SUMMARY
In the context of nutrition and health promotion, there are products called functional foods that have various beneficial effects in the body, in addition to those that are merely nutritional. Within these functional foods, among others, we can distinguish between probiotic and prebiotic compounds. The microorganisms most commonly used in probiotic foods belong to the genera Lactobacillus and Bifidobacterium .
In the present work we have studied the effect of diets supplemented with Lactobacillus casei or Bifidobacterium bifidum in animal development and especially on intestinal function, centered on its immunitary, digestive and absorptive activity of growing animals.
The bacterial strains used modify the activity of the small intestine of healthy mice, significantly affecting their enzymatic activity (sucrase, maltase and aminopeptidase) and nutrient uptake (galactose and glycic sarcosine), as well as intestinal immune activity (greater number of Peyer's plates). However, these effects do not seem to disturb the development of the growing animals since there are no significant differences in their body weight or blood parameters.
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These results show the possible beneficial effects in intestinal physiology and contribute to the knowledge of the possible mechanisms of action of probiotics, which could be used in the preventive treatment of different pathologies related to the digestive system.
ABSTRACT
The products called functional foods, which are taken in the nutritional value of the organism, are situated in the context of diet and health promotion. Amongst these functional foods we can distinguish, among others, between probiotic and prebiotic compounds. The micro-organisms most widely used in probiotic foods belong to the Lactobacillus and Bifidobacterium types .
In this article we have studied the effect of diets supplemented with Lactobacillus casei or Bifidobacterium bifidum on animal development and especially on the intestinal function, based on their immune, digestive and absorptive activity in growing animals.
The bacterium strains used modify the activity of the small intestine of healthy mice, significantly affecting their enzymatic activity (sucrase, maltase and aminopeptidase) and the collection of nutrients (galactose and glycilsarcosine), as well as the intestinal immune activity (higher number of Peyer's patches). However, these effects do not appear to disturb the development of the growing animals since no significant differences are appreciated in their body weight in their blood parameters.
These results make clear the possible beneficial effects on intestinal physiology and contribute to the understanding of the possible mechanisms of action of the probiotics, which could be used in the preventive treatment of different pathologies related to the digestive apparatus.
Introduction
In today's western society, due to the higher life expectancy, there is a growing interest in health and in all those factors that may affect it. One of the issues of greatest concern is that of food and its impact on health 1,2 . This has generated the new concept of "functional food" defined as "that which contains certain components, which has beneficial effects on one or more functions of the organism, helping to improve the welfare state and contributing to the reduction of the risk of diseases, in addition to its own nutritional effects. "
The current consumer prototype does not renounce food with easy and comfortable access, but also demands a series of qualities associated with fresh and natural products, without transformation or little modified industrially. This requires hygienic requirements (safety and innocuousness), nutritional requirements (reduced energy content and adequate supply of nutrients), organoleptic properties (appearance, texture, taste and rewarding odor) and a healthy or beneficial effect ( International Food Information Council Foundation ). In addition, the consumption of the so-called "Mediterranean diet", whose main components are legumes, vegetables, fruits and fermented dairy products, without forgetting others such as olive oil, red wine and fish, based on food and lifestyle Healthy, has shown its benefits on chronic diseases in the Western world, especially on certain types of cancer and cardiovascular diseases 3 . Thus, the scientific community generally believes that the benefits and benefits of the Mediterranean diet lie in its nutritional content, in particular the presence of prebiotic and / or probiotic components.
Currently, the intestinal microbiota is being reconsidered as an active factor in human health and disease, as is concluded in recent literature reviews on various physiological and pathological situations 4-7 . On the other hand, new paradigms are being proposed by the scientific community that allow relating nutrients, the immune system and cancer 8,9 or diet and antimutagen 10 . Undoubtedly, from the epidemiological point of view, cancer of the digestive system has a special incidence in Western society due to its progressive increase in recent decades. As a reference, the average European rate for colon and rectal cancer in 1998 was 32.7-53.4 per 100,000 inhabitants, respectively 11 , with a survival rate of less than 50% at 5 years (EUROCARE-3 study). In the particular case of Spain, in the year 2000, colon cancer accounted for almost 3% of all mortality in both sexes.
Products called functional foods are an important part in the context of nutrition and health promotion 12 . These are not only considered of interest to maintain health, but also to prevent and alleviate certain diseases 13-17 . However, the use of some probiotic microorganisms, recognized as safe, can have negative effects in immunosuppressed subjects 18 . The microorganisms most commonly used as probiotics belong to the genera Lactobacillus and Bifidobacterium 19 , known as lactic acid bacteria. The Lactobacillus group consists of three groups 20 : L. casei (L. casei, L. paracasei, L. rhamnosus and L. zeae), L. acidophilus (L. acidophilus, L. crispatus, L. johnsonii, and L. gasseri) and L. fermentum (L. fermentum and L. reuteri ).
The use of probiotics has been established and accepted to alleviate some of the aforementioned intestinal diseases 21,22 . Thus, L. gasseri and L. casei possess some immunoregulatory capacity, due to their action on B lymphocytes 23-25 and macrophages 26 . It has been demonstrated that the commensal flora of the intestine affects the expression of PPARg and that it is modified in patients with ulcerative colitis 27 . On the other hand, the use of Bifidobacterium bifidum has been shown to inhibit the growth of HT-29 and HCT-116 cells in vitro.
Scientific studies show that it is necessary to know the behavior and possible mechanisms of action of probiotics in healthy subjects. It is therefore of great interest to investigate the possible beneficial and / or harmful effects that these microorganisms may have on the organism before recommending its use as a therapeutic and / or preventive treatment in different pathological conditions related to the gastrointestinal tract.
Therefore, the objective of this work is to study whether the intake of diets supplemented with each of these probiotics ( L. casei or B. bifidum ) affects animal development and especially intestinal function, specifically immune, digestive and absorptive. These results will be decisive to be able to subsequently use these microorganisms as therapy in various pathological situations.
Material and methods
Obtaining and growing bacterial strains
The strains of Lactobacillus casei used in the present work have been isolated from kefir in the lactology laboratory of the Public University of Navarra 29 . On the other hand, strains of Bifidobacterium bifidum have been obtained from the Spanish Collection of Cultures Type CECT-870. Both strains have been grown in previously autoclaved milk (Skim milk, DIFCO) and have been cultured for 24 hours under aerobic conditions and 37ºC for Lactobacillus and for 72 hours, under anaerobic conditions (5% C02) and 39ºC for Bifidobacterium , until reaching both two a concentration of 10 8 cfu / ml.
Animals and diets
Swiss mice (Charles River Laboratory Animals, Barcelona) of 28 days and 20 g of initial body weight were randomly distributed in each of the three experimental groups (n = 30-20): control (C), Bifidobacterium bifidum (B) and Lactobacillus casei (L). They were placed in cages (n = 5) in a room at constant temperature (22 ± 2ÞC) and humidity and controlled photoperiod of 12 h of light and 12 h of darkness.
The three experimental groups were fed during four weeks with a commercial diet that covers the minimum nutritional requirements for the correct development of the growing animals (AIN-93). The probiotic factors under study ( Lactobacillus casei and Bifidobacterium bifidum ) were administered to the groups corresponding to a concentration of 108 cfu / ml dissolved in milk, so that the control group was also given the same amount of milk but without bacteria.
The body weight of the animals was recorded weekly. At the end of the experimental period the animals were anesthetized and sacrificed by decapitation. Blood was collected for subsequent serum determinations and different organs were extracted and weighed. The small intestine was collected, cleaned with cold saline and after counting in each of the fragments the number of Peyer's plates present, it was frozen at -80 C for the subsequent isolation of the brush border membrane vesicles (VMBC ), in which the determinations of the intestinal enzymatic activity were carried out, as well as the assays of uptake of D-galactose and glycic sarcosine. A small portion of colon (1 cm) was collected to be fixed and processed for histological analysis.
The committee of "Animal Ethics and Experimentation" of the Public University of Navarra reviewed and approved this protocol and the method of animal sacrifice according to the Canadian Council on Animal Care (1993).
Histological analysis
The colon samples collected for the histological study were immediately fixed in 4% formalin for 24 h. They were then dehydrated and after inclusion in paraffin, they were cut into 5 mm pieces and stained in hematoxylin-eosin for later microscopic analysis.
Obtaining VMBC from mouse intestine
The VMBCs were obtained from a jejunum fragment of each of the mice of the different experimental groups, following the method described by Shirazi-Beechey 30 .
The mucosa was resuspended in a medium containing 100 mM mannitol and 2 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at pH 7.1 with Tris-hydroxymethyl-aminomethane. The suspension was homogenized in a Potter-Elvehjem homogenizer (Braun, Melsungen, Germany) at 3,000 rpm at 4 ° C for 1 min. Next, MgCl 2 was added to a final concentration of 10 mM, and the mixture was incubated for 20 min in cold and continuous agitation. Subsequently, it was centrifuged at 2,000 xg for 15 min, and the supernatant was collected and centrifuged at 27,000 xg for 30 min. The pellet thus obtained was resuspended in a medium consisting of 100 mM mannitol, 0.1 mM magnesium sulfate, and 2 mM HEPES at pH 7.4 with Tris.
Finally, the mixture was centrifuged at 27,000 xg for 30 min and the pellet was resuspended in a medium containing 300 mM mannitol, 0.1 mM magnesium sulfate, and 10 mM HEPES at pH 7.4 with Tris.
A sample of the suspension thus obtained was used for the determination by Bradford of the amount of protein present and the measurement of the sucrase activity (EC3.2.1.48), in order to check its enrichment in the apical membrane which was found to be 5%. times compared to the initial homogenate.
Enzymatic activity
For each of the experimental groups, several aliquots of VMBC were thawed and unified and each of the enzymatic assays was carried out in triplicate. The activities of sucrase (EC 3.2.1.48) and maltase (EC 3.2.1.20) were determined according to the method described by Dahlqvist 31 . After incubation with the corresponding substrate for 30 min, the glucose released in each case was determined with the help of Kit A-510 from Sigma.
N-aminopeptidase activity (EC 3.4.11.2) was also determined using L-Gly-Pro-b-naphthylamide as substrate 32 .
Substrate capture studies
Uptake of sugar (D-galactose) and of a dipeptide (glycilsarcosine) in VMBC was measured by a slightly modified version of rapid filtration technique developed by Hopfer 33 . Each of the trials for each experimental group was performed in triplicate.
The uptake of D-galactose was determined at 37 ° C in the presence of a sodium gradient. The incubation medium used at pH 7.4 was composed of 0.1 mM D-galactose, 100 mM NaSCN, 100 mM mannitol, 0.1 mM MgSO4, 10 mM HEPES, and the radioactive substrate used as a tracer (1mCi / mL of D- [1- 14 C] galactose; Amersham Radiochemical Center, UK).
Glycilsarcosine uptake was determined at 27 ° C in a medium at pH 5.5 composed of 1 mM glycic sarcosine, 280 mM mannitol, 0.1 mM MgSO4, 1 mM MES, and the radioactive substrate used as a tracer (10 mCi / mL of [1- 3 H] glycic sarcosine, Moravek Biochemicals, Inc., Brea, CA, USA). At set times, the incubation was stopped by adding cold stop solution (150 mM KSCN, 0.25 mM floricrin, and 10 mM HEPES at pH 7.4 for the uptake assays of D-galactose and 210 mM KCl and 2 mM HEPES at pH 7.5 for the dipeptide uptake assays). The suspension is then passed through a cellulose nitrate filter (0.45 mm, 25-mm diameter, Sartorius, Edgewood, NY, USA) which is washed twice in cold solution and dissolved in scintillation liquid (HiSafe 3) to subsequently measure its radioactivity in a ß counter.
Analysis of protein expression by Western blot
The expression of the PEPT1 and SGLT1 transporters in the VMBC of the different experimental groups was measured by Western blot. The samples were diluted in a medium composed of 50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.05% bromophenol blue. After boiling for three minutes, they were loaded (50 μg) on a 12% polyacrylamide gel and separated by electrophoresis for 60 min. The proteins thus separated were then transferred to a nitrocellulose membrane (Hybond P., Amersham-Pharmacia Biotech, Barcelona) by electrophoresis at 250 mA / 100 V and 4 ° C for 2 h.
The membranes were blocked at room temperature for 90 min in Tris saline medium (100 mM Tris-HCl, pH 7.5, 0.9% NaCl) containing 6% skimmed milk powder. Both transporters were detected by incubating the membranes overnight at 4 ° C with the anti-rat PEPT1 antibodies donated by Dr. Smith (University of Michigan, MI, USA), and rabbit anti-SGLT1 (Chemicon) at a dilution of 1: 500. After successive washes in Saline Tris medium containing 0.1% Tween, the membranes were incubated with the secondary antibody (P0488, Dako Diagnostics, Barcelona) at a dilution of 1: 35,000 for one hour at room temperature. Finally, the immunoreactive proteins were visualized by chemiluminescence (Pierce Super Signal West Dura, Pierce Biotechnology, Rockford, IL, USA) and the bands were quantified by densitometry through the "Quantity One" program, version 4.5 (Bio-Rad).
Statistic analysis
The results are expressed as the value of the mean ± ESM. For the statistical analysis, the SPSS program (version 12.0) was used and the Kruskal Wallis test was applied with the corresponding a posteriori comparisons. The differences between the groups were considered statistically significant when p <0.05.
Results
Development of animals
The inclusion of both probiotic strains in the usual diet of the animals does not alter the body weight of the animals, indicating that they do not exert any harmful effect on the development of these animals. In addition, no differences were observed in the blood parameters measured or in the weight of the different extracted organs (jejunum, liver, spleen, pancreas and colon).
Therefore, we can deduce that the intake of Lactobacillus casei and Bifidobacterium bifidum does not modify the normal growth and development of these animals nor their metabolic state.
Intestinal morphology
Differences were observed regarding the morphology of the colon between the different experimental groups. Groups B and L, visually appear to have a greater number of lymphocytic infiltrates ( Fig. 1 ). On the other hand, in the jejunum, there is also a greater presence of Peyer's patches ( Fig. 2 ), being significantly higher (p <0.01) in the group fed with Lactobacillus and still higher in the group fed with Bifidobacterium ( p <0.001) than in the control group.
Nutrient uptake and enzymatic activity in VMBC
The effect of supplemented diets on the intestinal physiology of these animals was analyzed. As shown in Figure 3 in all assays of enzymatic activity performed (sucrase, maltase and aminopeptidase N) these activities appear increased. In the case of maltase and aminopeptidase N activities, this increase is clearer in the group fed with Bifidobacterium than in that fed with Lactobacillus .
On the other hand, the substrate uptake graphs show that the presence of bacteria in the diet of these animals decreases the uptake of D-galactose while increasing that of glycic sarcosine ( Fig. 4 ). This effect is appreciable only when the absorption of the nutrient is mediated by the corresponding transporter located in the apical membrane of the enterocyte, ie at short times and in the presence of a gradient of Na + or H + in each case. In longer periods of incubation (10 min and 60 min, respectively), both the sugar and the dipeptide cross the apical membrane through a diffusion process that is not altered by the presence of any of the bacterial strains in the diet.
Analysis of the expression of SGLT1 and PEPT1 by Western blot
In order to verify if these effects observed in the uptake of nutrients were related to changes in the expression of the transporters involved in it, the expression of the sodium-dependent sugar transporter SGLT1 as well as the dipeptide transporter PEPT1 was analyzed in the apical membrane of enterocytes by Western blot.
Figure 5 shows a clear increase in the expression of the PEPT1 transporter in groups B and L compared to the control group. Therefore, it seems that the increase in uptake of glycic sarcosine observed could be due to an increase in the expression of the transporter responsible for said uptake.
The expression of SGLT1 in both groups supplemented with the bacteria also appears increased with respect to the control. In this case, this increase in expression is accompanied by a decrease in the galactose uptake previously mentioned. This could be explained by a loss of affinity of the transporter for its substrate due to the presence of bacterial strains in the intestinal epithelium.
Discussion
In recent years, numerous investigations have analyzed the role of probiotic microorganisms (lactic acid bacteria and yeast) in the development of different chronic diseases (cancer, allergies ...), proving that they have a beneficial effect in preventing them 34,35 .
However, little is known about the effects that these types of organisms play in healthy individuals and their possible use as preventive and / or therapeutic agents. Therefore, it is necessary to analyze its effects in healthy individuals, and thus be able to recommend them with scientific basis as preventive therapy, knowing exactly the most effective doses and the possible side effects that they may have.
In the present work we have analyzed the effect of two probiotic strains (lactic acid bacteria) commonly used in the food industry ( Lactobacillus casei and Bifidobacterium bifidum ) in the development and intestinal physiology of healthy animals. The intestinal barrier is responsible for regulating the entry of nutrients and various substances to the body that will affect the nutritional status of the individual, whose correct maintenance is of vital importance when it comes to preventing the development of various diseases.
The choice of bacteria concentration used in this study as well as the duration of the experimental period has been based on previous scientific studies 36,37 .
The normal development of the animals and their metabolic status do not seem to be affected by the inclusion in the diet of both bacterial strains, suggesting that they could be used at the doses analyzed as a dietary supplement 38 .
However, both the intestinal morphology and the activity of the small intestine (enzymatic activity and absorption of nutrients), is affected by the consumption of both strains. Apparently these could activate the intestinal immune system, given that a greater number of Peyer's plaques are seen in the jejunum of these animals and a greater presence of lymphocytic infiltrates in their colon. This fact coincides with the possible immunomodulatory effect of probiotic microorganisms described in the literature 39,40 .
In terms of intestinal physiology, an enzymatic activity increased by the intake of both bacteria, both enzymes involved in the digestion of sugars (sucrase and maltase) and peptides (aminopeptidase N). However, while the absorption of the dipeptide glycosarcosine is increased in groups B and L with respect to its control, the absorption of D-galactose is diminished. These differences observed in nutrient uptake studies could be due to an effect of both strains on the expression levels of the respective transporters involved in each of the absorptive processes. To test this hypothesis, the expression of the Na + -dependent sugar transporter, SGLT1 as well as the Na + / H + -dependent dipeptide transporter PEPT1 was measured by Western blot. In these analyzes, changes in the expression levels of both proteins have been observed due to the ingestion of the bacterial strains, with a clear increase in the presence of both transporters in the apical membrane of the enterocytes.
The expression of both transporters is sensitive to diet 41-43 , thus a greater expression of them could be due to a greater presence of their respective substrates in the intestinal lumen. This fact could be due, in turn, to a greater digestive capacity of the intestine by an increase in its enzymatic activity, as described above.
These results coincide with the work of other authors who have also observed a greater enzymatic activity in the jejunum of rats fed a mixture of prebiotics and probiotics 44 . On the other hand, other authors, in in vitro studies performed on Caco-2 human colon adenocarcinoma cells, observed an activating effect of glycic sarcosine uptake after incubation with Lactobacillus casei 36 .
However, the increased expression of SGLT1 coincides with a lower uptake of D-galactose, suggesting that the transporter could see its affinity for the substrate altered and therefore introduce a smaller amount of it into the enterocyte. This loss of affinity could be due to alterations in the process of insertion of the protein in the apical membrane caused by the presence of bacterial strains or some of their metabolites. It is known that the intake of probiotics modifies the intestinal mucosa according to studies carried out in humans 45 and in animals 46,47 . Therefore, this fact could affect the mechanisms of insertion of the different transporters present in it, in turn altering its function 48 .
In conclusion, it seems that both bacterial strains modify the activity of the small intestine in healthy animals, increasing their immune and enzymatic activity, and altering the uptake of nutrients. These facts do not harm the normal development of these animals since no differences are observed neither in the biochemical parameters analyzed nor in their body weight.
In this sense, this study contributes to recommend safely the use of these strains in healthy animals and points to a beneficial role of these in those diseases that affect the gastrointestinal tract.