WO2014020209A1

WO2014020209A1 - Use of milk protein hydrolysates as gastrointestinal protectors

Info

Publication number: WO2014020209A1
Application number: PCT/ES2013/070550
Authority: WO
Inventors: Mª Isidra RECIO SÁNCHEZ; Mª Angeles Beatriz MIRALLES BURAGLIA; Daniel MARTÍNEZ MAQUEDA; Blanca HERNÁNDEZ LEDESMA; Sonia Cristina DE PASCUAL-TERESA FERNÁNDEZ; Lourdes Amigo Garrido; Mª del Rocío GIRÓN MORENO; Carlos GOICOECHEA GARCÍA
Original assignee: Consejo Superior De Investigaciones Científicas (Csic); Universidad Rey Juan Carlos
Priority date: 2012-07-30
Filing date: 2013-07-26
Publication date: 2014-02-06

Abstract

The present invention relates to the use of an isolated bioactive peptide which has stimulating activity for the secretion of mucins and/or opioid activity, which is included in an enzymatic hydrolysate of at least one milk protein selected from a whey protein hydrolysate or a milk casein hydrolysate, in which the structure thereof includes a sequence having at least two aromatic amino acids, wherein the first aromatic amino acid is a tyrosine in the first or second position relative to the N-terminal end of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine in the third or fourth position relative to the first tyrosine; in order to stimulate the formation of intestinal mucus and/or to promote gastrointestinal protection.

Description

USE OF HYDROLYZES OF MILK PROTEINS AS LEVEL PROTECTORS

GASTROINTESTINAL

DESCRIPTION

Technical sector

The present invention is within biology and medicine and can be applied to the food and pharmaceutical industry. The invention relates to the use of peptides and / or enzymatic hydrolysates of milk proteins to improve the protection of the intestinal tract by stimulating mucin secretion, and to the compositions containing said peptides. In addition, the present invention also collects new peptide sequences with opioid activity.

State of the art

During the last years, many of the investigations on the protein fraction of foods have focused on the study of biologically active peptides present in the sequence thereof. These peptides are in an inactive state when they are part of the precursor protein but can be released by enzymatic hydrolysis, either during gastrointestinal digestion or during food processing. Once released, the peptides can act as biologically active compounds in the body. Thus, for example, it has been shown that they can exert opiode, antihypertensive or antimicrobial activity; and there is increasing evidence that they may have activity in the immune system or improve the bioavailability of minerals.

The intestine is the organ responsible for the absorption of nutrients, it acts as a barrier, signal recognition and synthesis of bioactive compounds. These functions are regulated by hormones and cytokines, but it is known that certain components of the diet can also play a fundamental role in modulating these functions. Epithelial cells Intestinals, which are forming a monolayer that covers the entire surface of the gastrointestinal tract, are especially important in this modulation because they interact directly with the intestinal content, including food, its digested and bacterial flora. Therefore, the food, in addition to a source of nutrients, can act as a "modulator" of different physiological functions. The organ where this concept can be applied directly is the gastrointestinal tract because it is in contact with a large number of substances that we ingest. Modulation of intestinal functions through diet has recently been described as essential to improve human health (Shimizu and Son, 2007 Curr. Pharm. Design 13: 885-895).

A key function of the intestinal epithelium is to act as a physical barrier between the external and internal environment. The intestinal surface is covered by a mucous layer that plays an important physiological role that includes the lubrication of the cell surface, the defense against colonization by pathogenic bacteria and the protection against intestinal proteases. The main structural components of the mucus are the mucins, high molecular weight glycoproteins produced by the goblet cells of the epithelial surface. The epithelium of the intestine, both thin and thick, contains goblet cells that produce mucins. Mucin 2 is the predominant gel-forming mucin in the healthy intestine of human, rat and mouse. Any experimental or pathological situation that changes directly or indirectly the amount of mucin, its structure or the aqueous or ionic content will cause an alteration of the mucosal barrier. Gastric ulcer or ulcerative colitis are examples of the consequences of a failure in the expression and / or composition of the gastric or intestinal mucus, respectively.

Recent studies have shown that some components of the diet can have a positive influence on the characteristics of the intestinal mucus, producing an increase in the secretion or expression of mucins or even increasing the number of intestinal goblet cells, although the mode of The action of each compound may differ (Barceló et al., 2000 Gut 46: 218-224). For example, dietary fiber seems to favor the total mucin secretion capacity in the lumen of the small intestine, although the stimulating effect on secretion is dependent on both the quantity and quality of the ingested dietary fiber (Tanabe et al, 2005 J. Nutr. 135: 2431-2437) and may be due to an increase in the number of goblet cells

(Ito et al., 2009 J. Nutr. 139: 1640-1647). Regarding the genes encoding the mucins (MUC), it has been shown that low concentrations of butyrate stimulate the expression of MUC2

(Gaudier et al., 2004 Am. J. Physiol. Gastrointest. Líver Physiol. 278: G1168-G1174) while high concentrations of this short-chain fatty acid suppress expression, which can decrease the intestinal function of the barrier ( Burger Van Paasen et al., 2009 Biochem. J. 420: 211-219).

In the field of food proteins, the role of proteins and peptides in the regulation of different functions at the intestinal level is being investigated. In particular, Trompette et al., 2003 (J. Nutr. 133: 3499-3503) have found that some dairy peptides, specifically the YPFPGPI peptide, / 3-casomorphine 7, which corresponds to fragment (60-66) of the / Bovine 3-casein A2, induces mucin production in rat jejunum, by activating the enteric nervous system and interacting with opioid receptors. It was also found that an a-lactoalbumin hydrolyzate induced mucin release while neither casein nor a mixture of amino acids produced this effect. Since this secretory activity disappeared in the presence of naloxone, an opioid antagonist, it was suggested that secretion was produced by a nervous mechanism involving the activation of μ type opioid receptors (Claustre et al., 2002 Am. J. Physiol. Gastrointestinal. Liver Physiol. 283: G521-G528; Trompette et al., 2003 J. Nutr. 133: 3499-3503). The presence of these receptors in intestinal cells suggests the possibility that / 3-casomorphins, which occur in the intestinal lumen during digestion, can control the mucin production through direct action on intestinal goblet cells. Subsequently, it has been shown that the YPFPGPI peptide, / 3-casomorphine 7, induces the secretion of mucin 5AC in human colon goblet cells (HT29-MTX) (Zoghbi et al., 2006 Am. J. Physiol. Gastrointest. Liver Physiol. 290: G1105-G1113; Plaisancié et al. 2010 Int Patent Appl. WO2010 / 130956). However, not all opioid peptides have activity on the production of intestinal mucins. The luminal administration in rat jejunum of bovine peptides / 3-casomorphine-6 (YPFPGP), β-casomorphine 4 (YPFP) and / 3-casomorphine 4-NH ₂ (YPFF-NH ₂ ), although they have an affinity for opioid receptors (Teschemacher et al., 1997 Biopolymers 43: 99-117; Meisel and FitzGerald 2000 Br. J. Nutr. 84: S27-S31), do not induce mucin secretion (Trompette et al., 2003 J. Nutr. 133: 3499-3503).

On the other hand, in order to establish an association between mucin production and its biosynthesis, expression studies of the genes coding for mucins have been carried out. In rat intestinal cells (DHE) it has been shown that exposure to the YPFPGPI peptide, / 3-casomorphine 7, causes an increase in the expression of the main genes involved in the production of rat mucins (rMuc), rMuc2 and rMuc3. In human HT29-MTX cells, this peptide also results in an increase in the expression of the gene encoding the main secreted mucin, MUC5AC (Zoghbi et al., 2006 Am. J. Physiol. Gastrointestinal. Liver Physiol. 290: G1105- G1113). In an in vivo study, an increase in the expression of the rMuc3 and rMuc4 genes has been observed in rats given a diet rich in hydrolyzed caseins compared to a diet rich in amino acids and a protein-free diet (Han et al., 2008 J. Agrie. Food Chem. 56: 5572-5576).

However, although it is known that hydrolysates containing β-casomorphine 7 have mucin secretory activity, other peptide sequences that are present in milk casein hydrolysates and that are also responsible for inducing mucin secretion have not been identified. Therefore, the present invention provides new peptide sequences with stimulating activity. of the secretion of intestinal mucins in human intestinal cells. This biological activity would not have been previously described for these peptides, so it implies a new use of them. In addition, their production is described by enzymatic hydrolysis of low-cost food proteins (whey proteins, caseinates, etc.), resulting in hydrolysates containing the new peptides in relevant concentrations and therefore with protective activity at the intestinal level.

Description of the Invention

The present invention provides hydrolysates of whey and casein proteins that contain a series of peptides that positively affect the secretory activity of mucins, stimulating the formation of the intestinal mucosa and that affect the expression of the MUC5AC gene in intestinal secretory epithelial cells of mucins (HT29-MTX). New peptides derived from caseins capable of inducing mucin secretion and presenting opioid activity in guinea pig ileum are also described.

A first aspect of the invention relates to the use of at least one isolated bioactive peptide that: a) possesses mucin secretion stimulating activity and / or opioid activity; b) is present in an enzymatic hydrolyzate of at least one milk protein selected from a whey protein hydrolyzate (preferably a whey protein hydrolyzate with trypsin) or a milk casein hydrolyzate (preferably a casein hydrolyzate of pepsin milk); c) its structure comprises a sequence with at least two aromatic amino acids, where the first aromatic amino acid is a tyrosine located in the first or second position with respect to the N-terminal end of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine located in third or fourth position with respect to the first tyrosine (which means that between the first tyrosine residue and the second aromatic amino acid there is one or two separation amino acids); and where said structure is selected from SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12; for the manufacture of a pharmaceutical composition or a functional ingredient to stimulate the formation of the intestinal mucosa. The formation of said intestinal mucosa is due to a greater mucin secretion that is induced by the above isolated peptides. In fact, it has been proven that mucin formation is due to increased expression of the MUC5AC gene in secretory human goblet cells (HT29-MTX), when exposed to at least one of the isolated bioactive peptides. This stimulation of the formation of the intestinal mucosa by the bioactive peptides above, is useful to promote gastrointestinal protection.

Table 1 shows the list of the sequences of the bioactive peptides SEQ ID No: 1 to SEQ ID No: 12 contemplated within the scope of the present invention.

Table 1 .

YLLF SEQ ID No: 1 Amino acid sequence of fragment 102-105 of bovine / 3-lactoglobulin

YLLF-NH ₂ SEQ ID No: 2 Amino acid sequence of the aminated 102-105 fragment of the bovine / 3-lactoglobulin

YPSF-NH ₂ SEQ ID No: 3 Amino acid sequence of the amidated fragment 41-44 of the human / 3-casein

YPFV-NH ₂ SEQ ID No: 4 Amino acid sequence of the amidated fragment 51-54 of the human / 3-casein

YPFVE SEQ ID No: 5 Amino acid sequence of fragment 51-55 of the human / 3-casein

RYLGY SEQ ID No: 6 Amino acid sequence of fragment 90-94 of the Q _sl- bovine casein YLGY SEQ ID No: 7 Amino acid sequence of fragment 91-94 of the Q _sl- bovine casein

AYFYPEL SEQ ID No: 8 Amino acid sequence of fragment 143-149 of Q _sl- bovine casein

YFYPEL SEQ ID No: 9 Amino acid sequence of fragment 144-149 of the Q _sl- bovine casein

YFYPE SEQ ID No: 10 Amino acid sequence of fragment 144-148 of the bovine Q _s i-casein

YFYP SEQ ID No: 11 Amino acid sequence of fragment 144-147 of Q _sl- bovine casein

YFY SEQ ID No: 12 Amino acid sequence of fragment 144-146 of the Q _sl- bovine casein

Within the scope of the present invention, enzymatic hydrolyzate refers to the combination of a protein substrate that serves as a starting material and proteolytic enzymes in a suitable ratio and under specific conditions of pH and hydrolysis time that gives rise to peptides with the sequence desired.

The starting material of the present invention would be any suitable substrate comprising one or more proteins or peptides, of animal, plant, or microorganism origin, containing the amino acid sequence of the bioactive peptides of interest (SEQ ID No 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12, table 1), preferably milk, casein or whey protein concentrates. Since they are all milk sequences, it is obvious that any preparation containing the starting proteins could be used: β-lactoglobulin, β-casein, or ¾i-casein of different species, or peptides or fragments thereof of any size, alone or mixed with other proteins. For example: whole casein, caseinates and milk in its different forms of presentation, fermented milk products, hydrolysed milk proteins, milk by-products, dairy products for animal feed, etc. Said starting material is dissolved or dispersed, at an appropriate concentration, in water or in a buffer solution, at a pH suitable for the action of the proteolytic enzyme. Any proteolytic enzyme capable of breaking the protein present in the starting material and providing the peptides of interest may be used, but preferably pepsin at pH 2.0-3.0 or trypsin at pH 7.5-8.5. Proteolytic microorganisms that carried out fermentation of the substrate and hydrolysis of the protein could also be used.

The hydrolysis conditions: pH, temperature, enzyme-substrate ratio, reaction interruption etc., are optimized in order to select the most active hydrolysates. In a particular embodiment, the bioactive peptides are obtained using casein hydrolyzed with pepsin at pH 2.5, in an enzyme / substrate ratio 4/100 (w / w) and performing hydrolysis at 37 ° C, for a period of time between 10 minutes and 24 hours, but preferably for less than 6 hours. In another particular embodiment, the peptides are obtained by hydrolyzing a whey protein concentrate with trypsin at pH 8.0, in a 5/100 enzyme / substrate ratio (w / w) and performing hydrolysis at 37 ° C, over a period of time. of time between 10 minutes and 24 hours, but preferably for less than 4 hours.

Within the scope of the present invention, the pharmaceutical composition refers to the mixture of an active ingredient, or mixture of active ingredients, with suitable excipients (lactose, maltodextrin, etc.) to give oral administration forms, preferably tablets or capsules. . The active principle is understood as the individual peptides, the mixture thereof or the enzymatic hydrolysates that contain them. The doses of the peptides of interest may be between 1 to 10 mg / day, but preferably they will be administered at doses of 5 to 7 mg / day.

Within the scope of the present invention, functional ingredient refers to a food ingredient that in addition to exercising a Beneficial effect on the individual who consumes it for its nutritional value, satisfactorily demonstrates improving one or more functions in the body. The function in the organism of the functional ingredient is referred both to the improvement of a physiological function, and to the decrease in the risk of suffering from a disease. The functional ingredient may contain the individual peptides, the mixture thereof or the enzymatic hydrolysates that contain them, in addition to different excipients in order to improve their organoleptic or technological characteristics (lactose, fructose, starch, maltodextrins, etc.). This functional ingredient could be incorporated into different foods compatible with a healthy diet such as yogurts, juices, infant formulas, cereal bars, dairy preparations and desserts, powdered food preparations, etc.

The pharmaceutical composition or functional ingredient above are useful for stimulating the formation of the intestinal mucosa.

In a preferred embodiment, the isolated bioactive peptide for the manufacture of the pharmaceutical composition or functional ingredient comprises at least one peptide selected from at least one of the group consisting of: SEQ ID No: 1, SEQ ID No: 2 and a combination from both. Said peptides SEQ ID No: 1 and / or SEQ ID No: 2 (Table 2) are present in whey protein hydrolysates (preferably in whey protein hydrolysates with trypsin) and increase the secretory activity of mucins in producing intestinal epithelial cells. of mucins, and therefore, stimulate the formation of the intestinal mucosa. It should be understood that the use of a whey protein enzyme hydrolyzate, comprising at least one of the individual peptides SEQ ID No: 1, SEQ ID No: 2, or a combination thereof, is equally valid for the preparation of the pharmaceutical composition or functional ingredient.

Table 2. Sequences of dairy peptides with secretory activity of mucins derived from whey proteins. YLLF SEQ ID No: 1

YLLF-NH ₂ SEQ ID No: 2

The available technology allows the bioactive peptides identified in Table 2 (SEQ ID No: 1 and SEQ ID No: 2), upon knowing their sequence, can be obtained by chemical and / or enzymatic synthesis, hydrolysis of proteins of different species (cow , sheep, goat, etc.), hydrolysis of recombinant human proteins or by recombinant methods of heterologous production.

In another preferred embodiment, the isolated bioactive peptide for the manufacture of the pharmaceutical composition or functional ingredient comprises at least one peptide selected from at least one of the group consisting of: SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 and any combination of the same. Peptides SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and / or SEQ ID No: 12 (Table 3) are present in casein hydrolysates (preferably in casein hydrolysates of milk with pepsin) and increase the secretory activity of mucins in intestinal mucin-producing epithelial cells, thus stimulating the formation of the intestinal mucosa.

Table 3. Sequences of dairy peptides with secretory activity of casein-derived mucins.

YPSF-NH2 SEQ ID No: 3

YPFV-NH2 SEQ ID No: 4

YPFVE SEQ ID No: 5

RYLGY SEQ ID No: 6

YLGY SEQ ID No: 7

AYFYPEL SEQ ID No: 8

YFYPEL SEQ ID No: 9

YFYPE SEQ ID No: 10

YFYP SEQ ID No: 11 YFY SEQ ID No: 12

The available technology allows the bioactive peptides identified in Table 3 (SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12), upon knowing its sequence, can be obtained by chemical and / or enzymatic synthesis, hydrolysis of proteins of different species (cow, sheep , goat, etc.), hydrolysis of recombinant human proteins or by recombinant methods of heterologous production.

In another preferred embodiment, the isolated bioactive peptide for the manufacture of the pharmaceutical composition or functional ingredient comprises at least one peptide selected from at least one of the group consisting of: SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 and any combination thereof. The peptides SEQ ID No: 3, SEQ ID No: 4 and / or SEQ ID No: 5 are present in a hydrolyzate of the milk / 3-casein fraction (preferably in milk / 3-casein hydrolysates with pepsin) Therefore, for the preparation of the pharmaceutical composition or functional ingredient, as the invention is understood, it is possible to use at least one of the peptides defined by SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 , or any combination of said peptides, or an enzymatic hydrolyzate of the milk / 3-casein fraction comprising at least one of said peptides.

In another preferred embodiment, the isolated bioactive peptide for the manufacture of the pharmaceutical composition or functional ingredient comprises at least one peptide selected from at least one of the group consisting of: SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 and any combination thereof. Peptides SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and / or SEQ ID No: 12 are present in hydrolysates of the ¾i-casein fraction of milk (preferably in _sl -casein hydrolysates of milk with pepsin), and as mentioned above increase the secretory activity of mucins in intestinal epithelial cells producing mucins. In addition, any of the peptides SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 or a combination thereof, in addition to stimulating secretion activity of mucins, present opioid activity in guinea pig ileum preparations (Table 4). Accordingly and as in the previous cases, for the preparation of the pharmaceutical composition or the functional ingredient according to the invention, it is possible to use at least one of the individual peptides defined by SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, or any combination of said peptides, or an enzymatic hydrolyzate of the ¾i-casein fraction of the milk comprising at least one of said peptides. By possessing these opioid activity sequences, they could exercise different physiological functions related to the interaction with opioid receptors at the intestinal level such as delayed intestinal transit, activities related to the immune system, etc.

Table 4. Sequences of milk peptides derived from caseins with opioid activity.

It should be understood that with the use of at least one of the above isolated bioactive peptides, for the manufacture of a pharmaceutical composition or a functional ingredient to stimulate the formation of the intestinal mucosa (and consequently improve gastrointestinal protection), as described above. , the present invention also contemplates the protection of an isolated bioactive peptide that: a) possesses mucin secretion stimulating activity and / or opioid activity; b) is present in an enzymatic hydrolyzate of at least one milk protein selected from a whey protein hydrolyzate (preferably a whey protein hydrolyzate with trypsin) or a milk casein hydrolyzate (preferably a casein hydrolyzate of pepsin milk); c) its structure comprises a sequence with at least two aromatic amino acids, where the first aromatic amino acid is a tyrosine located in the first or second position with respect to the N-terminal end of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine located in third or fourth position with respect to the first tyrosine; and where said structure is selected from SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12; to stimulate the formation of the intestinal mucosa and / or promote gastrointestinal protection, and therefore for its use as a stimulant of the formation of the intestinal mucosa and / or as a gastrointestinal protector. However, it is possible that for its application, said peptide is preferably being part as a component of a pharmaceutical composition or a functional ingredient, as defined above.

Similarly, the use of the first aspect of the invention can also be protected as a method of treatment to stimulate the formation of the intestinal mucosa and / or favor gastrointestinal protection characterized in that it comprises administering to an individual a therapeutically effective amount of a peptide. isolated bioactive that: a) has mucin secretion stimulating activity and / or opioid activity; b) is present in an enzymatic hydrolyzate of at least one milk protein selected from a whey protein hydrolyzate (preferably a whey protein hydrolyzate with trypsin) or a milk casein hydrolyzate (preferably a milk casein hydrolyzate with pepsin); c) its structure comprises a sequence with at least two aromatic amino acids, where the first aromatic amino acid is a tyrosine located in the first or second position with respect to the N-terminal end of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine located in third or fourth position with respect to the first tyrosine; and where said structure is selected from SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No:

According to the present invention, the term "therapeutically effective amount" refers to the amount of the peptides of the invention calculated to produce the desired effect. Specifically, it refers to the amount of peptide, or mixture of peptides, or of the hydrolysates that contain the peptides, which is capable of inducing the secretion of mucins at the gastrointestinal level and thus contributing to the protection thereof. The doses of the peptides of interest may be between 1 to 10 mg / day, but preferably they will be administered at doses of 5 to 7 mg / day. The doses of the hydrolysates containing them may be between 0.5-5 g / day, but preferably they will be administered at doses of 1-2 g / day.

Detailed explanation of the figures

Figure 1. Effect of peptides SEQ ID No: 5 (A), SEQ ID No: 6 (B), SEQ ID No: 8 (C) at a concentration of 0.1 mM on mucin secretion in HT29- cells MTX determined by enzyme conjugated lectin assay (ELLA). The data are expressed as a percentage of mucin secretion versus control (untreated cells). Each point represents the mean + standard error of the average of three biological replicates in triplicate. Significant differences obtained through two-way ANOVA applying the Bonferroni test. *** P <0.001; * P <0.05. Figure 2. Effect of hydrolysates obtained from whey proteins (A), and caseins (B) in concentrations of 0.1 and 1.0% on the level of MUC5AC mRNA determined by quantitative PCR (RT-qPCR) between 2 and 24 hours of exposure. The data are expressed as relative expression level of MUC5AC versus the control (untreated cells), using cyclophilin as the reference gene. Each point represents the mean + the standard error of the average of three biological replicates in triplicate. Significant differences obtained through two-way ANOVA applying the Bonferroni test. * P <0.05.

Figure 3. Effect of peptides SEQ ID No: 2 (A), SEQ ID No: 6 (B) and SEQ ID No: 8 (C) in concentrations of 0.5, 0.1 and 0.05 mM on the MUC5AC mRNA level determined by quantitative PCR (RT-qPCR) between 2 and 24 hours of exposure. The data are expressed as relative expression level of MUC5AC versus the control (untreated cells), using cyclophilin as the reference gene. Each point represents the mean + the standard error of the average of three biological replicates in triplicate. Significant differences obtained through two-way ANOVA applying the Bonferroni test. ** P <0.01; * P <0.05.

Figure 4. Effect of peptides SEQ ID No: 9 (A) and SEQ ID No: 10

(B) in the preparation longitudinal fiber-myenteric plexus (FL-PM) of guinea pig ileum. Each point represents the average percentage inhibition of the electrically induced contractile response of 3-4 replicates + standard error of the mean in at least 3 different animals by the cumulative administration of the peptides.

Examples of embodiment of the invention

The invention will now be illustrated by tests carried out by the inventors, which show the specificity and effectiveness of the hydrolysates of whey proteins and caseins and the peptides they contain on mucin secretion and the expression of the MUC5AC gene in HT29-MTX cells. In addition, the opioid activity of some of them is illustrated in trials conducted in guinea pig ileum preparations. a) MATERIALS AND METHODS al) Peptides and hydrolysates

The β-casomorphine 7 bovine f (60-66) of the β-casein A2 of YPFPGPI sequence was purchased from Bachem (Bubendorf, Switzerland) and was used as a positive control. Peptides SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, and human and bovine oi-lactorphine peptides f (50-53) of the YGLF-NH ₂ sequence OIL-lactalbumin peptides , the deaminated form of the OI - bovine lactorphine f (50-53) of the YGLF sequence α-lactalbumin, bovine morphiceptin, f (60-63) of the YPFP-NH ₂ sequence β-casein A2, the neocasomorphine bovine f (114-119) of the β-casein of YPVEPF sequence and the bovine α-casein exorphine, f (91-96) of YLGYLE sequence, were obtained by solid phase synthesis (FMOC) in a 433A synthesizer (Applied Biosystems, Warrington, UK). The purity of all these synthetic peptides was greater than 90%, being verified by reverse phase liquid chromatography coupled to tandem mass spectrometry (HPLC-MS / MS).

In the preparation of whey protein hydrolyzate, a whey protein concentrate (Friesland Foods Domo, Zwolle, The Netherlands) was dissolved in 5% w / v water and heated at 90 ° C for 10 minutes. Hydrolysis was carried out by applying food grade porcine trypsin (Biocatalysts, Nantgarw, Wales, UK) with an enzyme-substrate ratio of 5% w / w, for 3 hours at 37 ° C and pH 8.0. The reaction was terminated by inactivation of the enzyme by heat treatment at 95 ° C for 15 minutes.

Casein hydrolyzate was obtained from commercial casein (Promilk 85, Arras Cedex, France) at 6% w / v in water with a final pH of 2.5. Hydrolysis was carried out with food grade pepsin (Biocatalysts) in an enzyme-substrate ratio of 2% w / w, being added twice (initial moment and three hours of hydrolysis). It was incubated at 40 ° C for 6 hours with final inactivation of the enzyme by heating at 80-85 ° C for 15 minutes. The peptides contained in the hydrolysates were identified by HPLC-MS / MS. a.2) Analysis via RP-HPLC coupled online to tandem mass spectrometry (RP-HPLC-MS / MS)

Esquire-LC equipment (Bruker Daltonik GMBH, Bremen, Germany) was used. The HPLC equipment (1100 series) consisted of a quaternary pump, an automatic injector, an eluent degassing system and a variable wavelength ultraviolet detector (Agilent Technologies, Waldbronn, Germany) coupled in line to a mass spectrometer of ionic trap Esquire 3000 (Bruker Daltonik). The column was a Hi-Pore C18 column (250 x 4.6 mm ID, 5 particle size) (Bio-Rad Laboratories, Richmond, CA, USA). Solvent A was a mixture of water and trifluoroacetic acid (1000: 0.37) and solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27). 50 μΐ of prepared sample was injected at a concentration of 4.5 mg / ml. A flow of 0.8 ml / min was used, with a linear gradient from 0% to 50% of solvent B in 60 minutes. The eluent was monitored at 214 nm and the sample flow to the mass spectrometer nebulizer was 275 μΐ / min. Electrospray ionization (ESI) used N2 as a nebulizer agent (8 L / min), fogging pressure 60 psi, drying temperature 350 ° C and a capillary voltage of 4 KV. The mass spectra were acquired in the range of 100-2500 m / z and a fragmentation ramp between 0.3 and 2 V was used. The Esquire ControlTM 5.0 program (Bruker Daltonik) was used as the data acquisition system and for To identify the sequence of the different peptides, the Data AnalysisTM 3.0, Sequence EditorTM 2.1 and BiotoolsTM 2.1 software were all used by Bruker Daltonik. a.3) Cell cultures

The HT29-MTX cell line derived from human colon adenocarcinoma was used, with mucin secretion capacity donated by Dr. Thécla Lesuffleur (INSERM UMR S 938, Paris, France). Cells were grown in Dulbecco-modified Eagle medium (DMEM) supplemented with 10% inactivated bovine fetal serum and 10 mL / L of penicillin-streptomycin at 37 ° C, in a humidified atmosphere with 5% C0 ₂ . Weekly passes were performed using 0.05% trypsin / EDTA (all reagents were from Gibco, Paisley, UK). The cells were seeded in 12-well plates with a density of 5><10 ⁵ cells per well (Nunc, Roskilde, Denmark). The cell line was used between passes 12 and 22. The experiments were performed 21 days after confluence. From 24 hours before the test, we worked with culture medium without serum or antibiotic to eliminate the interference of proteins or hormones. On the day of the test, the medium was removed, the cell monolayer was washed twice with phosphate buffered saline (PBS) and the medium was added with the peptides in concentration 0.05, 0.1 and 0.5 mM, incubating at 37 ° C for times between 30 minutes and 24 hours. In the control wells only the culture medium was added. At the selected times, supernatants were collected that were kept frozen at -70 ° C until analysis. Extraction and subsequent purification of cellular RNA was carried out with the Nucleospin® RNA II kit (Macherey-Nagel, Düren, Germany). a.4) Enzyme Conjugated Lectin Assay (Enzyme-Linked Lectin Assay; ELLA).

The quantitative determination of total mucins was performed by an ELLA assay based on the use of wheat germ agglutinin, which has shown a strong reactivity against specific sugars present in the mucins secreted by goblet cells. The total glycoprotein content in the samples studied was obtained by interpolation in a standard curve constructed from commercial porcine gastric mucin (Sigma, St Louis, CA, USA). The upholstery of the 96-well polystyrene plate was performed with the samples from the calibration line and with the samples to be tested dissolved in sodium carbonate buffer (0.5 M, pH 9.6), leaving to incubate overnight at 4 ° C The blockade was performed with bovine serum albumin (BSA) (Sigma) at 2% for 1 hour at 37 ° C. Biotinylated wheat germ agglutinin (Vector Laboratories, Peterborough, UK) was added in PBS-Tween-BSA (1: 1000), and the samples were incubated 1 hour at 37 ° C. Subsequently, to amplify the signal, the avidin-peroxidase complex (Vector laboratories) in PBS-Tween-BSA (1: 50,000) was added. Colorimetric determination was carried out by reading the absorbance at 492 nm in a Multiskan Ascent plate reader (Labsystems, Barcelona, Spain) using a solution of o-phenylenediamine dihydrochloride (OPD) (Dako, Glostrup, Denmark). The data of each sample tested was expressed as a percentage of glycoprotein secretion against its control sample at the same time and cell assay. a.5) Quantitative PCR assay

The RNA copy number was determined by quantitative PCR with real-time fluorescence measurement, using SYBR Green as a fluorophore in Lightcycler 480 (Roche, Mannhein, Germany) with 384 well plates. The cDNA was obtained from 375 ng of RNA by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. For MUC5AC (access code NCBI AJ001402), target gene, oligos 2870-2899 / 3109-3091 were used, while for the reference gene cyclophilin (access code NCBI Y00052) oligos 280-340 / 445- were used 421 (Zoghbi et al., 2006 Am. J. Physiol. Gastrointest. Líver Physiol. 290: 1105-1113). Each cDNA sample was studied in triplicate. Each reaction tube contained 5 μΐ of 2 ^χ SYBR Green real-time PCR Master Mix (Applied Biosystems), 0.25 μΐ to 10 μΜ of each of the specific oligos, 0.27 μΐ of cDNA and 4.23 of H ₂ 0. The amplification was started at 95 ° C for 5 minutes and 45 cycles at 95 ° C for 10 seconds, 60 ° C for 10 seconds and 72 ° C for 10 seconds. Controls were used to confirm the absence of oligo dimers and to verify that there was no DNA contamination. By analyzing the melting and electrophoresis curves in 2% agarose gel it was found that all the trials amplified a single product. To calculate the efficiency (E = 10-l / slope) a curve was determined with the number of cycles (Ct) obtained from the amplification of known amounts of cDNA. Relative expression values were calculated using the critical threshold method (Δ ACt). All experiments were performed in triplicate based on three biological replicas. a.6) Statistical analysis

Data were analyzed using two-way ANOVA, using GraphPad Prism software, followed by the Bonferroni test for individual comparisons. The differences between means and controls were considered significant with values of P <0.05 (*), P <0.01 (**) or P <0.001 (***). a.7) In vitro evaluation of the opioid activity of the peptides in the longitudinal fiber-myenteric plexus (FL-PM) preparation of guinea pig ileum

The possible opioid nature of the peptides has been studied by assessing their ability to inhibit electrically induced contractions of the longitudinal-myenteric fiber preparation of guinea pig ileum (FL-PM). For this study, female albino guinea pigs from Harían Ibérica (Castelar, Spain), weighing between 250 - 300 g, have been used. The FL-PM preparation was obtained from said intestinal segments, according to the modification of the technique described by Ambache (1954 J. Physiol. Lond. 125: 53-55) to isolate longitudinal fibers of rabbit ileum. The in vitro organ bath technique was used, in which the equipment used is made up of 10-ml glass cups that were filled with 5 ml of Krebs solution (NaCl 118, KC1 4.75; CaCl ₂ 2.54; KH ₂ P0 ₄ 1.19; MgS0 ₄ 1.2; NaHC0 ₃ 25; 11 mM glucose), which is continuously bubbled with carbon dioxide (95% 0 ₂ and 5% C0 ₂ ) and maintained at a temperature of 37 ° C. The preparation is then given a baseline tension of 1 g, allowed to rest for 15 minutes for stabilization and electrically stimulated continuously. The stimulation conditions were constant: simple square pulses of 0.3 Hz frequency, 2 ms duration and supramaximal voltage. The contractile activity of Preparations were recorded using a data recording and analysis system coupled to an isometric force transducer.

Once, after the beginning of the electrical stimulation, the preparations presented a stable contractile response, concentration-response curves were performed, by adding to the bath of organs of cumulative increasing doses of the peptides, spaced in 10 min (considered as the interval of time required for the peptides to exert their effect). The concentrations evaluated were: 6, 1 x 1CT ⁸ , 1.8 x 1CT ⁷ , 5.5 x 1CT ⁷ , 1.6 x 1CT ⁶ and 5 x 1CT ⁶ M. After administration of the last dose and after 10 min waiting, a single dose of the opioid antagonist naloxone (10 ^{~ 6} M) was added to check if their effect reversal took place and to confirm that the inhibitory effect was mediated by selective interaction with opioid receptors. In order to compare the effect of the new peptides with that of a known opioid agonist and with a food peptide with described opioid activity, in parallel to the evaluation of the new peptides, morphine and β-casomorphine curves were also performed 7 following the same protocol and have been used as reference standard curves.

The results are expressed as% contraction inhibition, considering as 100% the average amplitude of the last 5 contractions before the addition of the first dose of the curve. Each preparation was used to carry out a single concentration-response curve.

Example 1. Effect of different synthetic food peptides on the secretory activity of mucins in HT29-MTX cells.

Table 5 shows the maximum mucin secretion values in HT29-MTX cells after the addition of the peptides of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 at the concentration of 0.1 mM. In addition, Table 5 shows these values for six other peptides previously described as opioids. Eleven of the 14 food peptides tested resulted in a significant increase (P <0.001) of mucin secretory activity in HT29-MTX cells. The maximum secretion values ranged from 191 to 587% with respect to the control (untreated cells). It should be noted that SEQ ID No: 2 and SEQ ID No: 5 produced greater increases in mucin secretion than β-casomorphine 7.

Table 5. Maximum mucin secretion with respect to the control (untreated HT29-MTX cells) due to the addition of different synthetic food peptides in 0.1 mM concentration. Data obtained from three biological replicates in triplicate. Significant differences between mean and control values (untreated cells) using two-way ANOVA (Bonferroni test).

Mucina

Peptide

secreted

Protein or

or

Sequence Designation P food Control

SEQ ID Des-NH ₂ - / 3-lactorphine / 3 _B- lactoglobulin

163 <0,, 05 No: 1 bovine f (102-105)

SEQ ID / 3 _B -lactoglobulin

/ 3-bovine lactorphine 452 <o, 001 No: 2 f (102-105)

SEQ ID / 3 _H -casein f (41-

/ 3-human casorphine 215 <, 001 No: 3 44)

SEQ ID / 3 _H -casein f (51-

Valmuceptin 259 <, 001 No: 4 54)

SEQ ID / 3-casomorphine 5/3 _H -casein f (51-

339 <, 001 No: 5 human 55)

SEQ ID ¾j, -) -casein

- 191 <or, 001 No: 6 f (90-94)

SEQ ID ¾j, -) -casein

- 226 <o, 001 No: 7 f (91-94)

SEQ ID a _s iB ^_ casein

- 220 <o, 001 No: 8 f (143-149)

/ 3-casomorphine 7/3 _B- casein A2

YPFPGPI 282 <, 001 bovine f (60-66)

human α-lactorphine to _{H / B} -lactalbumin

YGLF '-NH ₂ 587 <o, 001 and bovine f (50-53) Des-NH ₂ - Q-lactorphine to _{H / B} -lactalbumin

YGLF 209 <0.001 human and bovine f (50-53)

/ 3 _B- casein A2

YPFP-NH? MMoorrffiicceeppttiinnaa bboovviinnaa 130 ns f (60-63)

Neocasomorphine / 3 _B -casein f (114-

YPVEPF 120 ns bovine 119)

α-casein exorphine o¾-casein f (91-

YLGYLE 104 ns

2-7 bovine 96)

Figure 1 shows the effect during the exposure time of the peptides of SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 8. at a 0.1 mM concentration on the secretion of mucins in HT29-MTX cells . SEQ ID No: 5 showed increased mucin secretion values at 30 minutes and 2 hours followed by a significant increase at 4 hours and 8 hours (Fig 1A). SEQ ID No: 6 and SEQ ID No: 8 showed absolute levels lower than those presented by the previously described but significant peptides at 2 and 4 hours, respectively (Fig IB and 1C).

These results show that not all opioid sequences induce mucin secretion. Thus, for example, bovine morphiceptin, bovine neocasomorphine and bovine α-casein 2-7 bovine peptides, which had previously been described as opioid agonist peptides (Teschemacher 2003 Curr. Pharm. Design 9: 1331-1344), did not stimulate production of mucins in intestinal epithelial cells. The YGLF-NH2 and YGLF sequences are capable of promoting mucin secretion. On the other hand, it is described for the first time that the peptides SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4 and SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 are capable of promoting the secretion of mucins, thus promoting a protective activity at the intestinal level.

Example 2. Effect of different milk protein hydrolysates derived from whey proteins and caseins on the expression of the MUC5AC gene of HT29-MTX cells.

In order to assess whether whey protein and casein hydrolysates affected the expression of the MUC5AC gene, performed stimulation tests of HT29-MTX cells after 2, 4, 8 and 24 hours of incubation with hydrolyzate concentrations of 0.1% and 1%. It was observed that whey protein hydrolyzate resulted in an increase in copies of MUC5AC between 4 and 8 hours. Baseline expression when the concentration of 1% was added at 4 hours was 1.53 times the baseline value (Figure 2A). Casein hydrolyzate resulted in an increase in copies of MUC5AC between 4 and 8 hours. The baseline expression when the 1% concentration was added at 4 hours was 1.86 times the baseline value (P <0.5) (Figure 2B).

These results indicate that both whey protein hydrolysates and casein hydrolysates tested produce overexpression of the MUC5AC gene in human goblet secreting cells.

Example 3. Effect of different milk peptides derived from whey proteins and caseins on the expression of the MUC5AC gene of HT29-MTX cells.

Concentrations between 0.05 and 0.5 mM of the peptides of SEQ ID No: 2, SEQ ID No: 6 and SEQ ID No: 8 were added to the cell incubation medium for times between 2 and 24 hours.

Figure 3 shows the results of quantitative PCR at concentrations of 0.05 mM, 0.1 mM and 0.5 mM. Treatment of cells with SEQ ID No: 2 caused a significant increase

(P <0.05) of the number of copies of the MUC5AC gene at 4 hours of exposure to the concentration of 0.5 mM (2.1 times the baseline expression) (Fig. 3A). Treatment of cells with SEQ ID No: 6 resulted in an increase in the number of mRNA copies at 4 hours

(between 1.5 and 1.7 times the baseline expression), although due to the variability found, this increase was not statistically significant (Fig. 3B). Treatment of the cells with SEQ ID No: 8 caused a significant increase (P <0.05) (1.9 times the baseline expression) of the number of copies of the MUC5AC gene at 4 hours of exposure to the concentration of 0 , 1 mM (Fig. 3C). The times in which overexpression is detected coincide with the time at which the cells treated with this peptide had shown a greater mucin secretion.

Example 4. Opioid activity of food peptides by guinea pig ileum assay.

Figure 4 shows the opioid agonist activity of peptides SEQ ID No: 9 and SEQ ID No: 10 determined in vitro by assay in the FL-PM preparation of guinea pig ileum. The peptides induced an inhibition of electrically induced contractions that ranged between 20 and 30% in the case of SEQ ID No: 9 (Fig 4A) and between 25 and 35% in the case of SEQ ID No: 10 (Fig. 4B), in the concentration 5.5 10 ^{~ 7} M. The effect of all the peptides was reversed with the administration of naloxone (10 ^{~ 6} M). In similar curves morphine resulted in a maximum inhibition of 75% while / 3-casomorphine 7 produced a maximum inhibition of 20%.

These results indicate that the sequences tested, whose opioid activity had not been previously described, produce an inhibition of the contraction in the FL-PM preparation of guinea pig ileum mediated by opioid receptors greater than that of β-casomorphine 7.

Claims

1. Use of at least one isolated bioactive peptide that: a) possesses mucin secretion stimulating activity and / or opioid activity; b) is present in an enzymatic hydrolyzate of at least one milk protein selected from a whey protein hydrolyzate or a milk casein hydrolyzate; c) its structure comprises a sequence with at least two aromatic amino acids, where the first aromatic amino acid is a tyrosine located in the first or second position with respect to the N-terminal end of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine located in third or fourth position with respect to the first tyrosine; and where said structure is selected from SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12; for the manufacture of a pharmaceutical composition or a functional ingredient to stimulate the formation of the intestinal mucosa.

2. Use according to claim 1, wherein the pharmaceutical composition or functional ingredient is to promote gastrointestinal protection.

3. Use according to one of claims 1 or 2, wherein said isolated peptide is present in a whey protein hydrolyzate, has mucin secretion stimulating activity and comprises a structure selected from the group consisting of: SEQ ID No: 1 , SEQ ID No: 2 and a combination of both.

4. Use according to one of claims 1 or 2, wherein said isolated peptide is present in a milk casein hydrolyzate, It has a mucin secretion stimulating activity and comprises a structure selected from the group consisting of: SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 and any combination thereof.

5. Use according to one of claims 1 or 2, wherein said isolated peptide is present in a hydrolyzate of the milk / 3-casein fraction, has mucin secretion stimulating activity and comprises a structure selected from the group consisting of in: SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 and a combination thereof.

6. Use according to one of claims 1 or 2, wherein said isolated peptide is present in a hydrolyzate of the _sl -casein fraction of milk, has mucin secretion stimulating activity and comprises a structure selected from the group consisting of : SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 and a combination thereof.

7. Use according to claim 6 wherein, when the peptide comprises a structure selected from the group consisting of: SEQ ID No:

8. SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 and a combination thereof; said peptide, in addition to mucin secretion stimulating activity, has opioid activity.

8. Isolated bioactive peptide that: a) has mucin secretion stimulating activity and / or opioid activity; b) is present in an enzymatic hydrolyzate of at least one milk protein selected from a whey protein hydrolyzate or a milk casein hydrolyzate; c) its structure comprises a sequence with at least two aromatic amino acids, where the first aromatic amino acid is a tyrosine located in first or second position with respect to the end N-terminal of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine located in third or fourth position with respect to the first tyrosine; and where said structure is selected from SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12; to stimulate the formation of the intestinal mucosa and / or promote gastrointestinal protection.

9. A method of treatment to stimulate the formation of the intestinal mucosa and / or promote gastrointestinal protection characterized in that it comprises administering to an individual a therapeutically effective amount of an isolated bioactive peptide that: a) possesses mucin secretion stimulating activity and / or opioid activity; b) is present in an enzymatic hydrolyzate of at least one milk protein selected from a whey protein hydrolyzate or a milk casein hydrolyzate; c) its structure comprises a sequence with at least two aromatic amino acids, where the first aromatic amino acid is a tyrosine located in the first or second position with respect to the N-terminal end of said sequence and the second aromatic amino acid is a phenylalanine or a tyrosine located in third or fourth position with respect to the first tyrosine; and where said structure is selected from SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: