Comparative Evaluation of Oral Administration of Probiotic Lactobacilli-fermented Milks on Macrophage Function

Probiotics & Antimicro. Prot. (2012) 4:173–179 DOI 10.1007/s12602-012-9107-x

Comparative Evaluation of Oral Administration of Probiotic Lactobacilli-fermented Milks on Macrophage Function Rajeev Kapila • Suman Kapila • Meena Kapasiya Divya Pandey • Ajay Dang • Vamshi Saliganti

•

Published online: 22 May 2012 Ó Springer Science + Business Media, LLC 2012

Abstract Six strains of lactobacilli belonging to three species (Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus helveticus) were evaluated for probiotic attributes viz. acid tolerance, bile tolerance and cell surface hydrophobicity. All the six strains exhibited probiotic attributes with considerable degree of variation. Three Lactobacillus strains selected on the basis of probiotic attributes were used for preparing three different fermented milks. In order to evaluate the effect of feeding these probiotic fermented milks on macrophage cell function, an in-vivo trial was conducted in mice for a period of 2, 5 and 8 days. The control group of mice was fed with skim milk. The phagocytic activity of macrophages increased significantly (P \ 0.05) on feeding fermented milk prepared using L. acidophilus, L. casei and L. helveticus as compared to milk group (control) on 2nd, 5th and 8th day of feeding, respectively. Likewise, the release of b-glucuronidase and b-galactosidase from peritoneal macrophages increased significantly (P \ 0.05) on 2nd, 5th and 8th day of feeding as compared to their respective control group (milk). The results thus depict that feeding of probiotic fermented milk enhances phagocytic activity of the macrophages. Keywords Probiotic

Lactobacilli  Macrophage  Phagocytosis 

R. Kapila  S. Kapila (&)  M. Kapasiya  D. Pandey  A. Dang  V. Saliganti NDRI, Karnal, Haryana, India e-mail: [email protected]

Introduction The science of probiotics, live microbial cultures which, when ingested in sufficient numbers, provide beneficial effects to the consumer beyond basic nutrition [1], may be traced to Metchinikoff in 1907. Various studies have indicated that probiotics may alleviate lactose intolerance; have a positive influence on the intestinal flora of the host; stimulate/modulate mucosal immunity; reduce inflammatory or allergic reactions; reduce blood cholesterol etc. [2, 3]. Considering this impressive list of potential health-promoting benefits, it is not surprising that there continues to be considerable interest in the use of probiotics as biotherapeutic agents [4, 5]. Furthermore, given a heightened awareness among consumers of the link between diet and health and the fact that probiotic-containing foods are generally perceived as ‘‘safe and natural,’’ the global market for such foods is on the increase, particularly dairy-based products marketed for the prophylaxis or alleviation of gastrointestinal disorders [6]. Naidu et al. [7] describes probiotics as microbial dietary adjuvants that beneficially affect the hosts’ physiology by modulating their mucosal and systemic immunity as well as improving the nutritional and microbial balance in their intestinal tracts. Vertebrate immune system can mount both innate and adaptive immune response in the event of infection by pathogenic microorganisms. Many types of immune cells are recruited to elicit an immune response and subsequently neutralize the pathogens. These cells include natural killer cells, macrophages, neutrophils, dendritic cells, lymphocytes and epithelial cells. These cells are quickly activated in the event of infection leading to production of an array of humoral mediators. Some may change their physiology and become phagocytic, yet others get involved in antibody synthesis and secretion.

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These activated immune cells may provide immediate protection against pathogens or promote specific immune responses. Therefore, these cells are useful in the probiotic enhancement of immunologic barrier in the animal gastrointestinal tract. At present, there is much evidence concerning the role of probiotics, especially lactic acid bacteria (LAB), in the maintenance of health or in the prevention of disease [8] and the modulation of immune system. Probiotics have profound effects on potentiating both arms of immune responses. The selection of potential probiotic strains that would be capable of performing effectively in the gastrointestinal tract is a significant challenge. Strain selection is generally based on in vitro tolerance of physiologically relevant stresses: e.g. low pH, elevated osmolarity and bile [9, 10]. Lactobacillus sp. has been used successfully as a probiotic in traditional milk products, as well as cheeses and a number of commercial fermented food products. Moreover, the beneficial effects of the probiotic strains vary not only at the species level, but at the strain level too. Hence, the main objective of this work was to evaluate the probiotic potential of six lactobacilli strains and to observe the effect of feeding fermented milk on macrophage function in mice.

Materials and Methods

Probiotics & Antimicro. Prot. (2012) 4:173–179

Bile Tolerance Tolerance for bile acids was tested as per the method of Gilliland et al. [12]. MRS broth supplemented with 0.5 %, 1 %, 1.5 % and 2 % w/v ox bile and without supplement as a control was inoculated with actively growing bacteria. Survival was evaluated using log-phase cultures by plate count on MRS agar, after 0, 1, 3 and 6 h of incubation at 37 °C in MRS broth containing bile salts. Cell Surface Hydrophobicity Hydrophobicity was determined according to the method described by Rosenberg et al. [13], with slight modifications. Active culture was harvested by centrifugation at 12,0009g for 5 min at 4 °C, washed twice and re-suspended in 5 mL phosphate urea magnesium sulphate buffer (pH 6.5). The initial absorbance (OD initial) of the cell suspension was adjusted to approx. 0.8–1.0. To 3 mL of bacterial suspension, 1.0 mL of n-hexadecane, xylene or octane was added slowly. The suspension was preincubated at 37 °C for 10 min followed by vortexing for 2 min. The hydrocarbon layer was allowed to rise completely. After 1 and 2 h, aqueous phase was removed carefully with a Pasteur pipette and the final absorbance (ODfinal) was measured at 620 nm using spectrophotometer. The decrease in the absorbance was taken as a measure of the cell surface hydrophobicity (%H) calculated with the following equation. ODinitial ODfinal  100 ODinitial

Bacterial Stains and Culture Condition

% Hydrophobicity ¼

The lactobacilli cultures used in this study were obtained from National Collection for Dairy Cultures, National Dairy Research Institute, Karnal. Lactobacillus casei NCDC17 and L. casei 19, L. acidophilus NCDC 15 and L. acidophilus VK2, L. helveticus NCDC 288 and L. helveticus NCDC 292 were stored at -80 °C in MRS broth supplemented with 20 % (v/v) glycerol. The cultures were activated prior to use by subculturing twice in MRS broth for 18 h at 37 °C. Fermented milks were prepared daily by inoculating sterile skim milk with each strain and incubating for 18 h at 37 °C. The bacterial number in fermented milk was determined by plate count on MRS plate, after aerobic incubation at 37 °C for 24–48 h.

where ODinitial and ODfinal are the absorbance before and after extraction with the three hydrocarbons.

Acid Resistance Resistance to acidic conditions [11] was tested with some modifications. The pH of MRS broth was adjusted to pH 2.0, pH 2.5, pH 3.5 and pH 6.5 with 1.0 M HCl. Survival was evaluated using the log-phase cultures by plating on MRS agar, after 0, 1 and 2 h of incubation at 37 °C in acidic MRS broth of different pH.

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In Vivo Study Twenty-four male albino mice procured from the small animal house of National Dairy Research Institute (NDRI), Karnal, Haryana, India, of 45 ± 10 days old with mean body weight of 22 ± 1.2 g (mean ± SD), were randomized into four groups (n = 4) consisting of six mice each on the basis of their body weight and age so that mean body weight and mean age of four groups did not differ(P [ 0.10) at the beginning of experiment (Table 1). In addition, 6 mice were killed on zero day. Three groups of animals were fed basal diet (protein 12 % and fat 10 %) along with milk fermented with three best cultures (L. casei, L.helveticus and L. acidophilus) from respective species of lactobacilli based on in vitro tests of previous experiment. These animals were fed for a period of 2, 5 and 8 days while one control group was fed with skim milk. The fermented milk contained 1 9 109 cells mL-1. 1.5 9 109 cells were fed to each mouse daily along with the basal diet. The study was

Probiotics & Antimicro. Prot. (2012) 4:173–179 Table 1 Grouping of animals

175

cell-free supernatant of the fermented milks of three cultures of lactobacilli (L. casei, L.helveticus and L. acidophilus) in order to establish the direct effect of active components produced by lactobacilli in milk on phagocytic activity of the macrophages.

Group

Diet

Control group 1 (Non fermented milk)

Basal diet ? skim milk

Probiotic fermented milk I

Basal diet ? probiotic fermented milk L. casei (LC) (NCDC-19)

Probiotic fermented milk II

Basal diet ? probiotic fermented milk L. helveticus (LH) (NCDC-292)

Results and Discussion

Probiotic fermented milk III

Basal diet ? probiotic fermented milk L. acidophilus(LA) VK2

Acid Resistance

approved by the Institute Animal Ethical Committee (IAEC). Macrophage Function Macrophage function was evaluated in terms of phagocytic activity, b-galactosidase and b-glucuronidase activity. The mice were killed by cervical dislocation on days 2, 5 and 8 post feeding, and the peritoneal cavity macrophages were collected with Dulbecco’s Modified Eagle Medium Ham’s F12 (without phenol red). The activities of secreted enzymes viz. b-galactosidase and b-glucuronidase were assayed in peritoneal fluid as per the method of Conchie et al. and Stossel [14, 15], respectively. Phagocytic activity of peritoneal macrophages was determined by Hay and Westwood [16] method. Briefly, peritoneal exudate containing macrophages (1 9 105 cells/ mL) was incubated at 37 °C for 2 h in 35-mm culture plates. Non-adherent cells were removed by decantation, and poured fresh medium (1 mL) and the culture plates again incubated at 37 °C for 2 h. The macrophages were then incubated with 100 lL of yeast cell suspension (108 cells/ mL) for 1 h in a humidified atmosphere (5 % CO2) at 37 °C. The medium was removed and the cells washed twice gently with culture medium and then incubated with 1 mL of tannic acid (1 %) for 1 min at room temperature. The cells were again washed with culture medium, dried in air, stained for 5 min with May-Grunwald’s eosin methylene blue modified stain, freshly diluted with Giemsa buffer (1:2), and then stained with Giemsa solution (freshly diluted with buffer) for 15 min. The extra stain was removed by washing the cells with Giemsa buffer. The phagocytosis was observed at 1,0009 magnification under oil immersion, and the following observations were recorded:

The effect of different pH on the viability of the six strains of lactobacilli is shown in Table 2. In general, all strains of lactobacilli showed lower viability in MRS broth at pH 2.0 than at pH 2.5, and 3.5. On one hand, there was a progressive reduction in viability at pH 2.0 with time in all the strains tested, but on the other hand, all strains maintained their viability with slight drop of about one log cfu/ml at pH 2.5 and 3.5 similar to that of control at pH 6.5. Though comparative analysis revealed that all strains survived at pH 2.0, but acid resistance was observed to be least (3–4 log cfu/mL) for both the strains of L. helveticus at pH 2.0 for 2 h of treatment as compared to 5–6 log cfu/mL for Table 2 Survival of selected Lactobacillus strains under acidic condition after 1 and 2 h of incubation in MRS broth Strain Lactobacillus acidophilus NCDC 15

Lactobacillus acidophilus VK2

Lactobacillus casei NCDC 17

Lactobacillus casei NCDC 19

Lactobacillus helveticus NCDC 288

Percent Phagocytosis ¼ Number of macrophages with yeast cell internalized per 100 macrophages In addition, in vitro phagocytic activity of the peritoneal macrophages collected from naı¨ve mice was also studied by incubating the macrophages with different concentration of

Lactobacillus helveticus NCDC 292

pH

0h

1h

2h

2.0

6.70

5.30

5.00

2.5

6.13

6.00

6.00

3.5

6.85

7.30

7.00

6.5

6.48

6.60

7.18

2.0

6.48

5.00

4.90

2.5

8.22

7.72

7.53

3.5

8.22

7.79

7.90

6.5

8.78

8.89

8.90

2.0

7.70

5.30

4.80

2.5

7.78

7.89

7.88

3.5

7.41

7.81

8.09

6.5

7.75

7.96

8.21

2.0

5.70

5.00

4.80

2.5

8.80

7.94

7.87

3.5 6.5

8.03 9.11

7.94 9.09

7.93 9.14

2.0

5.90

4.20

4.00

2.5

5.84

5.76

6.80

3.5

5.57

5.82

6.48

6.5

7.26

6.95

6.98

2.0

6.78

3.90

3.00

2.5

6.78

6.59

5.30

3.5

6.78

6.65

6.09

6.5

6.45

6.65

6.78

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176

Probiotics & Antimicro. Prot. (2012) 4:173–179

L.casei and L. acidophilus. In the present study, survival up to a log count of 6.0–8.0 at pH as low as 2.5 indicated good degree of acid tolerance. Survival at pH 2.5 and 3 is significant as ingestion with food or dairy products raises the pH in stomach to 3.0 or higher [17]. Bile Tolerance In general, all the strains exhibited good bile tolerance (Table 3). Bile salt tolerance of L. helveticus was observed to be reduced significantly (2.0–3.5 log cfu/mL) at different bile salt concentrations (0.5, 1.0, 1.5 and 2.0 %) as compared to the control (without bile salt). The other four strains of L.casei and L. acidophilus showed revival on 3 h of incubation after initial decline in viability. In the present Table 3 Survival of selected Lactobacillus strains under different bile concentrations after 1, 3 and 6 h of incubation in MRS broth Strains

Bile concentration

0h

1h

3h

Lactobacillus acidophilus NCDC 15

Control

5.28

5.54

5.68

5.89

0.5 %

5.32

5.26

5.32

5.61

Lactobacillus acidophilus VK2

Lactobacillus casei NCDC 17

Lactobacillus casei NCDC 19

Lactobacillus helveticus NCDC 288

Lactobacillus helveticus NCDC 292

a

No growth

123

6h

1%

5.00

5.10

5.23

5.32

1.5 %

4.60

4.50

4.90

5.89

2%

5.00

5.15

5.46

5.94

Control

7.98

8.05

8.31

8.48

0.5 %

7.23

7.69

7.83

7.90

1%

6.00

7.43

7.48

7.67

1.5 %

6.40

7.10

7.43

7.65

2%

7.40

7.00

7.11

7.40

Control 0.5 %

7.00 5.00

7.18 5.00

7.30 5.30

7.48 5.60

1%

5.00

5.48

5.00

5.48

1.5 %

6.30

6.95

6.00

6.00

2%

5.60

5.00

5.00

5.30

Control

8.03

8.13

8.24

8.48

0.5 %

6.78

7.41

7.67

8.19

1%

6.95

7.1

7.30

8.15

1.5 %

6.70

6.70

7.28

8.24

2%

6.30

6.00

7.15

Control

6.78

7.08

7.78

–a

0.5 %

3.48

2.11

1.85

–

1%

3.47

2.04

1.48

–

1.5 %

2.88

1.70

1.60

–

8.10

2%

2.88

2.00

2.08

–

Control 0.5 %

6.53 6.78

6.67 3.18

6.80 2.92

– –

1%

6.78

2.48

1.90

–

1.5 %

3.48

2.70

1.95

–

2%

3.22

3.18

2.95

–

study, L. casei (NCDC-19) and both the strains of L. acidophilus showed no remarkable reduction in cell viability at 0.5, 1, 1.5 % and 2 % bile salt concentrations even after 6 h as compared to control. The variation in bile sensitivity observed in this study is consistent with many reports [18, 19] for lactobacilli. A consensus, therefore, emerges that wide variation exists in the susceptibility of probiotic cultures to bile, and this property is species, as well as strain, specific [12]. Cell Surface Hydrophobicity The strains under study were evaluated for their cell surface hydrophobicity towards different hydrocarbons, that is, n-hexadecane, xylene and n-octane, which may reflect the colonization potential of the organism to intestinal lumen. All six strains showed variable degree of hydrophobicity with the organic solvents. As evident from Fig. 4, L. acidophilus (NCDC-15) has exhibited the highest (74.6 ± 2.6 %) affinity followed by L. helveticus (NCDC292) showing 35.45 ± 2.6 % hydrophobicity for n-hexadecane after 2 h of treatment. The remaining four strains of Lactobacillus have shown 5–20 % hydrophobicity in this organic solvent. In case of xylene, L. casei (NCDC-17) showed maximum affinity (90.95 ± 4.4 %) followed by either strains of L. helveticus with hydrophobicity of 34.45 ± 2.2 and 33.96 ± 2.5 %, respectively, on 2 h of incubation with this solvent. Affinity for n-octane was observed to be ranged between 23 and 35 % irrespective of the stains of lactobacilli studied. The variation in hydrophobicity to solvents and among strains has been explained by the fact that the adhesion depends upon the origin of strains as well as the surface properties [20] (Table 4). Phagocytic Activity The effect of feeding skim milk and different fermented milks with cultures of lactobacilli (L. casei, L. helveticus and L. acidophilus) on phagocytic activity is depicted in Fig. 1. Phagocytic activity was expressed by counting that number of macrophages with ingested yeast (Saccharomyces cerevisiae) cells (Fig. 2). In skim milk–fed group, there was no significant variation (P B 0.05) in the phagocytic activity up to the 8th day. But in fermented milk–fed groups, phagocytic activity increased significantly on 2nd, 5th and 8th day. The phagocytic activity was observed to be 72.6 ± 3.1, 68.6 ± 3.37 and 55.4 ± 3.2 %, respectively, on fifth day of feeding with L. acidophilus-, L. casei- and L. helveticus-fermented milk. Phagocytic activity declined on 8th day in comparison with that of 5th day, but still remained considerably high in L. acidophilusand L. casei-fed groups than control group. Similar observations were made by administering L. casei in mice

Probiotics & Antimicro. Prot. (2012) 4:173–179

177

Table 4 Cell surface hydrophobicity of selected Lactobacillus strains to selected hydrocarbons Strain

Hydrocarbon

Lactobacillus acidophilus NCDC 15

n-Hexadecane

1h

2h

45.00

74.60

Xylene n-Octane n-Hexadecane

Lactobacillus acidophilus VKK

% Hydrophobicity

6.70

29.80

25.24 6.66

35.00 7.00

9.63

16.20

Xylene n-Octane

Lactobacillus casei NCDC 17

13.40

24.83

6.25

15.80

Xylene

95.18

90.95

n-Octane

18.87

24.40

n-Hexadecane

5.00

7.00

Xylene

5.20

8.75

n-Octane

17.87

24.40

n-Hexadecane

10.33

20.33

Xylene

12.60

33.96

n-Octane

15.77

23.37

C

9.78

35.45

100ul

Xylene

22.32

34.45

n-Octane

24.91

28.31

n-Hexadecane

Lactobacillus casei NCDC 19 Lactobacillus helveticus NCDC 288 Lactobacillus helveticus NCDC 292

n-Hexadecane

Fig. 2 Macrophage showing engulfed yeast cells

80

70

e e

200ul

d

400ul

80

Phagocytic activity (%)

70 60

d

d

0 day 2 days 5 days 8 days

c c c

c

50

b

b

a 30

c c

50

b

b

40

ab

a 30

a

a a

a

20

b 40

Phagocytic activity (%)

60

a a a

10

20

0 C

10 0 C

Milk

LC

LH

LA

Fig. 1 Phagocytic activity of the peritoneal macrophages in mice after feeding probiotic fermented milks for period of 2, 5 and 8 days

by Peridgon et al., [21], but feeding of L.bulgaricus was not as effective in increasing the phagocytic activity of the peritoneal macrophages. The effect of incubating cell-free supernatant of three lactobacilli-fermented milks with peritoneal macrophages of naı¨ve mice on phagocytic activity is depicted in Fig. 3. The results showed that phagocytic activity increased significantly on the addition of different concentrations of cell-free supernatant of lactobacilli-fermented milk. The

Milk

LC

LH

LA

Fig. 3 Phagocytic activity of the peritoneal macrophages in naı¨ve mice on incubating macrophages with different concentrations of cellfree supernatant of fermented milk of lactobacilli cultures (in vitro assay)

increase was observed in a dose-dependent manner. However, it is difficult to correlate the in vitro activity with in vivo study because under in vivo conditions the probiotic bacteria and fermented milk bioactive components interact with various immune cells and generate interleukins for innate and adaptive responses. b-Glucuronidase The activity of b-glucuronidase in mice on zero day before start of experiment was 4.13 ± 1.32 nmoles/h/106 cells

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Probiotics & Antimicro. Prot. (2012) 4:173–179 35

100

30

2 days

70

Units /106 cells

Units/106 cells

2 days 5 days 8 days

80

8 days

20

c

15

0 day

d

5 days 25

d

90

0 day

c

60

40 30

ab

a

a a

a a

c

c

b

b

50

c

c

a a

20

b

10

b

b

ab 5

a

b

b

0 C

Milk

LC

LH

LA

Fig. 4 b-Glucuronidase activity of the peritoneal macrophages in mice after feeding probiotic fermented milks for a period of 2, 5 and 8 days. One unit is expressed as p-nitrophenol liberated per hour per 106 cells. The values are mean ± SEM for six mice per group

(Fig. 4) On feeding skim milk for 2 days, there was significant (P B 0.05) increase in the activity of b-glucuronidase, But, animals fed with L. casei-fermented milk for 2 days expressed remarkably (P B 0.01) increased (threefold) values. Though the release of b-glucuronidase declined sharply on days 5 and 8 on feeding either of the three cultures of lactobacilli, but still it was significantly higher than the control groups. b-Galactosidase The activity of b-galactosidase on zero day before starting the experiment was 26.52 ± 3.8 nmoles/h/106 cells (Fig. 5) and changed non-significantly on feeding skim milk. However, b-galactosidase increased significantly on the 2nd day of feeding fermented milk as compared to control groups. The animal groups fed on L. casei-fermented milk exhibited tremendous increase in the enzyme activity on the 2nd day followed by decline on days 5 and 8. Likewise, feeding of milk fermented with L. acidophilus released its higher quantities on day 2 than the subsequent days. In case of L. helveticus-fermented milk-fed group, b-galactosidase released observed to be considerably higher than control groups during week long feeding. Similar increase in the release of lysosomal enzymes has been reported earlier in peritoneal macrophages [21–24]. If we compare the lysosomal enzyme levels and phagocytic activity, the enzymes levels were higher on day 2. Phagocytic activity started increasing from day 2 onwards,

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Milk

LC

LH

LA

a

0 C

10

Fig. 5 b-Galactosidase activity of the peritoneal macrophages in mice after feeding probiotic fermented milks for a period of 2, 5 and 8 days. One unit is expressed as o-nitrophenol liberated per hour per 106 cells. The values are ±mean SEM for six mice per group

attained its peak on day 5. This suggests that there may be some correlation between the increased enzyme levels and phagocytic activity of macrophages. However, Schynder and Bagglioini [25] suggested that the enzyme release process cannot always be directly related to phagocytosis. Our results thus suggest that probiotic fermented milk enhances macrophage function which is the important component of primary defence in an organism. We are further trying to explore the mechanism by which macrophage function is enhanced by focusing on the expression of Toll-like receptors. Acknowledgments The authors acknowledge Dr. V. K. Kansal, ExPrincipal Scientist, NDRI for providing L. acidophilus VK2 strain. The financial aid to carry out this study by Department of Biotechnology (DBT), Government of India, is fully acknowledged.

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