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Influence of feeding fermented milkand non-fermented milk containingLactobacillus casei on immuneresponse in miceSuman Kapila a , P. R. Sinha a & Surjeet Singh aa Animal Biochemistry Division , National Dairy ResearchInstitute , Karnal, IndiaPublished online: 03 Apr 2007.

To cite this article: Suman Kapila , P. R. Sinha & Surjeet Singh (2007) Influence of feedingfermented milk and non-fermented milk containing Lactobacillus casei on immune response inmice, Food and Agricultural Immunology, 18:1, 75-82, DOI: 10.1080/09540100701317618

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Influence of feeding fermented milk andnon-fermented milk containing Lactobacillus casei

on immune response in mice

SUMAN KAPILA, P. R. SINHA, & SURJEET SINGH

Animal Biochemistry Division, National Dairy Research Institute, Karnal, India

(Received 4 January 2006; accepted 5 March 2007)

AbstractThe effect of feeding Lactobacillus casei in the form of fermented and non-fermented milk onnon-specific as well as humoral immune response in mice was investigated. Feeding of L. caseifermented milk (LcF), its cell free supernatant (LcS), and L. casei non-fermented milk (LcNF)to mice for period of two, five and eight days resulted in increased activity of b-galactosidase andb-glucuronidase in peritoneal macrophages in comparison to skim milk (control). Thephagocytic activity of peritoneal macrophages also increased significantly (p B0.01) in micefed on LcF, LcS and LcNF compared to skim milk-fed mice. The maximum rise in b-galactosidase, b-glucuronidase and phagocytic activity was on day 5 post-feeding. In challengestudies with Shigella dysenteriae , it was observed that colonization of S. dysenteriae in theintestine, liver and spleen was significantly less in mice fed on LcNF and LcF in comparison tomice fed on skim milk for a period of seven days before challenge. Levels of secretory antibodieswere higher in groups fed LcNF and LcF. The results suggest the immunomodulatory potentialof L. casei .

Keywords: Lactobacillus sp., Shigella dysenteriae, fermented milk, phagocytosis.

Introduction

Lactic acid bacteria (LAB) and cultured dairy products have been reported to possess

a beneficial effect on human health, particularly for the treatment of intestinal

disorder, attenuation of lactose intolerance, and hypocholesterolemic (Kapila et al.

2006), and anticarcinogenic activity. LAB also exerts positive effects on allergy and

experimental autoimmune diseases (du Toil et al. 1998; Salminen et al. 1998).

Moreover, reports indicate that fermented milk has a profound influence on the

immunological state of the host (Perdigon & Pesce de Ruiz Holgado 2000; Von der

Weid et al. 2001). It has also been demonstrated that LAB, particularly lactobacilli,

can confer protection against experimental infections by enteropathogens in mice

(Matar et al. 2001). Some lactobacilli, when administered orally to mice, activate

macrophages and induce the release of lysosomal enzymes (Singh & Kansal 2003).

Apart from non-specific immune response, ingestion of Lactobacillus casei protect

against infection with Salmonella typhimurium and Escerichia coli (Perdigon & Pesce de

Correspondence: Suman Kapila, Animal Biochemistry Division, National Dairy Research Institute, Karnal

132001, India. E-mail: suman_ndri@yahoo.com

ISSN 0954-0105 print/ISSN 1465-3443 online # 2007 Taylor & Francis

DOI: 10.1080/09540100701317618

Food and Agricultural Immunology, March 2007; 18(1): 75�82

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Ruiz Holgado 2000). The present study was carried out to elucidate the effect of

feeding L. casei NCDC19 in the form of fermented milk, non-fermented milk, and its

cell free supernatant on non-specific immune response (macrophage activity) and

specific immune response (secretory IgA antibodies) by using Shigella dysenteriae.

Materials and methods

Microorganisms

Lactobacillus casei (NCDC 19) and Saccharomyces cerevisiae (NCDC 40) used in this

study were obtained from the National Collection for Dairy Cultures, National Dairy

Research Institute, Karnal. The Lactobacillus culture was maintained in MRS broth,

and the yeast in Potato dextrose broth. An isolate of S. dysenteriae (All India Institute

of Medical Science, New Delhi) was grown in brain heart infusion broth at 378C for

18 h. Lactobacillus casei NCDC19 was used in the preparation of fermented milk

(LcF) by inoculating at 1% in sterile skim milk (10% non-fat milk) for 18 h at 378C.

The final product contained 1.3�109 cfu/g. Non-fermented milk (LcNF) was

prepared by growing L. casei in MRS. The cells were harvested by centrifugation,

and washed several times before suspending in sterile skim milk. The final product

contained 1.1�1.3�109 cfu/g. Cell free supernatant (LcS) was prepared by

centrifuging fermented milk at 10 000�g for 15 min at 48C, and by filtering the

supernatant through 0.22 u Millex GV disposable filter unit to remove cells.

Animals and feeding procedure

Swiss albino mice, weighing 30�40 g, were kept as per the norms of the Institutional

Animal Ethics Committee (IAEC). The mice were fed on a synthetic diet containing

10% fat and 15% protein ad libitum along with any of the above mentioned

supplements (5 g/day). For studying non-specific immune response, the animals

were divided into 12 groups of five mice each. Groups 1�3 were fed on LcF; groups

4�6 were given LcS; groups 7�9 were fed on LcNF, and Groups 10�12 received

equal amounts of skim milk for two, five and eight days, respectively.

Macrophage culturing and enzyme activity

Mice were sacrificed by cervical dislocation and the peritoneal cavity macrophages

were collected with 5 ml of Dulbecco’s Modified Eagle Medium (DMEM) Ham’s F12

(without Phenol red) following gentle massage of the abdomen. The medium was

supplemented with sodium bicarbonate (1.2 g/l), bovine serum albumin (1 g/l),

penicillin (200 U/ml), streptomycin (50 ug/ml) and the pH was adjusted to 7.2.

Macrophages were distributed into culture grade petri dishes and incubated in a

humidified atmosphere at 5% CO2 in air at 378C for 2 h to allow attachment of

adherent cells (Perdigon et al. 1986a). Non-adherent cells were removed. After

washing, the adherent macrophage were cultured in DMEM Ham’s F-12 medium for

18 h. The activities of the secreted enzymes were assayed in the culture supernatant as

well as in peritoneal fluid. b-Glucuronidase was assayed by the method of Stossel

(1980) and b-galactosidase according to Conchie et al. (1959).

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In vitro phagocytosis assay

The phagocytic activity was measured by putting aliquots of peritoneal macrophages

(105 cells/ml) to 35-mm Petri dishes. The cells were incubated at 378C for 2 h,

followed by washing with the medium. After 2 h incubation, 100 ul autoclaved yeast

cell suspension (108 cells/ml) was added and incubated for 1 h at 378C in a 5% CO2

humidified incubator. The plates were gently washed twice with culture medium and

treated with 1% tannic acid solution for 1 min, to allow for differential staining of

ingested and undigested yeast cells. The plates were washed with medium and air

dried before staining with May-Grunwald stain, freshly diluted with Giemsa buffer

(1:2), for 2 min and observed at 1000�magnification under oil immersion. The

percentage of macrophages with ingested bacteria was determined by counting 100

cells (Hay & Westwood 2002).

Challenge studies and colonization assays

Swiss mice were divided into four groups of 12 mice each. One group was fed on LcF,

second group on LcS, third group was given LcNF, and the fourth group was given

skim milk (5 g/day), in addition to a synthetic diet for seven consecutive days. After

feeding, the animals were challenged with 1�108 cfu S. dysenteriae cells, given orally

through a tube. Four mice in each group were sacrificed by cervical dislocation on

days 2, 5 and 8 post-challenge, and the number of viable bacteria in the intestine, liver

and spleen was determined. The organs were homogenized to a final volume of 5 ml in

0.1% peptone water using a Teflon homogenizer. The cell suspension was diluted in

peptone water plated on Salmonella-Shigella agar, and the number of colonies was

counted after 48 to 72 h of incubation at 378C.

Measurement of intestinal fluid antibodies by ELISA

Secretory immunoglobulin (s-IgA) anti-pathogen response was studied in the

intestinal fluid by ELISA in mice fed on skim milk, fermented milk, cell free

supernatant and non-fermented milk for seven consecutive days, and then challenged

with S. dysenteriae . The ELISA was performed on day 2, 5 and 8 post-challenge by the

method of Engwall and Perlmann (1971), and modified by Perdigon (1991a, b).

Intestinal fluid was collected as per the procedure described by Lim et al. (1981). The

intestine from gastro-duodenal to ileocaecal junctions was carefully removed, the

contents were washed out with 5 ml phosphate buffered saline (PBS; pH 7.2) and

centrifuged at 2000�g for 30 min.

The wells of microplates were coated in quadruplicate with 0.1 ml of bacteria (1�109 cells/0.1 ml). The antigen (Perdigon et al. 1991b) was allowed to bind to plastic

surface at 48C for 18 h. The non-coated antigen was removed by washing with PBS/T.

Free binding sites were blocked with 200 ul of blocking solution and incubated at RT

for 2 h with occasional shaking, Subsequently, the solution was removed and the wells

were washed three times with PBS/T. Diluted intestinal fluid (0.1 ml) containing

antibodies against S. dysenteriae was added to each well. The plate was incubated for 1

h at 378C with occasional shaking to allow antibodies to bind antigen. After Ag-Ab

reaction, the plate was washed three times with PBS/T, followed by the addition of 0.1

ml of goat antimouse (IgG, g-chain specific, Bangalore genei Pvt. Ltd., India)

peroxidase conjugate diluted 1:1000 to each well and incubated at 378C for 1 h with

Influence of feeding fermented milk and non-fermented milk 77

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occasional shaking. Unbound goat antimouse peroxidase conjugate was removed by

washing wells of ELISA plates three times with PBS/T followed by two times with

PBS. Substrate solution (0.1 ml) (0.04% o-phenylene-diamine hydrochloride, 0.012%

H2O2 in phosphate citrate buffer, pH 5.0) was added to each well, and the reaction

was carried out at RT for 30 min in darkness. The reaction was stopped with 0.1 ml of

H2SO4 (4N) and OD measured at 493 nm using ELISA plate recorder.

Statistical analysis

The results were expressed as mean9standard error of mean. Significance was tested

by using analysis of variance (ANOVA), and comparison between means was made by

critical difference (CD) value.

Results

Enzyme activity in macrophages

b-Galactosidase activity of peritoneal macrophages from mice fed on LcF for 2 days

was nearly three-fold higher than that of macrophages obtained from mice fed on skim

milk. Similarly, a four-fold increase in b-galactosidase activity was observed in LcNF-

fed mice compared to mice given skim milk, while this increase was comparatively less

in the LcS-fed group. After five and eight days feeding, a similar trend was observed in

b-galactosidase activity (Table I). In the case of cultured macrophage, an increase in b-

galactosidase activity was 6-, 12- and 15-fold higher in mice fed for two days on LcS,

LcF and LcNF, respectively, but no effect was observed in cultured macrophages in

mice fed for five and eight days.

Ingestion of LcF, LcNF and LcS for two days resulted in increased b-glucuronidase

activity in peritoneal fluid as well as in cultured macrophages in mice (Table II).

Phagocytic activity

The in vitro phagocytic activity of peritoneal macrophages in mice fed on LcF, LcNF

and LcS showed a peak on the fifth day, and thereafter declined on eighth day. There

was a significant (p B0.01) increase in phagocytic activity of macrophages in mice fed

on LcF, LcNF and LcS compared to macrophages in mice fed on skim milk for a

similar period (Figure 1).

Table I. b-galactosidase activity released by peritoneal macrophages of mice fed on different diets.

FeedingPeritoneal fluid Culture filtrate

period (day) Skim milk LcF LcS LcNF Skim milk LcF LcS LcNF

2 29a93 85b99 49a911 114c97 10a93 111c99 61b93 143d920

5 25a93 37ab97 32ab94 53b95 5a91 3a91 3a90 15a93

8 21a92 32ab96 23a92 51b93.1 6a91 7a91 3a91 6a91

The values are expressed in n mole of o -nitrophenol liberated per hour per 106 cells. The values are mean9

SEM for five mice per group. The values with the same symbols are statistically at par within a row whereas,

values with different symbols are significantly different at p B0.001. Lactobacillus casei fermented milk

(LcF), L. casei cell free supernatant (LcS) and L. casei non-fermented milk (LcNF).

78 S. Kapila et al.

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Effect of feeding L. casei fermented milk, cell free supernatant and non-fermented milk on the

growth of S. dysenteriae in the intestine, liver and spleen

The mice were fed on LcF, LcNF and LcS for a period of seven days before challenge

with S. dysenteriae , and a significant decrease in the colonization of pathogenic

bacteria was observed in the intestine, liver and spleen (Table III), in comparison to

mice fed on skim milk. The colonization of pathogen in the intestine on day 2 post-

challenge was one to two log units less in groups fed LcF, LcNF and LcS in

comparison to skim milk-fed group, and decreased over time.

Specific antibodies present in the intestinal fluid

It was observed that levels of S-IgA in the intestinal fluid were more or less similar in

all three groups on day 2 post-challenge (Figure 2). But on the fifth day, the levels of

S-IgA in the intestinal fluid was about two times higher in LcF-fed mice than skim

Table II. b-glucuronidase activity released by peritoneal macrophages of mice fed on different diets.

FeedingPeritoneal fluid Culture filtrate

period (day) Skim milk LcF LcS LcNF Skim milk LcF LcS LcNF

2 12a92 48c95 26b94 67d94 1a90 8b91 2a90 13c91

5 1a90 12b91 10ab92 14b91 0.3a90 0.7a90 0.5a90 2a90

8 1a90 12b90 9ab92 13b91 1a90 2a91 1a90 2a91

The values are expressed in n mole of p -nitrophenol liberated per hour per 106 cells. The values are mean9

SEM for five mice per group. The values with the same symbols are statistically at par within a row, whereas

values with different symbols are significantly different at p B0.001. Lactobacillus casei fermented milk

(LcF), L. casei cell free supernatant (LcS), L. casei non-fermented milk (LcNF).

0

10

20

30

40

50

60

70

80

2 5 8Feeding pe riod (Da ys)

% P

hag

ocy

tosi

s

Skim milk LcF LcS LcNF

Figure 1. Phagocytic activity of peritoneal macrophages in mice fed on skim milk, Lactobacillus casei

fermented milk (LcF), cell free supernatant (LcS) and non-fermented milk (LcNF) for a period of two, five

and eight days.

Influence of feeding fermented milk and non-fermented milk 79

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milk. Similarly, on day 8 post-challenge, the levels of S-IgA were more in LcF and

LcNF-fed mice. Overall production of IgA was more in LcF-fed group.

Discussion

We observed that the lysosomal enzymes activities of peritoneal macrophages and

cultured macrophages were significantly increased (Tables I and II) on day 2 after

feeding LcF, LcNF and LcF. But this effect was more in the case of LcNF-fed groups,

indicating that L. casei NCDC19 has immunomodulatory potential. Likewise, L. casei

CRL-431 (Perdigon et al. 1986a, 1988) and L. bulgaricus (Perdigon et al. 1986a,b;

Perdigon & Alvarez 1992) have also been reported to increase the release of lysosomal

enzyme from cultured as well as peritoneal macrophages.

Table III. Effect of feeding different diets for a period of seven days before challenge on protection against

Shigella dysenteriae in mice.

Intestine Liver Spleen

Days after

challenge

Skim

milk LcF LcS LcNF

Skim

milk LcF LcS LcNF

Skim

milk LcF LcS LcNF

2 10b90 8a91 9b90 9b91 6b90 6b91 6b90 5a91 0.0 0.0 0.0 0.0

5 9c91 8b90 8b90 7a90 7c90 6b90 6b90 5a90 6d91 4b90 5c90 3a90

8 4b90 1a90 4b92 1a90 5b90 3a91 3a90 3a90 2b91 1a90 3b90 1a90

The values are expressed in log cfu/ml and are mean9SEM for four mice per group. The values with the

same symbols are statistically at par within a row, whereas values with different symbols are significantly

different at p B0.05. Lactobacillus casei fermented milk (LcF), L. casei cell free supernatant (LcS), L. casei

non-fermented milk (LcNF).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

2 5 8Feeding Period (Days)

OD

493

nm

Skim milk LcF LcS LcNF

Figure 2. Levels of secretory-IgA in mice. IgA response was studied in mice fed on skim milk, Lactobacillus

casei fermented milk (LcF), cell free supernatant (LcS) and non-fermented milk (LcNF) for seven days.

Each group was challenged with Shigella dysenteriae . Y axis represents OD/100 min.

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The phagocytic activity of peritoneal macrophages was determined using yeast (S.

cerevisiae). It was observed that feeding of LcF, LcNF and LcS resulted in increased

phagocytic activity of peritoneal macrophages in comparison to macrophages obtained

from mice fed on skim milk, but this effect was not distinct in the group fed LcS. The

maximum phagocytosis (�70%) was observed on day 5 (Figure 1). The administra-

tion of L. casei enhanced phagocytic activity on the second and third day in mice

(Perdigon et al. 1986a). If we compare the lysosomal enzyme levels and phagocytic

activity, enzyme levels were higher on days 2 and 5. Phagocytic activity started

increasing from day 2 onwards, and attained its peak value on day 5. This suggests

that there may be some correlation between the increased enzyme levels and

phagocytic activity of macrophages. Similar results were observed by Perdigon et al.

(1986a) on feeding L. casei in mice, but feeding of L. bulgaricus was not as effective in

increasing the phagocytic activity of peritoneal macrophages, although levels of

lysosomal enzymes were high. On the other hand, Schynder and Baggiolini (1978)

suggested that the enzyme release process cannot always be directly related to

phagocytosis.

In this study, we observed a protective effect of feeding LcF, LcNF and LcS against

the colonization of S. dysenteriae in the intestine, liver and spleen. Colonization of

pathogenic bacteria in mice fed on LcF and LcNF was less in comparison to skim

milk-fed mice. This effect was observed only when mice were given LcF and LcNF for

a period of seven days before challenge, and prior feeding for two days was not

effective in protecting against S. dysenteriae (data not shown). Similarly, a period of

two days of feeding was not effective in the prevention of colonization of S.

typhimurium in the liver and spleen in mice (Perdigon et al. 1991b).

In the present study, it was observed that the feeding of LcF enhanced the levels of

S-IgA in the intestinal fluid, but when LcNF and LcS were fed, non-significant

variation in the levels of S-IgA were observed in comparison to skim milk-fed mice.

The colonization of S. dysenteriae was less in LcNF despite its low S-IgA levels,

whereas LcF group showed higher levels of S-IgA, but colonization of S. dysenteriae

was more; this fact is difficult to explain. Perdigon and Pesce de Ruiz Holgado (2001)

suggested that the induction of the inflammatory immune response by LAB would be

negative in the protection against enteropathogens. This may be due to an increase in

the permeability of the intestine that may cause colonization of the pathogen. In our

case, it may be possible that L. casei fermented milk might have shown mild

inflammatory response, and, therefore, it could not prevent colonization of S.

dysenteriae to the extent shown by LcNF. Feeding of LcF, LcNF and LcS has shown

enhanced immune response in comparison to milk. But, production of S-IgA was

higher in LcF, whereas LcNF showed better immunostimulatory response, such as

macrophage activity and decreased colonization to pathogen.

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