The effect of fermented milk containing lactobacillus

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THE EFFECT OF FERMENTED MILK CONTAINING LACTOBACILLUS CASEI ON THE IMMUNE RESPONSE TO EXERCISE

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P. PUJOL a,* , J. HUGUET b , F. DROBNIC a 9 M. BANQUELLSa, O. RUIZ a , P. GALILEA a , N. SEGARRA b , S. AGUILERA c , A. BURNAT c , J.A. MATEOSc and E. POSTAIREd a

Department of Physiology, Olympic Training Center, Sant Cugat del Vallés, Barcelona, Spain; b Laboratorio Echevarne, Barcelona, Spain; c Nutrition Service Danone S.A., Spain; d Nutrition Group, CIRDC, Danone, Le Plessis Robinson, Paris, France (Received 22 March 1999; Accepted 10 November 1999)

There is evidence that exhaustive exercise produces depression of the immune system, especially on the number and activity of Natural killer (NK) cells. On the other hand, fermented milk has been shown to moderate the immune response by inducing NK activity. The present work was carried out to determine if a Lactobacillus casei (LC) fermented milk supplemented diet would provide protection of the immune system against an exercise induced immune system depression of NK cells. Twenty-five athletes were selected out of 94 for their significant decrease in NK cell concentration compared with a normal basal concentration in plasma 2 h after an exercise stress test. Subjects ingested a daily fermented milk diet with LC for one month and a standard milk diet also for one month. After each phase of dieting, a subject was investigated before, 5 min and 2 h after an exercise stress test, testing for NK cells and IL-1β IL-6, IL-2, IFNγ, IgA, IgM, IgG, NK cells, CD8, CD4, CD3 and sIL-2 receptor. A significant smaller decrease of NK cell concentration after 2 h was found in the fermented milk feeding phase vs. the standard milk period. Keywords: Immune system; natural killer cells; Lactobacillus casei; fermented milk; intensive exercise

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INTRODUCTION There is growing interest in the effect of exercise on the immune system. This is mainly due to the fact that exhaustive exercise has been shown to depress the immune system and there are a number of reports implicating mild infectious disease with reduced performance by athletes. One of the constant findings in studies about immune depression and exhaustive exercise has been a decrease in the number and activity of Natural killer (NK) cells following intense and prolonged exercise (Kappel et al, 1991, Nieman et al, 1993, Pedersen et al, 1990; 1994, Shinkaiefû/., 1992). It has been postulated (Nieman et al, 1995; 1997, Northoff and Berg, 1991, Pedersen et al, 1994, Roitt et al, 1989, Shephard et al., 1996, Woods et al, 1999) that NK cells together with macrophages and peripheral blood mononuclear cells constitute a primary and extended defense mechanism. Several studies (Kato et al, 1983, Perdigón et al, 1986) performed on animals and humans suggest that lactic acid bacteria or fermented milks produce a beneficial effect on the immune system. Among these effects, the most constantly reported is an enhancement of NK cell activity, production of antibodies, and proliferation of T and B cells (Nieman et al, 1995; 1997, Pedersen et al, 1990; 1994, Weinstock et al, 1997, Woods etal, 1999). Lactobacillus casei {LC) is a microorganism used in fermented milks for human nutrition. LC has been reported to be partially resistant to gastric and bile acids and can be delivered to the gastrointestinal tract where, it has been demonstrated, to modify the intestinal milieu by providing protection against intestinal infection (Guérin et al, 1998). Part of the protective effect against enteropathogens has been attributed to its modulation of the immune response by acting to precipitate a delayed hypersensitivity, by antibody formation, and by induction of NK cell activity. Kato et al, 1983 reported induction of NK cell in mice by intraperitoneally-administered LC. Also Perdigón et al, 1986 demonstrated activation of macrophage function by LC administered orally and intraperitoneally. These authors suggested that lactobacilli could be useful as immuno therapeutic agents. The purpose of this study was to investigate the effect of ingestion of fermented milk with LC in a group of recreational athletes who presented a decrease in NK cell number following a very intense stress test.

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SUBJECTS AND METHODS


Subjects A group of 99 male and female subjects gave their written consent to participate in the study and the protocol received the approval of the Ethical Committee of Olympic Training Center (CAR). The subjects were recreational athletes who practiced aerobic physical activity during a minimum of three times a week, for more than 30 min at an intensity of 60% of their VO2max or more. All subjects were asked to refrain from taking any ergogenic aid they may have been using for at least two months previous to their being recruited for the study. Subjects with known hypersensitivity or intolerance to milk products in general were excluded, as were those who were on special diets, as for weight reduction, or because they were vegetarians. Also excluded were subjects who had experienced episodes of exercise induced asthma and those that had been on a course of antibiotics or other prescribed drug treatment within one month prior to the start of the study. Pregnant females were not eligible for the study. A urine pregnancy test was performed in all female subjects. The characteristics of the subjects are depicted in Table I. Design of the Study The study consisted of two parts. In the first part every subject performed a graded cycle ergometer exercise test in order to obtain his or her VO2maxFollowing a warm-up of 5 min with a power output of 50 W, the power was increased 30 W every 3 min until volitional fatigue. Throughout the test VO2, pCO 2 , pO 2 ventilation and respiratory quotient were recorded every 30 s and EKG was also continuously recorded. For blood lactate measurement 20 JXL of venous blood from the earlobe was taken immediately before the test and 5 min after the test. The tympanic membrane temperature was measured before the test and 5 min after the test. The lactate analysis was performed by a photoenzymatic method measured on a photometer 4020 Hitachi/Boehringer, Mannheim. The tympanic membrane TABLE I

Age (years) Body mass index (kg/m 2 ) VO2max (mL/min/kg)

Characteristics of subjects Mean

Range

27 22.44 43.39

18-41 19.08-27.42 33.6-77.9


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temperature was measured with an Infrared Temperature Scanner (Ototemp 3000 SD Natick, MA). After a rest of one week to ten days, a second stress test was performed on the cycle ergometer at 75% VC^max for 60 min. Prior to the maximal stress test, a personal and family health history was taken from each participant, together with a complete blood analysis including blood count, cholesterol, HDL, LDL, glucose, uric acid, urea, GOT, GPT, and proteins. Any subject who dropped out was replaced with another one. Each subject reported separately to the laboratory after a light breakfast (without milk or any other lactic products). After resting for approximately 20 min, each subject exercised at a power of 50 W for 5 min and thereafter at 75% of VChmax for 60min at 60rpm. During the test, ventilation ( V E ) , O 2 uptake (VO2) and CO 2 output (VCO2) were monitored at 30, 45 and 60min respectively with Jaeger Eos-Sprint equipment (Erich Jaeger Gmbh, Würzburg, Germany) calibrated prior to every test. This equipment is composed of an oxygen paramagnetic analyzer and a FleischType pneumotacograph. Heart rate from an EKG record was also continuously recorded throughout the test. Blood for analysis of NK cells and other immune parameters was drawn from the right antecubital vein with the subject lying recumbent, before the test, and twice more at 5 min, and 2 h after the test. Blood lactate concentration was measured before the test and 3 and 5 min after the test and tympanic temperature was measured before and 5 min after the test. The temperature measure was made because it has been shown that hyperthermia can induce an immunological response which resembles the change observed in relation to exercise (Hammami et aL, 1998, Pedersen et al, 1994). Second Phase of the Study Twenty five subjects out of 94 (5 subjects out of the 94 dropped out) were selected for the second phase of the study. There were no more dropouts. The selection was based on the fact that the subjects showed a significant decrease (more than 3%) in the number of NK cells compared with the basal cell concentration in plasma 2 h after the test. In the second phase of the study a selected cohort of subjects ingested 500 mL per day of fermented milk with LC during one month and also ingested 500 mL of milk per day for another month with a wash out period of 30 days in between. The fermented milk with LC was provided by Danone S.A. Actimel rM . The supply of 1 month's amount of fermented milk with LC to the subjects took place in spaced periods and interimly the milk was kept at 2-6°C. At the


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end of the total period all subjects performed a stress test of 60 min at 75% of their VC>2max on the cycle ergometer at 60rpm. In each test blood was taken from an antecubital vein before the test and again at 5 min and 2 h following the test, for analysis of various immune cell concentrations. A diet history was obtained from each subject two days before each test (both post-fermented milk with LC milk ingestion) and was analysed using the Professional Diet Balancer (Nutridata Software Co., Wappingers Falls, NY, USA) software package. This software has a data base of 1600 foods with 23 nutrient values provided for each food. The program compares the total of each nutrient reported by the subjects with the US recommended daily allowance (RDA) based on weight, age, sex and physical activity or energy expenditure. It was of interest to measure the percentage of carbohydrates in their total calorie intake. This is important since Nieman et al, 1997 demonstrated that carbohydrates affect natural killer cell redistribution. Fermented Milk with LC The fermented milk with LC was prepared with traditional yoghourt cultures (L. bulgaricus and S. thermophilus) in addition with LC (strain DN-114 001). The presence of at least 10 colony forming units per mL (cfu/mL) of yoghourt bacteria has been confirmed; it contained 3.2 x 108cfu/mL of LC, grown on acid MRS with OXGALL. The nutrient composition is: 3.7 g protein, 3.3 g fat, 5.2 g carbohydrates per 100 g. Methods Sample Collection and Serum Preparation Twenty mL of venous blood were drawn from each athlete. Ten mL were collected into commercially available heparinized tubes (Sarstedt, Germany) and lOmL were allowed to clot for 30 min at room temperature into lOmL tubes (Sarstedt, Germany). Tubes were centrifuged (1500 g) at room temperature and the serum was withdrawn. All samples were tested for immunoglobulins within the next 3 h and subsequently stored at —30°C before cytokine testing. Blood Cell Counts To determine lymphocyte subsets, 100 jiL of heparinized blood was mixed with 10 yiL of selected monoclonal antibodies (mab) conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE) or phycoerythrin-Texas

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FIGURE 1 Whole blood stained with CDS-FITC/CDló + CDSó-PE and analyzed on EPICS XL-MCL flow cytometer. Window acquired for Forward Scatter vs. Sample Size. Dot plot 2 displays FL1 (CD3-FITC) vs. FL2 (CD16 + CD56-PE) gated on lymphocyte region (A). Square Bl and linear region D displays the percentage of NK lymphocytes (CD 16/ CD56+ and CD3—) while square B4 and linear region C exhibits the percentage of T lymphocytes (CD34- and CD16/CD56-).

Red (ECD) in the following staining combinations: anti-CD4 rnab (PE)/ anti-CD8 mab (FITC)/anti-CD3 mab (ECD) from Coulter (Coulter Corp., USA), anti-CD19 mab (PE)/anti-HLADR mab (FITC)/anti-CD3 mab (ECD), anti-CD14 mab (PE)/anti-CD64 mab (FITC) from Immunotech (Immunotech. France) and anti-CD16 + CD56 mab (PE)/anti-CD3 mab (FITC) from Becton Dickinson (Becton Dickinson, USA) (Fig. 1). After a 15 min incubation in the dark, the samples were lysed with ImmunoPrep solution in the Q-Prep workstation (Coulter Corp., USA). The lysate was immediately analysed using Coulted Epics XL-MCL flow cytometer. The number of cells counted was 10,000 per sample. Findings were expressed as the percentage of cells yielding a specific fluorescence in a gated lymphocyte region, except for the CD14/CD64 that were gated in the monocyte region. Natural Killer Activity Peripheral blood mononuclear cells were isolated from heparinized blood by density gradient centrifugation. Eight mL of whole blood was diluted 1:1 with PBS. It was layered over 5mL of Lymphoprep (Nycomed, Norway) and centrifuged (600 g) for 20 min 20°C, The mononuclear cell layer was removed and washed twice with RPMI 1640 (GIBCO) supplemented with 10% AB human serum (GIBCO). The pellet was reconstituted at a concentration of 2 x 106 cells/mL (effector cells). NK activity was assessed by the EuTDA cytotoxicity assay. This method has been developed by Blomberg et al, 1996 and is based on loading target cells with acetoxymethyl ester of fluorescence enhancing ligand (bis (acetoxymethyl ) 2,2' : 6' ,2'-terpyridine- 6', 6"-dicarboxylate) ( B ATD A).



The ligand penetrates the cell membrane quickly. Within the cell the esterbonds are hydrolysed to form a hydrophilic ligand (2,2;: Q,Hf~ terpyridine6,6"-dicarboxylic acid (TDA) which no longer passes the membrane. After cytolysis the ligand is released and introduced to an Eusolution (Eu). The Eu and the ligand form a highly fluorescent and stable chelate (EuTDA). The measured signal correlates directly with the amount of lysed cells. These authors showed that in a NK cell assay the new EuTDA release assay shows good correlation with 54Cr-release assay. Two mL of K562 (ATCC-CCL 243) adjusted to 1 x 106 cells/mL were loaded with 5|iL of fluorescence enhancing ligand (BATDA) at 20°C during 15 min. The cells were washed 6 times with PBS with 10% BSA. Finally the pellet was adjusted with RPMI 1640 to 5 x 104 cells/mL (target cells). A hundred \iL of these cells were added per well to a microtiter plate (Sarstedt, USA) containing effector cells to give 40:1, 20: 1, 10:1 and 5:1 effector target rations. The microtiter plate was incubated for 2 h at 37°C in a 5% CO2 incubator. At the end of the incubation, the plate was centrifuged for 5 min at 500 g. Then 20 jiL of the supernatant was transfered to a flat-bottom plate (DELFIA microtitration plate); 200 |j,L of Eu solution was added to each well which were incubated for 15 min at room temperature on a shaker. Finally the fluorescence was measured on a 1232 DELFIA time-resolved fluorometer (Wallac). Controls included spontaneous release (100 (iL of RPMI 1640 medium added to the labelled target cells) and total release (100 jiL of RPMI 1640 medium supplemented with 10% Tween, added to the labelled target cells). Lysis percentage was calculated using the counts for each E : T ratio: ni,

. (test release — spontaneous release) % lysis = ^ — : J- x 100 (total release — spontaneous release) Results were normalized to lytic units (LU), calculated as the number of effector cells required to lyse 20% of 5 x 103 target cells, reported as the number of LU contained in 1 x 107 cells. Intraassay coefficient of variation was 3 %. Immunoglobulins

The quantitative determination of immunoglobulins (IgG, Ig A and IgM) was measured by nephelometry using BNII (Behring Nephelometer II). The reference curves are constructed by multi-point calibration based on an international standard IFCC CRM-470 (Community Bureau of References Commission of the European Communities, EUR 15243EN, ISSN1018-5593:1-172, 1993). In the serum assay protocol the serum samples are

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automatically diluted and measured; the dilutions used were 1:20 (IgA and IgM) and 1:400 (IgG). The results were expressed in mg/dL. Cytokimes Testing for Interleukines: IL-1 beta, sIL-2R and IL-6 was done by Chemi» luminescent Enzyme Immunometric Assay with an automatic analyzer IMMULITE (Diagnostic Products Corp. DPC, USA). The detection limits were: 5 pg/mL for IL-1 beta, 10 U/mL for sIL-2R and 2pg/mL for IL-6. IL-2 and INF gamma testing was done by ELISA Immunotech (Immunotech, France) according to the manufacturer's instructions. The detection limits were 10 pg/mL for IL-2 and 0.5 U/mL for INF gamma.


Statistical Analysis Variables measured on interval or dimensional scales were reported using central trend measures (mean and median) as well as scope or dispersion measures (standard deviation and quartiles). A Kolmogorov (see, e.g., Fleiss, 1986) test was performed to test normality of variables. Student's ¿-test for paired samples was performed to compare the two phases of experimentation when normality hypothesis could not be rejected. In those cases where normality hypothesis could be rejected, a nonparametric Wilcoxon test for paired samples was performed. Significance was accepted for p values lower than 5%. (SAS software, release 6:12; SAS Institute, Gary, NC was used).

RESULTS AND DISCUSSION Stress tests performed under both milk and fermented milk treatment showed the same degree of increase in tympanic temperature from baseline values to 5 min (Fig. 2). In no case did the temperature rise above 37°C. It has been demonstrated that rectal temperature is 1°C higher than tympanic temperature. It has been shown by Pedersen and Ullum, 1994 that a core temperature of 39°C can induce an alteration in the immune system that resemble the change observed in relation to exercise. Therefore, no interference with the immune system following exercise could have been due to the present increase of temperature observed in the present study. The baseline blood lactate concentration and the increase at 3 and 5 min after exercise did not show any significant difference between the two treatment schedules (Fig. 3).


Mean SD

N=25

38 37.5H 37

P