**3. Biological activities of GMP**

 The use of GMP is growing, since it is a bioactive peptide with unique nutritional and nutraceutical properties. Many biological activities of GMP have been reported, highlighting antimicrobial, anticariogenic, gastric acid inhibitory, cholecystokinin (CCK) releasing, prebiotic, and immune modulatory. Of particular interest is GMP's capacity to modulate the immune response, due to its potential use in treatment or prevention of different immunopathologies.

#### *Glycomacropeptide: Biological Activities and Uses DOI: http://dx.doi.org/10.5772/intechopen.82144*

 One of the first antimicrobial effects observed in GMP was due to its ability to bind cholera toxin and *Escherichia coli* enterotoxins. Chinese hamster ovary (CHO)-K1 cells undergo morphological changes in presence of cholera toxin and GMP at 20, 100, and 1000 μg/mL was able to suppress this morphological change by more than 70%. Treatment of GMP with proteinases lowered this activity, but removal of sialic acid abolished it [8]. Curiously, as GMP doses increased the inhibitory effect decreased. Authors demonstrated that sialic acid is mediating this inhibitory activity, so it could be inferred that GMP at high doses has less sialic acid available probably as consequence of its aggregation into polymers. Likewise, GMP has showed inhibitory activity on CHO-K1 cells morphological change induced by *E. coli* thermolabile enterotoxins [9]. Later, the binding ability of GMP to intestinal pathogenic bacteria was evidenced, mainly to entherohemorragic *E. coli* (EHEC O157). This activity decreased when the peptide was desialylated and peroxidated, showing that sialic acid is essential to GMP attachment. Besides, GMP prevented in a dose-dependent manner the adhesion of EHEC O157 to Caco-2 cells and when it was conjugated with xylooligosaccharide or carboxymethyldextran, the release of IL-8 by Caco-2 was also suppressed [10]. The same inhibitory effect on pathogenic bacteria adhesion to Caco-2 cells was corroborated with 3[H] thymidine-labeled enterophatogenic *E. coli* (EPEC) *Salmonella typhimurium* or *Shigella flexneri* [11]. Besides, it has been reported that GMP generates dose-dependent inhibition of enterotoxigenic *E. coli* (ETEC) K88 adhesion to ileal mucosa *in vitro* [12]. Finally, a recent report showed that GMP also prevents the attachment of several strains of EHEC and EPEC to Caco-2 and HT-29 mammalian cells [13]. In this study, it was demonstrated that GMP, beyond inhibiting bacterial adhesion, is able to maintain the structural integrity of tight junctions on Caco-2 monolayers, thus delaying the paracellular translocations of EPEC. However, it is important to clarify that authors did not measure the expression level of proteins associated with these junctions.

 There are several *in vivo* assays that show a probable protective effect of GMP against pathogenic bacteria. GMP has been reported to neutralize in 80% the rate of incidence of diarrhea induced by cholera enterotoxins LT-1 in mice, and abolished that induced by LT-2 [9]. On the other hand, the addition of GMP into diets of piglets diminished the bacterial count in mucosal scrapping and abated the attachment of ETEC K88 to the intestine, mainly on the ileal mucosa where the receptor for *E. coli* is located [12]. Moreover, a diet supplemented with 1% GMP protected weaning piglets from damage caused by *E. coli* infection, and prevented the reduction of growth, morphological damage, and the increase in intestinal permeability associated to infection [14].

 In association with this antimicrobial effect, an anticariogenic activity to GMP has been demonstrated. Firstly, *in vitro* assays using different products that contain GMP as an active principle showed that it could inhibit the adhesion of bacteria inducing dental plaque and carious in oral cavity to surface plastic, such as *Streptococcus mutans*, *S. sanguis*, and *Actinomyces viscosus* [15]. Later assays corroborated these results, as the incorporation of GMP with or without caseinophosphopeptide to salivary films modified the adhesion capacity of *S. sobrinus* and *S. mutans* to bovine enamel discs [16]. This antimicrobial effect was linked to a remineralization activity of GMP, as demonstrated later through an experimental protocol in human that showed that GMP alone or combined with xylitol promotes more remineralization than commercial fluoride toothpaste [17]. The anticariogenic and remineralization activity of GMP have been claimed in several patents [15, 17].

Several studies have related GMP with the inhibition of gastric secretion. First ones were mostly developed using dogs by a group of Russian researchers. The first evidence that GMP inhibits gastric secretion was showed by Shlygin and co-workers [18] using gastrin to evoke it. Subsequent works demonstrated similar effect using

 different gastric secretion stimulants [19]. Some years later, it was proposed that this inhibitory effect was caused by a GMP fragment rather than the whole molecule [20, 21]. Later, injecting dogs with a protein fraction obtained from the gastric content of unweaned rats, it was observed an inhibition in dog gastric secretion to a food stimulus [22]. This inhibitory action was similar to that induced by GMP in dogs. GMP was also demonstrated to inhibit gastric motility after its intravenous injection in dogs [23]. All these experiments point out that at physiological conditions GMP may be playing a crucial role in the preservation of active milk proteins in newborn animal during natural breast feeding. In addition to dogs, other experimental models such as rats, pigs, and calves and also isolated organs were used to demonstrate that GMP induces gastric secretion inhibition in association with a decrease in blood of some regulatory digestive hormones, as gastrin and CCK (as reviewed in [24]). However, variations in used gastric stimuli, GMP dose, and origin, via of administration and experimental approach may be the cause of the differences in the reported intensity to this GMP activity.

 Related with the effect of this bioactive peptide on digestive hormones, GMP has also been associated with appetite control. Several *in situ* studies with Wistar rats have suggested that glycosylated forms of GMP A variant could regulate food intake through CCK secretion, a hormone involved in satiety, as GMP stimulate its release [25, 26]. Nevertheless, in studies in human, GMP had no effect in the regulation of food intake over a short-term period [27], neither in the loss of body weight after 12 months of sustained consumption [28]. Likewise, GMP with different degree of glycosylation does not modify the concentration of CCK in human plasma [29]. As other authors have pointed out [27], these inconsistencies related to GMP's effect on CCK release are probably due to dose changes in both animal models and human trials.

 For many years, the prebiotic properties of GMP have been discussed. The first evidence that GMP might possess prebiotic activity arose with the bifidobacterial growth promoting effect of human's colostrums and milk by *in vitro* assays [30]. In an attempt to discern the component of the milk responsible for this activity, an important role of N-acetyl-glucosamine (GlcNAc) and oligosaccharides with GlcNAc [31] was pointed out. Subsequent investigations were quite contradictory, until the prebiotic activity of GMP was demonstrated for the first time in 1984 [32]. This work showed that human GMP is a promoter of bifidobacterial growth, although the effect was lost when it was hydrolyzed, as well as when bovine GMP was used, showing the importance of the GMP peptide chain to function as a prebiotic. Eight years later, it was reasserted that peptide fraction was decisive for the prebiotic effects on bifidus [33]; while other researchers leaned by sialic acid as the inductor of the effect [34]. Following this line, the supplementation of milk with 2% GMP increased the *in vitro* growth of *Bifidobacterium lactis* [35]. Besides, the addition of 2 mg/mL of cow GMP to the growth medium, promoted the growth of *Lactobacillus rhamnosus* and *Bifidobacterium thermophilum*, and apparently glycosylation was not an essential factor to carry out this function [36]. On the other hand, using an artificial colon model to simulate colonic fermentation, GMP was shown to modulate the gut microbiota of elderly subjects by promoting the growth of several health-relevant taxa, like *Coprococcus* and *Dorea*, both related to resistance to pathobiont colonization [37]. Finally, a recent *in vitro* research has shown that GMP promotes the growth of *Bifidobacterium longum* ssp. *infantis* in a dose-dependent manner and modulates its genes expression. Again, they found that the effect was lost with periodate GMP, suggesting that its activity is due to the oligosaccharides present in the molecule [38].

 In the last years, several research groups have demonstrated that oral treatment with GMP modifies *in vivo* the microbiota in gut using different experimental approaches. First works were developed in mice, and showed that after 15 days of

*Glycomacropeptide: Biological Activities and Uses DOI: http://dx.doi.org/10.5772/intechopen.82144* 

 GMP treatment, there was a significant increase in *Lactobacillus* and *Bifidobacteria*  in fecal samples, at the same time the number of *Enterobacteriaceae* and coliforms decreased [39]. Feeding mice for 8 weeks with a GMP-enriched diet reduced *Desulfovibrio* bacteria in normal and phenylketonuric mice; a bacteria that is associated with the pathogenesis of inflammatory bowel disease (IBD). Likewise, normal mice increased Firmicutes and phenylketonuric ones Bacteroidetes; specifically, the genus *Allobaculum* (associated with body weight loss [40]) and *Bacteroidales*, respectively [41]. In this case, the prebiotic property of GMP was associated with an increase in short chain fatty acid production, mainly acetate and propionate, that may has an important role in the regulation of immune response. So, although the prebiotic activity of GMP has been demonstrated, more research is needed to clarify whether the peptidic or carbohydrate fraction or both are involved in this bioactivity.

### **3.1 The immunomodulatory properties of GMP**

GMP has been shown to modulate the immune response in a number of different ways. First, we summarize literature reports about regulatory activity of GMP on immune cells demonstrated by *in vitro* assays, and later, we will focus on those studies in which the regulatory effect of GMP on immune response was analyzed in animal models.

 In relation to the immunomodulatory effects of GMP on immune cells, different *in vitro* approaches have corroborated the inhibitory action of GMP on splenocyte proliferative response to mitogens, and have shown that both sialic acid residues and polypeptide portions of GMP are essential in this inhibitory effect. In 1992, it was showed that the GMP fraction obtained from κ-casein inhibited proliferation of mouse splenocytes induced by *Salmonella typhimurium* lipopolysaccharide (LPS) [42]. Furthermore, GMP displayed an inhibitory activity on the proliferative response induced by concanavalin A (Con A) and phytohemagglutinin (PHA) [42, 43]. Initially, it was found that sialic acid was the key in this inhibitory activity, as it was lost after digestion with neuraminidase [44]. However, the inhibitory effect of GMP was increased after digestion with trypsin and pronase, which suggests that the peptidic chain is also involved in this immunomodulatory activity. In this regard, the same working group showed that the inhibitory effect on PHA-induced proliferative response is higher when the number of sialic acid residues is increased and that on LPS-induced proliferation is highest with a GMP fraction containing two sialic acid residues [45]. Both inhibitory effects decreased significantly after neuraminidase digestion. They also suggested that phosphate group at serine-149 plays a role in GMP binding to the mitogen receptor, as they observed a reduced inhibitory activity after GMP chymotrypsin digestion. Regarding to the associated mechanism to GMP inhibitory effect on splenocyte proliferation, it was showed that GMP stimulates the synthesis of a soluble inhibitory component, an interleukin (IL-1) receptor antagonist or IL-1ra [46, 47]. Moreover, GMP was able to bind to mouse CD4+ helper T cells and to suppress the expression of the IL-2 receptor on the cell membrane, inhibiting the PHA-induced proliferation of mouse splenocytes [48]. Subsequently, the inhibitory action of GMP on LPS-induced cellular proliferation was confirmed in mouse splenocytes, although they did not report any effect on PHAor Con A-stimulated cells [49]. But later, controversy about the effect of GMP on the *in vitro* proliferation of spleen cells was generated, as it was reported that GMP increases the proliferation response of lymphocytes stimulated by Con A [50]. In this study, an increase in Foxp-3 and IL-10 expression was also demonstrated. Besides, authors showed an inhibition on secretion of IFN-γ and TNF-α and on STAT4 activation when cells were stimulated by Con A in presence of GMP. The same team studied the action of GMP on monocyte cell line THP-1 and they found that GMP increases the secretion

of TNF, IL-1β, and IL-8 by THP-1 cells, and this effect is mediated via MAP kinase and NF-kB pathways [51].

On the other hand, GMP is also able to downregulate dendritic cell response to LPS by inducing a slight but significant decrease in the production of IL-6, IL-1β, and TNF-α, but without changing the production of IL-12 and IL-10 [49]. Strikingly, the regulatory effect of GMP on neutrophils is the opposite, as it improves proliferation and phagocytic activity of the human macrophage like cells U937 [52]. However, the observation that both polypeptide and carbohydrate portions are essential for GMP biological effects is reinforced in this study, as peptides of pepsin-digested GMP and sialic acid-rich GMP fractions significantly enhanced cell proliferation and phagocytic activities stimulated by non-digested or asialo-GMP on U937 cell. Also, an upregulatory effect of GMP on production of IgA by LPS-stimulated splenocytes has been reported, being correlated with an increase in the population of IgA positive cells [53].

 There are several studies that analyze the immunomodulatory activity of GMP on immune response when it is orally administered to experimental animals. In the context of splenocytes response to mitogens, two *in vivo* studies were carried out to analyze the possible immunomodulatory activity of GMP. First one was developed in 1998 and demonstrated that mice fed with a GMP-supplemented diet show an enhanced proliferative response of spleen cells to Con A, without generating significant changes in the response to LPS or PHA [54]. Later in 2012, it was showed that oral intake of GMP by rats reduces the proliferative response of splenocytes induced by Con A [55]. In both *in vivo* studies, animals were antigen-immunized because antibody response was also measured. All together *in vitro* plus *in vivo* studies, point out the inhibitory effect of GMP on splenocyte proliferation to mitogens. The opposite response reported by one *in vitro* [50] and *in vivo* [54] assay was quite possibly due to concentration-dependent effects or assay-used conditions.

The effect of orally administered GMP on humoral immunity has also been studied. Mice fed with GMP have shown suppressed levels of specific IgG to dietary and injected antigens, with no change in IgM, IgA, and IgE antibody response [54]. In this regard, a recent study showed that oral administration of GMP to mice resulted in a greater number of IgA positive plasma cells in the intestinal lamina propria [56]. All these results [54, 56] plus *in vitro* ones [53] about Igs production fit together, suggesting an immuno-suppressing activity of GMP on systemic humoral response, but an immuno-stimulating activity on humoral mucosal immunity.

 Martínez-Augustin and co-workers [57, 58] have studied the immunomodulatory action of GMP in experimental models of intestinal inflammation. They have demonstrated that GMP administered orally to rats exerts an anti-inflammatory effect in ileitis and colitis induced with trinitrobenzenesulfonic acid (TNBS); said anti-inflammatory effect shows a degree of efficacy similar to that of sulfasalazine, a drug widely used in the treatment of inflammatory bowel disease. GMP was shown to protect rats from TNBS-induced colonic and ileal inflammatory damage, by reducing the damage score and the extent of necrosis, and also by diminishing the increased alkaline phosphatase colonic activity and inducible oxide nitric synthase expression. IL-1β and IL-1ra messenger RNA levels were significantly decreased in colon as a consequence of GMP administration; and myeloperoxidase activity and levels of IL-1β and IL-17 were decreased in ileum. Initially, the authors assumed that the action mechanism of GMP was not related to anti-oxidative activity or to regulatory cell induction, as glutathione or TGF-β levels in colon and Foxp-3 in ileum were not affected [57, 58]. However, when GMP was orally administered to rats, an increase on Foxp3 expression in spleen cells was observed, although secretion of cytokines by *ex vivo* Con A-stimulated splenocytes did not change [50]. Putting together these results with the regulatory activity of

### *Glycomacropeptide: Biological Activities and Uses DOI: http://dx.doi.org/10.5772/intechopen.82144*

 GMP on monocytes (THP-1) and splenocytes cytokine response obtained by the same working group and previously mentioned in this review [50, 51], authors concluded that the intestinal anti-inflammatory action of GMP is likely to be mediated by the direct modulation of monocyte or splenocyte activity, especially by hampering the activation of Th1 cells while favoring the differentiation of Treg cells [50].

 In recent years, a Mexican laboratory led by Salinas [55, 59–61] has focused on the study of the immunomodulatory activity of GMP in experimental allergy models. They found that oral administration of GMP to rats before and during sensitization with allergen significantly reduces the level of allergen-specific IgE in serum, and also decreases the proliferative response and the production of IL-13 by splenocytes stimulated by the allergen [55]. Treatment of animals with GMP also protected them from systemic anaphylaxis as GMP administration increased survival rates and lessened signs of severity of anaphylactic shock. Moreover, GMP reduced the intensity of urticarial inflammatory reaction when sensitized animals were intradermically challenged with the allergen [55]. With these results, it was demonstrated the immunomodulatory properties of GMP on allergic sensitization and its beneficial effect on clinical signs associated to early-phase allergic reaction. Then, they investigated whether GMP may impact on late-phase and chronic inflammatory allergic reactions, using two experimental models that after repetitive exposure to allergens displayed local recruitment and activation of immune cells with persistent production of inflammatory mediators in affected tissues, together with substantial changes in the extracellular matrix and alterations in structural cells [62]. Specifically, they used experimental models of asthma and atopic dermatitis prophylactically administered with GMP, that is to say, prior to and during pathology establishment. As expected, GMP intake resulted in reduction of IgE titers in serum. In addition to this, in asthma model, GMP substantially decreased blood eosinophilia and suppressed the recruitment of inflammatory cells to the bronchoalveolar compartment. GMP also inhibited eosinophils infiltration, goblet cells hyperplasia, and collagen deposit in lung tissue [59]. Equivalent results were obtained in allergen-induced atopic dermatitis model, where GMP reduced the intensity of cutaneous inflammatory process and edema, abolished pruritus, and reduced eosinophils recruitment and mast cells hyperplasia in dermis [60]. In both models, expression of IL-5 and IL-13 was markedly inhibited in lung and skin, while expression of IL-10 was increased. Their research then turned to the mechanism by which GMP modulates the allergic response. They demonstrated that GMP administration increases the amount of *Lactobacillus* and *Bifidobacterium* present in gut of allergen-sensitized animals after 3 days of oral treatment, and that of *Bacteroides* after 17 days. Interestingly, this intestinal microbiota is associated with protection in allergy. GMP intake also increased the production of TGF-β by splenocytes of sensitized animals in response to allergen and impacted mast cell function, inhibiting their activation and also the release of histamine in response to allergens. No change in tissue mast cell number was found [61]. These results obtained in experimental allergy models again show a double way by means GMP exerts its control on inflammation; on one hand, through a direct modulation of immune cells activity involved in the process, and on other side by potentiating a regulatory microenvironment against the Th2-inflammatory one. More studies are needed to understand which immune cell receptors recognize GMP and which intracellular signals activate or inhibit.

Finally, there are few studies that analyze the role of GMP on cancer. In a rat model of pharmacological-induced colorectal cancer, oral administration of 100 mg/kg of GMP decreased the number of aberrant crypt foci although no effect was observed at doses of 10 and 50 mg/kg. On the other hand, there was no change in methylation and expression level of p16 and MUC2, two tumor suppressor genes [63]. Additionally,

through an *in vitro* assay GMP was showed to inhibit the expression of p65 NF-kB in human colorectal tumor HT-29 cells activated with LPS, key element in colorectal cancer induced by inflammatory bowel diseases [64].

Although more studies are needed in relation to some biological activities, current results propose GMP as a good candidate to be used as a functional ingredient in food industry.
