Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

Introduction

Lactic acid bacteria (LAB) present many important properties in food manufacturing, such as improvement of organoleptic and physical characteristics, production of lactic acid contributing to the extension of the shelf life, and reduction of lactose content. Several LAB are used in dairy industries as starter cultures as well as probiotics. Probiotics are defined as live microorganisms which when administered in adequate amounts confer health benefits to the host (WHO, 2002; Bertazonni-Minelli ; Oelschaeger, 2010; Todorov ). Probiotic beneficial effect for the host include suppression of growth of pathogens, control of serum cholesterol level, modulation of the immune system, improvement of lactose digestion, synthesis of vitamins, increase in bio-availability of minerals and possible anti-carcinogenic activity (Gomes and Xavier, 1999; Kailasapathy and Chin, 2000; Chan and Zhang, 2005). Bacteriocin producing LAB may also present a probiotic potential if capable of surviving the harsh conditions in the gastro-intestinal tract (GIT), including low pH and high concentrations of bile salts. As probiotics, these bacteria can confer health benefits to the host such as reduction of gastrointestinal infections and inflammatory bowel disease, modulation of the immune system, and defense against colonization by pathogenic microorganisms (WHO, 2002; Galdeano ; Oelschlaeger, 2010).

Aggregation of LAB is an important feature in evaluation of potential probiotic properties. While auto-aggregation may result in biofilm formation, co-aggregation with pathogens is important for elimination of non-desirable strains from the GIT (Todorov and Dicks, 2008). These capabilities are strain-specific and depend on species-specific surface proteins that recognize or bind to components in the environment. According to Kleerebezem , several genes encoding for surface proteins are homologous to genes coding for proteins with predicted functions, such as mucus-binding, aggregation-promotion and intracellular adhesion.

In the evaluation of a possible probiotic potential of LAB it is important to check for antibiotic resistance as they can act as potential reservoirs of resistance genes that can be transferred to other microorganisms, producing multidrug resistant strains (Dicks ). Probiotic consumers may be under treatment for a variety of illnesses, and the beneficial effects of the probiotic strain may be hampered by possible interactions with the medicaments used by these consumers. It should be emphasized that the interaction between antibiotics or other medicaments and probiotic bacteria in the GIT depends on their concentration in this environment (Todorov ; Todorov ), so establishing the Minimal Inhibitory Concentration (MIC) values play an important role for the proper evaluation of these interactions. Of higher importance are the drugs for treatment of chronic diseases normally used in long course treatments, since they may accumulate in the gastrointestinal tract and affect the viability of probiotic bacteria (Carvalho ).

Application of bacteriocinogenic strains for dairy product preservation is in agreement with consumers’ demands for foods that are naturally preserved. Additional claims of health-promoting benefits due to probiotic activity bring extra value to these foods. In this study we report results on the evaluation of the probiotic potential of the strain Lc. lactis DF04Mi.

Materials and Methods

Bacterial strains

The study was conducted with a bacteriocinogenic Lactococcus lactis subsp. lactis strain (Lc. lactis DF04Mi) isolated from raw goat milk (Furtado ). L. monocytogenes 711 (collection of University of São Paulo, Faculty of Pharmaceutical Sciences, Department of Food Science and Experimental Nutrition) and E. faecalis ATCC 19433 and Lactobacillus sakei ATCC 15521 (ATCC, Manassas, Virginia, USA) were used as co-aggregation partners. All strains were stored at −80 °C in presence of 15% glycerol.

Auto-aggregation and co-aggregation with L. monocytogenes 711, E. faecalis ATCC 19433 and Lb. sakei ATCC 15521

The cells of a culture of Lc. lactis DF04Mi grown in MRS broth (Difco) for 24 h at 37 °C were harvested by centrifugation (7,000 × g, 10 min, 20 °C), washed and resuspended in sterile saline (0.85% NaCl, w/v). One ml of the cell suspension adjusted to an OD660nm = 0.3 was transferred to a 2 mL sterile plastic cuvette and the OD660nm recorded, using a spectrophotometer (Ultraspec 2000, Pharmacia Biotech). After 60 min OD660nm readings were performed in the supernatant, after centrifuging the cultures at 300 × g for 2 min at 20 °C. Auto-aggregation was determined using the following equation (Todorov ):

OD0min and OD60min refer to the initial OD and the OD determined after 60 min, respectively.

For evaluation of co-aggregation strains Lc. lactis DF04Mi, E. faecalis ATCC 19433 and Lb. sakei ATCC 15521 were grown in 10 mL MRS at 30 °C and L. monocytogenes 711 in 10 mL BHI at 37 °C. Cells were harvested after 24 h (7,000 × g, 10 min, 20 °C), washed and re-suspended in sterile saline (0.85% NaCl). One ml of the cell suspension of Lc. lactis DF04Mi (adjusted to an OD660nm = 0.3) and 1 mL of co-aggregation partner (adjusted to an OD660nm = 0.3) were mixed and transferred to a 2 mL sterile plastic cuvette and the OD660nm recorded using a spectrophotometer. OD660nm readings were performed in the supernatant after 60 min and after centrifuging the suspension at 300 × g for 2 min at 20 °C. Co-aggregation was calculated using the following equation (Todorov ):

OD0min and OD60min refer to the OD determined immediately after mixing the cultures and the OD determined after 60 min, respectively. Experiments were conducted in triplicates on two separate occasions.

Effect of pH and bile on growth

Lc. lactis DF04Mi was grown in MRS broth (Difco) adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 9.0, 11.0 and 13.0 by adding 1 M HCl or 1 M NaOH before autoclaving. If needed, pH was re-adjusted after sterilization by adding sterile 1 M HCl or 1 M NaOH. The strain was also grown in MRS broth containing 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 2.0% and 3.0% (w/v) oxbile (Sigma). All tests were conducted in sterile flat-bottom 96-well microtiter plates (TPP, Switzerland). Each well was filled with 180 μL of the medium and inoculated with 20 μL of cultures of Lc. lactis DF04Mi activated in MRS broth (Difco) at 37 °C until presenting OD600nm = 0.2. Growth of Lc. lactis DF04Mi in the tested conditions was monitored through determination of OD600nm every hour for 12 h, using a microtiter plate reader (Versa-max, Sunnyvale, CA, USA). Cultures grown in non-modified MRS broth served as control. Experiments were done in triplicate.

Determination of cell surface hydrophobicity in Lc. lactis DF04Mi

The test for bacterial adhesion to hydrocarbons (BATH), described by Doyle and Rosenberg (1995) with some modification (Todorov ), was used. Lc. lactic DF04Mi was grown in MRS broth (Difco) at 37 °C for 18 h. Cells were harvested (6,700 g, 4 °C, 6 min), washed twice with sterile saline solution (0.85% NaCl), resuspended in the same solution and the optical density (OD580nm) was determined. An aliquot e of 1.5 mL cell suspension was added to 1.5 mL n-hexadecane (Sigma) and vortexed for 2 min. The phases were allowed to separate for 30 min at room temperature. One ml of the watery phase was removed and the optical density (OD580nm) determined. The experiment was repeated and the average optical density value determined. The percentage of hydrophobicity was calculated as follows:

Experiments with all strains were conducted in triplicate.

Resistance to drugs and antibiotics

Lc. lactis DF04Mi was tested for resistance to several drugs used in human and animal therapy, purchased in local drugstores (Sao Paulo, SP, Brazil) and solubilized in sterile water to achieve the desired concentration listed in Table 1. An overnight culture of Lc. lactis DF04Mi in MRS broth (Difco) was mixed with MRS soft agar (1.0%, w/v, Difco) in order to achieve a population of 106 cfu/mL and transferred to empty petri plates. After solidification of the agar 10 μL of each tested drug were spotted onto the surface. The plates were incubated at 37 °C for 24 h, examined for the presence of inhibition zones around the spotted drug, and those presenting inhibition zones larger than 2 mm diameter were subjected to the determination of the minimal inhibition concentration (MIC). For this test, serial two-fold dilutions of the drug were prepared in sterile water and 10 μL were spotted onto the surface of MRS soft agar plates, previously inoculated with strain Lc. lactis DF04Mi (approx. 106 cfu/mL). The plates were incubated for 24 h at 37 °C and examined for the presence of inhibition zones around the spots. The MIC corresponded to the highest dilution that resulted in inhibition halos of at least 2 mm diameter. In a similar experimental approach, susceptibility of strain Lc. lactis DF04Mi to antibiotics listed in Table 2 was tested by the disk diffusion test, using antibiotic disks from CEFAR (Sao Paulo, Brazil). The inhibitory effect of the antibiotics was expressed in millimeters of the inhibition zones.

Table 1

Effect of drugs on growth of Lactococcus lactis subsp lactis DF04Mi in MRS agar, presented as diameter of inhibition zones and Minimal Inhibitory Concentration (MIC).

Brazilian commercial name	Concentration (mg/mL)	Active substance	Medicament class	Inhibition zone (mm) [MIC (mg/mL)]
AAS	20	Acetylsalicylic acid	Analgesic / Antipyretic	-
Amoxil	100	Amoxicillin	β-Lactam antibiotic (Penicilin)	40 [ < 0.2]
Antak	30	Ranitidine hydrochloride	Histamine H2-receptor antagonist (proton pump inhibitor)	-
Arotin	4	Paroxetine	selective serotonin reuptake inhibitor (SSRI) antidepressant	-
Aspirina	100	Acetylsalicylic acid	Analgesic / Antipyretic	-
Atlansil	40	Amiodarone	Antiarrhythmic	12 [1.25]
Cataflam	10	Diclofenac potassium	Non-steroidal anti-inflammatory drug (NSAID)	20 [2.5]
Celebra	40	Celecoxib	NSAID	-
Clorana	5	Hydrochlorothiazide	Diuretic	-
Coristina R		Acetylsalicylic acid, Pheniramine maleate, Phenylephrine hydrochloride, Cafein	Analgesic / Antipyretic / Antihistaminic / Decongestant.	-
Diclofenaco*	10	Diclofenac potassium	NSAID	18 [0.16]
Diclofenaco*	10	Diclofenac potassium	NSAID	14 [0.32]
Dorflex	10	Orphenadrine citrate, Metamizole sodium, Cafein	Analgesic	-
Doxuran	0.8	Doxazosin	Antihypertensive / Treatment of prostatic hyperplasia	-
Dramin	20	Dimenhydrinate	Antiemetic	-
Fenergan	5	Promethazine hydrochloride	Antihistaminic	-
Fluimucil	8	Acetylcysteine	Mucolitic agent	-
Flutec	30	Fluconazole	Antifungal	-
Higroton	10	Chlorthalidone	Thiazide diuretic
Omeprazole	4	Omeprazole	Proton pump inhibitor	-
Neosaldina	60	Metamizole sodium, isometheptene mucate, cafein	Analgesic	-
Nimesulida	20	Nimesulide	NSAID	-
Nisulid	20	Nimesulide	NSAID	-
Redulip	3	Sibutramine hydrochloride monohydrate	Anorexiant / Sympathomimetic	-
Seki	3.54	Cloperastine	Antitussives (central and periferic mode of action)	-
Spidufen	120	Ibuprofen arginine	NSAID	20 [7.5]
Superhist	80	Acetylsalicylic acid, Pheniramine maleate, Phenylephrine hydrochloride	Analgesic / Antipyretic / Antihistaminic / Decongestant	-
Tylenol	150	Paracetamol	Analgesic / Antipyretic	-
Tylex	6	Paracetamol, Codein	Analgesic	-
Urotrobel	80	Norfloxacin	Antibiotic	10 [2.5]
Yasmin	0.6	Ethinylestradiol, drospirenone	Contraceptive	-
Zestril	4	Lisinopril	Antihypertensive (Angiotensin-converting enzyme (ACE) inhibitor)	-
Zocor	2	Simvastatin	Hypolipidemic	-
Zyrtec	2	Cetirizine hydrochloride	Antihistaminic	-

Produced by two different companies.

− = no inhibition.

Table 2

Effect of antibiotics on the growth of Lactococcus lactis subsp lactis DF04Mi, presented as diameter of the inhibition zone.

Antibiotic (μg/disk)	Classification (mode of action)	Inhibition zone (mm)
Amicacin (30)	Aminoglycoside (inhibits protein synthesis)	13
Ampicilin (10)	Penicillin / β-Lactam (interferes with bacteria cell wall synthesis)	30
Bacitracin (10)	Cyclic polypeptide (inhibits bacteria cell wall synthesis)	21
Cefazolin (30)	1st generation cephalosporin / β-Lactam (interferes with bacteria cell wall synthesis)	24
Cefepime (30)	4th generation cephalosporin / β-Lactam (interferes with bacteria cell wall synthesis)	34
Cefotaxim (30)	2nd generation cephalosporin / β-Lactam (interferes with bacteria cell wall synthesis)	33
Ceftazidim (30)	3th generation cephalosporin / β-Lactam (interferes with bacteria cell wall synthesis)	23
Ceftriaxon (30)	3th generation cephalosporin / β-Lactam (interferes with bacteria cell wall synthesis)	30
Cefuroxim (30)	β-Lactam (interferes with bacteria cell wall synthesis)	30
Ciprofloxacin (5)	Fluoroquinolone (inhibits the bacterial topoisomerase II)	18
Clindamicin (2)	Licosamide (inhibits protein synthesis)	25
Cloranfenicol (30)	Chloramphenicol (prevents peptide bond formation - inhibits protein synthesis)	28
Erytromicin (15)	Macrolide (inhibits protein synthesis)	28
Furazolidon (10)	Antibiotic / antiparasitic	21
Gentamicin (10)	Aminoglycoside (inhibits protein synthesis)	15
Imipenem (10)	Carbapenem / β-Lactam (interferes with bacteria cell wall synthesis)	32
Kanamicin (30)	Aminoglycoside (inhibits protein synthesis)	17
Metronidazol (50)	Nitroimidazole antibiotic (acts on DNA of microorganisms ameba, and protozoa)	0
Nalidixic acid (30)	Syntetic quinolone antibiotic (acts on DNA gyrase)	0
Neomicin (30)	Aminoglycoside (inhibits protein synthesis)	15
Nitrofurantoin (300)	Nitrofuran derivative (nucleic acid inhibitor)	22
Ofloxacin (5)	Licosamide (inhibits protein synthesis)	22
Oxacilin (1)	β-Lactam (interferes with bacteria cell wall synthesis)	15
Penicillin G (10)	Penicilin / β-Lactam (interferes with bacteria cell wall synthesis)	30
Rifampicin (30)	Semisynthetic compound derived from Amycolatopsis rifamycinica (inhibits DNA-dependent RNA polymerase)	17
Rifampicin (5)	Semisynthetic compound derived from Amycolatopsis rifamycinica (inhibits DNA-dependent RNA polymerase)	16
Streptomicin (10)	Aminoglycoside (inhibits protein synthesis)	20
Tetraciclin (30)	Tetraciclin (inhibits protein synthesis)	35
Tobramicin (10)	Aminoglycoside (inhibits protein synthesis)	15
Trimetoprim (5)	Trimetoprim (inhibts folate syntesis)	0
Vancomicin (30)	Glycopeptide (inhibits bacteria cell wall synthesis)	21

Results and Discussion

Bacteriocin produced by Lc. lactis DF04Mi presented a large spectrum of activity, inhibiting the growth of many food spoilage bacteria and foodborne pathogens. Similar results were recorded for the cell free supernatant and for the semi-purified bacteriocin. It is important to emphasize the bioactivity against L. monocytogenes, a foodborne pathogen of increasing importance. Antimicrobial activity against Gram-negative bacteria is also a relevant characteristic that was previously detected in other microorganisms including bacteriocins produced by Lc. lactis DF04Mi (Furtado ).

Auto-aggregation of Lc. lactis DF04Mi was low (12.2%) (Figure 1). Co-aggregation of Lc. lactis DF04Mi with the pathogens L. monocytogenes 711 and E. faecalis ATCC 19443 was 30.0% and 24.3%, respectively, which are lower values than for Lb. sakei ATCC 15521, a non-pathogen. The low levels of auto-aggregation and co-aggregation with pathogens may play an important role in preventing the formation of biofilms, and in this way eliminating the pathogens from the gastrointestinal tract.

Figure 1

Auto-aggregation of Lactococcus lactis subsp. lactis DF04Mi, Listeria monocytogenes 711, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19433 and co-aggregation of Lactococcus lactis subsp. lactis DF04Mi with Listeria monocytogenes 711, Lactobacillus sakei ATCC 15521 or Enterococcus faecalis ATCC 19433.

Good growth of Lc. lactis DF04Mi was recorded in MRS broth (Difco) when the initial pH was 6.0, 7.0 or 9.0 (Figure 2). Similar results were reported for Lc. lactis subsp. lactis HV219 (Todorov ). In another study, growth of several strains of Lb. plantarum, Lb. rhamnosus, Lb. pentosus and Lb. paracasei was suppressed at pH 3.0 and 4.0, while variable results were recorded in medium with an initial pH of 11.0 and 13.0, with poor growth recorded for strains Lb. paracasei ST242BZ and ST284BZ, Lb. rhamnosus ST462BZ, Lb. plantarum ST664BZ and Lb. pentosus ST712BZ at pH 13.0 (Todorov ).

Figure 2

Growth of Lactococcus lactis subsp. lactis DF04Mi in MRS containing increasing concentrations (0 to 3%) of oxbile (B) and at different pH (3 to 13) (A).

Lc. lactis DF04Mi grew well in the absence of oxbile, or when the concentration was below 3.0% (Figure 2). The bacteriocinogenic Lc. lactis HV219 strain was less tolerant to bile salts, as the growth of it was inhibited by concentrations of oxbile above 0.3% (Todorov ).

Other studies have also reported similar effects of oxbile and pH for different strains of Lc. lactis, such as Lb. plantarum 423, Lb. salivarius 241 and Lb. curvatus DF38 (Brink ). Haller reported variable results for different strains of Lb. plantarum when exposed to HCl (pH 2.0) and bile salts, and as many as 10% of Lb. plantarum cells, but less than 0.001% of Lb. sakei and Lb. paracasei cells survived these conditions. Although these assays cannot predict the behavior of the microorganism in the human body, the results are valuable in selecting Lactococcus spp. for probiotic applications, as resistance to low pH and high concentrations of bile salts is important for growth and survival of bacteria in the intestinal tract (Havenaar ; Carvalho ).

It has been suggested that there is a possible correlation between surface hydrophobicity of probiotic strains and the ability to adhere to the intestinal mucosa (Wadstrom ). Cell surface hydrophobicity defines a non-specific interaction between microbial and host cells. The initial interaction may be weak, is often reversible and precedes subsequent adhesion processes mediated by more specific mechanisms involving cell-surface proteins and lipoteichoic acids (Granato ; Rojas ; Roos and Jonsson, 2002). Bacterial cells with a high hydrophobicity usually form strong interactions with mucosal cells. Hydrophobicity values of 52.4% were recorded for Lc. lactis DF04Mi. Higher hydrophobicity values of 75% – 80% were recorded for strains Lb. rhamnosus ST461BZ, Lb. rhamnosus ST462BZ and Lb. plantarum ST664BZ and these values were higher than those reported for Lb. rhamnosus GG, well known commercial probiotic (55%) (Todorov ). Hydrophobicity varies among genetically close related species and even among strains of the same species (Schar-Zammaretti and Ubbink, 2003). Some strains with a high cell surface hydrophobicity were not able to properly adhere to Caco-2 and HT-29 cells (Todorov ). Strain Lb. pentosus ST712BZ, characterised with a relatively low hydrophobicity (38%) adhered to HT-29 cells at 63%. Only 32% adherence was recorded for Lb. rhamnosus GG (Todorov ). Although hydrophobicity may assist in adhesion, it is not a prerequisite for strong adherence to human intestinal cells.

The effect of drugs on the survival of probiotic strains is important for patients being treated for chronic diseases and taking probiotics. As shown in Table 1, Lc. lactis DF04Mi was inhibited by antiarrhrythmic drugs containing amiodarone, by non-steroidal anti-inflammatory drugs containing potassium diclofenac or ibuprofen arginine, and by the two tested antibiotics (amoxyl and urotrobel). The interference of anti-inflammatory drugs containing diclofenac on the viability of LAB detected in this study was also reported for other bacteriocinogenic LAB such as Lb. plantarum ST8KF and ST341LD, E. faecium ST311LD and Le. mesenteroides subsp. mesenteroides ST33LD (Todorov and Dicks, 2008), Lc. lactis subsp. lactis HV219 (Todorov ), Lb. casei Shirota and Lb. casei LC01 (Carvalho ) and Lb. casei Shirota (Botes ).

It should be pointed out that the activity of these medicaments against potential probiotic bacteria depends on the amount of the active compound that reaches the GIT, and that the correct evaluation of possible interactions between them depends on the determination of MIC of these medicaments (Todorov , 2008). Due to their long-term application, drugs may accumulate in the GIT and MIC be reached, affecting the viability of the probiotic cultures.

The non-steroidal anti-inflammatory drugs were the medicaments that affected Lc. lactis DF04Mi more significantly and potassium diclofenac presented MIC of 0.16 mg/mL and spidufen of 7.5 mg/mL. Considering that the recommended daily doses of these two drugs are 100–150 mg and 600 mg respectively, they will hardly affect the survival of Lc. lactis DF04Mi. Similar results were reported in other studies run with other probiotic LAB and gastrointestinal tract related bacteria (Botes ; Todorov and Dicks, 2008; Carvalho ; Todorov , 2008).

Except for metronidasole, nalidixic acid and trimetoprim, all the antibiotics tested (Table 2) in this study inhibited the growth of Lc. lactis DF04Mi to some extent. However, it is important to point out that nalidixic acid is an antibiotic mostly active against Gram-negative bacteria. Resistance of LAB to antibiotics is a controversial subject. It is important to keep in mind that antibiotic-resistant probiotic LAB may be responsible for horizontal transfer of resistance genes to other bacteria present in the human GIT (Dicks ). Resistance may be inherent to a bacterial genus or species, but may also be acquired through exchange of genetic material, mutations or incorporation of new genes (Ammor ). Teuber (1999) and Salyers suggested that starter cultures and probiotics may serve as vectors in the transfer of antibiotic resistant genes. Such transfer was documented in other bacterial groups by Levy and Marshall (2004) and Salyers .

Results gathered in this study indicate the potential application of Lc. lactis DF04Mi isolated from goat milk for manufacturing of novel and functional goat milk products, with increased safety as it is capable to produce bacteriocins with antilisterial activity and presents several features that suggest probiotic activity.