], wherein the food contains any one or more of plant product ingredient, animal milk product ingredient, animal meat product ingredient, functional additive ingredient, and any acceptable adjuvant.

[0028]

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

[0029]

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

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

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

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

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

[0034] (e) improving the duration of sleep bouts, preferably increasing the duration of sleep bouts;

[0035] (f) improving the thigmotaxis, preferably decreasing the 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 neural cells, preferably increasing the content of N-methyl-D-aspartate receptors in neural cells;

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

[0040]

[12] . The use according to

[11] , wherein the food product comprises any one or more of infant food, children food, adolescent food, pregnant and lactating women food, adult food, and elderly food.

[0041]

[13] . The use according to

[11] or

[12] , wherein the food product comprises any one or more of plant product ingredients, animal milk product ingredients, animal meat product ingredients, functional additive ingredients, and any acceptable adjuvants.

[0042]

[14] . The use according to any one of

[11] to

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

[0043] Effects of the invention

[0044] The present application provides that the combination of casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose can effectively improve sleep problems, and even can improve the anxiety state caused by these sleep problems. In particular, the nutritional composition comprising casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose provided by the present application and the food added with or using the same can improve the number of awakenings, the time of awakenings, the number of sleep rounds and the time of sleep rounds, improve the sleep structure from multiple dimensions, and improve the sleep quality. At the same time, the nutritional composition comprising casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose provided by the present application and the food added with or using the same can improve the body's hedonicity and effectively relieve the anxiety caused by sleep problems.

[0045] Further, the present application studies the physiological indicators related to sleep, and finds that the nutritional composition comprising casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose provided by the present application and the food added with or using the same can improve the levels of various biochemical indicators related to sleep in the body, specifically including improving the contents of melatonin and gamma-aminobutyric acid in brain tissue, and the contents of N-methyl-D-aspartate receptor and cyclic adenosine monophosphate in nerve cells.

[0046] And, when the mass ratio of casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose is within a certain range, the two have a synergistic effect. Especially in improving the physiological indicators related to sleep, the synergistic effect between casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose is more obvious. The present application unexpectedly finds that neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose and casein phosphopeptide can significantly amplify each other's improvement effect on sleep problems and / or anxiety state caused by sleep problems.

[0047] In addition, the casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lacto-N-neotetraose in the nutritional composition provided by the present application both have good safety, and can be suitable for use by people of all life stages, which can be applied to infant formula, nutritional supplements, health products, nutritional products and other forms of products. Compared with behavior therapy, improving sleep through dietary supplementation is easier to operate, and can be applied to families of various economic levels. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 : Comparison of improvement effects of CPP and LNnT in different proportions on the hedonicity of zebrafish after forming a composition. DETAILED DESCRIPTION

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

[0050] I. Definition of Terms

[0051] In the present application, "comprise", "have", "include" or "contain" can mean inclusive or open-ended and do not exclude additional, unrecited elements or method steps. At the same time, "comprise", "have", "include" or "contain" can also mean closed- ended, excluding additional, unrecited elements or method steps.

[0052] In the present application, "may" indicates both the meaning of performing a certain process and the meaning of not performing a certain process.

[0053] In the present application, "optional" or "optionally" indicates the use or non-use of certain substances, components, execution steps, applied conditions, and the like.

[0054] In the present application, "value A~value B", "value A-value B", "value A or more / less" indicates a numerical range including the end point values A and B.

[0055] In the present application, "about" is used to define the numerical range and parameters of the present application as approximate numerical values, and the relevant numerical values in the specific examples have been presented as accurately as possible. Unless otherwise explicitly stated, it should be understood that all ranges, numbers, numerical values and percentages used in the present application are modified by "about". Here, "about" generally means that the actual numerical value is within ±5%, ±3%, ±1% or ±0.5% of a certain numerical value or range. And the numerical values and numerical ranges appearing in the present application should be understood to include the systematic errors that are unavoidable in industrial production.

[0056] In the present application, "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", and the like mean that the specific elements (for example, features, structures, properties, and / or characteristics) described in relation to the embodiments are included in at least one embodiment described herein, and can be present in other embodiments or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0057] In the present application, the unit names used are international standard unit names, and if not specifically stated, "%" used means weight or mass percentage content.

[0058] In the present application, "infants" means the group of humans up to 3 years of age, wherein it includes infants from 0 to 6 months of age, older infants from 6 to 12 months of age and toddlers from 12 to 36 months of age.

[0059] In the present application, "children" means the group of humans from 3 to 6 years of age.

[0060] In the present application, "adolescents" means the group of humans from 7 to 18 years of age.

[0061] In the present application, "pregnant and lactating women" includes women in gestation and women in lactation.

[0062] In the present application, "elderly" means the group of humans over 41 years of age.

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

[0064] II. Nutritional composition

[0065] The nutritional composition provided in the present application comprises essential active ingredients as shown in (I) and (II) below:

[0066] (I) casein phosphopeptide,

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

[0068] wherein the neutral non-fucosylated human milk oligosaccharide comprises at least lacto-N-neotetraose.

[0069] The casein phosphopeptide (CPP) described in the present application is a casein-derived peptide having at least one phospho-serine (SerP) residue. The CPP preferably comprises at least one SerP per 20 amino acids, more preferably at least one SerP per 10 amino acids or even 1-3 SerP per 7 amino acids. The CPP preferably has a phosphorus content of 0.6 to 1.5 mass%. The CPP can be prepared by enzymatic hydrolysis of casein or caseinates, in particular of whole casein, alpha-casein, K-casein or beta-casein, using, for example, trypsin, pepsin, chymotrypsin, pancreatin or bacterial (Bacillus), fungal or plant endogenous and / or exo-proteases or mixtures thereof.

[0070] The lactose-N-neotetraose (LNnT) of the present application is a linear tetrasaccharide consisting of D-galactose, N-acetyl-D-glucosamine, D-galactose and D-glucose, the terminal D-galactose is connected to N-acetyl-D-glucosamine (GlcNAc) by a β-(1→4) bond, to D-galactose by a β-(1→3) bond, and to the reducing end D-glucose by a β-(1→4) bond. Its molecular formula is C 26 H 45 NO 21 The source of LNnT of the present application is not particularly limited, typically, it can be obtained by ordinary chemical synthesis method, microbial fermentation method, etc. In addition, LNnT can also be derived from animal milk such as cow milk, etc.

[0071] In some embodiments, in the nutritional composition of the present application, the (I) casein phosphopeptide and (II) neutral non-fucosylated human milk oligosaccharide containing at least lactose-N-neotetraose are the main active ingredients (active ingredients), that is, the nutritional composition of the present application mainly relies on the (I) casein phosphopeptide and (II) neutral non-fucosylated human milk oligosaccharide containing at least lactose-N-neotetraose to exert specific physiological activity functions, for example, to help improve sleep and / or help improve anxiety state caused by sleep problems. In other words, in some embodiments, the active ingredients (ingredients for exerting specific physiological activity functions, i.e. ingredients for exerting the effect of helping to improve sleep and / or helping to improve anxiety state caused by sleep problems) of the nutritional composition consist of the following (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 application finds that, compared with casein phosphopeptide and neutral non-fucosylated human milk oligosaccharide such as lactose-N-neotetraose alone, the nutritional composition provided by the present application can more effectively help improve sleep and / or help improve 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 oligosaccharide contained in the nutritional composition also contains lactose-N-tetraose.

[0074] In other embodiments, the nutritional composition contains substantially no neutral non-fucosylated human milk oligosaccharide other than lactose-N-neotetraose.

[0075] In other embodiments, the nutritional composition contains substantially no human milk oligosaccharide other than lactose-N-neotetraose.

[0076] In some embodiments, in the nutritional composition described in the present application, the lacto-N-neotetraose in the (I) casein phosphopeptide and (II) neutral non-fucosylated human milk oligosaccharide is the main effective component (active ingredient), that is, the nutritional composition described in the present application mainly relies on the casein phosphopeptide and lacto-N-neotetraose contained therein to exert a specific physiological activity function, for example, to help improve sleep and / or help improve the anxiety state caused by sleep problems.

[0077] In some embodiments, the nutritional composition contains the necessary active ingredients shown in (I) and (II) below: (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide is lacto-N-neotetraose.

[0078] In some embodiments, the active ingredients (ingredients for exerting a specific physiological activity function, i.e., ingredients for exerting the effect of helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems) of the nutritional composition consist of the components shown in (I) and (II) below: (I) casein phosphopeptide, (II) neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide is lacto-N-neotetraose.

[0079] In some embodiments, the nutritional composition contains active ingredients (ingredients for exerting a specific physiological activity function, i.e., ingredients for exerting the effect of helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems) and non-active ingredients (substances that do not exert the effect of helping to improve sleep and / or helping to improve the anxiety state caused by sleep problems). For example, the non-active ingredients can be other nutrients, any food acceptable adjuvant, and / or substances that are produced in the production or acquisition process of the active ingredients and cannot be effectively separated from the active ingredients or do not need to be separated. In some embodiments, the nutritional composition consists of the active ingredients and the non-active ingredients.

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

[0081] In some specific embodiments, in the nutritional composition, the mass ratio of the casein phosphopeptide to the lacto-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, 1 :15.0.8、1:14.9 or 1:15.0, etc.; preferably, the mass ratio of the casein phosphopeptide to the lacto-N-neotetraose is 1:(0.3-13.0); more preferably, the mass ratio of the casein phosphopeptide to the lacto-N-neotetraose is 1:(0.6-9.0); even more preferably, the mass ratio of the casein phosphopeptide to the lacto-N-neotetraose is 1:(0.8-7.0); further preferably, the mass ratio of the casein phosphopeptide to the lacto-N-neotetraose is 1:(1.0-3.0).

[0082] The present application does not make any particular limitation on the form of the nutritional composition, which can typically be a liquid or a solid, etc. From the perspective of production, transportation, storage and use convenience, the nutritional composition of the present application is preferably a powdered solid.

[0083] III. Use of the nutritional composition

[0084] The present application proposes that the casein phosphopeptide and the neutral non-fucosylated breast milk oligosaccharide such as lacto-N-neotetraose are compounded in a certain ratio, which can help to improve sleep and can help to improve the anxiety state caused by sleep problems, and there is a synergistic effect between the two substances.

[0085] In some embodiments, the sleep problems include an increase in the amount of wakeful activity during sleep, an increase in the number of awakenings, an increase in the time of awakenings, a decrease in the number of sleep bouts, and / or a decrease in the time of sleep bouts, etc.

[0086] The present application does not aim to treat or prevent diseases, and at the same time, the sleep problems and the anxiety state caused by sleep problems of the present application have not reached the level that can be recognized as diseases.

[0087] Based on this, the present application provides the use of the above-mentioned nutritional composition in the preparation of a foodstuff that helps to improve sleep and / or helps to improve the anxiety state caused by sleep problems.

[0088] In some embodiments, the improvement in sleep includes any one or more of an improvement in the amount of wakeful activity, an improvement in the number of awakenings, an improvement in the time of awakenings, an improvement in the number of sleep bouts, and an improvement in the time of sleep bouts.

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

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

[0091] In some embodiments, the improvement in the time of awakenings is an increase in the time of awakenings.

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

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

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

[0095] In some embodiments, the helping to improve the state of anxiety caused by sleep problems includes improving the tendency to touch.

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

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

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

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

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

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

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

[0104] In some embodiments, the improvement of sleep and / or the improvement of anxiety state caused by sleep problems includes the improvement of the content of melatonin and gamma-aminobutyric acid in brain tissue, and the content of N-methyl-D-aspartate receptor and cyclic adenosine monophosphate in nerve cells.

[0105] The present application is not particularly limited to specific food products containing or prepared using the nutritional composition described above.

[0106] In some embodiments, the food product described in the present application is in a liquid or solid form at room temperature.

[0107] In some embodiments, the food product described in the present application is a baby food, a child food, an adolescent food, a pregnant woman food, an adult food, or a senior citizen food.

[0108] In some embodiments, the food product described in the present application is a confectionery, such as a hard candy, a gel candy, a crisp candy, a pressed candy, an aerated candy, and the like.

[0109] In some embodiments, the food product described in the present application is a beverage, such as a carbonated beverage, a tea beverage, a coffee beverage, a fruit and vegetable juice beverage, a lactic acid bacteria beverage, and the like.

[0110] In some embodiments, the food product described in the present application is a dairy product, such as a milk powder, a cheese, a yogurt, a liquid milk, and the like.

[0111] In some embodiments, the food product described in the present application is a baked food, such as a bread, a cake, a biscuit, and the like.

[0112] In some embodiments, the food product described in the present application is a dietary supplement, such as a hard capsule, a soft capsule, a tablet, an oral liquid, a pill, a granule, a powder, and the like.

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

[0114] The present application does not particularly limit the absolute content of the lacto-N-neotetraose and the casein phosphopeptide in the food product, which should meet the requirements of the local food-related laws and regulations.

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

[0116] In addition to the above-described components in the nutritional composition, the food can further contain other ingredients, such as common food ingredients of proteins / amino acids, carbohydrates, fats, vitamins, minerals, etc.

[0117] Further, depending on the kind of food and the final needs of the target subject, in some embodiments, the food contains any one or more of the following ingredients: plant product ingredients, animal milk product ingredients, animal meat product ingredients, functional additive ingredients, and any acceptable adjuvants.

[0118] As for the plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, oranges, tangerines, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblics, and mulberries, or extracts thereof; fruit and vegetable materials such as onions, cucumbers, tomatoes, cauliflowers, red beetroots, spinach, kohlrabi, Brussels sprouts, garlic, basil, Oregon grass, or extracts thereof; cereals such as rice (indica rice, japonica rice, waxy rice), wheat (wheat, barley, oat, rye), corn, sorghum, millet, foxtail millet, yellow rice, buckwheat, soybeans, fava beans, peas, mung beans, adzuki beans, kidney beans, or extracts thereof; nut materials such as walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts, or extracts thereof; coffee or extracts thereof; and some medicinal and edible plant materials or extracts thereof.

[0119] As for the animal milk product ingredients, examples include fresh milk derived from cows, sheep, etc., and reprocessed milk products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, casein powder, etc.

[0120] As for the animal meat product ingredients, examples include meat product ingredients of pigs, cows, sheep, aquatic products, or poultry.

[0121] For the functional additive ingredients, examples can include vitamin supplements (e.g., vitamin A, beta-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 (e.g., iron, copper, manganese, zinc, cobalt, nickel, chromium, vanadium, fluorine, selenium, iodine, silicon, tin, etc.), nucleotide supplements (e.g., ), dietary fiber (e.g., inulin, konjac powder, galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, soybean polysaccharide, cyclodextrin, resistant dextrin, soybean fiber, etc.), functional polyunsaturated fatty acid supplements (e.g., arachidonic acid oil powder, docosahexaenoic acid oil powder, etc.), and the like.

[0122] For any acceptable adjuvant, examples can include solvents, antioxidants, antibacterial agents, thickeners, diluents, co-solvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweetening agents, food flavors, food colorants, and the like.

[0123] The embodiments of the present application will be described in detail below with reference to Examples and Experimental Examples, but those skilled in the art will understand that the following Examples and Experimental Examples are only for illustration of the present application and should not be considered as limiting the scope of the present application. In the Examples and Experimental Examples, the specific conditions not mentioned are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The materials or instruments used are commercially available conventional products unless otherwise specified.

[0124] The experimental instruments used in the Experimental Examples of the present application include: a zebrafish recirculating water culture system (ESEN-AW-S1, Beijing Aiseng 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 Paili Instrument Co., Ltd.); a conductivity meter (HM-100TDS, HM Digital Co., Ltd. of Korea); a thermometer (high precision, Oudashi Technology Co., Ltd.); an ultra-low temperature refrigerator (HYCD-205, Qingdao Haier Co., Ltd.); a mini centrifuge (LX-500, Anhui Zhongke Co., Ltd.); a zebrafish behavior analysis system (DanioVision, Noldus Co., Ltd. of the Netherlands).

[0125] The experimental animals used in the Experimental Examples of the present application include wild-type AB strain zebrafish, which are bred and maintained by the Precision Nutrition and Proactive Health Center of Yantai University:

[0126] (1) Rearing environment: Adult zebrafish were reared in a recirculating water system, with a 14 h / 10 h (light / dark) cycle, water temperature maintained at 28±0.5℃, pH 7.0-8.0, and electrical conductivity around 500 μs. Fresh brine shrimp eggs were fed twice a day.

[0127] (2) Zebrafish mating: Adult zebrafish were placed in a mating box with a ratio of 1:2 (male:female) after the end of feeding at night. The next morning, the isolation plate was removed, and the fish were allowed to mate. After 0.5-1 h, the fish were collected and placed in a recirculating water system. The embryos were collected using a filter and cultured in a glass dish containing E3 water at 28±0.5℃. Normal 6 h embryos were selected for subsequent studies on the effects of nutritional compositions on sleep and brain development mechanisms.

[0128] Example 1: Study on the improvement effect of nutritional compositions on sleep disorders in zebrafish

[0129] Zebrafish were induced to develop insomnia symptoms by p-chlorophenylalanine. In this example, the improvement effects of casein phosphopeptide (CPP) (purchased from Guangzhou Lvcui Biological Technology Co., Ltd.) and lacto-N-neotetraose (LNnT) (purchased from DMV-Fonterra Ingredients) on this insomnia symptom were investigated at different doses, alone or in combination.

[0130] 1. Experimental method

[0131] 1.1 Sleep deprivation model construction and experimental grouping

[0132] Randomly selected 4 dpf wild-type AB zebrafish were placed in a 6-well plate, with 30 fish per well. Blank control group (Comparative Example 1), model group (Comparative Example 2), positive control group (Comparative Example 3), and sample treatment groups (Comparative Examples 4-9 and Examples 1-9) were set up. The blank control group was normally cultured with E3 water (without any other treatment), and the other groups were treated with 2 mM p-chlorophenylalanine. The model group was not intervened, 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, and Examples 1-9 were treated with different proportions of CPP and LNnT. The grouping settings during the specific experiment are shown in Table 1.

[0133] Table 1. Design scheme for improving sleep with different nutritional substances

[0134]

[0135] 1.2 Zebrafish motor behavior evaluation

[0136] Randomly selected 4 dpf wild type AB strain zebrafish in 6-hole plate, each hole is handled 30 zebrafish. Respectively water soluble to give sample, each group 28℃ treatment 24 h, take out the treatment of juvenile fish, in 96-hole plate each hole put 1 young fish, open monitoring area bottom light, 96-hole plate is placed in behavior monitoring area 10 minutes makes it adapt to the environment, adjusts camera capture range to coincide with each hole, attempts to capture each hole zebrafish action trajectory success starts monitoring zebrafish juvenile fish behavior trajectory.Statistical processing result uses mean±SE to express.

[0137] (1) Autonomous activity: application behavior analysis instrument determines the activity amount (total movement distance) of zebrafish in the wakeful state within 60 min.

[0138] (2) Sleep structure: in zebrafish juvenile fish, the total activity time within each minute is less than 0.1 second, then it is considered to be in the sleep state; a continuous sleep time is defined as a sleep round. By analyzing the sleep round number of each treatment group zebrafish within 5 h under light and dark conditions, the characteristics of its sleep structure can be understood, and then its sleep quality is evaluated.

[0139] 2. Experimental results

[0140] (1) Zebrafish wakeful activity amount (activity distance) investigation result

[0141] The experimental results are shown in Table 2. As can be seen from the table, the average distance of wake-up activity of normal zebrafish with normal sleep behavior without any treatment (Comparative Example 1) is about 2840.84 mm, while the distance of wake-up activity after modeling treatment (Comparative Example 2) is as high as 4694.89 mm, and the difference is extremely significant (p<0.0001) after one-way ANOVA difference significance analysis, which indicates that the modeling is successful. At the same time, we give melatonin treatment (Comparative Example 3) as a positive control group, and find that the distance of wake-up activity of zebrafish after melatonin intervention is reduced to about 2445.74 mm, which is significantly lower than that of the model group (Comparative Example 2) (p<0.0001), and has no significant difference with the normal control group (Comparative Example 1) (p>0.05); Comparative Examples 4-6 are the distances of wake-up activity of zebrafish after low, medium and high dose CPP intervention, and the results show that the values are all reduced to about 3000 mm, and the difference significance analysis shows that the three comparative examples are significantly lower than the model group (Comparative Example 2) (p<0.0001); Comparative Examples 7-9 are the distances of wake-up activity of zebrafish after low, medium and high dose LNnT intervention, and the results show that the values of the three comparative examples are about 4200-4400 mm, which is higher than the value of CPP intervention alone, but is still lower than the model group, and the difference significance analysis shows that Comparative Example 7 has no significant difference with the model group (p>0.05), while Comparative Example 8 (p<0.05) and Comparative Example 9 (p<0.05) are significantly lower than the model group. The above results show that CPP intervention alone can reduce the night activity distance of zebrafish, while LNnT intervention at low dose does not show obvious effect, but intervention at medium and high doses can reduce the night activity distance of zebrafish. When CPP and LNnT are combined in different proportions, it is found that the distance of wake-up activity of zebrafish after composition intervention is reduced to 2400-2700 mm, and the difference significance analysis shows that the distances of wake-up activity of zebrafish in the nine composition intervention groups are significantly lower than those of the model group (p<0.0001), and have no significant difference with the positive control group treated with melatonin (Comparative Example 3) (p>0.05). This shows that the improvement of the distance of wake-up activity of zebrafish by the composition (Examples 1-9) formed by combining CPP and LNnT in different proportions is significantly higher than the effect of single substance intervention (Comparative Examples 4-9), and the difference significance analysis results also prove this point, and the specific results are shown in Table 3. Therefore, under appropriate doses, single CPP and LNnT can improve the distance of wake-up activity of sleep disorder zebrafish, and the combination of the two can significantly amplify the improvement of the distance of wake-up activity of sleep disorder zebrafish by single substance, and the two have synergistic effect. In addition, it is further found that there is no significant difference among the nine examples.

[0142] Table 2 Influence of different nutrients or combinations on the wake-up activity of sleep disorder zebrafish

[0143]

[0144] Table 3 Difference significance analysis of the improvement of the distance of the zebrafish wakefulness activity by the monomer and composition of the nutrient substance

[0145]

[0146]

[0147] (2) Zebrafish sleep structure investigation results

[0148] The number of sleep rounds at night was used to evaluate the improvement of the sleep structure by CPP and LNnT monomers or the compound formed by the two in different proportions. The experimental results are shown in Table 4. It can be seen from the table that the number of sleep rounds at night of the normal zebrafish is about 776 times on average (Comparative Example 1), and the number of sleep rounds at night is significantly reduced to about 249 times after modeling (Comparative Example 2) (p<0.0001), which indicates that the modeling is successful; the number of sleep rounds at night is significantly increased to about 829 times after the melatonin intervention (Comparative Example 3). Comparative Examples 4-6 are the number of sleep rounds of zebrafish after intervention by CPP at low, medium and high doses, and it can be seen that the number of sleep rounds of zebrafish is significantly increased with the increase of the concentration of CPP, and the number of sleep rounds of the three doses is significantly higher than that of the model group (Comparative Example 2) (p<0.0001); Comparative Examples 7-9 are the number of sleep rounds of zebrafish after intervention by LNnT at low, medium and high doses, and it is found that the average is 371, 424 and 345 respectively, which is slightly improved compared with the number of sleep rounds of the model group, but the effect is slightly worse than that of CPP at medium and high doses; Comparative Examples 1-9 are the number of sleep rounds of zebrafish after intervention by the composition formed by the combination of CPP and LNnT in different proportions, and it can be seen from Table 4 that the number of sleep rounds of zebrafish in the nine groups is more than 800 times, which is significantly higher than that of Comparative Examples 4-9, and the specific difference significance analysis results are shown in Table 5, and the combination of CPP and LNnT can significantly amplify the improvement of the number of sleep rounds of zebrafish with sleep disorders by single CPP or LNnT, and the two have a synergistic effect. At the same time, it is found through difference significance analysis that Example 8 is significantly higher than the positive control group (Comparative Example 3) treated by melatonin, and the number of sleep rounds of the other eight groups has no significant difference (p>0.05) with the positive control group (Comparative Example 3), which indicates that the composition formed by CPP and LNnT can significantly improve the number of sleep rounds at night of zebrafish with sleep disorders, and the effect is best when CPP and LNnT are combined at a ratio of 1:0.91.

[0149] Table 4 Effect of different nutrients or combinations on the sleep structure of zebrafish with sleep disorders

[0150]

[0151] Table 5 Analysis of the significance of the difference in the improvement of the sleep cycle number of zebrafish by the monomer and composition of nutrients

[0152]

[0153]

[0154] The above results show that CPP and LNnT have significant efficacy in improving sleep disorders in the ratio range of 1:0.41-11, specifically by reducing the amount of wakefulness activity of zebrafish and increasing the number of sleep cycles of zebrafish, especially in Example 8, i.e. when CPP and LNnT form a composition at a ratio of 1:0.91, the intervention effect is best.

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

[0156] 1. Experimental method

[0157] 1.1 Sleep deprivation model construction and experimental grouping

[0158] The same as Experimental Example 1.

[0159] 1.2 Evaluation of zebrafish motor behavior

[0160] Randomly select 4 dpf wild type AB strain zebrafish in a 6-well plate, and treat 30 zebrafish in each well. Respectively, water-soluble samples are given, each group is treated at 28°C for 24 h, and the treated larvae are taken out.

[0161] Thigmotaxis detection: a 12-well plate with a diameter of 24 mm is divided into a central region and a total region, the central region has a diameter of 12 mm, 1 test fish is placed in each well and allowed to move freely in the center. Zebrafish larvae are first adapted to a dark environment for 5 min in the behavior observation system, and then tested under the condition of 15 min light-dark alternation (dark 5 min, light 5 min, dark 5 min alternation). The thigmotaxis behavior of zebrafish is measured continuously for 15 min. The distance and time of zebrafish swimming in the two regions are counted, and the distance percentage of the central region in the total region is taken as an index to represent the thigmotaxis of zebrafish. The results are statistically processed and expressed as mean±SE.

[0162] 2. Experimental results

[0163] Previous studies have shown that after sleep deprivation, people's perception and evaluation of their own and others' emotions tend to be negative, which further leads to anxiety behavior. In this experimental example, the thigmotaxis index is used to evaluate the anxiety-like behavior of zebrafish after sleep disorders. Specifically, by investigating whether zebrafish will spontaneously approach the edge zone of the container in a new environment, the anxiety level of zebrafish is reflected.

[0164] In the experiment, the 12-well plate was divided into two observation areas, the center area and the edge area, with the center area being half of the whole area. In this experiment, the proportion of the distance traveled by the zebrafish in the edge area was used to reflect the anxiety level. The experimental results are shown in Table 6. As can be seen from the table, the proportion of the distance traveled by the zebrafish in the edge area of the normal control group (Comparative Example 1) was 61.70%, while the proportion of the distance traveled by the zebrafish in the edge area of the model group (Comparative Example 2) increased significantly to 92.05%, and there was a very significant difference (p<0.0001) compared with the control group, which indicated that the zebrafish in the model group had developed severe thigmotaxis. The thigmotaxis of the positive control group (Comparative Example 3) treated with melatonin was significantly reduced to 64.95%, and there was a very significant difference (p<0.0001) compared with the model group (Comparative Example 2), but there was no significant difference (p>0.05) compared with the blank control group (Comparative Example 3); Comparative Examples 4-6 were the effects of CPP low, medium and high dose intervention on the thigmotaxis of sleep-disordered zebrafish. As can be seen from Table 6, the proportion of the distance traveled by the zebrafish in the edge area of the three groups after intervention was reduced to 75%-90% compared with the model group, and after significant difference analysis, it was found that except for Comparative Example 4, which had no significant difference (p>0.05) compared with the model group, Comparative Examples 5 and 6 were significantly lower than the model group; Comparative Examples 7-9 were the effects of LNnT low, medium and high dose intervention on the thigmotaxis. The results showed that the proportion of the distance traveled by the zebrafish in the edge area of the low dose group (Comparative Example 7) was slightly lower than that of the model group, but after significant difference analysis, it was found that there was no significant difference (p>0.05) compared with the model group, while the mean distribution of the medium and high dose groups was reduced to 79.01% and 72.48% respectively, and after significant difference analysis, it was found that there was a significant difference (p<0.0001) compared with the model group. This indicates that CPP and LNnT alone at low dose have no significant effect on improving the thigmotaxis of zebrafish, but at medium and high doses, they can significantly reduce the thigmotaxis; Examples 1-9 were the effects of LNnT combined with different ratios on the thigmotaxis of sleep-disordered zebrafish. As can be seen from Table 6, the intervention of the composition reduced the proportion of the distance traveled by the zebrafish in the edge area to different degrees, and after 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 effect of the composition was better than that of CPP and LNnT alone, and after significant difference analysis, it was found that Examples 1-9 were significantly lower than Comparative Examples 4-9 (see Table 7 for significant difference analysis results), i.e. the combination of CPP and LNnT can significantly amplify the effect of CPP or LNnT alone on improving the thigmotaxis of sleep-disordered zebrafish, and there is a synergistic effect between them. Further comparison of the proportion of the distance traveled by the zebrafish in the edge area of the nine examples showed that the results were as follows: Figure 1As shown in the figure, it can be seen that Example 8 is significantly lower than the other 9 groups (p<0.0001). This shows that CPP and LNnT have the best effect on improving the anxiety caused by sleep disorders in zebrafish after being compounded at a ratio of 1:0.91.

[0165] Table 6 Effect of different nutrients or combinations on the thigmotaxis of sleep-disordered zebrafish

[0166]

[0167] Table 7 Analysis of the significance of the effect of nutrient monomers and combinations on the thigmotaxis of zebrafish

[0168]

[0169]

[0170] The above results show that CPP and LNnT have a significant effect on improving the anxiety caused by sleep disorders in the ratio range of 1:0.41-11, which is manifested by reducing the distance of zebrafish activity in the marginal area (thigmotaxis), especially Example 8, i.e., the intervention effect is best when CPP and LNnT are compounded at a ratio of 1:0.91.

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

[0172] 1. Experimental method

[0173] 1.1 Sleep deprivation model construction and grouping

[0174] The same as Experimental Example 1.

[0175] 1.2 Biochemical index detection

[0176] Randomly select 4 dpf wild type AB strain zebrafish in a 6-well plate, and treat 30 zebrafish in each well. Respectively, water-soluble samples are given, and after 24 h of treatment at 28°C, 0.01 M 1xPBS is added to lyse the zebrafish, and the lysate containing zebrafish tissue homogenate is collected to determine the content of γ-aminobutyric acid (GABA), melatonin (MT), cyclic adenosine monophosphate (cAMP) and glutamate receptor (NMDAR) using an ELISA kit. The specific detection method is as follows:

[0177] 1) Kit name: Fish Gamma-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 method: 30 zebrafish larvae were collected in each group in a centrifuge tube, the residual liquid was sucked out, and an appropriate amount of phosphate buffered saline solution (PBS) was added, according to 1:9 (w / v), i.e. 1 gram of tissue + 9 mL of PBS, to obtain 10% of the homogenate lysate, which was frozen at -20°C for standby.

[0179] 3) Experimental steps:

[0180] ① Take out the required micro-well enzyme-labeled plate strip from the aluminum foil bag after equilibration at room temperature for 20 min, and put the remaining plate strip back into 4°C with a self-sealing bag.

[0181] ② Set the standard sample hole and the sample hole, and add different concentrations of standard sample 50 μL to each standard sample hole;

[0182] ③ Add 10 μL of the sample to be tested to the sample hole, and then add 40 μL of sample diluent; the blank hole is not added.

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

[0184] ⑤ Discard the liquid, pat dry on a blotting paper, fill each hole with washing solution, stand for 1 min, shake off the washing solution, pat dry on a blotting paper, and repeat the plate washing 5 times (or use a plate washer to wash the plate).

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

[0186] ⑦ Add 50 μL of stop solution to each hole, and measure the OD value of each hole at 450 nm within 15 min.

[0187] 4) Result judgment: Draw a standard sample linear regression curve with the standard sample concentration as the abscissa and the corresponding OD value as the ordinate, and calculate the concentration value of each sample according to the curve equation.

[0188] 2. Experimental results

[0189] (1) Changes in the contents of MT and GABA in zebrafish tissues

[0190] Melatonin (MT) is an endogenous sleep regulator, which has been proven to correct sleep-wake rhythm and improve sleep quality. Mechanistically, on the one hand, melatonin can activate MT1 and MT2 receptors to regulate biological clock, play a role in sedation and induce sleep; on the other hand, the daytime and nighttime secretion levels of melatonin affect the content of gamma-aminobutyric acid (GABA) in the brain, thereby affecting the sleep regulation cycle. Therefore, the content changes of MT and GABA in the tissues of each group of zebrafish were investigated, and the experimental results are shown in Table 8.

[0191] As can be seen from Table 8, the content of MT in the brain tissue of normal zebrafish is about 168.86 pg / mL on average, but the content of MT in the brain tissue of zebrafish with sleep disorders is significantly reduced to 101.14 pg / mL, which has a significant difference (p<0.001) with the blank control group, which shows that MT is a susceptible index for evaluating sleep. Comparative examples 4-6 are the content of MT in zebrafish after intervention with low, medium and high doses of CPP, and from Table 8 it can be seen that the content of MT increases with the increase of CPP concentration, and when the high dose is intervened, it can reach 168.69 pg / mL, and after significant difference analysis, it is found that it is significantly higher than the model group (p<0.0001); Comparative examples 7-9 are the content of MT in zebrafish after intervention with low, medium and high doses of LNnT, and from the table it can be seen that the content of MT in the three groups is in the range of 125-138 pg / mL, and after significant difference analysis, it is found that the three are significantly higher than the model group (p<0.005, p<0.01, p<0.0001); Examples 1-9 are the content of MT in zebrafish after intervention with the composition formed by the combination of CPP and LNnT in different proportions, and from Table 8 it can be seen that the content of MT is higher than 190 pg / mL, which has a very significant difference (p<0.0001) compared with the model group, and compared with the comparative examples, it is found that the effect of the composition is significantly higher than that of the two monomer comparative examples, that is, the combination of CPP and LNnT can significantly amplify the improvement effect of single CPP or LNnT on the decrease of MT content in zebrafish with sleep disorders, and the specific results are shown in Table 9, which shows that CPP and LNnT have a synergistic effect. And by further comparing the content of MT in each example, it is found that the content of MT in example 8 is as high as 211.14 pg / mL, which shows that when CPP and LNnT are compounded to form a complex at a ratio of 1:0.91, it has the best effect of improving the content of MT in zebrafish.

[0192] In the central nervous system of mammals, the sleep-aiding mechanism of GABA is mainly achieved by regulating the excitability of neurons. After GABA binds to the receptors on neurons, it can inhibit the firing frequency of neurons, thereby producing a calming and sleep-inducing effect. Table 8 shows the differences in GABA content in the brain tissues of zebrafish in each group. As can be seen from Table 8, the average GABA content in the brain tissues of normal zebrafish is 21.04 μmol / L, but the GABA content in zebrafish with sleep disorders decreases rapidly to 13.71 μmol / L, and this difference is significant (p<0.0001), which indicates that the modeling is successful, and the GABA content in the body of sleep-disordered subjects is significantly reduced. Comparative Examples 4-6 represent the GABA content in zebrafish after intervention with CPP at low, medium and high doses. As can be seen from Table 8, as the dose of CPP intervention increases, the GABA content 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 intervention with LNnT at low, medium and high doses. As can be seen from the table, the value is slightly lower than when CPP is intervened alone, but is still higher than the model group, and the difference is significant (p<0.001); Examples 1-9 are the effects of the combination of CPP and LNnT at different ratios on the GABA content in zebrafish after intervention. The results show that the GABA content is increased to the range of 24.09-27.02 μmol / L, and after significant difference analysis, it is found that each group is significantly higher than the model group, and the specific significant difference results are shown in Table 10. As can be seen from Table 10, the combination of CPP and LNnT can significantly amplify the improvement effect of single CPP or LNnT on the decrease of GABA content in sleep-disordered zebrafish, i.e. the two have a synergistic effect. In particular, in Example 8, the GABA content in zebrafish can be significantly increased to 27.02 μmol / L.

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

[0194]

[0195] Table 9 Significant difference analysis of the MT content in zebrafish in the comparative examples and examples

[0196]

[0197]

[0198] Table 10 Significant difference analysis of the GABA content in zebrafish in the comparative examples and examples

[0199]

[0200]

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

[0202] It is known that NMDA receptors (NMDARs) are the receptors of excitatory neurotransmitter glutamate, and D-serine activates NMDA receptors. It is a common belief that excitatory neurons should lead to wakefulness, and inhibitory neurons should lead to sleep, but the postdoctoral research paper “D-Serine made by serine racemase in Drosophila intestine plays a physiological role in sleep” of the Beijing Brain Science and Brain-like Research Center, Peking McGovern Institute for Brain Research, Rao Yi Laboratory in 2019 showed that D-serine promotes sleep through NMDA receptors, which apparently proves that the molecular and cellular mechanisms of sleep are completely opposite to the general idea. This study also found that the level of NMDAR in the nerve cells of normal zebrafish (Comparative Example 1) was about 1397.87 μg / mL (the experimental results are shown in Table 11), but after modeling (Comparative Example 2), the level of NMDAR in the nerve cells of zebrafish with sleep disorders directly decreased to 854.59 μg / mL, which had a very significant difference (p<0.001) with the normal control group (Comparative Example 1), indicating that the modeling was successful, and as the sleep disorder was disturbed, the level of NMDAR in the zebrafish body was significantly reduced. Comparative Examples 4-6 show the level of NMDAR in the nerve cells of zebrafish after intervention with CPP at low, medium and high doses. As can be seen from the table, they are all higher than the model group, with NMDAR levels of 1096-1249 μg / mL, and after significant difference analysis, it is found that they are all significantly higher than the model group (p<0.05, p<0.001, p<0.0001); Comparative Examples 7-9 are the levels of NMDAR in the nerve cells of zebrafish after intervention with LNnT at low, medium and high doses. As can be seen from Table 11, their values are lower than when CPP is intervened alone, but higher than the model group, and after significant difference analysis, LNnT at low and medium doses has no significant difference with the model group (p>0.05), and LNnT at high dose is significantly higher than the model group (p<0.001). This shows that CPP alone has the effect of improving the level of NMDAR in the nerve cells of zebrafish, and LNnT has this effect at high doses. Examples 1-9 show the effect of combined intervention of CPP and LNnT in different proportions on the level of NMDAR in the nerve cells of zebrafish. The results show that the combined intervention is significantly better than the single intervention (Examples 1-9 are significantly higher than Comparative Examples 4-9), and the specific significant difference analysis is shown in Table 12, that is, the combination of CPP and LNnT can significantly amplify the improvement of NMDAR level in the nerve cells of sleep disturbed zebrafish by single CPP or LNnT, and there is a synergistic effect between them.Further comparing the 9 examples, it is found that the NMDAR in Example 8 can reach 1536.5 μg / mL, having the best effect of improving the NMDAR level, and after difference significance analysis, it is found that Example 8 is 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, and plays a role in promoting anti-anxiety and enhancing sleep through the cAMP / PKA-CREB-BDNF signaling pathway. The experiment found that the level of cAMP in the nerve cells of normal zebrafish (Comparative Example 1) was 21.83 nmol / L, but the level of cAMP in the nerve cells of zebrafish with sleep disorders after modeling (Comparative Example 2) rapidly decreased to 14.14 nmol / L, which was significantly different from the control group (Comparative Example 1) (p<0.0001), indicating that the content of cAMP in the nerve cells significantly decreased with the occurrence of sleep disorders. Comparative Examples 4-6 are the levels of cAMP in the nerve cells of zebrafish after intervention with low, medium and high doses of CPP (results shown in Table 11), and the content of cAMP is significantly improved, and has a trend of increasing with the increase of the dose of CPP intervention. After difference significance analysis, it was found that only in the medium and high doses, it was significantly higher than the model group (p<0.0001), which indicated that the improvement of cAMP in the nerve cells of zebrafish with sleep disorders by CPP alone was only effective in the medium and high doses, and there was no significant improvement in the low dose; Comparative Examples 7-9 are the content of cAMP in the nerve cells of zebrafish after intervention with low, medium and high doses of LNnT, and the results show that the content of cAMP is higher than that of the model group, but after difference significance analysis, it was also found that only in the medium and high doses, there was a significant difference with the model group (p<0.05; p<0.0001), and there was no significant difference in the low dose (p>0.05). Examples 1-9 are the levels of cAMP in zebrafish with sleep disorders after intervention with compositions formed by CPP and LNnT in different proportions. As can be seen from Table 11, the level of cAMP after intervention with the composition is higher than 24 nmol / L, and after difference significance analysis, it was found that the nine examples were all significantly higher than the model group (p<0.0001), and significantly higher than Comparative Examples 4-9, indicating that the combination of CPP and LNnT can significantly amplify the improvement of cAMP level in the nerve cells of zebrafish with sleep disorders by single CPP or LNnT, and there is a synergistic effect between the two, and the specific difference significance is shown in Table 13. Further comparative analysis of the nine examples found that the content of cAMP in Example 8 can reach 28.44 nmol / L, which is the best.

[0204] Table 11 Comparison of NMDAR and cAMP content in the nerve cells of zebrafish in each group

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[0206] Table 12 Difference significance analysis of NMDAR level in the nerve cells of zebrafish in comparative examples and examples

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[0209] Table 13 Analysis of significant differences in cAMP levels in zebrafish nerve cells in the examples and comparative examples

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[0211]

[0212] The above results show that the composition formed by compounding CPP and LNnT can improve sleep, and after intervention of the composition formed by compounding CPP and LNnT, the content levels of key nerve factors MT and GABA mediating sleep in brain tissue are significantly improved. Mechanism exploration shows that the composition can improve sleep by activating excitatory neurotransmitter receptors NMDAR in the nervous system and regulating the cAMP / PKA-CREB-BDNF signal pathway. In particular, Example 8, when CPP and LNnT are compounded at a ratio of 1:0.91 to form a composition, the effect is best.

Claims

1. Use of a nutritional composition in the manufacture of a food for improving sleep and for improving an anxiety state caused by a sleep problem; the nutritional composition comprising an active ingredient, and the active ingredient of the nutritional composition consisting of components represented by (I) and (II) below: (I) a casein phosphopeptide, (II) a neutral non-fucosylated human milk oligosaccharide; wherein the neutral non-fucosylated human milk oligosaccharide is lacto-N-neotetraose; and, in the nutritional composition, the mass ratio of the casein phosphopeptide to the lacto-N-neotetraose is 1 : (0.1-10.0); the improvement of sleep including any one or more of improvement in the amount of wakeful activity and improvement in the number of sleep bouts; the improvement of the anxiety state caused by a sleep problem including improvement in the tendency to touch, the improvement in the tendency to touch being a decrease in the tendency to touch; the improvement of sleep further including any one or more of improvement in the number of awakenings, improvement in the time of awakenings, and improvement in the time of sleep bouts; the improvement of sleep and / or the improvement of the anxiety state caused by a sleep problem including improvement in the content of melatonin and / or gamma-aminobutyric acid in brain tissue; the improvement of sleep and / or the improvement of the anxiety state caused by a sleep problem including improvement in the content of N-methyl-D-aspartate receptors and / or cyclic adenosine monophosphate in nerve cells; the food including any one or more of infant food, child food, adolescent food, pregnant and lactating woman food, adult food, and elderly food; the food containing any one or more of plant product ingredients, animal milk product ingredients, animal meat product ingredients, functional additive ingredients, and any acceptable adjuvants; in the food, the content of the casein phosphopeptide is 50-300 mg / 100 g, and the content of the lacto-N-neotetraose is 0.05-2 g / 100 g, based on the total dry matter content of the food.

2. The use according to claim 1, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

3. The use according to claim 1 or 2, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

4. The use according to any one of claims 1 to 3, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

5. The use according to any one of claims 1 to 4, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

6. The use according to any one of claims 1 to 5, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

7. The use according to any one of claims 1 to 6, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

2. Use according to claim 1, characterized in that, 8. The use according to any one of claims 1 to 7, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

3. Use according to claim 1, characterized in that, 9. The use according to any one of claims 1 to 8, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

4. Use according to claim 1, characterized in that, 10. The use according to any one of claims 1 to 9, wherein the food is a food for improving sleep and for improving an anxiety state caused by a sleep problem.

5. Use according to any one of claims 1 to 4, characterized in that, ​ 6. Use according to any one of claims 1 to 4, characterized in that, ​ 7. Use according to any one of claims 1 to 4, characterized in that, ​

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