Lysozyme hydrolysate for proactive inhibitory control and control of impulsivity

Abstract

To provide a non-therapeutic method, composition, food product, nutraceutical, feed product, and the like for improving or maintaining (protecting) cognitive functioning.SOLUTION: A non-therapeutic method for improving cognitive functioning in a subject, the method comprising administering to the subject a composition comprising a subtilisin A hydrolysate of lysozyme. The improvement of cognitive functioning is one or more selected from the group consisting of improving inhibitory control, improving attention, improving control of impulsivity, and improving arousal, in a non-therapeutic method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a non-therapeutic method of improving or maintaining cognitive function in a patient, the method comprising administering to the patient a composition comprising a subtilidin A hydrolysate of lysozyme. Further, the present invention relates to a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof. Additionally, the present invention relates to the use of a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement, and / or feed product comprising a subtilidin A hydrolysate of lysozyme in a non-therapeutic method of improving or maintaining cognitive function in a patient, or a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement, and / or feed product comprising a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof. In particular, the present invention relates to non-therapeutic methods of improving or maintaining impulse control and / or improving or maintaining inhibitory control in a patient and / or to a subtilisin hydrolysate of lysozyme for use in a method for treating impaired impulse control and / or impaired inhibitory control in a patient in need thereof. [Background technology]

[0002] Modern society is an environment that increasingly demands near-constant vigilance and attention from human patients. At school, work, or university, while navigating busy traffic, at home, and when exposed to several (social) media sources; adequate cognitive function is both a prerequisite and an advantage for patients who must navigate all sources of signals, information, temptations, and sexual seduction. Patients may be at risk of insufficient reaction time whenever their work or environment demands faster reaction times. Social media, (video, internet) games, alcohol, cigarettes, real-life, real-time social interactions and encounters, etc., are all examples of situations requiring proper impulse control, adequate forward inhibitory control, and / or attention. Therefore, both impulse control and sustained attention are examples of core cognitive functions that play a critical role in daily functioning.

[0003] Healthy human patients experience a natural, gradual, and steady decline in cognitive function over time as they age throughout adult life. Because cognitive functions, such as those related to inhibitory control, forward-looking inhibitory control, attention, sustained attention, impulse control, alertness, and sustained vigilance, naturally decline over time, appropriately receiving, acquiring, and responding to information and signals of all types and sources therefore requires increased effort as a result of aging. For example, older adults may suffer from feeling lost or experience feelings of anxiety when interacting with (car) traffic, public transportation, crowds, etc., due to the decline in cognitive function. For example, over the years, professionals such as teachers, security guards, and bus drivers may experience the unpleasant effects of gradual decline in cognitive function when interacting intensively with young people.

[0004] Apart from the fact that cognitive function declines in healthy patients as a reality of life, some health problems and pathological conditions are associated with (the risk of) cognitive decline, such as inhibitory control, forward inhibitory control, attention, sustained attention, impulse control, alertness, and sustained wakefulness.For example, cognitive decline is observed in human patients suffering from one or more of the following: hyperactivity, neurodegenerative disease, Alzheimer's disease, dementia, Parkinson's disease, obsessive-compulsive disorder, obsessive-compulsive disorder, Tourette's syndrome, attention deficit hyperactivity disorder, psychiatric disorders, trichotillomania, and addiction, such as drug, narcotic, nicotine, alcohol, gaming, and gambling addiction.In particular, reduced or impaired forward inhibitory control, especially impaired impulse control, is an aspect that interferes with the daily life of these patients.In addition, patients suffering from one or more of attention deficit hyperactivity disorder, psychiatric disorders, schizophrenia, and hyperactivity experience the disadvantages of reduced cognitive function, such as impaired sustained attention, for example, impaired overall alertness.

[0005] Therefore, there is still a need to find a solution that improves cognitive functions in healthy patients, such as cognitive functions related to inhibitory control, forward inhibitory control, attention, sustained attention, impulse control, arousal, and sustained wakefulness. There is also still a need to find a solution that improves cognitive functions that have declined or are declining in healthy patients, such as cognitive functions related to inhibitory control, forward inhibitory control, attention, sustained attention, impulse control, arousal, and sustained wakefulness, the decline of which is associated with the natural phenomenon of cognitive deterioration as a result of aging. In addition, there is also a need to find a solution that makes it possible to improve or maintain cognitive functions in patients, or to stop or reverse the decline of cognitive functions in patients, such as cognitive functions related to inhibitory control, forward inhibitory control, attention, sustained attention, impulse control, arousal, and sustained wakefulness, the decline of which is associated with health problems or diseases associated with said cognitive decline.

[0006] Patent Document 1 relates to a composition for preventing or treating neurodegenerative diseases, particularly dementia such as Alzheimer's disease. The composition contains a lysozyme hydrolysate. The effect of the composition on the ability to perform normal daily tasks is evaluated using the mouse burrowing test [Non-Patent Documents 1-3]. Burrowing in mice is an activity of daily living (ADL: self, social, and domestic environment care) similar to ADL in humans. Disruption of the ability to perform ADL is an early sign of neurodegenerative diseases such as Alzheimer's disease. Patent Document 2, for example, relates to a composition for use in preventing or treating alertness, vigilance, and cognition, where cognition is defined as a complex set of skills related to areas such as problem solving, learning, memory, and language. The composition disclosed in Patent Document 2 may contain a lysozyme hydrolysate. In Patent Document 2, for example, the effects of the composition on alertness, vigilance, and cognition are assessed by applying the Mackworth Clock test [Non-Patent Document 4] and the Critical Tracking task [Non-Patent Documents 5 and 6]. The Mackworth Clock test is designed to define vigilance and sustained attention, where vigilance describes the psychological readiness to receive and respond, which, unlike attention, does not necessarily have to be consciously experienced [Non-Patent Document 4]. The Critical Tracking task requires and tests focused attention and visual-motor control [Non-Patent Document 6]. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2017 / 144443 [Patent Document 2] International Publication No. 2009 / 133055 [Non-patent literature]

[0008] [Non-Patent Document 1] Deacon, R., "Assessing burrowing, nest construction, and hoarding in mice," Journal of Visualized Experiments, January 2012, Vol. 59, e2607, pp. 1-12. [Non-patent document 2] Jirkof, P., "Burrowing and nest building behavior as indicators of well-being in mice," Journal of Neuroscience Methods, 2014, 234, 139-146. [Non-patent document 3] Deacon, R.M.J., "Burrowing: A sensitive behavioral assay, tested in five species of laboratory rodents," Behavioral Brain Research, 2009, 200:128-133. [Non-patent document 4] Mackworth, N.H., "The breakdown of vigilance during prolonged visual search," Quarterly Journal of Experimental Psychology, 1948, Vol. 1, pp. 6-21. [Non-patent document 5] Jex, H.R., McDonnell, J.D., and Phatak, A.V., “A “critical” tracking task for man-machine research related to the operator’s effective delay time,” NASA contractor report NASA CR-616, November 1966, pp. 1–105. [Non-patent document 6] Petzoldt, T., Bellem, H., and Krems, J.F., “The critical tracking task—A potentially useful method to assess driver distraction?” Human Factors, 2014, Vol. 56, No. 4, pp. 784–803. Summary of the Invention [Problem to be solved by the invention]

[0009] The first goal of the present invention is to provide a composition, food product, dietary supplement, feed product, etc. for improving or maintaining (protecting) cognitive function. The cognitive function is preferably any one or more of inhibitory control, forward inhibitory control, attention, sustained attention, impulsive control, and alertness, more preferably any one or more of forward inhibitory control, sustained attention, and impulsive control. Specifically, the cognitive function is inhibitory control, forward inhibitory control, and / or impulsive control. More specifically, the cognitive function is all of inhibitory control, preferably forward inhibitory control, attention, preferably sustained attention, impulsive control, and alertness.

[0010] The object of the present invention is to provide a composition for improving or protecting cognitive function, wherein the cognitive function is any one or more of inhibitory control, attention, impulsive control, alertness, forward inhibitory control, and sustained attention, particularly inhibitory control, forward inhibitory control, and / or impulsive control.A further object of the present invention is to provide a composition for improving cognitive function in healthy individuals, wherein the individual is preferably a human patient.A further object of the present invention is also to provide a composition for use in a method for treating impaired cognitive function in a patient in need thereof, such as a human patient.Treating impaired cognitive function in a patient in need thereof means any one or more of reversing already-deteriorated cognitive function, improving the current state of cognitive function, improving cognitive function, preserving or protecting cognitive function, etc., wherein the cognitive function is any one or more of inhibitory control, forward inhibitory control, attention, sustained attention, impulsive control, and alertness, more preferably any one or more of forward inhibitory control, sustained attention, and impulsive control.Specifically, the cognitive function is inhibitory control, forward inhibitory control, and / or impulsive control. More particularly, the cognitive functions are inhibitory control, preferably forward inhibitory control, attention, preferably sustained attention, impulsivity control, and vigilance. [Means for solving the problem]

[0011] At least one of the above objects is achieved by providing a protein lysate of the present invention. More particularly, at least one of the above objects is achieved by providing a lysozyme lysate of the present invention, the lysate being a subtilidin A hydrolysate. In particular, at least one of the above objects is achieved by providing a composition comprising a subtilidin A hydrolysate of lysozyme.

[0012] A first aspect of the present invention relates to a non-therapeutic method of improving cognitive function in a patient, said method comprising administering to said patient a composition comprising a subtilidin A hydrolysate of lysozyme. Subtilidin A is a serine endopeptidase. Improving cognitive function can be an increase in the quality or degree of cognitive function for the benefit of the patient involved and / or for the benefit of patients interacting with the patient involved, and / or maintaining cognitive function at a current level, thereby preventing a deterioration or decline in cognitive function, for example as a result of the patient's natural aging process.

[0013] Preferred is a non-therapeutic method for improving cognitive function in patients, wherein improving cognitive function is any one or more of improving inhibitory control, improving attention, improving impulsive control, and improving alertness.In particular, improving inhibitory control is improving forward inhibitory control, and / or improving attention is improving sustained attention.Preferred is the non-therapeutic method of the present invention, wherein improving cognitive function is improving impulsive control and / or improving (forward) inhibitory control.More preferred is the non-therapeutic method of the present invention, wherein improving cognitive function is improving all of (forward) inhibitory control, (sustained) attention, impulsive control, and alertness.

[0014] The improvement in attention, such as sustained attention, and / or vigilance, such as general alertness, under the influence of ingestion of a subtilidin A hydrolysate of lysozyme, as assessed by administration of the Sustained Attention Test (SAT), i.e., a psychomotor vigilance test established for human patients, is at least 0.5% for sustained attention, and, for example, at least 1%, at least 2%, at least 2.5%, or at least 3% for general attention. For example, attention, such as sustained attention, and / or vigilance, such as general alertness, improves by 0.5%-10%, or 1%-3%, under the influence of daily ingestion of a subtilidin A hydrolysate of lysozyme, e.g., daily ingestion of a food product containing the subtilidin A hydrolysate of lysozyme. In particular, such improvement is achieved when a human patient, e.g., a healthy human patient, ingests a subtilidin A hydrolysate of lysozyme according to the present invention.

[0015] The improvement in cognitive control, such as proactive cognitive control (or proactive inhibitory control), and / or impulse control (control of impulses) under the influence of ingestion of subtilisin A hydrolysate of lysozyme, when assessed by application of an anticue task as established for human patients ["Switch hands! Mapping temporal dynamics of proactive manual control with anticues", JJ Adam, S. Jennings, TJ H Vand'Eerdt, PPM Hurks, PW M Van Gerven, Acta Psychologica, 2015, 161, pp. 137-144], is at least 0.5% for cognitive control, e.g., proactive cognitive control, and at least 1%, at least 2%, at least 2.5%, or at least 3% for impulse control. For example, forward cognitive control and / or impulse control improve by 0.5%-10%, or 2%-3%, under the influence of daily intake of a subtilisin A hydrolysate of lysozyme, e.g., daily intake of a food product containing a subtilisin A hydrolysate of lysozyme. Typically, the forward cognitive control is forward inhibitory control.

[0016] In embodiments, in non-therapeutic methods of improving cognitive function in a patient, the patient is a human patient and / or a healthy patient, preferably a healthy human patient, such as a hyperactive patient. The healthy patient may be diagnosed with metabolic syndrome.

[0017] In particular, in non-therapeutic methods of improving cognitive function in a patient, the patient is a male, preferably a human male, more preferably a human male at least 60 years old, preferably at least 65 years old, more preferably at least 67 years old, such as 60-105 years old, or 67-75 years old. One embodiment is a non-therapeutic method of improving cognitive function in a patient, wherein the patient is a human patient, more preferably a healthy human patient at least 60 years old, preferably at least 65 years old, more preferably at least 67 years old, such as 60-105 years old, or 67-75 years old.

[0018] A further aspect of the present invention relates to a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof. Preferred is a subtilidin A hydrolysate of lysozyme for use in said method, wherein the impaired cognitive function is any one or more of impaired inhibitory control, impaired forward inhibitory control, and impaired impulsivity control. The improvement in cognitive control, e.g., proactive cognitive control (or proactive inhibitory control) and / or impulse control (control of impulsivity), under the influence of ingestion of a subtilisin A hydrolysate of lysozyme, when assessed by application of an anti-cue task as established for human patients ["Switch hands! Mapping temporal dynamics of proactive manual control with anticues", JJ Adam, S. Jennings, TJH Vend'Eerdt, PPH Furks, PW M Van Gerven, Acta Psychologica, 2015, vol. 161, pp. 137-144], is at least 0.5% for cognitive control, e.g., proactive cognitive control, and at least 1%, at least 2%, at least 2.5%, or at least 3% for impulse control. For example, forward cognitive control and / or impulse control improve by 0.5%-10%, or 2%-3%, under the influence of daily intake of a subtilisin A hydrolysate of lysozyme, e.g., daily intake of a food product containing subtilisin A hydrolysate of lysozyme. Typically, the forward cognitive control is forward inhibitory control.

[0019] In particular, the subtilisin A hydrolysate of lysozyme is for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient in need thereof is suffering from one or more of hyperactivity, neurodegenerative diseases, Alzheimer's disease, dementia, Parkinson's disease, obsessive-compulsive disorder, Tourette's syndrome, attention deficit hyperactivity disorder, psychiatric disorders, trichotillomania, and addictions, such as addictions to drugs, narcotics, nicotine, alcohol, gaming, and gambling. The patient in need thereof is preferably a human patient. The impaired cognitive function to be treated is particularly impaired inhibitory control, impaired forward inhibitory control, and / or impaired impulse control, more preferably a combination of impaired inhibitory control, impaired forward inhibitory control, impaired impulse control, impaired attention, impaired sustained attention, and impaired alertness.

[0020] Also preferred is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the impaired cognitive function is any one or more of impaired attention, impaired sustained attention, and impaired vigilance. The improvement in attention, such as sustained attention, and / or vigilance, such as general vigilance, under the influence of ingestion of the subtilidin A hydrolysate of lysozyme, as assessed by administration of the Sustained Attention Test (SAT), i.e., a psychomotor vigilance test established for human patients, is at least 0.5% for sustained attention, and, for example, at least 1%, at least 2%, at least 2.5%, or at least 3% for general attention. For example, attention, such as sustained attention, and / or alertness, such as general wakefulness, improves by 0.5%-10%, or 1%-3%, under the influence of daily intake of a subtilidin A hydrolysate of lysozyme, e.g., daily intake of a food product containing subtilidin A hydrolysate of lysozyme. In particular, said improvement is achieved when a human patient, e.g., a healthy human patient, ingests a subtilidin A hydrolysate of lysozyme according to the present invention.

[0021] In particular, the subtilisin A hydrolysate of lysozyme is used in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient in need thereof suffers from any one or more of attention deficit hyperactivity disorder, psychotic disorder, schizophrenia, and hyperactivity.The patient in need thereof is preferably a human patient.The impaired cognitive function to be treated is particularly impaired attention, impaired sustained attention, and / or impaired vigilance, more particularly a combination of impaired inhibitory control, impaired forward inhibitory control, impaired impulsive control, impaired attention, impaired sustained attention, and impaired vigilance.

[0022] One embodiment is a subtilisin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient is a male, preferably a male human, more preferably at least 60 years old, preferably at least 65 years old, more preferably at least 67 years old, such as 60-105 years old, or 67-75 years old.

[0023] The present inventors have surprisingly found that daily intake of subtilisin A hydrolysate of lysozyme for at least four weeks improved cognitive function, particularly inhibitory control and / or impulsive control, more particularly a combination of inhibitory control, forward inhibitory control, impulsive control, attention, sustained attention, and alertness. See examples in the Examples section below.

[0024] According to embodiments of the present invention, in a non-therapeutic method of improving cognitive function in a patient, at least 30% by weight of a subtilidin A hydrolysate of lysozyme is lysozyme peptides having a molecular weight of less than 0.5 kDa. Further, for a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, at least 30% by weight of the subtilidin A hydrolysate of lysozyme is lysozyme peptides having a molecular weight of less than 0.5 kDa.

[0025] Preferred is a non-therapeutic method for improving cognitive function in a patient, said method comprising administering to said patient a composition comprising a subtilidin A hydrolysate of lysozyme, wherein the degree of hydrolysis of the lysozyme is at least 12%, preferably 15%-50%, more preferably 18%-40%, most preferably 19%-30%, e.g., 21%. Also preferred is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the degree of hydrolysis of the lysozyme is at least 12%, preferably 15%-50%, more preferably 18%-40%, most preferably 19%-30%, e.g., 21%.

[0026] For a preferred non-therapeutic method of improving cognitive function in a patient, the daily dose of subtilidin A hydrolysate of lysozyme is 0.5-20 grams per day, preferably 1-10 grams per day, more preferably 2-8 grams per day, for example, 5 grams per day. The daily dose is preferably administered to the patient as a single dose per day. Preferred is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the daily dose of subtilidin A hydrolysate of lysozyme is 0.5-20 grams per day, preferably 1-10 grams per day, more preferably 2-8 grams per day, for example, 5 grams per day. The daily dose is preferably administered to the patient as a single dose per day. Preferred is a daily dose of less than 10 grams, for example, 1-9.5 grams, as a single dose per day. Preferably, a human patient, e.g., a healthy human patient, is administered a daily dose of between 0.5 grams and less than 10 grams of subtilidin A hydrolysate of lysozyme, although higher doses, such as up to 20 grams, are also applicable. Typically, a daily dose is administered to a patient in need thereof, e.g., a (healthy) human patient, in accordance with the present invention for 4 weeks, preferably at least 8 weeks, 16 weeks, 40 weeks, 1 year, 2 years, 5 years, or for the remainder of the patient's life. Beneficial effects on cognitive function (e.g., improved impulsivity control, improved inhibitory control) are achieved after at least 4 weeks of daily ingestion of 0.5 grams to 20 grams, preferably less than 0.5-10 grams, of subtilidin A hydrolysate of lysozyme in accordance with the present invention. It is part of the present invention that the subtilidin A hydrolysate of lysozyme is provided once daily, or, for example, twice daily, e.g., in two equal doses, accumulating to 0.5-20 grams / day, or 1-10 grams / day.

[0027] One aspect of the invention relates to the use of a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement, and / or feed product comprising a subtilisin A hydrolysate of lysozyme in a non-therapeutic method of improving cognitive function in a patient. The subtilisin A hydrolysate of lysozyme is the hydrolysate of any of the foregoing aspects and embodiments of the invention.

[0028] One aspect of the present invention relates to a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement, and / or feed product comprising a subtilidin A hydrolysate of lysozyme for use in a method for the treatment of impaired cognitive function in a patient in need thereof. The subtilidin A hydrolysate of lysozyme is a hydrolysate of any of the foregoing aspects and embodiments of the invention. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 shows a flow chart of the study design; the study is a randomized, double-blind, placebo-controlled, crossover clinical trial. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition The term "inhibitory control," or "response inhibition," as used herein, has its ordinary scientific meaning and, as used herein, refers to the cognitive processes, more specifically, the executive functions, that enable individuals to inhibit their impulses and natural, habitual, or dominant behavioral responses to stimuli in order to select more appropriate behaviors consistent with achieving their goals. Self-control is an important aspect of inhibitory control. "Improving inhibitory control," therefore, refers to an increased inhibition of a patient's impulses. "Impaired inhibitory control," therefore, refers to a patient's reduced ability to inhibit impulses and to inhibit natural, habitual, or dominant behavioral responses to stimuli in order to select more appropriate behaviors consistent with achieving their goals.

[0031] The term "attention," as used herein, has its ordinary scientific meaning and herein refers to the behavioral and cognitive process of selectively focusing on a discrete aspect of information, whether considered subjective or objective, while ignoring other perceptible information. It is a state of wakefulness. It is the clear and vivid possession by the mind of one of what appears to be several simultaneous objects or trains of thoughts. Focusing, concentrating the mind, is of its essence. "Improving attention," therefore, relates to increasing concentration and selective focus.

[0032] The terms "impulsivity" or "impulsiveness," as used herein, have their ordinary scientific meaning and, as used herein, refer to a tendency to act impulsively, exhibiting behavior characterized by little forethought, reflection, or consideration of consequences. Impulsive acts are typically poorly considered, prematurely expressed, overly risky, or inappropriate for the situation, and they often result in undesirable outcomes that jeopardize long-term goals and strategies for success. "Improving impulse control," therefore, refers to, among other things, a decreased tendency to act impulsively. "Impaired impulsive control," therefore, refers to a situation in which a patient has an increased tendency to act impulsively and increasingly exhibits behavior characterized by little or no forethought, reflection, or consideration of consequences.

[0033] The term "alertness," as used herein, has its ordinary scientific meaning and herein refers to a state of active attention with high sensory arousal, e.g., alertness and readiness to encounter danger or emergency, or rapidity to perceive and act. Alertness is related to psychology as well as physiology. "Improving alertness" therefore refers to enhancing a state of active attention, increasing sensory arousal. "Impaired alertness" therefore refers to a state of short or no active attention.

[0034] The term "progressive inhibitory control," as used herein, has its ordinary scientific meaning and refers herein to how a patient prepares to stop an upcoming response tendency, i.e., anticipatory arrest. Arrest refers to the behavioral outcome when a patient stops an initial response tendency. "Improved prospective inhibitory control," therefore, relates to more effective and / or faster preparation to stop an upcoming response tendency. "Impaired prospective inhibitory control," therefore, relates to a reduced possibility or tendency, or even no possibility or tendency, for a person to prepare to stop an upcoming response tendency, resulting in delayed or no anticipatory arrest.

[0035] The terms "sustained attention" or "attention span," as used herein, have their ordinary scientific meaning and refer herein to the amount of focused time a person can devote to a task without becoming distracted. Distractibility occurs when an individual is uncontrollably drawn to some other activity or sensation. The ability to focus and sustain attention on a task is important for the achievement of a person's goals. "Improving sustained attention," therefore, refers to increasing the amount of focused time a person can devote to a task without becoming distracted and / or making it easier for a person to spend time on a task without becoming distracted. "Deteriorated sustained attention," therefore, refers to an environment in which a person can spend less focused time on a task without becoming distracted, or even no focused time at all.

[0036] The term "food" has its ordinary scientific meaning throughout and refers herein to a liquid, semi-liquid, or solid food product suitable for human and / or animal consumption.

[0037] The term "functional food" has its ordinary scientific meaning throughout the text and refers herein to a food product containing one or more active ingredients, in particular a subtilisin A hydrolysate of lysozyme according to the present invention as the active ingredient.

[0038] The term "food supplement" has its ordinary scientific meaning throughout the text and refers herein to a supplement applicable for human and / or animal consumption that contains an appropriate amount of one or more subtilisin A hydrolysates of lysozyme according to the present invention as a functional ingredient. The supplement may be in the form of a pill, sachet, powder, etc.

[0039] The term "patient" has its ordinary scientific meaning throughout and, as used herein, refers to any member of the class Mammalia, including, without limitation, humans, non-human primates, livestock, domestic animals, and laboratory animals.

[0040] The term "lysozyme hydrolysate" as used herein as a general term refers to a hydrolysate, eg, prepared in vitro, of lysozyme.

[0041] "Metabolic syndrome" refers to a number of interrelated clinical disorders, including obesity, insulin resistance and hyperinsulinemia, glucose intolerance, hypertension, dyslipidemia (hypertriglyceridemia and low HDL cholesterol levels), as described, for example, in Moller and Kaufman (Annual Rev. of Medicine, 2005, Vol. 56, pp. 45-62). In the context of the present invention, metabolic syndrome is preferably diagnosed by impaired glucose regulation / insulin resistance and two or more other criteria from the following: 1) impaired glucose regulation / insulin resistance, 2) abdominal obesity, 3) hypertriglyceridemia, 4) low HDL cholesterol levels, or 5) microalbuminuria, where the clinical features are further defined according to the WHO definition, as described in Moller and Kaufman.

[0042] "Degree of hydrolysis" is the percentage of peptide bonds in a protein hydrolysate that are broken.

[0043] Detailed Description of the Invention While the present invention has been described in relation to several embodiments, it is intended that alternatives, modifications, permutations, and equivalents thereof will become apparent to those skilled in the art upon reading the specification and studying the drawings. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0044] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.

[0045] Furthermore, the terms first, second, third, etc. in the description and claims are used to distinguish between like elements and do not necessarily dictate a sequential or chronological order. The terms are interchangeable under appropriate conditions, and embodiments of the invention may operate in orders other than those described or illustrated herein.

[0046] The embodiments of the invention described herein may be combined and operated in conjunction, unless otherwise specified.

[0047] Furthermore, although various embodiments may be referred to as "preferred" or "eg" or "for example" or "in particular," these are to be understood as representative ways in which the invention may be practiced, rather than as limitations on the scope of the invention.

[0048] The term "comprising," as used in the claims, should not be construed as being limited to the subsequently listed elements or steps: it does not exclude other elements or steps. It should be construed as specifying the presence of a feature, integer, step, or ingredient, as stated, but does not exclude the presence or addition of one or more other features, integers, steps, or ingredients, or groups thereof. Thus, the scope of the phrase "a composition comprising A and B" should not be limited to a composition consisting only of components A and B; rather, for the present invention, the recited components of the composition are only A and B, and further, the claims should be construed to include equivalents of those components.

[0049] In addition, 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 there is one and only one element. The indefinite article "a" or "an" therefore normally means "at least one."

[0050] The present inventors have surprisingly found that administering a composition containing a protein lysate to a human patient in a randomized, double-blind, placebo-controlled, crossover study results in improved cognitive function. In particular, the present inventors have found in the clinical trial that cognitive functions related to inhibitory control and / or attention, such as forward inhibitory control and sustained attention, are improved in human patients administered an effective amount of subtilisin A hydrolysate of lysozyme.

[0051] A first aspect of the present invention relates to a non-therapeutic method for improving cognitive function in a patient, comprising administering to the patient a composition containing a subtilidin A hydrolysate of lysozyme. In particular, an effective amount of subtilidin A hydrolysate of lysozyme is administered to the patient. For example, the patient is in need of immediate improvement in cognitive function. The inventors surprisingly found that daily ingestion of a composition containing subtilidin A hydrolysate of lysozyme resulted in improved cognitive function. Improvement was already evident after a time course of four weeks of daily ingestion. In a crossover clinical trial, patients showed improvements in cognitive function, i.e., inhibitory control, attention, impulse control, and alertness, after four weeks of ingestion of 5 grams of subtilidin A hydrolysate of lysozyme. Switching to daily ingestion of a placebo after a 2-8 week, most frequently a 4-week washout period, resulted in a decline in cognitive function to the level measured at the beginning of the initial 4-week ingestion period (see Figure 1 for a scheme of the clinical trial setup conducted). Patients who then started on daily ingestion of a placebo over 4 weeks showed no improvement in cognitive function, whereas switching to daily ingestion of a composition containing a subtilisin A hydrolysate of lysozyme, e.g., 200 ml of an aqueous solution containing 5 grams of the hydrolysate, resulted in an improvement in cognitive function after 4 weeks compared to the cognitive function measures measured at the beginning of the 4-week period and compared to patients who received a placebo over the same time course.

[0052] Preferred is the non-therapeutic method for improving cognitive function in patients, wherein improving cognitive function is any one or more of improving inhibitory control, improving attention, improving impulsive control and improving alertness.More preferred is the non-therapeutic method for improving cognitive function in patients, wherein improving cognitive function is any one or more of improving inhibitory control, improving impulsive control, and preferably said patient is a human patient, for example, a healthy human patient.Even more preferred is the non-therapeutic method for improving cognitive function in patients, wherein improving cognitive function is all of improving inhibitory control, improving attention, improving impulsive control and improving alertness.

[0053] In particular, improving inhibitory control is improving forward inhibitory control, and / or improving attention is improving sustained attention.

[0054] In an embodiment, in a non-therapeutic method for improving cognitive function in a patient, the patient is a human patient and / or a healthy patient, preferably a healthy human patient. The patient is, for example, a healthy human patient who is a hyperactive patient.

[0055] The improvement in attention, such as sustained attention, and / or alertness, such as general alertness, under the influence of ingestion of a subtilidin A hydrolysate of lysozyme, as assessed by administration of the Sustained Attention Test (SAT), i.e., a psychomotor vigilance test as established for human patients, is at least 0.5% for sustained attention, and e.g., at least 1%, at least 2%, at least 2.5%, at least 3% for general attention. For example, attention, such as sustained attention, and / or alertness, such as general alertness, improves by 0.5%-10%, or 1%-3%, under the influence of daily ingestion of a subtilidin A hydrolysate of lysozyme, e.g., daily ingestion of a food product containing subtilidin A hydrolysate of lysozyme.

[0056] The improvement in cognitive control, such as forward cognitive control, and / or impulse control under the influence of ingestion of a subtilisin A hydrolysate of lysozyme, when assessed by application of an anticue task as established for human patients, is at least 0.5% for cognitive control, e.g., at least 1%, at least 2%, at least 2.5%, at least 3% for impulse control. For example, forward cognitive control and / or impulse control improves by 0.5%-10%, or 2%-3% under the influence of daily ingestion of a subtilisin A hydrolysate of lysozyme, e.g., daily ingestion of a food product containing a subtilisin A hydrolysate of lysozyme.

[0057] Daily ingestion of a composition comprising a subtilidin A hydrolysate of lysozyme results in improved cognitive function in areas of cognition related to self-control, impulse control, sustained attention, and sustained wakefulness. In particular, daily ingestion of a composition comprising a subtilidin A hydrolysate of lysozyme results in improved cognitive function in areas of cognition related to self-control and impulse control. Therefore, the benefits of daily ingestion of a composition comprising a subtilidin A hydrolysate of lysozyme for a patient, preferably a healthy patient, e.g., a healthy human patient, are many and varied. In particular, when subtilidin A hydrolysate of lysozyme is ingested (daily) by a human patient for at least four weeks, preferably at least 0.5 grams / day and up to 20 grams per day, preferably less than 10 grams / day, e.g., 5 grams / day. For example, improved and increased alertness is beneficial in many everyday environments, such as while navigating busy traffic, attending a school class, a workplace workshop, a business meeting, a lecture at a university or conference, driving a truck overnight, or during an intercontinental airplane flight; for security guards, working as a ground controller at an airport or port, performing complex surgical procedures, during extended court proceedings, etc. Improved impulse control can contribute to an overall improved quality of life for both the patient and those with whom they interact. For example, improved impulse control can reduce aggression, for example, during everyday traffic, in crowded places, etc. Similar to the benefits associated with improved alertness for patients, improved attention, e.g., prolonged and / or uninterrupted attention and / or more focused, intense attention, also provides many similar benefits to patients.

[0058] Under the influence of intake of subtilisin A hydrolysate of lysozyme, preferably for at least four weeks, preferably daily, further benefits of improved attention, such as sustained attention, and / or alertness, such as general alertness, and / or cognitive control, such as forward-looking cognitive control, and / or impulse control are evident, for example, for students, such as schoolchildren or university students, while studying and preparing for exams, while attending lectures and courses, and while sitting for exams, and thus generally when intellectual tasks are to be performed. Typically, students are 17-26 years old, for example, 18-24 years old. Improved attention, such as sustained attention, and / or alertness, such as general alertness, and / or cognitive control, such as forward-looking cognitive control, and / or impulse control, for example, help students learn and / or absorb new knowledge while attending classes, and further help students recall acquired knowledge, for example, while taking exams or when subjected to (oral) tests.

[0059] Further benefits of improved attention, such as sustained attention, and / or alertness, such as general alertness, and / or cognitive control, such as proactive cognitive control, and / or impulse control under the influence of intake, preferably daily intake, of a subtilisin A hydrolysate of lysozyme are evident, for example, for sportsmen / sportswomen when playing or training for or during competitions at any level, and particularly when playing or training for and during competitions at the top level, and / or when performing in tournaments, such as Olympic Games, World Championships, etc. Improved attention, such as sustained attention, and / or alertness, such as general alertness, and / or cognitive control, such as proactive cognitive control, and / or impulse control, support the sportsman or sportswoman, for example, during their daily exercise routine and when spending several hours each day that require dedication and endurance, and are helpful to them, for example, while concentrating on winning through competitions and participating in matches.

[0060] Further benefits of improved attention, such as sustained attention, and / or alertness, such as general alertness, and / or cognitive control, such as proactive cognitive control, and / or impulse control, under the influence of intake, preferably daily intake, of subtilisin A hydrolysate of lysozyme, are evident, for example, for children, adolescents, young adults, and adults suffering from ADHD (attention deficit hyperactivity disorder), particularly for young adults suffering from ADHD, for example, those aged 14-22, preferably 15-20, or 16-20, e.g., 16-18. Improved attention, such as sustained attention, and / or alertness, such as general alertness, and / or cognitive control, such as proactive cognitive control, and / or impulse control, are beneficial for patients suffering from ADHD, for example, young adults suffering from ADHD, for example, while attending classes at school, concentrating on homework, studying, hobbies, social interactions, games, eating dinner, etc.

[0061] One embodiment is a non-therapeutic method of improving cognitive function in a patient, wherein the patient is a human patient selected from a student, e.g., a schoolchild or university student, an athlete or sportswoman, e.g., a top athlete or sportswoman, and from children, adolescents, young adults, and adults suffering from ADHD.

[0062] The present inventors have surprisingly found that daily ingestion of a composition containing subtilidin A hydrolysate of lysozyme, for example, at a daily dose of less than 10 grams / day, e.g., 1-9 grams / day, for at least 4 weeks, had a beneficial effect on cognitive function, reversing approximately 1-2 years of the natural decline in cognitive function due to normal aging in human patients. In other words, the magnitude and extent of improvement in any one or more of impulsivity, alertness, forward inhibitory control, and sustained attention after ingestion of a composition containing subtilidin A hydrolysate of lysozyme was similar to the patient's cognitive function as if he or she were 1-2 years younger. The decline in cognitive function over adult life is a normal and natural phenomenon. By daily ingestion of a composition containing subtilidin A hydrolysate of lysozyme, this natural deterioration in cognitive function is delayed by 1-2 years. One embodiment is a non-therapeutic method of improving cognitive function in a patient, wherein the patient is an adult human patient who is at least 18 years of age. Thus, improvement of up to 1-2 years of improved impulse control and / or improved inhibitory control, equivalent to reversal of the decline in impulse control and / or decline in impulsivity control due to normal aging in a human patient, is achieved by daily ingestion of less than 10 grams of subtilisin A hydrolysate of lysozyme according to the present invention for at least 4 weeks.

[0063] Furthermore, the present invention relates to a non-therapeutic method for improving cognitive function in a healthy human patient diagnosed with metabolic syndrome, wherein a composition comprising a subtilidin A hydrolysate of lysozyme is administered to the patient, particularly an effective amount of a subtilidin A hydrolysate of lysozyme is administered to the patient.

[0064] Preferably, the patient to whom the composition containing subtilidin A hydrolysate of lysozyme is administered needs to maintain or improve cognitive function or to prevent cognitive decline. As described above, daily ingestion of a composition containing subtilidin A hydrolysate of lysozyme by a human patient improves cognitive function and reverses the natural decline in cognitive function that occurs over 1-2 years. The human patient is a healthy patient and / or is 50 years of age or older, e.g., 60 years of age or older, 65-100 years of age, or over 75 years of age. Preferably, the patient needs improvement or reversal of already-decreased cognitive function, i.e., improvement of already-decreased or worsened cognitive function, such as sustained attention, forward inhibitory control, alertness, and impulsivity control. Typically, the patient is a human patient 65 years of age or older. Also preferred is a non-therapeutic method of improving cognitive function in a patient, wherein the patient is a human child, a human patient under the age of 18, or a human patient aged 18-65. For example, the human patient may be a student, such as a university student, an adolescent, a young adult, a young adult or adult suffering from ADHD, a sportsman or sportswoman, especially an elite sportsman or sportswoman.

[0065] Without wishing to be bound by theory, improved cognitive function in patients administered a composition containing a subtilidin A hydrolysate of lysozyme is related to improved peripheral vascular function induced by ingestion of the composition containing subtilidin A hydrolysate of lysozyme. The improved cognitive function observed in human patients includes, among other improvements, improved impulse control and improved sustained attention. The inventors measured these positive effects of ingestion of a composition containing subtilidin A hydrolysate of lysozyme on cognitive function in a series of cognitive tests, as detailed in the Examples section below: the Sustained Attention Test (SAT), known in the art, a vigilance task designed to assess an individual's ability for sustained attention, and an anti-cue task, known in the art, a reaction time task that assesses forward-looking cognitive control, particularly related to impulse control. Surprisingly, the improvements in cognitive function observed in clinical trials and in human patients who were subjected to daily intake of a composition containing subtilidin A hydrolysate of lysozyme declined again after the daily intake was discontinued (patients were switched to daily placebo intake after a 2-8 week washout period). Therefore, again without wishing to be bound by theory, sustained improvements in cognitive function, such as impulse control, forward inhibitory control, alertness, and sustained attention, occur and are maintained when a composition containing subtilidin A hydrolysate of lysozyme is taken daily and chronically. Long-term daily intake, for example by a human patient for at least 4 weeks, of a composition containing subtilidin A hydrolysate of lysozyme, preferably in an amount of less than 10 grams per day, e.g., 0.5-9.5 grams daily, then results in maintaining improved peripheral vascular function in association with adequate blood circulation through the brain, ultimately resulting in the observed beneficial effects of the composition on cognitive function.

[0066] One embodiment is a non-therapeutic method of improving cognitive function in a healthy human patient, wherein improving cognitive function is either improving forward inhibitory control or improving sustained attention in the patient, preferably improving forward inhibitory control. One embodiment is a non-therapeutic method, wherein forward inhibitory control is impulse control and / or wherein sustained attention is alertness, e.g., general alertness.

[0067] In particular, in non-therapeutic methods for improving cognitive function in a patient, the patient is a male, preferably a human male, more preferably at least 60 years old, preferably at least 65 years old, more preferably at least 67 years old, e.g., 60-105 years old, or 67-75 years old. In particular, elderly men aged 67 years or older benefit from the cognitive function-restoring effect of daily ingestion of a composition containing a subtilidin A hydrolysate of lysozyme. Comparing men and women divided into six groups based on gender and age (under 55 years old, 56-66 years old, and over 67 years old), men aged 67 years or older benefit to a greater extent from daily ingestion of a composition containing a subtilidin A hydrolysate of lysozyme. The beneficial effect was approximately doubled in these elderly men.

[0068] A further aspect of the present invention relates to a subtilidin A hydrolysate of lysozyme for use as a pharmaceutical. A further aspect relates to a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof. The patient may be a patient diagnosed with metabolic syndrome, i.e., a human patient. Another aspect is the use of a subtilidin A hydrolysate of lysozyme or a composition containing a subtilidin A hydrolysate of lysozyme for the manufacture of a (nutritional) composition for treating impaired cognitive function in a patient, preferably a human patient. In particular, an effective amount of subtilidin A hydrolysate of lysozyme is administered to the patient. For example, the patient is in need of direct improvement of cognitive function. For example, the patient is in need of direct improvement or reversal of already-decreased cognitive function. Further preferred is a subtilidin A hydrolysate of lysozyme for use in a method for preventing, preventing, improving, or slowing further decline of impaired cognitive function in a patient in need thereof. Also preferred is a subtilidin A hydrolysate of lysozyme for use in a method for preventing, preventing, improving or slowing cognitive decline in a patient in need thereof. The present invention also relates to a method for improving or reversing impaired cognitive function in a patient suffering from said impaired cognitive function, wherein a composition comprising subtilidin A hydrolysate of lysozyme is administered to the patient. In particular, an effective amount of subtilidin A hydrolysate of lysozyme is administered to the patient. Preferably, the patient suffering from said impaired cognitive function is in need of (direct) improvement of cognitive function or in need of (direct) halting (further) decline of cognitive function.

[0069] Preferred is a subtilisin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the impaired cognitive function is any one or more of impaired inhibitory control, impaired forward inhibitory control, and impaired impulsivity control.

[0070] Also preferred is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the cognitive function is any one or more of inhibitory control, forward inhibitory control, and impulsivity control. Preferably, the patient in need thereof is a human patient. Preferably, the patient in need thereof is administered a daily dose of 0.5-20 grams, preferably less than 10 grams per day of subtilidin A hydrolysate of lysozyme, and preferably, the patient in need thereof is administered a single dose of subtilidin A hydrolysate of lysozyme as a single dose or as multiple doses per day, for example, two half doses per day, for at least 4 weeks, for example 6 months, or 4 weeks to 10 years, or for the remaining life of the patient, e.g., human patient.

[0071] The improvement in attention, such as sustained attention, and / or alertness, such as general alertness, under the influence of ingestion of a subtilidin A hydrolysate of lysozyme, as assessed by administration of the Sustained Attention Test (SAT), i.e., a psychomotor vigilance test as established for human patients, is at least 0.5% for sustained attention, and e.g., at least 1%, at least 2%, at least 2.5%, at least 3% for general attention. For example, attention, such as sustained attention, and / or alertness, such as general alertness, improves by 0.5%-10%, or 1%-3%, under the influence of daily ingestion of a subtilidin A hydrolysate of lysozyme, e.g., daily ingestion of a food product containing subtilidin A hydrolysate of lysozyme.

[0072] The improvement in cognitive control, such as forward cognitive control, and / or impulse control under the influence of ingestion of a subtilidin A hydrolysate of lysozyme, when assessed by administration of an anti-cue task as established for human patients, is at least 0.5% for cognitive control, e.g., at least 1%, at least 2%, at least 2.5%, or at least 3% for impulse control. For example, forward cognitive control and / or impulse control improves by 0.5%-10%, or 2%-3%, under the influence of daily ingestion of a subtilidin A hydrolysate of lysozyme, e.g., daily ingestion of a food product containing a subtilidin A hydrolysate of lysozyme.

[0073] In particular, the composition comprising or the subtilidin A hydrolysate of lysozyme is for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient in need thereof is suffering from any one or more of hyperactivity, neurodegenerative diseases, Alzheimer's disease, dementia, Parkinson's disease, obsessive-compulsive disorder, Tourette's syndrome, attention deficit hyperactivity disorder, psychiatric disorders, trichotillomania, or addiction, such as addiction to drugs, narcotics, nicotine, alcohol, gaming, or gambling. Furthermore, the subtilidin A hydrolysate of lysozyme is for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient in need thereof is suffering from any disease associated with reduced, worsened, or impaired cognitive function. For example, the patient, such as a human patient, is suffering from reduced cognitive function due to (previous) trauma, infection, inflammation, ischemia, stroke, cardiovascular disease, etc. Cognitive functions are in particular inhibitory control and / or impulsive control, and preferably cognitive functions are a combination of inhibitory control, impulsive control, alertness and attention.

[0074] Also preferred is a subtilidin A hydrolysate of lysozyme, or a composition comprising subtilidin A hydrolysate of lysozyme, for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the impaired cognitive function is any one or more of: worsened or impaired attention, worsened or impaired sustained attention, and worsened or impaired vigilance.

[0075] In particular, the subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient in need thereof is suffering from any one or more of attention deficit hyperactivity disorder, psychotic disorder, schizophrenia, and hyperactivity. In addition, the subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient in need thereof is suffering from any disease or disorder associated with impaired attention, deteriorated or impaired sustained attention, and impaired or impaired wakefulness, other than these aforementioned health problems and diseases.

[0076] Preferably, the patient is a healthy patient, e.g., a human patient aged 50 years or older, e.g., 60 years or older, 65-100 years old, or over 75 years old. Preferably, the patient is a healthy patient. Preferably, the patient requires improvement or reversal of already-decreased cognitive function, i.e., improvement of already-decreased or worsened cognitive function, such as sustained attention, proactive inhibitory control, alertness, or impulsivity control. In particular, the patient requires improvement or reversal of already-decreased cognitive function, i.e., improvement of already-decreased or worsened cognitive function, such as sustained attention, proactive inhibitory control, or impulsivity control, or requires improvement or reversal of already-decreased cognitive function, i.e., improvement of a combination of already-decreased or worsened cognitive function, such as sustained attention, proactive inhibitory control, alertness, or impulsivity control. Typically, the patient is a human patient aged 65 years or older. Preferably, the human patient is a healthy human patient. Also preferred is a subtilisin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient is a human child under the age of 18, or wherein the patient is a human patient between the ages of 18-65.

[0077] One embodiment is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the patient is a male, preferably a male human, more preferably a male human at least 60 years of age, preferably at least 65 years of age, more preferably at least 67 years of age, for example 60-105 years of age, or 67-75 years of age. As outlined herein above, elderly males aged 67 years and older in particular will benefit from daily ingestion of a composition comprising a subtilidin A hydrolysate of lysozyme, when improvement of cognitive function is assessed.

[0078] According to embodiments of the present invention, in a non-therapeutic method of improving cognitive function in a patient, at least 30% by weight of a subtilidin A hydrolysate of lysozyme is lysozyme peptides having a molecular weight of less than 0.5 kDa. Further, for a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, at least 30% by weight of the subtilidin A hydrolysate of lysozyme is lysozyme peptides having a molecular weight of less than 0.5 kDa.

[0079] Preferred is a non-therapeutic method of improving cognitive function in a patient, said method comprising administering to said patient a composition comprising a subtilidin A hydrolysate of lysozyme, wherein the degree of hydrolysis of lysozyme is at least 12%, preferably 15%-50%, more preferably 18%-40%, most preferably 19%-30%, e.g., 21%. Also preferred is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the degree of hydrolysis of lysozyme is at least 12%, preferably 15%-50%, more preferably 18%-40%, most preferably 19%-30%, e.g., 21%.

[0080] According to the present invention, the composition comprises a subtilidin A hydrolysate of lysozyme. Thus, lysozyme is hydrolyzed using subtilidin A. Subtilidin A is an endopeptidase, more specifically a serine protease, and even more specifically a subtilase. Subtilidin A is commercially available, among others, under the trademark Alcalase. The composition is, for example, a nutritional composition or a food supplement.

[0081] How to obtain a subtilidin A hydrolysate of lysozyme is known in the art, and is described, for example, in WO 2006 / 009448 and Example 1.

[0082] Lysozyme is commercially available, for example, from Bouwhuis Enthoven (100% pure protein). Lysozyme can also be extracted from eggs and subsequently purified. In one embodiment of the present invention, the lysozyme subjected to subtilisin A hydrolysis is preferably purified lysozyme. The concentration of lysozyme in egg white is high (3-4%) compared to other lysozyme sources. A routinely used process for lysozyme purification is cation exchange chromatography. Lysozyme binds to an ion exchanger at its native pH (pH 9). This can be performed in a stirred tank reactor or a chromatography column. After elution from the adsorbent particles with salt, lysozyme is sufficiently pure for food applications. For further purification, anion exchange chromatography at pH 4 can be applied to obtain highly pure lysozyme suitable for pharmaceutical applications. The advantages of this purification process are that the starting material (egg white) remains unchanged, the process can be easily upscaled, only one adsorption step is required for food applications, and biological activity is maintained. Therefore, in one embodiment according to the present invention, the lysozyme to be subjected to subtilidin A hydrolysis is purified using cation exchange chromatography.

[0083] The subtilidin A hydrolysate of lysozyme preferably comprises a high proportion of di- and / or tripeptides, preferably at least 10, 20, 30, 40, 50, 60, 70% or more by weight of the subtilidin A hydrolysate of lysozyme, more preferably at least 20%, even more preferably at least 30%, and most preferably at least 40% by weight of the subtilidin A hydrolysate of lysozyme. In one embodiment, peptides with longer chain lengths and higher molecular weights, e.g., 4, 5, 6, 7, 8 or more amino acids, are therefore preferably present in an amount less than 60% by weight of the subtilidin A hydrolysate of lysozyme.

[0084] In one embodiment, the subtilidin A hydrolysate of lysozyme preferably comprises peptides less than 0.5 kD in a proportion of at least 25, 30, 40, or 45% by weight of the subtilidin A hydrolysate of lysozyme, preferably the subtilidin A hydrolysate of lysozyme comprises at least 30%, most preferably at least 40% by weight of peptides less than 0.5 kD. Preferably, the subtilidin A hydrolysate of lysozyme comprises at most 80, 70, 60, or 50% by weight of peptides less than 0.5 kD.

[0085] In one embodiment, a subtilisin A hydrolysate of lysozyme contains a distribution of fragments with molecular weights of <0.5 kD: 0.5 to 1.0 kD: >1 kD in the following ratio: 30-70: 70-15: 70-15. As a guide, fragments less than 0.5 kD correspond to fragments of less than 4-5 amino acids, while fragments of about 0.5-1.0 kD correspond to 4-9 amino acids, and fragments greater than 1 kD correspond to peptide fragments of more than 9 amino acids.

[0086] Preferably, the degree of hydrolysis of the subtilisin A hydrolysate of lysozyme is at least 15%, more preferably at least 20%, and most preferably at least 30%. The degree of hydrolysis is preferably measured using the TNBS method, e.g., as described in WO 2006 / 009448. The degree of hydrolysis is the difference between the number of free amino groups in the hydrolysate and the number of free amino groups in the intact protein, divided by the total amount of peptide bonds in the hydrolysate / intact protein. Generally, the degree of hydrolysis is expressed as a percentage, and therefore the resulting value is multiplied by 100%.

[0087] The molecular weight distribution of the subtilidin A hydrolysate of lysozyme is preferably 30-70% of the target peptides less than 0.5 kD and 15-70% of the peptides between 0.5 kD and 1 kD, based on the molecular weight of the subtilidin A hydrolysate of lysozyme. The molecular weight distribution, peptide chain length distribution, and maximum peptide weight of the hydrolysates can be determined by methods known in the art, such as SDS-PAGE analysis, HPLC analysis, MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry as described by Kaufmann, J. Biotechn. 41, 1995, pp. 155-175, and Soeryapranata et al., J. Food Sci. 67, 2002, pp. 534-538), HP-GPC (high-performance gel permeation chromatography as described by Terheggen-Largo et al., BMC Pediatrics 2, 2002, pp. 10), and Edman degradation (Siemensma et al., Trends Food Sci Technology 4, 1993, pp. 16-21). The maximum peptide molecular weight of the target protein is preferably less than 10 kD.

[0088] Alternatively, compositions similar to the subtilisin A hydrolysate of lysozyme of the present invention can be produced de novo using chemical synthesis methods. In particular, di- and tri-peptide libraries can be produced by combinatorial chemistry, thereby generating all possible combinations of di- and tripeptides. From this pool, or di- and tri-peptide library, a mixture can be constructed that has essentially the same activity on cognitive function as the hydrolysate described above.

[0089] Compositions containing subtilisin A hydrolysate of lysozyme preferably improve cognitive function, prevent cognitive decline, or improve or reverse already-declined cognitive function after regular, preferably daily, ingestion of an effective amount. In the context of this specification, an effective amount or effective dose of a composition is the amount of the composition required to achieve the claimed effect of the present invention, e.g., an in vivo therapeutic or prophylactic effect. The effective amount required to be administered to a patient depends on several factors, such as the patient (e.g., human or animal), the dosage form (daily, several times daily, weekly), and the composition and / or texture of the product. Determining an effective amount using routine experimentation is within the skill of the art. Preferably, an effective amount is at least 0.1 grams, more preferably at least 0.5 grams, even more preferably at least 1 gram, even more preferably at least 2 grams, and most preferably at least 4 grams of the claimed composition per day. For example, an effective amount is about 5 grams, e.g., 5.0 grams, administered to a patient, e.g., once daily. Preferably, the effective amount is at least 7 mg / kg of patient body weight per day, more preferably at least 14 mg / kg per day, even more preferably at least 28 mg / kg, and most preferably at least 56 mg / kg of patient body weight per day. For example, an effective amount is between 40 mg / kg of patient body weight per day and 100 mg / kg of patient body weight per day, e.g., for a patient weighing between 50 kg and 120 kg. Typically, the daily dose of subtilisin A hydrolysate of lysozyme for a human patient is less than 10 grams, e.g., 0.5-9 grams per day.

[0090] Thus, provided is a composition comprising a subtilidin A hydrolysate of lysozyme produced as described above or as described in Example 1, and optionally further enriched and / or purified for use according to the present invention. Also provided is a composition, particularly a food product, functional food product, and / or food supplement composition, comprising a suitable amount of subtilidin A hydrolysate of lysozyme for use according to the present invention.

[0091] Food supplements and food products containing at least one lysozyme subtilidin A hydrolysate according to the present invention can be prepared as known in the art. The food supplement may be in any dosage form, such as tablets, pills, powder sachets, gels, capsules, etc. Intake by patients is preferably oral. The food product may be in the form of a drink, solid or semi-solid food, such as a snack, dessert, sauce, whole meal, etc. Preferably, the food product is a product that is consumed regularly, preferably daily, such as a staple food, e.g., bread, noodles, soft drinks, dairy products, e.g., cheese, yogurt, fermented dairy products, milk, butter, etc. The lysozyme subtilidin A hydrolysate may therefore be added to a food base or incorporated into the food product during its manufacturing process. Accordingly, any existing food or food supplement product containing lysozyme subtilidin A hydrolysate for use according to the present invention is encompassed herein.

[0092] In a preferred embodiment, the food product is a drink, preferably based on fruit or vegetable juice, although milk-based drinks are also included. The drink can be prepared in a daily dose volume of 50 ml, 100 ml, 150 ml, 200 ml, or more. Preferred is an aqueous drink prepared containing, for example, 5 grams of lysozyme subtilisin A hydrolysate, or less than 10 grams, e.g., 0.1-9 grams, in a 200 ml daily dose volume. It is understood that the food supplement or food product can further contain additional food-grade ingredients, including, but not limited to, flavorings, vitamins, minerals, stabilizers, emulsifiers, other bioactive ingredients, food bases / nutrients, such as proteins, carbohydrates, and / or lipid components. The lysozyme subtilisin A hydrolysate can be added at any stage during the normal manufacturing process of the food product / food supplement.

[0093] The food product / food supplement may also contain other inactive ingredients and carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, such as carboxymethylcellulose (CMC), magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate, etc. It may also contain water, electrolytes, essential and non-essential amino acids, trace elements, minerals, fiber, sweeteners, flavors, colorants, emulsifiers, and stabilizers (e.g., soy lecithin, citric acid, esters of mono- or di-glycerides), preservatives, binders, flavors, etc.

[0094] For food supplements in pill, tablet, or capsule form, coatings may be added that alter the location and / or time of release of the hydrolysates in vivo. Controlled release formulations are known in the art.

[0095] A (food) product containing subtilidin A hydrolysate of lysozyme or subtilidin A hydrolysate of lysozyme may be taken prophylactically by a patient at risk of cognitive decline. The cognitive function is either forward inhibitory control or sustained attention in the patient. Furthermore, forward inhibitory control is, for example, impulse control, and / or sustained attention is, for example, general alertness.

[0096] In one embodiment, the composition according to the present invention is an aqueous composition.The composition according to the present invention can be a ready-to-use solution, from which daily dose is obtained or prepared by dilution as a stock solution, or it can be a dry composition, from which daily dose is obtained by adding liquid.The composition can also be used as a dry substance and mixed with foodstuffs.Those skilled in the art are familiar with these and other methods of administering the composition to patients who need it.

[0097] Preferably, the composition is taken either as a food supplement, for example in the form of a tablet, sachet, etc., or as a functional food in the form of a drink, semi-solid or solid food product.

[0098] For a preferred non-therapeutic method of improving cognitive function in a patient, the daily dose of subtilidin A hydrolysate of lysozyme is 0.5-20 grams per day, preferably 1-10 grams per day, or less than 10 grams per day, more preferably 2-8 grams per day, for example, 5 grams per day. The daily dose is preferably administered to the patient as a single dose per day. Preferred is a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the daily dose of subtilidin A hydrolysate of lysozyme is 0.5-20 grams per day, preferably 1-10 grams per day, more preferably 2-8 grams per day, for example, 5 grams per day. The daily dose is preferably administered to the patient as a single dose per day. For example, a single daily dose may be administered to a patient as a 50-300 ml, e.g., 100 ml, 125 ml, 200 ml, 250 ml, aqueous drink, with a volume of about 200 ml containing about 5 grams of subtilisin A hydrolysate of lysozyme being preferred.

[0099] One aspect of the invention relates to the use of a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement, and / or feed product comprising a subtilidin A hydrolysate of lysozyme in a non-therapeutic method of improving cognitive function in a patient. The subtilidin A hydrolysate of lysozyme is a hydrolysate of any of the above-mentioned aspects and embodiments of the invention.

[0100] One aspect of the present invention relates to a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement, and / or feed product comprising a subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof, wherein the subtilidin A hydrolysate of lysozyme is a hydrolysate of any of the above-mentioned aspects and embodiments of the invention.

[0101] To the inventors' surprise, they are the first to establish that daily ingestion of less than 10 grams of subtilisin A hydrolysate of lysozyme for at least four weeks results in improved cognition, wherein the improved cognitive function is, for example, improved impulsivity control, improved inhibitory control, or a combination of improved impulsivity control, improved inhibitory control, improved alertness, and improved attention, which to the inventors' knowledge has not previously been established in the prior art.

[0102] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way. [Example]

[0103] Example 1: Preparation of subtilisin A hydrolysate of lysozyme SDS-PAGE (molecular weight determination): The molecular weight of the peptides was determined by SDS-PAGE. Samples and molecular weight markers (10-250 kDa) were loaded onto a 4-20% Criterion TGX gel. Subsequently, a voltage was applied to the gel, and after migration of the samples, the gel was stained with Coomassie Blue.

[0104] Bulk density: Bulk density was measured in a 50 mL graduated cylinder. The cylinder was filled with 50 mL of powder and weighed. The cylinder was tapped once. Bulk density is expressed in g powder / mL.

[0105] Molecular size distribution (size exclusion chromatography (SEC)): The sample was injected onto a Superdex peptide column. Potassium phosphate and sodium sulfate buffers in water were used as eluents. Detection was performed using refractive index and UV 220 nm. The column was calibrated with a mixture of peptide and maltose.

[0106] A 5% (w / v) solution of lysozyme in water (100% protein content, Bauhaus Enthoven, Raalte, The Netherlands) was prepared, and the pH was adjusted to between 7.5 and 8.5 with 3 M KOH. Hydrolysis was initiated by adding Alcalase™ (Novozymes) to a final concentration of 4% protein basis. The solution was incubated at 60°C for a total of 6 hours under continuous stirring. The Alcalase™ was then inactivated by raising the temperature to 90°C for 15 minutes. The solution was then cooled to 2°C and stored overnight under continuous stirring.

[0107] The resulting hydrolysate solution was filtered through a 10 μm filter followed by a 1 μm filter. The filtrate was then heated at 135° C. for 15 seconds and concentrated to a dry matter of 57° Brix (approximately 45% dry matter) in a Niro evaporator at 90° C. and a flow of 3300 L / h. After evaporation, the product was spray dried to give a powder with very good flow properties as evidenced by visual observation.

[0108] The final product had the following properties: white powder, good solubility, 21% degree of hydrolysis (TNBS method), and a maximum molecular weight of less than 10 kDa. The size distribution of the peptides was as follows: 46% <500 Da, 23% 500-1000 Da, and 32% >1000 Da.

[0109] Similarly, a 5% (w / v) solution of lysozyme in water (100% protein content, Bauhaus Enthoven, Raalte, The Netherlands) was prepared and the pH was adjusted to 8.0 with 3 M KOH. Hydrolysis was initiated by adding Alcalase™ (Novozymes) to a final concentration of 4% protein basis. The solution was incubated at 60°C for a total of 5 hours under continuous stirring. Alcalase™ was inactivated by raising the temperature to 90°C for 15 minutes. The solution was then cooled to 6°C and stored overnight under continuous stirring.

[0110] The resulting hydrolysate solution was filtered through a 100 μm filter followed by a 1 μm filter. The filtrate was then heated to 135°C for 15 seconds and concentrated to a dry matter of 56.5°Brix (approximately 45% dry matter) in a Niro evaporator at 90°C and a flow rate of 3300 L / h. After evaporation, the product was spray-dried to obtain a powder with very good flow properties, as evidenced by visual observation. Evaluation of the particle size distribution showed that, on a logarithmic scale, the majority of particles had a Gaussian distribution around 120 micrometers (between 40 and 400 micrometers), with a relatively small fraction of particles larger than 500 micrometers. The size distribution: d(0,1) was 68 micrometers in the final product, d(0,5) was 124 micrometers, and d(0,9) was 242 micrometers. The particle size distribution of the powder was measured by laser diffraction technique using a Malvern Mastersizer 2000. Prior to measurement, the powder samples were dispersed in isopropanol. The bulk density of the resulting white hydrolysate powder was 0.42 g / mL.

[0111] The final product had the following properties: white powder, good solubility, 21% degree of hydrolysis, and a maximum molecular weight of less than 10 kDa. The size distribution of the peptides was as follows: 46% <500 Da, 23% 500-1000 Da, and 32% >1000 Da. Degree of hydrolysis: The degree of hydrolysis (DH) was measured by the orthophthaldialdehyde (OPA) method. The amount of free amino groups was measured by staining with orthophthaldialdehyde and spectrophotometric detection at 412 nm. DH is defined as: [(total number of free amino groups) - (number of free amino groups in intact protein))] * [100%] / [total amount of peptide bonds]. The dry matter content was 97.90% (moisture content was 2.10%). Dry matter (DM): The dry matter content was measured by an infrared moisture meter (Sartorius). 2 mL of sample was spread on a filter paper and weighed. The sample was then dried until a constant weight was achieved. DM = (weight after drying) / (weight before drying). * 100%.

[0112] Example 2: Clinical Trials The study involved was a randomized, double-blind, placebo-controlled, crossover clinical trial. Figure 1 shows an overview of the clinical trial protocol. The purpose of the study was to assess the effect of a composition containing a subtilisin A hydrolysate of lysozyme on cognitive function, more specifically, improving inhibitory control and attention, e.g., forward inhibitory control and / or sustained attention, over a 4-week period in patients, where forward inhibitory control is impulse control and / or sustained attention is vigilance, e.g., general alertness. Tests to assess cognitive function were conducted prior to the 4-week period (time points V3 and V6 in Figure 1 ) and again at the end of the 4-week period (time points V5 and V8 in Figure 1 ) in which patients were administered a composition containing a subtilisin A hydrolysate of lysozyme or a placebo daily. All measurements were performed in otherwise healthy patients who met the criteria for a diagnosis of metabolic syndrome. Each 4-week intervention period was typically separated by a 4-week washout period, but this period could be extended or shortened to be between 2 and 8 weeks.

[0113] A composition containing lysozyme subtilisin A hydrolysate or placebo was provided as a readily soluble white powder in 5 g aliquots in sachets. Patients were asked to dissolve the powder in approximately 200 mL of water prior to ingestion. A placebo was also provided as a powder similar in color and taste to the hydrolysate. 68 patients were randomized at the start of the study.

[0114] The first cognitive function test administered was the Sustained Attention Test (SAT), a vigilance task designed to assess an individual's ability to sustain attention, used to assess reaction time and to assess attention. The SAT is a psychomotor vigilance test, also used to assess sustained attention and general alertness. For the SAT, the preparation intervals were 1-2, 3-4, 5-6, 7-8, and 9-10 s. The psychomotor vigilance test is a simple task designed to assess sustained attention. Patients were instructed to press a button as soon as a stimulus appeared. The stimulus consisted of a square in the center of the screen turning green. In this test, the duration of a single trial consisted of 8 min. During this 8-min period, 80 stimuli appeared, with the inter-stimulus interval varying randomly between 1 and 10 s. The first time patients had to perform the test, four demonstration trials were used to demonstrate the principles of the test. After the demo trial, eight practice trials had to be performed, followed by 80 formal test trials.

[0115] The second cognitive test administered was the anticue task, a four-choice reaction time (RT) task using left and right anticues to indicate the mirror-image response hand. Specifically, the anticue required participants to prepare the fingers of the hand opposite the cue (counter-correspondence mapping). For an overview of the anticue task, see "Switch hand! Mapping temporal dynamics of proactive manual control with anticues," JJ Adam, S. Jennings, TJ H Vovend'Eerdt, PPM Hurks, and PWM Van Gerven, Acta Psychologica, 2015, 161, pp. 137–144. The anticue task assesses proactive cognitive control, particularly as it relates to impulse control. The anticue task examines the efficiency and time course of proactive inhibitory manipulations, and in this regard, the anticue task focuses on elucidating response conflict in the manual motor system. The anticue task therefore assesses impulse control, reaction time, and executive function. In this clinical trial, both an anticue setting (the "uncued anticue test") and an anticue:cue setting (the "cued anticue test") were used. For both settings, improvements in reaction time were assessed under the influence of the ingestion of the lysozyme subtilisin A hydrolysate of the present invention ("Treatment" in Figure 1) or a placebo. The fixation time was approximately 750 msec, the gap time was approximately 750 msec, and the preparation interval (target interval) was 100 msec, 150 msec, 250 msec, 450 msec, or 850 msec.

[0116] Statistical Analysis: Cognitive test data were analyzed using a dummy stepwise paired General Estimation Equations procedure with identification as the link, in which the change value was treated as the dependent parameter, while sex, age, body mass index, subsequent product intake order, treatment, warm-up / target interval, interval-by-treatment interaction, and the value at the first warm-up interval were applied as independent parameters. Treatment was also evaluated as an independent parameter without adding other parameters. Model fit was assessed using the Wald Chi-square test. Outliers were identified using the Grubbs test. Because no missing values were recorded and no dropouts were observed, the intention-to-treat (ITT) population was analyzed with and without outliers. Two-sided estimation was applied throughout the study, and significance was determined using a p-value of 0.05. Statistical analyses were performed using STATA, version 12 (StataCorp, College Station, TX, USA) and GraphPad, version 6 (GraphPad Prism, La Jolla, CA, USA).

[0117] Study population Population-based The study population consisted of otherwise healthy male and female patients between the ages of 18 and 75 who met criteria for metabolic syndrome. Patients were weight-stable, non-smokers.

[0118] The inclusion criteria for a clinical trial participant to be deemed suitable for enrollment in the study and met by patients enrolled in the clinical trial were as follows: - Able to give written informed consent, -Between 18 and 75 years old, - Generally in good health as determined by the investigator, - are non-smokers, - Stable weight (<5% change) in the 3 months prior to study entry - Meet harmonized criteria for the presence of metabolic syndrome as agreed upon by the International Diabetes Federation (IDF), the National Heart, Lung and Blood Institute, the American Heart Association, the World Heart Federation, the International Atherosclerosis Society, and the International Association for the Study of Obesity, defined as having at least three of the following five risk factors: Central obesity (waist circumference >94 cm in men or >80 cm in women) or BMI >30 kg / m Elevated triglycerides (>1.7 mmol / L (150 mg / dL)) Decreased HDL cholesterol [<1.03 mmol / L (40 mg / dL) in men and <1.29 mmol / L (50 mg / dL) in women] Elevated fasting plasma glucose >5.6 mmol / L (100 mg / dL) Elevated blood pressure (systolic blood pressure >130mmHg or diastolic blood pressure 85mmHg)

[0119] See also Table 1 for characteristics of clinical trial patients.

[0120] Exclusion criteria Presented human patients were excluded if they met any of the following criteria; 1. You are under 18 years old or over 75 years old, 2. Women who are pregnant, breastfeeding, or may wish to become pregnant during the study 3. Hypersensitivity to any of the test product's ingredients (i.e., egg protein); 4. Significant acute or chronic comorbidities, such as cardiovascular disease, chronic kidney disease (CKD), gastrointestinal disorders, endocrinological disorders, immunological disorders, metabolic disorders, or any condition that, in the investigator's judgment, contraindicates participation in the study; 5. Have any condition or have taken any medication that the investigator believes would interfere with the objectives of the study, pose a safety risk, or complicate the interpretation of the study results; including diuretics, antihypertensives, and other medications that otherwise interfere with the renin-angiotensin-aldosterone system (RAAS), such as ACE-inhibitors, angiotensin receptor blockers, direct renin inhibitors or aldosterone receptor inhibitors, and cholesterol-lowering agents, such as statins; 6. Receiving nonsteroidal anti-inflammatory drugs (NSAIDs) within 2 weeks of the baseline visit or for the duration of the study 7. Having either type I or type II diabetes. 8. Intake exceeding the recommended alcohol guidelines, i.e., >21 alcohol units / week for men and >14 alcohol units / week for women; 9. History of illegal drug use, 10. Individuals who, in the opinion of the investigator, are unlikely to attend the trial due to poor attendance or for any reason; 11. Patients may not receive treatment containing an investigational drug, 12. If the patient has participated in recent experimental trials, these must have been completed at least 30 days prior to the start of this study, or if the patient has donated blood at a blood bank within the 8-week period prior to the start of the trial. 13. Have malignant disease or concomitant end-stage organ disease.

[0121] The active ingredient, subtilisin A hydrolysate of lysozyme, was manufactured by NIZO Food Research (Ede, The Netherlands), which allows for the production and testing of spray-dried products. The active ingredient sachet contained: subtilisin A hydrolysate of lysozyme, citric acid, flavoring, acesulfame K, sucralose, and quinine HCl. The placebo sachet contained: maltodextrin, flavoring, citric acid, cloudifier, tartaric acid, malic acid, acesulfame K, sucralose, caramel E150a, and quinine HCl. Each sachet was dissolved in approximately 200 mL of water prior to ingestion. The placebo sachet contained potato starch maltodextrin as an inert filler instead of the hydrolysate. The active and placebo powders were similar in color and taste. The flavour of both products was bitter lemon. The sachets were manufactured in accordance with Generally Accepted Procedures and Good Manufacturing Practices. Manufactured in accordance with U.S. Food and Drug Administration (FDA) 100% FDA Approved ...

[0122] SAT results All 68 clinical trial participants were evaluated as a single group of patients. See Tables 2-4 for the measured values. A decrease was observed in reaction time values between intervals 1 (1-2 seconds) and 2 (3-4 seconds), while reaction times measured for each study arm remained approximately constant between intervals 2 and 3 (5-6 seconds), between 3 and 4 (7-8 seconds), and between 4 and 5 (9-10 seconds). The measured test results showed that the change in reaction time was significantly greater (shorter time required after 4 weeks) in the treatment group (patients administered lysozyme hydrolysate) than in the placebo group.

[0123] Comparing the test results measured on day 0 with those measured four weeks after treatment for the five preparation intervals, patients receiving the lysozyme hydrolysate of the present invention had a reaction time 5-10 msec shorter after four weeks of treatment than patients receiving a placebo. This reaction time reduction represents an improvement of approximately 1-3%, or approximately 1.3-2.7%. Furthermore, the change in reaction time was significantly greater in the group of patients receiving the lysozyme hydrolysate than in the placebo group (shorter reaction times were required after four weeks of treatment). Therefore, daily intake of a composition containing a lysozyme subtilisin A hydrolysate results in a significant reduction in the time required to complete the SAT task compared to daily intake of a placebo.

[0124] [Table 1]

[0125] [Table 2]

[0126] [Table 3]

[0127] [Table 4]

[0128] Uncued Anti-Cue Test Results Changes in reaction times (msec) between day 0 (time points V3 and V6 in Figure 1) and week 4 (end of the intervention period: time points V5 and V8 in Figure 1) for five target intervals of 100 msec, 150 msec, 250 msec, 450 msec, and 50 msec were assessed for a group of healthy patients who received a placebo daily for 4 weeks and a group of healthy patients who received a composition containing subtilisin A hydrolysate of lysozyme ("Treatment" in Figure 1). See Tables 5-7 for datasets related to the Uncued-Anticue test.

[0129] When five target intervals were evaluated, administration of a placebo for four weeks had no effect on reaction time. Therefore, placebo intake for four weeks had no effect on human patients' performance in the uncued-anticue test. In contrast, ingestion of a composition containing a subtilisin A hydrolysate of lysozyme of the present invention by treatment-group patients resulted in a reduction in the reaction time required to complete the task. The improvement in reaction time under the influence of lysozyme hydrolysate intake was nearly identical for the five target intervals evaluated, approximately 10.4 msec. This reduction in reaction time represents an improvement of approximately 2-3%. When five different target intervals were considered, the difference in change between day 0 and four weeks after treatment between placebo-group and treatment-group patients remained constant (p<0.009). When all clinical trial participants (male and female) were evaluated, improvement in reaction time was inversely correlated with age: the older the patient, the smaller the decrease in reaction time (p<0.001).

[0130] The results obtained using the left and right hands are combined before calculating the difference in mean interaction and reaction times between day 27 ("d27") and day 0 ("d0").

[0131] [Table 5]

[0132] [Table 6]

[0133] [Table 7]

[0134] Cue-Anti-Cue Test Results Changes in reaction times (msec) between day 0 (time points V3 and V6 in Figure 1 ) and 4 weeks (end of the intervention period: time points V5 and V8 in Figure 1 ) at five target intervals of 100 msec, 150 msec, 250 msec, 450 msec, and 50 msec were evaluated for a group of healthy patients who received a placebo daily for 4 weeks and a group of healthy patients who received a composition containing a subtilisin A hydrolysate of lysozyme ("Treatment" in Figure 1 ). The measured data points are summarized in Tables 8 and 9. When the five target intervals are considered, administration of placebo for 4 weeks had a negative effect on reaction times. That is, for the five target intervals, reaction times were 0-7 msec slower after 4 weeks of treatment with placebo compared to reaction times on day 0, before treatment with placebo began. Ingestion of a placebo for four weeks therefore did not positively affect the performance of human subjects in the cued-anticue test. In contrast, ingestion of a composition containing a subtilisin A hydrolysate of lysozyme of the present invention by treatment subjects for four weeks resulted in a decrease in the reaction time required to perform the task when all five target intervals were evaluated.

[0135] The improvement (i.e., reduction) in reaction time under the influence of ingestion of subtilisin A hydrolysate of lysozyme was approximately the same for the first four evaluated target intervals, approximately 12 msec (between 11.5 msec and 12.7 msec). For the 850 msec target interval, the improvement in reaction time after 4 weeks was approximately 2.6 msec compared to the reaction time on day 0. On average, the improvement in reaction time after 4 weeks was approximately 10 msec compared to the reaction time on day 0, combining the durations of the five priming intervals. This reduction in reaction time represents an improvement of approximately 2-3%.

[0136] Thus, the intake of lysozyme hydrolysate for 4 weeks reduces the reaction time required for the test when compared to placebo, which appears to increase the required reaction time to some extent. When five different target intervals are considered, the difference in the change values between day 0 and 4 weeks after treatment between placebo and treated patients remains constant (p<0.009). When all participants (male and female) in the clinical trial were evaluated, it was observed that the improvement in reaction time was inversely correlated with age: the older the patient, the lower the decrease in reaction time (p<0.001).

[0137] When considering male humans aged 67 or older (67-75 years) (14 males), the improvement in reaction time (i.e., shorter reaction times) was greater for reaction times assessed after 4 weeks of lysozyme hydrolysate ingestion compared to the improvement in reaction time when all 68 patients were considered, and compared to reaction times on day 0, before the start of the 4-week ingestion. That is, the improvement compared to placebo averaged approximately 32 msec and varied between 22 and 40 msec when five target intervals were assessed. For the 68 patients evaluated as a whole in a single group, the improvement compared to placebo averaged approximately 13 msec and varied between 9 and 16 msec when five target intervals were assessed. Therefore, when considering improvements in cognitive function, older male patients benefit more from treatment with lysozyme hydrolysate than younger males and women of all ages included in the clinical trial.

[0138] The results obtained using the left and right hands are combined before calculating the difference in mean interaction and reaction times between day 27 ("d27") and day 0 ("d0").

[0139] The improvement in reaction time in the cued-anti-cue test, in terms of reaction time shortening by approximately 10 msec under the influence of ingestion of a composition containing the subtilidin A hydrolysate of lysozyme of the present invention, is comparable in absolute terms to the increase in reaction time seen in human patients over a period of approximately 1 to 2 years. Thus, ingestion of a lysozyme hydrolysate, i.e., the subtilidin A hydrolysate of lysozyme of the present invention, has an effect comparable to arresting the (natural) deterioration of cognitive function that occurs over 1 to 2 years of aging.

[0140] [Table 8]

[0141] [Table 9]

Claims

1. 1. A non-therapeutic method of improving cognitive function in a patient, said method comprising administering to said patient a composition comprising a subtilisin A hydrolysate of lysozyme.

2. 2. The non-therapeutic method of claim 1, wherein improving cognitive function is one or more of improving inhibitory control, improving attention, improving impulsivity control, and improving alertness.

3. 3. The non-therapeutic method according to claim 2, wherein improving inhibitory control is improving forward inhibitory control and / or improving attention is improving sustained attention.

4. The non-therapeutic method according to any one of claims 1 to 3, wherein the patient is a human patient and / or a healthy patient, preferably a healthy human patient, such as a hyperactive patient.

5. 5. The non-therapeutic method according to any one of claims 1 to 4, wherein the patient is a male, preferably a male human, more preferably at least 60 years old, preferably at least 65 years old, more preferably at least 67 years old, such as 60-105 years old, or 67-75 years old.

6. A subtilidin A hydrolysate of lysozyme for use in a method for treating impaired cognitive function in a patient in need thereof.

7. 7. A subtilisin A hydrolysate of lysozyme for use in the method of claim 6, wherein the impaired cognitive function is any one or more of impaired inhibitory control, impaired forward inhibitory control, and impaired impulsive control.

8. 8. A subtilisin A hydrolysate of lysozyme for use in the method of claim 6 or 7, wherein the patient in need thereof is suffering from any one or more of hyperactivity, neurodegenerative diseases, Alzheimer's disease, dementia, Parkinson's disease, obsessive-compulsive disorder, obsessive-compulsive disorder, Tourette's syndrome, attention deficit hyperactivity disorder, psychiatric disorders, trichotillomania, addiction, such as addiction to drugs, narcotics, nicotine, alcohol, games, gambling.

9. 7. A subtilisin A hydrolysate of lysozyme for use in the method of claim 6, wherein the impaired cognitive function is any one or more of impaired attention, impaired sustained attention, and impaired vigilance.

10. 10. A subtilisin A hydrolysate of lysozyme for use in the method of claim 6 or 9, wherein the patient in need thereof is suffering from any one or more of attention deficit hyperactivity disorder, psychotic disorder, schizophrenia, hyperactivity.

11. 11. A subtilidin A hydrolysate of lysozyme for use in the method according to any one of claims 6 to 10, wherein the patient is a male, preferably a male human, more preferably a male human at least 60 years old, preferably at least 65 years old, more preferably at least 67 years old, such as 60-105 years old, or 67-75 years old.

12. 12. A subtilidin A hydrolysate of lysozyme for use in a non-therapeutic method according to any one of claims 1-5, or a method according to any one of claims 6-11, wherein at least 30% by weight of the subtilidin A hydrolysate of lysozyme are lysozyme peptides with a molecular weight of less than 0.5 kDa.

13. 13. A subtilidin A hydrolysate of lysozyme for use in a non-therapeutic method according to any one of claims 1 to 5 or 12, or in a method according to any one of claims 6 to 12, wherein the degree of hydrolysis of the lysozyme is at least 12%, preferably 15%-50%, more preferably 18%-40%, most preferably 19%-30%, for example 21%.

14. 14. A subtilidin A hydrolysate of lysozyme for use in a non-therapeutic method according to any one of claims 1 to 5 or 12-13, or a method according to any one of claims 6 to 13, wherein the daily dose of said subtilidin A hydrolysate of lysozyme is 0.5 grams to 20 grams per day, preferably 1 gram to 10 grams per day, more preferably 2 grams to 8 grams per day, for example 5 grams per day, and is preferably administered to the patient as a single daily dose.

15. 15. A dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement and / or feed product comprising a subtilidin A hydrolysate of lysozyme according to any one of claims 1 to 14 for use in a non-therapeutic method according to any one of claims 1 to 5, 12 to 14, or a dietary supplement, food product, functional food, foodstuff, food ingredient, food supplement and / or feed product comprising a subtilidin A hydrolysate of lysozyme according to any one of claims 1 to 14 for use in a method according to any one of claims 6 to 14.

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