Effects of Feeding Lactobacillus spp . on the Level of Cell Glutathione Sulphydryl and Immunoglobulin M in ICR Mice

Effects of feeding seven strains of Lactobacillus spp. on the level of cell glutathione sulphydryl (GSH) in spleen, liver and erythrocyte of the ICR mice and on the level of immunoglobulin M in the spleen were determined. The level of cell glutathione sulphydryl in the spleen was dependent on the strain of Lactobacilli, it was significantly higher in the mice fed with L. casei CU 001, L. rhamnosus CU 02, L. acidophilus NCFM and L. casei YIT9018 (p>0.05). The level of cell glutathione sulphydryl in the liver increased in the mice fed with L. casei YIT9018, L. acidophilus NCFM, L. casei CU 001 (p>0.05), the level of glutathione sulphydryl of the erythrocyte showed significantly higher value than control mice when fed with L. acidophilus NCFM, L. casei YIT9018, L. casei CU 001 (p>0.05). The level of immunoglobulin M in the spleen of ICR mice expressed as the plaque count revealed significantly higher value than the control mice when fed with L. casei CU 001, L. acidophilus NCFM and L.casei YIT 9018. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 3 : 415-419)


INTRODUCTION
Lactobacillus spp.are one of the important component group of intestinal microflora which could afford the host animal not only with specific or nonspecific immunopotentiating capacity but with an antagonistic activities against pathogenic bacteria (Crawford, 1979;Perdigon et al., 1990;Yoon and Won 2002).Whey proteins consisting of alpha-lactalbumin and beta-lactoglobulin could be hydrolyzed and synthesize tripeptide glutathione, an important antioxidant defence mechanism in living cell, which can be used in the tissue as an antioxidant removing hydroxyl ions in the cell to form paired sulfhydryl molecule (Bounous et al., 1983;Bounous and Gold 1991;Tammy and Taylor, 1994).They could stabilize DNA molecule during the course of cell multiplication (Anderson, 1985;Carol et al., 1993) and it has been known that sulfur containing amino acids cysteine, glutamic acid and glycine are important constituents synthesizing glutathione molecule which could play an improtant role in the immune system as a precursor of immunoglobulin (Rose, 1980;Bounous et al., 1989;Yihong et al., 1998).
This study was aimed for finding out the effects of lactobacilli on the synthesizing glutathione molecules in the laboratory animal ICR mouse system, based on the consideration that the proteolytic activities of Lactobacilli are variable depending on the species and strains of the lactobacilli (Salminen et al., 1996).

Bacterial strains and media
The strains and sources of bacteria used in this study are given in Table 1.Lactobacillus spp.were cultured in MRS broth (Difco, USA) at 37°C and maintained in 11% skim milk containing 0.75 M adonitol at -70°C.

Mice
Male specific pathogen free ICR mice were obtained from Daehan Biolink (Choongbook, Korea) and were acclimated for at least 1 wk before use.The mice were kept under the condition of 12 h light-dark cycle at a controlled temperature (22±2°C) and were supplied with water and feed ad libitum and experimental design is shown in Table 2.

Plaque forming cell assay
Ig M plaque forming cell were determined by the procedures described by Cunningham and Szenberg (1968).On the fifth day after immunization, spleen cell suspensions were prepared by gently tamping the spleen through a 50 mesh stainless steel screen, and collecting the cells in Earle's balanced salt solution (BSS, Sigma E3261, USA) supplemented with 10% heat inactivated calf serum.The spleen cells were washed with BSS and made up to 15ml with BSS.Sheep red blood cells were washed twice with BSS and made up to a 20% concentration.Guinea pig serum (Komed Ltd.Seoul, Korea) as a source of complement was diluted 1/15 with BSS.All stock solutions were kept on ice water until used.The test consisted of mixing 0.05 ml of spleen cells, 0.15 ml of sheep red blood cells complement solution in a test tube at 37°C.The whole mixture was immediately withdrawn and put into slide chambers, sealed with warm paraffin wax, and incubated at 37°C for 60 min.The number of plaque forming cells was counted and the total number of plaque forming cells per spleen was estimated by multiplying in each sample (0.05 ml spleen cells) by 300.Plaque forming cells have been expressed per total organ, since this appears to reflect more accurately the functional status of the spleen per se (Cunningham, 1965).

Determination of cell glutathione of spleen, liver and erythrocyte
Spleen and liver was washed in ice-cold 0.9% NaCl.Ninety mg of tissue was homogenized by homogenizer, centrifuged at 3,000×g for 10 min at 4°C, took 50-100 µl of supernatant as a sample and determined the content using assay kit (Calbiochem, USA) following the manufacturers directions.The total volume was adjusted to 900 µl with buffer (Solution 3), 50 µl of solution R1 was added and mixed thoroughly.After adding 50 µl of solution (R2) and mixing thoroughly, the reaction mixture was incubated at 25°C in the dark and A 420 was measured (Anderson, 1989).500 µl of whole blood was taken from the heart into the heparin treated tube, centrifuged at 2,500×g for 10 min at 4°C and erythrocyte pellet was collected, glutathione in the erythrocyte was determined by the assay kit (Calbiochem, USA).The results were expressed as µmol/g.

Statistical analysis
Within the same treatment group, for the comparison of the glutathione and IgM values between the treatments, values were compared using SAS Duncan's multiple-range test.

Effects of feeding Lactobacillus spp. on the level of spleen cell glutathione sulfhydryl (GSH)
The spleen glutathione levels were shown in Figure 1.Mice fed with control diet containing whey protein concentrate and basal diet (20/80) was determined as containing 4.03 µmol/g of reduced glutathione per spleen, whereas mice fed with the diet fortified whey protein isolate (30/70) contained 4.14 µmol/g of glutathone were higher than that of Bounous et al. (1989).The difference between the values of mice fed on control diet and mice fed the fortified diet with whey protein was not statistically significant.
The glutathione contents in spleen of mice fed on the diet fortified with Lactobacilli (2×10 9 cfu/ml) were higher than that of control mice fed on control diet.But all treatments do not show statistically significant difference, only mice fed on L. casei CU 001, L. rhamnosus CU 02, L. acdidophilus NCFM and L. casei YIT 9018 had significantly higher in spleen glutathione than mice fed on control diet (p<0.05).Beneficial activities of lactic acid bacteria could be explained on the basis of the boosting effect of cell glutathione, an antioxidant in cell, which neutralize free radicals by handing off an electron then pairing off to remain neutral themselves.The destructive hydroxyl radicals is changed to H 2 O by getting electron from glutathione sulfhydroxyl molecule (Anderson, 1985).Many kinds of reactive oxygen species can be formed in the human body (oxidative stress), free radicals can damage or destroy cell walls causing apoptosis and disrupt DNA, then pathogenesis could be proceeded.Existence of enough antioxidants is effective for the reduction of reactive radicals by scavenging reactive oxygen species, otherwise glutathione depletion and the subsequent low stores of protein thiol result in both calcium release from intracellular calcium and cytotoxicity (Anderson,1985;Bounous and Gold, 1991) Elevated scavenging of reactive oxygen by glutathione would be a good trait of Lactobacilli.

Effects of feeding Lactobacillus spp. on the level of liver cell glutathione sulfhydryl (GSH)
The liver glutathione levels were shown in Table 3.In the liver cell of mice fed on control diet, the concentration of reduced glutathione was 9.31 µmol/g, whereas that in the liver cell of mice fed on the diet fortified with whey protein isolate (30/70) was 10.12 µmol/g.The difference between whey protein fortified diet and control diet showed no statistical significance, a similar degree of glutathione increasing effects were shown in the group fed on L. acidophilus 4356 and L. rhamnosus GG ATCC 5310.
All the Lactobacilli treated groups except those above two groups significantly higher liver glutathione level than control (p<0.05), and they could be divided into two groups on the basis of glutathione level.The modrately higher group includes L. helveticus CU 631 and L. rhamnosus CU 02, and intensively higher group is L. casei CU 001, L. acidophilus NCFM, and L. casei YIT9018.Those effects by the Lactobacilli were dependent on the species or strains of lactic acid bacteria.average level of glutathione in the liver cells were different, approximately twice of that in spleen, the glutathione level increasing effects of Lactobacilli were similar with those in spleen showing the different glutathione synthesis capability in metabolism of each lactobacilli (Fahey et al., 1978).

Effects of feeding Lactobacillus spp. on the level of erythrocyte cell glutathione sulfhydryl (GSH)
The erythrocyte glutathione levels are shown in Figure 2. Mice fed on control diet showed containing whey 2.33 µmol/g of reduced glutathione, whereas erythrocyte of mice fed on the diet fortified with whey protein isolate (30/70) contained 2.70 µmol/g, significantly higher than that of control diet..
The difference between the increased value by feeding whey protein fortified diet and that of control diet showed statistical significance.
All the lactobacilli treated group showed significantly higher erythrocyte glutathione level than control (p<0.05).The treated group could be divided into three groups on the basis of glutathione level, a modrately higher group including L. helveticus CU 631 and L. rhamnosus ATCC 5310, a higher group of L. acidophilus ATCC 4356 and L. rhamnosus CU02, and an intensively higher group consisting L. casei CU 001, L. acidophilus NCFM, and L. casei YIT9018.Those effect of Lactobacilli on glutathione content in erythrocyte were similar in spleen and liver showing that the value of erythrocyte glutathione level seemed to be the indicative to those of spleen and lever, and could be used as an indicative values.
The level of cellular glutathione depends upon the supply of its constituent amino acids, of which cysteine is usually limiting, and the upper level of cellular glutathione is controlled by feedback inhibition of gammaglutamylcysteine synthetase by glutathione.Thus enhancement of glutathione levels by the lactic acid bacteria could be achieved by the supply of cysteine by the variable proteolytic activity of lactobacilli (Fahey et al., 1978;Johnston et al., 1993).

Enhancement of Ig M level by feeding the Lactobacilli
Immunoglobulin M levels in the spleen of mice was determined by the IgM plaque forming cell assay methods (Cunningham and Szenberg, 1968) and the results are shown in Figure 3. Mice fed with control diet containing whey protein isolate and basal diet (20/80) were determined as containing 128×10 3 plaque forming cells per spleen, whereas mice fed with the diet fortified whey protein isolate (30/70) contained 132×10 3 plaque forming cells per spleen.The difference between the increased value by feeding whey protein fortified diet and that of control diet showed no statistical significance.The results of control diet and control diet and diet with Lactobacilli (2×10 9 cfu/ml) revealed significantly different.All the lactobacilli treated group showed significantly higher plaque forming cells than control (p<0.05),L. casei CU 001, L. acidophilus NCFM exerted a most intensive immunomodulating effect.Those effects by the Lactobacilli were dependent on the species or strains of lactic bacteria.As dietary cysteine content is a rate limiting substrate for the synthesis of glutathione, which is necessary for the proliferation of lymphocyte, modulation of intracellular glutathione by feeding lactic acid bacteria may have affected immune responsiveness enhancing immunomodulatory activity.
In vivo administration of GSH has been demonstrated to enhance the activation of cytotoxic T-cells, but depletion of GSH intracellularly inhibited the activation of lymphocytes, increased susceptibilities of human lymphoid cells to radiation, suggesting that intracellular GSH can modulate the function of immune cells, and a deficiency in GSH may contribute to the immunodeficiency (Roederer et al., 1992).There seemed a tendency that a higher GSH level pararelled a higher level of IgM forming plaque cells in this experiment.

Table 1 .
Sources of strains of Lactobacillus spp.used in this investigation

Table 2 .
Experimental design of mouse trial

Table 3 .
Liver glutathione (GSH) contents of mice fed with whey protein isolates (WPI) and administrated with Lactobacillus spp.