SIMULACIÓN SUCESIVA Y CONVENCIONAL

PREPARACIÓN DE LAS CÉLULAS

Se siembra un inoculo (2mL de células en MRS al 20% v/v de glicerol) de las cepas de estudio en 500 mL de caldo MRS con L-cisteína (0.5 g/L) y se incuba a 37 °C por 24 h para obtener una densidad celular de 109-1010 UFC/mL. Las células fueron recuperadas por centrifugación a 8000 rpm (3578 g) por 10 min a 4 °C y después el paquete celular se resuspendió en peptona salina (PS, 1 g/L de peptona, 8.5 g/L NaCl), se volvió a centrifugar bajo las mismas condiciones. Las células lavadas fueron suspendidas en un total de 10 mL de PS y almacenada a 4 °C hasta su uso. La suspensión de células frescas fueron preparadas para cada experimento y enumeradas por vaciado en placa en agar MRS con L-cisteína (0.5 g/L). Las placas fueron incubadas anaeróbicamente a 37 °C por 3 dias usando el GasPack Plus system.

ENSAYO DE SOBREVIVENCIA DE CÉLULAS LIBRES Y MICROENCAPSULADAS EN JUGO GÁSTRICO SIMULADO Y JUGO INTESTINAL.

El jugo gástrico simulado fue preparado por disolución de pepsina (a una concentración final de 0.3 g/L) en una solución salina (0.2% NaCl, p/v) y ajustando el pH a 2.0 con HCl concentrado. La mezcla fue esterillizada por filtración a través de una membrana (0.45 µm, Nalgene Brand Products, Nalgene Co., Rochester, NY, USA). Los autores señalan, sin embargo, que la simulación de los jugos gástricos de los estudios in vitro tiende a sobrestimar las pérdidas de viabilidad que se producirían in vivo, ya que es sabido que la presencia de componentes de los alimentos temporalmente puede elevar el pH gástrico (Mainville, Arcand, y Farnworth, 2005).

El jugo intestinal simulado fue preparado de acuerdo al método de Huang y Adams (2004). La pancreatina y las sales biliares fueron suspendidas en regulador de fosfatos (PB, 0.05 mol/L

NaH2PO4, pH 8.0) a una concentración final de 1 g/L y 4.5 g/L, respectivamente. A mezcla fue

ajustada a pH de 7.4 con 0.1 mol/L NaOH y esterilizado por filtración. Ambos, jugos gástricos e intestinales fueron preparados en fresco para ser usados en el mismo día.

Las suspensiones celulares lavadas de la cepa de estudio (en este caso B. adolescentes) (0.5 mL) fueron añadidos a 4.5 mL de jugos gástricos a 37 °C, se mezclan bien con vortex por 10 s y se incuba por 5, 30, 60 y 120 min a 37 °C con agitación periódica y monitoreo del pH en el jugo gástrico. La sobrevivencia de las bacterias después de cada intervalo de tiempo establecido fue enumerada mediante cuenta por vaciado en placa en agar MRS-cys a 37 °C por 72 h como se describió anteriormente.

SURVIVAL AND STABILITY ASSAY OF FREE AND MICROENCAPSULATED B. ADOLESCENTIS AFTER SEQUENTIAL INCUBATION IN SIMULATED GASTRIC AND INTESTINAL JUICES.

Las suspensiones celulares lavadas de la cepa de estudio (en este caso B. adolescentes) (0.5 mL) fueron añadidos a 4.5 mL de jugos gástricos a 37 °C, se mezclan bien con vortex por 10 s y se incuba por 60 min a 37 °C. Los jugos intestinales simulados templados a 37 °C fueron entonces añadidos y el pH se ajusta a 7.4 con 0.1 mol/L NaOH. El volumen final de la mezcla fue llevada a 10 mL con regulador de fosfatos e incubados por 60, 120 y 240 min a 37 °C con agitación periódica. La sobrevivencia de las bacterias después de los tiempos del sistema secuencial de incubación fueron enumerados mediante cuenta por vaciado en placa en agar MRS-cisteína a 37 °C durante 72 h como se describió anteriormente.

SIMULACIÓN SUCESIVA

El modelo gastro-intestinal más reciente desarrollado por Mainville et al. (2005) simula el estómago y duodeno. Cada vaso-recipiente está equipado sondas de temperatura y pH y los puertos de entrada para el medio, HCl, NaOH y la bilis de buey. El medio en cada reactor se mantiene en suspensión por un agitador magnético y la temperatura se mantenga a 37 ° C con un baño de agua recirculador.

Para determinación de actividad antimicrobiana, las cepas fueron lavadas en solución salina esteril (0.75% p/v NaCl) mediante centrifugación a 8000 g x15 min a 18°C.

DISEÑO DE UN MODELO GASTROINTESTINAL INFANTIL

The GIM in Fig. 1 was developed to simulate nutrient flow through an infant gastro-intestinal tract, aged between 6 and 12 months, and isa combination of the models described by Molly et al. (1993) and Mainville et al. (2005). The GIM consists of four vessels, representingthe stomach, duodenum, jejunum, and ileum. Pancreatic juice and bile salts [2.4 g NaHCO3, 0.18 g pancreatin (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 1.2 g Ox-bile (Oxoid Ltd, Hampshire, England)] were kept in a separate vessel. The reservoir vessel contained 400 ml sterile saliva buffer (1.24 g NaCl, 0.44 g KCl, 0.04 g CaCl2, 0.24 g NaHCO3). Vessels were connected with autoclavable Nalgene tubing. Peristaltic pumps were linked to a LabPro Interface system (Vernier Software & Technology, Beaverton, USA), controlled by a computer program developed by J. Kistner and R. Kistner (Information Technology, University of Stellenbosch). All components, except the computer, were housed in a 37 °C room. The substrate was pumped from the reservoir to the stomach, andthen to the duodenum, jejunum and ileum at specific time intervals (Table 1). The pancreatic juice and bile salts (200 ml) were injectedinto the duodenum after 2.5 h. The flowspeed of the peristaltic pumps was controlled at 104 ml/min, as used by Van den Driessche et al.(1999). The pH in the stomach was gradually decreased to 3.7 with the addition of 0.5 M HCl to simulate milk digestion in infants. The pH in the duodenum and jejunum was kept at 6.5 by the addition of 0.5 M NaOH and in the ileum at 6.0 by adding 0.5MHCl. Changes in pH were monitored with pH probes (Vernier Software & Technology, Beaverton, OR) linked to peristaltic pumps.

2.4. Preparation of starter cultures and operation of the GIM

E. mundtii ST4SA and L. plantarum 423 were inoculated, separately, into 10 ml MRS broth (Biolab) and incubated at 37 °C for 18 h. The cultures were then transferred to 200 ml MRS broth (Biolab) and incubated at 37 °C to OD600=1.5. The cells were harvested (8000 ×g, 15 min, 18 °C), washed with half-strength saliva buffer (Marteau et al., 1997) and the pellets resuspended in 10 ml of the same buffer. These cell suspensions were used to inoculate the reservoir (400 ml saliva buffer), supplemented with either 58.2 g N1 or 65.3 g L1. Final cell numbers in the reservoir were 1.0×108 cfu/ml and the pH adjusted to approximately 6.8 with sterile 0.5 M NaOH. In a separate experiment, the reservoirwas inoculated with a combination (1:1) of strains ST4SA and 423 (total=1×108 cfu/ml). Conditions were the same. Samples were collected with sterile tubing (1 mm diameter)connected to a 1 ml sterile syringe. Viable cell numbers of strains ST4SA and 423were determined by plating onto Enterococcus SpecificAgar (Difco, Becton, Dickinson and Company, Le Pont de Claix, France) and MRS agar (Biolab), respectively. All plates were incubated at 37 °C for 48 h. Antimicrobial activity was determined using the agar spot method, as described before.

Most conventional methodologies involve growing the cells to be studied, centrifugating and resuspending the cells in an acidic milieu, with or without washing, at pH values varying between 1.0 and 3.0 or incubating bacteria in a medium containing F0.3% Oxgall bile, porcine bile, or bile salts (Lankaputhra and Shah, 1995; Charteris et al., 1998; Prasad et al., 1998; Jacobsen et al., 1999).

SIMULACIÓN SUCESIVA

The simulator of the human intestinal microbial ecosystem (SHIME) was developed to simulate the entire human gastrointestinal system (Molly et al., 1993); it is inoculated with human fecal material to establish a microbial population that resembles that found in the human gastrointestinal tract. The original model consisted of five reactors representing the duodenum/jejunum, ileum, caecum and ascending colon, transverse colon, and the descending colon. A sixth reactor was later added to simulate the stomach. This model has been used to investigate interactions of probiotic bacteria with the human intestinal microflora (Molly et al., 1996; Nollet et al., 1997), and the effects of probiotic bacteria and synbiotic products on the human gastrointestinal microbiota (Kontula et al., 1998; Alander et al., 1999; Gmeiner et al., 2000). Microbial populations, bacterial enzymes, volatile fatty acids, and gas production have been measured in the various parts of model in response to probiotic, prebiotic, and symbiotic treatments. The TNO model of the upper GI tract contains four chambers to simulate the stomach, duodenum, jejunum, and ileum (Minekus et al., 1995). HCl is added to the stomach chamber so that the pH drops over time from pH 4.8 to 1.7. Marteau et al. (1997) have used this model to study the survival of four different lactic acid bacteria. Mattila-Sandholm et al. (1999) reported that the TNO model and the SHIME model were not able to adequately select for probiotic strains. They reported that probiotics with known clinical survival did not survive well in the TNO model, and that the SHIME model gave conflicting results. Morelli (2000) observed that even these more sophisticated models still do not adequately simulate the ingestion of a probiotic product, nor use proper physiologically relevant substances to control pH or simulate human bile.

The ideal in vitro model simulates the events of food ingestion and digestion, and allows for the addition of a food matrix or a meal before or along with the probiotic strain to be tested. In addition, it should be able to distinguish between organisms

sensitive to the gastrointestinal transit conditions and those that have been shown to be resistant in vivo.

2.3. Assessment of survival by conventional procedures

An inoculum of each organism containing 1_109 CFU/ml was prepared. Each inoculum (0.1 ml) was used to inoculate test tubes containing 9.9 ml of the appropriate growth media acidified to pH 2.0 with HCl, or growth media containing 0.3% Oxgall bile (Difco) The tests tubes were kept at 37 8C and samples were taken at inoculation time (time 0), and at 15, 30, 45, and 90 min for enumeration of bacteria. Serial dilutions of each sample were prepared using a saline solution containing 0.9% NaCl and 0.1% bacto peptone (Difco). The pour plate method was used and Petri dishes were incubated at the optimum growth temperature of each strain. The percent survival rate was determined by comparing the growth in the acidified medium or the médium containing bile to the results obtained in the standard medium (control) at the same time. Each experiment was carried out three times.

2.4. Survival of bacteria in the dynamic model system

Irradiated kefir (412 ml) served as the food matrix. Kefir produced by Les Produits de Marques Liberte´ (Candiac, QC, Canada) was irradiated at 10 kGy. Dosimeters were used to monitor the dose received. The temperature of the irradiator was controlled at 5F1 8C. Microbiological analyses were carried out to ensure that the kefir microflora was dead when the test bacteria were added (Mainville et al., 2001). The temperature of kefir when added to the model was 6F2 8C. The survival of the individual strains was tested by mixing a selected strain into the irradiated kefir just before the beginning of the experiment. A post facto experiment showed that the irradiated kefir did not affect the survival of the bacteria under study during a time frame similar to the duration of the dynamic model study. A 10-ml culture of the strain was centrifuged at 1430_g (model GS-6R, Beckman) for 10 min, at 20 8C. The supernatant was discarded and the pellet was resuspended in 5 ml of irradiated kefir.

The cells were then added to the remaining kéfir volume to obtain 107 CFU/ml of kefir. The 90-min study period began when the kefir started to enter the stomach reactor. The kefir was added at a rate of 100 ml/min, simulating the in vivo consumption rate of the product. The model (Fig. 1) consisted of two 1-l jacketed glass beakers (Kontes, Vineland, NJ, USA) representing the stomach and the duodenum. A cover was designed to accommodate a Radiometer Copenhagen bRed RodQ pH electrode (London Scientific, London, ON, Canada), a temperature probe, and entry ports for kefir and HCl delivery into the stomach reactor. For the duodenum reactor, there were three entry ports for stomach digesta, NaOH, and Oxgall bile. A magnetic stir bar was placed inside each vessel and agitation was controlled via a magnetic stirrer plate. The temperature inside the reactors was controlled by circulating water at 37 8C through the jacketed beakers. Peristaltic pumps (Minipuls 3, Gilson, Middleton, WI, USA) were used to control the delivery of the products to be added, as well as the emptying rate of the stomach reactor into the duodenum reactor. The emptying rate of the stomach reactor was controlled to approximate the conditions in the stomach following yoghurt consumption in humans (Berrada et al., 1991). The quantity of kefir remaining in the stomach reactor was calculated by considering the rate of addition of each solution into, and the rate of emptying from, the stomach vessel. HCl addition in the stomach vessel was controlled to reproduce the Ph curve found in humans during and after milk consumption (Minekus et al., 1995). The pH of the duodenum vessel was maintained at pH 6.5. At the beginning of the meal delivery, the stomach reactor contained 17.5 ml of 150 mmol HCl to simulate the cephalic phase of acid secretion (Guyton and Hall, 2000). The rate of HCl delivery was 3.5 ml/min until the stomach reactor reached a pH of 3.0,

at which time it was lowered to 0.9 ml/min, to simulate gastrin inhibition. 1 M NaOH was added to the duodenum reactor (0.65 ml/min) to maintain the pH at 6.5 during the experiment. At time 0, the duodenum reactor contained 7 ml of a 4% Oxgall bile solution. The Oxgall solution was pumped into the duodenum reactor for the first 30 min, and then the solution was reduced to 2% for the remainder of the experiment in order to have Oxgall in the duodenum at close to 0.3% throughout the experiment. The model was attached to a Bioge´nie ID system (Ste-Foy, QC, Canada) to record pH and temperature values for the 90-min study period. The survival of each strain was measured by counting the live bacteria in a 1-ml sample taken at 30, 60, and 90 min from the two reactors. The experiment was performed four times for each bacterial strain. Survival was calculated as the ratio of the number of viable cells determined at each sampling time to the number of cells that would have been present in each reactor if all the cells had survived. The maximum number of bacteria that could be present in each reactor at each sampling time was calculated by integration of the following differential equations representing mass balance of bacteria and of the total volume in each reactor, with the assumption that bacteria are perfectly dispersed in the reactor fluid.

VP=volume of liquid in a reactor [ml] at time t [min]; Qin=sum of all fluids coming in [ml/min]; Qout= fluid pumped out [ml/min]; Cin=number of bacteria/ml of each fluid coming in a reactor; C=number of bacteria/ml within the liquid phase in a reactor. Results were reported as percent (%) survival or log removal. Log removal is defined as the log (base 10) of the survival ratio. About 1 log removal corresponds to 10% survival, 2 log removal to 1% survival, etc. This transformation linearizes the data to facilitate statistical analysis.

MÉTODO CONVENCIONAL

2.4. Concentrated cell culture preparation

Erlenmeyer flasks (1000 mL) containing 200 mL of the MRS broth (De Man et al., 1960), were inoculated with 1.5 mL of a glicerol stock culture and incubated at 37 _C ± 1 _C in a rotatory shaker (MA 830, Marconi, Piracicaba, Brazil) at 180 rpm and grown to optical density (OD, 600 nm) of 1.0. The cells were harvested by centrifugation (Hitachi, Himac CR 21E, Tokio, Japan) at 5000g for 10 min at 4 _C. Cell pellets were washed twice with 0.9% (w/v) sodium chloride solution and were finally re-suspended in 10 mL of 0.9% (w/v) sodium chloride solution. These cell suspensions were used as free cells or were aseptically mixed with 40 mL of polymer solution, according Table 1, and were applied to the immobilization system. The average cell concentrations of these polymer-probiotic mixtures were approximately 5 _ 1012 CFU mL_1.

2.7. Resistance to gastrointestinal media

2.7.1. Preparation of simulated gastric and intestinal juices

The simulated juices were prepared according to Charteris et al. (1998) and Michida et al. (2006). Simulated gastric juices (SGJ) were prepared by suspending pepsin (P7000, 1:10,000) in sterile sodium chloride solution (0.5%, w/v) to a final concentration of 3 g L_1 (1038 U mL_1) and adjusting the pH to 2.0 with concentrated HCl or sterile 0.1 mol L_1 NaOH. Simulated intestinal juices (SIJ) were prepared by suspending pancreatin USP (P-1500) in sterile sodium chloride solution (0.5%, w/v) to a final concentration of 1 g L_1, with 4.5% bile salts (Oxoid, Basingstoke, UK) and adjusting the pH to 8.0 with sterile 0.1 mol L_1 NaOH. Both solutions were filtered for sterilization through a 0.22 lm membrane.

2.7.2. Cell tolerance to gastrointestinal

The tolerance of free and immobilized cells of L. plantarum on simulated gastric and intestinal juices was determined using the adapted method described by Charteris et al. (1998). The tests were performed using a series of sterile Falcon tubes of 15 mL, one for each sampling point (see times of sampling bellow). Two different conditions were tested: in the first and second tests, 0.4 mL of the suspension of either immobilized or free cells were mixed with 1.8 mL of SGJ or SIJ, gently mixed and incubated for 120 min at 37 _C ± 1 _C. The control for these tests (CONT) was done by incubating 0.4 mL of either free or immobilized cells in 1.8 mL sterile sodium chloride solution (0.5 %, w/v) for 120 min at 37 _C ± 1 _C. After the addition of free cells or immobilized to SGJ and SIJ, the pH of these were corrected to 2.0 and 8.0, respectively, with sterile 0.1 mol L_1 NaOH or concentrated HCl. Aliquots of 1 mL were removed at 0, 30, 60, and 120 min (for all trials) for the determination of total viable counts.

2.11. Determination of total viable counts

Total viable counts of L. plantarum were determined by a pour plate method using MRS agar (except to yogurt trials) after serial 10-fold dilutions in peptone water. Plates were incubated at 37 _C for 48 h.

CONVENCIONAL (CONC POR ABSORBANCIA)

2.1. Strain and growth conditions

L. lactis G50 (FERM P-18415) was maintained by the International Patent Organism Depository (IPOD; Tsukuba, Japan). The strain wassubcultured by transferring 1% (v/v) inoculum to lactose-free M17 broth (Becton, Dickinson and Company, MD, USA) supplemented with 0.5% glucose (GM17) and incubating for 18 h at 30 °C. Under in vitro conditions simulating the GI tract, we tested the survival (resistance to lysozyme, low pH, and bile) and carbohydrate preference of lactic acid bacterial strains other than G50 to serve as reference control strains.

2.2. Resistance of cells grown on different carbon sources to GI tract stresses

Resistance to lysozyme was determined as previously described (Kimoto-Nira et al., 2008). The cells grown with different carbón sources were harvested by centrifugation for 15 min at 1840 g at 4 °C; the pellets were washed with water. One milligram of whole cells (wet weight) was suspended in 60 mM phosphate buffer (pH 6.2) supplemented with 1% NaCl and then incubated with 100 μg/ml lysozyme (egg white lysozyme, Wako Pure Chemical, Osaka, Japan) at 37 °C. The degree of cell lysis was determined by the change in absorbance at 620 nm in a Spectronic 20 spectrophotometer (Bausch & Lomb, Rochester, NY, USA). Values obtained after 1 h were expressed as a percentage of the initial value at 0 h. To determine resistance to low pH, washed bacterial cells were suspended in 0.85% NaCl (pH 6.5) and samples were plated onto GM17 agar plate for lactococci or MRS agar plate for the Lactobacillus strain, and the cells were resuspended to 0.85% NaCl adjusted to pH 2.5 (Maffei and Nobrega, 1975) with 1 N HCl. The cells were then incubated at 37 °C for 90

min (Berrada et al., 1991) and then plated onto GM17 or MRS agar plates. The number of colonies was counted following an overnight incubation at 30 °C for lactococci or 37 °C for the Lactobacillus strain.

Bile tolerance was measured by two methods; growth and survival study. For growth study, oxgall (dehydrated bile, Becton, Dickinson and Co.), dissolved in distilled water and sterilized by autoclaving at 121 °C for 15 min, was added to carbohydrate-supplemented mediato a final concentration of 0.3% (v/v). These media were inoculated with strain G50 as described previously and then cultivated. Wecultured the bacteria at 37 °C, not at the optimal growth temperatura of 30 °C for lactococci, to simulate intestinal conditions. Bacterial growth with or without bile was measured as the absorbance of cultures at 620 nm relative to uninoculated broth, and was expressed as the hours required for the optical density (OD) to increase by 0.3 units (Buck and Gilliland, 1994). For survival study, washed cells ofthe tested strains were suspended in 50 mMsodium phosphate buffer (pH 7.0) and oxgall solution was added to a final concentration of 0.3% (v/v). The mixtures were then incubated in a water bath at 37 °C. Samples were taken at 0 and 3 h, and plated onto GM17 or MRS agar plates (Kimoto et al., 2002). Colonies were counted after incubation overnight at 30 °C or lactococci for 37 °C for the Lactobacillus strain.

INTRODCUCCIÓN

Varios Compartimentos (estómago, duodeno, yeyuno, íleon y caecum) fueron virtualmente definidos en este modelo in vitro, con tiempos especificados de incubación para cada compartimento. El tiempo total de incubación fue establecido a 10.25 h con 2 h para el estómago, 0.25 h para el duodeno, 4h para el yeyuno, 3h para el íleon y 1 h para el ciego (intestino ciego). Diferentes valores de pH fueron utilizados en cada compartimento de el modelo in vitro.

DESCRIPCIÓN DEL MODELO IN VITRO

El mecanismo experimental consiste de un vaso de vidrio rodeado por una chaqueta termostática con doble entrada (vaso de vidrio doble pared con una entrada y una salida para control de temperatura por recirculación de agua a 37°C). El equipo se mantuvo con agitación magnética constante (120 rpm) para reproducir los movimientos dinámicos previstos por los músculos del tracto digestivo (fenómeno peristáltico en el intestino). Una bomba peristáltica con termostato circula agua a 37 °C en el circuito. Un potenciómetro controla termostáticamente los valores de pH en cada etapa del proceso, y fue calibrado antes de cada experimento. Los experimentos fueron hechos en una campana de flujo laminar.

CONDICIONES EXPERIMENTALES EN ESTE MODELO IN VITRO

La temperatura fue mantenida a 37°C para reproducir la temperatura del cuerpo humano. El amortiguador de fosfatos salino (PBS) fue utilizado como fluido gastrointestinal simulado (SGIF) y está compuesto por 8 g/L de NaCl, 0.2 g/L de Na2HPO4 y 1.44 g/L de KH2 PO4. El tiempo de incubación de las bacterias encapsuladas dentro del SGIF fue adaptado de los estudios de Graf, Brinch, y Madsen (2000). Los valores de pH fueron ajustados conforme a los cambios en el pH que pueden ser encontrados en la alimentación y ayunas en humanos, como sugiere Vandamme, Lenourry, Charrueau, y Chaumeil (2002). Tres posibles situaciones de SGIF en humanos fueron definidos, en relación al pH (humanos en un estado de ayuno sin comida, humanos en un estado de alimentación recibiendo una comida estándar y humanos en un estado de alimentación recibiendo una comida copiosa). Una comida copiosa es más abundante en cantidad y calidad que una comida estándar. Las condiciones experimentales en este modelo in vitro se resumen en el cuadro 1.

aEl modelo de tracto gastrointestinal consta de 5 compartimentos con un tiempo específico de incubación para cada compartimento. Los valores de pH del fluido gastrointestinal simulado (SGIF: amortiguador de fosfatos salino, 500 mL) en cada compartimento fue considerado como valores potenciales de pH del contenido gastrointestinal (sin comida, con comida estándar o con una comida copiosa o abundante). Se realizaron dos tipos de experimentos, con y sin adición de enzimas y sales biliares. La pepsina fue añadida en el estómago mientras que la pancreatina y las sales biliares fueron añadidas en el duodeno. Las enzimas y las sales biliares estuvieron presentes en los otros compartimentos (yeyuno, íleon e intestino ciego).

ENSAYOS IN VITRO DE LIBERACIÓN Y VIABILIDAD EN EL MODELO DEL TRACTO GASTROINTESTINAL

Los ensayos se llevaron a cabo con las esferas de alginato y proteína de suero lácteo que contienen las bacterias inmovilizadas. Los controles fueron las bacterias enumeradas en PBS a pH de 6.5 después del tiempo de incubación total de 10.25 h. Después de ajustar a varios valores de pH del estómago (estómago, 1.8, 2.5 o 3.0) con HCl (37%), las esferas de alginato-proteína de suero lácteo (1 g) fueron añadidas dentro de SGIF (500 mL) durante 2 h (tiempo de incubación en el estómago). A continuación el pH fue gradualmente ajustado con 10 mol/L de NaOH para llegar a los valores en el duodeno (duodeno, 4.0, 5.5 o 6.0). Después de 0.25 h de incubación, una modificación en el pH dio los valores del yeyuno (yeyuno, 6.0, 6.5 o 7.0) mantenido durante 3 h seguido por los del íleon (íleon, 7.0, 7.5 o 8.0 durante 4 h) y ciego (ciego, 7.0, 7.5 o 8.0 durante 1 h). Después de cada periodo de incubación, 1 mL de el SGIF fue mezclado con 9 mL de NaCl (9 g/L). Se llevaron a cabo diluciones seriadas de la mezcla en NaCl, y 0.1 mL de una dilución adecuada fue dispersada sobre cajas petri con agar MRS. Después de 48 h de incubación a 37 °C, el número de unidades formadoras de colonias por caja petri fue utilizado para calcular la concentración bacteriana. El factor de dilución apropiado, el volumen de SGIF (500 mL) y la masa de las esferas (1 g) fueron utilizados para convertir las UFC/mL en UFC/g. Los valores de UFC fueron expresados como la medición de 9 mediciones microbiológicas con réplicas de 3 experimentos. Los experimentos fueron repetidos con la adición de enzimas y sales biliares. La pepsina (3 g/L) de la mucosa del estomago porcino (Sigma-Aldrich) fue añadida al inicio de la incubación en el estómago. La pancreatina (10 g/L) del páncreas de bovino (Sigma-Aldrich) y sales biliares (3 g/L, mezcla de ácido cólico y deoxicólico) (Fluka, Buchs, Switzerland) fueron añadidos al inicio de la incubación en el duodeno.

RESULTADOS

El tiempo de incubación de 10.25 h fue más alto comparado a estos estudios previos. El tiempo máximo de incubación de 6 h (3 h en el estómago, 3 h en el intestino delgado) y 6.5 h (0.5 h en el estómago, 6 h en el intestino delgado) fueron respectivamente reportados por Michida et al., (2006) y Picot and Lacroix (2004).

La pepsina fue añadida dentro del SGIF al comienzo de la incubación en el estómago. Después de 2 h de incubación, las sales biliares y la pancreatina fueron añadidos dentro del SGIF. Los diferentes tiempos de incubación correspondientes al duodeno (0.25 h), yeyuno (3 h), íleon (4 h) y ciego (1 h) fueron mantenidos, y la cantidad de bacterias fueron enumeradas.

De acuerdo con las normas para la evaluación de los probióticos en alimentos reportado por un grupo de trabajo mixto (FAO/WHO) (Chesson et al., 2002), dos de las pruebas in vitro más utilizados actualmente son la resistencia a la acidez gástrica y las sales biliares, ya que se basa tanto en la supervivencia como en estudios de crecimiento.

METODOLOGÍA

Se utilizó un diseño Plackett-Burman para seleccionar los parámetros que pueden afectar la sobrevivencia de las cepas probióticasdurante el paso al estomago simulado (Plackett y Burman, 1946). Este diseño estadístico permite la identificación de variables de respuesta para los principales efectos en un modelo de estudio con relativamente pocos experimentos. En este estudio, un diseño de 8 corridas fue utilizado para analizar las variables significativas que afectan la sobrevivencia de las bacterias probióticas en experimentos in vitro. Las variables incluidas: efecto protector de 15% p/v de leche descremada, composición del medio (MRS o solución electrolítica), presencia de lisozima (100 ppm), presencia de pepsina (0.3%), y pH del medio.

Análisis de viabilidad de las BAL al paso sucesivo por saliva artificial, jugos gástricos y jugos intestinales

Se adoptó un modelo experimental estómago/intestino para analizar la supervivencia de las posibles cepas probióticas, de acuerdo al modelo desarrollado por Vizoso et al., (2006) con algunas modificaciones. De un cultivo en caldo MRS de 24 h a 37 °C se toma 1 mL para realizar cuenta enplaca por diluciones seriadas. Del cultivo anterior se toma una muestra con 1x109 UFC/mL, se inocula nuevamente en caldo MRS y se determinan las UFC/mL para cuantificar la concentración de microorganismos a tiempo 0. Para simular la dilución y una posible hidrólisis de las bacterias en la cavidad oral humana, la suspensión de caldo

MRS + bacterias se diluyó 1:1 en una solución electrolítica esteril que simula la saliva (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2 y 1.2 g/L de NaHCO3) con una concentración final de lisozima de 100 ppm y se incubó durante 5 min a 37 ° C. Se determinará cuenta viable por diluciones seriadas para monitorear la concentración en UFC/mL en agar MRS. Posteriormente la muestra se diluye 3:5 con jugo gástrico artificial, que consiste en la solución electrolítica mencionada anteriormente ajustada a pH 2.5 y con 0.3% de pepsina (Sigma). Si es necesario, ajustar el pH a 2.5 con HCl 5 M. Después de 1 h de incubación a 37 °C, otra alícuota de 1 ml se retiró y se determinó cuenta viable en agar MRS. Para simular la dilución en el intestino delgado, el volumen restante de la simulación en jugo gástrico se diluye 1:4 con la secreción artificial duodenal (pH 7.2) que consiste en 6.4 g/L NaHCO3, 0.239 g/L KCl, 1.28 g/L NaCl y 0.1% de pancreatina. De esta última etapa, se probarán cuatro experimentos, tres con 0.5% de sales biliares diferentes (oxgall, ácido deoxicólico y ácido turocólico) y uno al que se le adicionará colesterol. A tiempos de 2 y 3 h se tomarán alícuotas de 1 mL para determinar por cuenta viable en agar MRS, las UFC/mL finales.

Finalizado el pase sucesivo por las condiciones gastrointestinales simuladas, las cepas que sobrevivieron serán sembradas masivamente, propagadas y conservadas para las posteriores pruebas.

Vizoso, P. M. G., Franz, C. M. A. P., Schillinger, U. and Holzapfel, W. H. 2006. Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products. Int. J. Food Microbiol. 109: 205-214.