ES2392906B1 - GALACTO-OLIGOSACCHARIDES DERIVED FROM MULTIFUNCTIONAL LACTULOSE WITH IMMUNOMODULATING AND PREBIOTIC ACTIVITY - Google Patents

Abstract

Multifunctional lactulose-derived galacto-oligosaccharides with immunomodulatory and prebiotic activity. # The present invention claims the multifunctional character of lactulose-derived galactooligosaccharides (GOS-Lactulose) using in vivo (Wistar rats) and in vitro models using intestinal cell lines of origin. human (Caco-2 and HT29-MTX) validated as a model of intestinal epithelium. The main advantages derived from the use of this product are multiple and of great added value: improved prebiotic properties that are additional to those of the commonly used commercial prebiotics. This product is obtained either by direct transgalactosylation of lactulose using a {be} -galactosidase from Aspergillus oryzae, or by transgalactosylation of lactose and subsequent chemical isomerization. The GOS-Lactulose object of the patent could be described as second generation prebiotics and, therefore, of high added value and superior to that of the conventional prebiotics already marketed as lactulose or GOS derived from lactose.

Description

The present invention is directed to the food sector, more specifically to the functional food sub-sector, and to the pharmaceutical industry sector.

STATE OF THE PREVIOUS TECHNIQUE

The physiological state of the large intestine, and in particular the colon, is of great importance for human health, due in large part to the metabolic activities of the microbiota that colonizes it. A large number of diseases (diarrhea, ulcerative colitis, inflammation, cancer, etc.) are related to alterations in the composition of the colon microbiota. Furthermore, it is well known that strains of the genera Bifidobacterium and Lactobacil / us can exert beneficial effects on these disorders by modulating the host's physiological, metabolic and immunological functions. Since the concept of "prebiotic" was introduced more than a decade ago, referring to oligosaccharides resistant to gastrointestinal digestion that beneficially affect the individual by selectively stimulating the growth and / or activity of beneficial bacterial species in the intestine [Gibson & Roberfroid, 1995], there is considerable interest in their use as food ingredients in response to the high demand in the market for healthy foods and food supplements. Therefore, research in this field has focused on the development and characterization of new prebiotics that meet the necessary requirements to be considered as such. These properties are based on being resistant to gastrointestinal digestion, the ability to be fermentable and selectively favor the growth and / or activity of beneficial bacterial species in the colon (mainly Bifidobacterium spp. And Lactobacil / us spp.) With the ultimate goal of improve gastrointestinal health. The main prebiotic ingredients commercially available on the market are based on the following carbohydrates: i) lactulose (lactose isomer) which is probably the first carbohydrate used as a bifidogenic factor for human consumption [Méndez & Olano, 1979]; ii) galactooligosaccharides (GOS) obtained after an enzymatic transgalactosylation process from lactose; iii) fructooligosaccharides (FOS) produced by the enzymatic action of fructosyltransferase on sucrose; and iv) oligofructose obtained from the partial hydrolysis of inulin. To evaluate the prebiotic activity of nondigestible carbohydrates, a series of aspects are considered, including: quantitative changes in the microbial population, formation of fermentation end products, as well as the fermentation speed. However, it is believed that a large part of the commercially available prebiotic carbohydrates so far rapidly ferment in the large intestine such that their action takes place mainly in the proximal areas [Rastall et al., 2000; McBain & Macfarlane, 2001; Tzortzis et al., 2005]. These results partly explain the current great interest in having a "second generation" of new prebiotic ingredients that have a series of properties such as:

i)

be active at low doses;

ii)

no side effects;

iii)

to have a speed fermentation slower so that they can

present a greater persistence in the large intestine and, especially, in the colon since it is this intestinal region that presents the highest incidence of digestive and oncological pathologies [Roberfroid, 2007]; iv) have great selectivity regarding the control of the intestinal microbiota (for example, being able to be metabolized by specific bifidobacteria); v) possessing additional biological activity exerting beneficial effects on specific physiological functions and / or reducing the risk of disease, for example through its effect on the displacement of pathogens and the regulation of the function of the immune system [Gibson et al., 2005 ].

Improving the properties of the commercialized prebiotics, so that they more selectively favor the growth and activity of beneficial microorganisms (Bifidobacterium spp. And Lactobacilus spp.) In the colon, would be very advantageous to reinforce their physiological function in the intestinal epithelium. and the immune system associated with the intestine and, thus, improve the host's defenses against pathogenic microorganisms. Along these lines, efforts are being made

important ones aimed at the identification of prebiotic oligosaccharides with added bioactivities, although for the moment the results obtained have had limited success [Ouwehand et al., 2005]. As an example, a mixture of commercial lactose-derived GOS was shown to have affinity for membrane receptors from commensal microorganisms such as Escherichia coli; Although this fact causes less interaction and adhesion with intestinal (Caco-2) and liver (HepG2) cells of human origin [Shoaf et al., 2006], it is also necessary to use high concentrations of the prebiotic, which makes it difficult to use it in the practice has physiologically relevant effects [Macfarlane et al., 2008]. Taking this background into account, new products have been developed based on mixtures of prebiotic ingredients with probiotic bacteria [Patents WO 2010081126, WO 2009071578] and / or compounds with immunological properties [Patent EP 2014181] with the aim of reinforcing the gastrointestinal health of the consumer . However, these products based on multiple ingredients usually present a high cost in the market and, in addition, in certain cases it is difficult to guarantee the survival of certain probiotic bacteria in food matrices, as well as after the digestion process. Therefore, the production of prebiotic carbohydrates with added beneficial functions would present a potential interest for the food and / or pharmaceutical industry. On the other hand, a series of new galactooligosaccharides obtained by transgalactosylation from lactulose have recently been synthesized using 13-galactosidases from Kluyveromyces Jactis (Martínez-Villaluenga et al., 2008), or Aspergillus acu / eatus (Cardelle-Cobas and col., 2008a), as well as from the isomerization with sodium aluminate of the galactooligosaccharides obtained from lactose with the 13-galactosidase of Kluyveromyces lactis (Cardelle-Cobas et al., 2008b). Subsequently, an in vitro study carried out with human faeces has shown the potential bifidogenic power of these new galactooligosaccharides (Cardelle-Cobas et al., 2009), although in this study the pattern of carbohydrates obtained after transgalactosylation was neither determined nor was the effect evaluated thereof on other bacterial groups present in the intestinal microbiota and which may also have an effect on the gastrointestinal health of the individual. However, to explore the

possible application of these new GOS as prebiotic carbohydrates, it is necessary to study the obtained oligosaccharide profile, its digestibility (closely related to its structural composition), as well as its ability to modulate the intestinal microbiota with in vivo tests and its effect on the biodiversity of groups bacterial beneficial for health. Likewise, and with the objective of satisfying the current demand for second-generation prebiotics, it would be convenient to evaluate possible added bioactivities not previously studied, such as their ability to interact with probiotic and / or immunomodulatory bacteria.

BRIEF DESCRIPTION OF THE INVENTION

The inventors have demonstrated the multifunctional character of lactulose-derived galactooligosaccharides (GOS-Lactulose) using in vivo (Wistar rats) and in vitro models using human intestinal cell lines (Caco-2 and HT29-MTX) validated as a model of intestinal epithelium.

The main advantages derived from the use of this product are multiple and of great added value:

1.-Very high resistance to gastrointestinal digestion in vivo, being much superior to commercial prebiotics (GOS-Lactose).

2.-Immunomodulatory capacity.

•

Increased gene expression of interleukins that are anti-inflammatory or involved in the regulation of inflammation such as IL-6 and IL-10.

•

reduction of the production of TNF-a and IL-1J3 factors (pro-inflammatory) by intestinal cells.

•

increased expression of nuclear transcription factor (NFKB) involved in various processes of intracellular signaling, growth and survival

mobile.

3.-Greater ability to interact with probiotic bacteria.

4.-Decreased interaction and possible survival of typical intestinal pathogenic bacteria.

5.-Prebiotic properties in the large intestine (cecum and colon) at low doses (1%, w / w) and after a 14-day intake period.

6.-Bifidogenic activity much superior to prebiotics known and already marketed as lactose-derived galacto-oligosaccharides (GOS-Lactose) evaluated under the same conditions of animal experimentation.

7.-Greater biodiversity in terms of specific bifidobacterial strains that metabolize GOS-Iactulose compared to commercial prebiotics (Lactulose and GOS-Lactose).

8.-Prebiotic effect without side effects (relative to the weight gain, the daily amount ingested of diet or the relative weights of organs such as the stomach, liver and spleen).

Until now, there was only in the literature data on the synthesis and potential in vitro bifidogenic character of GOS-Lactulose obtained with ~ galactosidases from Kluyveromyces lactis and Aspergillus aculeatus (Cardelle-Cobas et al., 2009). However, the present patent application provides a large number of data showing, on the one hand, a series of advantages of GOS-Lactulose, synthesized using a J3-galactosidase from Aspergillus oryzae, compared to prebiotics already marketed and, on the other hand part, a series of added bioactivities not described so far. These results guarantee that the GOS-Lactulose object of the patent could be described as second generation prebiotics and, therefore, of high added value and superior to that of the conventional prebiotics already marketed as lactulose or GOS derived from lactose.

Specifically, the resistance to digestion of GOS-Lactulose throughout the intestinal tract was very high, with no ileal digestibility (0%), while commercially available prebiotic carbohydrates (which had also previously been eliminated) of mono- and disaccharides) had a digestibility of around 42% under the same experimental conditions. Consequently, GOS-Lactulose fermentation in the large intestine of the animals studied caused a significant increase in the number of bifidobacteria in the cecum, as well as in the relative percentage of bifidobacteria with respect to total bacteria in the cecum and colon, but not after the intake of commercial prebiotics (GOS derived from lactose). Furthermore, it was observed that the fermentation of GOS-Lactulose favored a significant increase in population diversity of the group of bifidobacteria with respect to treatments with lactulose or GOS-Lactose; This increase has been described by the scientific community as promoting intestinal health. These positive data regarding prebiotics already commercialized cannot be deduced from the background published so far. Likewise, the greater biodiversity of bifidobacteria that metabolize GOS-Lactulose opens great expectations when it comes to designing broader spectrum prebiotics that counteracts the genetic variability that exists between individuals. The pre-biotic effect of GOS-Lactulose is complemented by a series of added bioactive functions that represent an additional benefit for consumer health. Thus, analyzes of gene expression in the intestine sections (ileum and colon) of these animals also show that the administration of GOS-Lactulose induces an increased expression of anti-inflammatory or implicated interleukin (IL) genes. in the regulation of inflammation such as IL-6 and IL-10. In vitro studies have also shown a selective interaction between GOS-Lactulose and different bacterial species of the genus Bifidobacterium (B. longum, B bifidum and B. animalis), a fact that may favor their activity in the intestine. However, the interaction with other bacteria with greater pathogenic potential, including Bacteroides spp. and Escherichia coli and typical intestinal pathogens (Listeria monocytogenes and Sa / monel / a enterica) are lower. Furthermore, GOS-Lactulose works by directly inhibiting the inflammatory response in intestinal cells caused by the pathogen L. monocytogenes, for example by reducing the production of TNF-a by intestinal cells, which would favor the restoration of homeostasis in the Intestinal mucosa. Therefore, GOS-Lactulose have characteristics that give it a unique added value, since its prebiotic effect is increased compared to others

5 commercial products and exerts additional beneficial physiological effects by regulating the synthesis and expression of markers of immune function that can improve host defenses and reduce chronic inflammation processes, as well as reduce the interaction and possible survival of pathogenic bacteria. On the other hand, indicate that no antecedents have been found in the

1O few studies exist on GOS derived from lactulose for these additional beneficial effects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of lactulose-derived galacto-oligosaccharides (GOS-Lactulose) with improved and additional prebiotic properties than commonly used commercial prebiotics. This product is obtained either by direct transgalactosylation of lactulose using a 13-galactosidase from Aspergil / us oryzae, or

20 by transgalactosylation of lactose and subsequent chemical isomerization. After obtaining the product free of digestible carbohydrates after treatment with activated carbon, its prebiotic effect and bioactivity in physiological processes have been studied. GOS-Lactulose are composed of a complex mixture or combination

25 of polymerization degree oligosaccharides ~ 2. Specifically, and based on the identification made by ESI-MS (Figure 1), the main oligosaccharides were tri- and disaccharides, followed by tetra- and pentasaccharides, respectively. In this way, the GC-MS identification and characterization of the majority fractions, di- and trisaccharides, were carried out, which were

30 are characterized by the presence of galactobioses and galactosyl glucoses, as well as galactosyl-fructose disaccharides with 1-1, 1-3, 1-5 bonds and trisaccharides such as 6'-galactosyl-lactulose, as shown in Table 1 Although the chemical structure of two trisaccharides from GOS-Lactulose obtained by other enzymes had previously been studied, an exhaustive characterization of the composition of these oligosaccharides had not been carried out, which will influence their digestibility and activity.

1.-In vivo evaluation of digestibility. Growing male Wistar rats divided into four groups were used in the in vivo studies, respectively supplied i) the control diet, ii) the control diet + 1% GOS-Lactulose + 0.2% Cr20 3 (non-digestible marker), iii) the control diet + 1% commercial prebiotic carbohydrates (GOS-Lactose) + 0.2% Cr20 3, iv) the control diet + 1% Lactulose + 0.2% Cr203 for a period of 14 days with a daily intake of 14 grams. After sacrifice, the ileum, cecum, and colon were dissected and washed with sterile distilled water to collect the intestinal contents of each section. GOS derived from lactulose did not affect rat growth, intake, relative efficiency, or the relative weight of organs such as the stomach, liver, or spleen. The digestive survival of GOS-Lactulose in the terminal ileum was evaluated by GC-MS by identifying and quantifying the oligosaccharides and the content of Cr20 3 present in the diets, in the ileum and in faecal samples. As can be seen in Figures 2 and 3, each and every one of the carbohydrates that make up the product object of the patent reached the terminal ileum and was not present in the faeces of the animals. These results indicate the high digestive survival of GOS-Lactulose, as well as its complete fermentation in the large intestine, essential characteristics of prebiotic compounds. The apparent ileal digestibility of the GOS-Lactulose was null, while it is worth noting the high ileal digestibility values (-42%) obtained by the commercially available prebiotics (GOS derived from lactose). To confirm the considerable degree of digestibility of commercial prebiotics, Figure 4 shows the disappearance of a significant number of trisaccharides (those chromatographic peaks marked with an asterisk) in the ileal content of the experimental animals after ingestion. As additional proof of the digestibility of GOS-Lactose, Figure 5 shows the appearance of some disaccharides in the ileum sample that are a direct consequence of the digestibility of the higher molecular weight GOS-Lactose since, as previously commented, GOS-Lactose does not originally contain mono- or disaccharides when eliminated by

size exclusion chromatography before they were incorporated into the supplied diet.

2.-In vivo evaluation of prebiotic activity.

Growing male Wistar rats divided into four groups were used in the in vivo prebiotic activity studies under the conditions indicated in the previous section. The study of the effect of GOS-Lactulose on the modulation of intestinal bacterial populations (total bacteria, Bacteroides sp., Lactobacil / us spp., Bifidobacterium spp., Clostridium group

coccoides / Eubacterium rectum / e and group of C / ostridium leptum) was performed using quantitative PCR (qPCR) in different sections of the intestine (ileum, cecum and colon). As Table 2 illustrates, a significant bifidogenic effect was found in the cecum of the group of animals that ingested GOS-Lactulose compared to the control, while no increase in bifidobacteria was observed in the group that consumed commercial lactose-derived GOS. On the other hand, a significant increase in the percentage of bifidobacteria with respect to total bacteria in the cecum and colon of the group of animals that consumed GOS-Lactulose could also be observed (Table 3). Furthermore, it is worth noting a significant increase in beneficial bacteria corresponding to the Clostridium coccoides / Eubacterium rectum / e group, with relevance in gastrointestinal health. It is worth highlighting the studies carried out regarding the effect of GOS-Lactulose on the intestinal biodiversity of the group of bifidobacteria, comparing them with that obtained after ingestion of lactulose and GOS-Lactose. As Figure 6 shows, a significantly higher number of species corresponding to the group of bifidobacteria responded to GOS-Lactulose fermentation, compared to conventional prebiotics such as Lactulose and GOS-Lactose. As a result, increased enrichment (number of electrophoretic bands) was observed in bifidobacteria present in the cecum of rats treated with GOS-

Lactulose compared to those treated with lactulose, GOS-Lactose and control diet (Table 4). The Shannon and Evenness population diversity indices showed higher values in the case of cecal samples from rats fed with GOS-Lactulose. In addition, similarity studies showed that the cecum bifidobacteria population in rats fed GOS-Lactulose are very different from those fed

lactulose, GOS-Lactose and control diet (Figure 7). Taken together, these results clearly show the power higher carbohydrate prebiotic subject of the patent (GOS-Lactulose) versus commercially available prebiotics (GOS-Lactose) under them

5 conditions of experimentation. Therefore, GOS-Lactulose could exert a prebiotic effect in a greater number of individuals and at lower doses than those used for already commercial prebiotics.

3.-In vitro study of the interaction with intestinal bacteria.

1 O 3. 1. Molecular recognition by membrane receptors in probiotic, commensal and intestinal pathogens. Different probiotic bacteria (B. animalis subsp lactis Bb12 and Lactobacillus rhamnosus GG ATCC 53103), bifidobacteria (Bifidobacterium longum CECT 7347, Bifidobacterium animalis subsp. Animalis,

15 IATA-A2, Bifidobacterium bifidum CECT 7365) and commensals (Bacteroides fragilis [IATA-SAE2, -CAQ2, -CBS1, -CAR1] and Escherichia coli [IATA-CBL2, -CBM1, -CBD1 O, -CCD2]) isolated from healthy individuals, as well as intestinal pathogens (Listeria monocytogenes CECT 935 and Salmonella enterica CECT 443). The interaction of GOS-Lactulose with intestinal bacteria was evaluated

20 by means of competitive inhibition assays of the microbial recognition sites of a-and J3-N-acetylgalactosamine and galactopyranosyl residues present in mucins and glycosaminoglycans of the extracellular membrane of intestinal cells. The degree of interaction was estimated as a reduction in the percentage of fluorescence detected in mucin-coated wells (Type 11).

25 with respect to the total fluorescence added in each well, which is equivalent to the bacteria adhered to the mucin layer with respect to the total added. The results obtained (Figure 8) show the high degree of molecular recognition of GOS-Lactulose by probiotics and bifidobacteria, a fact that supports and explains the prebiotic effect of GOS-Lactulose observed

30 in in vivo studies, while the absence of effect on the adhesion of commensal bacteria and intestinal pathogens suggest the non-use of GOS-Lactulose by these microorganisms.

3. 2. In vitro effect on the intestinal inflammatory response.

The invasive intestinal pathogen Listeria monocytogenes CECT 935 was used to assess the effect of GOS-Lactulose on the production of cytokines such as tumor necrosis factor (TNF) -ae interleukin (IL) -113 -in human cell cultures of intestinal origin (Caco-2 line) validated as models of intestinal epithelium. Figure 9 shows the inhibition (p = 0.001) of TNFa and IL-113 production by GOS-Lactulose. These results indicate that GOS-Lactulose reduces the inflammatory effect of L. monocytogenes CECT 935 by reducing the synthesis of pro-inflammatory cytokines that contribute to increasing intestinal permeability.

4.-In vivo study of immunomodulatory activity.

In the studies, growing male Wistar rats divided into four treatment groups were used as indicated in section 1. After the end of the treatment period, the animals were anesthetized (40 mg sodium pentobarbital 1 kg body weight) to carry out the obtaining blood by intracardiac puncture, which was rapidly frozen in liquid nitrogen, and dissection of intestinal tissue. In samples of small intestine sections, reverse expression transcription and real-time PCR were used to monitor the expression (mRNA) of markers of homeostatic control of inflammation and immune response. The biomarkers analyzed included nuclear transcription factor (NFKB), pleiotropic interleukins (IL) such as IL-6, IL-1 O, and IL-15, and proinflammatory drugs such as TNFa. The results obtained (Figure 1 O) show that the administration of GOS-Lactulose induces the expression of IL-6 and IL-10, which may favor the regulation of inflammatory processes at the intestinal level. The inclusion of GOS-Lactulose in the diet of the animals caused an increased expression of NFKB, involved in various processes of intracellular signaling, growth and cell survival. Quantification (ELISA) of TNFa in peripheral blood samples confirmed that the inclusion of GOS-Lactulose in the diet of the animals does not alter the baseline parameters of this inflammation activation marker with respect to the control group.

The data provided shows that the compounds object of this application (GOS-Lactulose) present an added value to their prebiotic effect (greater persistence in the intestinal tract and stimulating effect of the growth of bifidobacteria, as well as a greater biodiversity) with an additional effect by modulate immune function. All this makes this product an attractive alternative to be used as second-generation prebiotics in a

5 wide number of commercial products such as baby food, dietary supplements, pet food, etc. Applications could also be presented in the field of the pharmaceutical and / or nutraceutical industry

Therefore, the present invention refers to a combination of galacto-

1O bioactive oligosaccharides derived from lactulose or GOS-Lactulose (glycosyl-fructose) for use as multifunctional food ingredients, preferably with prebiotic and immunomodulatory activity.

In a preferred embodiment of the invention, said combination of galacto-

Bioactive oligosaccharides is obtainable through a process that comprises carrying out a transgalactosylation reaction of lactulose preferably catalyzed by f3-galactosidase, and more preferably said enzyme comes from Aspergil / us oryzae.

In another embodiment of the invention, said combination of bioactive galacto-oligosaccharides is obtainable through a method that comprises carrying out a lactose transgalactosylation reaction catalyzed preferably by f3-galactosidase, and more preferably said enzyme comes from Aspergillus oryzae. A more preferred embodiment subsequently adds an isomerization

25 chemistry of the lactose derivative obtained.

A subject of the present invention is also the above-referenced bioactive galacto-oligosaccharide combination characterized in that said immunomodulatory activity preferably occurs with increasing

30 population of bifidobacteria and / or induce the expression of biomarkers involved in homeostatic control of inflammation and / or activation of the immune system.

In a preferred embodiment of the invention, said combination of bioactive galacto-oligosaccharides is characterized in that said prebiotic activity preferably occurs in the large intestine of a subject after ingestion of a dose of said combination. In an even more preferred embodiment, said intake is at least 1% w / w, and more preferably over a period of at least 14 days.

In another preferred embodiment of the invention, said combination of bioactive galacto-oligosaccharides is characterized in that they have digestive survival of up to 100% in the large intestine of a subject after ingestion of said combination.

More preferably, the combination of bioactive galacto-oligosaccharides is characterized in that after ingestion of said combination, the fermentation, preferably complete, of the subject also occurs in the large intestine of the subject.

Even more preferably, the combination of bioactive galacto-oligosaccharides is characterized in that after the ingestion of said combination, a prebiotic effect also occurs, increasing the concentration of bifidobacteria in the large intestine of the subject with respect to said concentration of bifidobacteria before ingestion. .

In another even more preferred embodiment, the combination of bioactive galacto-oligosaccharides is characterized in that the prebiotic effect also leads to an increase in the bifidobacteria biodiversity of the subject's large intestine with respect to said bifidobacteria biodiversity before ingestion.

A last preferred embodiment of the invention refers to the previously described bioactive galacto-oligosaccharide combination characterized in that it can also produce an inhibitory effect on the colonization of pathogenic bacteria of the subject's intestine.

In the present invention, the term "bioactive galacto oligosaccharides" or "bioactive galacto oligosaccharide mixture" refers to lactulose-derived galacto oligosaccharides (GOS-Lactulose), specifically glycosyl-fructose and derivatives, with improved and additional properties to those of commonly used commercial prebiotics.

In the present invention the term "Food ingredient" refers to one of the components or the main constituent of any mixture or combination that constitutes a commercial food.

10 In the present invention, the term "carbohydrates with immunomodulatory activity" refers to carbohydrates with the ability to interact and / or regulate the activation of the immune system, with a potential favorable effect on host defenses and the control of chronic inflammation processes. .

In the present invention the term "Carbohydrates with prebiotic activity" refers to oligosaccharides resistant to gastrointestinal digestion that selectively stimulate the growth and / or activity of bacterial species of the intestinal tract, considered beneficial that can exert beneficial effects on the health of the host .

20 When the present invention talks about "Inducing the expression of biomarkers involved in the control of inflammation and the immune response", he is referring to the positive effect of oligosaccharides on the increase of RNA (messenger) of regulatory molecules of the processes of

25 inflammation and activation of the immune response.

In the present invention the term "Subject" refers to any animal, preferably a mammal, more preferably it refers to Wistar rats used as an experimental animal model.

In the present invention the term "digestive survival" refers to the property of carbohydrates to be resistant to the acid conditions and hydrolytic activities present in the gastrointestinal tract, being able to reach the large intestine in an active way and in physiologically significant amounts.

In the present invention the term "complete fermentation" refers to the complete metabolism of the carbohydrates subject to the patent by the intestinal microbiota.

In the present invention the term "bifidogenic effect" refers to compounds with the ability to selectively stimulate the growth of bifidobacteria (beneficial bacterial species in the colon).

In the present invention the term "Biodiversity of bifidobacteria of the large intestine of the subject" refers to the variability of the mentioned bacterial group, this being a direct consequence of the metabolism of prebiotic compounds. Therefore, it refers to the largest variety of identified Bifidobacterium species (mainly Bifidobacterium animalis and Bifidobacterium pseudo-longum).

In the present invention the term "Positive interaction on the growth and activity of prebiotic bacteria" refers to the selective stimulation of the metabolism and growth of bacteria considered beneficial (preferably Bifidobacterium spp. And Lactobacillum spp.) By prebiotic carbohydrates.

In the present invention the term "Negative interaction on the growth, activity and survival of pathogenic bacteria" refers to the inhibitory effect of prebiotic carbohydrates on the metabolic activity, growth and intestinal colonization of intestinal pathogens.

DESCRIPTION OF THE FIGURES

Figure 1 shows the spectrum obtained after ESI-MS analysis of the oligosaccharides that make up the GOS-Lactulose.

Figure 2 shows the chromatograms obtained after GC-MS analysis of the disaccharide content: A) of GOS-Lactulose in the terminal ileum of the group of rats that consumed this product, B) of GOS-Lactulose in the diet supplied, and C ) of GOS-Lactulose in the faeces of the group of rats that

5 consumed this product.

Figure 3 shows the chromatograms obtained after GC-MS analysis of the trisaccharide content: A) of GOS-Lactulose in the terminal ileum of the group of rats that consumed this product, B) of GOS-Lactulose in the diet

1st supplied, and C) of GOS-Lactulose in the faeces of the group of rats that consumed this product.

Figure 4 shows the chromatograms obtained after GC-MS analysis of the trisaccharide content: A) of GOS-Lactose (commercial prebiotics) in the

15 terminal ileum of the group of rats that consumed this product, B) of GOS-Lactose (commercial prebiotics) in the diet supplied, and C) of GOS-Lactose (commercial prebiotics) in the feces of the group of rats that consumed this product. The peaks marked with asterisks correspond to the trisaccharides that have been digested after ingestion.

Figure 5 shows the chromatograms obtained after GC-MS analysis of the disaccharide content obtained after the digestion of the trisaccharide fraction: A) of GOS-Lactose (commercial prebiotics) in the terminal ileum of the group of rats that consumed this product. , and C) of GOS-Lactose (prebiotics

25 commercial) in the faeces of the group of rats that consumed this product.

Figure 6 shows a denaturing gradient electrophoresis gel (DGGE) of bifidobacteria isolated from cecum samples from groups of rats consuming GOS-Lactulose, Lactulose and GOS-Lactose,

30 respectively.

Figure 7 shows the bifidobacteria similarity dendogram obtained after DGGE analysis of samples from cecum of rats treated with a control diet, lactulose, GOS-Lactose or GOS-Lactulose after 14 days of treatment. In the upper part of the figure the scale of

Intensity between samples.

Figure 8 shows the degree of interaction of GOS-Lactulose with different

5 probiotic bacteria (Lactobacillus rhamnosus GG ATCC 53103 and Bifidobacterium animalis subsp lactis Bb12), bifidobacteria (Bifidobacterium / ongum CECT 7347) and commensals (different strains of Bacteroides and cochis / Eschis) / healthy as well as intestinal pathogens -Listeria monocytogenes CECT 935 and Sa / monella

1O enterica CECT 443-obtained from the Spanish Type Culture Collection (CECT). The results are shown as the interval of variation of the measurements and mean value (n = 4).

Figure 9 shows the effect of GOS-Lactulose on the production of

15 tumor necrosis-a and interleukin-1 J3, both with proinflammatory activity, triggered by the invasive intestinal pathogen Listeria monocytogenes (CECT 935) in human cell cultures of intestinal origin (Caco-2 line). The results are shown as the interval of variation of the measurements and mean value.

Figure 10 shows the effect of GOS-Lactulose on the expression (mRNA) of immunological markers in ileum sections of experimental animals (Wistar rats). The results are shown as relative expression with respect to J3-actin.

Table 1 shows the GC-MS identification of the GOS-Lactulose disaccharide and trisaccharide fraction.

Table 2 shows the effect of the control or 1% supplemented diet

30 of GOS-Lactulose, Lactulose or GOS-Lactose (commercial prebiotics) after ingestion over a period of 14 days on the different bacterial populations in the different intestinal sections studied (ileum, cecum and colon).

Table 3 shows the effect of the control diet or that supplemented with 1% GOS-Lactulose, Lactulose or GOS-Lactose (commercial prebiotics) after ingestion over a period of 14 days in the relative percentage of bifidobacteria with respect to total bacteria in the different intestinal sections studied

5 (ileum, cecum and colon).

Table 4 shows the degree of enrichment (defined as the number of electrophoretic bands corresponding to the genus Bifidobacterium after DGGE analysis), as well as the Shannon and Evenness indices (relative to diversity

1st population) of samples from the cecum of rats treated with a control diet, lactulose, GOS-Lactose or GOS-Lactulose after 14 days of treatment.

BIBLIOGRAPHY

Cardelle-Cobas, A., Martínez-Villaluenga, C., Villamiel, M., Olano, A., Corzo, N. (2008a). Synthesis of oligosaccharides derived from lactulose and Pectinex Ultra SP-L. Journal of Agriculture / and Food Chemistry 56, 3328-3333. Cardelle-Cobas, A., Corzo, N., Villamiel, M., Olano, A. (2008b). lsomerization of lactose-derived oligosaccharides: A case study using sodium aluminate. Journal of Agriculture / and Food Chemistry 56, 10954-10959. Cardelle-Cobas, A., Fernández, M., Salazar, N., Martínez-Villaluenga, C., Villamiel, M., Ruas-Madiedo, P., de los Reyes-Gavilán, C. G. (2009). Bifidogenic effect and stimulation of short chain fatty acid production in human faecal slurry cultures by oligosaccharides derived from lactose and lactulose. Journal of Dairy Research 76, 317-325. Gibson, G. R., Roberfroid, M. B. (1995). Dietary modulation of the human colonic microbiota -introducing the concept of prebiotics. Journal of Nutrition 125, 1401-1412. Gibson, G. R., McCartney, A. L., Rastall, R. A. (2005). Prebiotics and resistance to gastrointestinal infections. British Journal of Nutrition 93, S31-S34. Macfarlane, G. T., Steed, H., Macfarlane, S. (2008). Bacterium! metabolism and health-related effects of galacto-oligosaccharides and other prebiotics. Journal of Applied Microbe / Ogy 104, 305-344.

-Martínez-Villaluenga, C., Cardelle-Cobas, A., Olano, A., Corzo, N., Villamiel, M., Jimeno, M. L. (2008). Enzymatic synthesis and identification of two trisaccharides produced from lactulose by transgalactosylation. Journal of Agriculture / and Food Chemistry 56, 557-563. McBain, A. J., Macfarlane, G. T. (2001). Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacteria! ecosystems. Journal of Medica / Microbiology 50, 833-842. Méndez, A., Olano, A. (1979). Lactulose: A review on sorne chemical properties and applications in infant nutrition and medicine. Dairy Science Abstract 41, 531-535.

Ouwehand, A. C., Derrien, M., de Vos, W., Tiihonen, K., Rautonen, N. (2005). Prebiotics and other microbial substrates for gut functionality. Current Opinion in Biotechnology 16, 212-217. Rastall, R. A., Gibson, G. R., Fuller, R., Gaskins, H. R. (2000). Colonic functional foods. In Functional foods -Concept to product. pp. 71-93. Edited by Glenn R Gibson and Christine M Williams. Woodhead Publishing You (Cambridge, United Kingdom). Roberfroid, M. (2007). Prebiotics: the concept revisited. Joumal of Nutrition 137, 8308-8378.

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8hoaf, K., Mulvey, G. L., Armstrong, G. D., Hutkins, R. W. (2006). Prebiotic galactooligosaccharides reduces adherence of enteropathogenic Escherichia coli to tissue culture cells. lnfection and lmmunity 74, 6920-6928. Tzortzis, G., Goulas, A. K., Gee, J. M., Gibson, G. R. (2005). A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo. Joumal of Nutrition 135, 1726-1731. WO 2010081126. Compositions comprising probiotic and prebiotic components and mineral salts, with lactoferrin. Inventors: Longoni, V., Penci, M.

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Patent WO 2009071578. Composition based on probiotic bacteria in association with a prebiotic and use thereof in the prevention and / or treatment of respiratory pathologies and / or infections and in the improvement of intestinal functionality. Inventors: Mogna, G., 8trozzi, G. P., Mogna, L.

-Patente EP 2014181. Compositions based on prebiotic and immunogenic ingredients for the prevention and treatment of gastroenteric disorders caused by dysbiosis and / or alterations of the normal intestinal flora. Inventor: DI Pierro, F.

EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and are not to be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting the field of application thereof.

Example 1.

1.-Obtaining GOS-Lactulose by enzymatic transgalactosylation of lactulose and subsequent treatment with activated carbon.

Prepare a 450 g / L solution of lactulose in 50 mM sodium phosphate buffer and 1 mM magnesium chloride at pH 4.5, add 8 U / mL of ~ -galactosidase from Aspergil / us oryzae and incubate at 60 ° C for 8h with 300 rpm orbital shaking At the end of the reaction time, the enzyme is inactivated, keeping the sample at 1 00 ° C for 5 min. This mixture of sugars derived from lactulose is purified to obtain fractions enriched in oligosaccharides with prebiotic properties. For this, the sample is diluted with water to a concentration of sugars of 50 g / L, activated carbon is added at a concentration of 75 g / L and it is kept under stirring for 30 min, the mixture is filtered and washed with 500 mL of water, in this way practically all monosaccharides (97%) and a large part of disaccharides (65%) are eliminated. The oligosaccharides adsorbed on the activated carbon are released with 250 mL of a 50% ethanol-water mixture, keeping it stirred for 30 min and filtering it. Subsequently, the alcoholic solution with the sugars is evaporated or lyophilized, obtaining the product as a concentrated liquid (syrup) or powder. The final product has a high percentage of oligosaccharides (with a degree of polymerization ~ 3), this being around 70%. Activated carbon can be reused for further purifications.

2.-Study of the in vivo prebiotic activity of GOS-Lactulose.

2. 1. Experimental design. Growing male Wistar rats (approximate initial weight of 40-45 gr) are used, kept in metabolic cages under controlled conditions of temperature (25 ° C), humidity (50%) and light (12-hour cycles). Animals are pre-adapted to the experimental conditions with a control diet (AIN-93G, Testdiet) for a period of 6 days prior to the start of the experimental phase. Next, four groups of rats are established (n = 1 O per group) to which they are supplied respectively i) the control diet, ii) the control diet + 1% GOS-Lactulose + 0.2% Cr20 3 (as a non-digestible marker ), iii) the control diet + 1% commercial lactose-derived GOS (GOS-Iactose) + 0.2% Cr203, iv) the control diet + 1% lactulose + 0.2% Cr203 for a period of 14 days, the daily intake being 14 grams . At the end of the experimentation period, the animals are fed 14 hours, they are fed 4 grams of the corresponding diet and they are sacrificed under general anesthesia (40 mg sodium pentobarbital 1 kg body weight). Finally, the ileum, cecum, and colon are dissected and washed with sterile distilled water to collect the intestinal contents of each section, which are frozen, lyophilized, and stored at -80 o C for further analysis.

2.2. Quantitative determination and evaluation of in vivo digestive survival of prebiotic carbohydrates in the terminal ileum. The identification and quantification of oligosaccharides present in the diets, in the ileum and in faecal samples is carried out by GC-MS after its derivatization to trimethylsilyl eximas. The apparent ileal digestibility (%) of the different oligosaccharides used in this invention is calculated according to the expression: [(Pf / Cr20 3f) - (Pi / Cr203i)] / (Pf / Cr203f), where Pf and Pi represent the amount of oligosaccharide (mg per 100 mg sample) in the diet and ileal samples, respectively, while Cr203f and Cr203i represent the indigestible marker concentrations (mg per 100 mg) in the diet and in the ileal content.

2. 3. Effect of prebiotic carbohydrates on the modulation of intestinal bacterial populations. Total DNA extraction is carried out from lyophilized samples of intestinal content (ileum, cecum and colon) using QIAamp DNA stool kit (Quiagen, West Sussex, UK). Eluted DNA is treated with RNase and

Analyzed using a NanoDrop spectrophotometer. The purified DNA is stored at -20 ° C until use. The different bacterial groups under study, which included total bacteria, Bacteroides spp., Lactobacillus spp., Bifidobacterium spp., Group C / ostridium coccoides / Eubacterium rectum / e, group Clostridium leptum and enterobacteria are analyzed in samples of intestinal content by PCR quantitative (qPCR). For this, specific primers of the bacterial group based on specific sequences of the 16S rRNA gene are used. QPCR assays are carried out in multiwell plates using an iQ5 Cycler Multicolor detection system. The bacterial concentration of each sample is expressed as log1 0 of the copy number by interpolating the Ct values of the intestinal samples in the standard calibration curves obtained for each bacterial group. The data obtained is statistically analyzed (multivariate analysis) in order to determine the prebiotic potential of the different oligosaccharides. The population diversity of the genus Bifidobacterium is analyzed by DGEE, using specific primers (Bif164-f and Bif662-GC-r). This technique provides a pattern or profile of genetic diversity, and can be applied to studies of population structure and dynamics in digestive ecosystems. The results obtained show that each treatment causes a stable and repeatable band pattern of the genus Bifidobacterium. Dendograms are obtained from DGGE using the Dice similarity index and the UPGMA algorithm (Unweighted Pair Group Method using Arithmetic averages). In addition, diversity indices (enrichment, Shannon and Evenness) derived from the DGGE band profile are analyzed based on the experimental diets or the intestinal section sampled in rats.

3.-In vitro study of the interaction with intestinal bacteria.

3. 1 Molecular recognition by membrane receptors in probiotic microorganisms. diners and intestinal pathogens. The studies have been carried out with probiotic bacteria (8. animalis subsp. Lactis Bb12 and Lactobacillus rhamnosus GG ATCC 53103), commensals (Bifidobacterium longum CECT 7347, Bifidobacterium animalis subsp. Animalis IATA-A2, Bifidobacterium bifidum CECT 765, is [IATA-SAE2, -CAQ2, -CBS1, -CAR1] and Escherichia coli [IATA-CBL2, -CBM1, -CBD10, -CCD2])

obtained from healthy individuals and intestinal pathogens (Salmonella enterica CECT 443 and Listeria monocytogenes CECT 935) from collection. The microorganisms, grown in specific culture media for 20 h, were isolated by centrifugation (1,500 x g 15 min) and washed (x3) with a phosphate buffer solution (PBS) (pH 7). The cells were incubated (37 ° C / 30 min) with a fluorescein solution (75 J. µM) and washed with PBS. Subsequently, the cells were resuspended at a concentration of approximately 1 colony forming unit (cfu) / ml (optical density 0.5 ~ .600 nm) in PBS. The bacterial suspensions were mixed with the GOS-Lactulose (0.3% w / v), described in previous sections of the present application, and incubated (37 ° C / 1 h) in 96-well plates with immobilized mucin (Type 11) . Immunization of mucin was carried out by incubating (4 ° C / 24h) the plates with a solution (0.5 mg / mL) of mucin (Type 11) in PBS. The degree of molecular recognition and interaction of GOS-Lactulose with intestinal bacteria was evaluated according to the criteria detailed in the description of the invention.

3. 2 In vitro effect on the intestinal inflammatory response.

The effect of GOS-Lactulose on the production of pro-inflammatory biomarkers was evaluated in cultures of intestinal cells of human origin (Caco-2) widely accepted as a model of intestinal epithelium by presenting the characteristics of mature human enterocytes. In the experiments, the Caco-2 cells were seeded at an initial density of 5 x 1 04 cells / cm2 and grown for 5 days, with changes of culture medium (Dulbecco's Modified Eagle Medium) every 2 days. The studies used 20h cultures of the collectable invasive intestinal pathogen - Listeria monocytogenes CECT 935 - grown in Brain-Heart broth. The bacteria were collected by centrifugation and washed with a phosphate buffer solution (PBS) (pH 7). Bacterial suspensions were prepared, in duplicate, equivalent to 1 os cfu / mL and only one of them was mixed with the GOS-Lactulose (0.3% w / v). The mixtures were incubated (37 ° C / 95% C02 / 3h) with the cell culture and the supernatant was subsequently obtained (9300 x g / 10 min).

The inflammatory response in intestinal cells was monitored by quantifying the concentration of tumor necrosis factor (TNFa) and interleukin (IL) -1p. Quantification of TNFa and IL-1 p was carried out in aliquots (1 00 ~ L) of the supernatant using ELISA techniques (eBiosciences, sensitivity 4 pg / mL) according to the manufacturer's instructions.

4.-In vivo study of immunomodulatory activity.

The study was carried out on growing male (Wistar) rats that were randomly distributed into different groups (n = 15) administered: i) control diet, ii) control diet + 1% GOS-Lactulose + 0.2% Cr203 (non-digestible marker), and iii) the control diet + 1% commercial prebiotic carbohydrates + 0.2% Cr203, over a period of 14 days. After the treatment period, the animals were sacrificed before anesthesia (40 mg sodium pentobarbital 1 kg body weight) and the intestine was dissected, the sections of which were collected in a cryoprotective medium for the RNA and were immediately frozen in liquid nitrogen. In the homogenized samples (Tissue lyser, Qiagen) the total RNA content was extracted using a commercial kit (RNA mini kit, Qiagen) for this purpose, according to the manufacturer's instructions. From aliquots (1 µg) of RNA, cONA was obtained by reverse transcriptase reaction (AMV Reverse Transcriptase, Promega). Subsequently, changes in the expression (mRNA) of biomarkers of homeostatic control of inflammation and immune response were monitored with real-time PCR techniques. Monitored biomarkers included pleiotropic interleukins (IL) (IL-6, -10, and -15), nuclear transcription factor kappa B -NFKB, and tumor necrosis alpha (TNFa), for which primer sequences were designed (primer) using the GenBank databases (NCBI).

Example 2.

1.-Obtaining GOS-Lactulose by enzymatic transgalactosylation of lactose and subsequent isomerization and treatment with activated carbon.

Aspergillus oryzae and incubated at 60 ° C for 3h with orbital shaking at 300

r.p.m. After the reaction time, the enzyme is inactivated by keeping the sample at 1 00 ° C for 5 min. This mixture of sugars derived from lactose is isomerized with sodium aluminate under the following conditions: the mixture is diluted with water to a concentration of 100 g / L of sugars, sodium aluminate is added in a 1/1 ratio (w / w) and it is kept at 40 ° C under stirring for 12 h. The reaction is stopped by immersion in ice-cold water and subsequent neutralization with sulfuric acid and centrifuged at 9000g for 1 O min. This isomerized mixture is treated with activated carbon as described in Example 1.

2.-Study of the in vivo prebiotic activity of GOS-Lactulose.

2. 1. Experimental design. Growing male Wistar rats (approximate initial weight of 40-45 gr) are used, kept in metabolic cages under controlled conditions of temperature (25 ° C), humidity (50%) and light (12-hour cycles). Animals are pre-adapted to the experimental conditions with a control diet (AIN-93G, Testdiet) for a period of 6 days prior to the start of the experimental phase. Next, four groups of rats are established (n = 10 per group) who are respectively supplied with i) the control diet, ii) the control diet + 1% GOS-Lactulose + 0.2% Cr203 (as a non-digestible marker), iii) the control diet + 1% commercial lactose-derived GOS (GOS-Iactose) + 0.2% Cr20 3, iv) the control diet + 1% lactulose + 0.2% Cr203 for a period of 14 days, with a daily intake of 14 grams. At the end of the experimentation period, the animals are fed 14 hours, they are fed 4 grams of the corresponding diet and they are sacrificed under general anesthesia (40 mg sodium pentobarbital 1 kg body weight). Finally, the ileum, cecum, and colon are dissected and washed with sterile distilled water to collect the intestinal contents of each section, which are frozen, lyophilized, and stored at -80 o C for further analysis.

2.2. Quantitative determination and evaluation of in vivo digestive survival of prebiotic carbohydrates in the terminal ileum. The identification and quantification of oligosaccharides present in the diets, in the ileum and in faecal samples is carried out by GC-MS after their derivatization to trimethylsilyl oximes. The apparent ileal digestibility (%) of the different oligosaccharides used in this invention is calculated according to the expression: [(Pf / Cr20 3f) - (Pi / Cr203i)) / (Pf / Cr203f), where Pf and Pi represent the amount of oligosaccharide (mg per 100 mg sample) in the diet and ileal samples, respectively, while Cr203f and Cr203i represent the indigestible marker concentrations (mg per 100 mg) in the diet and in the ileal content.

2. 3. Effect of prebiotic carbohydrates on the modulation of intestinal bacterial populations. Total DNA extraction is carried out from lyophilized samples of intestinal content (ileum, cecum and colon) using QIAamp DNA stool kit (Quiagen, West Sussex, UK). Eluted DNA is treated with RNase and analyzed using a NanoDrop spectrophotometer. The purified DNA is stored at -20 ° C until use. The different bacterial groups under study, which included total bacteria, Bacteroides spp., Lactobacil / us spp., Bifidobacterium sp., Group Clostridium coccoides / Eubacterium rectum / e, group C / ostridium leptum and enterobacteria are analyzed in samples of intestinal content using quantitative PCR (qPCR). For this, specific primers of the bacterial group based on specific sequences of the 168 rRNA gene are used. QPCR assays are carried out in multiwell plates using an iQ5 Cycler Multicolor detection system. The bacterial concentration of each sample is expressed as log of the number of copies by interpolating the Ct values of the intestinal samples in the standard calibration curves obtained for each bacterial group. The data obtained is statistically analyzed (multivariate analysis) in order to determine the prebiotic potential of the different oligosaccharides. The population diversity of the genus Bifidobacterium is analyzed by DGEE, using specific primers (Bif164-f and Bif662-GC-r). This technique provides a pattern or profile of genetic diversity, and can be applied to studies of population structure and dynamics in digestive ecosystems. The results obtained show that each treatment causes a stable and repeatable band pattern from the Bifidobacteria group. Dendograms are obtained from DGGE using the Dice similarity index and the UPGMA algorithm (Unweighted Pair Group Method using Arithmetic averages). In addition, diversity indices (enrichment, Shannon and Evenness) derived from the DGGE band profile are analyzed based on the experimental diets or the intestinal section sampled in rats.

3.-In vitro study of the interaction with intestinal bacteria.

3. 1 Molecular recognition by membrane receptors in probiotic microorganisms. diners and intestinal pathogens. The studies have been carried out with probiotic bacteria (B. animalis subsp. Lactis Bb12 and Lactobacil / us rhamnosus GG ATCC 53103), commensals (Bifidobacterium longum CECT 7347, Bifidobacterium animalis subsp. Animalis IATA-A2, Bifidobacterium bifidum CECT 765, fragilis [IATA-SAE2, -CAQ2, -CBS1, -CAR1] and Escherichia coli [IATA-CBL2, -CBM1, -CBD10, -CCD2]) obtained from healthy individuals and intestinal pathogens (Salmonel / a enteric CECT 443 and Listeria monocytogenes CECT 935) for collection. The microorganisms, grown in specific culture media for 20 h, were isolated by centrifugation (1,500 x g 15 min) and washed (x3) with a phosphate buffer solution (PBS) (pH 7). Cells were incubated (37 ° C / 30 min) with a fluorescein solution (75 µM) and washed with PBS. Subsequently, the cells were resuspended at a concentration of approximately 108 colony forming units (cfu) / ml (optical density 0.5 ~ .600 nm) in PBS. The bacterial suspensions were mixed with the GOS-Lactulose (0.3% w / v), described in previous sections of the present application, and incubated (37 ° C / 1 h) in 96-well plates with immobilized mucin (Type 11) . Immunization of mucin was carried out by incubating (4 ° C / 24h) the plates with a solution (0.5 mg / mL) of mucin (Type 11) in PBS. The degree of molecular recognition and interaction of GOS-Lactulose with intestinal bacteria was evaluated according to the criteria detailed in the description of the invention.

3. 2 In vitro effect on the intestinal inflammatory response.

The effect of GOS-Lactulose on the production of pro-inflammatory biomarkers was evaluated in cultures of intestinal cells of human origin (Caco-2) widely accepted as a model of intestinal epithelium by presenting the characteristics of mature human enterocytes.

In the experiments, the Caco-2 cells were seeded at an initial density of 5 x 1 04 cells / cm2 and grown for 5 days, with changes of culture medium (Dulbecco's Modified Eagle Medium) every 2 days. The studies used 20h cultures of the collectable invasive intestinal pathogen - Listeria monocytogenes CECT 935 - grown in Brain-Heart broth. The bacteria were collected by centrifugation and washed with a phosphate buffer solution (PBS) (pH 7). Bacterial suspensions were prepared, in duplicate, equivalent to 108 cfu / mL and only one of them was mixed with GOS-Lactulose (0.3% w / v). The mixtures were incubated (37 ° C / 95% C02 / 3h) with the cell culture and the supernatant was subsequently obtained (9300 x g / 10 min). The inflammatory response in intestinal cells was monitored by quantifying the concentration of tumor necrosis factor (TNFa) and interleukin (IL) -1p. TNFa and IL-1p were quantified in aliquots (100 J.LL) of the supernatant using ELISA techniques (eBiosciences, sensitivity 4 pg / mL) according to the manufacturer's instructions.

4.-In vivo study of immunomodulatory activity. The study was carried out on growing male (Wistar) rats that were randomly distributed into different groups (n = 15) administered: i) control diet, ii) control diet + 1% GOS-Lactulose + 0.2 % Cr20 3 (non-digestible marker), and iii) the control diet + 1% commercial prebiotic carbohydrates + 0.2% Cr20 3, over a period of 14 days. After the treatment period, the animals were sacrificed before anesthesia (40 mg sodium pentobarbital 1 kg body weight) and the intestine was dissected, the sections of which were collected in a cryoprotective medium for the RNA and were immediately frozen in liquid nitrogen. In the homogenized samples (Tissue lyser, Qiagen) the total RNA content was extracted using a commercial kit (RNA mini kit, Qiagen) for this purpose, according to the manufacturer's instructions. CNA was obtained from aliquots (1 µJg) of RNA by reaction of the reverse transcriptase (AMV Reversa Transcriptase, Promega). Subsequently, changes in the expression (mRNA) of biomarkers of homeostatic control of inflammation and immune response were monitored with real-time PCR techniques. Monitored biomarkers included pleiotropic interleukins (IL) (IL-6, -10, and -15), the factor

nuclear transcription kappa B -NFKB and tumor necrosis alpha (TNFa), for

which primer sequences (primer) were designed using the bases of

data from the GenBank (NCBI).

Claims (9)

one.

A combination of bioactive galacto-oligosaccharides derived from lactulose (glycosyl-fructose) for use as food ingredients with prebiotic and immunomodulatory activity.

2.

The combination of bioactive galacto-oligosaccharides according to claim 1, characterized in that it is obtainable through a process comprising carrying out a transgalactosylation reaction of lactulose catalyzed by ~ -galactosidase from Aspergillus oryzae.

3.

The combination of bioactive galacto-oligosaccharides according to claim 1, characterized in that it is obtainable through a process comprising carrying out a transgalactosylation reaction of lactose catalyzed by ~ -galactosidase from Aspergillus oryzae and subsequent chemical isomerization of the lactose derivative obtained.

Four.

The combination of bioactive galacto-oligosaccharides according to claims 1-3 characterized in that said immunomodulatory activity occurs by increasing the population of bifidobacteria and / or inducing the expression of biomarkers involved in the homeostatic control of inflammation and activation of the immune system.

5.

The bioactive galacto-oligosaccharide combination according to claims 1-3 characterized in that said prebiotic activity occurs in the large intestine of a subject after ingestion of a dose of said combination in at least 1% w / w for at least 14 days.

6.

The combination of bioactive galacto-oligosaccharides according to claims 1-3 characterized in that they have a digestive survival of up to 100% in the large intestine of a subject after ingestion of said combination.

7.

The combination of bioactive galacto-oligosaccharides according to claims 1-3, characterized in that after the ingestion of said combination, the complete fermentation of the same takes place in the large intestine of the subject.

8. The bioactive galacto-oligosaccharide combination according to claims 1-3, characterized in that after the ingestion of said combination a prebiotic effect occurs, increasing the concentration of bifidobacteria in the large intestine of the subject with respect to said concentration of 1 Or bifidobacteria before ingestion. 9. The combination of bioactive galacto-oligosaccharides according to claim 8 characterized in that the prebiotic effect also leads to an increase in the bifidobacteria biodiversity of the subject's large intestine 15 regarding said bifidobacteria biodiversity before ingestion. 1O. The combination of bioactive galacto-oligosaccharides according to claims 1-3 characterized in that it produces an inhibitory effect in the colonization of pathogenic bacteria of the subject's intestine.