The incidence of obesity and the number of people considered overweight has increased over the last decade. Since obesity and being overweight are associated with a variety of diseases such as cardiovascular problems, hypertension and atherosclerosis, this increase is a major health concern. Obesity is also increasingly recognized as a serious problem for animals and particularly for pets such as dogs and cats.

Several solutions have been proposed in order to control weight. Factors responsible for overweight are of three types: (i) individual or animal specific factors (age, physiological status, activity); (ii) diet specific factors (nutrient balance); and (iii) feeding management and environmental factors with a high importance of feeding or eating practices. Therefore, there have been incessant researches on the mechanism of appetite regulation through increasing satiety, which is known as the most effective way of controlling food intake.

Intestinal hormones secreted by enteroendocrine cells (EECs) modulate physiological processes like food intake regulation by paracrine, endocrine and neuronal pathways f Among these hormones, cholecystokinin (CCK) and glucagon like-peptide 1 (GLP-1) are released by the “intestinal sensing” phenomenon, a nutrient recognition at the apical side of the EECs ² . The GLP-1 regulates blood glucose via its incretin action and promotes satiety and food intake decrease via its anorexigenic properties. Peptide hormone CCK retards gastric emptying, stimulates pancreatic secretion and decreases food intake. GLP-1 and CCK are thus now well recognized as being usable to increase satiety and also to prevent and treat obesity.

GLP-1 has a short lifetime as being inactivated by the dipeptidyl peptidase 4 enzyme (DPP -IV) which is a serine exopeptidase existing in a soluble form (plasma, urine, amniotic fluid) as well as in a membranous form located at the cell surface of the tissues (intestine, kidneys, liver...) ³ . Inhibition of DPP -IV results in the prolongation of the circulating half-life of the GLP-1, thereby increasing its effects.

Although a great number of molecules or compositions have been suggested to be active in stimulating the release of one of the aforementioned satiety peptides, only very few of them have been derived from natural products and/or can be used in food products. Chemical compounds, such as e.g. valine-pyrrolidide ⁴ , Lys[Z(N02)]-thiazolidide and Lys[Z(N02)]- pyrrolidide ⁵ are non-natural and have the disadvantage of often having to be administered by injection, and they may result in side effects as chemical drugs often do. Food protein hydrolysates are well-used food ingredients and are of natural origin. It is well recognized that proteins exert a satiety effect greater than carbohydrates or fat. The intensity of this satiety effect highly depends on protein nature and source ⁶ . In particular, proteins are commonly known as precursors of a range of biologically active peptides which can act on GLP-1, CCK stimulation and/or DPP -IV inhibition. Bioactive peptides are latent in intact proteins but can be released through in vitro or in vivo enzymatic hydrolysis from the parent food proteins. In particular, the process of gastrointestinal (GI) digestion is able to release bioactive peptides that might exert significant physiological effects on energy homeostasis. Hydrolysed protein samples are highly complex and can contain up to hundreds of different molecules, which makes it difficult to identify bioactive compounds.

An object of the present invention is to provide new solutions for increasing satiety, thereby contributing to the reduction of food intake, the prevention or the treatment (esthetic and/or medical) of obesity and the maintenance of overall health conditions.

There is also a need to provide products which are of natural origin, and which can easily be added into edible compositions.

Finally, there is also a need for products having an acceptable taste, especially to animals, while providing good satiety effects.

The present invention addresses this need by providing novel protein hydrolysates and peptides inhibiting DPP -IV and/or stimulating the secretion of CCK and/or GLP-1 by intestinal enteroendocrine cells.

DEFINITIONS

Unless specifically stated otherwise, percentages are expressed herein by weight of a product reference.

In the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1- 1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Moreover, the term “at least” encompasses the hereafter cited value. For example, “at least 5%” has to be understood as also encompassing “5%”.

Moreover, in the present invention or disclosure, measurable values, such as an amount, have to be understood as encompassing standard deviations which can easily be determined by the skilled person in the technical domain of reference. Preferably, these values are meant to encompass variations of ±5%.

As used throughout, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references "a", "an", and "the" are generally inclusive of the plurals of the respective terms. For example, reference to "a method" or "a food" includes a plurality of such "methods" or "foods". Similarly, the words "comprise", "comprises", and "comprising" are to be interpreted inclusively. Likewise, the terms "include", "including" and "or" should all be construed to be inclusive. All these terms however have to be considered as encompassing exclusive embodiments that may also be referred to using words such as “consist of’.

The methods and compositions and other embodiments exemplified here are not limited to the particular methodologies, protocols, and reagents that are described herein because, as the skilled artisan will appreciate, they may vary.

Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by the skilled artisan in the field(s) of the invention or disclosure, or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention or disclosure, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein. The term "polypeptide" or "protein" means a polymer of amino acids. The term encompasses naturally occurring and non-naturally occurring (synthetic) polymers and polymers in which artificial chemical mimetics are substituted for one or more amino acids. The term also preferably encompasses fragments and variants that have the same or substantially the same physicochemical properties and perform the same or substantially the same function as the original sequence. The term encompasses polymers of any length, preferably polymers containing from about 2 to 1000 amino acids, more preferably from about 5 to 500 amino acids. In an embodiment, a polypeptide refers to a peptide, i.e. a polypeptide comprising two or more amino acids linked by a carboxyl group of one amino acid to an amino group of another. In an embodiment, such a peptide comprises up to 10 amino acids. The terms “polypeptide” and “peptide” can thus be interchangeably used. A “bioactive peptide” is a peptide that exerts a positive biological effect due to its health promoting properties, typically when released through in vitro or in vivo enzymatic hydrolysis from the parent proteins.

By the term “free peptide”, it is meant herein a peptide that is individually present as unbound ingredient in a product or a composition. Free peptides do not form part or are not contained into proteins or polypeptides. When contained in a product (a protein hydrolysate or an edible composition), the free peptides are not expected to significantly react in situ with any other ingredient that may also be present. They are thus comprised as such in a product (such as a protein hydrolysate or an edible composition).

In the context of the present invention or disclosure, the term “polypeptide” or “peptide” encompasses “functional variants thereof’ and “functional fragments thereof’. Although specific peptides have been identified, it is envisioned that similar peptides will provide the same biological activity, such as satiety. Amino acid sequences containing conservative substitutions, deletions or insertions (herein referred to as “variants”) or being truncated (herein referred to as “fragments”), and having the same or substantially the same biological activity as the parent amino acid sequence, are thus encompassed. In other words, in an embodiment, a “polypeptide” refers to one in the group consisting of the polypeptide under consideration, functional variants thereof, and functional fragments thereof. In another embodiment, a “polypeptide” strictly refers to the polypeptide under consideration.

The percent identities referred to in the present context are determined on the after optimal alignment of the sequences to be compared, which may therefore comprise one or more insertions, deletions, truncations and/or substitutions. This percent identity may be calculated by any sequence analysis method well-known to the person skilled in the art. The percent identity may be determined after global alignment of the sequences to be compared of the sequences taken in their entirety over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch ⁷ . For amino acid sequences, the sequence comparison may be performed using any software well- known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix. Preferably, the percent identify as defined in the context of the present invention or disclosure is determined via the global alignment of sequences compared over their entire length. Non-limiting examples of conservative amino acid substitution are given in Table 1 below.

Table 1 As used herein, the term "isolated," with respect to peptides, refers to material that has been removed from its original environment, if the material is naturally occurring. For example, a naturally-occurring protein or peptide present in a living animal is not isolated, but the same peptide, which is separated from some or all of the coexisting materials in the natural system, is isolated. Such isolated peptide could be part of a composition and still be isolated in that the composition is not part of its natural environment. An "isolated" peptide may include material that is synthesized or produced by recombinant DNA technology.

The term “molecules” refers to amino acids and/or (poly)peptides and/or proteins depending on the value of molecular weight. These molecules are restricted to those which are soluble in water. The molecular weight distribution is typically determined by size exclusion chromatography (SEC), such as the one disclosed in Guerard et al., 2001 ⁸ .

The term “edible composition” herein refers to products intended for consumption by humans or animals, in particular used for food or drink for human or animals (herein referred as “food product” and “food supplement”), products used as components of any such food product or supplement (“food ingredients”).

Typically, a food product is used primarily for nutrition, taste, or aroma or components of that product. The term "pet food" or “pet food product” means a food product intended for consumption by a pet. For instance, a “dog food” or a “dog food product” means a composition intended for consumption by a dog. As another example, a “cat food” or a “cat food product” means a composition intended for consumption by a cat.

Nutritionally-balanced pet foods are widely known and used in the art. A "nutritionally- complete", "nutritionally-balanced" or "complete and nutritionally-balanced food" is one that contains all known required nutrients for the intended recipient or consumer of the food, in appropriate amounts and proportions based, for example, on recommendations of recognized or competent authorities in the field of companion animal nutrition (such as AAFCO). Such foods are therefore capable of serving as a sole source of dietary intake to maintain life, without the addition of supplemental nutritional sources. Conversely, treats, drinks and beverages are pet foods which are not encompassed within the terms "nutritionally-balanced pet foods".

There are three main categories or classes of nutritionally-balanced pet foods depending on their moisture content, which is either low or medium or high:

- dry or low moisture-containing products (having less than about 14% moisture), such as dry kibbles; - canned or wet or high moisture-containing products (having more than about 50% moisture, typically 70 to 80%), such as loaf (terrines, pates, mousses, and the like), chunk-in-“X” products (chunks in a “X” preparation, such as chunk-in-jelly products, chunk-in-gravy products, and the like;

- semi-moist or semi-dry or soft dry or soft moist or intermediate or medium moisture- containing products (having from about 14 to about 50% moisture).

The term “kibble” used herein refers to particulate chunks or pieces formed by either a pelleting or extrusion process. Typically, kibbles are produced to give dry and semi-moist pet food, preferably dry pet food. The pieces can vary in sizes and shapes, depending on the process or the equipment. For instance, kibbles can have spherical, cylindrical, oval, or similar shapes. They can have a largest dimension of less than about 2 cm for example.

The term “treat” (or “biscuit” or “snacks”) means any food item that is designed to be fed to a pet, preferably at non-meal time, by the owner to help, promote or sustain a bonding process between a pet and its owner. Treats are a subset of pet food and are not intended to provide a complete and balanced diet. Typically, treats make up to 10% of the pet’s daily caloric intake.

The term "food supplement" or “dietary supplement” or “supplement” is known in the art as any food component which provides specific nutritional components and does not provide the full energy value required (i.e. generally less than 2000 or 2500 kcal/day for humans). More particularly regarding animals, a “pet supplement” means a product that is intended to be eaten in addition to the normal animal diet. Supplement is not intended to diagnose, treat, cure, or prevent any disease. Dietary supplements may be in any edible form, e.g., solid, liquid, gel, paste, tablets, capsules, powder, and the like.

Components of foods products or food supplements are designated herein under the term “food ingredient”.

The term "treatment" and derived terms mean reversing, alleviating, stopping or preventing a disorder as defined in the context of the invention or disclosure and/or at least one symptom linked to said disorder. The term "treatment" also refers to a prophylactic treatment which can delay the onset of the above-mentioned diseases or disorders.

The term “medicament” includes products intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in human or animals and products intended to affect the structure or function of the body of human or animals. An “enhanced feeling of satiety” or “induced satiety” as referred to herein means a more pronounced and/or quicker feeling of satiety (satiation) and/or a longer lasting feeling of satiety after eating (satiety). Such effects typically extend the time elapsed between meals and can result in a smaller amount of food and/or number of calories being consumed daily and/or per meal etc. The references herein to satiety include both what is strictly referred to as "satiation" and "satiety", including respectively "end-of-meal" satiety and "between-meals" satiety.

The terms “obesity” and “overweight” refer to an excess of body fat in proportion to lean both mass. Obesity is variously measured in terms of absolute weight, weightheight ratio, distribution of subcutaneous fat, and societal and esthetic norms. A common method of measure for humans is body mass index (BMI). The BMI refers to the ratio of body weight (expressed in kilograms) to the square of height (expressed in meters). In accordance with the U.S. Centers for Disease Control and Prevention (CDC), an overweight adult has a BMI of 25 kg.m ² to 29.9 kg.m ² , and an obese adult has a BMI of 30 kg.m ² or greater. BMI of 40 kg.m ² or greater is indicative of morbid obesity or extreme obesity. For children, the definitions of overweight and obese take into account age and gender effects on body fat. Regarding animals, in particular dogs and cats, body weight relative to an animal ’ s optimal weight (relative body weight - RBW) can be used as a defining criterion for obesity. Dogs and cats are classified as overweight when their body weight exceeds their optimal body weight by 10 to 20%, and as obese when RBW is above 20% ⁹ . Another method consists of a body condition scoring, combining dogs and cats visual assessment with body palpation and resulting in a body condition score (BCS) for evaluating pet body fat. Overweight may be defined by BCS greater than 5 and obesity by BCS greater than 7 ¹⁰ . Disease risk associations with higher BCS in adult dogs and cats appear to increase for overweight dogs and cats having a BCS above 6/9, respectively ¹¹ .

As used herein, a hydrolysate displays "DPP -IV inhibiting activity" when the DPP -IV (dipeptidyl peptidase-4) inhibition assay shows an IC50 (i.e. the concentration of inhibitor that inhibits 50% of the DPP -IV activity) of at most 10 mg.mL ¹ , more preferably at most 7 mg.mL more preferably at most 5 mg.mL ¹ , more preferably 4 mg.mL ¹ , more preferably at most 2 mg.mL ¹ , more preferably at most 1 mg.mL ¹ , and most preferably at most 0.8 mg.mL ¹ . A peptide displays "DPP -IV inhibiting activity" when the DPP -IV (dipeptidyl peptidase-4) inhibition assay shows an IC50 (i.e. the concentration of inhibitor that inhibits 50% of the DPP- IV activity) of at most 10 mM, more preferably 5 mM and most preferably at most 0.1 mM.

As used herein, a hydrolysate or peptide displays "CCK stimulation” or “CCK release” when stimulation by intestinal cells of secretion of endogenous CCK molecules occurs. Typically, in the CCK secretion stimulation assay, a concentration of CCK of at least 10 pM, more preferably at least 12 pM, more preferably at least 14 pM is measured.

As used herein, a hydrolysate or peptide displays "GLP-1 stimulation” or “GLP-1 release” when stimulation by intestinal cells of secretion of endogenous GLP-1 molecules occurs. Typically, in the GLP-1 secretion stimulation assay, a concentration of GLP-1 of at least 120 pM, more preferably at least 150 pM, more preferably at least 200 pM is measured.

As used herein, the term “palatability” refers to the overall willingness of a pet to eat a certain pet food and be satisfied by that food. Whenever a pet shows a preference, for example, for one of two or more pet foods, the preferred pet food is more “palatable”, and has “enhanced palatability”. Such preference can arise from any of the pet’s senses, but typically is related to, inter alia, taste, aroma, flavour, texture, smell and/or mouth feel. The term “flavour-enhancer” is often used in place of “palatability-enhancer”, for convenience.

Different methods exist to assess palatability. Examples of such methods involve exposure of pets to pet foods either simultaneously (for example, in side-by-side, free-choice comparisons, e.g., by measuring relative consumption of at least two different pet foods in a so-called “two- bowl” test), or sequentially (e.g., using single bowl testing methodologies). It can advantageously be determined by at least a "two-bowl test" or "versus test" in which the animal has equal access to both compositions. Advantageously, expert panels of pets are used in order to guarantee repeatable and fine measurement of palatability performance for pet food products.

“Coating”, as used herein, refers to the topical deposition of a product onto the surface of a food or supplement, such as by spraying, dusting, and the like.

“Inclusion” as used herein, refers to the addition of a product in the core of a food or supplement, such as pet food or pet supplement. For example, inclusion of a product in a pet food can be made by mixing it with other pet food ingredients, before further processing steps for obtaining the final pet food product (including thermal treatment and/or extrusion and/or retorting, etc).

The term “animal” means herein any non-human animal including livestock and pets, such as without limitation, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, birds, horses, cows, goats, sheep, donkeys, pigs, and the like. DE T ATT, ED DESCRIPTION INCLUDING DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present inventors have now identified bioactive peptides from protein hydrolysates that stimulate the cellular release of satiety peptide hormones CCK and/or GLP-1 and/or that inhibit DPP -IV. These peptides or hydrolysates are particularly effective in inducing satiety.

Peptides and hydrolysates

Amino sequences of these bioactive peptides are listed in Table 2.

Table 2

It is herein disclosed a polypeptide (or a peptide) comprising an amino acid sequence chosen from DLVDK, PSLVH, LKPT, LTDY, GPFPLLV, MDLP, DLDL, FAMD, VADTMEVV, CSSGGY, VAPEEHPT, FPYPLPRPL, VLPL, VLEL, LTLL, VPLM, VDGL, LLLE, DGLL, MEMT, EYLPV, PPTT and FPKATG.

A first aspect of the present invention relates to a peptide consisting of at least one amino acid sequence chosen from DLVDK, PSLVH, LKPT, LTDY, GPFPLLV, MDLP, DLDL, FAMD, VADTMEVV, and CSSGGY.

The present inventors have demonstrated that peptides GPFPLLV, MDLP, DLDL, FAMD, VADTMEVV, CSSGGY, VAPEEHPT, MEMT, EYLPV, PPTT and FPKATG perform at least in reducing the activity of DPP -IV. Advantageously, it has been observed that these peptides are able to cross the intestinal barrier, simulated in vitro, and reach the basolateral side. Peptides PSLVH, DLVDK, VLPL, FPYPLPRPL, VLEL and LTLL perform at least in stimulating the CCK release. Peptides LKPT, LTDY, VPLM, DGLL, LLLE, VDGL, LTLL and VLPL perform at least in stimulating the GLP-1 release.

As an example, the amino acid sequence is chosen from DLVDK, PSLVH, LKPT, LTDY, GPFPLLV, MDLP, DLDL, FAMD, FPYPLPRPL, VLPL, VLEL, LTLL, VPLM, VDGL, LLLE, DGLL, MEMT, EYLPV and PPTT. It was found that these peptides in particular are very potent CCK and/or GLP-1 releaser compounds and/or DPP -IV inhibitory compounds, and are therefore of particular interest. One skilled in the art is however aware of the fact that for example the compounds showing only moderate DPP -IV inhibiting activity or CCK releasing activity or GLP-1 releasing activity may have substantially improved activities in vivo.

As another example, the amino acid sequence is chosen from FPYPLPRPL, VLPL, LTLL, PSLVH, LLLE and VDGL, preferably from FPYPLPRPL, VLPL and LTLL. It was found that these peptides in particular are very potent CCK and GLP-1 releaser compounds.

As yet another example, the amino acid sequence is chosen from VAPEEHPT, FPYPLPRPL and FPKATG.

As yet another example, the amino acid sequence is chosen from VAPEEHPT, DLDL and DLVDK. As defined above, functional variants having the same or similar functions can be encompassed. In particular, the polypeptide can comprise an amino acid sequence having more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95% identity to an amino acid sequence as above described and having the same or similar function to said amino acid sequence as above described.

As another example, the peptide consists of an amino acid sequence as above described.

The polypeptide/peptide as disclosed or claimed herein can be isolated. The polypeptide/peptide as disclosed or claimed herein can be purified. The polypeptide/peptide as disclosed or claimed herein can be natural. Alternatively, the polypeptide/peptide as disclosed or claimed herein can be synthetic.

A polypeptide/peptide as disclosed or claimed herein can be used as such or used in the form of the base or a salt thereof. Examples of salts include, without limitation, acetate, chloride, sulfate, and phosphate salts.

The polypeptides/peptides as disclosed or claimed herein can be substantially pure. For the uses described herein including the therapeutic purposes, it is not critical to have a high purity of a specific polypeptide/peptide. Thus, the polypeptides/peptides as disclosed or claimed herein can be purified to at least about 90 to 95% purity, yet to at least about 98% purity. Purity may be assessed by e.g. high-performance liquid chromatography (HPLC) coupled to a detector adapted to the detection of peptides or at a lesser extent amino-terminal amino acid sequencing.

It is also disclosed herein a protein hydrolysate comprising at least one polypeptide/peptide as above described. The protein hydrolysate can thus comprise at least one amino acid sequence chosen from DLVDK, PSLVH, LKPT, LTDY, GPFPLLV, MDLP, DLDL, FAMD, VADTMEVV, CSSGGY, VAPEEHPT, FPYPLPRPL, VLPL, VLEL, LTLL, VPLM, VDGL, LLLE, DGLL, MEMT, EYLPV, PPTT and FPKATG, and preferably from DLVDK, PSLVH, LKPT, LTDY, GPFPLLV, MDLP, DLDL, FAMD, FPYPLPRPL, VLPL, VLEL, LTLL, VPLM, VDGL, LLLE, DGLL, MEMT, EYLPV and PPTT.

According to the present disclosure or invention, the protein hydrolysate comprises at least 2, preferably at least 3, more preferably at least 4 different amino acid sequences selected from the above lists.

In particular, said at least one amino acid sequence is in the protein hydrolysate in the form of a free peptide. Yet more particularly, the amino acid sequence is chosen from VAPEEHPT, DLDL and DLVDK.

The presence of such polypeptides/peptides can be demonstrated by means of common protein or peptide fractionation and analysis techniques, such as for example mass spectrometry, HPLC or other fractionation techniques in combination with e.g. sequential Edman degradation, mass spectrometry or specific affinity assays targeted towards a certain peptide, such as an enzyme- linked immunosorbent assay (ELISA).

A protein hydrolysate of the disclosure or the invention can be of any form, liquid or solid, such as in a powder form. For example, for powder form, a moisture content inferior to 8%, and preferably from 2 to 7%, is typically obtained.

According to the present disclosure, any protein source able to provide one or more peptide sequence(s) as listed above can be used. The protein can be derived from one protein source or may be derived from more than one protein source. Examples of such protein sources are microorganisms (yeast, bacteria, fungi), plants (e.g. soy, pea, cotton, corn, wheat), animals, fishes, and from animal derived protein sources such as milk or egg. In particular, the protein hydrolysate as herein disclosed can be obtained from a protein source chosen from a fish and/or an animal.

According to the present invention, the protein source is a fish, more precisely of the Cichlidae fish Family (hereafter named “Cichlidae protein source”), preferably Nile tilapia ( Oreochromis niloticus niloticus). Indeed, several of the amino acid sequences identified herein are found in Nile tilapia protein, and are thus readily obtainable. Alternatively, as an example, the protein source is an animal, in particular poultry. Indeed, several of the amino acid sequences disclosed herein are found in poultry protein, and are thus readily obtainable.

It is also envisioned that combinations of a marine protein source and an animal protein source can be combined with each other, and/or can be combined with at least one other protein source. A protein hydrolysate can thus be obtained from a fish and an animal as herein described.

Polypeptides/peptides or protein hydrolysates of the invention can advantageously be used for inducing satiety or for the prevention and treatment of DPP -IV, CCK or GLP-1 mediated conditions such as for example obesity, type II diabetes and other disorders.

Fish protein hydrolysates of the invention

According to the invention, the protein hydrolysate is a Cichlidae fish Family hydrolysate.

A second aspect of the present invention thus relates to a protein hydrolysate obtained from a protein source of the Cichlidae fish Family comprising at least one peptide chosen from DLVDK, PSLVH, LKPT, LTDY, GPFPLLV, MDLP, DLDL, FAMD, VADTMEVV, and CSSGGY.

Another aspect of the present invention relates to a protein hydrolysate which:

(i) is obtained from a protein source of the Cichlidae fish Family; and

(ii) has:

- the following molecular weight distribution: at least 65% of peptides with a molecular weight of less than 1000 Da, and preferably less than 90% of peptides with a molecular weight of less than 1000 Da; at least 13% of peptides with a molecular weight between 1000 and 3000 Da, and preferably less than 23% of peptides with a molecular weight between 1000 and 3000 Da; at least 1.5% of peptides with a molecular weight between 3000 and 5000 Da, and preferably less than 7% of peptides with a molecular weight between 3000 and 5000 Da; at least 0.5% of peptides with a molecular weight between 5000 and 10000 Da, and preferably less than 9% of peptides with a molecular weight between 5000 and 10000 Da; less than 3% of peptides with a molecular weight greater than 10000 Da, and preferably at least 0.3% of peptides with a molecular weight greater than 10000 Da; and/or - a fat content from 5% to 15%, and/or

- a protein content of more than 70%, and/or

- a degree of hydrolysis (DH) of more than 15%, and

(iii) comprises at least one amino acid sequence chosen from DLVDK, PSLVH, DLDL, MDLP, VADTMEVV, VAPEEHPT, GPFPLLV, LKPT, LTDY, FAMD, and CSSGGY.

Amino acid sequences are preferably chosen from DLVDK, PSLVH, DLDL, MDLP, VADTMEVV, VAPEEHPT, GPFPLLV, LKPT, LTDY, FAMD and CSSGGY, and preferably from DLVDK, PSLVH, DLDL, MDLP, GPFPLLV, LKPT, LTDY and FAMD.

As yet indicated, a specific protein hydrolysate is obtained from a protein source of the Cichlidae fish Family (hereafter named “Cichlidae hydrolysate”) and has:

- the following molecular weight distribution: at least 65%, preferably at least 70%, more preferably at least 72% of molecules with a molecular weight of less than 1000 Da, and preferably less than 90%, more preferably less than 85%, and even more preferably less than 83% of molecules with a molecular weight of less than 1000 Da; at least 13% of molecules with a molecular weight between 1000 and 3000 Da, and preferably less than 23%, more preferably less than 20% of molecules with a molecular weight between 1000 and 3000 Da; at least 1.5%, preferably at least 1.8% of molecules with a molecular weight between 3000 and 5000 Da, and preferably less than 7%, more preferably less than 5%, and even more preferably less than 4% of molecules with a molecular weight between 3000 and 5000 Da; at least 0.5%, preferably at least 1% of molecules with a molecular weight between 5000 and 10000 Da, and preferably less than 9%, more preferably less than 5%, and even more preferably less than 4% of molecules with a molecular weight between 5000 and 10000 Da; less than 3%, preferably less than 2.5%, more preferably less than 2% of molecules with a molecular weight greater than 10000 Da, and preferably at least 0.3% of molecules with a molecular weight greater than 10000 Da; and/or

- a fat content from 5% to 15%, preferably from 5% to 10% (% by weight of hydrolysate), and/or - a protein content of more than 70%, preferably from 70% to 85%, preferably more than 75%, more preferably from 75% to 85% (% by weight of hydrolysate), and/or

- a DH of more than 15%.

As yet defined above, the term “molecules” refers to amino acids and/or (poly)peptides and/or proteins depending on the value of molecular weight.

As taught hereafter and further illustrated in the Examples below, this specific Cichlidae hydrolysate shows a significant DPP -IV inhibiting activity as well as a significant CCK and GLP-1 release activity (see Example 2.1.3 and Table 3). Protein that passes into the intestine after a meal experiences digestion in particular by gastric enzymes (pepsin) and is further digested in the upper intestine by pancreatic digestive enzymes (pancreatin). It has been demonstrated in vitro that this Cichlidae protein hydrolysate subjected to digestion by pepsin and pancreatin increases the ability to induce GLP-1 releasing activity on enteroendocrine cells, and does not lose the ability to induce CCK releasing activity on enteroendocrine cells (see Example 2.1.3 and Table 3). The present inventors observed that the Cichlidae hydrolysate shows significantly higher CCK induction in STC-1 cells compared to intact Cichlidae proteins before the action of gastrointestinal enzymes. In the same way, results obtained by the inventors indicate the higher potential of the Cichlidae hydrolysate on DPP -IV inhibition activity than Cichlidae raw material, even before the action of gastrointestinal enzymes (see Example 2.1.4 and Table 4). Moreover, the present inventors observed that the in vitro digested Cichlidae hydrolysate shows significantly higher GLP-1 induction in STC-1 cells compared to in vitro digested intact Cichlidae proteins (not previously hydrolyzed). Finally, it was shown in vivo that this hydrolysate provides an increased satiety in an animal (see Example 2.3- Table 13 for cats and Example 2.4- Table 14 for dogs).

This Cichlidae hydrolysate of the invention also demonstrates additional advantages, such as an increased solubility, digestibility and reduced allergenicity risks (relative to intact Cichlidae proteins) as well as a good acceptance level indicating that it would be a suitable ingredient in foods. In particular, it was demonstrated that this Cichlidae hydrolysate does not decrease palatability of foods. Thus, the Cichlidae protein hydrolysate of the invention is a promising novel ingredient that can be incorporated into a wide variety of foods, in particular pet foods, or used alone by individuals seeking foods with enhanced satiety effects to help manage food intake, preserve or increase lean body mass and/or maintain or lose non-lean body mass. In a particularly preferred embodiment, the Cichildae protein source is Nile tilapia (Oreochromis niloticus niloticus).

The Cichlidae protein hydrolysate of the invention advantageously comprises from 3 to 15% of minerals (% by weight of dry matter). Preferably, the mineral fraction contains at least one macroelement selected from the group consisting of magnesium, phosphorus, potassium, calcium and sodium.

The Cichlidae protein hydrolysate of the invention has a DH of more than 15%, the DH being measured with an OPA-method ¹² .

As used herein, the “protein content” includes total nitrogen organic content, i.e. peptides and amino acids content.

In a particular embodiment, proteins of the Cichlidae protein hydrolysate of the invention comprise at least 85%, preferably at least 90% of soluble proteins (% based on total protein weight). Preferably, proteins of the Cichlidae protein hydrolysate of the invention comprise less than 99% of soluble proteins. The proportion of soluble proteins is typically determined as in the Examples.

Preferably, the Cichlidae protein hydrolysate of the invention comprises up to 25%, in particular up to 20% of free amino acids (% based on hydrolysate weight).

It also has advantageously the following amino acid composition: from 9.0% to 10.5% glutamic acid, from 5.5% to 7.0% aspartic acid, from 4.5% to 5.5% lysine, from 4% to 5.5% leucine, from 4.5% to 5.0% arginine, from 4.5% to 6.0% alanine, from 2.5% to 4.0% valine, from 2.5% to 5.0% isoleucine, from 6.5% to 8.0% glycine, from 2.5% to 3.5% threonine, from 2.0% to 3.0% serine, from 1.5% to 2.5% tyrosine, from 2.5% to 3.0% phenylalanine, from 1.5% to 2.5% methionine, 4.0% to 5.5% proline, 1.0% to 2.0% histidine, 0.3% to 0.7% cysteine, expressed as percentage by weight relative to the total weight of hydrolysate.

Hydrolysis potentiates the stimulation of CCK and GLP-1 and the inhibition of DDP-IV and enables to generate bioactive peptides.

As above explained, a Cichlidae protein hydrolysate comprises at least one amino acid sequence chosen from DLVDK, PSLVH, DLDL, MDLP, VADTMEVV, VAPEEHPT, GPFPLLV, LKPT, LTDY, FAMD and CSSGGY. Preferably, a Cichlidae protein hydrolysate, such as the one above described comprises the advantageous combination of DLVDK, PSLVH, DLDL, MDLP, VADTMEVV, VAPEEHPT, GPFPLLV, LKPT, LTDY, FAMD and CSSGGY. Indeed, the present inventors have observed that these Cichlidae bioactive peptides become active through digestion and are involved in biological functions. More particularly, the inventors demonstrated that peptides having the amino acid sequences VAPEEHPT, DLVDK and DLDL are contained as such in the Cichlidae hydrolysate and are advantageously resistant to digestion. In particular, the Cichlidae protein hydrolysate thus comprises at least one peptide consisting of the amino acid sequence chosen from VAPEEHPT, DLVDK and DLDL. Bioactive peptides having amino acid sequences PSLVH, MDLP, VADTMEVV, LKPT, LTDY, FAMD, CSSGGY and GPFPLLV form part of polypeptides in the Cichlidae hydrolysate. Enzymatic hydrolysis of certain proteins may be problematic due to amino acid composition. For example, proteins may be resistant to enzymatic hydrolysis or only allow for incomplete hydrolysis. However, the inventors demonstrated that these peptides are released and become active during gastrointestinal digestion of a Cichlidae protein hydrolysate as above described. In other words, the gastrointestinal digestion enables the release of these peptides by breaking down peptide bonds. This combination of peptides in a protein hydrolysate is particularly advantageous as they stimulate the cellular release of more than one satiety peptide hormone, and more particularly of both CCK and GLP-1, as above explained.

Animal protein hydrolysates of the disclosure

Regarding this alternative example (protein source being an animal, preferably poultry), amino acid sequences can be chosen from VLEL, VLPL, FPYPLPRPL, LTLL, FPKATG, PPTT, MEMT, DGLL, VPLM, LLLE, VDGL and EYLPV, and preferably from VLEL, VLPL, FPYPLPRPL, LTLL, PPTT, MEMT, DGLL, VPLM, LLLE, VDGL and EYLPV, and even more preferably from VLPL, FPYPLPRPL and LTLL.

An animal protein hydrolysate which is particularly able to stimulate the secretion of CCK and GLP-1 and inhibit DPP -IV is disclosed hereafter. This poultry hydrolysate is obtained by enzymatic hydrolysis of at least poultry, and has:

- the following molecular weight distribution: at least 90% of molecules with a molecular weight of less than 1000 Da, and preferably less than 99% of molecules with a molecular weight of less than 1000 Da; at least 1.5%, preferably at least 2% of molecules with a molecular weight between 1000 and 3000 Da, and preferably less than 7%, more preferably less than 5% of molecules with a molecular weight between 1000 and 3000 Da; less than 2%, preferably less than 1% of molecules with a molecular weight greater than 3000 Da, and preferably at least 0.2% of molecules with a molecular weight greater than 3000 Da; and/or

- a fat content from 10% to 22%, preferably from 12% to 20% (% by weight of hydrolysate), and/or

- a protein content of more than 40%, preferably of more than 50% (% by weight of hydrolysate), and/or

- a DH of more than 68%.

As taught hereafter, this specific poultry hydrolysate shows a significant DPP -IV inhibiting activity as well as a significant CCK and GLP-1 release activity. It has been demonstrated in vitro that this animal protein hydrolysate subjected to digestion by pepsin and pancreatin increases the ability to induce CCK and GLP-1 releasing activity on enteroendocrine cells. The present inventors observed that the poultry hydrolysate shows significantly higher CCK and GLP-1 induction in STC-1 cells compared to intact poultry proteins, as well as a higher inhibition of DPP -IV, before the action of gastrointestinal enzymes. Moreover, the present inventors observed that the in vitro digested poultry hydrolysate shows significantly higher CCK induction in STC-1 cells compared to in vitro digested intact animal proteins (not previously hydrolyzed).

Parameters such as DH, molecular weight distribution etc. are determined as above described regarding the Cichlidae hydrolysate.

Preferably as above explained, a poultry protein hydrolysate comprises at least one amino acid sequence chosen from VLEL, VLPL, FPYPLPRPL, LTLL, FPKATG, PPTT, MEMT, DGLL, VPLM, LLLE, VDGL and EYLPV. Bioactive peptides having amino acid sequences VLEL, VLPL, FPYPLPRPL, LTLL, FPKATG, PPTT, MEMT, VPLM, LLLE, VDGL, EYLPV form part of polypeptides in the poultry hydrolysate of the disclosure. The present inventors have observed that these amino sequences form part of polypeptides and that peptides are released during gastrointestinal digestion. In other words, the gastrointestinal digestion enables the release of these bioactive peptides by breaking down peptide bonds. These bioactive peptides become active through digestion and are involved in biological functions. This combination of peptides in such a protein hydrolysate is particularly advantageous as they stimulate the cellular release of more than one satiety peptide, and more particularly of both CCK and GLP-1, as above explained. Methods of preparation of protein hydrolysates and polypeptides/peptides

Methods of preparation of protein hydrolysates

One aspect of the invention provides a process for preparing a protein hydrolysate. The process generally comprises contacting a protein source with one or more enzymes that breaks some peptide bonds and enables to generate bioactive peptides.

The present invention also relates to a method for obtaining a protein hydrolysate as above described having inhibitory properties of DPP -IV and/or stimulatory properties of CCK secretion and/or GLP-1 at the level of intestinal cells, and which can for example exert a satiating effect as defined above. The method comprises the steps of: a) hydrolyzing the protein source with at least one peptidase at a temperature of between 25 °C and 80 °C, for 30 min to 7 hours, b) inactivating said at least one peptidase, for example at a temperature of between 80 °C and 110 °C for 10 to 60 minutes.

The process can comprise before step a) an optional step of grinding the protein source.

The process can also comprise before step a) an optional step of mixing or dispersing the protein source in water, for example in an amount of 5-40% (% by total weight of raw material).

In particular, a protein source of the Cichlidae fish Family is used. The whole fish or a part of the fish, such as viscera, head, bones, skin, fillet, and preferably viscera, head and/or bones can be used as protein source. In a particular embodiment, the proportion of viscera used as protein source is controlled, so as to limit autolysis by endogenous enzymes. For example, the protein source contains less than 30% of viscera.

Yet in particular, poultry is used as the protein source.

Hydrolysis is performed by using at least one peptidase. The hydrolysis reaction is generally initiated by adding the at least one peptidase to the protein source. In step a) of hydrolysis, the at least one peptidase can be added before and/or during obtaining the temperature of hydrolysis.

A peptidase can be endogenous (for example coming from viscera) or exogenous. Preferably, at least one exogenous peptidase is used. Typically, an exogenous peptidase is chosen from endopeptidases, exopeptidases or peptidases having both activities. More preferably, at least one endopeptidase is used. Endopeptidases act preferentially in the inner regions of peptide chains away from the N- and C-terminus. Several endopeptidases are suitable for use to practice the invention. For example, the endopeptidase can be chosen from serine peptidase, subtilisin peptidase, cysteine peptidase. In a further embodiment of the invention, the endopeptidase can be combined with at least one exopeptidase. Generally, exopeptidases act only near the ends of polypeptide chains at the N- or C-terminus. For example, the exopeptidase can be chosen from aminopeptidases and carboxypeptidases. The at least one peptidase used must be compatible with a dietary use, in particular for dogs or cats.

The amount of peptidases added to the protein source can and will vary depending upon the desired DH and the duration of the hydrolysis reaction. The amount of peptidases may range from about 0.05% to 3% of hydrolysate.

Preferably, said step a) is performed so as to obtain a hydrolysate having a DH of more than 15%, the DH being measured with an OPA-method (11).

The method can also comprise a step consisting of separating the hydrolysate from the rest of the reaction mixture obtained from step b). Preferably, the process for preparing the Cichlidae hydrolysate as above described comprises a step of separation.

The separation of the hydrolysate from the rest of the reaction mixture can be carried out by centrifugation and/or by filtration. Preferably, the separation of the protein hydrolysate obtained may be carried out with a horizontal centrifuge which performs a three-phase separation, which enables to obtain a soluble part and fat reduced fraction as a hydrolysate according to the invention.

The method can also comprise one or more additional steps of pH adjustment. The pH can be adjusted by any appropriate compound that is acceptable for use in pet food, such as phosphoric acid, caustic soda, etc.

A pH adjustment can be performed during step a), so as to optimize the hydrolyzing activity of the enzyme. Typically, said pH adjustment is performed so as to adjust the pH to a pH value which is optimal pH value or pH range according to the enzyme manufacturer’s recommendations. That will be readily apparent to those skilled in the art.

In particular, said method comprises, in addition, a step of drying the thus obtained hydrolysate. A moisture content inferior to 8%, and preferably from 2 to 7%, is typically obtained. An object of the present disclosure relates to a hydrolysate obtained by one of the methods as aforementioned.

The protein hydrolysates as disclosed or claimed herein may be fractionated by means of extraction, precipitation, filtration, ultrafiltration, nanofiltration, microfiltration or chromatography (preferably ion exchange or affinity chromatography), or any combination of the above techniques, as to (further) isolate the DPP -IV inhibiting activity and/or the CCK and/or GLP-1 release activity. As such, a fraction comprising a mixture of polypeptides/peptides or even single polypeptides/peptides may be identified as having an increased activity compared with the starting protein hydrolysates. Such mixture of polypeptides/peptides or single polypeptides/peptides are also encompassed in the present disclosure.

Methods of preparation of polypeptide s/peptides

Polypeptides/peptides may also be prepared by means of recombinant DNA technology or by synthetic or transgenic means. The polypeptides/peptides described herein may be produced by means of recombinant nucleic acid techniques. In general, a nucleic acid sequence encoding the desired peptide is then inserted into an expression vector, which is in turn transformed or transfected into host cells.

As an alternative, the polypeptides/peptides are produced by synthetic means, for example by using solution and solid phase synthesis using traditional tert-butyloxycarbonyl (BOC) or fluorenylmethoxycarbonyl (FMOC) protecting groups. The different techniques available so far are part of the common general knowledge.

The polypeptides/peptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction. Additional purification may be achieved by conventional chemical purification means, such as HPLC. Other methods of purification, including barium citrate precipitation, are known in the art, and may be applied to the purification. Uses of hydrolysates or polypeptides/peptides

The inventors observed that stimulation of secretion of CCK and/or GLP-1 and/or inhibition of DPP -IV can be obtained with protein hydrolysates of the disclosure or invention, such as a fish protein hydrolysate or an animal protein hydrolysate as above described. The inventors observed that these hydrolysates potentiate the stimulation of CCK and GLP-1 hormone secretion during digestion and inhibit DPPIV activity.

The inventors also identified polypeptides/peptides displaying CCK and/or GLP-1 release activity and/or DPP -IV inhibiting activity.

The hydrolysates and polypeptides/peptides according to the invention can be used for pharmaceutical or non-pharmaceutical uses.

Hydrolysates or polypeptides/peptides of the invention will thus increase satiety in a subject, so that said subject will feel less hungry and have a reduced food intake. In particular subjects being overweight, such as e.g. obese subjects or subjects being only slightly overweight, will benefit from stimulation of CCK and/or GLP-1 and/or inhibition of DPP -IV by administration of the one or more protein hydrolysates according to the present invention. The products can however also be employed as to retain a certain weight as to not get overweight, and may therefore be used to stabilize and/or improve the body weight as for cosmetic/esthetic purposes, i.e. for stabilizing and/or improving appearance. Increasing satiety can contribute to the reduction of body weight. According to another aspect, the present invention provides the (non-pharmaceutical) use of a protein hydrolysate or a polypeptide/peptide of the invention for inducing or increasing satiety in a human or animal.

The invention also concerns a method for inducing or increasing satiety in a human or animal, the method comprising the step of administering a protein hydrolysate or a polypeptide/peptide of the invention to a human or animal.

In a particular embodiment, a protein hydrolysate or a polypeptide/peptide of the invention induces an enhanced feeling of satiety during its consumption.

According to another aspect, the present invention provides the (non-pharmaceutical) use of a protein hydrolysate or a polypeptide/peptide of the invention for improving or controlling perception of body image, and/or to controlling body weight, and/or to controlling calorie intake.

The invention also concerns a method (i.e. cosmetic and/or esthetic method) for improving or controlling perception of body image, and/or to controlling body weight, and/or to controlling calorie intake in a human or animal, the method comprising the step of administering a protein hydrolysate or a polypeptide/peptide of the invention to a human or animal.

Preferably regarding this non-pharmaceutical use, the subject is not obese and/or healthy (unsuffering from any of the above-mentioned disorders).

In an embodiment, the subject is an animal, more preferably a pet such as a dog or a cat. Pets refer to domesticated and companion animals. In particular, the pet can have a BCS inferior to 6 and/or a RBW inferior to 10%.

In another embodiment, the subject is a human.

Advantageously for these non-therapeutic uses, a protein hydrolysate or a polypeptide/peptide of the invention can be in an edible composition. An edible composition may be any food product, food ingredient or food supplement. The edible compositions of the present invention are intended for consumption by a human or animal. Preferably, the edible composition is used in a weight loss or weight control plan. The edible composition comprising the protein hydrolysate or the polypeptide can be administered to the subject prior to or during a meal. A particular embodiment thus relates to an edible composition comprising at least one polypeptide/peptide or at least one protein hydrolysate as above described, wherein said edible composition is a food product, food ingredient or food supplement.

When intended for humans, the edible composition may be a food product, such as jam, beverage, marmalade, dairy product (such as milk or cheese), meat product, poultry product, fish product, vegetable-based soup, or bakery products, or a supplement.

The edible composition can then comprise a polypeptide/peptide or protein hydrolysate of the invention in an amount sufficient to provide from 20 mg to 1000 mg, preferably from 50 mg to 500 mg per kg body weight of a human. In a particular embodiment, an edible composition is intended for animals, preferably for pets, preferably for cats or dogs. Advantageously, inventors observed that products of the invention do not decrease food palatability to pets. The edible composition can be a pet food, a pet food ingredient or a pet supplement.

The edible composition can then comprise a polypeptide/peptide or protein hydrolysate of the invention in an amount sufficient to provide from 50 mg to 10 g, preferably from 200 mg to 6 g per kg body weight of an animal.

In a particular embodiment, the edible composition is a pet food ingredient. An example of pet food ingredient can be a palatability-enhancer for pet food.

In a particular embodiment, the edible composition is a pet supplement.

In a particular embodiment, the edible composition is a pet food. Examples of pet food are dry pet foods - such as kibbles -, wet pet foods - such as a loaf or a chunk-in-X product -, semi-moist pet foods, treats. In a particular embodiment, the pet food is a dog food, typically a kibble. In a particular embodiment, the pet food is a cat food, typically a kibble.

According to a particular embodiment is provided herein a method for preparing a pet food, comprising: i) providing a hydrolysate or a polypeptide/peptide as above described; ii) providing a pet food preparation comprising at least one pet food ingredient; iii) adding said hydrolysate or polypeptide/peptide to said pet food preparation; and iv) obtaining said pet food.

In particular, the hydrolysate or polypeptide/peptide may be added to said pet food preparation at step iii) by coating or by inclusion, preferably by inclusion. For example, the hydrolysate can be added in an amount of 0.05 to 60%, preferably 0.5 to 40%, preferably 0.5 to 20% (% by weight of pet food).

A particular embodiment of the present invention relates to a kit comprising, in one or more containers in a single package: at least one polypeptide/peptide or protein hydrolysate as above described, one or more ingredients for pet, preferably selected in the group consisting of cereals, fats, palatability-enhancers, animal or fish meals, nutrients, carbohydrates, preservatives, texturing agents and/or anti -oxidants. Another aspect of the invention relates to the therapeutic use of a protein hydrolysate or a polypeptide/peptide as above defined.

The invention refers to a protein hydrolysate or a polypeptide/peptide of the invention, for use as a medicament.

A particular embodiment relates to a protein hydrolysate or a polypeptide/peptide of the invention for use in eliciting CCK release, GLP-1 release, or combinations thereof, from a cell comprising contacting said cell with a protein hydrolysate or a polypeptide/peptide as above described.

The invention also relates to a method of eliciting CCK release, GLP-1 release, or combinations thereof, from a cell comprising contacting said cell with a protein hydrolysate or a polypeptide/peptide as above described.

A particular embodiment relates to a protein hydrolysate or a polypeptide/peptide of the invention for use in the treatment of a condition mediated by GLP-1 and/or CCK and/or DPP- IV.

The invention also relates to a method of treating a condition mediated by GLP-1 and/or CCK and/or DPP -IV.

More particularly, polypeptides/peptides or hydrolysates as above described comprising one or more of amino acid sequences chosen from DLDL, MDLP, VADTMEVV, VAPEEHPT, GPFPLLV, FAMD, CSSGGY, FPKATG, PPTT, EYLPV and MEMT can be used in the treatment of a condition mediated by DPP -IV.

More particularly, polypeptides/peptides or hydrolysates as above described comprising one or more of amino acid sequences chosen from DLVDK, PSLVH, VLEL, VLPL, FPYPLPRPL and LTLL can be used in the treatment of a condition mediated by CCK.

More particularly, polypeptides/peptides or hydrolysates as above described comprising one or more of amino acid sequences chosen from LKPT, LTDY, VPLM, LLLE, VDGL, LTLL, DGLL and VLPL can be used in the treatment of a condition mediated by GLP-1.

The invention also provides a method for treating obesity, type II diabetes, type I diabetes, renal diseases, steatosis, neurodegenerative diseases, anti-inflammatory troubles or atherosclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of a protein hydrolysate or a polypeptide/peptide as above defined. The invention also relates to the use of a protein hydrolysate or a polypeptide/ppetide of the invention for preparing a medicament for treating obesity, type II diabetes, renal diseases, steatosis, neurodegenerative diseases, anti-inflammatory troubles or atherosclerosis.

The invention also relates to a protein hydrolysate or a polypeptide/peptide of the invention for use in the treatment of obesity, type II diabetes, type I diabetes, renal diseases, steatosis, neurodegenerative diseases, anti-inflammatory troubles or atherosclerosis.

The involvement of DPP -IV, CCK and GLP-1 in the pathogenesis of these disorders is well described in the literature and these disorders are therefore the major target of the hydrolysate or polypeptide/peptide according to the present invention.

Type II diabetes is characterized by resistance to insulin and by a decrease of insulin secretion, such that the body does not respond to insulin appropriately, resulting in hyperglycemia. It is often accompanied by obesity. As GLP-1 contributes to normalization of blood glucose levels as well as to the control of satiety and obesity (body weight), an increase of GLP-I levels is expected to contribute to prophylaxis and/or treatment of type II diabetes.

The involvement of GLP-1 is recognized regarding obesity, type II diabetes, but also regarding renal diseases ^13,14 , steatosis ¹⁵ , neurodegenerative diseases - such as Alzheimer disease - ¹⁶ , anti-inflammatory troubles ¹⁷ or atherosclerosis ¹⁸ .

The involvement of DPP -IV is recognized regarding obesity, type II diabetes, but also regarding renal diseases type I diabetes ¹⁹ , renal diseases ¹⁴ , neurodegenerative diseases - such as Alzheimer disease - ²⁰ or atherosclerosis ¹⁸ .

In a particular embodiment, the protein hydrolysate or the polypeptide/peptide can be administered to the subject prior to or during a meal.

Advantageously for these therapeutic uses, a protein hydrolysate or a polypeptide/peptide of the invention can be in a pharmaceutical composition. The pharmaceutical composition may be in the form of a solution or as a solid depending on the use and/or the mode of application and/or the mode of administration. Advantageously, it is administered orally. It can comprise ingredients normally used in this type of formulation such as binders, flavorings, preservatives or colorings and, in the case of medicaments, may be in the form of tablets, granules or capsules. Compositions according to the invention can also be in the form of drinks, or in the form of suspensions or syrups. A particular embodiment thus relates to a pharmaceutical composition comprising at least one polypeptide/peptide or at least one protein hydrolysate as above described, preferably combined with a pharmaceutically acceptable carrier and/or excipient.

The skilled person is able to determine the appropriate amount of protein hydrolysate or polypeptide/peptide to be added in the composition as well as the posology, for example regarding the type of composition, the type and seriousness of the pathological condition to be treated, the weight and/or sex of the subject.

The following examples are employed to further illustrate the present invention and disclosure, but are in no way meant to limit the scope thereof.

EXAMPLES

1. MATERIAL AND METHODS

1.1. In vitro assays on hydrolysates and synthetic peptides

1.1.1. Hydrolysates

A tilapia hydrolysate (HI) was prepared according to the following process: an aqueous solution containing water (about 20%) and heads, bones and viscera of tilapia (about 80%) was heated to 55 °C and maintained at this temperature during lh and hydrolyzed with an alkaline microbial endopeptidase (about 0.2%). The enzyme was inactivated by heating the obtained mixture at 95 °C during 30 min. A step of separation by centrifugation occurred and the non fatty soluble part obtained was dried by atomization.

A poultry hydrolysate (H2) was prepared according to the following process: poultry raw material was heated to 60 °C and maintained at this temperature during 6 h and hydrolyzed with 3 endo- and exopeptidases. The enzymes were inactivated by heating the obtained mixture at 100 °C during 30 min. The obtained mixture was dried by spray-drying.

Various parameters were analyzed:

- DH was measured by using the OPA method, as described in Nielsen et al (11).

The moisture content was determined by using the method of Commission Regulation (EC) No 152/2009 of 27 January 2009.

The total protein content was determined by using the Dumas method, based on NF EN ISO 16634-1. The amount of soluble proteins was determined by solubilizing the sample in ultrapure water, separating the aqueous phase by centrifugation, and by applying the method of Kjedahl on this aqueous phase (Commission Regulation (EC) No 152/2009 of 27 January 2009).

The amount of fats has been determined by using the method of Commission Regulation (EC) No 152/2009 of 27 January 2009.

1.1.2. Preparation of synthetic peptides

Synthetic peptides were purchased from GeneCust (Boynes, France) with 95% of purity. Then, peptides were solubilized in ultrapure water at respective concentrations of 10 mM for DPP-IV inhibition and 20 mM for CCK and GLP-1 assays, except for peptide LLLE which was solubilized at 20 mM with ultrapure water containing 50% of dimethyl sulfoxide (DMSO).

1.1.3. Size exclusion chromatography (SEC) by fast protein liquid chromatography (FPLC)

The peptide molecular weight distribution of hydrolysates was obtained by SEC as disclosed in Guerard et al., 2001 (10).

1.1.4. Simulated gastrointestinal digestion (SGID) of fish by-product

Simulated digestions of hydrolysates were performed according to the protocol described by Caron et al. with some modifications in order to mimic the dog digestion ²¹ . Briefly, the three first steps of the digestive tract (mouth, stomach and small intestine) were simulated using a static mono compartmental process in a reactor under constant magnetic stirring. To mimic the dog digestive process, the temperature was maintained at 39°C during all the simulation. Two grams of hydrolysates were solubilized in 16 mL of salivary fluid at pH 7.0 without salivary enzyme. Samples of 4 mL (mouth) were collected after 2 min. 24 mL of gastric fluids were then added decreasing the pH solution to 2 and porcine pepsin was added in a 1:40 (w/v) Enzyme/Substrat ratio (enzymatic activity > 2000 U.mg ¹ of dry weight). In vitro gastric digestion was performed over 2 h during when the pH was monitored and maintained at 2.0. Samples of 4 mL (stomach) were collected after 2 h and directly heated at 95 °C during 10 min to stop hydrolysis. 36 mL of intestinal fluid and 4 mL of 1 M NaHCCb solution were added to reach the pH to 6.8. Pancreatin was added in a 1:50 (w/v) ratio enzyme / substrate (enzymatic activity 100 U.mg ¹ of dry weight) and intestinal digestion was carried out over 4 h and samples collected and heated as described above in order to inactivate the enzymes. All hydrolysate samples were then centrifuged at 13000 x g for 10 min. Supernatants were collected and kept at -20 °C for further analysis.

1.1.5. Cell culture conditions

The Caco-2 cell line was purchased from Sigma-Aldrich (Villefranche sur Saone, France) and the STC-1 cell line was a grateful gift received from Corinne Grangette (Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL, France). Cells were grown in flask of 75 cm ² at 37 °C, 5% CO2 atmosphere in Dulbecco’ s modified Eagle’ s Medium (DMEM, 4,5 g.L ¹ glucose) supplemented with 10% of fetal bovine serum, 100 U.mL ¹ of penicillin, 100 pg.mL ¹ of streptomycin and 2 mM of L-glutamine. Caco-2 and STC-1 cells were weakly and twice a week subcultured, respectively. All cells used in this study were between the 40 and the 50 passages for Caco-2 cells and between the 19 and 42 passages for STC-1 cells.

1.1.6. CCK and GLP-1 secretion study

When 80-90% confluence were reached, STC-1 cells were trypsinized and seeded at a density of 40000 cells/well in 24-well culture plates (Thermofisher Scientific) allowing to reach 60- 80% confluence. Cell culture medium was removed from each well and replaced with hydrolysates (1% w/v) or peptides (1 mM) diluted in Hepes buffer (4.5 mM KC1, 1.2 mM CaCL, 140 mM NaCl and 20 mM Hepes, pH 7.4). Hepes buffer was used as negative control. After 2h of incubation at 37 °C, 5% CO2 atmosphere, supernatants were collected on ice, centrifuged (1 500 x g for 5 min) and stored at -20 °C for further CCK and GLP-1 concentration measurements using GASK-PR (Cisbio, Codolet, France) and GLP-1 active (Merck, Molsheim, France) kits, respectively. The analyses were carried out according to the protocols supplied by the manufacturers.

1.1.7. DPP-IV activity assay (in situ Caco-2 cells)

In situ method using confluent Caco-2 cells described by Caron et al. was slightly modified and used to study DPP-IV activity ²² . A substrate solution (Gly-Pro-AMC) and peptides and digest dilutions were prepared in phosphate-buffered saline (PBS) buffer (IX, pH 7.4).

Briefly, after 7 days of growth, Caco-2 cells were trypsinized and seeded at a density of 8,000 cells/well in 96-well optical black plates (Nunc, ThermoFisher Scientific, Rochester, NY, USA). After 7 days, culture media were removed from wells and the cells were washed with 100 pL of PBS buffer (pH 7.4). Then, 100 pL of PBS were added to the wells followed by 25 pL of hydrolysates diluted in PBS at increasing concentrations (3.47; 6.95 and 13.89 mg.mL ¹ , respectively representing 0.2%, 0.5% and 1% w/v) or 25 pL of synthetic peptides dilutes in PBS at increasing concentrations (between 0.2 and 1.5 mM) or 25 pL of PBS buffer (control wells). After 5 min of incubation at 37 °C, 50 pL of substrate solution (Gly-Pro-AMC, 1 mM) were added to each well. Fluorescence was recorded every 2 min for lh at 37 °C using a Xenius XC spectrofluorometer (Safas Monaco, Monaco). Excitation and emission wavelengths were set to 260 and 480 nm, respectively. Voltage was set to 300 V. The percentage of the DPP -IV activity inhibition was defined as the percentage of DPP -IV activity inhibited by a given concentration of digest or diprotin A (commercial DPP -IV peptide inhibitor) as positive control compared with control buffer response. The concentration of the digest required to obtain 50% inhibition of the DPP -IV activity (IC50) was determined by plotting the % of DPP -IV activity inhibition as a function of sample final concentration natural logarithm. IC50 were expressed in mg.mL ¹ (w/v) and mM for the digests and synthetic peptides, respectively.

1.1.8. DPP-IV activity assay (in vitro acellular assay)

DPP -IV inhibitory activity of digests and of synthetic peptides was measured in triplicates. Digests were diluted in 0.1 M Tris-HCl buffer pH 8.0 at final concentrations ranging from 1.29 to 2.57 mg.mL ¹ and synthetic peptides from 0.4 to 1 mM. In a 96-well microplate, 25 pL of diluted samples were added to 75 pL 0.1 M Tris-HCl buffer pH 8.0, 25 pL of DPP-IV (0.018 units. mL ¹ prepared in Tris-HCl buffer) and 50 pL of the substrate Gly-Pro-p- nitroanilide (1 mM prepared in Tris-HCl buffer) were then added for lh at 37 °C under gentle stirring. Absorbance was recorded at 405 nm at 2 min intervals using a ELx808 Absorbance Reader (BioTek Instruments Inc., USA). The % DPP-IV inhibition was defined as the percentage of DPP-IV activity, inhibited by a given concentration of digest/peptide compared with control response. The concentration of the digest/fraction required to cause 50% inhibition of the DPP- IV activity (IC50) was determined by plotting the % DPP-IV inhibition as a function of sample final concentration natural logarithmic. IC50 were expressed in mg.mL ¹ (w/v) and mM for the digests and synthetic peptides, respectively. The tripeptide Ile-Pro-Ile (diprotin A) was used as reference inhibitor. 1.1.9. Passage through the intestinal barrier model

To obtain an intestinal barrier model, Caco-2 cells were cultivated on insert (D = 4.2 cm; pore size = 3 pm, //reference : 353092, Dutscher, Issy-les-Moulineaux, France) in a Compagnon 6 wells plate (Dutscher, Issy-les-Moulineaux, France). To this end, Caco-2 cells are seeded at a density of 84000 cells by insert in 2 mL of DMEM medium. A volume of 2.5 mL of DMEM medium was added in each well of the plate. Cells were incubated at 37 °C during 15 days and the medium on apical and basolateral sides replaced every 2 days.

The day of the experiment, a transport medium Hepes-Hanks Salt Solution (HBSS) was prepared and filtered on polyvinylidene difluoride (PVDF) filter 0.2 pm. Samples were diluted at 4 mg.mL ¹ in the transport medium. The apical and basolateral sides of each well were washed with 500 pL and 1 mL of transport medium (heated at 37 °C), respectively. Then 1 mL of transport medium at 37 °C was added in the apical side and 2.5 mL in the basolateral side. Plate was incubated at 37 °C, 5% of CO2 during 30 min and the supernatant was removed and replaced with 1 mL of samples or pre-heated transport medium (for the control). Kinetic was performed with the sampling of 100 pL in apical and basolateral sides at 15 min, 250 pL in apical and 1 mL in basolateral sides at 60 min and the resting of supernatant in the apical (650 pL) and in the basolateral sides (1.4 mL) at 120 min of incubation at 37 °C, 5% CO2.

1.1.10. Simulated gastrointestinal digestion of tilapia by-product raw materials vs tilapia hydrolysate HI

The stimulation of the secretion of active GLP-1 induced by the tilapia hydrolysate and its raw material was measured by a radioimmunoassay as described previously in section 1.1.6. The control (representing the basal level of intestinal hormone secretion) was composed of a Hepes buffer at pH 7.4. In this experiment, the tilapia hydrolysate HI and its raw materials were subjected to a gastrointestinal simulated digestion (“SGID”), as detailed in 1.1.4. The samples were incubated at 1% (dry weight/volume).

1.1.11. Comparison of different marine hydrolysates on CCK and GLP-1 secretion and DDP-IV inhibitory activity

Two marine hydrolysates H3 and H4 were prepared according to conventional hydrolysis process, H3 was obtained from tuna byproducts and H4 was obtained from wild white fish byproducts. Those hydrolysates were compared to tilapia hydrolysate HI. Measurements of CCK concentrations, GLP-1 concentrations, and DDP-IV inhibition were carried out as detailed in sections 1.1.6 (for GLP-1 and CCK stimulation) and 1.1.8. (for DPP -IV inhibition). 1.1.12. Statistical analyses

In vitro test: data presented are mean ± standard deviation (SD). To compare GI hormone secretion levels induced by hydrolysates and peptides, a one-way ANOVA using general linear model and pairwise comparisons with Dunnett ok Tukey tests were performed using GraphPad Prism. Values and means not sharing an identical letter on tables were considered as significantly different for a p value < 0.05.

1.2. In vivo assays- Cats

1.2.1. Preparation of kibbles containing hydrolysates Nutritionally-balanced cat kibbles suitable for consumption by pets were prepared by using an extrusion process comprising the steps of mixing the kibble ingredients, preconditioning, extruding and drying.

The kibble ingredients (rice, corn, wheat, com, fiber source, flax, poultry meal by-products, vitamins, minerals, amino acids) and the hydrolysate (if added) were fed into a preconditioner before entering an extruder with jackets for cooling or heating. Cooling water was constantly passed through the jackets. The extruded material was passed through a die and cut into homogenous spherical shape. They were thus dried in a hot air dryer and the resulting diets had less than 8% moisture. They were stored at ambient temperature before being tested. These dry kibbles were then coated with 6% poultry fat and with 2% of a commercial palatability enhancer.

The metabolizable energy value of the control, i.e. the pet food without hydrolysate (PFO) was 2580 kcal.kg ¹ . The metabolizable energy value of the pet food with 5% of the tilapia hydrolysate HI (PF1) was 2510 kcal.kg ¹ .

1.2.2. Determination of palatability Operating method of the test:

The palatability of two products was assessed at Panelis (France), using a two-bowl test (two short meals per day for two days) on 40 cats. Identical amounts of food product A and food product B were weighed out and placed in identical bowls. The amount present in each ration enables the daily requirements to be met. If animals have higher or lower consumption compared to predetermined values (which are function of, e.g., the animal weight and/or metabolism), they are not taken into account into statistical treatment. The final intake of each food was measured. The results are shown as relative consumption ratios of A or B.

Statistical analysis

Statistical analysis was used to determine if there was a significant difference between the 2 ratios. A Student’s t-test with 3 error thresholds, namely 5%, 1% and 0.1%, was performed.

Significance levels are noted as below:

NS not significant (p > 0.05)

* significant (p < 0.05)

** highly significant (p < 0.01)

*** very highly significant (p £ 0.001).

1.2.3. Determination of the satiating effect

The method described in the patent application WO2015155177 has been used in order to assess the satiating effect of the pet food PF1.

A feeding trial was performed in a randomized monadic way, with the 2 different pet foods PF0 and PF1. Each pet food was offered in a single bowl, in a balanced serving order, and tested by a panel of 40 cats during 2 days. The food was weighed out and placed in feeding bowls. The amount offered enabled the daily energy requirements of the pets to be met. The bowl was presented to the pets in a feeding system comprising all necessary equipment to collect and preferably record the relevant data. Pets had free access to the distributed food and free access to drinking water throughout the duration of the test. Tests began at 11 o’clock. Only one pet food was available to the pets per day of the test. Before starting any pet food evaluation, the pets were split in two groups, such that one group received the control pet food and the other group received the candidate pet food; in the second part of the procedure, the pet food allocation was inverted and the test was then repeated.

The measured parameters were the quantities of pet foods consumed and temporal data associated with these quantities. From these parameters, different criteria (also referred to herein as “crit”) were calculated:

- total calorie consumption throughout the feeding trial (tC); duration between the start of the feeding trial and the first feeding event (DFE1); and - total number of feeding events with low or no consumption during the feeding trial (NbFElow); calorie consumption at the first feeding event (CFE1).

For comparing results obtained for the different pet foods, a statistical analysis was performed with a student’s T-test. Typical significance levels for statistical tests were noted as below:

NS not significant (p > 0.05)

* significant (p < 0.05)

** highly significant (p < 0.01)

*** very highly significant (p < 0.001).

For each of these criteria, Acritmean was calculated: Acritmean = crit(PFl) - crit(PFO) for all pets. It is then possible to conclude on the palatability and/or satiating effect of the pet food tested:

- if AtC _mean ³0 and either ADFEl _mean >0 or ANbFElow _mean >0: the pet food tested has no calorie intake reducing effect for pets (AtC _mean ³0) and is not as palatable as the control pet food (ADFEl _mean >0 and ANbFElow _mean >0);

- if AtCmean³0, ADFEl _m ean£0, and ANbFEl owm .- : the pet food tested has no calorie intake reducing effect for pets (AtCmean³0) although it is at least as palatable as the control pet food (ADFElmean£0 and ANbFEl OWmean£0);

- if AtC _mean <0, and either ADFEl _mean >0 or ANbFEl ow _mean >0: the pet food tested is not as palatable as the control pet food (ADFEl _mean >0 and ANbFEl ow _mean >0) although it has a calorie intake reducing effect for pets (AtC _mean <0);

- if AtCmean<0, ADFEl _m ean£0, and ANbFEl owm .- : the pet food tested has a calorie intake reducing effect for pets (AtCmean<0) and as it is at least as palatable as the control pet food (ADFElmean£0 and ANbFEl owmean£0). Then, if ACFEl _m ean³0, the calorie intake reducing effect is due to a satiating effect and not to a hypocaloric effect. 1.3- In vivo assays- Dogs

1.3.1- Preparation of kibbles containing hydrolysates

Nutritionally-balanced dog kibbles suitable for consumption by pets were prepared by using an extrusion process comprising the steps of mixing the kibble ingredients, preconditioning, extruding and drying.

The kibble ingredients (com, wheat, fiber source, poultry meal by-products, vitamins, minerals, amino acids) and the hydrolysate (if added) were fed into a preconditioner before entering an extruder with jackets for cooling or heating. Cooling water was constantly passed through the jackets. The extruded material was passed through a die and cut into homogenous spherical shape. They were thus dried in a hot air dryer and the resulting diets had less than 8% moisture. They were stored at ambient temperature before being tested. These dry kibbles were then coated with 6% poultry fat and with 2% of a commercial palatability enhancer.

The metabolizable energy value of the control, i.e. the dog pet food without hydrolysate (PFDO) was 3635 kcal.kg ¹ . The metabolizable energy value of the dog pet food with 5% of the tilapia hydrolysate HI (5PFD1) was 3550 kcal.kg ¹ . The metabolizable energy value of the dog pet food with 7.5% of the tilapia hydrolysate HI (7.5PFD1) was 3628 kcal.kg ¹ .

1.3.2- Determination of palatability

Operating method of the test:

The palatability of two products was assessed at Panelis (France), using a two-bowl test (two short meals morning or afternoon) on 40 dogs. Identical amounts of food product A and food product B were weighed out and placed in identical bowls. The amount present in each ration enabled the half daily requirements to be met. If animals had higher or lower consumption compared to predetermined values (which were function of, e.g., the animal body weight and/or metabolism), they were not taken into account for any statistical treatment. The final intake of each food was measured. The results are shown as relative consumption ratios of A or B.

Statistical analysis

Statistical analysis was used to determine if there was a significant difference between the 2 ratios. A Student’s t-test with 3 error thresholds, namely 5%, 1% and 0.1%, was performed.

Significance levels are noted as below: NS not significant (p > 0.05)

* significant (p < 0.05)

** highly significant (p < 0.01)

*** very highly significant (p £ 0.001)

1.3.3- Determination of the satiating effect

A monadic feeding trial was performed to evaluate 2 levels of HI inclusion (5 and 7.5%). 38 dogs were allocated in three groups, according to body weight and tested successively 3 diets on 4 consecutive meals, i.e. the testing period for each pet food was composed of 2 days with 2 meal occasions per day. The 3 different dog pet foods were PFD0 (Control Pet Food), 5PFD1 (5% HI Pet Food) and 7.5PFD1 (7.5% HI Pet Food). Each pet food was offered in a single bowl with a balanced serving order. There was at least a 1-day wash out period between 2 testing periods. Tests began on day 1 with the first meal at 7:45 am (meal 1) and then 2 ^nd meal (meal 2) at 15: 15 pm. Day 2 the 3 ^rd meal (meal 3) started at 7:45 am and then the 4 ^th meal (meal 4) started at 15:15 pm. Meal 4 had a food quantity increased by 20%. The amount of food offered enabled to satisfy dogs’ daily metabolizable energy requirements. Dogs had free access to drinking water throughout the duration of the test.

The measured parameter was the quantity of pet foods consumed. From this parameter, total calorie intake by kg metabolic weight was calculated per testing period (tCmw);

A statistical analysis was performed with a Student’s t-test to compare intakes. Typical significance levels for statistical tests are noted as below:

NS not significant (p > 0.05)

* significant (p < 0.05)

** highly significant (p £ 0.01)

*** very highly significant (p £ 0.001).

2. RESULTS

2.1. In vitro assays on hydrolysates

2.1.1. Characterization of hydrolysates

The tilapia hydrolysate HI obtained has a moisture content of 5.3%, a DH of 28%, a protein content of 80.6% in which 90.3% are soluble proteins, a fat content of 7.2%, a pH of 5.9. The peptide molecular weight distribution of the tilapia hydrolysate HI is as follows: 75.5% with a molecular weight of less than 1000 Da, 18% with a molecular weight between 1000 and 3000 Da, 3.5% with a molecular weight between 3000 and 5000 Da, 2.9% with a molecular weight between 5000 and 10000 Da, and 3% with a molecular weight between 5000 and 20000 Da.

The tilapia hydrolysate HI contains at least the following amino acid sequences: DLVDK, PSLVH, DLDL, MDLP, VADTMEVV, VAPEEHPT, GPFPLLV, LKPT, LTDY, FAMD and CSSGGY.

The poultry hydrolysate H2 obtained has a moisture content of 1.9%, a DH of 80.5%, a protein content of 60.6 % in which 98.5% are soluble proteins, a fat content of 15.3%.

The peptide molecular weight distribution of the poultry hydrolysate H2 is as follows: 97.3% with a molecular weight of less than 1000 Da, 2.3% with a molecular weight between 1000 and 3000 Da, 0.3% with a molecular weight between 3000 and 5000 Da, 0.25% with a molecular weight between 5000 and 10000 Da and 0.1% with a molecular weight between 5000 and 20000 Da.

The poultry hydrolysate H2 contains at least the following amino acid sequences: VLEL, VLPL, FPYPLPRPL, LTLL, FPKATG, PPTT, MEMT, DGLL, VPLM, LLLE, VDGL and EYLPV.

2.1.2. Passage through the intestinal barrier

Peptides GPFPLLV, MDLP, DLDL, FAMD, VADTMEVV, CSSGGY, VAPEEHPT, MEMT, EYLPV, PPTT and FPKATG were identified in the basolateral side, meaning that they crossed the intestinal barrier model. This unexpected result is paramount since peptides harboring DPP- IV inhibitory activity are not always able to cross the intestinal barrier. Indeed, the haemorphin LLVVH4 evidenced after the simulated gastrointestinal digestion (or “SGID”) of bovine haemoglobin and characterized as promising DPP -IV inhibitory peptide in a first study was shown not to be able to cross Caco-2 cell monolayer in a second study ^23,24 . Hence, because peptides according to the invention are able to cross intestinal barrier, these are particularly interesting for use in-vivo.

2.1.3. In vitro cell culture test: effect of hydrolysates on the stimulation of CCK and GLP-1 secretion and on the inhibition of DPP-IV during GI digestion The hydrolysates HI and H2, which contain peptides according to the invention or the disclosure, were tested for their ability to stimulate the secretion of CCK and GLP-1 at 1.0% (w/v) by STC-1 enteroendocrine cells after a stimulated digestion. DPP -IV activity inhibition was determined with the IC50 which indicates the minimal concentration to inhibit by 50% the DPP -IV activity.

The results concerning the secretions of CCK and GLP-1 (expressed in fold of control (FOC)) and the inhibition of DPP -IV activity by the hydrolysates HI and H2 are presented in Table 3. Values are means ± SD and means without a common letter are different (p < 0.05). For sake of clarity, statistical treatments were carried out independently for each hydrolysate HI or H2. Table 3

The STC-1 cell exposure to HI and H2 induced a significant increase in CCK and GLP-1 release.

HI led to a higher stimulating effect on CCK secretion for mouth and stomach samples. The intestinal digest of HI very highly stimulated the secretion of GLP-1 (45 fold of control (FOC)). Action of digestive enzymes thus greatly potentiates the stimulation of GLP-1 release. In contrast, other work showed that a salmon skin gelatin hydrolysate lost its GLP-1 stimulatory activity and had a significantly lower DPP -IV inhibitory activity after SGID ²⁵ . The results obtained with the tilapia hydrolysate according to the invention, demonstrate the particular interest of its intestinal digest on the secretion of GLP-1.

H2 highly stimulated CCK secretion for mouth, stomach and intestine samples. The inventors also observed that this specific hydrolysate H2 stimulates 2 times more the GLP-1 secretion than non-hydrolyzed tilapia by-products. This confirms the interest of hydrolyzing raw material in order to generate bioactive peptides which are often resistant to digestion. Moreover, the intestinal digest of H2 very highly stimulated the secretion of GLP-1 (21 FOC). Similar observation can be made from a work showing the better angiotensin-converting enzyme (ACE)-inhibitory activity of a land snail by-product hydrolysate compared to that obtained with its raw material, before and after SGID ²⁶ . In the same way, another work evidenced the better potential of a cuttlefish byproduct hydrolysate to in vitro inhibit DPP -IV activity and stimulate GLP-1 secretion than its raw material before and after SGID ²⁷ .

The results also show the high potential of HI and H2 to inhibit DPP -IV activity from the start of the digestive process and even before gastrointestinal enzyme actions. Indeed, the calculated IC50 is steady during in vitro GI digestion.

2.1.4. In vitro cell culture test: effect of tilapia hydrolysate HI vs tilapia by-product raw materials on the stimulation of GLP-1 secretion

GLP-1 secretion by STC-1 cells was increased after the gastrointestinal digestion. As shown in the Table 4, the action of gastrointestinal enzymes potentiates the stimulation of GLP-1 secretion both for the raw material and the tilapia hydrolysate HI (dose of 1% dry weight/vol). The effect of gastrointestinal enzymes on GLP-1 bioactivity was particularly significant for the tilapia hydrolysate HI: measured GLP-1 concentrations increased from 5.27 ± 0.35 times the control for the mouth digest to 10.23 ± 2.32 times the control for the gastric digest and to 44.93 ± 3.02 times the control for the intestinal digest.

Remarkably, the intestinal digest of tilapia hydrolysate stimulated GLP-1 secretion twice as much as the intestinal digest of its raw material, confirming the interest of the hydrolysate in generating bioactive peptides after gastro-intestinal digestion. Table 4

2.1.5. In vitro cell culture test: comparison of tilapia hydrolysate HI to other marine hydrolysates on the stimulation of CCK and GLP-1 secretions and DDP-IV inhibition

The results concerning the inhibitory potential of DPP -IV activity (mg.mL ¹ ) and the secretions of CCK and GLP-1 (FOC, control Hepes buffer) by different marine hydrolysates (1% w/v) are presented in the Table 5. Values are means ± SD.

Table 5 HI was compared with two other marine hydrolysates H3 and H4: when HI was applied to STC-1 cell lines, its bioactive potential was higher for CCK (3.84 FOC ± 0.46) and GLP-1 (17.19 FOC ± 0.59) secretions than for H3 (1.30 FOC ± 0.27 and 1.57 FOC ± 0.90, for CCK and GLP-1 respectively) and H4 (11.07 FOC ± 0.64 and 2.78 FOC ± 0.86, for CCK and GLP- 1 respectively). Moreover, its DPP -IV inhibitory potential was also higher when compared to other marine hydrolysates (lower IC ₅₀ of 3.8 mg. mL ¹ ). These results highlight the particular interest of the HI bioactive potential in comparison to other marine sources, especially because it can stimulate 2 satiating hormonal peptides (CCK and GLP-1) while having a good DPP -IV inhibitory effect. From these results, it can be hypothesized that the hydrolysate according to the invention is of particular interest in the regulation of energy homeostasis. Other hydrolysates made from different raw materials and processed differently do not show this activity.

2.2. In vitro assays using synthetic peptides

2.2.1. Effect of synthetic peptides on the stimulation of CCK secretion

Synthetic peptides according to the invention (Table 6) or according to the disclosure (Table 7) were tested for their ability to stimulate the secretion of CCK in STC-1 enteroendocrine cells.

The results concerning the secretions of CCK by peptides (1 mM) are presented in Tables 6 and 7. Values are means ± SD and means without a common letter are different (p < 0.05).

Table 6

Table 7 The STC-1 cell exposure to synthetic peptides PSLVH, DLVDK, VLPL, FPYPLPRPL, LTLL, VLEL induced a significant increase in CCK release.

2.2.2. Effect of peptides on the stimulation of GLP-1 secretion

Synthetic peptides according to the invention (Tables 8 and 9) or according to the disclosure (Table 10) were tested for their ability to stimulate the secretion of GLP-1 in STC-1 enteroendocrine cells.

The results concerning the secretions of GLP-1 by peptides (1 mM) are presented in Tables 8, 9 and 10. Values are means ± SD and means without a common letter are different (p < 0.05).

Table 8

Table 9

Table 10

The STC-1 cell exposure to synthetic peptides LKPT, LTDY, VPLM, LLLE, DGLL, LTLL, VDGL, VLPL, LTLL induced a significant increase in GLP-1 release.

2.2.3. Effect of synthetic peptides on the inhibition of DPP-IV

The capacity of peptides to inhibit the DPP-IV activity was determined with the IC ₅₀ which indicates the minimal concentration to inhibit by 50% the DPP-IV activity.

The results concerning the inhibition of DPP-IV activity by peptides are presented in Table 11.

Table 11

The results show the interesting potential of synthetic peptides to inhibit DPP -IV activity, and in particular of DLDL, MDLP, FAMD, GPFPLLV, PPTT, MEMT and EYLPV having an IC ₅ o lower than 1 mM.

2.3. In vivo assays on hydrolysates- Cats

The results regarding the two-bowl test (palatability) are presented in Table 12.

Table 12 Dry cat foods directly comprising the tilapia hydrolysate HI are at least as palatable as cat foods, and can even be more palatable, than a control pet food.

The results concerning the criteria tC, DFE1, and NbFElow and CFE (for all pets) are presented in Table 13. Table 13

As shown in Table 13, AtC _mean <0, ADFEl _mCan = 0, and ANbFElow _mean = 0: PF1, which comprises the tilapia hydrolysate HI, has a calorie intake reducing effect on pets (AtC _mean <0) and as it is at least as palatable as the control PFO (ADFE l _mCan = 0 and ANbFElow _mean = 0). Moreover, CFE1 was not significantly different between PFO and PF1, showing that the caloric intake reducing effect of PF1 is due to a satiating effect and not a hypocaloric effect.

2.4. In vivo assays on hydrolysates- Dogs

The results regarding the two-bowl test (palatability) are presented in Table 14.

Table 14

As shown in Table 14, consumptions were not significantly different between dog foods PFDO (control dog food) and 5PFD1 (dog food comprising 5% of hydrolysate HI), and between 5PFD1 (dog food comprising 5% of hydrolysate HI) and 7.5PFD1 (dog food comprising 7.5% of hydrolysate HI). Consumption was significantly different between PFDO (control dog food) and 7.5PFD1 (dog food comprising 7.5% of hydrolysate HI)..

The results show that the incorporation of hydrolysate HI maintains dog food palatability and may even increase it depending on the incorporated rate. The results concerning the criteria tCmw are presented in Table 15.

Table 15

As shown in Table 15, total calorie consumption by metabolic weight is significantly higher for the control diet PFDO than for the experimental diets with tilapia hydrolysate 5PFD1 and 7.5 PFD1. The results show that the incorporation of hydrolysate HI, either at 5% or 7.5% in the dog food, allows to obtain a caloric intake significantly reduced. This demonstrates that hydrolysate HI has a satiating effect.

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