Use of collagen hydrolysate as an active ingredient in improving sleep

Abstract

The present invention relates to the use of collagen hydrolysate as an active ingredient in improving sleep. Collagen hydrolysate is preferably used in the absence of other sleep-enhancing compounds in order to achieve maximum sleep-enhancing effect and / or not to counteract the inherent sleep-enhancing effect of collagen hydrolysate. The present invention further relates to a composition comprising at least 97.5% by weight, preferably 97.5% by weight, of collagen hydrolysate as an active ingredient, and 2.5% by weight or less, preferably 2.5% by weight, of one or more additional compounds that can regulate sleep. The present invention also relates to a sleep-improving composition that consists essentially of collagen hydrolysate and / or is essentially free of additional compounds that can regulate sleep.

Description

[Technical field]

[0001] The present invention relates to active ingredients in improving sleep.The active ingredients can be administered as an oral supplement. [Background technology]

[0002] Sleep is an important aspect of health and quality of life and is part of a fundamental physiological rhythm that is characterized in humans by three states: wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep.

[0003] Sleep problems are an increasing problem worldwide. Sleep disorders can occur as a disruption of normal sleep patterns and / or sleep-related cognitive behaviors. It is believed that 30-50% of individuals wake up too frequently during the night.

[0004] Furthermore, it is estimated that 30-50% of individuals experience waking in the morning feeling unrefreshed or experience impaired cognitive performance, which appears to be related to sleep deprivation and / or poor quality sleep. Impairments in cognitive function are most evident with respect to attention, concentration, and other cognitive domains such as working memory, reaction time, susceptibility to cognitive interference, episodic memory, and impulse inhibition.

[0005] Thus, sleep and sleep quality can be characterized as an overall experience taking into account various parameters such as the time needed to fall asleep (sleep onset), whether one wakes up frequently during the night (fragmented sleep, sleep awakenings), waking up too early (final awakening too early), waking up unrefreshed, daytime sleepiness, and waking up with cognitive impairment.

[0006] Depending on the severity and duration, sleep disorders can manifest as either non-pathological (ie, as a non-medical condition) or pathological (ie, as a medical condition).

[0007] Even in non-pathological situations, improved sleep can lead to improved overall well-being, health, and performance. For example, acute sleep deprivation and partial sleep restriction can induce stress associated with prolonged wakefulness, and reduced sleep quality is known to affect emotional instability and irritability, and to reduce positive mood. Furthermore, improved sleep has been found to promote learning ability (Stickgold et al. Nat Neurosci. 2000 December; 3(12): 1237-8), improve the activity of the immune system and host defense (Irwin et al. FASEB J. 1996 April; 10(5): 643-53.), and reduce the risk of impaired glucose tolerance and diabetes (Gottlieb et al. Arch Intern Med. 2005 Apr. 25; 165(8): 863-7). Therefore, the reasons why (further) improvements in sleep and sleep quality may be beneficial may be different, even in the absence of sleep-related medical conditions.

[0008] Sleep improvement may also be required by medical conditions (i.e., sleep disorders). Insomnia is the most common sleep disorder and is typically classified as chronic insomnia (e.g., for durations greater than 3 weeks), short-term insomnia (e.g., for durations of 1-3 weeks), or transient insomnia (e.g., durations less than 1 week) (Roth, Int. J. Clin. Pract. 2001;(Suppl.):3-8). Individuals suffering from insomnia can also be classified based on when the sleep difficulties most frequently occur, namely, as (1) sleep onset insomnia (difficulty falling asleep); (2) sleep maintenance insomnia (difficulty staying asleep); (3) terminal insomnia (early morning awakening with inability to return to sleep).

[0009] Some ingredients are administered to enhance sleep. For example, benzodiazepines are frequently used as sleep enhancers and exert their pharmacological action by interacting with benzodiazepine binding sites associated with gamma aminobutyric acid (GABA) receptors. GABA is the major inhibitory neurotransmitter in the central nervous system. GABA plays a role in regulating conditions such as sleep, anxiety, convulsions, depression, psychosis, and cognition in mammals. Well-known benzodiazepines include diazepam (Valium), lorazepam (Temesta), clonazepam (Rivotril), and alprazolam (Xanax). The stress-reducing and sleep-enhancing effects of GABA have also been established, and therefore GABA is found in a variety of sleep-enhancing compositions. Other well-known active ingredients used to enhance sleep include sedative antidepressants, antipsychotics, antihistamines, non-benzodiazepine GABA modulators, melatonin receptor agonists, serotonin receptor agonists and glucocorticoid inhibitors (e.g., phosphatidylserine).

[0010] A limitation of current sleep enhancers is that many of them have highly undesirable side effects including one or more of the following: abnormal thinking and behavioral changes, hallucinations, complex behaviors such as sleep-driving, amnesia, anxiety, suicidal thoughts, withdrawal symptoms, depressant effects, impaired motor or cognitive performance, tastelessness, headaches, parasomnias such as sleepwalking, anaphylaxis and anaphylactoid reactions, and / or weight gain.

[0011] Furthermore, existing sleep enhancers may help improve sleep onset or sleep duration, but may not improve overall sleep quality sufficiently or may have side effects.For example, benzodiazepines and other GABA modulators can typically promote sleep onset, but still have limited effectiveness in promoting sleep throughout the night, do not have the benefit of reducing early awakening, and typically even worsen behavioral and cognitive functions in the morning.Classical benzodiazepines are also known to typically reduce slow wave sleep (SWS) and rapid eye movement (REM) sleep.

[0012] It is an object of the present invention to provide a safer and improved sleep enhancer. Summary of the Invention

[0013] The present inventors have found that ingestion of collagen hydrolysate improves overall sleep. Ingestion of collagen hydrolysate as the sole active ingredient results in improved overall sleep compared to ingestion of collagen hydrolysate in combination with known sleep enhancing compounds such as gamma-aminobutyric acid, L-theanine, 5-hydroxytryptophan, and / or L-tryptophan.

[0014] Without being bound by theory, it has been found that collagen hydrolysate has a strong sleep-enhancing effect intrinsically.However, the sleep-enhancing effect of collagen hydrolysate seems to be counteracted by known sleep-enhancing compounds such as gamma-aminobutyric acid (GABA), L-theanine, 5-hydroxytryptophan, and / or (L-)tryptophan, especially GABA.

[0015] The findings of the present inventors are surprising because of the existing belief that collagen hydrolysate is not effective as a sleep enhancer when administered alone.Therefore, in the art, collagen hydrolysate is combined with other sleep enhancer compounds to have sleep enhancer effect.For example, US Patent No. 10568939 discloses that collagen peptide has no effect on sleep onset, duration or quality when used alone, and therefore should be combined with known sleep compounds.

[0016] With this invention, the inventors have overcome existing preconceptions in the art.

[0017] Collagen hydrolysate has been found to be particularly effective in improving sleep in physically active individuals.Firstly, the overall sleep quality of athletes is significantly improved by the intake of collagen hydrolysate.Therefore, collagen hydrolysate is preferably consumed by physically active individuals, especially athletes.

[0018] Furthermore, it was found that taking collagen hydrolysate already resulted in a measurable improvement in sleep within one week. In comparison, when collagen hydrolysate was combined with GABA, the sleep effect was delayed. Thus, collagen hydrolysate has a faster benefit as a sleep enhancer when taken alone and without other sleep enhancers.

[0019] Administration of collagen hydrolysate relatively close to bedtime, for example within 4 hours before bedtime, more preferably within 2 hours before bedtime (e.g., 2 hours to 30 minutes before bedtime), has been found to be most beneficial in the context of the present invention. Without wishing to be bound by theory, the inventors have found optimal bioavailability of collagen hydrolysate and its derived components following oral ingestion relatively close to bedtime, which is associated with improved sleep (e.g., compared to oral ingestion earlier).

[0020] The inventors found that the use of collagen hydrolysate without other known sleep enhancing compounds was safe and caused the least amount of discomfort, such as fatigue, mood or psychomotor related. In particular, the use of collagen hydrolysate alone resulted in the highest neurocognitive performance after sleep.

[0021] The use of collagen hydrolysate as a sleep enhancer in the present invention is generally considered to be safe and can be used both therapeutically or non-therapeutically (e.g., without the need for a medical prescription), e.g., as a daily supplement.

[0022] In one aspect, the present invention relates to the use of collagen hydrolysate as an active ingredient in improving sleep.

[0023] In one aspect, the present invention relates to a collagen hydrolysate for use as an active ingredient in ameliorating sleep disorders such as selected from the group consisting of insomnia, restless legs syndrome, narcolepsy and sleep apnea.

[0024] In one aspect, the present invention relates to a composition suitable for improving sleep, comprising at least 97.5% by weight, preferably 97.5% by weight, of collagen hydrolysate as active ingredient and up to 2.5% by weight, preferably 2.5% by weight, of one or more further compounds that modulate sleep, relative to the active ingredients and / or compounds capable of modulating sleep in the composition.

[0025] In one aspect, the present invention relates to a sleep-improving composition that consists essentially of collagen hydrolysate and / or is essentially free of additional compounds capable of regulating sleep. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Use in the context of the present invention relates to use in improving sleep.

[0027] The use of collagen hydrolysate in sleep enhancing formulations is already known in the art, but it is used in combination with sleep enhancing compounds such as GABA receptor agonist and / or melatonin receptor agonist.Therefore, the consensus in the art is that collagen hydrolysate alone cannot enhance sleep, but it can regulate the activity or effectiveness of other sleep enhancing compounds.However, the inventors have found that the overall improvement of sleep is the highest when collagen hydrolysate is used as the main or only active ingredient for regulating sleep.

[0028] In the context of the present invention, collagen hydrolysate is preferably the active ingredient, more preferably the active ingredient in improving (eg enhancing) sleep and / or improving sleep disorders.

[0029] In the context of the present invention, the term "active ingredient" refers to a component of a dosage form that performs a biological function or induces or affects (e.g., enhances or inhibits) a physiological process in some way when administered. "Activity" refers to the ability to perform a function or induce or affect a process. Active ingredients are distinguishable from excipients, such as carriers, vehicles, diluents, lubricants, binders, disintegrants, fillers, and other formulation aids, as well as encapsulating or other protective components. Compounds that can regulate sleep (e.g., enhance sleep) are examples of active ingredients. In the context of the present invention, active ingredients are not compounds that can regulate sleep, but rather can be, for example, ingredients that have a positive effect on general health or performance.

[0030] (Collagen hydrolysate of the present invention) The term "collagen" in the context of the present invention refers to an amino acid sequence comprising a repeat (Gly-XY) sequence, preferably comprising at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 300, or 400 repeats of the sequence Gly-XY, where X and Y are amino acid residues independently selected from each other, but more preferably X and / or Y are proline. "Collagen" preferably has a sequence found in native collagen of one or more animal species. Additionally or alternatively, "collagen" can refer to the full length sequence or fragments or subunits thereof of (native) collagen, preferably one or more of types I to XXVII collagen, more preferably one or even more of types I, II, III, V or X collagen, even more preferably one or more of types I, II or III collagen. For example, the term "collagen" can refer to alpha-1 (I), alpha-2 (I), alpha-1 (II) or alpha-1 (III) chains, or fragments thereof. The term "collagen" encompasses the triple helical structure formed by the three subunits present in native collagen.

[0031] The term "collagen hydrolysate" in the context of the present invention means a mixture of short chains of amino acids (dipeptides, tripeptides, oligopeptides, polypeptides) resulting from (partial) hydrolysis from native (full-length) collagen, such as by enzymatic hydrolysis. The degree of hydrolysis affects the average molecular weight of the final product (expressed in Daltons, Da). The term "collagen hydrolysate" may be used interchangeably and synonymously with the terms "hydrolyzed collagen" or "collagen peptides". "Collagen hydrolysate" in the context of the present invention encompasses collagen that has undergone hydrolysis or partial hydrolysis. Collagen hydrolysates may be one or more of enzymatic hydrolysates, alkaline hydrolysates and acid hydrolysates. Additionally or alternatively, collagen hydrolysates may be obtained by one or a combination of the following processes: alkaline hydrolysis, acid hydrolysis and enzymatic hydrolysis. "Collagen hydrolysate" in the context of the present invention may be produced from collagen-containing material in a one-step process or via an intermediate gelatin stage (i.e., resulting in "hydrolyzed gelatin"). The term "collagen hydrolysate" therefore encompasses hydrolyzed gelatin (i.e., hydrolyzed gelatin). The term "gelatin" in the context of the present invention means collagen in a (irreversible) form obtained by partial hydrolysis of collagen. Depending on the process used, two types of gelatin are generally obtained, namely type A (acid hydrolysis) and type B (alkaline hydrolysis). In the context of the present invention, "gelatin" can refer to either type A or type B gelatin, or a combination thereof. Depending on the physical and chemical method of partial hydrolysis, the molecular weight of the peptides can be within a wide range (e.g. 10 kDa to 95 kDa). Partial hydrolysis provides gelatin with the ability to retain water and its gelling ability, and typically distinguishes it from "collagen peptides" or "collagen hydrolysates", i.e. products obtained by complete hydrolysis of collagen. The term "hydrolyzed gelatin" in the context of the present invention means a product obtained by hydrolysis of gelatin, resulting in a lower molecular weight of gelatin.The hydrolysis reaction to obtain hydrolyzed gelatin involves the breaking of one or more peptide bonds by the addition of one molecule of water. Hydrolyzed gelatin is distinguished from gelatin inter alia in terms of its gelling ability, i.e. hydrolyzed gelatin has reduced / no gelling ability and gelatin has gelling ability. Gelatin may be hydrolyzed by acid (hydrogen ions), i.e. to obtain "acid hydrolyzed gelatin". Gelatin may be hydrolyzed by alkali (hydroxyl ions), i.e. to obtain "alkaline hydrolyzed gelatin". Gelatin may be hydrolyzed by one or more enzymes (e.g. pepsin, trypsin), i.e. to obtain "enzymatically hydrolyzed gelatin".

[0032] "Collagen" or "collagen hydrolysate" in the context of the present invention may include proteins or peptides that are artificially synthesized, such as by recombinant or chemical synthesis. Recombinant synthesis encompasses that the protein or peptide is encoded by recombinant DNA expressed in an expression system. The expression system for the recombinant peptide may be a cell such as a bacterial cell (e.g., Escherichia coli, Bacillus subtilis species), a yeast cell [e.g., Saccharomyces cerevisiae, Pichia pastoris or Ogataea angusta (Hansenula polymorpha), Candida bodinii], a fungal cell (e.g., Aspergillus oryzae, Aspergillus niger, Trichoderma reesei), a mammalian cell (e.g., CHO cell, HeLa cell, HEK293 cell, NS0, Sp2 / 0), an insect cell or a plant cell (e.g., tobacco, cereals, legumes, fruits, vegetables). Synthetic synthesis encompasses one or more of solid phase synthesis, chemoenzymatic synthesis, Merrifield synthesis and Bailey peptide synthesis.

[0033] In various embodiments of the invention, the collagen hydrolysate may be produced by enzymatic or partial enzymatic hydrolysis of collagen, the enzymes used for this purpose may be one or more selected from the group consisting of serine proteases, alkaline proteases, neutral proteases, flavour proteases, complex proteases, thiol proteases, bromelain, metalloproteases, proteases, carboxypeptidases, pepsin, chymotrypsin, trypsin, cathepsin K, chymotrypsin, papain and subtilisin. In one embodiment, the hydrolysis, preferably the enzymatic hydrolysis, is carried out at a pH of 5 to 8, preferably at a pH of 6 to 7. In one embodiment, the hydrolysis, preferably the enzymatic hydrolysis, is carried out at a temperature of 55° C. to 70° C., preferably at a temperature of 60° C. to 65° C. In one embodiment, the hydrolysis, preferably the enzymatic hydrolysis, is carried out for a period of 3 to 8 hours, preferably for a period of 4 to 7 hours, more preferably for a period of 5 to 6 hours.

[0034] A collagen hydrolysate in the context of the present invention may be derived from one or more types of collagen selected from collagen types I to XXVII, preferably one or more of collagen types I, II, III, V or X, more preferably one or more of collagen types I, II or III. Additionally or alternatively, a collagen hydrolysate in the context of the present invention may be a mixture of two or more types of collagen, preferably two or more of collagen types I, II and III.

[0035] The collagen and / or collagen hydrolysate disclosed herein may be derived from any one or more animals or animal species, such as bovine, porcine, chicken, and fish. In one embodiment, the collagen is derived from bovine. In one embodiment, the collagen is derived from porcine. In one embodiment, the collagen is derived from fish. In one embodiment, the collagen is derived from chicken. In various embodiments, the collagen is a mixture of collagen from different sources, such as collagen from multiple animal species and / or collagen from different tissues. For example, the collagen disclosed herein may be a mixture of two or more collagens selected from the group consisting of fish collagen, porcine collagen, chicken collagen, and bovine collagen.

[0036] Collagen in the context of the present invention may be derived from one or more tissues selected from the group consisting of skin, scales, antlers, processes (e.g., humps), horns, head, brain, neck, ears, eyes, nose, tongue, lips, mouth, esophagus, trachea, sternum, larynx, bronchi, limbs, feet, toes, palms, nails, bones, cartilage, bone marrow, joints, membranes, back, ligaments, tendons, ribs, diaphragm, muscles, skeletal muscles, smooth muscles, intestines, blood vessels, bladder, stomach, aorta, heart, liver, kidneys, chest, lungs, spleen, pancreas, eggs, sperm, testes, ovaries, nerves, gallbladder, and abdomen. The term "skin" disclosed herein includes "hide", i.e., the outer covering of a large animal such as a cow (species) or any other large animal. The terms "skin" and "hide" may be used interchangeably herein and may refer to the outer covering of an animal regardless of size.

[0037] In a preferred embodiment, the collagen taught herein is derived from skin and / or skin connective tissue. In a preferred embodiment, the collagen taught herein is derived from cartilage. In a preferred embodiment, the collagen taught herein is derived from bone. In a preferred embodiment, the collagen taught herein is derived from sternum. The collagen disclosed herein can be a mixture of collagens derived from two or more tissues and / or two or more animals. In one embodiment, the collagen disclosed herein is a mixture of two or more collagens selected from the group consisting of skin collagen, cartilage collagen, sternal cartilage and bone collagen.

[0038] It is believed that sleep improvement can be achieved using collagen hydrolysates from various sources with a wide range of molecular weights, for example, in the range of 1000Da to 9000Da (typical for collagen hydrolysates). Evidence that various collagen hydrolysates may be suitable for improving sleep was found by carrying out a study aimed at investigating the single-dose bioavailability of collagen hydrolysates from various sources (bovine, fish and porcine) with various average molecular weights (2000Da and 5000Da for bovine collagen hydrolysate). This study showed that the uptake of collagen hydrolysate in blood appears similar for collagen hydrolysates from porcine, bovine and fish sources, and for collagen hydrolysates with a relatively high average molecular weight (5000Da) or a relatively low average molecular weight (2000Da).

[0039] The collagen hydrolysate may have an average molecular weight in the range of 500 Da to 25000 Da, for example 1000 Da to 15000 Da or 2000 Da to 10000 Da. The collagen hydrolysate may have an average molecular weight of at least 500 Da, or 600 Da, or 700 Da, or 800 Da, or 900 Da, or 1000 Da, or 1100 Da, or 1200 Da, or 1300 Da, or 1400 Da, or 1500 Da, or 1750 Da, or 2000 Da, or 2250 Da, or 2500 Da, or 2750 Da, or 3000 Da, or 3250 Da, or 3500 Da, or 4000 Da, or 4500 Da, or 5000 Da, or 5500 Da. Additionally or alternatively, the collagen hydrolysate may have an average molecular weight of 10000 Da or less, or 9500 Da or less, or 9000 Da or less, or 8750 Da or less, or 8500 Da or less, or 8250 Da or less, or 8000 Da or less, or 7750 Da or less, or 7500 Da or less, or 7250 Da or less, or 7000 Da or less, or 6750 Da or less, or 6500 Da or less, or 6250 Da or less, or 6000 Da or less, or 5750 Da or less, or 5500 Da or less, or 5250 Da or less, or 5000 Da or less, or 4500 Da or less, or 4000 Da or less, or 3500 Da or less, or 3000 Da or less, or 2500 Da or less.

[0040] Collagen hydrolysates having an average molecular weight of more than 1000 Da and less than 6000 Da are believed to be most beneficial in the context of the present invention and for overall sleep enhancement, particularly those in the range of 1500 Da to 3000 Da. In a preferred embodiment, the collagen hydrolysate has an average molecular weight in the range of 500 Da to 7500 Da, more preferably 1000 Da to 6000 Da, and even more preferably 1500 Da to 3000 Da.

[0041] The average molecular weight in the context of the present invention is preferably the weight average molecular weight (Mw).

[0042] A preferred method for determining the (average) molecular weight and / or molecular weight distribution of collagen hydrolysates is by High Performance Size Exclusion Chromatography (HPSEC). The following protocol is the preferred HPSEC protocol:

[0043] An Agilent HPLC, 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) equipped with a TSKgel SWXL precolumn and a G2000SWXL column (Tosoh Bioscience) is used. The analysis is performed using WinGPC software (PSS). The eluent is 100 mM phosphate buffer pH 5.3. The sample is eluted from the column (e.g., 0.5 mL / min, isocratic) and monitored by UV detection (e.g., 214 nm, analysis time: 40 min per injection + 180 min equilibration). Calibration is performed using a Narrow Calibration Standard (Low FILK).

[0044] Use of Collagen Hydrolysate in Conjunction with Other Active Ingredients In one embodiment, the composition (for use according to the invention) contains no other active ingredients in addition to the collagen hydrolysate.

[0045] (In certain embodiments, the collagen hydrolysate may be combined with one or more other active ingredients (e.g., vitamins, minerals, fiber, etc.) for use according to the invention, e.g., that have a general positive effect on health or performance.)

[0046] In one embodiment, the composition comprises one or more other active ingredients in addition to the collagen hydrolysate, the active ingredient preferably not being a compound capable of regulating sleep.

[0047] Use of Collagen Hydrolysate in Conjunction with Other Compounds Capable of Modulating Sleep Without being bound by theory, it has been found that collagen hydrolysate has a strong sleep-enhancing effect intrinsically.Although the exact reason is still unclear, the present inventors have found that the sleep-enhancing effect of collagen hydrolysate can be counteracted by other known sleep-enhancing compounds, in particular gamma-aminobutyric acid (GABA), L-theanine, 5-hydroxytryptophan, and / or (L-)tryptophan.Therefore, collagen hydrolysate is preferably administered to individuals without additional sleep-enhancing compounds.

[0048] In one embodiment, the collagen hydrolysate is used in the absence of additional compounds and / or active ingredients capable of regulating sleep. In one embodiment, the collagen hydrolysate is administered in a composition consisting (essentially) of collagen hydrolysate and / or a composition that is (essentially) free of additional compounds and / or active ingredients capable of regulating sleep.

[0049] In one embodiment, the use of the present invention relates to the use of a composition comprising collagen hydrolysate and essentially free of GABA or containing 1% by weight or less, or 0.1% by weight or less, or 0.01% by weight or less of GABA relative to the total amount of active ingredients and / or compounds capable of regulating sleep.

[0050] In one embodiment, the use of the present invention relates to the use of a composition comprising collagen hydrolysate and essentially free of melatonin or comprising less than 1% by weight, or less than 0.1% by weight, or less than 0.01% by weight of melatonin relative to the total amount of active ingredients and / or compounds capable of regulating sleep.

[0051] In one embodiment, the use of the invention relates to the use of a composition comprising collagen hydrolysate and essentially free of L-theanine or comprising 1% by weight or less, or 0.1% by weight or less, or 0.01% by weight or less of L-theanine relative to the total amount of active ingredients and / or compounds capable of regulating sleep.

[0052] In one embodiment, the use of the present invention relates to the use of a composition comprising collagen hydrolysate and essentially free of 5-hydroxytryptophan or comprising 1% by weight or less, or 0.1% by weight or less, or 0.01% by weight or less of 5-hydroxytryptophan relative to the total amount of active ingredients and / or compounds capable of regulating sleep.

[0053] In one embodiment, the use of the present invention relates to the use of a composition comprising collagen hydrolysate and essentially free of tryptophan or containing 1% by weight or less, or 0.1% by weight or less, or 0.01% by weight or less of tryptophan relative to the total amount of active ingredients and / or compounds capable of regulating sleep.

[0054] The terms "consisting essentially of collagen hydrolysate" or "essentially free of further compounds" (other than collagen hydrolysate) in the context of the present invention mean that further substances or compounds other than collagen hydrolysate may be present, but that said further substances or compounds cannot be measured according to standard techniques in the art and / or are, for example, below a certain threshold value, preferably below 0.1 wt.-%, more preferably below 0.01 wt.-%, even more preferably below 0.001 wt.-% and most preferably below 0.0001 wt.-% based on the total weight or total active ingredients in the composition.

[0055] The collagen hydrolysate of the present invention is preferably a compound capable of regulating sleep, preferably capable of improving sleep. The term "further compound capable of regulating sleep" means any compound capable of regulating sleep that is not a collagen hydrolysate.

[0056] When collagen hydrolysate is referred to as a "compound" in the context of the present invention, this refers to the total mixture of amino acid chains (e.g., peptides) that collagen hydrolysate is composed of.For example, if a particular composition contains 10% by weight of collagen hydrolysate, this means that the total mixture of amino acid chains (e.g., peptides) of collagen hydrolysate in the composition is 10% by weight.

[0057] For example, if a composition contains 10% collagen hydrolysate by weight and 10% GABA by weight (as the only two active ingredients in the composition), the composition would be considered to contain 50% collagen hydrolysate by weight and 50% GABA relative to the compound capable of regulating sleep in the composition.

[0058] The term "compound capable of regulating sleep" or "active ingredient capable of regulating sleep" in the context of the present invention refers to a compound or mixture of compounds that has been demonstrated to be effective in improving sleep, for example in terms of improving one or more of sleep onset (time), sleep duration, sleep quality, sleep pattern and cognitive function (after sleep), and / or improving daytime sleepiness and / or wakefulness after sleep onset. "Compound capable of regulating sleep" encompasses a compound or mixture of compounds that act on the sleep-wake cycle and / or that affect the activity of neurotransmitters, hormones and / or that bind to specific receptors that regulate the sleep-wake cycle. Examples of "compounds capable of regulating sleep" in the context of the present invention include gamma aminobutyric acid (GABA), GABA receptor agonists (e.g., benzodiazepines, baclofen, pregabalin, vigabatrin), enhancers of endogenous GABA receptor agonists (e.g., GABA analogs, topiramate, gabapentin, tiagabine, valproic acid, phenobarbital), melatonin, precursors of melatonin (e.g., (L-)tryptophan), melatonin receptor agonists (rozerem, valdoxan, hetrioz, 6-chloromelatonin, 2-(2-phenylethyl)benzofuran), luzindole, 4-phenyl-2-propenoyl benzoyl propenoate ... Included are pyonamide tetralin, enhancers of endogenous melatonin (e.g., 5-HTP, L-theanine, magnesium, vitamin B6, valerian or valerian root), serotonin, precursors of serotonin (e.g., L-tryptophan), serotonin receptor agonists (e.g., lysergic acid diethylamide, psilocybin, buspirone, sumatriptan), enhancers of endogenous serotonin (e.g., fenfluramine, lexapro, fluoxetine, sertraline), glucocorticoid inhibitors, glucocorticoid receptor antagonists, inhibitors of endogenous glucocorticoids, and anti-insomnia agents.Enhancers of endogenous serotonin in the context of the present invention are preferably selective serotonin reuptake inhibitors such as one or more selected from the group consisting of fluoxetine, sertraline, paroxetine, fluvoxamine, citalopram, escitalopram, vilazodone, vortioxetine, dapoxetine and nefazodone.

[0059] In the context of the present invention, a "compound capable of regulating sleep" is preferably a "sleep-regulating compound" in the composition, which means that the compound has a demonstrated effect in regulating sleep as part of the composition of the present invention.For example, if the sleep regulation (e.g., sleep improvement) by GABA is measured by a suitable method disclosed herein, then GABA is a sleep-regulating compound in the composition.In this example, this can be done, for example, by comparing the effect of a composition with or without GABA (as a control) on sleep.

[0060] An "enhancer" of an endogenous compound in the context of the present invention means that the level of the compound is increased when the "enhancer" is administered (e.g., orally), and the increase is preferably at least 5%, more preferably at least 25%, even more preferably at least 50%, most preferably at least 100%, and / or a statistically significant measurable increase. For example, L-theanine intake is known to enhance GABA levels in the body. Thus, L-theanine is an example of an enhancer of endogenous GABA in the context of the present invention.

[0061] The compound capable of regulating sleep may be derived from a plant and / or may be a plant extract. For example, melatonin is also found in certain plants, and therefore, in the context of the present invention, may be a compound derived from a plant and / or may be a plant extract. The compound capable of regulating sleep, derived from a plant and / or a plant extract, may be selected from the group consisting of, for example, melatonin, valerian (root), chamomile, lavender, passion flower, lemon balm, skullcap, kava, horsetail (equicetum), tocotrienol, resveratrol (3,5,4'-trihydroxy-trans-stilbene), cannabidiol, astaxanthin, beta-carotene, camu camu (myrciaria dubia), hemp, and ashwagandha.

[0062] In a preferred embodiment, the compound and / or active ingredient that can regulate sleep is 5-hydroxytryptophan (i.e., 5-HTP).5-HTP is the precursor of serotonin, a neurotransmitter that plays an important role in regulating sleep.When 5-HTP is taken as a supplement, it is converted into serotonin in the body, and can then help promote sleep.

[0063] In a preferred embodiment, the compound and / or active ingredient capable of regulating sleep is GABA or a GABA analogue.

[0064] In a preferred embodiment, the compound and / or active ingredient that can regulate sleep is L-theanine.L-theanine is an amino acid that is commonly used as a dietary supplement to promote relaxation and improve sleep.L-theanine is believed to act by increasing the levels of GABA, serotonin, and dopamine in the brain, which are neurotransmitters that play an important role in regulating sleep.

[0065] In a preferred embodiment, the compound and / or active ingredient that can regulate sleep is (L-) tryptophan.(L-) tryptophan is an essential amino acid that is the precursor of serotonin, a neurotransmitter that plays an important role in regulating sleep.When tryptophan is taken as a supplement, it is converted into 5-HTP in the body, then into serotonin, and finally into melatonin.

[0066] In a preferred embodiment, the compound and / or active ingredient that can regulate sleep is melatonin.Melatonin is a hormone that is believed to regulate the body clock and plays an important role in sleep.It is produced by the pineal gland in the brain, and its level increases as it gets dark in the evening and decreases as it gets light in the morning.Melatonin is believed to help regulate the sleep-wake cycle and promote the feeling of sleepiness and drowsiness.

[0067] In a preferred embodiment, the compound and / or active ingredient capable of regulating sleep is an anti-insomnia agent.

[0068] The term "antinsomnia agent" in the context of the present invention means a compound, preferably a drug, used to treat insomnia, including, but not limited to, barbiturates such as secobarbital, amobarbital, butalbital, cyclobarbital, pentobarbital, allobarbital, barbital, butobarbital, mephobarbital, phenobarbital, butabarbital, and vinylbital; quinazolinones and derivatives such as chloroqualone, diproqualone, etaqualone (Aolane, Atinazone, Ethinazone), mebroqualone, mecloqualone (Nuvallene, Kasphene), and methaqualone (Quaalude); benzodiazepines such as clorazepate, diazepam, flurazepam, halazepam, prazepam, lor ... non-benzodiazepines such as zaleplon and eszopiclone; antihistamines such as diphenhydramine and doxylamine; melatonin; antidepressants such as trazone, amitriptyline, doxepin, trimipramine, mianserin and mirtazapine; antipsychotics such as chlorpromazine, clozapine, olanzapine, quetiapine, risperidone, zotepine, clonidine, guanfacine, cannabidiol, tetrahydrocannabinol, suvorexant, pregabalin, gabapentin, phenibut, imagabalin. The term "anti-insomnia agent" encompasses any agent that requires prescription by a medical health care professional and / or a sleep specialist.

[0069] (Administration of collagen hydrolysate) In a preferred embodiment, the collagen hydrolysate disclosed herein is administered orally.

[0070] The term "administer" or "administration" in the context of the present invention includes providing a compound, substance, or composition to a subject who will consume it. A subject who consumes a compound, substance, or composition may administer it to himself / herself. In such cases, the term "administer" may be read as "ingest." The term "administer" includes self-administration.

[0071] Without wishing to be bound by theory, the inventors have found optimal bioavailability of collagen hydrolysate and its derived components following oral ingestion relatively close to bedtime, which is associated with improved sleep (e.g., compared to earlier oral ingestion).

[0072] It is most beneficial in the context of the present invention and for overall improved sleep if the collagen hydrolysate (daily dose) is administered relatively close to bedtime, such as 4 hours to 15 minutes, more preferably 2 hours to 30 minutes before bedtime. In a preferred embodiment, the collagen hydrolysate (daily dose) is administered within 4 hours, or within 3 hours, or within 2 hours, or within 2.5 hours, or within 1 hour, or within 45 minutes, or within 30 minutes, or within 15 minutes before bedtime.

[0073] It should be noted that "going to bed" refers to the moment an individual goes to bed to sleep or in anticipation of sleeping, regardless of the time of sleep onset.

[0074] The term "daily dose" in the context of the present invention means the total dry weight administered to a subject per day.

[0075] The term "unit dose" in the context of the present invention means the total dry weight administered to a subject in a single dose. A unit dose is typically a pre-prepared form (e.g., a pre-filled dose) that is ready for administration. A unit dose may be identifiable, for example (as well as) from the product packaging or label. Examples of unit dosage forms are individual tablets, pre-filled sachets, individual capsules, bulk powders, liquid solutions, emulsions or suspensions.

[0076] It is most beneficial in the context of the present invention and overall improvement of sleep when a relatively low dose of collagen hydrolysate is administered, for example 50g or less, preferably 30g or less, more preferably 20g or less, most preferably 15g or less. In a preferred embodiment, the use of collagen hydrolysate disclosed herein comprises administering collagen hydrolysate in an amount of 2g to 50g, preferably 5g to 30g, even more preferably 10g to 20g, the dose being a unit dose and / or daily dose, preferably a daily dose. Without being bound by theory, the inventors have found that a relatively low dose of collagen hydrolysate can improve sleep without providing unnecessary calories and / or promoting weight gain (which may be considered especially for individuals who do not need weight gain and / or who are not physically active).

[0077] In different embodiments, the daily dose of collagen hydrolysate is at least 1 g, at least 2 g, at least 3 g, at least 4 g, at least 5 g, at least 10 g, at least 15 g, at least 20 g, at least 25 g, at least 30 g, at least 35 g, at least 40 g, at least 45 g, or at least 50 g, where the daily dose is the total dry weight administered to the subject per day. Additionally or alternatively, in different embodiments, the daily dose of collagen hydrolysate as disclosed herein is 50 g or less, 45 g or less, 40 g or less, 35 g or less, 30 g or less, 25 g or less, 20 g or less, 15 g or less, 14 g or less, 13 g or less, 12 g or less, 11 g or less, 10 g or less, 5 g or less, or 1 g or less, where the daily dose is the total dry weight administered to the subject per day.

[0078] A daily dose of collagen hydrolysate is most beneficial in the context of the present invention and for overall improved sleep when administered as a single unit dose, preferably relatively close to bedtime as disclosed herein.

[0079] The inventors have found that the overall sleep-enhancing effect of collagen hydrolysate occurs relatively quickly when it is administered without other additives known to enhance sleep, particularly without GABA receptor agonists and / or melatonin receptor agonists, and when collagen hydrolysate is administered sufficiently frequently, for example at least once every other day, or more preferably every day.In addition, or alternatively, the effect of collagen hydrolysate in improving overall sleep is seen most quickly when it is administered for 4 consecutive days, particularly for 7 consecutive days (consecutively).

[0080] In one embodiment, the collagen hydrolysate is administered daily or every other day.

[0081] In one embodiment, the collagen hydrolysate is administered at least once daily.

[0082] In one embodiment, the collagen hydrolysate administration regimen comprises administering the collagen hydrolysate for at least 2 consecutive days, preferably at least 4 consecutive days, more preferably at least 7 consecutive days.

[0083] In one embodiment, the collagen hydrolysate is administered for at least 1 consecutive week, preferably at least 2 consecutive weeks, more preferably at least 4 consecutive weeks.

[0084] The term "consecutive" or "consecutively" used in the context of administration of collagen hydrolysate means that administration follows a sequence without gaps in a given period.For example, "collagen hydrolysate is administered for 4 consecutive days" means that collagen hydrolysate is administered at least once a day for 4 consecutive days (e.g., on Monday, Tuesday, Wednesday, and Thursday of the same week), regardless of the number of administrations per day or the total number of administrations.For example, "collagen hydrolysate is administered for 4 consecutive weeks" means that collagen hydrolysate is administered at least once a week for 4 consecutive weeks, regardless of the number of administrations per day, per week, or the total number of administrations.

[0085] (Collagen hydrolysate preparation) The collagen hydrolysate in the context of the present invention may be provided in a food formulation, a food supplement formulation or a pharmaceutical formulation, preferably in a food supplement formulation.

[0086] The collagen hydrolysates disclosed herein can be provided in a solid dosage form such as a capsule, tablet, or powder, preferably a powder.

[0087] The collagen hydrolysates disclosed herein may be provided in formulations such as drinkable solutions or suspensions, beverages such as beer, syrups, artificially flavored beverages, carbonated beverages, (water-soluble) powder mixtures, (water-soluble) pastes, (water-soluble) powders, (water-soluble) tablets, (water-soluble) pills, (water-soluble) dragees, (water-soluble) caplets, (water-soluble) sachets, or (water-soluble) capsules. Additionally or alternatively, the collagen hydrolysates taught herein may be present in functional foods, such as juices, shakes, dairy drinks, yogurt, yogurt drinks, sweets, energy bars, nutritional bars, slimming bars, or confectioneries such as gummies or center-filled gummies.

[0088] (Use in physically active individuals) The present inventors have found that the collagen hydrolysate of the present invention is particularly effective in improving overall sleep in physically active individuals, especially athletes.Very physically active individuals, especially athletes, may suffer from lack of sleep or even insomnia-related symptoms, such as daytime fatigue or daytime sleepiness.It seems that such physically active individuals can greatly benefit from the administration of collagen hydrolysate according to the present invention.

[0089] In a preferred embodiment, the collagen hydrolysate is administered to a physically active individual.

[0090] In a preferred embodiment, the collagen hydrolysate is administered to an individual who exercises regularly, for example at least once per week, or at least twice per week, or at least three times per week, or at least four times per week, or at least five times per week, or at least six times per week, or at least seven times per week, and / or from 1 to 10 times per week, or from 2 to 7 times per week, or from 3 to 5 times per week. Additionally or alternatively, in a preferred embodiment, the collagen hydrolysate is administered to an individual who engages in a minimal amount of exercise, for example, for at least 2 hours per week, or at least 4 hours per week, or at least 6 hours per week, or at least 8 hours per week, or at least 10 hours per week, or at least 12 hours per week, or at least 14 hours per week, or at least 16 hours per week, or at least 18 hours per week, or at least 20 hours per week, and / or 1-24 hours per week, or 3-20 hours per week, or 5-16 hours per week, or 7-14 hours per week.

[0091] The term "exercise" in the context of the present invention refers to any physical activity that can be planned, structured, and has an end or intermediate goal (e.g., improving or maintaining physical fitness). The term "exercise" encompasses all professional or non-professional activities, including sports, strength or resistance training, and endurance or aerobic activities. Additionally or alternatively, "exercise" may include endurance training. The term "endurance training" refers to training the aerobic system, as opposed to the anaerobic system, by performing physical exercise at an increased heart rate for an extended period of time (e.g., at least 30 minutes at 70% of maximum heart rate (MHR)), for example, during walking, running, jogging, cycling, swimming, jumping rope, etc.

[0092] In one embodiment, a physically active individual is defined according to at least a moderate or average physical activity score, preferably a high physical activity score, such as by the Physical Activity Questionnaire (PAQ). The PAQ is preferably one or more of the following:

[0093] (i) The International Physical Activity Questionnaire-Short Form (IPAQ-SF), generally considered the most frequently used and validated PAQ (e.g., the IPAQ-SF reported by Ahmad et al. Baltic Journal of Health and Physical Activity 2015;7(1):29-41);

[0094] (ii) the Global Physical Activity Questionnaire (GPAQ), designed by the World Health Organization (WHO) for chronic disease risk factor monitoring and considered the most widely used PAQ internationally;

[0095] (iii) the European Health Survey-Physical Activity Questionnaire (EHIS-PAQ), an EU-wide surveillance system in all member states. (Semper et al.Validity and Reliability of International Physical Activity Questionnaires for Adults across EU Countries:Systematic Review and Meta Analysis.Int.J.Environ.Res.Public Health 2020,17,7161)

[0096] For example, individuals who score as engaging in at least "moderate" physical activity preferably fall into a two or three categorical score using the IPAQ-SF. The following categorical scores are used in the IPAQ-SF: 1. Low: No activity is reported, or some activity is reported but not enough to meet Category 2 or Category 3. 2. Moderate: Meet the following three criteria: (i) at least 20 minutes of vigorous activity per day for 3 or more days; (ii) at least 30 minutes of moderate intensity activity and / or walking per day for 5 or more days; or (iii) any combination of walking, moderate intensity or vigorous intensity activity achieving at least 600 metabolic equivalent task (MET) minutes per week for 5 or more days. 3. High: Any one of the following two criteria: (i) vigorous intensity activity for at least 3 days and accumulatively at least 1500 MET-min / week; (ii) any combination of walking, moderate intensity, or vigorous intensity activity for at least 7 days and accumulatively at least 3000 MET-min / week.

[0097] In a preferred embodiment, the collagen hydrolysate is administered to an athlete. In one embodiment, an "athlete" is an individual who meets at least three, and preferably all, of the following criteria: 1) an individual is engaging in exercise aimed at improving their own performance / outcome; 2) the individual participates in a sports competition; 3) the individual is officially registered with a local, regional or national sports federation; 4) Individuals have athletic training and sports as their main activity or focus of personal interest, devoting several hours to these activities for all or a significant part of the day, in excess of the time allocated to other types of professional or leisure activities.

[0098] (Sleep-enhancing effects of collagen hydrolysate) In one embodiment, the collagen hydrolysate is used to improve, preferably reduce, sleep onset time. In one embodiment, the collagen hydrolysate is used to improve, preferably increase, sleep duration (after sleep onset). In one embodiment, the collagen hydrolysate is used to improve, preferably increase, sleep pattern (after sleep onset). In one embodiment, the collagen hydrolysate is used to improve, preferably increase, sleep quality (after sleep onset). In one embodiment, the collagen hydrolysate is used to improve, preferably reduce, wakefulness after sleep. In one embodiment, the collagen hydrolysate is used to improve, preferably reduce, daytime sleepiness.

[0099] Sleep improvement can be determined using parameters familiar to those skilled in the art, such as one or more of sleep onset latency (SOL), wake after sleep onset (WASO, e.g., minutes or hours), total sleep time (TST, e.g., minutes or hours), sleep efficiency (SE, %), number of awakenings (nWAK), number of awakenings per hour of sleep (WAKI), time in bed (min), total sleep time (min or hours), sleep latency N2 (min or hours), deep sleep latency (min or hours), total sleep time (min or hours), sleep stage change (count), sleep stage change (count / hour), REM stage sleep # (%), N1 stage sleep # (%), N2 stage sleep # (%), N3 stage sleep # (%), night sleep # (%), wake (count), wake (count / hour). Sleep may additionally or alternatively be assessed according to the number of nights requiring sleep medication to assist with sleep, improvement in daytime functioning (e.g., as assessed by the Work and Social Adjustment Scale), improvement in various quality of life measures (e.g., using the 36-item Short Form Survey (SF-36) and / or reduction in daytime sleepiness (e.g., as assessed by the Karolinska Sleepiness Scale or the Epworth Sleepiness Scale).

[0100] In one embodiment, collagen hydrolysate is used to improve, preferably reduce, wakefulness after sleep, preferably as determined by polysomnography (PSG).

[0101] The term "sleep pattern" in the context of the present invention refers to the time spent in each sleep stage, including the relative percentage of the duration of each stage compared to the duration of other stages, as measured, for example, by spectral analysis of electroencephalograms. Parameters of "normal" and "abnormal" sleep patterns for a particular population are well known to those skilled in the art (see, for example, Latta et al., 2005, Sleep 28:1525-1534). In mammals, sleep is typically divided into two broad types: rapid eye movement (REM) sleep and non-rapid eye movement (NREM) sleep. NREM can be further divided into three stages: N1, N2, and N3 (or delta or slow wave sleep). Sleep progresses in cycles of REM and NREM. The amount of deep sleep (stage N3) is greater in the early sleep cycle, while the proportion of REM sleep increases in the later sleep cycle just before natural awakening. In humans, each sleep cycle lasts an average of 90 to 110 minutes. REM sleep accounts for 20%-25% of total sleep time in most adults. Criteria for REM sleep include rapid eye movement and rapid low voltage EEG. In the context of the present invention, improving sleep patterns encompasses increasing the amount, frequency and / or duration of REM sleep. A subject's sleep patterns can be monitored overnight using electrodes and electroencephalograms (EEG) or (wearable) devices designed to monitor and measure various physiological parameters.

[0102] In one embodiment, collagen hydrolysate is used to improve, and preferably reduce, daytime sleepiness.

[0103] Sleep improvement in the context of the present invention includes an improvement, preferably a reduction, of daytime sleepiness, more preferably morning or afternoon sleepiness. Daytime sleepiness can be measured by the Karolinska Sleepiness Scale (KSS). The KSS measures the subjective level of sleepiness at a particular time during the day. The KSS has been used in studies of shift work, jet lag, and driving performance. The KSS spans nine levels (1=very alert, 2=very alert, 3=alert, 4=rather alert, 5=not alert or sleepy, 6=some signs of sleepiness, 7=sleepy, but no effort to stay awake, 8=sleepy, some effort to stay awake, 9=very sleepy, effort to stay awake, fighting sleep). In the context of the present invention, KSS measurements are preferably started in the morning after sleep and repeated throughout the morning and afternoon, for example every hour. Improved sleep can be defined, for example, according to a reduction in KSS score by one or more points at one or more time points.

[0104] In one embodiment, the collagen hydrolysate is used to improve, preferably increase, cognitive function, preferably cognitive function after sleep. In one embodiment, the collagen hydrolysate is used to improve, preferably reduce, cognitive impairment, preferably cognitive impairment after sleep.

[0105] The term "after sleep" in the context of the present invention preferably refers to the morning and / or the first 4 hours (e.g., the first 3 hours, the first 2 hours, the first hour, the first 30 minutes) after the normal sleep time. For example, if an individual normally sleeps until 9:00 a.m., the "after sleep" time is the period until 1:00 p.m. For example, if an individual normally sleeps until 6:00 a.m., the "after sleep" time is the period until 10:00 a.m.

[0106] In the context of the present invention, cognitive function may be established according to one or more of any suitable tests used to determine cognitive impairment, such as the Reaction Time Test, Choice Reaction Time Test, Digit Span Test and Stroop (Colors & Words) Test, and / or the Montreal Cognitive Assessment (MoCA).

[0107] Reaction times can be determined, for example, by asking participants to press the space bar on a keyboard when a green circle appears on a blank screen, and reaction times can be expressed as the fastest or average time (in ms or s).

[0108] Choice reaction times can be determined, for example, by asking participants to press an arrow key that matches a direction on a screen, and choice reaction times can be the fastest or average time (in ms or s).

[0109] Digit span testing (in arbitrary units) can involve, for example, asking participants to enter numbers in the correct order that correspond to a string of 3-14 digits displayed on a screen.

[0110] The Stroop test can involve, for example, measuring a baseline level when participants are presented with color names (written in black) on a white background and instructed to correctly identify the color name by pressing the right to left arrow.

[0111] A Stroop test can involve measuring interference levels, for example, when participants are presented with colored names on a white background and instructed to correctly identify the font color while ignoring incongruent colors named on the screen (e.g., if "green" is written in red font, red is the correct answer). In a Stroop test, participants are instructed to respond as quickly as possible using the left or right arrow keys, and outcomes are the percentage of correct responses and / or the mean reaction time (in ms or s) for correct responses relative to baseline and interference levels.

[0112] In one embodiment, the collagen hydrolysate is used to improve, preferably increase, cognitive function (after sleep), the improvement of cognitive function being preferably established according to the Stroop Color & Words Test, more preferably according to the Stroop Color & Words Test.

[0113] In one embodiment, the collagen hydrolysate is used to inhibit cognitive interference (after sleep), where inhibition of cognitive interference is defined according to the Stroop test, more preferably according to the Stroop Color & Words Test.

[0114] The term "improvement" in the context of the present invention means an improvement relative to a control or reference (population) not receiving the collagen hydrolysate of the present invention and / or an improvement compared to the situation before the administration of the collagen hydrolysate of the present invention is started, etc. The improvement can be determined, for example, by measurements made daily, and the average measurement for one week can be compared to the average measurement before the start of treatment, or the average measurement of the control or reference (population) to determine the improvement in sleep. The improvement can be an improvement of at least 1%, for example at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the corresponding level in the control or reference and / or compared to the level before the administration of the collagen hydrolysate of the present invention is started. Additionally or alternatively, a level can be considered to be improved when, for example, it is significantly improved based on an appropriate statistical test, compared to the level in the control or reference and / or compared to the level before the administration of the collagen hydrolysate of the present invention is started. Improvement can mean an increase in one or more of "sleep onset time", "wake after sleep", "sleep duration", "sleep quality", "sleep pattern", "daytime sleepiness", "cognitive function after sleep", "daytime function", and "quality of life measures" disclosed herein. Improvement can mean an improvement in cognitive function, preferably cognitive function after sleep, using a suitable test, for example, as disclosed herein. Improvement can include a reduction in scores on cognitive impairment tests, such as the Montrell Cognitive Assessment (MoCA). Improved sleep can mean a reduction in AIS from a value of 6 or more (i.e., indicating the presence of insomnia) to a value of less than 6 (i.e., indicating the absence of insomnia). Improved sleep can mean a reduction in AIS of at least 1 point, for example, a reduction of 1 point, 2 points, 3 points, 4 points, 5 points, 6 points, 7 points, 8 points, 9 points, 10 points, 11 points, 12 points, 13 points, 14 points, 15 points, 16 points, 17 points, 18 points, 19 points, 20 points, 21 points, 22 points, or 23 points.Improved sleep can mean a decrease in the KSS of at least 1 point, for example a decrease of 1 point, 2 points, 3 points, 4 points, 5 points, 6 points, 7 points, or 8 points.

[0115] In a preferred embodiment, the collagen hydrolysate improves one or more selected from the group consisting of sleep onset time, sleep duration, sleep quality, sleep pattern and cognitive function after sleep.

[0116] In a preferred embodiment, the collagen hydrolysate improves wakefulness after sleep onset and / or daytime sleepiness.

[0117] (Individuals who would benefit from improved sleep) The collagen hydrolysate of the invention is suitable for any individual who would benefit from and / or wish to improve their sleep, including individuals who suffer from sleep disorders, individuals who suffer from chronic sleep deprivation, or individuals who do not have (chronic) sleep problems. The term "sleep deprivation" means that the sleep duration is less than the average sleep requirement of the individual concerned. The use of the collagen hydrolysate can be considered either therapeutic or non-therapeutic depending on the relevant context and subject. Specialized physicians and / or sleep specialists can decide on a case-by-case basis whether the lack of sleep and / or the desire to improve sleep is a therapeutic or non-therapeutic intervention (in other words, whether the person falls into the group of healthy subjects or the group of pathological subjects). Therapeutic use encompasses use in pathological situations, such as in cases of sleep disorders and / or chronic sleep deprivation. Therapeutic use is usually in individuals with sleep-related symptoms of pain and distress and / or in individuals in whom lack of sleep may pose (serious) health and psychological risks, which can be established for example by specialized physicians and / or sleep specialists. Therapeutic use is typically in individuals who are administered sleep enhancing drugs such as anti-insomnia drugs, for example after consulting a specialized physician and / or sleep specialist. Non-therapeutic use encompasses use in non-pathological situations, such as individuals who do not suffer from pathological sleep, but who can benefit from improved sleep, i.e., "sleep healthy" individuals, such as when there is sleep deprivation and / or more sleep is desired to improve overall well-being. In "sleep healthy" individuals, the sleep deprivation may be of such a nature or severity that it disappears naturally over time (i.e., it is not chronic). Additionally or alternatively, in "sleep healthy" individuals, the sleep deprivation may be such that they generally do not seek help from specialized physicians and / or sleep specialists. Collagen hydrolysate in the context of the present invention may provide one or more of the following benefits in "sleep healthy" individuals: improve general performance, improve cognitive function (e.g., attention, concentration, memory), increase productivity in daily life, increase (cosmetic) appearance.

[0118] In one embodiment, the use of the collagen hydrolysate of the present invention is therapeutic. In one embodiment, the use of the collagen hydrolysate of the present invention is non-therapeutic, for example cosmetic and / or to improve general well-being or function. In one aspect, the present invention relates to a method of treatment comprising administering a collagen hydrolysate to an individual experiencing sleep deprivation.

[0119] (Use in Sleep Disorders) In one aspect, the present invention relates to a collagen hydrolysate and / or a composition comprising a collagen hydrolysate for use in improving a sleep disorder.

[0120] The use in sleep disorders is preferably a therapeutic use.

[0121] In one aspect, the present invention relates to a method for improving, e.g. preventing and / or treating, a sleep disorder, comprising administering to an individual a collagen hydrolysate and / or a composition comprising a collagen hydrolysate.

[0122] In one embodiment, the sleep disorder is insomnia. Those skilled in the art are aware of the various types of insomnia and their classification (Fietze et al. Front Psychiatry. 2021 Jun 29; 12: 683943). The term "insomnia" in the context of the present invention encompasses acute insomnia and chronic insomnia. "Chronic insomnia" in the context of the present invention is a sleep disturbance that occurs at least three nights per week and lasts for at least three months. "Acute insomnia" in the context of the present invention is insomnia that is not chronic insomnia.

[0123] The preferred method of determining whether an individual has insomnia is by the Athens Insomnia Scale (AIS). The AIS is measured by assessing eight factors (sleep induction, night-time awakening, final awakening, total sleep time, sleep quality, daytime well-being, daytime functional ability, and daytime sleepiness) rated on a scale of 0 to 3, and sleep is finally assessed from the cumulative score of all factors and reported as the individual's sleep outcome. A cutoff score of 6 or more on the AIS is used to establish a diagnosis of insomnia (Soldatos et al. Journal of Psychosomatic Research, 48(6), 555-560).

[0124] In one embodiment, the presence of insomnia is defined by an AIS of 6 or more, for example 6-24, or 12-24, or 18-24.

[0125] Insomnia and similar sleep disorders may alternatively or additionally be diagnosed by a clinical interview with a psychologist and / or may involve the completion of self-report questionnaires and sleep diaries. Suitable questionnaires for determining the presence of insomnia in the context of the present invention are the Insomnia Severity Index (Bastien et al., 2001, Sleep Med 2:297-307) and the Insomnia Symptoms Questionnaire (Spielman et al., 1987, Sleep 10:45-56).

[0126] In one embodiment, the sleep disorder is restless legs syndrome. The presence of restless legs syndrome can be determined by a specialized physician and / or sleep specialist, such as according to the criteria established by the International Restless Legs Syndrome Study Group. a) a strong, often irresistible urge to move the legs, usually accompanied by an unpleasant sensation b) symptoms begin or worsen while at rest, e.g., while sitting or lying down; c) symptoms are partially or temporarily relieved by activities such as walking or stretching; d) Symptoms are worse at night, e) The symptoms cannot be explained solely by another medical or behavioral condition.

[0127] In one embodiment, the sleep disorder is narcolepsy. The presence of narcolepsy can be determined by a specialized physician and / or sleep specialist, such as based on one or more of a sleep history, a sleep record, a sleep test (e.g., polysomnography), multiple sleep latency tests, a genetic test, and a lumbar puncture.

[0128] In one embodiment, the sleep disorder is sleep apnea. The presence of sleep apnea may be determined by a specialized physician and / or sleep specialist, such as based on an overnight polysomnography and / or a home sleep test.

[0129] (Collagen hydrolysate-based composition) In one embodiment, the composition (for use) comprises one or more further compounds capable of regulating sleep.

[0130] In a preferred embodiment, the composition (for use) relates to a composition comprising at least 60% by weight, preferably at least 65% by weight, more preferably at least 70% by weight, even more preferably at least 75% by weight of collagen hydrolysate as active ingredient, based on the total weight of active ingredients and / or the total weight of compounds capable of regulating sleep.

[0131] In a preferred embodiment, the composition of the invention (for use) relates to a composition comprising collagen hydrolysate as active ingredient in an amount of at least 90% by weight, preferably at least 95% by weight, more preferably at least 97.5% by weight, even more preferably at least 99% by weight, based on the active ingredients and / or compounds capable of regulating sleep in the composition, the composition may comprise one or more further compounds capable of regulating sleep.

[0132] In one embodiment, for example, a composition for use as disclosed herein consists (essentially) of collagen hydrolysate relative to the active ingredients and / or compounds capable of regulating sleep in the composition.

[0133] In one embodiment, for example, the compositions for the uses disclosed herein are (essentially) free of further compounds and / or active ingredients capable of regulating sleep.

[0134] In one embodiment, for example, a composition for use as disclosed herein comprises at least 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99.99%, or 100% collagen hydrolysate by weight relative to the active ingredients and / or compounds capable of regulating sleep in the composition.

[0135] In one embodiment, for example, a composition for a use disclosed herein comprises at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70% or 80% by weight of one or more further compounds and / or active ingredients capable of regulating sleep.

[0136] In one embodiment, the present invention relates to a composition suitable for the use disclosed herein, comprising at least 70% by weight, preferably at least 80% by weight, more preferably at least 97.5% by weight, even more preferably at least 99% by weight of collagen hydrolysate as active ingredient and one or more further compounds capable of regulating sleep, based on the total weight of the active ingredients and / or compounds capable of regulating sleep in the composition.

[0137] In one embodiment, the composition of the present invention (for use) is a sleep improving composition.In one embodiment, the composition of the present invention (for use) is suitable for improving sleep.In one embodiment, the composition of the present invention is suitable for improving one or more of the following: wakefulness at sleep onset, sleep time, sleep quality, sleep pattern, daytime sleepiness, and cognitive function after sleep.

[0138] In various embodiments, the present invention relates to uses of the collagen hydrolysates disclosed herein, including administering the collagen hydrolysates in (sleep enhancing) compositions disclosed herein.

[0139] (General definition) The terms "comprising" or "to comprise" and their conjugations are used in the context of the present invention in an open-ended sense to indicate that the items following the word are included but not to exclude items not specifically mentioned.

[0140] Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0141] In the context of the present invention, a level is considered to be "increased" or "decreased" if it is at least 1% higher or lower (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher or lower) than the corresponding level in the control or reference, respectively. Additionally or alternatively, a level in a sample may be considered to be increased or decreased if it is statistically significantly higher or lower, respectively, compared to the level in the control or reference (including compared to an earlier time point), regardless of the magnitude of the change. The term "reduce" may be used interchangeably with the term "decrease" in the context of the present invention. [Brief description of the drawings]

[0142] [Figure 1A] Concentration-time curves of free Hyp response [μg / mL] after ingestion of the studied products (mean, SD). n=6, CH=collagen hydrolysate; LMW=low molecular weight; HMW=high molecular weight. [Figure 1B] Concentration-time curves of total Hyp response [μg / mL] after ingestion of the study products (mean, SD). n=6, CH=collagen hydrolysate; LMW=low molecular weight; HMW=high molecular weight.

[0143] Working Example

[0144] Example 1 Example 1 examines the effect of collagen hydrolysate (CH) administration on sleep in men with sleep disorders.

[0145] method Sample size estimation Sample size was determined from a priori power analyses (G*power 3.1.9.2, Microsoft Windows) for the two primary outcome measures: sleep onset latency (SOL) and sleep efficiency (SE) measured by polysomnography (PSG). Sample size was estimated using a mean difference of 10 min (10 min SD) for SOL and 4% (4% SD) for SE. With two-sided and an alpha of 0.05, it was calculated that 10 or more participants were required to achieve 80% statistical power.

[0146] participants Thirteen athletic men with sleep disorders provided written informed consent to participate in this study. Participant characteristics are shown in Table 1. Height and weight were recorded at the time of habituation. Inclusion criteria were: 1) male, 18-35 years of age; 2) Athens Insomnia Scale (AIS) score ≥ 6, indicating difficulty sleeping; 2) Morningness-Eveningness (MEQ) score 31-69, indicating someone who is neither an extreme morning person nor an extreme evening person; and 3) scheduled exercise ≥ 3 times per week in the past 6 months. Participants completed a health history questionnaire and were excluded if they used sleeping medications, had a diagnosed sleep disorder, reported excessive use of substances that negatively affect sleep such as drugs, alcohol, or smoking, had regularly used anti-inflammatory medications (within 2 weeks of participation), had food allergies, or had any other contraindications to the study procedures.

[0147] [Table 1]

[0148] Experimental design Prior to the supplementation trial, participants completed a familiarization trial in which their physical activity and sleep were monitored over 7 days and nights at home. This was to characterize their exercise and sleep schedules (i.e., bedtime and wake-up time) for the supplementation trial and to familiarize them with the protocol and equipment. No supplements were consumed during these 7 days. Sleep was monitored by actigraphy and subjective sleep quality for 7 days, followed by PSG for the final 2 nights (nights 6 and 7).

[0149] Following this habituation trial, participants were randomized in a double-blind fashion to consume 15 g of CH (collagen hydrolysate) in 200 ml, or placebo (PLA), 1 hour before bedtime for 7 days (trials were separated by 7 days). CH was given for 7 days, as this period of consumption has previously been shown to positively affect physiological markers.

[0150] Sleep monitoring during both trials was the same as in the habituation trial. Surveys to assess fatigue and sleepiness were also completed daily. On day 7, participants reported to the laboratory in the evening (≥16:00), venous blood and urine samples were taken, a telethermography pill (E-celcisus Performance Pill, Body Cap, France) was consumed, and a battery of cognitive function tests were performed. Participants returned to the laboratory after an overnight fast on the morning of day 8 (09:00–10:00) to take another blood and urine sample and to repeat the cognitive function tests. Participants were instructed to maintain the same bedtime and wake-up times as in the habituation week. On day 7 of both trials, participants followed a low-protein diet and consumed a standardized dinner. No exercise was allowed on day 7. Participants were instructed to repeat the food intake and physical activity patterns recorded during the habituation week.

[0151] Administration Participants consumed 15 g / day of CH (molecular weight approximately 2000 Da) derived from bovine hide or an inactive, taste-matched placebo control as a 200 ml liquid bolus without food, 1 hour before scheduled bedtime (recorded in a habituation trial), for 7 days. Both supplements were provided in identical opaque bottles by Rousselot BV (Ghent, Belgium). Investigators remained blinded until data analysis was completed. Participants reported no adverse effects from the supplements.

[0152] Athens Insomnia Scale and Morningness-Eveningness Questionnaire The AIS is an 8-item survey assessing sleep difficulties over the past month. A score of ≥6 indicates poor sleep (Soldatos et al. Journal of Psychosomatic Research, 48(6), 555-560). The AIS is based on the International Classification of Diseases, 10th Revision (ICD-10) diagnostic criteria and has good reliability and validity in adult populations. It also has good reliability in athletic populations. The MEQ measures circadian rhythm and distinguishes individuals into morning and evening types. A score of <31 indicates extreme evening type; a score of 69 indicates extreme morning type. Extreme morning types or evening types were excluded to increase the homogeneity of the sample.

[0153] Sleep and training diaries, and actigraphy Using a sleep diary, participants self-reported their time in bed, lights out, SOL, number of awakenings, awakening length, awakening time, and wake-up time during the trial. Any daytime sleep was also recorded. A training diary was used to record the intensity and amount of scheduled exercise completed each day. Intensity (1 = very low intensity, 10 = maximum intensity) was multiplied by duration (min) to obtain a composite score of training load. Participants also wore a wristwatch actigraph (ActiGraph GT9X Link, Actigraph, FL, USA) on their non-dominant arm to measure physical activity levels and sleep. Participants were instructed not to remove the watch except for showering or bathing. If the watch was removed at any other time, they were instructed to record this in the sleep diary. Actigraphy data were sampled in 30-s epochs and analyzed using the Cole Kripke algorithm (Cole et al., 1992) in ActiLife software (ActiGraph, FL, USA). Lights off, wake and rise times from the sleep diary were used for data analysis.Sleep variables analyzed from actigraphy were total time in bed (TIB), total sleep time (TST), SOL, SE, wake after sleep onset (WASO) and mean wake time.Physical activity was analyzed as the total time (min) spent in moderate to vigorous activity using the cut-off points suggested by Freedson and colleagues (Medicine&Science in Sports&Exercise,30(5),777-781).

[0154] Polysomnography Polysomnographic sleep was measured on the sixth and seventh nights of each trial using ambulatory monitoring (Embletta MPR with ST+Proxy, Natus Medical, CA, US). The sixth night was used for habituation collection only and was not used for analysis. Six electroencephalogram (EEG) channels (F3-M2; F4-M1; C3-M2; C4-M1; O1-M2 and O2-M1), two electrooculogram (EOG) channels (LEOG-M2 and REOG-M1), submandibular electromyogram (EMG) (three EMG electrodes) and PSG units were installed in the experimental room approximately 6-8 h before bedtime. All EEG sites were positioned using the international 10-20 electrode placement. PSG was scheduled to begin recording 30 min before bedtime. All recordings were collected at home. Participants were instructed to sleep in the same bed in the same environmental conditions and to be exposed to the same mental stimuli (e.g., phone use, television) 1 h before going to sleep for each trial. Any deviations were reported in a subjective sleep diary. Home-based PSG is considered valid and reliable as a clinical-based PSG setting. Recordings were then exported in EDF format, downloaded into Domino software v3.0.0.3 (SOMNOmedics, Germany) and manually scored in 30-s epochs using standard adult criteria from the American Academy of Sleep Medicine (AASM) ("AASM Scoring Manual-American Academy of Sleep Medicine", n.d.) by a registered sleep physiologist with over 20 years of experience. Each recording was labeled with a different, non-identifiable code so that the scorers (RNK and KA) were blinded to the identity and trial of the participants. Similar to actigraphy, lights off was marked when the participant first attempted to go to sleep and lights on was marked when the participant last woke up (both from the sleep diary). To assess intrarater reliability, three blinded PSG recordings were scored twice by RNK. To assess interrater reliability, KMA independently scored three PSGs previously scored by RNK.Each recording was scored for outcomes: time in bed (min), total sleep time (min), sleep latency (min), sleep efficiency (%), sleep latency N2 (min), deep sleep latency (min), REM sleep latency (min), total sleep duration (min), sleep stage change (counts), sleep stage change (counts / hr), awakening (counts), awakening (counts / hr), awakening after sleep onset (min), REM stage sleep # (%), N1 stage sleep # (%), N2 stage sleep # (%), N3 stage sleep # (%), night sleep # (%), awakening (counts), awakening (counts / hr).

[0155] Subjective Survey Subjective sleep quality was recorded on a 7-point Likert scale (1 = extremely bad, 7 = extremely good). Sleepiness was measured using the Karolinska Sleepiness Scale (KSS, 1 = extremely alert, 10 = extremely sleepy, unable to stay awake). Fatigue was measured on a 100 mm visual analogue scale (0 mm = not at all tired, and 100 mm = extremely tired). Sleep quality was recorded on awakening, and KSS and fatigue scores were recorded on awakening at 14:00 and 1 hour before going to bed. These were recorded daily with the average values ​​over the 7 days used for analysis.

[0156] Cognitive function Participants completed a battery of cognitive tests on a laptop that took approximately 10 minutes. Three to five practice trials were given before every test. The first test was a simple reaction time (RT), in which participants were instructed to press the space bar when a green circle appeared on a blank screen. The second test was a choice RT, in which participants were presented with a right or left arrow and had to press the arrow key that matched the direction on the screen. For both, the mean and fastest reaction times for correct responses were analyzed. There were 10 and 14 attempts for simple RT and choice RT, respectively. Participants then completed a digit span test, which measures attention and short-term recall. To complete this test, a series of 3-14 digit numbers was projected on the screen and participants were then instructed to type the numbers in the correct order. The length of the longest correct span across 12 trials was used for the analysis. Finally, participants completed the Stroop color word test, which measures attention and processing speed but primarily measures the ability to inhibit cognitive inference. For the baseline level, participants were presented with color names (written in black) on a white background and instructed to correctly identify the color name by pressing the right to left arrow. For the interference level, participants were presented with colored names on a white background and asked to correctly identify the font color while ignoring the incongruent color named on the screen (e.g., if "green" is written in red font, red is the correct answer). Participants had to respond as quickly as possible using the left or right arrow key. Outcomes were the percentage of correct responses, the mean RT (ms) of correct responses, and baseline (14 trials) and interference (20 trials) levels.

[0157] Dietary management Participants were instructed to record their food intake on day 7 of the habituation trial and to repeat this intake in the two supplementation trials. On this day, they were instructed to limit their intake of protein-rich foods (a list was provided), to avoid caffeine consumption after 11:00, and to attend the laboratory ≥2 h after a meal. Participants were given a low-protein dinner for home consumption consisting of 1 × 250 g pack of White Golden Vegetable Rice (Tesco, PLC, Herts, UK) and 2 × 37 g Nutri-Grain Blueberry Bars (Kellogg, Michigan, USA). After this meal, they avoided any food or liquid, except water and their respective supplement, from 1 h before sleep until after the laboratory visit on day 8.

[0158] Blood and urine samples Blood and urine samples were collected on the evening of day 7 and the morning of day 8. Urine samples were collected in plastic containers. Venous blood samples were obtained by venipuncture in 1x10 ml vacutainers for serum and 1x10 ml vacutainers coated with EDTA for plasma. The EDTA vacutainers were immediately centrifuged at 3000 rpm (4°C) for 10 min and the serum vacutainers were left for 30 min before centrifugation. Urine samples and plasma and serum supernatants were subsequently stored in aliquots at -80°C. Samples were analyzed for high-sensitivity c-reactive protein (hs-CRP), interleukin-6 (IL-6), cortisol, and noradrenaline, normetanephrine, as these are sensitive to changes in sleep quality (Faraut et al., 2012). IL-6 was analyzed in plasma using a commercially available ELISA kit (R and D Systems, MN, USA). The interassay coefficient of variation (CV) was <6.9%.

[0159] Measurement of serum cortisol, urinary noradrenaline and normetanephrine by LC-MS / MS The liquid chromatography tandem mass spectrometry (LC-MS / MS) method was performed with a Waters Acquity I-class UPLC system coupled to a Xevo TQ-XS tandem mass spectrometer (Waters Corp., Milford, MA, USA) operated in positive electrospray mode. Serum cortisol was extracted using an Extrahera™ automation system (Biotage, Uppsala, Sweden) under positive pressure provided by a nitrogen generator. In a 2 mL well plate, 200 μL of calibration standards (Chromsystems, Munich, Germany NIST SRM971 traceable), quality control substances and serum samples were added to each well along with 50 μL of an internal standard solution containing cortisol-d4 (IsoSciences, King of Prussia, PA, USA). Then, 200 μL of isopropanol:water 50:50 (v / v) was added to dissociate the bound proteins. After mixing, the samples were loaded onto an ISOLUTE® Supported Liquid Extraction (SLE+) 400 μL plate (Biotage). Elution was performed by adding 750 μL of methyl tertiary butyl ether (MTBE) in two cycles. The eluents were collected in the corresponding deep-well plate. At each stage, positive pressure was applied to remove residual solvent. The samples were then dried to completion under a gentle stream of nitrogen gas heated to 60° C., then reconstituted with 100 μL of 50:50 methanol / water before being vortexed and sealed. 10 μL of the extract was injected into the LC-MS / MS. Chromatographic separation was achieved using a CORTECS™ Core-Shell C18 50×2.1 mm, 2.7 μm, reversed phase (Waters Corp., Milford, MA, USA) column heated to 30° C. The mobile phase used was (A) water and (B) methanol in 0.1% formic acid, pumped at a flow rate of 0.4 mL / min at 70:30% (A:B), gradually increased to 100% (B), then returned to the starting gradient in 4 min. Tandem mass spectrometry detection was based on mass-to-charge (m / z) precursor to product ion transitions specific for each compound, i.e., cortisol (363.3>121.2) and cortisol-d4 (367.3>121.2).The interassay CV for serum cortisol was <6.0% over the assay range of 0.3 nmol / L to 806 nmol / L.

[0160] Urinary noradrenaline and normetanephrine were extracted using solid-phase extraction (Chromsystems Biogenic amino#80600, Munich, Germany) according to the manufacturer's instructions. The assays were calibrated using human urine-based calibration standards and controls (Chromsystems) traceable to certified reference materials. m / z transitions were noradrenaline (152.1>107.1), normetanephrine (166.1>106.1), noradrenaline-d6 (158.1>111.1) and normetanephrine-d3 (169.1>109.1). Interassay CV for noradrenaline was <6.7% over an assay range of 23.6 nmol / L to 4421 nmol / L; normetanephrine CV was 3.4% to 6.2% over an assay range of 20.5 nmol / L to 10311 nmol / L. Urine results from LC-MS / MS analysis were adjusted for variations in renal function by dividing by urinary creatinine. Urine creatinine was analyzed using a Roche second generation kinetic colorimetric assay based on the Jaffe method performed on a COBAS® C501 analyzer (Roche, Burgess Hill, UK). Interassay CV was <2.1%. Final results are expressed as nmol / L per mmol / L creatinine. Data for noradrenaline are not presented due to analytical difficulties in some samples.

[0161] High-sensitivity serum C-reactive protein (hs-CRP) Serum hs-CRP was measured using a particle-enhanced immunoturbidimetric assay analyzed on a COBAS® C501 analyzer (Roche, Burgess Hill, UK). Interassay CV was <2.6% over the assay working range of 1.43 nmol / L to 190 nmol / L.

[0162] Data analysis Data analysis was performed using SPSS (IBM SPSS Statistics for Windows, version 27.0, IBM Corp., Armonk, NY). Normality was assessed by plotting residuals against predicted values, checking histograms of residuals and skewness and kurtosis, as well as homogeneity of variance. Variables collected by PSG and actigraphy, sleep quality, and physical activity were measured using paired t-tests. Any variables not normally distributed were analyzed with nonparametric Wilcoxon signed rank tests. Actigraphy and subjective sleep data are the average of 7 nights of supplementation. Core temperature, KSS, fatigue, and all cognitive and hematological outcomes were analyzed with linear mixed models. Time, condition (CH vs. CON), and time*condition interaction effects were included as fixed factors, and participant was included as a random factor. Models were run using a maximum retention approach. Thus, random intercepts and random slopes were included for all within-subject variables. Covariance type was scaled identity, and model estimation was restricted maximum likelihood. Bonferroni correction was used to adjust post hoc tests for multiple testing. Intra- and inter-rater reliability for PSG variables was assessed with intraclass correlation coefficients (ICC) using absolute agreement and two-way mixed-effects models (as in SPSS). Values ​​<0.50 were considered poor reliability, 0.50–0.75 were considered moderate reliability, 0.75 and 0.90 were considered good reliability, and >0.90 was considered excellent reliability. P<0.05 was considered statistically significant. Effect sizes in Hedges' g are reported for pairwise comparisons between conditions.

[0163] result The main finding was that 7 days of CH supplementation improved overall sleep compared to placebo control, with the greatest effects seen in 1) reduced awakenings as measured by PSG and subjective sleep diary, and 2) improved cognitive performance as measured by the Stroop test.

[0164] The frequency of awakenings was significantly less in CH (1.3 ± 1.5 counts) vs. CON (1.9 ± 0.6 counts) (P = 0.025; g = 0.539). There was a time * condition interaction effect for correct responses in the baseline Stroop test (P = 0.007), and post-hoc tests revealed that participants' responses were more accurate during CH trials (P = 0.009; g = 0.573).

[0165] This study suggests that CH may be used as a non-pharmacological strategy to enhance sleep in individuals with sleep disorders, particularly those who are physically active, such as athletes.

[0166] Example 2 Example 2 examines the effects of various collagen hydrolysate-based formulations on recreationally active individuals with sleep disorders.

[0167] method Example 2 uses a similar experimental design as Example 1.

[0168] participants Participants were recreationally active, normal-weight male adults (ages 18-30) with sleep disorders based on an Athens Insomnia Scale (AIS) threshold of ≥6.

[0169] Formulation and Administration Drinks (volume 150 ml) are prepared based on the formulation according to Table 2. CH and placebo controls are the same as in Example 1. The total amount of sleep enhancing active ingredient or placebo is always 15 g. The dose is administered 60 minutes before bedtime each day for 7 days.

[0170] [Table 2]

[0171] Bioavailability The bioavailability of collagen hydrolysates after oral administration was determined in plasma samples.

[0172] Sleep parameters Sleep parameters are measured according to Example 1.

[0173] The number of awakenings is measured by PSG and subjective sleep diary.

[0174] Cognitive performance is measured by the Stroop test.

[0175] result The greatest overall improvement in sleep was seen when 100% collagen hydrolysate was administered as the active ingredient without other additives known to enhance sleep (Table 3).

[0176] The effect of 100% collagen hydrolysate is already measurable within one week, but this effect is delayed when combined with other additives.

[0177] Participants taking formulations #3-6 reported more complaints about fatigue, mood, or psychomotor and neurocognitive performance.

[0178] [Table 3]

[0179] The overall improvement in sleep has been found to be related to the amount of collagen hydrolysate in the composition (compared to other sleep enhancing compounds).

[0180] Plasma profiles showed that oral collagen hydrolysate administered relatively close to bedtime was favorable and bioavailability correlated with improved sleep.

[0181] Example 3 Example 3 shows the effect (of collagen hydrolysates obtained from different sources or having different molecular weights).

[0182] (the purpose) To investigate the single-dose bioavailability of collagen hydrolysates of different average molecular weights from different sources.

[0183] Similar bioavailability of collagen hydrolysate is believed to indicate similar functionality, such as in improving sleep.

[0184] method Bioavailability was assessed by evaluating the incorporation of free and peptide-bound hydroxyproline (Hyp) as a marker amino acid.A randomized, double-blind, crossover clinical trial was conducted in healthy volunteers.

[0185] A single dose of 10 g of collagen hydrolysate was provided at low average molecular weight (2000 Da). For bovine collagen hydrolysate, a single dose of 10 g of either low average molecular weight (2000 Da, "LMW") or high average molecular weight (5000 Da, "HMW") was provided. Blood was sampled for analysis over a 6-hour period following collagen hydrolysate ingestion.

[0186] result FIG. 1 shows the concentration-time curves of the free Hyp response (A) or total Hyp response (B) following ingestion of the study product.

[0187] Ingestion of 10 g of collagen hydrolysate increased plasma free Hyp concentration (ΔC max ) was found to be significantly increased, with mean coefficients of 7.2 for fish, 9.9 for porcine and 6.2 for both bovine low molecular weight (LMW) and high molecular weight (HMW) collagen hydrolysates. Regarding the bioavailability of free Hyp, there were no significant differences between the increments of area under the curve (iAUC) of the investigated products. In addition, the Hyp content in blood samples after total hydrolysis was determined. Significantly higher concentrations of total Hyp were determined compared to the iAUC of free Hyp and total Hyp. Total Hyp concentrations in plasma (ΔC max) was also greatly increased for either the fish or porcine collagen hydrolysates, as well as for both the bovine collagen hydrolysate LMW and the bovine collagen hydrolysate HMW.

[0188] Overall, the results show that uptake of collagen hydrolysates in blood appears similar for collagen hydrolysates from porcine, bovine and fish sources, and for hydrolysates with relatively high average molecular weight (5000 Da) and relatively low average MW (2000 Da).

[0189] A similar approach was used to study glycine and proline, which showed no significant differences between the uptake kinetics of different collagen hydrolysate sources or molecular weights, reaching a maximum between 60 and 130 min. max and T max ), the intake of free amino acids is very comparable between collagen hydrolysates of different animal sources and different molecular weights.

Claims

1. 1. Use of collagen hydrolysate as an active ingredient in the non-therapeutic improvement of sleep, wherein said collagen hydrolysate is administered in a composition comprising at least 70% by weight of collagen hydrolysate relative to the total weight of active ingredients in the composition.

2. 2. The use of claim 1, wherein the collagen hydrolysate improves one or more selected from the group consisting of sleep onset time, sleep duration, sleep quality, sleep pattern and cognitive function after sleep.

3. 3. The use according to claim 1 or claim 2, wherein the collagen hydrolysate improves wakefulness after sleep onset and / or daytime sleepiness.

4. Use according to any one of claims 1 to 3, wherein the amount of collagen hydrolysate in the composition is at least 80% by weight relative to the compound capable of regulating sleep in the composition.

5. Use according to any one of claims 1 to 4, wherein the amount of collagen hydrolysate in the composition is at least 97.5% by weight relative to the compound capable of regulating sleep in the composition.

6. Use according to any one of claims 1 to 5, wherein the amount of collagen hydrolysate in the composition is at least 99% by weight relative to the compound capable of regulating sleep in the composition.

7. The use according to any one of claims 1 to 6, wherein the collagen hydrolysate is administered in a composition consisting essentially of collagen hydrolysate and / or essentially free of further compounds capable of regulating sleep.

8. The use according to any one of claims 1 to 6, wherein the composition does not contain any other active ingredients in addition to the collagen hydrolysate.

9. The use according to any one of claims 1 to 6, wherein the composition comprises one or more other active ingredients in addition to the collagen hydrolysate.

10. 10. The use according to claim 9, wherein the composition comprises one or more further compounds capable of regulating sleep.

11. The further compound capable of regulating sleep is Gamma aminobutyric acid (GABA) receptor agonists and / or enhancers of endogenous GABA receptor agonists; Melatonin, melatonin precursors, melatonin receptor agonists, and / or enhancers of endogenous melatonin; Serotonin, precursors of serotonin, serotonin receptor agonists and / or enhancers of endogenous serotonin; and Antiinsomnia drugs, The use according to claim 10, selected from the group consisting of:

12. 12. The use according to claim 10 or claim 11, wherein the further compound capable of regulating sleep is one or more selected from the group consisting of GABA receptor agonists, melatonin, L-theanine, 5-hydroxytryptophan and tryptophan.

13. The use according to any one of claims 10 to 12, wherein said further compound capable of regulating sleep is derived from a plant and / or is a plant extract.

14. 10. The use according to any one of the preceding claims, wherein the collagen hydrolysate is administered within 4 hours, preferably within 2 hours, before going to bed.

15. The use according to any one of the preceding claims, wherein the collagen hydrolysate is administered in a daily dose of 2 to 50 g, preferably in a daily dose of 10 to 20 g.

16. 10. The use according to any one of the preceding claims, wherein the collagen hydrolysate is administered daily or every other day.

17. 10. The use according to any one of the preceding claims, wherein the collagen hydrolysate is administered for at least one week.

18. 10. The use according to any one of the preceding claims, wherein the collagen hydrolysate has an average molecular weight of from 1000 to 7500 Da.

19. 10. The use according to any one of the preceding claims, wherein the collagen hydrolysate is administered to physically active individuals, preferably athletes.

20. A composition comprising a collagen hydrolysate for use in improving a sleep disorder, the composition comprising at least 70% by weight of collagen hydrolysate based on the total weight of active ingredients in the composition.

21. 21. The composition for use according to claim 20, wherein the sleep disorder is selected from the group consisting of insomnia, restless legs syndrome, narcolepsy and sleep apnea.

22. 22. The composition for use according to claim 20 or claim 21, wherein the collagen hydrolysate improves one or more selected from the group consisting of sleep onset time, sleep duration, sleep quality, sleep patterns and cognitive function after sleep.

23. A composition for use according to any one of claims 20 to 22, wherein the collagen hydrolysate improves wakefulness after sleep onset and / or daytime sleepiness.

24. A composition for use according to any one of claims 20 to 23, comprising at least 80% by weight of collagen hydrolysate relative to the compound capable of regulating sleep in said composition.

25. A composition for use according to any one of claims 20 to 24, comprising at least 97.5% by weight of collagen hydrolysate relative to the compound capable of regulating sleep in said composition.

26. A composition for use according to any one of claims 20 to 25, comprising at least 99% by weight of collagen hydrolysate relative to the compound capable of regulating sleep in said composition.

27. 27. The composition for use according to any one of claims 20 to 26, wherein the collagen hydrolysate is administered in a composition consisting essentially of collagen hydrolysate and / or essentially free of further compounds capable of regulating sleep.

28. A composition for use according to any one of claims 20 to 26, wherein the composition does not contain any other active ingredients in addition to the collagen hydrolysate.

29. A composition for use according to any one of claims 20 to 26, wherein the composition comprises one or more other active ingredients in addition to the collagen hydrolysate.

30. 30. The composition for use according to claim 29, wherein the composition comprises one or more further compounds capable of regulating sleep.

31. The composition for use according to claim 30, wherein said one or more further compounds capable of regulating sleep are as defined in any one of claims 11 to 13.

32. A composition for use according to any one of claims 20 to 31, administered as defined in any one of claims 14 to 17.

33. A composition comprising a collagen hydrolysate as an active ingredient in an amount of at least 97.5% by weight relative to the compound capable of regulating sleep in the composition, and one or more additional compounds capable of regulating sleep.

34. 34. The composition of claim 33 comprising at least 99% by weight collagen hydrolysate.

35. The composition according to claim 33 or claim 34, wherein said further compound capable of regulating sleep is as defined in any one of claims 11 to 13.

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