Protective effect and cytokine production of a Lactobacillus plantarum strain isolated from ewes’ milk cheese

ARTICLE IN PRESS

International Dairy Journal 14 (2004) 29–38

Protective effect and cytokine production of a Lactobacillus plantarum strain isolated from ewes’ milk cheese Ana I. Haza, Adriana Zabala, Paloma Morales* ! y Bromatolog!ıa III, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain Departamento de Nutricion Received 2 January 2003; accepted 29 June 2003

Abstract Ten lactic acid bacteria strains were isolated from a rippened ewes’ milk cheese and tested for stimulation or inhibition of the viability of Vero and myeloma cells. None of the strains significantly affected the viability of Vero cells. However, an isolate (CBL/J) showed a strong inhibition of the cellular viability (108 cfu mL
1, 10.1% of survival on myeloma cells). Isolate CBL/J was submitted to a characterisation scheme and identified as Lactobacillus plantarum by DNA sequencing of 16S rRNA Polymerase Chain Reaction (PCR) products. Lb. plantarum CBL/J showed a protective effect against three N-nitrosamines [N-nitrosopyrrolidine (NPYR), N-nitrosodibuthylamine (NDBA) and N-nitrosopiperidine (NPIP)] but not against N-nitrosodimethylamine (NDMA). The highest protective effect was observed against NPIP at populations of 107 cfu mL
1 (110.9% of survival on Vero cells). To test the effect of Lb. plantarum CBL/J on cytokine production, [tumor necrosis factor alpha (TNF-a), interleukin-1b (IL-1b) and interleukin-8 (IL-8)], the human macrophage cell line (THP-1) was cultured in the presence and absence of lipopolysaccharide (LPS). Lb. plantarum CBL/J induced IL-1b release when cells were stimulating with and without LPS. However, IL-8 production was not observed in the presence of LPS and TNF-a release was only produced in the presence of the LPS. r 2003 Elsevier Ltd. All rights reserved. Keywords: Lb. plantarum CBL/J; inhibition of the cellular viability; N-nitrosamines; cytokines

1. Introduction Lactic acid bacteria (LAB) are widely used in the dairy industry for the manufacture of cheese and yoghurt. Certain LAB, often members of the Lactobacillus genus, are believed to have a positive influence on the health of the consumer (De Roos & Katan, 2000; Bengmark, Garc!ıa de Lorenzo, & Culebras, 2001). Possible health effects include immune system stimulation, cholesterol lowering and prevention of cancer recurrence (Fuller, 1993; Lee & Salminen, 1995; Elmer, Surawicz, & McFarland, 1996). Lactobacillus plantarum is widespread in nature, colonizing habitats as diverse as the gastrointestinal tract of animals, fresh and fermented vegetables, meat, fish and dairy products (Sharpe, 1981; Kandler & Weiss, 1986). The occurrence of Lb. plantarum strains in these habitats is mostly fortuitous, but they are increasingly *Corresponding author. Tel.: +34-1-394-37-47; fax: +34-1-3943743. E-mail address: [email protected] (P. Morales). 0958-6946/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0958-6946(03)00146-8

used as starters for several food and feed fermentations (Gilliland, 1984; Vescovo, Torriani, Dellaglio, & Botazzi, 1993). The role played by Lb. plantarum in the development of cheese flavour during maturation and ageing is still not well understood. Nevertheless, the use of selectived strains of Lb. casei and/or Lb. plantarum as adjunct cultures has been claimed repeteadly to improve and accelerate flavour development (Khalid & Marth, 1990; Beresford, Fitzsimons, & Cogan, 1999; Kiernan, Beresford, O’Cuinn, & Jordan, 2000). Lactic acid bacteria or a soluble compound produced by the bacteria may interact directly with tumor cells in culture and inhibit their growth (Hirayama & Rafter, 2000). The antiproliferative effect of fermented milk on the growth of a human breast cancer cell line (MCF7) and a human colon cancer cell line (HT-29) has been described by Biffi, Coradini, Larsen, Riva, and Di Fronzo (1997) and Baricault et al. (1995). Some model experiments in vitro or in animals suggested the potencial of certain LAB to inactivate carcinogens, proffering them a role as cancer preventive agents (Fernandes, Chandan, & Shahani, 1992; Rafter, 1995; Hosoda,

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Hashimoto, He, Morita, & Hosono, 1996; Pool-Zobel et al., 1996; El-Nezami, Kankaanp.aa. , Salminen, & Ahokas, 1998). The increasing appreciation of the importance of N-nitrosamines as potential human carcinogens has stimulated intense research on protective dietary factors in chemical carcinogenesis (Mart!ınez, Cambero, Ikken, Haza, & Morales, 2000; Mart!ınez et al., 1998). The feeding of a Lactobacillus acidophilus culture to rats challenged with the colon carcinogen 1,2-dimethylhydrazine (DMH) has been shown to decrease the incidence of colon tumors (Goldin, Gualtieri, & Moore, 1996). Wolloski, Seung-Taek, Bakalinsky, and PoolZobel (1999) found that Lb. bulgaricus 191R applied orally to rats could prevent 1,2-dimethylhydrazineinduced DNA breaks in the colon in vivo, whereas Streptococcus thermophilus CH3 was not effective. However, in vitro, both of these strains prevented DNA damage induced by N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG). Recently, Balansky et al. (1999) reported the protective effect of a milk product fermented by two Lb. bulgaricus strains (LBB.B144 and LBB.B5) towards the tumorogenic activity of 1,2dimethylhydrazine in rats and diethylnitrosamine in the Syrian golden hamster. They concluded that some fermented milk products might exert a significant chemoprevention of cancer in humans. The ability of LAB and their cell wall components to induce cytokine release by human peripheral blood mononuclear cells has been extensively studied (Heumann, Barras, Severin, Glauser, & Tomasz, 1994; Mancuso, Tomasello, von Hunolstein, Orefici, & Teti, 1994; Muller-Alouf . et al., 1994; Soell et al., 1995). Many components of gram positive bacterial cell wall (capsular polysaccharides, peptidoglycans, and lipoteichoic acids) have been involved in cytokine induction by macrophage cells (Bhakdi, Klonisch, Nuber, & Fischer, 1991; Keller, Fischer, Keist, & Basseti, 1992). In addition, the lipopolysaccharide (LPS) of gram-negative bacteria (Betz-Corradin et al., 1992; Heumann et al., 1992) induces the production of the proinflammatory cytokines, tumor necrosis factor alpha (TNF-a) and interleukin-1b (IL-1b) as well as interleukin-8 (IL-8). The induction of an inflammatory reaction in response to infection is to a large extent attributed to the effects of IL-1b (Dinarello, 1994). IL-8 is also produced by macrophages together with IL-1b and TNF-a soon after infection or tissue injury (Daig et al., 1996). Macrophages are important regulatory cells that play a role in cell-mediated immunity as antigen-presenting cells and as inflammatory, tumoricidal and microbiocidal cells (Abbas, Lichtman, & Pober, 1994). These activities are mediated through the release of different cytokines, and therefore cytokine production can be taken as indicative of the degree of macrophage activation (Cavaillon, 1994).

In this paper, we report the characterisation and identification of a bacterial strain, Lactobacillus plantarum CBL/J, isolated from ewes’ milk cheese and its protective effect against the cytotoxicity of N-nitrosamines in Vero cells. The production of TNF-a; IL-1b and IL-8 by human macrophages as induced by Lb. plantarum CBL/J was also evaluated. This evaluation was performed as an initial step to establishing rational criteria for screening and selecting LAB food microorganisms with human probiotic properties.

2. Materials and methods 2.1. Microorganisms A rippened ewes’ cheese manufactured with no added starter cultures, was used as the LAB source. A sample of the cheese was collected under sterile conditions. After homogenization in 1% w/v peptone, supplemented with 0.8% w/v NaCl, appropriate dilutions were spread onto the surface of five MRS (de Man, Rogosa, Sharpe) agar and five RCM (Reinforced clostridial medium) agar (Oxoid, Ltd., Basingstoke, Hampshire, England) plates. The plates were incubated for 2 days at 32 C. The RCM plates were incubated anaerobically using the Oxoid Anaerobic System. Then, 10 colonies, one per plate, were randomly selected. As all the isolates showed optimal growth in MRS broth incubated at 32 C under aerobiosis, these conditions were chosen for routine propagation. 2.2. Characterisation and identification of the isolate CBL/J Strain CBL/J was selected among the initial isolates as they provided the most interesting results in the cell culture assays described below. Subsequently, they were examined by microscopy to determine cell morphology and tested for oxidase and catalase activities and growth in MRS broth (aerobic and anaerobic conditions). Fermentation patterns were obtained with the API 50 CH rapid fermentation strips (BioMe! rieux, Madrid, Spain) in Lactobacillus Identification medium (API 50 CHL medium; BioMe! rieux Madrid, Spain), as specified by the manufacturer. Antibiotic susceptibility was determined by a disc diffusion assay as previously described by Charteris, Kelly, Morelli, and Collins (1998). The antimicrobial susceptibility discs were obtained from BioMe! rieux and included erythromycin (15 mg), penicillin G (10 units), cefoxitin (30 mg), gentamicin (10 mg), ciprofloxacin (5 mg), nalidixic acid (30 mg), chloramphenicol (30 mg), ampicillin (10 mg), tetracycline (30 mg), vancomycin (30 mg) and trimethoprim (1.25 mg) combined with sulfamethoxazole (23.75 mg).

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Identification of the strain was performed by DNA sequencing of polymerase chain reaction (PCR) products containing 16S rRNA sequences of the CBL/J strain at the Department of Product Functionality, NIZO Food Research (Ede, The Netherlands). 2.3. Chemicals N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibuthylamine (NDBA) and N-nitrosopiperidine (NPIP) were purchased from SigmaAldrich, Inc. (Milwaukee, Wisconsin 53233). Standard solutions of NDMA and NPYR (10 mg mL
1) were prepared in milli Q water (Millipore, Japan), and standard solutions of NDBA and NPIP (400 mg mL
1) in dimethylsulfoxide (DMSO, Merck, Darmstadt, Germany). N-nitrosamines are potent carcinogenic agents; safety precautions were taken for proper handling and disposal of the chemicals. The N-nitrosamines tested were chosen because they are the most frequently occurring volatile nitrosamines in foods (Tricker & Preussmann, 1991).

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typhimurium lipopolysaccharide (LPS, 0.4 mg mL
1; Sigma). Triplicates cultures of the cell line were exposed to live Lb. plantarum CBL/J at 105, 106, 107 and 108 cfu mL
1 and were incubated for 2 days at 37 C and 100% humidity in 5% CO2. After incubation, supernatants were harvested and stored at
20 C for cytokine assay.

2.5. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) cell culture assay . Cell proliferation kit I (Boehringer Mannheim, GmbH, Germany) was used to test the effect of the isolated strains on the viability of Vero and myeloma cells and to test the protective effect of Lb. plantarum CBL/J against the cytotoxicity of volatile N-nitrosamines. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out in 96 (inhibition of the cell viability asay) and 48-well (protective effect against N-nitrosamines) tissue culture microtitre plates (Nunc, Roskilde, Denmark).

2.4. Cell cultures 2.6. Inhibition of the cellular viability Vero cells (African green monkey kidney cells) are defined as a continuous cell line with a fibroblastic-like morphology. They were cultured in Dulbecco’s Modified Eagle medium supplemented with 10% v/v heat inactivated foetal calf serum, 50 IU mL
1 penicillin, 50 mg mL
1 streptomycin and 1% v/v l-glutamine. The myeloma cell line P3  63-Ag8.653 from BALB/c mice was kindly donated by the Instituto Llorente (Madrid, Spain). Myeloma cells were cultured in RPMI1640 medium supplemented with 15% v/v heatinactivated foetal calf serum, penicillin (50 IU mL
1), streptomycin (50 mg mL
1) and 1% v/v l-glutamine. All media and supplements required for the growth of both cell lines were purchased from Gibco Laboratories (Life Tecnologies, Inc., Gaithersburg, MD 20884-9980). Cell cultures were incubated at 37 C and 100% humidity in a 5% CO2, atmosphere. The THP-1 human macrophage cell line (ATCC TIB 202) was obtained from the American Type Culture Collection (10801 University Blvd. Manassas, VA 20110-2209). Cells were maintained in RPMI-1640 medium supplemented with 10% v/v heat-inactivated fetal calf serum, penicillin (50 IU mL), streptomycin (50 mg mL
1), 1% v/v l-glutamine and 2-mercaptoethanol (50 mm) (Sigma-Aldrich, Inc., Milwaukee, Wisconsin 53233). All media and supplements required for the growth of the cell line were purchased from Gibco Laboratories, except where otherwise noted. THP-1 cells were cultured at a final density of 5  105 cells mL
1 in 96-well flat-bottomed tissue culture plates (Costar, Cambridge, MA) with and without Salmonella

Vero or myeloma cell suspension (100 mL; 106 cells mL
1) were dispensed into each well and the plates were incubated for 24 h at 37 C. After incubation, 100 mL of an RPMI-1640 suspension containing live bacteria were added to the wells. The bacterial suspension was prepared by centrifugation of the cultures at 10 000g for 5 min. The pellet was washed three times with Hank’s saline solution (Gibco Laboratories, Life Tecnologies, Inc., Gaithersburg, MD 20884-9980) and then suspended in RPMI-1640. The final bacterial cell counts tested ranged from 106 to 108 cfu mL
1. For each concentration, 24 wells were evaluated. Plates were incubated for 24 h at 37 C and 100% humidity in a 5% CO2 atmosphere. After incubation, 10 mL stock MTT solution (0.5 mg mL
1) were added to each well and the plates were incubated for 4 h at 37 C and 100% humidity in a 5% CO2 atmosphere. In viable cells, the yellow tetrazolium salt, MTT, is converted into a purple formazan substrate by the mitochondrial enzyme, succinate dehydrogenase (SDH). To dissolve the dark formazan crystals, 100 mL solubilization solution were added to each well and the plates were incubated overnight at 37 C and 100% humidity. After incubation, the contents of the plates were thoroughly mixed for 5 min on a plate shaker. The optical density (OD) of each well was read at 620 nm (test wavelength) and 690 nm (reference wavelength) by an ELISA with a built-in software package for data analysis (iEMS Reader MF, Labsystems, Helsinki, Finland).

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2.7. Protective effect of Lb. plantarum CBL/J against N-nitrosamines Vero cell suspensions (200 mL; 106 cells mL
1) were dispensed in each well, and plates were incubated for 24 h at 37 C. After incubation, 100 mL of each concentration of bacterial tested (106–108 cfu mL
1) and 100 mL of each N-nitrosamine (1 mg mL
1) were added to the wells. For each concentration (106– 108 cfu mL
1), 24 wells were evaluated. Plates were incubated for 24 h at 37 C and 100% humidity in a 5% CO2 atmosphere. After incubation, 20 mL of Stock MTT solution (0.5 mg mL
1) was added to each of the culture wells, and the MTT assay was performed as described above, in Section 2.6. For all bacterial cell counts, N-nitrosamines, negative and positive controls were evaluated in three independent assays. Values presented in this paper are mean 7standard error of the mean. Vero cells without bacterial suspensions and without N-nitrosamines were considered as negative controls, and Vero cells with Nnitrosamines, as positive controls. The percentage survival of Vero cells (% SDH) is defined as the ratio of the number of Vero cells in the presence of bacterial cells to the number in the absence of bacterial cells; it is defined by the following relationship: % SDH activity=100ðA1 =A0 Þ; where A1 is the absorbance of the Vero cells exposed to the bacterial cultures, and A0 is the absorbance of negative control. 2.8. Cytokine quantitation Cytokine production in culture supernatants was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well tissue microtitre plates (Nunc, Roskilde, Denmark) were coated overnight at 4 C with 50 mL per well of monoclonal mouse antihuman IL-1b antibodies (5 mg mL
1) and TNF-a and IL-8 (1 mg mL
1) (Biosource International, Inc., California) as capturing antibodies diluted in 0.1 m sodium bicarbonate buffer (pH 8.2). Plates were washed three times with 0.01 m phosphate-buffered saline (PBS) (pH 7.2) containing 0.2% (v/v) Tween 20 (PBST) to remove excess capture antibody. To reduce non-specific binding, wells were blocked with 200 mL of PBST containing 3% (w/v) bovine serum albumin (PBST-BSA) (Merck, Darmstadt, Germany) for 30 min at 37 C and washed four times with PBST. Standard recombinant human IL-1b; TNF-a and IL-8 (Biosource International Inc., California), or samples, diluted in PBS, were added in 50 mL aliquots to appropriate wells. Plates were incubated for 1 h at 37 C. After washing four times with PBST, 50 mL per well of biotynilated monoclonal mouse anti-human IL-1b antibodies (2 mg mL
1), TNF-a (1 mg mL
1) and IL-8 (0.1 mg mL
1) (Biosource International, Inc., California) were used as the detection

antibodies diluted in PBST-BSA. Plates were incubated with shaking 1 h at room temperature. The plates were washed six times with PBST and incubated with 50 mL per well of streptavidin-horseradish peroxidase conjugate (1.5 mg mL
1 in PBSTBSA) at room temperature with shaking for 1 h. After washing six times, 100 mL per well of TMB substrate (tetrametyl benzidine; Roche Diagnostics, Spain) was added to the plates and 100 mL of stopping reagent consisting of 1 m H2SO4 was added to each well to stop the reaction. Absorbance was measured at 450 nm with an ELISA iEMS Reader MF (Labsystem, Helsinki, Finland). Cytokines were quantitated from standard curves by using the Genesis program. 2.9. Statistical analysis The Student’s t-test was used for statistical comparisons and results are expressed as means7standard errors. Differences were considered significant at po0:05:

3. Results 3.1. Characterisation and identification of isolate CBL/J As shown in Table 1, the isolate CBL/J was a Grampositive bacillus with the ability to grow at temperatures ranging from 4 C to 45 C, in 6.5% w/v NaCl broth and in the presence of 2% w/v bile. The isolate was catalase, oxidase and Voges-Proskauer negative, and did not show urease activity. It fermented lactose, glucose and galactose like typical Lactobacillus strains. Most of these characteristics, together with its carbohydrate fermentation pattern, suggested that this isolate could belong to the genus, Lactobacillus. The 16S rRNA gene sequence analysis successfully confirmed the taxonomic characterization of this strain. In this work, the isolate CBL/J was identified as Lactobacillus plantarum by PCR method and was shown to be sensitive to all the antibiotics tested (Table 2), apart from nalidixic acid, gentamicin and cefoxitin. 3.2. Effect of Lactobacillus plantarum CBL/J on the viability of Vero and myeloma cells The 10 LAB isolates were assayed for inhibition or stimulation of cellular viability of myeloma and Vero cells by the MTT assay. Stimulation or inhibition of viability of Vero cells was not observed with any of the isolates tested under the conditions of the assay (data not shown). A strong inhibition of the myeloma cells viability was found with the Lb. plantarum CBL/J at the bacterial populations used. The effect of Lb. plantarum CBL/J on

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Table 1 Phenotypic characteristics of Lactobacillus plantarum CBL/J isolated from ewes’ milk cheesea,b,c

Table 2 Sensitivity and resistance of Lb. plantarum CBL/J to different antibioticsa,b

Reaction or characteristic

Antibiotics

Antibiotic concentration

Reaction of CBL/J

Erythromycin (El5) Penicillin G (P10) Ciprofloxacin (CIP5) Chloramphenicol (C30) Trimethoprim/ Sulfamethoxazole (SxT25) Nalidixic acid (NA30) Ampicillin (AM10) Tetracycline (TE30) Vancomycine (VA30) Gentamicin (GM10) Cefoxitin (FOX30)

15 mg 10 Units 5 mg 30 mg 1.25 mg 23.75 mg 30 mg 10 mg 30 mg 30 mg 10 mg 30 mg

S I S S S S R S S S R R

Test

CBL/J

Test

CBL/J

Morphology Gram reaction Catalase Oxidase Urease Voges-Proskauer CO2 production H2S production Glycerol Erythritol d-Arabinose l-Arabinose Ribose d-Xylose l-Xylose Adonitol b-Methylxyloside Galactose d-Glucose d-Fructose d-Mannose l-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol a-Methyl-d-mannoside a-Methyl-d-glucoside N-acetylglucosamine Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose

Rod +





W
W + + W


+ + + + W W + W + + W + + + + + + + + +

Melibiose Saccharose Trehalose Inulin Melezitose D-Raffinose Starch Glycogen Xylitol b-Gentibiose d-Turanose d-Lyxose d-Tagatose d-Fucose l-Fucose d-Arabitol l-Arabitol

+ + + W + +
W + W + W +





Gluconate 2-Ketogluconate 5-Ketogluconate Growth: At 4 C At 8 C At 24 C At 32 C At 37 C At 45 C In 2% (w/v) bile In 6.5% (w/v) NaCl

W

+ + + + + + + +

a

+=positive reaction;
=negative reaction; W=weak reaction. For growth:+, indicates growth. c See text for details.

a b

Percentage of survival (% SDH activity)

Reaction or characteristic

S=sensitive; R=resistant; I=intermediate; ND=not determined. Units, as defined by supplier.

120 ***

***

100 ***

*** ***

80 60 40 20

***

0

Control

106

107

108 -1

Lb. plantarum CBL/J population (cfu mL )

Fig. 1. Stimulation or inhibition of the survival of Vero (&) and myeloma (’) cells by Lactobacillus plantarum CBL/J isolated from ewes’milk cheese. See text for details of cell cultures and their growth. Asterisk indicates significant difference from control (pp0:001). The data presented are the means of 72 values from three independent replicate experiments; standard error was in the range of 1–5%.

b

the viability of Vero and myeloma cells is shown in Fig. 1. At populations of 106–108 cfu mL
1, the viability of Vero cells was not greatly influenced by Lb. plantarum CBL/J (105.3–89.7% of survival respectively; pp0:001). However, a strong inhibition of the viability of myeloma cells was observed at populations of 108 cfu mL
1 (10.1%; pp0:001).

3.3. Protective effect of Lactobacillus plantarum CBL/J against cytotoxicity of N-nitrosamines The protective effect of Lb. plantarum CBL/J isolated from ewes’ milk cheese, against cytotoxicity of four Nnitrosamines was evaluated in Vero cells by MTT assay.

The effect of Lb. plantarum CBL/J, on the cytotoxic effects of four N-nitrosamines on Vero cells is shown in Fig. 2. It is clear that the effect of Lb. plantarum CBL/J depended on population and the type of N-nitrosamine. At cell populations in the range 106–108 cfu mL
1Lb. plantarum CBL/J showed a significant protective effect against N-nitrosopyrrolidine (NPYR), N-nitrosodibuthylamine (NDBA) and N-nitrosopiperidine (NPIP). However, the protective effect was most pronounced at bacterial cell populations of 106–107 cfu mL
1. With Nnitrosamine NDBA, Lb. plantarum CBL/J provided a protective effect at populations of 106–107 cfu mL
1, but had a very marked inhibitory effect on the survival of Vero cells when the population was 108 cfu mL
1. In contrast to the effect noted with NDBA and NPYR, the addition of Lb. plantarum CBL/J inhibited the survival of Vero cells in the presence of NPIP, with the

ARTICLE IN PRESS A.I. Haza et al. / International Dairy Journal 14 (2004) 29–38 120

1 .5

100

***

*** ***

***

***

80

***

60

40

Interleukin 1β ((IL1-β) (ng mL-1)

Percentage of survival (% SDH activity)

34

*** 1

***

***

*** ***

0 .5

**

*** 0

20

(A)

0

1 .5 ***

120

*** *

100

***

*** *** *

***

***

***

80

60

Interleukin 8 (IL-8) (ng mL-1)

Percentage of survival (% SDH activity)

***

1

*** 0 .5

** 0

40

***

(B)

***

***

o

r

***

105

** 106

*** 107

*** 108

Co 105 106 107 108 -1 Lb. plantarum CBL/J population (cfu mL )

20

0

Control

106

108

107 -1

Lb. plantarum CBL/J population (cfu mL )

Fig. 2. Protective effect of Lactobacillus plantarum CBL/J against cytoxicity of different N-nitrosamines: NDMA (’), NPYR (&), NDBA ( ), and NPIP ( ) on the survival of Vero cells by MTT assay. Asterisk indicates significant difference from control (pp0:001). The data presented are the means of 72 values from three independent replicate experiments; standard error was in the range of 1–3%.

magnitude of the inhibition increasing with the bacterial population. 3.4. Effect of Lactobacillus plantarum CBL/J on IL-1b, IL-8 and TNF-a production The effect of Lb. plantarum CBL/J on the production of IL-1b; IL-8 and TNF-a was evaluated with the cultures of THP-1 cells incubated with or without LPS stimulation. The experiments were performed with 105, 106, 107 or 108 live bacteria mL
1. Cytokine secretion in the culture supernatants was measured by ELISA. The effect of Lb. plantarum CBL/J on the production of interleukins-1b (IL-1b) and 8 (IL-8) by THP-1 culture cells, in the absence or presence of LPS, which was added as a stimulator, is shown in Fig. 3. Lb. plantarum CBL/J induced IL-1b release in the THP-1 cells when cultured with or without LPS (Fig. 3A). However, IL-1b production was better in the presence of the stimulator. In both the stimulated and unstimulated THP-1 cells, optimal IL-1b production occured at 107 cfu mL
1 (1.30 and 0.90 ng mL
1; respectively), but decreased at 108 cfu mL
1 (1.06 and 0.60 ng mL
1). Lb. plantarum

Fig. 3. Effect of live Lactobacillus plantarum CBL/J on the production of interleukin-1 (IL-1b) (A) and interleukin-8 ( IL-8) (B) by THP-1 cells (human macrophage cell line). Cells (5  105 cells mL
1) were cultured with 0.4 mg mL
1 (’) or without (&) lipopolysaccharide (LPS) for 48 h. Asterisks indicate significant difference from control (C0, THP-1 cells without the added live bacterial suspension). pp0:001; pp0:01: The data presented are the means of 96 values from three independent replicate experiments; standard error was in the range of 0.005–0.01.

CBL/J stimulated the production of IL-8 to a greater degree than LPS addition (Fig. 3B). Increasing the populations of bacterial strains up to 108 cfu mL
1 increased IL-8 production by 0.80 ng mL
1. In the presence of LPS, the addition of Lb. plantarum CBL/J did not stimulate the production of IL-8. Fig. 4 shows the effect of Lb. plantarum CBL/J on the production of the tumor necrosis factor, TNF-a: Upon concurrent stimulation with LPS (0.4 mg mL
1), the addition of Lb. plantarum CBL/J significantly increased the production of TNF-a with response increasing more than proportionally with increase in bacterial cell population. The maximum TNF-a induction occurred at 108 cfu mL
1 (1.56 ng mL
1). In unstimulated THP-1 cultures, Lb. plantarum CBL/J induced little or no TNF-a production. No detectable TNF-a was observed in the control samples.

4. Discussion The aims of the present study was to test the inhibition of the Vero and myeloma cell viability by 10 LAB isolated from ewes’ milk cheese. Since isolate CBL/J provided the best results, it was submitted to a

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Tumor necrosis factor-α ((TNF- α) (ng mL-1)

A.I. Haza et al. / International Dairy Journal 14 (2004) 29–38

2 ** *

1 .5 1

*** ***

0 .5

***

***

0 Co

***

***

***

105 106 107 108 L. plantarum CBL/J population (cfu mL-1)

Fig. 4. Effect of live Lactobacillus plantarum CBL/J on production of tumor necrosis factor alpha (TNF-a by THP-1 cells (human macrophage cell line). Cells (5  105 cells mL
1) were cultured with 0.4 mg mL
1 (’) or without (&) lipopolysaccharide (LPS) for 48 h. Asterisks indicate significant difference from control (C0, THP-1 cells without the added live bacterial suspension) pp0:001: The data presented are the means of 96 values from three independent replicate experiments; standard error was in the range of 0.005–0.01.

rigorous characterisation and identification. Subsequently, the protective effect of Lb. plantarum CBL/J against cytotoxicity of N-nitrosamines and its induction on TNF-a; IL1-b and IL-8 production were evaluated. On the basis of the carbohydrate fermentation profile and other characteristics (Table 1), the isolate CBL/J could be classified as Lactobacillus. However, the need for correct identification, has led to several taxonomic studies over the last ten years dealing with the taxonomic status of these species using molecular methods. PCR amplification and sequencing of 16S rRNA genes, is an effective method for identifying LAB strains associated with food products (Coccolin, Manzano, Cantoni, & Comi, 2000). In this work, the isolate of CBL/J was unambiguously identified as Lactobacillus plantarum by the PCR method. Our results indicate that Lb. plantarum CBL/J showed a strong inhibition of the cellular viability on myeloma cells (Fig. 1). This effect is dose-dependent (Fig. 1) and suggests that inhibition of the cellular viability may be attributed to the fact that LAB may bind to the membrane receptor sites of the susceptible cells. The use of cultured tumoral cells may be a useful tool to further study the effect of LAB and LAB-fermented foods on cancer cell growth and differentiation (Baricault et al., 1995). Cultured human colon cancer cell lines (HT-29), human breast cancer cell lines (MCF7) and various murine tumor cell lines (Yac-1, P815) were used to study the effect of fermented milks by strains of Lb. helveticus, Lb. acidophilus, Lb. casei and Bifidobacterim (Biffi et al., 1997; Fichera & Giese, 1994). All studies concluded that, different strains of bacteria have different capacities to interfere with the growth of

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tumoral cells, ranging from no effect to a very significant effect (e.g., both B. infantis and Lb. acidophilus, give approximately 85% inhibition). In previous work, we reported the antiproliferative effect of two Lb. sakei strains of meat origin (Zabala, Haza, & Morales, 2003). Results revealed that Lb. sakei CBL/K resulted in a 66.8% decrease in the myeloma cell number, while the most efficient strain in lowering the myeloma growth was Lb. sakei CBL/H (82.4% of inhibition). In this paper, Lb. plantarum CBL/J showed a marked inhibition of the cellular viability on myeloma cells (89.9% of inhibition). Subsequently, the protective effect of Lb. plantarum CBL/J against cytotoxicity of N-nitrosamines was evaluated. Lb. plantarum CBL/J exhibited a protective effect against cytotoxicity of the N-nitrosamines NPYR, NDBA and NPIP, but not against NDMA (Fig. 2). The protective effect of Lb. plantarum CBL/J against NPYR and NPIP was found at all of the populations in the range 106–108 cfu mL
1. However, the protective effect against NDBA was observed at populations of 106 and 107 cfu mL
1 only; a population of 108 cfu mL
1 increased the cytotoxic activity of this N-nitrosamine. One feasible mechanism of the cytotoxic action is that LAB may interact with the enzymes systems catalizing the metabolic activation of the N-nitrosamines. The microsomal cytochrome P450 (CYP) form, has been shown to play a pivotal role in the metabolic activation of N-nitrosamines. For instance, CYP2E1 metobilizes NDMA and NPYR, while, CYP2B1 enzyme activity is responsible for the metabolism of NDBA and NPIP. The different CYP forms, concommintantly with the LAB-binding, could explain the increase of the cytotoxic activity of NDBA (Parkinson, 1996). A potential function of LAB strains may be the capacity to reduce the carcinogenic or toxic effect of food carcinogens by binding to them or metabolically transforming them into less toxic and carcinogenic degradation products. El-Nezami et al. (1998) observed that the ability of two strains of Lactobacillus rhamnosus (LBGG and LC705) to bind aflatoxin B1 (a common food carcinogen) depended on incubation temperature and bacterial population. In a recent study, these authors concluded that surface components of these bacteria are involved in binding of aflatoxin B1 and, in all cases, binding is of a reversible nature; the stability of the complex formed depends on strain, treatment used, and environmental conditions (Haskard, El-Nezami, Kankaanp.aa. , Salminen, & Ahokas, 2001). Hosono, Tanabe, and Otani (1990), observed different binding properties of lactic acid bacteria isolated from kefir milk to heterocyclic amines. They assumed that these differences in the binding abilities among the mutagens were due to the chemical nature of the heterocyclic amines and cell wall components of the LAB. It has been suggested that the site of binding

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cells is mainly attributed to the cell wall skeleton of gram-positive bacteria (Zhang & Ohta, 1991b), and the outer membrane of gram-negative bacteria (Zhang & Ohta, 1991a). Binding activity is attributed to the amino group of mutagens and the differences seen in the binding action suggests that the extent of binding is attributed to the type of mutagens rather than the bacteria isolated (Rajendran & Ohta, 1998). It has been reported that cell wall peptidoglycans and polysaccharides are the two most important elements responsible for the binding (Zhang & Ohta, 1991b; Sreekumar & Hosono, 1998). Lb. plantarum CBL/J has a protective effect against the N-nitrosamines NPYR, NDBA and NPIP but not against NDMA. The fact that the protective activity of Lb. plantarum CBL/J on Vero cells varied with the type of N-nitrosamine suggests that the protective effect depended on various factors, such as the chemical structure of the N-nitrosamines which affect their binding to the bacterial cell. Currently, there is no direct evidence that LAB can inhibit carcinogenesis in humans. However, LAB can influence several mechanisms possibly linked to carcinogenesis such as preventing mutations, binding mutagens, inhibiting bacterial enzymes that form carcinogens from procarcinogens in the colon, decreasing pH in the colon lumen or activating the immune system (Wolloski et al., 1999). Studies are in progress to identify strains with high binding activity and to elucidate the mechanisms of binding. Finally, to investigate the role of cytokines in interactions between Lb. plantarum CBL/J and the immune systems, we tested the production of TNF-a; IL-1b and IL-8 from the THP-1 human macrophage cell line. Our results indicated that Lb. plantarum CBL/J induced significantly higher amounts of the interleukins TNF-a and IL-1b in the presence of LPS stimulation than in its absence. LPS, a bacterial endotoxin, is known to be primarily responsible for septic shock produced both in humans and in animals during gram-negative bacterial infections (Heumann et al., 1994). There is also general agreement that LPS is a potent stimulator of a range of cells inducing the synthesis of many cytokines including pro-inflammatory IL-1, IL-8 and TNF (Henderson & Wilson, 1996). In the present work, LPS augmented the production of TNF-a and IL-1b; but not IL-8. These results are consistent with previous reports showing that Lactobacillus spp. enhance production of cytokines in the presence of LPS (Mar!ın, Tejada-Simon, Murtha, Ustunol, & Pestka, 1997; Henderson & Wilson, 1996). The maximum TNF-a induction (Fig. 4) occurred at 108 cfu mL
1 Lb. plantarum CBL/J (1.56 ng mL
1). The positive effects of TNF-a include the activation of the immune system against pathogenic infections. However, high concentrations of TNF-a cause cachexia, tissue

injury, disseminated intravascular coagulation, and shock (Abbas et al., 1994). Our data demonstrate that Lb. plantarum CBL/J induced the production of IL-1b by THP-1 cells in the absence or presence of LPS. However, only IL-8 release was produced in the absence of the LPS (Fig. 3). Thus, this bacteria can directly induce the production of the two cytokines by human macrophages. Nevertheless, the production of IL-8 by Lb. plantarum CBL/J in the absence of LPS disagrees with the findings of Lammers et al. (2002) who reported that lactobacilli and bifidobacteria strains did not induce interleukin 8. In conclusion, the results showed that Lb. plantarum CBL/J isolated from ewes’ milk cheese induced the in vitro production of several cytokines by human macrophages. The in vitro approaches employed in this work should be useful in further characterisation of the effects of lactobacilli of food origin on the gut and immune system. Acknowledgements This work has been supported by Grant ALI98-0693 from the Comision Interministerial de Ciencia y Tecnolog!ıa (CICYT, Spain). References Abbas, A., Lichtman, A. H., & Pober, J. S. (1994). Cytokines. In W. B. Saunders, Cellular and molecular immunology (pp. 240–260). Philadelphia: Saunders. Balansky, R., Gyosheva, B., Ganchev, G., Mircheva, Z., Minkova, S., & Georgiev, G. (1999). Inhibitory effects of freeze-dried milk fermented by selected Lactobacillus bulgaricus strains on carcinogenesis induced by 1,2-dimethylhydrazine in rats and by diethylnitrosamine in hamsters. Cancer Letters, 147, 125–137. Baricault, L., Denariaz, G., Houri, J. J., Bouley, C., Sapin, C., & Trugnan, G. (1995). Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on cancer cell growth and differentiation. Carcinogenesis, 16, 245–252. Bengmark, S., Garc!ıa de Lorenzo, A., & Culebras, J. M. (2001). Use of ! pro-, pre and synbiotics in the ICU- future options. Nutricion Hospitalaria, XVI(6), 239–256. Beresford, T., Fitzsimons, N. A., & Cogan, T. M. (1999). Non-starter lactic acid bacteria growth in cheese contribution to flavour development? Dairy Industry International, 64, 19–21. Betz-Corradin, S., Mauel, J., Gallay, P., Heumann, D., Ulevitch, R. J., & Tobias, P. S. (1992). Enhancement of murine macrophage binding of and response to bacterial lipopolysaccharide (LPS) by LPS-binding protein. Journal of Leukocyte Biology, 52, 363–368. Bhakdi, S., Klonisch, T., Nuber, P., & Fischer, W. (1991). Stimulation of monokine production by lipoteichoic acids. Infection and Immunity, 59, 4614–4620. Biffi, A., Coradini, D., Larsen, R., Riva, L., & Di Fronzo, G. (1997). Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line. Nutrition and Cancer, 28, 93–99. Cavaillon, J. M. (1994). Cytokines and macrophages. Biomedicine and Pharmacotherapy, 48, 445–453. Charteris, W. P., Kelly, P. M., Morelli, L., & Collins, J. K. (1998). Antibiotic susceptibility of potentially probiotic Lactobacillus species. Journal of Food Potection, 61, 1636–1643.

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