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All issues Volume 90 / No 4 (July–August 2010) Dairy Sci. Technol., 90 4 (2010) 469-476 Full HTML
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Dairy Sci. Technol.
Volume 90, Number 4, July–August 2010
Special Issue: Selection of papers from the 4th International Dairy Federation Dairy Science and Technology Week,
21-25 April 2009, Rennes, France
Page(s) 469 - 476
DOI https://doi.org/10.1051/dst/2010009
Published online 18 March 2010

© INRA, EDP Sciences, 2010

1. INTRODUCTION

There is evidence that several foods or foods ingredients provide a benefit beyond the nutrients they contain. These substances are defined as functional food, and their putative biological effects have been extensively studied. To date, antihypertensive and immunomodulatory bioactivities are frequently exploited in the production of foodstuffs formulated to provide putative health benefits [11, 18].

Interestingly, angiotensin-I converting enzyme (ACE)-inhibitory and immunomodulatory properties seem to be associated, possibly because both are correlated to the presence of short-chain peptides [22].

So far, lactic acid bacteria have been preferred to other microorganisms to produce fermented milks rich in ACE-inhibitory activity [6, 23], in particular Lactobacillus helveticus [16, 20, 21], Lactobacillus delbrueckii subsp. bulgaricus (L. delb. bulgaricus) and Lactococcus lactis subsp. cremoris (L. lactis cremoris) [10]. Moreover, some bacterial strains, mostly lactic acid bacteria, release components during fermentation that possess immunomodulatory activity [24, 26]. Lactic acid bacteria fermentation products potentiate the cell-mediated immune response by increasing the proliferative response of lymphocytes to concanavalin A (conA), a known activator of lymphocyte proliferation [5]. In addition, some findings suggest that milk fermented by Lactobacillus strains can modulate the immune response against breast cancer cells in mice [26] and improve innate-defense capacity in human being [24].

However, species other than those belonging to Lactobacillus genus are often isolated from dairy products, that may possess interesting properties [6, 12, 23]. We were interested in Enterococcus faecalis because it is an enterococcal species frequently found in dairy products, traditional cheeses in particular, where it may play an important role in determining cheese taste and texture [1, 27].

The aim of our study was to measure the ACE-inhibitory and immunomodulatory bioactivities in milk fermented with E. faecalis TH563 and compare them to those generated by L. delb. bulgaricus LA2. These strains belong to a panel of 14 bacterial strains (7 L. delb. lactis, 2 L. delb. bulgaricus, 1 L. helveticus, 2 L. paracasei and 2 E. faecalis) representing species that are frequently isolated from traditional dairy products of North Eastern Italy [2] and showing different degrees of proteolytic activity.

Although E. faecalis is reported to generate fermented milk with ACE-inhibitory activity [17, 19, 25], few information about its ability to generate immunomodulatory activity is available. On the contrary, L. delb. bulgaricus is commonly used as starter culture for the production of yogurt and fermented milks.

2. MATERIALS AND METHODS

2.1. Bacteria culture

E. faecalis TH563 and L. delb. bulgaricus LA2 were evaluated for their proteolytic activity as described by Hull [13] and in accordance with IDF, Standard 149A [14].

Lactobacilli were propagated in MRS (de Man, Rogosa and Sharpe) broth (Biolife, Milan, Italy) for 24 h at 44 °C, while enterococci were propagated in M17 broth (Difco Laboratories, Detroit, Michigan) for 24 h at 37 °C. Revitalised microorganisms were used to inoculate (1%, v/v) 10 mL of sterilised skim milk (Biolife, Milan, Italy), which was incubated for 24 h at 44 °C (lactobacilli) and 37 °C (enterococci). One millilitre of these milk pre-cultures was used to inoculate 100 mL of skim milk. Incubation was carried out under sterile conditions at 44 °C (lactobacilli) and 37 °C (enterococci).

2.2. Separation of the peptide fraction

Fermented milk samples were centrifuged at 20 000× g for 15 min at 15 °C (J2-21 Beckman Coulter centrifuge, JA 20 rotor, Fullerton, CA, USA) to remove bacterial debris. The supernatant was filtered with Amicon Centricon Ultra15 (molecular weight cut-off 5000 g·mol−1; Millipore, Billerica, MA, USA) by centrifugation at 3200× g for 40 min at 15 °C. The fraction with molecular weight lower than 5000 g·mol−1 (5000 g·mol−1 fraction) was stored at −20 °C and used for further analyses. The concentration of peptides in the 5000 g·mol−1 fractions was spectrophotometrically determined by the method of Layne [15].

2.3. ACE-inhibitory activity

The ACE-inhibitory activity of the 5000 g·mol−1 fractions was measured by the method of Cushman and Cheung [4], as modified by Nakamura et al. [20]. An Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech, NJ, USA) was used to measure the optical density of each 5000 g·mol−1 fraction.

Each test was performed in triplicate, and the measured absorbance was used for the calculation of the percentage of ACE inhibition (% ACE-I) as follows: %   ACE - I = 100 × ( B - A ) / ( B - C ) , $$ \%\enspace \mathrm{ACE}-\mathrm{I}=100\times (B-A)/(B-C), $$where A is the optical density of the samples in the presence of ACE, B is the optical density of the total activity and C is the optical density of the blank. Data were subjected to the analysis of variance, and the differences between mean values were analysed by the test of Duncan (SPSS Inc., Chicago, IL, USA).

2.4. Bovine peripheral blood lymphocytes proliferation

Ten millilitres of 5000 g·mol−1 fraction of fermented milk by E. faecalis TH563 and 30 mL of 5000 g·mol−1 fraction of fermented milk by L. delb. bulgaricus LA2 were dried under vacuum, and the obtained powders were dissolved in 5 mL of complete medium prepared as follows: RPMI-1640 medium (Sigma, St. Louis, MO, USA) containing 10% of heat-inactivated new-born calf serum (NCS, Sigma, St. Louis, MO, USA), 2 mmol·L−1 of L-glutamine (Sigma, St. Louis, MO, USA), 100 μg·mL−1 of streptomycin and 100 U·mL−1 of penicillin (Sigma, St. Louis, MO, USA). The concentration of peptides in the 5000 g·mol−1 fraction for the proliferation test was determined spectrophotometrically as described by Layne [15]. The 5000 g·mol−1 fractions were sterilised by filtration (0.22 μm filters) and stored at −20 °C until use.

To evaluate the immunomodulatory activity of the 5000 g·mol−1 fractions, bovine peripheral blood lymphocytes (BPBL) were isolated from whole heparin-anticoagulated blood of nine non-pregnant, non-lactating dairy cows without clinical symptoms by density gradient centrifugation using the Lymphoprep reagent (AXIS-SHIELD PoC AS, Oslo, Norway). Cells were suspended in complete medium in the presence of 2 μg·mL−1 of conA (Sigma, St. Louis, MO, USA) as mitogen and were incubated at 37 °C in 5% CO2. After 24 h of differentiation, non-adherent BPBL were separated from adherent leukocytes and tested for viability with Trypan blue staining. Viable BPBL were adjusted at a density of 3 × 106 cells·mL−1 in complete medium and incubated for 48 h in a 96-well microplate (100 μL cell suspension per well) with or without conA (2 μg·mL−1, positive control) and in the presence of increasing concentrations (from 0 μg·mL−1 to 100 μg·mL−1) of each fermented milk. At the end of the incubation period, proliferation test was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation test, following the manufacturer’s instructions. Briefly, MTT powder (Sigma, St. Louis, MO, USA) was dissolved in Hanks’ Balanced Salt Solution (Gibco Invitrogen, UK) (5 mg·mL−1), added to the cells (15 μL per well) and incubated for 3 h to allow the reductases of living cells to convert the MTT into the insoluble formazan. The formazan was then eluted with 10% (v/v) Triton X100 (Sigma, St. Louis, MO, USA), and the absorbance was measured at a wavelength of 570 nm with background subtraction at 630 nm using a microplate reader (Spectra Count, Packard Bioscience).

Each cell proliferation test was performed in triplicate. The results were expressed as the percentage of the optical density observed in the conA-treated BPBL (% conA). Relative variations of cellular proliferation produced by each fermented milk were analysed using a generalised linear model (GLM, SPSS Inc., Chicago, IL, USA). Differences between mean values were analysed by the Dunnett test (SPSS Inc., Chicago, IL, USA).

3. RESULTS AND DISCUSSION

Milk fermented by E. faecalis TH563 showed a significantly (P < 0.05) higher ACE-inhibitory activity (69.43 ± 3.12%) than L. delb. bulgaricus LA2 (60.86 ± 1.01%). The persistency of high ACE-inhibitory values up to 1:50 dilution for E. faecalis TH563 indicated an enzyme saturation effect that disappeared at 1:100 dilution. On the contrary, ACE-inhibitory activity in milk fermented by L. delb. bulgaricus LA2 was significantly reduced to very low levels when the 5000 g·mol−1 fraction was diluted 10-fold (P < 0.05) (Fig. 1).

thumbnail Figure 1.

ACE-inhibitory activity of the 5000 g·mol−1 fraction obtained after Amicon Ultra15 filtration of fermented milks. ACE-inhibitory activity was expressed as the % ACE-I. Milk fermented by E. faecalis TH563 (dark grey bars) showed a higher ACE-inhibitory activity if compared to L. delb. bulgaricus LA2 (light grey bars). Results are presented as means ± SEM of three independent experiments. Different superscripts indicate statistically different means (P < 0.05; Duncan test).

Even if strains of E. faecalis have been reported to possess high proteolytic activity [27], the ability to produce fermented milks with ACE-inhibitory activity has been scarcely documented [19, 25]. In the present experiment, ACE-inhibitory activity seemed to be positively related to the proteolytic activity of the strain of interest. In fact, E. faecalis TH563 showed a higher proteolytic activity (0.292 mg of tyrosine·mL−1) and peptide concentration (14.78 mg·mL−1) in the 5000 g·mol−1 fraction than L. delb. bulgaricus LA2 (proteolytic activity: 0.100 mg of tyrosine·mL−1, peptide concentration: 4.89 mg·mL−1), suggesting potentially greater ability to produce small peptides, which are mainly responsible for ACE-inhibitory activity [29].

The peptide concentration in the samples for MTT was 30.43 mg·mL−1and 37.72 mg·mL−1 for E. faecalis TH563 and L. delb. bulgaricus LA2, respectively.

The 5000 g·mol−1 fraction obtained from the milk fermented by E. faecalis TH563 did not significantly affect BPBL proliferation either with or without the mitogen conA (Fig. 2a). The 5000 g·mol−1 fraction obtained from the milk fermented by L. delb. bulgaricus LA2 was able to decrease the conA-induced BPBL proliferation when added at 5 μg·mL−1 (P < 0.001), 25 μg·mL−1 and 50 μg·mL−1 (P < 0.01) peptide concentration, but not at 100 μg·mL−1 (Fig. 2b). At this concentration, other factors might be present in a sufficient concentration to counteract the inhibitory effect on BPBL proliferation. Moreover, it is difficult to explain how fermented milks could modulate the cells of the immune system, and it is even more complicated to identify specific components produced during milk fermentation responsible for these immunomodulatory activities. Fermented milks are complex matrices, rich not only in proteins and peptides but also in sugars, fat, minerals and polysaccharides of the bacterial membrane that can contribute to the whole immunomodulatory effect. In this regard, it was demonstrated that milk fatty acids produced during fermentation affect cellular proliferation [7].

thumbnail Figure 2.

Dose-response effect of 5000 g·mol−1 fraction obtained after Amicon Ultra15 filtration from milk fermented by E. faecalis TH563 (a) or L. delb. bulgaricus LA2 (b) by MTT proliferation test, in the presence (■) or in the absence (●) of the mitogen concanavalin A (conA). The data were expressed as the percentage of the optical density observed in conA-treated BPBL cultured without fermented milk but in the presence of conA (positive control). Results are presented as means ± SEM of nine independent experiments for each strain. Asterisks indicate means significantly different from the positive control (*P < 0.001; **P < 0.01; Dunnett t-test).

When the milk fermented by L. delb. bulgaricus LA2 was administered without conA, it did not affect BPBL proliferation, although a slight increase in BPBL proliferation was observed at a peptide concentration of 5 μg·mL−1 (Fig. 2b).

The results of this experiment were in agreement with the hypothesis of Fujiwara et al. [9] suggesting that immunomodulatory activity is essentially expressed by strains of lactobacilli. Conversely, the immunomodulatory activity was not associated with ACE-inhibitory activity, differently from the assumption of Narva et al. [22].

The preliminary results of our work suggest that the presence of E. faecalis strains in traditional cheeses, where they play an important role in determining cheese taste and texture [1, 27], could contribute to generate dairy products with ACE-inhibitory activity. E. faecalis strains are not usually employed in the production of dairy foods since some of them can harbour potential virulence factors or antibiotic resistance [28], and their presence in the food system is still a matter of controversy due to their pathogenic potential [8]. Thus, E. faecalis strains should be evaluated for safety aspects before being used in the food industry. E. faecalis TH563 does not carry vanA or vanB genetic determinants for vancomycin transferable antibiotic resistance [2], but in order to completely assess its safety as adjunct culture in fermented milk, the strain should be tested for the absence of other potential virulence factors such as haemolysin, aggregation substances, surface proteins ace and esp [3].

Finally, it would be interesting to evaluate if milk fermented with both E. faecalis TH563 and L. delb. bulgaricus LA2 as mixed culture could generate a fermented milk showing both ACE-inhibitory and immunomodulatory activities.

Acknowledgments

This work was supported by a grant from the Agriculture Assessorship of the Province of Vicenza, Italy.

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All Figures

thumbnail Figure 1.

ACE-inhibitory activity of the 5000 g·mol−1 fraction obtained after Amicon Ultra15 filtration of fermented milks. ACE-inhibitory activity was expressed as the % ACE-I. Milk fermented by E. faecalis TH563 (dark grey bars) showed a higher ACE-inhibitory activity if compared to L. delb. bulgaricus LA2 (light grey bars). Results are presented as means ± SEM of three independent experiments. Different superscripts indicate statistically different means (P < 0.05; Duncan test).
In the text
thumbnail Figure 2.

Dose-response effect of 5000 g·mol−1 fraction obtained after Amicon Ultra15 filtration from milk fermented by E. faecalis TH563 (a) or L. delb. bulgaricus LA2 (b) by MTT proliferation test, in the presence (■) or in the absence (●) of the mitogen concanavalin A (conA). The data were expressed as the percentage of the optical density observed in conA-treated BPBL cultured without fermented milk but in the presence of conA (positive control). Results are presented as means ± SEM of nine independent experiments for each strain. Asterisks indicate means significantly different from the positive control (*P < 0.001; **P < 0.01; Dunnett t-test).
In the text