The present invention relates to the field of nutrition. In particular it relates to rice protein hydrolysates and to their use as preventers and/or therapeutic agents for the obesity.

BACKGROUND ART Obesity is a significant clinical problem that contributes to life-threatening diseases such as diabetes and atherosclerosis. The identification of pathways leading to increased adipose tissue formation, and reversal of lipid stores in adipose tissue, raises the prospect of preventing or reversing obesity through pharmacological or nutritional means.

Obesity is the most prevalent nutritional disorder among children and adolescents throughout the world. Notwithstanding recent reports suggesting a levelling off of the prevalence of obesity in some countries, the burden of paediatric obesity for society is still high. In addition to short-term

complications such as psychosocial disturbances or orthopaedic problems, the origins of potential long-term metabolic consequences are also

identifiable in many obese children. It is well established that obesity is a multifactor disease in which genetic as well as psychological and

environmental causative factors are implicated, with diet and physical inactivity looming large. For all these reasons, the paediatric community insists in that nutrition and lifestyle education aimed at the prevention of obesity should be included in the routine care of children not only by general paediatricians, but also by parents and other professionals around the children.

On the other hand, it is well established and known that breastfeeding is protective against obesity, but not all children can be breastfed by their mothers, or they need supplemental food intake (infant formulas) apart of the breast milk.

Considering children and adolescents as the most interesting groups for the early prevention of obesity, the importance of this pathology in other collectives is also of great interest.

With the aim of fighting obesity and also to get a lean figure many dietetic foods are currently in the market. Also diet protocols well-known by the users are applied in which normally the balance of fat intake and carbohydrate intake is regulated. In document WO 2004024177 a composition with at least 10 % of protein hydrolysate from non-mammalian source is disclosed as a natural solution to the problem of loosing weight. The document mentions as possible non-mammalian protein hydrolysates produced from vegetable protein such as wheat, maize, pea, rice, soy, barley, oats, potato, and mixtures thereof. Examples of compositions are made with hydrolysed pea protein. Nonetheless, no data with one of the exemplified compositions is provided. The inventors of WO 2004024177 indicate that people consuming a composition comprising 10 % by weight of a generic protein hydrolysate felt a long lasting satiety and stopped eating even in ad libitum situations, without giving raise to an increase of the blood insulin levels.

Another protein hydrolysate composition aiming fighting the obesity epidemic is disclosed in document WO 2010078461 . In this case compositions containing soy protein hydrolysate are proposed to induce the secretion of cholecystokinin and thereby, to promote satiety when consumed.

In the field of infant nutritional formulas focusing on the treatment and/or prevention of obesity, the document ES 2350907 T3 is to be mentioned. This document discloses a nutritional composition comprising 35 to 60 wt. % fat,

25 to 75 wt. % digestible carbohydrates, 5 to 16 wt. % protein, and a non- digestible fermentable carbohydrate selected from the group consisting of polyfructose and galactooligosaccharides, wherein the protein comprises a) at least 25 wt. % peptides with a chain length of 2 to 30 amino acids based on dry weight of protein; b) at least 50 wt. % mammalian milk derived proteins, based on weight of protein; c) casein and whey in a weight ratio casein:whey of 10:90 to 90:10; and d) less than 15 wt. % free amino acids based on the weight of protein source. This composition is used to reduce the levels of blood glucose and insulin, which are thought to be responsible of obesity derived from insulin-resistance.

In ES 2350907 T3 the major protein components in the nutritional composition is non human milk. Rice protein and rice protein hyrolysates can also be included, although no examples and data are shown.

As can be deduced from the documents above, there are needed

compositions aiming the prevention or treatment of obesity in children, as well as in other collectives. In particular, for children with specific obese trends, there exists a need of infant formulas allowing the prevention or the treatment of this pathology.

SUMMARY OF THE INVENTION

The inventors have developed a process for obtaining protein hydrolysates from rice, which rice protein hydrolysates are capable of inducing lipolysis in adypocites and to avoid excess of fat accumulation. These hydrolysates are useful in the prevention and/or the treatment of obesity when they are administered, for example, as components of infant formulas.

Thus, a first aspect of the invention is a process for obtaining a rice protein hydrolysate comprising the steps of: a) adding a rice protein source in a liquid medium at a final protein

concentration comprised between 5 % and 20 % weight/volume (w/v) to obtain a reaction mixture;

b) adjusting the reaction mixture of step a) to a pH comprised between 6 and 8;

c) performing the protein hydrolysis reaction of the resulting mixture from step b) by adding an amount of an enzymatic system, said enzymatic system

having endoprotease and/or exoprotease activity, and

comprising one or more enzymes, each one of the enzymes being in a concentration between 0.01 g/100 ml of reaction mixture and

10.0 g/100 ml of reaction mixture,

at a constant pH and temperature, the temperature being comprised between 30°C and 55°C; and

d) deactivating the enzymatic system to stop the protein hydrolysis

reaction when the degree of hydrolysis (DH) of the mixture is comprised between 4 and 17, wherein said DH is calculated by means of the following formula: DH = B x Nb x (1/a) x 1/MP x 1/Htot x 100; wherein

B is the volume in millilitres (ml) of consumed base used for titrating released amino groups during protein hydrolysis,

Nb is the normality of the base used for titrating,

1/a is the average degree of dissociation of the amino groups related with the pK of said amino groups at a particular pH and temperature, MP is the amount in grams of the protein source in the reaction mixture, and

Htot in milliequivalents per g (meq/g) is the sum of the millimoles of individual amino acids per gram of protein associated with the source of protein.

The process of the invention is interesting since, apart from giving raise to products with interesting anti-obesity properties, it is in addition technical and instrumentally of low cost.

Another aspect of the invention is a rice protein hydrolysate obtainable by a process as defined above.

The rice protein hydrolysate "obtainable by" the process of the invention is used here to define the rice protein hydrolysate by the process for obtaining it and refers to the product obtainable by the preparation process comprising the steps a), b), c), and d) as defined above. For the purposes of the invention the expressions "obtainable", "obtained" and equivalent expressions are used interchangeably, and in any case, the expression "obtainable" encompasses the expression "obtained"

Proteins are important constituents of the human diet, since they comprise a principal source of nitrogen and essential amino acids. Proteins are used in many different food products, ranging from dairy products to beverages, dietary and medical products. For some food applications proteins are hydrolysed, amongst others for hypoallergenic infant nutrition, for nutrition for patients with digestion disorders and for sports nutrition. Proteolysis

procedure can also modify technological aspects of food proteins as solubility, viscosity, emulsion capacity and stability, foam and gel properties.

Hydrolysis of proteins with specific proteolytic enzymes and subsequent fractionation may result in the isolation of fractions with particular nutritional characteristics. Two types of characteristics can be differentiated:

a) Protein fractions with a relatively high or low content of specific amino acids.

b) Bioactive peptides with particular amino acid sequences, which are inactive in the intact protein molecule and become bioactive after their release from the intact molecule through the action of digestive enzymes in the body or through the action of proteolytic enzymes in food processing.

Most of the bioactivities of protein are latent, being absent or incomplete in the native proteins and only fully manifested upon proteolytic digestion to release and activate encrypted bioactive peptides from within. Proteolysis may release bioactive peptides during gastrointestinal transit or during food processing. Digestive enzymes, naturally occurring enzymes in foods, microbial enzymes, and starter lactic acid bacteria, can generate bioactive peptides during food digestion or fermentation thereby enhancing functional properties of the containing elaborated food. These rice protein hydrolysates of the invention may be used as components of other compositions, such as an edible composition. Another aspect of the invention is thus an edible composition comprising the rice protein

hydrolysate as defined above, together with appropriate amounts of other edible ingredients.

Another aspect of the invention is a nutritional composition which comprises a nutritionally effective amount of the rice protein hydrolysate as defined above.

The term "edible ingredient" as used herein refers to compounds, materials or compositions which are commonly used in the preparation of foods, nutritional compositions or supplements, such as vitamins, carbohydrate sources, lipid sources, minerals, etc. The term "nutritionally effective amount" as used herein, means an amount of an active agent high enough to deliver the desired benefit, but low enough to avoid serious side effects within the scope of nutritionist judgment.

The rice protein hydrolysate obtainable by the process provided herewith is also applicable to pharmaceutical compositions. Thus, another aspect is a pharmaceutical composition which comprises a therapeutically effective amount of the rice protein hydrolysate together with appropriate amounts of pharmaceutical acceptable excipients and/or carriers.

In this regard, the pharmaceutical product may be prepared in any suitable form which does not negatively affect to the bioavailability of the hydrolysate forming the composition of the invention. Selection of the excipients and the most appropriate processes for formulation in view of the particular purpose of the composition is within the scope of the person skilled in the art of pharmaceutical technology.

The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (either a human or non-human animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.

The term "pharmaceutically effective amount" as used herein, means an amount of an active agent high enough to deliver the desired benefit, but low enough to avoid serious side effects within the scope of medical judgment.

Finally, another aspect of the invention is a rice protein hydrolysate as defined above for use in the prevention and/or treatment of obesity.

This aspect can also be formulated as a method for the treatment or prevention of obesity in a subject suffering or with tendency to obesity, which comprises administering to said subject a therapeutically effective amount of the rice protein hydrolysate of the invention.

The invention aims moreover a rice protein hydrolysate for use in the prevention and/or treatment of obesity. In particular, another object of the invention is a rice protein hydrolysate for use in the prevention and/or treatment of obesity, said hydrolysate obtainable by enzymatic hydrolysis and with a degree of hydrolysis (DH) comprised between 4 and 17, preferably between 5 and 17, being the degree of hydrolysis (DH) measured as indicated above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar diagram showing the glycerol concentration (nmol/ml) generated by adipocytes put into contact with rice protein hydrolysates of the invention. G means glycerol; in the X-axis Nl means non-inducers; PC means positive control (Isoproterenol 10μΜ); RI-0 means non hydrolyzed protein; Rl- N3 means rice protein hydrolysate obtainable with Neutrase 0.8L™ digestion at point ¾ DHmax; RI-A1 means rice protein hydrolysate obtainable with Alcalase 2.4L™ digestion at point ¼ DHmax; RI-A2 means rice protein hydrolysate obtainable with Alcalase 2.4L™ digestion at point ½ DHmax; Rl- A3 means rice protein hydrolysate obtainable with Alcalase 2.4L™ digestion at point ¾ DHmax; RI-A4 means rice protein hydrolysate obtainable with Alcalase 2.4L™ digestion at DHmax; RI-NZ1 means rice protein hydrolysate obtainable with Novozyme FM 2,0L™ digestion at point ¼ DHmax; RI-NZ2 means rice protein hydrolysate obtainable with Novozyme FM2,0L™ digestion at point ½ DHmax; RI-NZ3 means rice protein hydrolysate obtainable with Novozyme FM2,0L™ digestion at point ¾ DHmax; RI-NZ4 means rice protein hydrolysate obtainable with Novozyme FM2,0L™ digestion at DHmax; RI-F3 means rice protein hydrolysate obtainable with

Flavourzyme 1000L™ digestion at point ¾ DHmax; RI-PE3 means rice protein hydrolysate obtainable with Porcine PEM™ digestion at point ¾ DHmax; RI-PR3 means rice protein hydrolysate obtainable with Protamex™ digestion at point ¾ DHmax; and RI-FP3 means rice protein hydrolysate obtainable with FlavoPro Whey™ digestion at point ¾ DHmax.

FIG. 2 is another bar diagram showing the glycerol concentration (nmol/ml) generated by adipocytes put into contact with the rice protein hydrolysates of the invention. In particular, with the rice protein hydrolysates being filtered. Each abbreviation in the X-axis corresponds to the same meaning as in FIG. 1 .

FIG. 3 is a graphic showing the percentage of fluorescence detected in Caenorhabditis elegans plated on Petri dishes containing the Nile Red dye. Y- axis shows the percentage of fluorescence detected with respect to the controls (worms plated on NG media). Each abbreviation in the X-axis corresponds to the same meaning as in FIG. 1 . Orl means Orlistat; %F means percentage of fluorescence.

DETAILED DESCRIPTION OF THE INVENTION Following definitions are added in order to make more comprehensive the present invention.

In the context of this document "protein hydrolysates" are defined as mixtures of polypeptides, oligopeptides and amino acids that are manufactured from protein sources, using partial hydrolysis. Partial hydrolysis refers to not complete (100%) peptide bonds breakdown. Protein hydrolysates can be produced by acid and alkaline hydrolysis, by heat treatment and by incubation with enzymes. As compared with the chemical process, proteolysis by enzymes has several advantages. These include mild process circumstances, specificity, high reaction velocity and a lot of choices. The processes used are common processes used in the food industry using food grade source materials, processing aids and appropriate equipments. The enzymes used are digestive proteolytic enzymes (such as pepsin, chymotrypsin, and trypsin) obtained from animals, or food grade enzymes obtained from edible parts of plants and from microorganisms with an accepted safe use in human nutrition. Enzymes from edible parts of plants and animals are generally considered as posing no health problems. Regarding enzymes from

microorganisms, the source organism should not be pathogenic and should not produce toxic compounds that remain in the final product. The result of enzymatic proteolysis (the peptide composition of a hydrolysate) depends on three main factors: a) the protein substrate; b) the type of protease(s) used; and c) the hydrolysis conditions. The expression "rice protein source" refers to the protein fraction of rice. This protein fraction includes peptides, whole proteins and some minor

compounds which are naturally accompanying the said proteins in the native or crude rice.

"Endoproteases" , also named "endopeptidases" or "endoproteinases" are proteolytic peptidases that break peptide bonds of non-terminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from their end-pieces. For this reason, endopeptidases cannot break down peptides into monomers, while exopeptidases can break down proteins into monomers. A particular case of endopeptidase is the oligopeptidase, whose substrates are oligopeptides instead of proteins. "Exoproteases" or "exopeptidases" are proteolytic peptidases that break terminal peptide bonds and can break down proteins into monomers. When in the context of the present invention it is stated that an enzyme has "mainly exoprotease activity", is to be understood that the enzyme is catalogued as capable of breaking terminal peptide bonds, although it can have a residual

endoprotease activity too.

The term "Adipolysis" refers to "lipolysis", the degradation of triglyceride stores, in differentiated adipocytes. Several compounds, including

isoproterenol and tumour necrosis factor-a (TNF-a) have been shown to stimulate adipolysis in differentiated 3T3-L1 and primary human adipocytes. Isoproterenol is a non selective agonist of the beta-adrenergic class of

GPCRs, which stimulate cAMP levels in adipocytes. Subsequent activation of protein kinase A (PKA) by elevated cyclic adenosine monophosphate (cAMP) results in phosphorylation of perilipin, which is a protein located at the surface of the lipid droplet. Although perilipin inhibits basal lipolysis by non-hormone sensitive lipases, phosphorylated perilipin recruits the hormone-sensitive lipase (HSL) to the surface of the lipid droplet. HSL cleaves triglycerides into their constituent fatty acids and free glycerol, which can be assayed as a marker of adipolysis. Although the mechanism by which TNF-a induces adipocyte lipolysis has yet to be completely elucidated, activation of the MAPK family, down regulation of subunit i of G-alpha protein (Gai), and/or down regulation of perilipin appear to play a role. In addition, extracellular glucose is required for the TNF-a-mediated adipocyte lipolysis. Glycerol generated by triglyceride breakdown is released into the extracellular space. Extracellular glycerol is easily assayed by incubation with glycerol kinase (to produce glycerol phosphate), glycerol phosphate oxidase (to produce H ₂ O ₂ ), and horseradish peroxidase in the presence of a colorimetric substrate.

The expression "between X and Y", associated to the definition of an interval in which X and Y are end-point numeric values, refers according to the present invention to a range including all the values of the interval as well as the end-points of the same. For example, the expression "between 4 and 17" includes the values 4 and 17, as well as, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 and 16.

The inventors have developed a rice protein hydrolysate which has the ability of preventing obesity in terms that it is able to reduce fat (lipid) accumulation and to induce lipolysis. The rice protein hydrolysate is obtainable by the process disclosed above, which includes the steps of mixing a rice protein source with an enzymatic system having endoprotease and/or exoprotease activity, and comprising one or more enzymes; and let to act this enzymatic system under constant temperature and pH, until a desired degree of hydrolysis is reached. As will be illustrated in the examples below, the rice protein hydrolysate may be added in an edible composition or in a nutritional composition, for instance in an infant formula, thus allowing the prevention of obesity in infants with trend to this pathology. In a preferred embodiment, the final protein concentration in step (a) is comprised between 5 % and 15 % weight/volume (w/v).

In a preferred embodiment, the process for obtaining the rice protein hydrolysate includes the step of mixing a protein concentrate of rice as rice protein source. A rice protein concentrate is any type of concentrated rice containing 40-90% by weight of protein. Preferably the protein concentrate contains 80 % by weight of protein. Other rice protein sources that can be processed by the process of the invention include rice flour. In another preferred embodiment, the liquid medium wherein the rice protein source is added is selected from the group consisting of water and a saline solution. In a most preferred embodiment, the liquid medium is water. In a preferred embodiment the rice protein source is added in the liquid medium in a final concentration selected from the group consisting of 5 % (w/v), 6 % (w/v), 7 % (w/v), 8 % (w/v), 9 % (w/v), 10% (w/v), 1 1 % (w/v), 12 % (w/v), 13 % (w/v), 14 % (w/v), 15 % (w/v), 16 % (w/v), 17 % (w/v), 18 % (w/v), 19 % (w/v), and 20 % (w/v) by weight/volume to obtain a reaction mixture. Preferred concentrations are those comprised between 5 % (w/v) and 10 % (w/v). The most preferred concentration expressed as weight/volume of the rice protein source is 8 % (w/v).

In another preferred embodiment the pH of the reaction mixture is adjusted in step b) with and organic or inorganic acid, such as HCI or acetic acid.

In a preferred embodiment, the process of obtaining a rice protein hydrolysate comprises adjusting pH to 7 in step b).

Preferred amounts of each one of the enzymes in the enzymatic system are in a concentration between 0.01 g/100 ml of reaction mixture and 10.0 g/100 ml of reaction mixture. In a most preferred embodiment the amount of each enzyme is comprised between 0.01 g/100 ml of reaction mixture and 2.0 g/100 ml of reaction mixture. In another most preferred embodiment the amount of each enzyme is comprised between 0.1 g/100 ml of reaction mixture and 2.0 g/100 ml of reaction mixture. Most preferred between 0.2 g/100 ml of reaction mixture and 0.8 g/100 ml of reaction mixture. Yet in a most preferred embodiment, the amounts of each one of the enzymes are selected from 0.3 g/100 ml of reaction mixture, 0.4 g/100 ml of reaction mixture, 0.5 g/100 ml of reaction mixture, 0.6 g/100 ml of reaction mixture, and 0.7 g/100 ml of reaction mixture. The reaction mixture includes all the components to perform the enzymatic hydrolysis, such as a liquid medium, especially water, the rice protein source, and the enzyme or mixture of enzymes (enzymatic system).

The expression "one or more enzymes" in the enzymatic system means that step c) of the process is carried out in the presence of one, two, three, four, five or six enzymes. Preferably only one enzyme is used in the process for obtaining the rice protein hydrolysate of the invention. Nonetheless, when more than one enzyme is used, the hydrolysis of the protein source for multiple sites is obtained.

Preferably, the enzymatic system comprises one enzyme, which has either endoprotease activity, exoprotease activity, or both activities.

In another preferred embodiment, the enzymatic system of step c) comprises two enzymes, each one encompassing either endoprotease activity, exoprotease activity, or both activities.

In another embodiment, the process for obtaining a rice protein hydrolysate comprises the steps of: a) adding a rice protein source in a liquid medium at a final protein

concentration comprised between 5 % and 20 % weight/volume (w/v) to obtain a reaction mixture;

b) adjusting the reaction mixture of step a) to a pH comprised between 6 and 8;

c) performing the protein hydrolysis reaction of the resulting mixture from step b) by adding an amount of an enzymatic system, said enzymatic system having mainly exoprotease activity, and comprising one or more enzymes, each one of the enzymes being in a concentration between 0.01 g/100 ml of reaction mixture and 10.0 g/100 ml of reaction mixture,

at a constant pH and temperature, the temperature being comprised between 30°C and 55°C; and

d) deactivating the enzymatic system to stop the protein hydrolysis

reaction when the degree of hydrolysis (DH) of the mixture is comprised between 4 and 17.

In one embodiment, when the enzymatic system has mainly exoprotease activity, the deactivating step is performed when the degree of hydrolysis (DH) of the mixture is 4 or 5, being preferred a DH of 5.

In yet another embodiment, which can be in combination with other embodiments of the invention, the step c) of protein hydrolysis with the enzymatic system is performed only once.

Also in another preferred embodiment of the process of the invention, the enzymatic system is deactivated in step d) to stop the protein hydrolysis reaction when the degree of hydrolysis (DH) of the mixture is comprised between 9 and 17. In other words, the enzymatic system is let to act in step d) until a degree of hydrolysis (DH) comprised between 9 and 17 is achieved. Then the enzymatic system is deactivated. Preferred degrees of hydrolysis (DH) are 9, 10, 1 1 , 12, 13, 14, 15, 16, and 17, being most preferred a degree of hydrolysis (DH) selected from de group consisting of 9, 12 and 17.

The degree of hydrolysis (DH) is the percentage of broken peptide-bonds due to the action of the enzymatic system. A value of 100% means that the total potential peptide-bonds in the protein source are broken, thus leading to free amino acids. As above indicated, DH is calculated by means of a formula considering the volume in millilitres (ml) of consumed base used for titrating released amino groups during protein hydrolysis, the normality of the base used for titrating, the average degree of dissociation of the amino groups related with the pK of said amino groups at a particular pH and temperature, the amount in grams of the protein source in the reaction mixture, and the sum of the millimoles of individual amino acids per gram of protein associated with the source of protein. In a preferred embodiment the progress of the hydrolysis reaction until a desired DH is achieved is followed by the pH-stat technique. This technique allows, in turn, the maintenance of the constant pH during the reaction of protein hydrolysis. The evaluation of the progress of hydrolysis is performed by titrating the released amino groups from the reaction with an alkaline solution. The technology is widely explained by Adler-Nissen, J. et al., in

Enzymatic Hydrolysis of Food Proteins. Elsevier Applied Science Publishers, London, UK 1986, pp. 122-124. Also in Spellman et al., "Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat processes for quantification of degree of hydrolysis", International Dairy Journal - 2003, Vol. 13, pp. 447-453. pH-stat is also disclosed by Navarrete del Toro et al, "Evaluation of the Progress of Protein Hydrolysis. Protein Hydrolysis using the pH-STAT technique", Current Protocols in Food Analytical Chemistry -2002, B2.2.1 - B2.2.14.

According to the pH-stat technique, the enzymes can work at constant pH and temperature during the entire process, so that no buffering is needed. The automated pH-stat process gives a direct measurement of the percentage of hydrolyzed peptide bonds, which is indicated as the degree of hydrolysis (DH).

For the maintenance of the constant pH during the protein hydrolysis reaction other several techniques or means may be used. Usual means include the presence of a pH-meter in the reaction mixture, and a supplier of an alkaline solution, which is regularly added while the enzymatic hydrolysis is performed and acidification of the reaction mixture occurs. If needed, and in the case a deregulation of pH occurs, also a supplier of an acid solution is included to readjust pH. In such a situation also solutions of HCI and/or of acetic acid may be used.

The temperature in which step c) of the process is carried out is comprised between 30 °C and 55 °C. Although the enzymatic system used in the process of the invention, comprising one or more enzymes, is active at these temperatures, the selection of the optimal temperature is encouraged in function of the composition of the enzymatic system having endoprotease and/or exoprotease activity used. The selection of optimal parameters when an enzymatic reaction is to be carried out allows getting optimal reaction times of enzymatic hydrolysis.

The control of the constant temperature in the process is performed automatically using a reactor with thermostat devices and temperature probes. Means to control the temperature in these kinds of reactors include heat exchangers, electrical resistances, and thermometers.

In a preferred embodiment, when the enzymatic system has endoprotease activity, it includes one or more enzymes selected from the group consisting of an alkaline protease, preferably an alkaline protease of Bacillus genus, a metalloprotease, trypsin, chymotripsin, and mixtures of all these enzymes. If an alkaline protease of Bacillus genus is employed, subtilisin, also identified by EC NUMBER 3.4.21 .62 is the most preferred. In another preferred embodiment, when the enzymatic system has

exoprotease activity (mainly exoprotease activity), it comprises a mixture of enzymes or a protease complex with endoprotease and exoprotease activity. They enzymes are, preferably, of fungal origin.

In a most preferred embodiment, the process of the invention is carried out using an alkaline protease and the temperature of the reaction is maintained at 50 °C.

In another most preferred embodiment, the enzyme is a mixture of trypsin and chymotripsin, and the temperature of the reaction is maintained at 37 °C.

In another embodiment, the process according to any of the above-referred embodiments and aspects of the invention, is carried out in such a way that in step c) the enzymatic system consists in an endoprotease enzyme, which is an alkaline protease, and in step d) the deactivation of the enzymatic system to stop the protein hydrolysis reaction is performed when the degree of hydrolysis (DH) of the mixture is 17, yielding to a protein hydrolysate that comprises at least the following peptides: QQQQP (SEQ ID NO: 1 ), QQFGNL

(SEQ ID NO: 2), KFPILN (SEQ ID NO: 3), RERFQ (SEQ ID NO: 4), RSQNIF (SEQ ID NO: 5), DTYNPR (SEQ ID NO: 6), RVRQNI (SEQ ID NO: 7),

SQKFPIL (SEQ ID NO: 8), RALPVDVV (SEQ ID NO: 9), and NSQKFPIL (SEQ ID NO: 10).

In another embodiment of the process according to the invention, in step c) the enzymatic system consists in the endoproteases trypsin and chymotripsin, and in step d) the deactivation of the enzymatic system to stop the protein hydrolysis reaction is performed when the degree of hydrolysis (DH) of the mixture is 9, yielding to a protein hydrolysate that comprises at least the following peptides: QLTGR (SEQ ID NO: 1 1 ), NEFVR (SEQ ID NO: 12), LGQNIR (SEQ ID NO: 13), VSHLAGK (SEQ ID NO: 14), VSHIAGK (SEQ ID NO: 15), NPQAYR (SEQ ID NO: 16), RVIEPR (SEQ ID NO: 17), GLLLPHY (SEQ ID NO: 18), LQAFEPI (SEQ ID NO: 19), SQAGTTEF (SEQ ID NO: 20), NIDNPNR (SEQ ID NO: 21 ), SGFSTELL (SEQ ID NO: 22), DFLLAGNK (SEQ ID NO: 23), QGDVIALPA (SEQ ID NO: 24), QNALLSPF (SEQ ID NO: 25), GLSLLQPY (SEQ ID NO: 26), FDEKNEL (SEQ ID NO: 27), ALPN DYLAN (SEQ ID NO: 28), QKFKDEH (SEQ ID NO: 29), DGVLRPGQL (SEQ ID NO: 30), FRDEHQK (SEQ ID NO: 31 ), IQAFEPIR (SEQ ID NO: 32), IQAFEPLR (SEQ ID NO: 33), and ALPVDVVANAYR (SEQ ID NO: 34). In another embodiment of the process according to the invention, in step c) the enzymatic system consists in an endoprotease enzyme, which is an active site-mutated alkaline protease, and in step d) the deactivation of the enzymatic system to stop the protein hydrolysis reaction is performed when the degree of hydrolysis (DH) of the mixture is 12, yielding to a protein hydrolysate that comprises at least the following peptides: TPIQY (SEQ ID NO: 35), LDPRQ (SEQ ID NO: 36), IQGTGVL (SEQ ID NO: 37), QYIAIK (SEQ ID NO: 38), PRGLLLP (SEQ ID NO: 39), IFAAGQY (SEQ ID NO: 40), GNNNRAQ (SEQ ID NO: 41 ), NEFVRQ (SEQ ID NO: 42), RALPNDVL (SEQ ID NO: 43), AFEPIRSV (SEQ ID NO: 44), NDQRGEIV (SEQ ID NO: 45), NDQRGEII (SEQ ID NO: 46), and KNNRGEEI (SEQ ID NO: 47).

In a preferred embodiment, the active site-mutated alkaline protease is the endopeptidase known as Novozym FM 2,0L™. This mutated form has an activity being 17 % lower than the non-mutated form.

In another embodiment of the process according to the invention, in step c) the enzymatic system consists in an exoprotease enzyme derived from Aspergillus spp, said exoprotease being able to produce non-bitter, non- gelling protein hydrolysate from whey protein, and in step d) the deactivation of the enzymatic system to stop the protein hydrolysis reaction is performed when the degree of hydrolysis (DH) of the mixture is 5, yielding to a protein hydrolysate that comprises at least the following peptides: QYYP (SEQ ID NO: 48), EPIRS (SEQ ID NO: 49), GNKRNP (SEQ ID NO: 50), LPHYTN (SEQ ID NO: 51 ), GLQLLKP (SEQ ID NO: 52), FDGVLRPG (SEQ ID NO: 53), GQNIRQY (SEQ ID NO: 54), and LPHYTN GAS (SEQ ID NO: 55).

In a preferred embodiment, the exoprotease enzyme derived from Aspergillus spp, is the exoprotease known as FlavorPro Whey 750P™ from Biocatalyst Ltd. (UK).

In a preferred embodiment the step of deactivating the enzyme to stop the protein hydrolysis reaction is performed once a desired DH is obtained, by raising the temperature from 80 °C to 100 °C. An increase of temperature comprised between these values provokes the enzymatic system

denaturalization and/or deactivation. Other suitable ways to deactivate the enzymatic system, and thus stopping the protein hydrolysis reaction, include filtering the rice protein hydrolysate with the desired DH through a filter allowing the retention of the enzymatic system. Depending on the molecular weight of the enzyme or enzymes in the enzymatic system, membranes with adequate molecular weight cut-offs are used for separating the enzyme/s from the hydrolysate.

In a preferred embodiment, the process of obtaining a rice protein hydrolysate includes an additional step wherein the hydrolysate is filtered through a filter or a membrane with a Molecular Weight Cut-Off equal or lower than 3000 Daltons.

With the process of the invention, several rice protein hydrolysates with the capability of inducing lipolysis are obtained. It is worth mentioning that the induction of lipolysis is a good help to reduce the fat tissue accumulation, thus being useful to treat obese subjects, or to prevent obesity in subjects with a particular susceptibility to develop such pathology. Then, with the rice protein hydrolysates of the invention the risk of developing obesity is reduced, and this is highly relevant if the consumption of the hydrolysate is initiated at early ages (infants).

In addition, further in vivo data with animals, in particular with, Caenorhabditis elegans (C. elegans) demonstrate that the rice protein hydrolysates of the invention reduce lipid or fat tissue accumulation or absorption.

A preferred embodiment of the invention is a rice protein hydrolysate obtainable by a process wherein the rice protein source is mixed at a final protein concentration of 8 % weight/volume in a liquid medium being water; pH is adjusted to 7; an endoprotease, preferably alkaline protease, is added in a final concentration comprised between 0.01 g/100 ml of reaction mixture and 2.0 g/100 ml of reaction mixture; the enzymatic system is let to act at constant pH (pH=7) and temperature, in particular at 50 °C, until a degree of hydrolysis of 17 is obtained; and then said enzymatic system is deactivated.

In another preferred embodiment, the rice protein hydrolysate is obtainable by a process wherein the rice protein source is mixed at a final protein

concentration of 8 % weight/volume in a liquid medium being water; pH is adjusted to 7; an endoprotease, preferably a mutated alkaline protease, is added in a final concentration comprised between 0.01 g/100 ml of reaction mixture and 2.0 g/100 ml of reaction mixture; the enzymatic system is let to act at constant pH (pH=7) and temperature, in particular at 50 °C, until a degree of hydrolysis of 12 is obtained; and then said enzymatic system is deactivated.

Also another preferred embodiment is a rice protein hydrolysate obtainable by a process wherein the rice protein source is mixed at a final protein

concentration of 8 % weight/volume in a reaction mixture that comprises water; pH is adjusted to 7; an endoprotease, preferably a mixture of trypsin and chymotrypsin, is added as enzymatic agent in a final concentration comprised between 0.2 g/100 ml of reaction mixture and 0.8 g/100 ml of reaction mixture, preferably 0.3 g/ 100 ml of reaction mixture; the enzymatic system is let to act at constant pH (pH=7) and temperature, in particular at 37 °C, until a degree of hydrolysis of 9 is obtained; and then said enzymatic system is deactivated.

Another also preferred embodiment of the invention is a rice protein

hydrolysate obtainable by a process wherein the rice protein source is mixed at a final protein concentration of 8 % weight/volume in a reaction mixture that comprises water; pH is adjusted to 7; a protease complex including mixtures of endoproteases and exoproteases from fungal origin is added in a final concentration comprised between 0.2 g/100 ml of reaction mixture and 0.8 g/100 ml of reaction mixture, preferably 0.3 g/ 100 ml of reaction mixture; the enzymatic system is let to act at constant pH (pH=7) and temperature, in particular at 50 °C, until a degree of hydrolysis of 5 is obtained; and then said enzymatic system is deactivated. Although a rice protein hydrolysate obtainable by the process disclosed above may be directly used, after deactivating the enzymatic system, other optional steps for dehydrating the obtained mixture may be applied. Examples of dehydrating processes include lyophilisation, spray-drying and drum- drying, among others.

If the rice protein hydrolysates are not dehydrated, they are kept and processed to avoid deterioration, for instance, by means of ultra-high temperature processing (UHT) or ultra-pasteurization.

All the hydrolysates obtainable by the process of the invention may be used as active ingredients in food (edible) compositions, in nutritional compositions and in pharmaceutical compositions.

In the sense of the present invention an edible composition, also termed a food product, includes a milk product, a baby cereal, a yogurt, a curd, a cheese (e.g. quark, cream, processed, soft and hard), a fermented milk, a milk powder, a milk based fermented product, an ice-cream, a fermented cereal based product, a milk based powder, a beverage, a dressing, and a pet food. Examples of other food products are meat products (e.g. liver paste, frankfurter and salami sausages or meat spreads), chocolate spreads, fillings (e.g. truffle, cream) and frostings, chocolate, confectionery (e.g. caramel, fondants or toffee), baked goods (cakes, pastries), sauces and soups, fruit juices and coffee whiteners. However, the term "food product" is used herein in its broadest meaning, including any type of product, in any form of presentation, which can be ingested by an animal. Particular embodiments of nutritional compositions are a dietary supplement, an additive, and an infant formula. Dietary supplements intend to supply nutrients, (vitamins, minerals, fatty acids or amino acids) that are missing or not consumed in sufficient quantity in a person's diet (infants, pregnant women, elderly people, etc). In a particular embodiment, the rice protein hydrolysate of the invention is homogenized with other ingredients, such as other cereals or powdered milk to constitute an infant formula.

In a preferred embodiment the nutritional composition is an infant formula. It is of especial interest the inclusion of the rice protein hydrolysates of the invention in infant formulas, because these infant formulas can be

administered to children, which are known to have predisposition to become obese. The finality is preventing them to be obese people during childhood, young and in adult age. In addition, the use of rice protein hydrolysates in infant formulas is also useful for other interesting properties of the rice.

In another preferred embodiment, the nutritional composition comprises other edible ingredients selected from the group consisting of nucleotides, polyunsaturated fatty acids (PUFAS), long-chain polyunsaturated fatty acids (LC-PUFAS), triglycerides, preferably medium-chain triglycerides (MC-TG), probiotic agents, prebiotic agents, carbohydrates, minerals, vitamins and mixtures thereof.

When the protein hydrolysates of the invention are used as a component of an edible or nutritional composition, the same are added in a percentage by weight comprised between 2 % and 90 %. Preferably, between 5 % and 70 %. Most preferably between 10 % and 40 %, being specially preferred between 20 % and 30 %.

Pharmaceutical compositions comprising the rice protein hydrolysate of the invention may be prepared in form of tablets, dried oral supplements, dry tube feeding, etc.

The edible compositions, the nutritional compositions and the pharmaceutical compositions comprising the rice protein hydrolysate of the invention, may further comprise, in preferred embodiments, the probiotic strain

Bifidobacterium longum biovar infantis strain, deposited with the Accession Number CECT 7210.

In a preferred embodiment the amount of the strain CECT 7210 in the edible compositions of the invention and in the nutritional compositions of the invention is comprised between 10 ⁵ cfu/g and 10 ⁹ cfu/g of the composition. The strain is preferably present in an amount of 10 ⁷ cfu/g of the composition.

In another preferred embodiment, the amount of the strain CECT 7210 in the pharmaceutical composition of the invention is comprised between 10 ⁷ cfu/g and 10 ¹¹ cfu/g of the composition, being preferred an amount of 10 ⁹ cfu/g of the composition.

The rice protein hydrolysates of the inventions, as well as any derivative product comprising it (edible, nutritional or pharmaceutical composition) are useful for preventing and/or treating obesity in subjects with especial predisposition. Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES

Example 1 . Preparation of "protein hydrolysates" with lipolytic activity

A Rice Protein Concentrate (Aminofood RIS80, Pevesa, Sevilla-Spain), in powder form was dissolved in de-mineralized water. Final protein

concentration in the reaction mixture was 8 % (w/v). The solution was further adjusted to pH 7.0 using 1 M Hydrochloric Acid.

Nine food grade commercial proteases (food grade proteases) were used: Neutrase 0.8L™, Alcalase 2.4L™, Novozym FM 2,0L™, Flavourzyme

1000L™, Porcine PEM™, Pancreas Trypsin™, Novozym 37005™ and Protamex™ from Novozymes (Bagsvaerd- Denmark) and FlavourPro Whey 750P™ from Biocatalysts (Pare Nantgarw- Wales [UK]). Temperature and pH conditions for each enzyme are shown in Table 1 .

Table 1 : Temperature and pH used for each enzyme.

°C PH

Neutrase 0.8L™ 50 7

Alcalase 2.4L™ 50 7

Novozym FM2,0L TM 50 7 Flavourzyme 1000L™ 50 7

Porcine PEM™ 37 7

Pancreas Trypsin Novo 37 7

6,0S™

Novozym 37005™ 50 7

Protamex™ 50 7

FlavorPro Whey 750P™ 50 7

These commercial proteases correspond to the following enzymes:

Alcalase 2.4L™ and Neutrase 0.8L™ are the major proteases from Bacilli. Alcalase 2.4L™ is and alkaline protease also named subtilisin. According to the Enzyme Commission number (EC number) it is identified with the number EC 3.4.21 .62. Neutrase 0.8L™ is a metalloprotease, also named neutral protease or bacillolysin, identified as EC 3.4.24.28.

The values X.XL indicated beside the enzyme name correspond to the Anson units (AU) at which they are supplied. The skilled in the art adapt then (by dilution) the amount to be used in the hydrolysis process.

Novozym FM 2.0 L™ corresponds to a mutated form of the enzyme Alcalase 2.4L™, with an activity being 17 % lower then the non-mutated form.

Porcine PEM™ corresponds to a mixture of Trypsin /chymotrypsin. Novozym 37005 is an endoprotease cleaving specifically glutamyl (Glu) residues (Kakudo et al., "Purification, Characterization, Cloning, and

Expression of a Glutamic Acid-specific Protease from Bacillus licheniforrnis ATCC 14580, The Journal of Biological Chemistry - 1992, Vol. 267, No. 33, pp. 23782-23788). Protamex™ is a mixture of Alcalase™ and Neutrase™ (EC numbers:

3.4.21 .62 and 3.4.24.28).

FlavorPro™ 750P and Flavourzyme™ are endo- and exoproteases, usually from fungal origin. Flavourzyme™ is obtained from Aspergillus oryzae. Flavor Pro™ 750P is derived from Aspergillus spp. These enzymes have mainly an exoprotease activity. Hydrolysis process:

Hydrolysis were underwent using a Laboratory fermentor (Lambda

Instruments, Zurich, Switzerland) as chemostat (pH-Stat). The pH-stat technique monitors the course of the reaction in which each peptide bond is hydrolyzed by proteases. pH-stat evaluates the progress of hydrolysis by titrating the released amino groups with an alkaline solution (Adler-Nissen, 1986- supra). Enzymes work at constant pH and temperature during the entire process, so that no buffering is needed. The automated pH-stat process gives a direct measurement of the percentage of hydrolyzed peptide bonds, the degree of hydrolysis (DH). The DH is calculated using the following equation:

DH = Bx Nb x -x x x lOO

where B (ml) is the volume of base consumed, Nb is the normality of the base (alkaline solution) used for tritrating the released amino groups (a 1 M NaOH solution was used), 1/a is the average degree of dissociation of the a-amino groups related with the pK of the amino groups at particular pH and temperature, MP (g) is the amount of the protein in the reaction mixture, and htot (meq/g) is the sum of the millimoles of individual aminoacids per gram of protein associated with the source of protein used in the experiment. For each protease tested (see Tables 2 and 3) four different samples were obtained with four different degrees of hydrolysis. At each sample point, 2 aliquots of 10 ml were taken and protease activity was stopped keeping samples at 100 °C during 10 minutes. Each sample was labelled using Rl as rice protein concentrate abbreviation followed by protease abbreviation and a number 1 to 4 indicating each sample point. As a general rule, first sample was taken at ¼ of DHmax (maximum DH), second sample at ½ of DHmax , third sample at ¾ DHmax and forth sample at DHmax. A non hydrolyzed sample of rice protein solution was kept and processed the same way as other samples (RI-0). Tables 2 and 3 show the total final working volume and amount of enzyme used to hydrolyse the rice protein concentrate. Table 2. Amount of enzyme in ml in the reaction mixture.

Proteases for hydrolysis of rice protein (Rl) (ml in the reaction mixture)

[Prot] Working Neutrase Alcalase Novozym Flavourzymel OOOL g/100 volume 0.8L™ 2.4L™ FM2,0L™ ™ (F)

ml (ml) (N) (A) (NZ)

8 400 1 ,4 2,8 2,8 2,8

Given specific activity of each of the enzymes (expressed in Anson Units (AU)/ml), Enzyme to substrate ratio (E/S) expressed in AU/g protein would be: 0.21 (0.02-1 .4) for Alcalase 2.4L™, 0.035 (0.01 -0.25) for Neutrase 0.8L™, 0.175 (0.017-1 .2) for Novozym FM 2.0L™ and 875 (90- 5800) for

Flavourzyme 1000L™. The intervals indicated in the parenthesis are the ranges between which the enzyme units (Enzyme to substrate ratio (E/S) expressed in AU/g protein AU/g) may be comprised.

Table 3. Amount of enzyme in grams (g) in the reaction mixture

Proteases for hydrolysis of rice protein (Rl) (g in the reaction mixture)

[Prot] Working Porcine Pancreas Novozym Protamex™ FlavorPro g/100 volume PEM™ Trypsin 37005™ (PR) Whey ml (ml) (PE) Novo (NV) 750P™

6,0S™ (FP)

(PT)

8 400 1 ,2 1 ,2 1 ,2 1 ,2 1 ,2

Example 2. Determination of adipolysis stimulation by rice protein

hydrolysates. Cell preparation and differentiation

3T3-L1 (ATCC CL-173) cells were propagated maintaining the cells in

DulbeccoA/ogt modified Eagle's minimal essential medium-DMEM /10% calf serum (not fetal calf serum). Plate cells at 4 x 10 ⁵ per T75 flask, and passage every third day. Cells were not allowed to become confluent. Stocks of frozen cells were prepared at the earliest passage possible, and also use thawed cells were used at the earliest passage possible. Cells were trypsinized and neutralized by addition of 3 volumes of DMEM/10% calf serum and were counted. 30000 cells/ml in DMEM/10% calf serum (not fetal calf serum) solution was prepared and plated in 96 -well plate (200 μΙ per well) and incubated (37°C, 5% CO ₂ ) until cells were confluent (2 days). Media was removed and 200 μΙ of Adipogenesis Initiation Media per well were added. Adipogenesis Initiation Media consisted in DMEM/10% fetal calf serum/0.5 mM IBMX/1 μΜ dexamethasone. IBMX is 3-isobutyl-1 -methylxanthine. After 2 other days of incubation media was again removed and 200 μΙ Adipogenesis Progression Media per well were added. Adipogenesis Progression Media consisted in DMEM/10% fetal calf serum/10 μg/mL insulin. After 2 days media was replaced with 200 μΙ of Adipogenesis Maintenance Media (DMEM/10% fetal calf serum). Cells were viewed under an inverted microscope; to observe visible lipid droplets (media has to be replaced every 48-72 h). Differentiated cells after 1 1 days of incubation were used for lipolysis assay.

Lipolysis assay:

Media was removed and cells washed twice with 100 μΙ aliquots of wash solution. After washing, 50 μΙ of test compound (Protein hydrolysates) per well were added. For a positive control, 50 μΙ/well Isoproterenol Positive Control Working Solution (10 μΜ) were added. For a negative control, 50 μΙ/well Incubation Solution + 2% BSA with no additions were added.

Cells were incubated for 24 h hours and culture supernatants were collected. 25 μΙ_ each of blank (Incubation Solution + 2% BSA), Glycerol Standard

Curve (0.4 μg glycerol/mL - 26 μg glycerol/mL) and culture supernatant samples into separate wells of a 96-well microtiter plate were dispensed and 200 μΙ Free Glycerol Assay Reagent per well was added. The Free Glycerol Assay Reagent comprised 0.75 mM ATP, 3.75 mM Magnesium salt, 0.188 mM 4-aminoantipyrine, 2.1 1 mM sodium-N-ethyl-N(3-sulfopropyl) m-anisidine,

1 .25 units/mL microbial glycerol kinase, 2.5 units/mL microbial glycerol phosphate oxidase, 2.5 units/mL horseradish peroxidase, Buffer pH 7.0, 0.05% sodium azide.

After 15 minutes incubation at room temperature absorbance was read in spectrophotometric plate reader at 560 nm. The amount (nmol/ml) of glycerol in each sample were determined by extrapolation into the Glycerol Standard Curve.

Protein hydrolysates screening results: When crude (non-filtered) protein hydrolysates were tested as putative lipolysis stimulators, thirteen different protein hydrolysates showed glycerol release capacity (adipolysis activation) over a known lipolysis activator (Isoproterenol). The results can be seen in FIG. 1 . This figure shows the concentration of glycerol calculated as indicated above, and which serves as indicator of adipolysis (lipolysis) observed in the 3T3-L1 (ATCC CL-173) cells (adipocytes). The threshold value of the positive control is indicated by the black dark line. Table 4 shows the characteristics of those crude protein hydrolysates from Example 1 (Degree of hydrolysis and enzyme used to obtain them) that showed significant lipolysis activation results (over the positive control threshold).

Table 4. DH of rice protein hydrolysates

Hydrolysate DH (Degree of

Used Enzyme

code hydrolysis)

RI-N3 6 Neutrase 0.8L™

RI-A1 5

RI-A2 10

Alcalase 2.4L™

RI-A3 15

RI-A4 17

RI-NZ1 4

RI-NZ2 8

Novozym FM2,0L™

RI-NZ3 12

RI-NZ4 15

RI-F3 6 Flavourzyme 1000L™

RI-PE3 9 Porcine PEM™

RI-PR3 9 Protamex™

5 FlavorPro Whey

RI-FP3 750P™ Example 3A. Filtered protein hydrolysates and lipolysis activity. Crude protein hydrolysates of Example 2, which showed a lipolysis activity over the threshold of the positive control (inductor= isoproterenol) were filtered using a Macrosep 3K Omega centrifuge filter (Pall Corporation, Port Washington, NY-USA) following provider instructions. Retentate was discarded and filtrates were used to assay lipolysis activation capacity as described previously. The amount per well of each filtered hydrolysate tested was the same than that used for crude hydrolysates (50 μΙ/well).

After filtering, all hydrolysates kept capacity to activate lypolisis compared to non inducers. The results are depicted in FIG. 2, wherein the glycerol concentration detected in the supernatant of 3T3-L1 cells (determined as in Example 2) serves as indicator of adipolysis (lipolysis) promoted by the filtered rice protein hydrolysates.

Four of filtered protein hydrolysates activated significantly the lipolysis over Isoproterenol values. These rice protein hydrolysates were RI-A4, RI-NZ3, Rl- PE3 and RI-FP3.

Experimental data of Examples 2 and 3 allow concluding that rice protein hydrolysates of the invention, obtainable by the digestion of rice protein sources with enzymes at constant pH (from 6 to 8) and temperature (from 30 °C to 55 °C) until a degree of hydrolysis (DH) comprised between 4 and 17, preferably between 5 and 17 (both end-points included) is achieved, are suitable compositions to be used for activating lipolysis and, thus to reduce lipid stores favouring obesity. Preferred hydrolysates are those in which DH is 12 or 17, and are obtained by digesting the protein source with an alkaline protease.

Another preferred hydrolysate is the one in which DH is 9, and it is obtained by digesting the protein source with a mixture of trypsin and chymotrypsin. Yet another preferred hydrolysate is the one in which DH is 5, and it is obtained by digesting the protein source with an exoprotease from fungal origin, preferably from Aspergillus oryzae. Example 3B. Peptide characterization of the rice protein hydrolysates

With the aim of better characterizing the rice protein hydrolizates that activated significantly the lipolysis over Isoproterenol values even after being filtered, an analysis by liquid chromatography coupled to mass spectrometry was done.

The selected filtered rice protein hidrolysates were RI-A4, RI-NZ3, RI-PE3 and RI-FP3, disclosed in Example 1 and FIG. 1 . The samples were diluted in water ½. Solutions were centrifuged at 4000 ^χ g for 30 minutes.

An HPLC analysis was done in an HPAgilent 1 100 System (Agilent

Technologies) equipped with a quaternary pump (Agilent Series 1 100). As acquisition data system it was used the software ChemStation (Agilent Technologies). The used column was a reverse phase Hi-Pore C18 column (250 x 4,6 mm d.i., 5 μιτι of particle size) (Bio-Rad Laboratories, Richmond, CA, EEUU), and to increase sensitivity a low dimensioned column was used (150 mm x 2.1 mm Inertsil 5 ODS3 C18 with 5 μιτι of particle size (Varian, Bergen op Zoom, The Netherlands)). Solvent A was a mixture of water and trifluoroacetic acid (1000:0,37), and solvent B was a mixture of acetonitrile and trifluoroacetic acid (1000:0,27). HPLC equipment was coupled to a mass spectrometry detector type Esquire-3000 (Bruker Daltonik GmbH, Bremen, Alemania). Filtered rice protein hydrolysates were eluted through the first column with a flow rate of 0.8 ml/min, with a lineal gradient of 0% to 45% of solvent B in A in 65 minutes. With the second column, the gradient was 0% to 45% of solvent B in A in 120 minutes. Solvent absorbance was monitored at a wavelength of 214 nm, and at the detector onset, the flow was passed to the mass spectrometry nebulizer (flow 0.2 ml/min). N ₂ and helium were used as nebulizer and dryer gases in the mass spectrometer at a pressure of 5 ^χ 10-3 Pa and the mass spectra were acquired in a range from 100-1500 m/z.

Capillary was maintained with a voltage of 4 kV. The signal of the analysis was the mean of 25 spectra, and for the MS(n) analysis a mean of 5 spectra was used, being n the number of generations of ions to be analysed. The intensity limit to perform MS(n) analysis was of 10000. Precursor ions were isolated in a range of 4 m/z, and they were fragmented with a voltage slope from 0.3 to 2.0 V. Spectral data were processed and transformed to mass values with the Data Analysis (version 3.0, Bruker Daltoniks) software. The software BioTools (version 2.1 , Bruker Daltoniks) was used to process MS(n) spectra, and to perform the peptide sequencing.

Following are exposed the results for each analysed rice protein hydrolysates A_Peptide sequences in RI-A4 by HPLC coupled in tandem to mass spectrometry:

Experimental Theorical

Mass Mass

(Daltons;Da) (Daltons;Da) ^a Sequence Fragment β_ ₇ _ c v _QQ QQQQP (SEQ 19 KDA GLOBULIN (124 -

627.5 627.298 _{|D NQ: 1) 128)} τ _ης -^ _ς QQFGNL (SEQ PROLAMIN PPROL 14P (85 - iva.i /ua.J a , _{d N0:} 2) 90)

KFPII IS 9FO Iri GLUTELIN B1 (316 - 321)

730.4 730.437 ^ ^ ^w GLUTELIN B2 (312 - 317)

j GLUTELIN B4, B5 (317-322)

734.5 734.382 19 KDA GLOBULIN (37 - 41)

763.7 763.398 ^Νθ'δ^ ⁰ GLUTELIN A1 (199 - 204)

7fid A 7fid w DTYNPR (SEQ GLUTELIN A1 y A2 (304 - 309)

ID NO: 6) GLUTELIN A3 (303 -308)

784 ς 7Rd 1 _Q 7 ^RVR Q ^NI ( ^SE Q GLUTELIN A1 y A2 (292 - 297)

ID NO: 7) GLUTELIN A3 (291 -296)

SO FPII ^FO GLUTELIN B1 (314 - 320)

831.8 831.485 fn n 0 GLUTELIN B2 (310 - 316)

' GLUTELIN B4, B5 (315-321) fifi ₇ ^l7 RALPVDVV GLUTELIN B1 (421 -428)

(SEQ ID NO: 9) GLUTELIN B2 (417 - 424)

NSQKFPIL GLUTELIN B1 (313 - 320)

(SEQ ID NO: GLUTELIN B2 (309 - 316) Experimental Theorical

Mass Mass

(Daltons;Da) (Daltons;Da) ^a Sequence Fragment

10) GLUTELIN B4, B5 (314-321) ^a Theoretrical mass mean

b This column shows the mature proteins (with all the genetic possible variants, if any) that include the detected peptides. Parenthesis show the amino acids fragments referred to the entire protein.

B_Peptide sequences in RI-PE3 by HPLC coupled in tandem to mass spectrometry:

Experimental Theoretical

Mass Mass ³

(Daltons;Da) (Daltons;Da) Sequence Fragment"

19 KDA GLOBULIN (33 -

573.3 573.407 QLTGR (SEQ ID NO: 11)

37)

PROLAMIN PPROL

663.3 663.474 NEFVR (SEQ ID NO: 12)

14PJaponica (29 - 33) PROLAMIN PPROL

699.4 699.547 LGQNIR (SEQ ID NO: 13)

14PJaponica (5 - 10) GLUTELIN B1 (410-416) GLUTELIN B2 (406-412)

710,4 710.4075 VSHLAGK (SEQ ID NO: 14)

GLUTELIN B4, B5 (411 - 417)

GLUTELIN A1, A2 (414 -

710.4 710.432 VSHIAGK (SEQ ID NO: 15)

420)

GLUTELIN A1, A2 (188 - 747.4 747.543 NPQAYR (SEQ ID NO: 16)

193)

GLUTELIN A1.A2 (63-

768.5 768.569 RVIEPR (SEQ ID NO: 17) 68)

GLUTELIN A3 (62 - 67) GLUTELIN A1.A2 (69- 811.5 811.69 GLLLPHY (SEQ ID NO: 18) 75)

GLUTELIN A3 (68 - 74) GLUTELIN A1.A2 (27- 816.4 816.438 LQAFEPI (SEQ ID NO: 19) 33)

GLUTELIN A3 (26 - 32) GLUTELIN A1.A2 (38-

839.4 839.58 SQAGTTEF (SEQ ID NO: 20) 45)

GLUTELIN A3 (37-44) GLUTELIN A1.A2 (296-

841.4 841.728 NIDNPNR (SEQ ID NO: 21)

300)

GLUTELIN A1, A2 (205-

852.4 852.714 SGFSTELL (SEQ ID NO: 22)

212)

GLUTELIN A1, A2 (179 -

876.5 876.744 DFLLAGNK (SEQ ID NO: 23)

186)

882.4 882.481 QGDVIALPA (SEQ ID NO: GLUTELIN A1, A2 (136 - Experimental Theoretical

Mass Mass ³

(Daltons;Da) (Daltons;Da) Sequence Fragment"

24) 144)

GLUTELIN A1 , A2 (338 - 345)

888.5 888.726 QNALLSPF (SEQ ID NO: 25)

GLUTELIN A3 (337 - 344)

GLUTELIN A1 , A2 y A3

889.5 889.13 GLSLLQPY (SEQ ID NO: 26)

(242-249)

GLUTELIN B1 , B2 (45 -

893.4 893.413 FDEKNEL (SEQ ID NO: 27)

51 )

ALPNDVLAN (SEQ ID NO:

925.5 925.928 GLUTELIN A1 (426 - 434)

28)

930.5 930.689 QKFKDEH (SEQ ID NO: 29) GLUTELIN A1 (122 - 128)

DGVLRPGQL (SEQ ID NO: GLUTELIN B1 (371 - 379)

953.5 953.529

30) GLUTELIN B2 (367 - 375)

GLUTELIN B1 , B2, B4,

958.4 958.462 FRDEHQK (SEQ ID NO: 31 ) B5 (1 19 - 125)

GLUTELIN A3 (68 - 74) GLUTELIN A1 , A2 (27 -

972.5 972.886 IQAFEPIR (SEQ ID NO: 32) 34)

GLUTELIN A3 (26 - 33) GLUTELIN B1 , B2, B4,

972.5 972.886 IQAFEPLR (SEQ ID NO: 33)

B5 (26-33)

ALPVDVVANAYR (SEQ ID GLUTELIN B1 (422 - 433)

1286.7 1286.698

NO: 34) GLUTELIN B2 (418 - 429)

Theoretical mass mean

This column shows the mature proteins (with all the genetic possible variants, if any) that include the detected peptides. Parenthesis show the amino acids fragments referred to the entire protein.

C_Peptide sequences in RI-NZ3 by HPLC coupled in tandem to mass spectrometry:

Experimental Theoretical

Mass Mass ³

(Daltons;Da) (Daltons;Da) Sequence Fragment"

GLUTELIN A1 (458

620.3 620.582 TPIQY (SEQ ID NO: 35)

- 462)

GLUTELIN A1 , A2

627.3 627.526 LDPRQ (SEQ ID NO: 36)

(173-177)

GLUTELIN B5

686.2 686.396 IQGTGVL (SEQ ID NO: 37) ENDOSPERM (84 -

90) Experimental Theoretical

Mass Mass ³

(Daltons;Da) (Daltons;Da) Sequence Fragment

GLUTELIN B1 (398-

403)

734.4 734.456 QYIAIK (SEQ ID NO: 38)

GLUTELIN B2 (394- 399)

GLUTELIN A1,A2

764.3 764.503 PRGLLLP (SEQ ID NO: 39) GLUTEUN A3 (66-

72)

19 KDA GLOBULIN

768.4 768.515 IFAAGQY (SEQ ID NO: 40)

(158-164)

772.4 772.48 GNNNRAQ (SEQ ID NO: 41) ^^ ¹ ^ ^B1 ^ ^179"

PROLAMIN 14P

791.4 791.556 NEFVRQ (SEQ ID NO: 42)

(29-34) oaa K _M , RALPNDVL (SEQ ID NO: GLUTELIN A1 (425- oyb.o oyb.o 4 432)

GLUTELIN A1, A2

917.5 917.831 AFEPIRSV (SEQ ID NO: 44)

(29-36)

NDQRGEIV(SEQ ID NO: GLUTELIN A1 , A2, y^y.D a^a.o/ _4G) A3 (230-237)

GLUTELIN B1 (229-

943.5 943.748 NDQRGEII (SEQ ID NO: 46) ¾ _ΤΕΙ _| _{Ν B4} , B5

(227-234)

958.4 958.483 KN^NRGEEI (SEQ ID NO: ₍₄₄₀

447)

a Theoretical mass mean

b This column shows the mature proteins (with all the genetic possible variants, if any) that include the detected peptides. Parenthesis show the amino acids fragments referred to the entire protein.

D_Peptide sequences in RI-FP3 by HPLC coupled in tandem to mass spectrometry: Experimental Theoretical

Mass Mass ³

(Daltons;Da) (Daltons;Da) Sequence Fragment"

GLUTELIN B1 (460 - 463)

569.5 569.249 QYYP (SEQ ID NO: 48) GLUTELIN B2 (456 - 459)

GLUTELIN A1 , A2 (31 -

600.5 600.323 EPIRS (SEQ ID NO: 49)

35)

GLUTELIN A1 , A2 (184 -

684.5 684.367 GNKRNP (SEQ ID NO: 50)

189)

GLUTELIN A1 , A2 (72 -

743.5 743.360 LPHYTN (SEQ ID NO: 51 )

77)

GLUTELIN B1 (241

767.7 767.491 GLQLLKP (SEQ ID NO: 52) GLUTELIN B2 (239

245)

GLUTELIN B1 (370

859.8 859.455 FDGVLRPG (SEQ ID NO: 53) GLUTELIN B2 (366

373)

PROLAMIN PPROL

877.8 877.441 GQNIRQY (SEQ ID NO: 54)

14PJaponica (6 - 12)

LPHYTNGAS (SEQ ID NO: GLUTELIN A1 , A2 (72 -

958.8 958.451

55) 80)

' Theoretical mass mean

This column shows the mature proteins (with all the genetic possible variants, if any) that include the detected peptides. Parenthesis show the amino acids fragments referred to the entire protein.

Example 4. Edible composition with anti-obesity properties in Caenorhabditis elegans. Caenorhabditis elegans wild type N2 Strain was used. Age-synchronized adults were obtained from gravid adults and embryos were plated on Petri dishes with Nematode Growth medium (NG medium) containing Nile Red dye (0.5 micrograms/ ml). Rice protein hydrolysates as indicated in Table 5 below and a culture of

CECT-7210 strain were overlaid on plates seeded with OP50 E. coli strain before transferring worms. OP50 E. coli was used as normal nematode diet. Fluorescence quantification was conducted after growth at 20 °C during 3 days. The following feedings, as listed in Table 5, were evaluated:

Table 5: Experimental data for studying fat absorption in an in vivo model:

Nile red stains stored fat. Then fluorescence measured in the worms is proportional to accumulated body fat.

Orlistat, a drug whose primary function is to prevent the absorption of fat, was used as a reference compound.

As can be deduced from FIG. 3, all evaluated rice protein hydrolysates of the invention and CECT-7210 strain were able to significantly reduce (p<0.01 ) fluorescence amount compared to Control (NG medium). Values beside % F indicate the percentage of detected fluorescence.

RI-FP3, RI-PE3 hydrolysates and the strain CECT-7210 were able to produce statistically significant reductions of stored fat in C. elegans similar to those obtained with Orlistat (a known inhibitor of fat absorption). RI-A4 and RI-NZ3 hydrolysates were also able to significantly reduce stored fat.

Other Bifidobacterium sp strain and other protein hydrolysate showed a non significant fluorescence reduction, 93.8 % and 93.7 % fluorescence amounts respectively (data not shown). All these data allow concluding that the rice protein hydrolysates of the invention can be used as indregients in edible or nutritional compositions with the aim of reducing the fat absorption. If in addition, the compositions comprise an effective amount of the strain Bifidobacterium longum biovar infantis CECT 7210, the fat absorption will be also significantly reduced.

Example 5. Infant formulas comprising the rice protein hydrolysates of the invention.

Next Table 6 shows the composition of a powder product elaborated with the rice protein hydrolysates of the invention and its nutritious components, adjusted with the purpose of adapting to the characteristics of composition of maternal milk of beginning for the feeding of the suckling baby until 6 ^th month.

Table 6

COMPONENT 100 g powder

Proteins from rice protein hydrolysate 12.0 g

of the invention

FAT 25.5 g

Middle-chain triglycerides (MCT) 5.4 g

Linoleic acid 3290 mg

a-linoleic acid 281 mg

CARBOHYDRATES 56.6 g

Maltodextrin 44.7 g

Corn starch 12.0 g

Choline 50 mg

Taurine 35 mg

Inositol 25 mg

L-carnitine 10 mg

MINERALS 2.5 g

Sodium 225 mg

Potassium 450 mg

Chlorine 330 mg

Calcium 450 mg

Phosphorous 250 mg

Iron 5.0 mg Magnesium 45 mg

Zinc 4.0 mg

Copper 320 meg

Iodine 100 meg

Manganese 150 meg

Selenium 10 meg

Fluoride 275 meg

VITAMINS

Vitamin A 450 meg (1500 IU)

Vitamin D 7.5 meg (300 IU)

Vitamin E 10.0 meg (14.9 IU)

Vitamin K 40 meg

Vitamin B1 500 meg

Vitamin B2 600 meg

Vitamin B6 400 meg

Vitamin B12 1 .0 meg

Vitamin C 70 mg

Folic acid 60 meg

Pantothenic acid 3.2 mg

Niacine 5.0 mg

Biotine 12 meg

NUCLEOTIDES

Cytidine-5'-monophosphate 8.1 mg

Uridine-5'-monophosphate 6.5 mg

Adenosine-5'-monophosphate 3.0 mg

Guanosine-5'-monophosphate 2.0 mg

lnosine-5'-monophosphate 2.0 mg g means grams; mg is milligrams; IU is international units, meg is micrograms (M9)-

Next Table 7 shows another composition of a powder product elaborated with the rice protein hydrolysates of the invention and its nutritious components. It is a Follow-on formula for the feeding of suckling babies from 6 ^th to 36 ^th month. Table 7

COMPONENT 100 g powder

Proteins from rice protein hydrolysate 14.0 g

of the invention

FAT 22 g

Middle-chain triglycerides (MCT) 4.8 g

Linoleic acid 2904 mg a-linoleic acid 253 mg

CARBOHYDRATES 57.5 g

Maltodextrin 45.5 g

Corn starch 12.0 g

Choline 50 mg

Taurine 33 mg

Inositol 25 mg

L-carnitine 10 mg

MINERALS 3.5 g

Sodium 260 mg

Potassium 620 mg

Chlorine 500 mg

Calcium 500 mg

Phosphorous 330 mg

Iron 7.5 mg

Magnesium 50 mg

Zinc 4.0 mg

Copper 330 meg

Iodine 100 meg

Manganese 150 meg

Selenium 10 meg

Fluoride 275 meg

VITAMINS

Vitamin A 450 meg (1500 IU)

Vitamin D 7.5 meg (300 IU)

Vitamin E 10.0 meg (14.9 IU)

Vitamin K 42 meg

Vitamin B1 520 meg

Vitamin B2 620 meg Vitamin B6 420 meg

Vitamin B12 1 .0 meg

Vitamin C 70 mg

Folic acid 60 meg

Pantothenic acid 3.2 mg

Niacine 5.0 mg

Biotine 12 meg

NUCLEOTIDES

Cytidine-5'-monophosphate 7.7 mg

Uridine-5'-monophosphate 6.2 mg

Adenosine-5'-monophosphate 2.8 mg

Guanosine-5'-monophosphate 1 .8 mg

lnosine-5'-monophosphate 1 .8 mg

Any of the rice protein hydrolysates of the invention, preferably those disclosed in the Examples, are used for the manufacture of the infant formulas disclosed in Tables 6 and 7.

REFERENCES CITED IN THE APPLICATION

- WO 2004024177

- WO 2010078461

- ES 2350907 T3

- Adler-Nissen, J. et al., in Enzymatic Hydrolysis of Food Proteins.

Elsevier Applied Science Publishers, London, UK 1986, pp. 122-124. Spellman et al., "Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat processes for quantification of degree of hydrolysis", International Dairy Journal - 2003, Vol. 13, pp. 447-453.

- Navarrete del Toro et al, "Evaluation of the Progress of Protein

Hydrolysis. Protein Hydrolysis using the pH-STAT technique", Current Protocols in Food Analytical Chemistry -2002, B2.2.1 - B2.2.14.

- Kakudo et al., "Purification, Characterization, Cloning, and Expression of a Glutamic Acid-specific Protease from Bacillus licheniforrnis ATCC 14580, The Journal of Biological Chemistry - 1992, Vol. 267, No. 33, pp. 23782-23788