ABSTRACT
The market for probiotic foodstuffs is one of the most dynamic in the functional foods area. Eleven probiotic strains are currently available in the Chilean local market, mainly in dairy products and infant formulas. These strains are Lactobacillus casei CRL431, L. acidophilus NCFM, L. johnsonii NCC533 (Lal), L. acidophilus La5, L. rhamnosus HN001 (DR20), L. rhamnosus GG, L. paracasei ST11, Bifidobacterium animalis spp. lactis 430, B. animalis spp. lactis DN173101, B. longum BB536 and B. animalis spp. lactis Bb12, the later being used in several products under different brands. Briefly presents the scientific evidence describing the different metabolic activities and their effects on human health, which support their use in human nutrition and probiotics.
SUMMARY
The market for probiotic products is one of the most dynamic in functional foods. In Chile, at present, 11 probiotic strains are being marketed in foods, mainly dairy products and infant formulas and foods. They are Lactobacillus casei CRL431, L. acidophilus NCFM, L.johnsonii NCC533 (Lal), L. acidophilus La5, L. rhamnosus HN001 (DR20), L. rhamnosus GG, L. paracasei ST11, Bifidobacterium animalis spp. lactis 430, B. animalis spp. lactis DN173 010, B. longum BB536 and B. animalis spp. lactis Bb12, which, unlike the others, is present in several products of different brands. Taking into account that the benefits of probiotics are strain-specific, this article summarizes the scientific evidence describing the different activities of these strains and their effects on health and therefore support their use as probiotics.
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INTRODUCTION
Probiotics are innocuous microorganisms that are incorporated into food and, once ingested, survive in the consumer's digestive tract where they regulate the intestinal microbiota and exert beneficial effects on their health (1). They differ from prebiotics that are "non-living food components (mainly dietary fibers), whose consumption confers a benefit to the health of the host in association with the modulation of the microbiota." Probiotics are mainly lactic bacteria belonging to the genera Lactobacillus or Bifidobacterium, which, for the most part, have been isolated from depositions of healthy individuals. These bacteria are screened, evaluating their ability to resist the acidic pH of the stomach and digestive enzymes and bile salts of the intestine, and adhere to mucus or intestinal epithelial cells, all properties that favor its survival and permanence in the digestive tract (2). The selection process also assesses in these strains the existence of functional activities such as antioxidant, anti-inflammatory, immunostimulatory, antitumor, analgesic, antibacterial, among others activities that allow the selected strain to modulate physiological functions in the host and exert its health effects. It should be noted that such properties are strain-specific, ie, that a particular strain exerts only some of all properties described for probiotics. For example, the functional properties of L. rhamnosus GG, L. rhamnosus DR20 and L. rhamnosus LCR35 are different even though these three strains belong to the same genus ( Lactobacillus) and to the same species ( rhamnosus ).
Probiotics can be considered as "functional ingredients" that are used to "functionalize" food, that is to say add a defined functional property that gives added value to the product. Food products containing probiotics therefore fall into the category of "functional foods", since they provide benefits for the health of the consumer, beyond the nutritional benefits of the food that contains them (3).
The world probiotic market is booming and has one of the highest growth rates in the global market for "functional foods" (4). The number of new products with probiotics is increasing every year, and although the main sector associated with the use of probiotics is still dairy products, the progress of microbiology and food technology (and in particular microencapsulation processes ), are allowing the incorporation of these microorganisms to products as varied as juices, ice cream, cereals, and also mayonnaise, chocolate and cookies (5).
Because probiotic properties are specific-strain, it is important to label these products well with the name of the strain (s) present (not just the genus name and species) in such a way that both the health professionals as consumers can differentiate between the different products available depending on the strain they contain.
In Chile, the first food product with probiotics was "Uno al día", which was launched in 1998 by Soprole. Since then, other products have come out in the local market in such a way that at present, all major dairy companies in the country have probiotic products. Table 1 shows the probiotic products currently present in the national market, with the name of the company that markets them and the one that produces the strain present in the product, in addition to the message that supports its commercialization.
Scientific evidence supporting the health effects of probiotic strains marketed in Chile in foods
Lactobacilus casei CRL 431. This strain stands out as the only probiotic strain of Latin American origin marketed internationally. In fact, it has been isolated and characterized by the Lactobacillus Reference Center (CERELA), in Tucumán, Argentina. This strain is used in the Sancor LecheBio in that country and is currently marketed and distributed by the international firm Christian Hansen. Most of the studies performed with CRL 431 have been in animal models, showing that this strain is capable of stimulating both local and systemic immunity and of inhibiting the growth of different enteropathogens (6,7).
On the other hand, thanks to its β-galactosidase activity that remains functional in the human digestive tract, it improves lactose tolerance in hypolactanic individuals (8). The consumption of a commercial product with L. casei CRL 431 and a strain of L. acidophilus has also been shown to decrease bacterial overgrowth at the intestinal level in humans (9). This property makes it interesting for individuals with irritable bowel syndrome or those who consume proton pump inhibitors, situations that predispose to bacterial overgrowth.
In children with gastroenteritis, consumption of the same product significantly reduced the number of stools, duration of diarrhea and episodes of vomiting, compared to placebo (10). Finally, it has been observed that the administration of L. casei CRL 431 increases the immune response to the oral polio vaccine in healthy adults, suggesting an adjuvant effect in the stimulation of the intestinal immune response (11).
Lactobacillus acidophilus La5 . Few studies have been done with La5 and most of them have associated this strain with others, making it difficult to assess its effect per se. In association with L. bulgaricus, B. lactis Bb-12 and S.thermophilus and in the presence of oligofructose, La5 modulates the intestinal microbiota and decreases bacterial translocation in colectomized patients, suggesting that these strains contribute to stabilize intestinal barrier function 12). In a placebo-controlled clinical trial conducted in patients with collagenous colitis, administration of La5 and Bb12 for 12 weeks significantly decreased the frequency of stool deposition and the number of days with fluid deposition per week (13). The consumption of a yogurt with LA5 and Bb12 by subjects colonized by H. pylori decreased urease activity, indicating that this product reduced the density of the pathogen in the stomach; the consumption of this yogurt also increased the efficiency of antibiotic treatment in subjects colonized by the pathogen (14,15). However, it is likely that this effect is mainly due to Bb12 since only this strain could inhibit H. pylori in in vitro assays.
Bifidobacterium lactis 420 . In association with L. acidophilus 74-2, consumption of yogurt with B. lactis 420 for three weeks (300 g / d) in healthy volunteers increased the phagocytic activity of neutrophils, thereby modulating the nonspecific cellular immune response; also decreased triglyceridemia by 11.6% in these subjects (16). On the other hand, the 8-week consumption of B. lactis 420 along with L. paracasei LPC-37 and L. acidophilus 74-2 increased the phagocytic activity of monocytes and granulocytes and the CD57 + lymphocyte count of healthy subjects, while that in patients with atopic dermatitis, these probiotics decreased the CD4 + CD54 + lymphocyte count and the allergic symptomatology index (SCORAD) (17). In vitro studies have shown that this strain decreases the expression of the cyclooxygenase-inducible isoform (cox2) while increasing the expression of its constitutive isoform, cox-1 in human intestinal cells Caco-2 (18); this finding is of particular interest because these enzymes play an important role in the health of the gastrointestinal mucosa, being the overexpression of cox-2 associated with inflammatory and tumoral pathologies.
Bifidobacterium animalis spp. lactis DN-173 010 . Their presence has been detected in the depositions of volunteers consuming yogurt with this strain both by culture and by molecular methods (19). Several clinical studies have been conducted to evaluate the effect of its consumption on intestinal transit and digestive comfort in humans. A double-blind, randomized controlled trial of 36 healthy women showed that consumption of 125g / d for 10 days of yogurt with DN-173 010 reduced colonic transit time without affecting fecal bacterial mass or bile acids secondary (20). The effect on intestinal transit time was confirmed in other clinical trials (21,22). In another study of 274 adults with irritable bowel syndrome with constipation, six-week consumption of a product with DN-173010 significantly improved abdominal distension and the "Health Related Quality of Life" index compared to control group (23). In addition, an increase in the frequency of stools in the subgroup of more constipated subjects (<3 stools / week) was observed. On the other hand several studies indicate that this strain prevents the development of preneoplastic lesions in animal models of colonic tumor and decreases procarcinogenic enzymatic activities (β-glucuronidase) of the colonic microbiota (24). DN-173 010 also decreases the proliferation of the human intestinal tumor cell line HT-29 and increases its differentiation, two cellular parameters strongly associated with the process of tumorization (25). These studies suggest that consumption of B. lactis DN-173 010 could be a protective factor against the development of colorectal cancer in humans. On the other hand, the consumption of B. lactis DN-173 010 for two weeks in healthy volunteers significantly decreased saliva levels of S. mutans, a pathogen involved in the development of dental caries (26).
Lactobacillus rhamnosus HN001 . Contrary to most probiotics, L. rhamnosus HN001 (also called DR20) is a strain that was isolated in New Zealand from Cheddar cheese. In vitro studies and animal models have shown their ability to interfere with pathogenic microorganisms such as salmonella or enterotoxigenic E. coli (27) and their presence at the fecal level has been confirmed in healthy volunteers after being consumed in a dairy product (28) . However, most of the information available on the effects of this strain in humans relates to its ability to enhance the individual's defenses, more particularly in subjects with mild immunosuppression. Indeed, several clinical trials in older adults have consistently shown that consumption of products with DR20 stimulates the cytotoxic activity of NK (Natural-Killer) cells, which are involved in the body's defense against tumors and infections and the phagocytic activity of monocytes and neutrophils from the peripheral blood (29-32); this effect is even greater in subjects older than 70 years, that is, those with a higher degree of immunosuppression.
A clinical trial was recently conducted in 382 preschoolers in Santiago to evaluate the protective effect of HN001 consumption on acute respiratory infection episodes during winter months in 2006. Although no changes were observed in incidence, duration and intensity of episodes of respiratory infection, a higher concentration of secretory immunoglobulin-A was observed in a subgroup of children, indicating the ability of this strain to stimulate the local immune system (33). Finally, administration of HN001 to mothers with a history of atopy during the gestation period and then to infants for 6 months resulted in a lower prevalence of eczema compared to those receiving placebo (34).
Lactobacillus johnsonii NCC 533 . Also called Lal, it is one of the most studied probiotic strains. It adheres to intestinal cells mainly thanks to its lipoteicoic acid (27) and remains alive in the consumer's digestive tract, where it can modulate the intestinal microbiota, as shown in a study carried out in Chilean volunteers (35).
The Lal genome has recently been described indicating that this microorganism has several characteristics that describe it more adapted to the small intestine than to the colon (36). Of interest is its ability to synthesize high-molecular-weight fructooligosaccharides (37), a phenomenon that may explain its capacity (similar to that of prebiotics) to stimulate the growth of bifidobacteria in the colon, as has been shown in humans (35 , 38).
Lal inhibits both growth and adhesion to intestinal cells of a large number of enteropathogens including the parasite Giardia lamblia and the bacterium Helicobacter pylori, a pathogen that specifically colonizes the human stomach (27). This latter effect is probably mediated by the production of lactic acid, H 2 0 2 , bacteriocins and / or by extracellular expression of the heat stress protein GroEL capable of adding H. pylori (39, 40). Clinical trials in Santiago-settled schoolchildren show that Lal interferes with H. pylori, reducing the gastric density of the pathogen (41). This effect requires the regular consumption of the probiotic, as children tend to recolonize rapidly after consuming the probiotic (42). In adults, Lal consumption decreases the gastric inflammation caused by the pathogen (43).
On the other hand, the effect of Lal on immunity has also been studied; the administration of this probiotic for three weeks increases the phagocytic activity of circulating leukocytes, an effect that remains during the 6 weeks after the cessation of its intake (44). Lal also moderately increases circulating levels of IgA (45). These antibacterial and immunostimulatory effects may explain the lower infection rate and better nutritional status seen in hospitalized elderly patients over 70 years of age who received enteral nutrition with Lal for 12 weeks (5.7% vs 17.3% in the control group) ( 46). Two clinical trials in patients with Crohn's disease were unable to demonstrate a protective effect of Lal consumption (2x10 -10 10 CFU / day) versus endoscopic disease recurrence (47,48).
Lactobacillus acidophilus NCFM . Of human origin, isolated in the 70's at the University of North Carolina (USA), is one of the first probiotic strains characterized. Its properties and effects on health have been described in more than 75 publications and its genome has recently been sequenced (49, 50). Its permanence in the digestive tract could be favored by its capacity of adhesion to intestinal epithelial cells (51). It produces hydrogen peroxide (H 2 0 2 ) and bacteriocins that inhibit the growth of pathogens such as S. typhimurium, S. aureus, E. coli, L. monocytogenes and C. albicans (27, 52). Such antimicrobial activities would allow NCFM to modulate the composition of the microbiota and decrease the risk of gastrointestinal infection and diarrhea. Goldin et al. In the 1980s showed that NCFM consumption decreases those colon bacterial populations involved in the expression of procarcinogenic enzyme activities azoreductase, nitroreductase and p-glucuronidase (53). NCFM also exerts a protective effect on animal models of colonic cancer (54). L. acidophilus NCFM has a β-galactosidase activity that remains active in the gut; this activity facilitates the digestion of lactose and decreases the digestive symptoms in hypolactanic subjects who consume dairy products with this probiotic (55). Administration of NCFM stimulates secretory IgA production in mice (56). More novel is the recent observation that NCFM has analgesic activity, which results in a reduction of abdominal visceral hypersensitivity. In fact, Rousseau et al. (57) have shown for the first time that NCFM increases in a dose-dependent manner and up to 40% the pain threshold in an animal model of visceral hypersensitivity, which is equivalent to the effect obtained with 1mg / kg of morphine administered by subcutaneous route. This analgesic effect would be due to the fact that this probiotic stimulates in more than 50 times the expression of opioid and cannabinoid receptors involved in the regulation of the nociceptive (pain) system at the intestinal level. To date, L. acidophilus NCFM is the only probiotic for which this property has been described. It could explain the relieving effect of certain probiotic strains in individuals with visceral hypersensitivity such as those with irritable bowel syndrome, but also in infants with colic and in children with recurrent abdominal pain.
Bifidobacterium animalis spp. lactis BM2 . It is one of the most widely used strains in the world. In addition to its use in dairy products, it has also been incorporated into milk powder and infant formulas; in some products Bb12 comes along with other probiotic strains of the genus Lactobacillus like LA5 or LGG. There are a large number of studies that support the properties of Bb12, many of them in newborns and in premature babies, which supports the safety of this strain (58). In preterm infants with antibiotics, the administration of this probiotic resulted in a greater weight gain compared to the children in the control group, in addition to a lower concentration of fecal calprotectin (a marker of inflammation), a higher fecal concentration of IgAs and fatty acids volatiles and a more acidic fecal pH (59). This probiotic also contributes to regulate the intestinal microbiota in these children (60). These observations may explain the results of clinical trials showing that the administration of Bb12 prevents necrotizing enterocolitis in very low birth weight preterm infants (61). On the other hand, the immunostimulating properties of both local and systemic Bb12 have been confirmed in several studies (62). These properties may explain its positive effect in children with allergies; the supplementation of hydrolysed formulas with B. lactis Bb12 results in an earlier recovery than in those receiving the control formula without probiotics (2 months vs. 6 months) (63). Several studies with Bb12 also suggest a moderate preventive effect against the development of acute diarrhea in children including that caused by rotavirus (64-67). On the other hand, the consumption of cereals fermented with Bb12 by older adults living in households normalized their digestive function, compared to a placebo group (68)
Lactobacillus rhamnosus GG . It is by far the best probiotic studied, with over 100 published clinical trials that have studied its effects on health. Numerous studies have evaluated the effects of LGG on diarrhea. A meta-analysis of 8 randomized trials (988 children) that analyzed the effect of LGG in the treatment of acute diarrhea showed that LGG did not affect the volume of deposition emitted but significantly reduced the duration of episodes of diarrhea (-1.1 day), particularly in those produced by rotavirus (-2.1 days), in addition to decreasing the risk of diarrhea> 7 days (RR = 0.25) and duration of hospitalization (-0.58 days) (69). Two other meta-analyzes concluded that LGG could also exert a preventive effect on diarrhea (antibiotic-associated diarrhea and traveler's diarrhea) (70,71). Such an effect could be due to the antibacterial (27) or immunostimulating properties of LGG. It should be noted, however, that LGG does not exert an inhibitory effect against H. pylori; however, delivered concurrently with antibiotic treatment, tends to decrease the adverse effects associated with its administration (40)
LGG has been tested in the nutritional management of patients with chronic inflammatory diseases of the digestive tract or with allergy. Daily administration of LGG along with mesalazine to patients with ulcerative colitis did not reduce the risk of relapse but increased the duration of the remission period compared to mesalazine alone (72). In patients with Crohn's disease, a meta-analysis performed from 7 clinical trials concluded that LGG does not affect either the risk of relapse or the duration of remission in these patients (73).
On the other hand, the perinatal administration of LGG in infants with a family history of atopy reduced the incidence of atopic eczema during the first 4 years of life compared to those who did not receive the probiotic (RR = 0.57, [0.33-0.97]) (74 ). The consumption of LGG by allergic children reduces the intensity of symptomatology and also modulates the immune system, increasing the concentration of IgAs at the intestinal level and decreasing the faecal levels of markers of inflammation such as TNF- α and α 1 -antitrypsin (75) . The effect of LGG on these pathologies may be due to the fact that it modulates the Th1 / Th2 profile of the lymphocyte populations and the cytokines released by them (76) and / or because it stabilizes the gastrointestinal barrier function by limiting the passage of antigens and other macromolecules from the lumen to the mucosa (77). The effect of the consumption of yogurt with LGG has also been evaluated in 59 women with constipation, showing a relieving effect of the adverse digestive symptoms associated with an increase in fiber intake (78). It should be noted that LGG, however, does not ferment lactose and therefore does not improve tolerance to lactose in hypolactase subjects.
LGG consumption in patients with cystic fibrosis affects both the severity of intestinal inflammation and abdominal pain; this probiotic also decreases the incidence and duration of Pseudomonas infection , and improves lung function and weight gain in these subjects. (79). In relation to this point, the protective effect of LGG against episodes of acute respiratory infection has been evaluated in 571 children from 18 kindergartens in Finland. Compared to the placebo group, children receiving the probiotic had fewer days of absence due to illness (4.9 vs 5.8 days, p = 0.03), a 17% decrease in children who had otitis as a complication of infection and the number of days with antibiotic treatment (80). This effect may be due to the fact that LGG can interfere with the presence of potential pathogens (S. aureus, S. pneumoniae and beta-hemolytic streptococci) in the upper respiratory tract (81).
Finally, the long-term effect of LGG consumption on the risk of dental caries has also been studied through a randomized, controlled trial carried out over 7 months in 18 kindergartens in Finland (82). The oral health and the concentration of S. mutans were evaluated in the children at the beginning and at the end of this period. The risk of dental caries was 44% lower in the group receiving the probiotic (OR = 0.56, p = 0.01) as well as lower pathogen counts in these children, indicating that LGG may have oral health benefits.
Lactobacillus paracasei NCC2461 (ST11 ) . Most studies published with ST11 have been performed in animal models. They show that this strain stimulates populations of CD4 + lymphocytes producing TGF6 and IL-10, cytokines involved in oral tolerance phenomena to antigens (83). In an animal model of visceral hypersensitivity induced by antibiotic in mouse, ST 11 also attenuates the sensation of pain (84); however, the modulation of the opioid or endocannabinoid receptors was not evaluated with this strain. On the other hand, administered simultaneously with long chain polyunsaturated fatty acids and prebiotics, ST11 reverses the negative effect of neonatal stress on growth and intestinal barrier function (85).
Bifidobacterium longum BB536 . A randomized, double-blind, controlled clinical trial evaluated the effect of yogurt consumption on B. longum BB536 (2x100g / day) (or its respective control) in 40 subjects with a cedar pollen allergy for 14 weeks. The product significantly decreased ocular symptoms and reduced (but not significantly) nasal and pharyngeal symptoms p = 0.044). At 4 weeks of treatment, an increase in circulating levels of IFNγ and a decrease in specific IgE levels was observed (86). These results were confirmed by other clinical trials; could be due to modulation of Th lymphocyte balance, probably by activation of the innate immunity receptor TLR9 (Toll Like Receptor-9) by CpG dinucleotide sequences of the BB536 bacterial DNA (87). On the other hand a study in 6 healthy volunteers showed that the consumption of a yogurt with BB536 (250 ml / d) for 2 weeks increased the proportion of bifidobacteria and of lactobacilli in the fecal microbiota as well as the levels of volatile fatty acids, whereas the levels of clostridia and potentially toxic substances such as ammonium, indole and p-cresol decreased, contributing to the generation of a healthier colonic environment (88).
In sum, at present there are in Chile different commercially available probiotic products, which differ from one another for the strain (s) they contain. The activities of probiotics are strain-specific which explains the variability of their effects on human health. These effects should be supported by scientific evidence that includes, in particular, clinical trials, but also provides information on their mechanisms of action. As summarized in Table 3 , probiotic consumption may be useful in reducing the risk of diarrhea, reducing allergy severity, alleviating gastrointestinal symptoms in subjects with irritable bowel syndrome, improving lactose tolerance in hypolactemic subjects, and to stimulate immunity in subjects with moderate immunity, so that their regular consumption represents a benefit for the health of the consumer.