World J Microbiol Biotechnol DOI 10.1007/s11274-014-1671-7
ORIGINAL PAPER
Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods Silvia-Simona Grosu-Tudor • Mihaela-Marilena Stancu Diana Pelinescu • Medana Zamfir
•
Received: 10 January 2014 / Accepted: 14 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures. Keywords Lactic acid bacteria Bacteriocins Antibacterial effect Characterization Fermented foods
S.-S. Grosu-Tudor M.-M. Stancu M. Zamfir (&) Department of Microbiology, Institute of Biology Bucharest of the Romanian Academy, Splaiul Independentei No. 296, P.O. Box 56-53, 060031 Bucharest, Romania e-mail: [email protected] D. Pelinescu Department of Genetics, Faculty of Biology, University of Bucharest, Intrarea Portocalilor 1-3, 060101 Bucharest, Romania
Introduction Lactic acid bacteria (LAB) constitute an important group of generally recognized as safe (GRAS) microorganisms found in very diverse environments, including cereals, fruits and vegetables, milk and meat, playing an essential role in the fermentation of these substrates and for the manufacture of many fermented foods and beverages (Doyle et al. 2013). Since their discovery, LAB have been extensively studied from different perspectives, and impressive amount of data are available regarding, among others, their contribution to the nutritional and organoleptic characteristics of the final products, and to the improvement of the shelf life of fermented foods, due to the production of a wide variety of compounds (organic acids, ethanol, hydrogen peroxide, bacteriocins, antibiotic-like peptides etc.), acting in a synergistic way to prevent or eliminate microbial contamination (Reis et al. 2012). Among these, bacteriocins, produced by members of most LAB genera, including lactobacilli, lactococci, pediococci, leuconostocs, streptococci, and enterococci, have been thoroughly characterized, both biochemically and genetically (Cleveland et al. 2001; Macwana and Muriana 2012). They are ribosomally synthetized antimicrobial peptides or proteins that inhibit growth of other bacteria, usually closely related to the producing organism (De Vuyst and Leroy 2007). The antibacterial spectrum includes, however, several spoilage microoorganisms and food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus (De Vuyst and Leroy 2007; Sip et al. 2012; Zendo 2013). Based on their biochemical and molecular characterization, bacteriocins have been classified into three major classes (Nes et al. 1996): heat-stable, lanthionine containing bacteriocins (class I); small, heat-stable, non-lanthionine containing
123
World J Microbiol Biotechnol
bacteriocins (class II); and large, heat-labile antimicrobial proteins (class III). Bacteriocin production can be considered as an advantage and a functional role for LAB strains to be used in the food industry, to improve food quality and safety (De Vuyst and Leroy 2007; Parada et al. 2007). Moreover, bacteriocin production by probiotic LAB may play an important role during in vivo interactions occurring in the human gastrointestinal tract (De Vuyst et al. 2004). Naturally occurring antimicrobial compounds, such as bacteriocins, received increasing attention for food preservation due to consumers’ demand for minimally processed foods, without chemical preservatives, still safe and able to maintain good shelf life (Ponce et al. 2008). On the other hand, the emergence of bacterial resistance and multiple resistance to antibiotics represents a major public health problem and intensive efforts have been devoted to the development of alternative antimicrobial agents (Galvin et al. 1999). LAB bacteriocins may have potential applications as selective antimicrobials, with little or no effect on the normal microbiota of the host (Birri et al. 2012). Having in mind the importance and potential applications of bacteriocin-producing strains, it is understandable the permanent concern of researchers to find new LAB strains producing bacteriocins with antibacterial activity towards undesirable pathogenic or food spoilage bacteria. Functional strains with the proper physiological and metabolic features have been successfully isolated from natural habitats or from traditional fermented foods (Leroy and DeVuyst 2004). Romanian traditional fermented dairy products have been shown to be rich sources for bacteriocin and exopolysaccharide producing LAB strains (Zamfir et al. 1999; Grosu-Tudor et al. 2013). In the present study, strains isolated from fermented dairy products, but also from plant-origin materials, were used for the screening of antimicrobial activity and bacteriocin production. One of the main plant-origin source was bors, an easily produced sour liquid that results from a fermentation process of wheat bran and maize flour, used to sour the typical Romanian soups named ‘‘ciorbe’’. The information about this product are so far at empirical level, but it is well known that consumption of bors has many beneficial health effects. The article describes our results concerning the selection of some novel bacteriocin-producing LAB strains and the characterization of their antimicrobial compounds.
Materials and methods Lactic acid bacteria LAB used throughout this study have been isolated from Romanian artisan dairy products (105 strains, Grosu-Tudor
123
et al. 2013), fermented vegetables (43 strains, Wouters et al. 2013), and from other several products of plant origin, such as fresh fruits and vegetables, and bors (126 strains, results not published). MRS medium (de Man et al. 1960) was used for the isolation and subsequent growth of these strains. Strains were stored at -80 °C in MRS broth, containing 25 % (v/v) of glycerol as a cryoprotectant. To obtain fresh cultures from the freezed stocks, strains were propagated twice in MRS medium at 37 °C before the experiments. Screening of LAB for antibacterial activity Antibacterial activity was assayed by directly spotting 10 ll of the cell free culture supernatant (CFCS), obtained by centrifugation (13,0009g, 10 min, 4 °C), onto fresh lawns (soft agar media) of the indicator strains (De Vuyst et al. 1996). The bacterial strains used as indicator microorganisms for the evaluation of antimicrobial activities were as follows: Enterococcus faecalis LMG 16216, Enterococcus faecium LMG 14255, Lactobacillus brevis LMG 6906, Lactobacillus delbrueckii subsp. bulgaricus LMG 6901T, Lactobacillus helveticus 102, Lactobacillus sakei LMG 13558, Leuconostoc mesenteroides LMG 13562, Pediococcus pentosaceus LMG 13561, Bacillus cereus CBAB, Bacillus subtilis ATCC 6633, Eschercihia coli ATCC 25922, Salmonella enterica ATCC 14028, and Staphylococcus aureus ATCC 25923. LAB strains were grown in MRS medium, the two Bacillus strains in Luria– Bertani medium (Bertani 1951), while the other strains in Brain Heart Infusion medium (BHI, Merck, Darmstadt, Germany). Corresponding solid media were prepared with 1.5 % (w/v) agar and the soft agar media used for overlays were prepared with 0.7 % (w/v) agar. Evidence of bacteriocin production LAB strains showing an inhibitory effect against one or more indicator strains have been further subjected to various tests in order to establish the nature of the inhibitory compound. Strains were cultivated in MRS broth at 20, 28, 37 and 42 °C. Cells were removed by centrifugation (13,000g, 10 min, 4 °C) and the pH of the CFCS was adjusted to pH 7.0 with 1 mol l-1 NaOH. Antibacterial activity was assayed quantitatively by an agar spot test (De Vuyst et al. 1996). Briefly, serial twofold dilutions in water of the bacteriocin sample were spotted (10 ll) onto fresh indicator lawns of Lact. delbrueckii subsp. bulgaricus LMG 6901T. The activity was defined as the reciprocal of the highest dilution which demonstrated complete inhibition of the indicator lawn and was expressed in activity units (AU) per millilitre of culture medium. The specific activity was determined by dividing the
World J Microbiol Biotechnol
bacteriocin activity with the total protein concentration, as determined by the method described by Whitaker and Granum (1980). The influence of several proteolytic enzymes, such as tripsin (Sigma-Aldrich Chemie GmbH, Germany), pronase E (Merck KGaA, Darmstadt, Germany), proteinase K (Carl Roth, Karlsruhe, Germany), and pepsin (Sigma-Aldrich) was further investigated. Enzymes were dissolved in 0.2 mol l-1 sodium phosphate buffer (pH 7.5), except pepsin, which was dissolved in 0.2 mol l-1 HCl/KCl buffer (pH 2.0) and were added to the CFCS, at a final concentration of 1 mg ml-1. The inhibitory activity was tested after incubation for 2 h at 37 °C. The CFCS diluted with the buffers used to dissolve the enzymes, and enzymes solutions without CFCS served as controls. Identification of the bacteriocin-producing LAB strains
Table 1 Inhibitory spectrum of the bacteriocins isolated from Lact. amylolyticus P40 and P50, Lact. oris P49, L. lactis 19.3, Lact. plantarum 26.1, and Ent. durans 41.2 Indicator strainb
P40
P49
P50 19.3a
26.1
41.2
Lactobacillus acidophilus 5e2
-
-
-
?
±
-
Lactobacillus brevis LMG 6906
-
-
-
-
±
?
Lactobacillus brevis 403
-
-
-
??
-
-
Lactobacillus brevis 530
-
-
-
??
-
-
Lactobacillus delbrueckii subsp. bulgaricus LMG 6901T
??
?
?
??
?
?
Lactobacilus pentosus 265
-
-
-
?
-
-
Lactobacillus plantarum 788
-
-
-
?
-
±
Lactobacillus plantarum 3
-
-
-
-
-
±
Lactobacillus plantarum 198
-
-
-
?
±
-
Lactobacillus plantarum 619
-
-
-
?
-
-
Lactobacillus rhamnosus L
-
-
-
-
-
±
Lactobacillus sakei subsp. sakei LMG 13558 Leuconostoc citreum 96
-
-
-
?
-
-
-
-
-
?
?
?
Leuconostoc mesenteroides LMG 13562
-
-
-
?
-
?
Lactobacillus helveticus 102
The taxonomic affiliation of isolated bacteria was determined on the basis of their 16S rRNA sequence. PCR amplification of 16S rRNA gene, purification and sequencing of amplification products were performed as previously described by Stancu (2012). The products of the sequencing reactions were analyzed with an automatic Applied Biosystems 3500 Genetic Analyzer (Applied Biosystem/Hitachi, Tokyo, Japan). DNA sequencing runs were assembled using the BioEdit software. The new sequences of isolated bacteria were compared to those from databases using the BLAST search program.
Leuconostoc mesenteroides 197
-
-
-
±
-
?
Leuconostoc mesenteroides 355
-
-
-
?
?
-
Bacteriocins isolation and partial purification
Leuconostoc mesenteroides/ pseudomesenteroides 246 Pediococcus pentosaceus LMG13561
-
±
-
??
-
??
Enterococcus faecalis LMG 16216
-
-
-
-
-
-
Enterococcus faecalis LMG 13566
-
-
-
?
-
-
Enterococcus faecium LMG 14255
-
-
-
?
?
-
Enterococcus faecium LMG 14203
-
-
-
?
?
?
Lactococcus lactis 17
-
-
-
-
?
?
Lactococcus lactis subsp. diacetylactis 21
-
-
-
?
-
?
Bacillus cereus 53
-
-
-
?
-
-
Bacillus cereus CBAB
-
-
-
??
-
-
Bacillus subtilis ATCC 6633
-
-
-
??
-
-
Escherichia coli 159
-
-
-
?
-
-
Salmonella arizonae 18
-
-
-
?
-
-
Listeria monocytogenes ATCC 1911
-
-
-
±
-
-
The bacterial strains were grown for 12 h in MRS medium, at their optimum temperature previously determined for bacteriocin production. The CFCS were neutralized and precipitated with ammonium sulphate (40 % saturation), overnight, with gentle agitation. The floating pellicle formed during this step was collected after centrifugation (10,000g, 10 min, 4 °C), dissolved in 5 mmol l-1 potassium phosphate buffer (pH 6.5), and then extracted with 15 volumes of a mixture of chloroform/methanol (2/1, v/v). After l h at 4 °C, the white precipitate formed in the organic phase was centrifuged for 15 min at 13 000 g and resuspended in ultrapure water. Characterization of bacteriocins The heat sensitivity and pH stability of the bacteriocins were firstly tested. The concentrated bacteriocins (CFCS precipitated with ammonium sulphate) were used for these tests. Heat resistance was tested at 60 °C for 10, 30, and 60 min, at 100 °C for 10, 30, 60, and 120 min, and at 121 °C for 15 min. To test the influence of pH, the
Leuconostoc citreum 167
Staphylococcus aureus 54
-
-
-
-
-
–
Staphylococcus aureus ATCC 6538
-
-
-
-
-
-
123
World J Microbiol Biotechnol Table 1 continued Indicator strain
b
P40
P49
P50 19.3
a
26.1
41.2
Klebsiella pneumoniae 2
-
-
-
?
?
?
Lactobacillus amylolyticus P40
-
-
-
?
?
-
Lactobacillus oris P49
-
-
-
?
?
-
Lactobacillus amylolyticus P50
-
-
-
-
-
-
Lactococcus lactis 19.3
-
-
-
?
±
?
Lactobacillus plantarum 26.1 Enterococcus durans 41.2
-
-
-
??
?
?
-
-
-
?
±
?
a
?? = large inhibition zones, over 20 mm in diameter; ? = clear inhibition zones, less than 20 mm in diameter; ± = less clear inhibition zones; - = no inhibition
b
LMG Laboratorium voor Microbiologie, Universiteit Gent, Belgium, ATCC America Type Culture Collection; CBAB Centre of Applied Biochemistry and Biotechnology Bucharest, Romania
bacteriocin samples were adjusted to pH values between 2.0 and 10.0 with HCl or NaOH, and allowed to stand for one hour before testing the inhibitory activity. The requirement of disulfide bridges for bacteriocin activity was determined by measuring the activity in the presence of 10 mmol l-1 dithiothreitol (DTT) (Kemperman et al. 2003). To establish the inhibitory spectrum of the bacteriocins, 10 ll of the concentrated samples were spotted onto indicator lawns of several lactic acid bacterium strains and other Gram-positive and Gram-negative bacteria (Table 1). These lawns were prepared by propagating fresh bacterial cultures to an optical density (600 nm) of about 0.5 and adding 100 ll of the cell suspension to 3.5 ml of overlay agar (top layer). Overlaid agar plates were incubated for 24 h at the optimal growth temperature for each indicator strain. To estimate the molecular mass of the bacteriocins, tricine sodium dodecylsulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) was carried out, according to the method of Scha¨gger and von Jagow (1987). Polyacrylamide concentrations in the stacking gel and separating gel were 9.6 and 16.0 %, respectively. Electrophoresis was conducted at a constant voltage of 30 V in the stacking gel and 90 V in the separating gel. After the run (10 h), the gel containing the bacteriocin samples was washed during 5 h with sterile ultrapure water that was replaced every hour. Finally, the gel was transferred to an MRS plate and overlaid with a top layer of soft MRS agar inoculated with the indicator strain, Lact. delbrueckii subsp. bulgaricus LMG 6901T. Another part of the gel, containing both bacteriocins and the standard proteins (broad range protein molecular weight markers, Promega GmbH, Mannheim, Germany; polypeptide SDS-PAGE
123
molecular weight standards, BioRad Laboratories, Hercules CA), was stained with Coomassie Brilliant Blue. Bacteriocin assay in various combinations and in co-cultures of the producing strains Partially purified bacteriocins, extracted with chloroform/ methanol, were mixed, in different combinations of two bacteriocins. In one set of experiments combinations were made with similar amounts of proteins of each bacteriocin sample used in the mixture, while in the second set of experiments, with similar bacteriocin activities. For the cocultivation studies, MRS medium was inoculated with equal proportions of inoculum of two strains. Each of the producing strain was co-cultivated with the sensitive strain Lact. delbrueckii subsp. bulgaricus LMG 6901T, but also various combinations between the producing strains were done. Only combinations of strains without killing effect against each other, as determined by the spot on the lawn method (Tagg et al. 1976), were used. As controls, the pure cultures of the tested strains were used. Bacteriocin assay was done using the cell-free culture supernatant of the mixed/pure cultures.
Results Screening for antibacterial activity; evidence of bacteriocin production None of the 274 tested strains showed inhibitory activity against the pathogens Salm. enterica ATCC 14028, E. coli ATCC 25922 and Staph. aureus ATCC 25923, as shown from the screening of the corresponding CFCS. There were, however, 53 LAB strains which inhibited the growth of the two Bacillus strains used as indicator (results not shown). Moreover, six of these strains were able to repress the growth of one or more LAB strains used as indicator. Three of these strains (isolated from bors) were only active against Lact. delbrueckii subsp. bulgaricus LMG 6901T and the two bacilli, while the other three strains (isolated from fermented dairy products), were active against the two bacilli and several LAB indicator strains. The results are included in Table 1, presenting the inhibitory spectrum of the six LAB strains. The pH adjustment of the CFCS resulted in the loss of antibacterial activity against the two bacilli (except in the case of one strain, 19.3), but did not affect the activity of the six selected strains against the other LAB indicators. In order to establish if the inhibitory compound is of proteinaceous nature, the influence of several proteolytic enzymes on the activity towards the sensitive strain Lact. delbrueckii subsp. bulgaricus LMG 6901T was investigated
World J Microbiol Biotechnol Table 3 Influence of growth temperature on bacteriocin production
1
1
P50
7
2 3
6 4
5
Strain
41.2
20 °C
28 °C
37 °C
42 °C
Lactobacillus amylolyticus P40
n.g.
400
400
200
Lactobacillus oris P49
n.g.
200
200
n.d.
Lactobacillus amylolyticus P50
n.g.
6,400
200
200
Lactococcus lactis 19.3
25,600
25,600
400
n.g.
Lactobacillus plantarum 26.1
200
800
100
n.d.
Enterococcus durans 41.2
n.g.
n.d.
200
200
7
2
6
3 4
5
Fig. 1 Influence of proteolytic enzymes on the antibacterial activity of strains Lact. amylolyticus P50 (left) and Ent. durans 41.2 (right). 1 CFCS, 2 neutralized CFCS, 3 CFCS treated with pepsin, 4 CFCS ? HCl/KCl buffer (pH 2.0), 5 CFCS treated with proteinase K, 6 CFCS ? sodium phosphate buffer (pH 7.5), 7 CFCS ? pronase E
Inhibitory activitya (AU/ml)
n.g. no growth, n.d. not detected a
Lact. delbrueckii subsp. bulgaricus LMG 6901T was used as indicator strain
Bacteriocins isolation and purification Table 2 Taxonomic affiliation of the six bacteriocin-producing LAB strains Strain name
Closest bacterial strain name (accession number)
Percentage of nucleotide identity (%)
Lactobacillus amylolyticus P40
Lactobacillus amylolyticus strain TUST015 (KC456629.1)
93
Lactobacillus oris P49 Lactobacillus amylolyticus P50
Lactobacillus oris strain F0423 (HM596285.1) Lactobacillus amylolyticus strain TUST015 (KC456629.1)
98
Lactococcus lactis 19.3
Lactococcus lactis strain MNC26 (JQ754457.1)
87
Lactobacillus plantarum 26.1
Lactobacillus plantarum strain LS13 (JQ236619.1)
88
Enterococcus durans 41.2
Enterococcus durans strain ex14 (KF317884.1)
99
99
(Fig. 1). The inhibitory activity of all the six strains was inactivated by the treatment with proteinase K and pronase E. Additionally, the activity of strain 41.2 was also inactivated by pepsin and tripsin and the actvity of strain 26.1 was inactivated by tripsin. Based on these results, the six strains were selected to be used in further experiments, as bacteriocin-producing LAB. Identification of the LAB strains Based on the analysis of the 16S rRNA gene sequences, the six selected strains have been shown to belong to Lactobacillus, Lactococcus and Enterococcus genera. Table 2 shows the identity of these strains and the similarity to other bacterial strains from the public databases.
As a preliminary test, we have studied the influence of the incubation temperature on the growth and bacteriocin activity of the six selected strains (Table 3). For strains Lact. amylolyticus P40 and Lact. oris P49, similar bacteriocin activities were determined at 28 and 37 °C. These two strains did not grow at 20 °C, while at 42 °C the bacteriocin activity was lower, or not detectable. For the bacteriocin isolation step, these two strains were incubated at 37 °C. Strain Lact. amylolyticus P50 grew well at 37 and 42 °C, with a corresponding bacteriocin activity of 200 AU ml-1. The growth of this strain was neglictible after 48 h of incubation at 20 °C and very pour at 28 °C. The bacteriocin activity of the CFCS of the culture obtained at 28 °C was very high; however, due to the slow growth at this temperature, for the further studies we have decided to grow the strain at 37 °C. The same incubation temperature was used for strain Ent. durans 41.2, for which the bacteriocin activity was similar at 37 and 42 °C. This strain was not able to grow at 20 °C, while the bacteriocin activity at 28 °C was not detectable. On the contrary, strains L. lactis 19.3 and Lact. plantarum 26.1 showed the highest bacteriocin activities when incubated at 28 °C. At 37 °C, the inhibitory activity was much lower, while at 42 °C, the growth was very slow and no activity could be detected. For these two strains, the temperature used for the bacteriocin isolation was 28 °C. Bacteriocins were firstly concentrated by ammonium sulphate precipitation (40 % saturation) and then extracted/ precipitated with a mixture of chloroform/methanol. The activities recovered in each purification step, for each strain, are shown in Table 4. Characterization of bacteriocins The concentrated bacteriocins (ammonium sulphate precipitates) were used for a further characterization,
123
World J Microbiol Biotechnol Table 4 Bacteriocin activity recovered during the purification steps Purification stepa
Volume (ml)
P40
P49
P50
19.3
26.1
41.2
Culture supernatant
500
400
200
200
25,600
800
200
AS precipitation
20
25,600
3,200
3,200
6,553,600
6,400
1,600
C/M—bacteriocin activity
3
25,600
3,200
3,200
6,553,600
3,200
1,600
C/M—protein concentration
1.02
0.94
0.99
0.94
0.61
0.91
C/M—specific activity
25,098
3,404
3,232
6,971,915
5,246
1,758
a
AS ammonium sulphate precipitation, C/M chloroform/methanol extraction/precipitation
Table 5 Influence of heat treatment on bacteriocin activity Temperature/time
P40
P49
P50
19.3
26.1
41.2
25,600
3,200
3,200
25,600
6,400
1,600
10 min
800
400
1,600
25,600
6,400
1,600
30 min 60 min
100 0
200 100
0 0
25,600 25,600
6,400 6,400
800 800
10 min
200
200
0
12,800
6,400
800
30 min
0
100
0
12,800
6,400
800
Control 60 °C
100 °C
121 °C
Inhibitory activitya (AU/ml)
60 min
0
0
0
12,800
3,200
800
120 min
0
0
0
6,400
1,200
400
15 min
0
0
0
6,400
a
800 \100
T
Lact. delbrueckii subsp. bulgaricus LMG 6901 was used as indicator strain
regarding the heat resistance and pH stability, influence of DTT on their activity, the inhibitory spectrum, and the estimation of the molecular weight. As an exception, in the case of strain 19.3, the CFCS with pH adjusted to 7.0 was used, since the activity of this bacteriocin is very high (25,600 AU ml-1 before concentration). Based on their heat resistance, the six bacteriocins could be classified into two groups: heat resistant (strains L. lactis 19.3, Lact. plantarum 26.1, and Ent. durans 41.2) and heat sensitive (strains Lact. amylolyticus P40 and P50, and Lact. oris P49). The first group retained at least 50 % of the activity after incubation at 100 °C for 2 h (Table 5). Within this group, the bacteriocin produced by L. lactis 19.3 showed a strong heat resistance, 25 % of its activity being recovered after 15 min of incubation at 121 °C. In the second group, there are the other three bacteriocins, produced by the strains isolated from bors, strongly inactivated by the heat treatment. The activities were mostly lost after incubation at 60 °C for 30 min. The incubation at 100 °C had even stronger effect, for instance in the case of Lact. amylolyticus P50, the activity was lost after 10 min of incubation (Table 5). The three heat resistant bacteriocins were also stable in a very wide pH range (activity was detected at pH between
123
2.0 and 10.0). The other three bacteriocins kept their activity at pH between 4.0 and 10.0. DTT did not destroy the activity of any of the six bacteriocins (results not shown), indicating that, most probably, they do not contain disulfide bridges that are essential for the antibacterial activity. The inhibitory spectrum of the six bacteriocins, as determined for the concentrated samples, is presented in Table 1. Again, we could make a distinction between a group of bacteriocins with a very narrow inhibitory spectrum (restricted to only one or two LAB strains), represented by the compounds that were also heat labile, and a group with a broad inhibitory spectrum, represented by the other three bacteriocins. Within this second group, we can underline the bacteriocin produced by L. lactis 19.3, able to inhibit the growth of 27 of the 36 LAB strains used as indicators, among which B. cereus 53, B. cereus CBAB, B. subtilis ATCC 6633, Listeria monocytogenes ATCC 1911 and the two Staph. aureus strains used in this test. It is was also observed that incubation of this bacteriocin at 60 and 100 °C resulted in a faster decrease of the activity when tested against Staph. aureus ATCC 6538, compared with the activity towards Lact. delbrueckii subsp. bulgaricus LMG 6901T (results not shown). Molecular mass estimation The partially purified bacteriocins (concentrated with ammonium sulphate, followed by extraction with chloroform/methanol) were subjected to Tricine-SDS-PAGE and after migration, the gels were extensively washed and overlaied with indicator lawns. After 24 h of incubation, a clear inhibition zone could be observed for each sample (Fig. 2). In case of L. lactis 19.3 and Lact. plantarum 26.1, this zone corresponded to a very low molecular mass (between 3,496 and 6,512 Da), while for the other four bacteriocins, the molecular mass was estimated to about 35,000 Da). In some gels, for the line corresponding to strain Lact. amylolyticus P40, a second inhibition zone, very weak, corresponding to a low molecular mass (about 6,500 Da), could be detected (Fig. 2).
World J Microbiol Biotechnol
a
MM
P40
P49
P50
41.2
P40
P49
P50
41.2
50,000 35,000 25,000
15,000 10,500
MM
b
26.1
19.3
19.3
c
+41.2
19.3
26.1
19.3 +41.2
26,625 16,950
26,625
14,437 6,512 3,496
16,950 14,437
6,512
Fig. 2 Tricine-SDS-PAGE of the bacteriocins isolated from the six selected strains (gels overlaied with the indicator lawn, L. delbrueckii subsp. bulgaricus LMG 6901T—left side pictures, and stained with Coomassie Brilliant Blue—right side pictures). a Lact. amylolyticus P40, Lact. oris P49, Lact. amylolyticus P50, and Ent. durans 41.2; the arrow indicates the weak band detected in some cases in the
bacteriocin sample isolated from Lact. amylolyticus P40; b Lact. plantarum 26.1, L. lactis 19.3, and a mixture of the two bacteriocins isolated from L. lactis 19.3 and Ent. durans 41.2, showing two clear inhibition bands; c L. lactis 19.3; MM molecular weight markers (see ‘‘Materials and methods’’)
123
World J Microbiol Biotechnol Table 6 Inhibitory activity of various mixtures of bacteriocins Combination of bacteriocins
Ratioa
Individual activities (AU/ml)
P40 ? P49
1:1
12,800 ? 1,600
1:8
1,600 ? 1,600
51,200
16
P40 ? P50
1:1
12,800 ? 1,600
102,400
7
1:8
1,600 1 1,600
409,600
128
P49 ? P50
1:1
1,600 1 1,600
409,600
128
19.3 ? P40
1:1
3,278,800 ? 12,800
6,553,600
2
19.3 ? P49
1:256 1:1
12,800 ? 12,800 3,278,800 ? 1,600
102,400 6,553,600
4 2
12,800
4
Combined activity (AU/ml) 102,400
Approximate fold of increase 7
1:2048
1,600 ? 1,600
1:1
3,278,800 ? 1,600
1:2048
1,600 ? 1,600
51,200
16
1.6:1
1,600 ? 8,000
51,200
5
8:1
1,600 ? 1,600
6,400
2
26.1 ? P49
1.5:1
1,600 ? 1,066
51,200
19
1:1
1,600 ? 1,600
204,800
64
26.1 ? P50
1.5:1
1,066 1 1,600
614,400
1:1
1,600 ? 1,600
25,600
1:1
800 ? 12,800
102,400
1:16
1,600 ? 1,600
1,600
19.3 ? P50 26.1 ? P40
41.2 ? P40 41.2 ? P49
6,553,600
2
230 8 8 0.5
1:1
800 ? 1,600
1,600
0.7
1:2
800 ? 800
1,600
1
41.2 ? P50
1:1 1:2
800 ? 1,600 800 ? 800
3,200
1
19.3 ? 26.1
1:1.5
2,185,866 ? 1,600
19.3 ? 41.2 26.1 ? 41.2
1,600 409,600
1 0.2
1:2048
1,600 ? 1,600
1:1
3,278,800 ? 800
800
0.3
409,600
0.1
1:4096
800 ? 800
400
0.3
1.6:1
1,600 ? 500
1,600
0.8
1:3
800 ? 800
3,200
2
Bold values indicate the combinations of bacteriocins with the highest increase in activity compared with the cumulative activities a
Ratio was calculated to get similar protein concentrations of each bacteriocin (first set of experiments) or similar activities of each bacteriocin (second set of experiments); Lact. delbrueckii subsp. bulgaricus LMG 6901T was used as indicator strain
Bacteriocin assay in various combinations and in co-cultures of the producing strains The activities of the various mixtures of the three bacteriocins produced by the strains isolated from bors were significantly higher (up to 128 times) comparing with the cumulative activities of the single bacteriocins, especially when the combinations were made with similar bacteriocin activities (Table 6). In case of the three strains isolated from fermented dairy products, the combination of their produced bacteriocins resulted in a slight decrease of the inhibitory activity. However, some combinations among bacteriocins produced by strains isolated from
123
dairy products and bors, respectively, seemed to be more effective than the single bacteriocins. For instance, an increase of about 230 times of the activity was observed in the case of a mixture of the two bacteriocins produced by Lact. plantarum 26.1 and Lact. amylolyticus P50 (Table 6). Co-cultivation of two bacteriocin-producing strains, or in the presence of the sensitive strain resulted, in most of the cases, in a decrease of the inhibitory activity (results not shown). There was only one strain, Lact. oris P49, which seemed to produce more bacteriocins when it was co-cultivated in the presence of the sensitive strain (400 AU ml-1 compared with 100 AU ml-1 for the mono-culture).
World J Microbiol Biotechnol
Discussion The present study describes six new LAB strains isolated from Romanian traditional fermented foods that are able to produce bacteriocins with various characteristics. A large number of LAB strains (274 isolates) have been tested for the ability to produce bacteriocins. They have been isolated from various types of fermented foods, such as dairy products, fermented vegetables and bors, but also from fresh vegetables, fruits and flowers. The isolation and screening of microorganisms from natural sources has always proved to be a successful way for obtaining industrially important strains or strains with valuable medical applications (Todorov and Dicks 2006; Yang et al. 2012). However, only a limited number of strains (about 20 %) showed antimicrobial activity, especially against Bacillus strains and other LAB used as indicators. In most cases, the antimicrobial activity might be due to the production of organic acids, since the pH neutralization resulted in the loss of this effect. The antibacterial activity was shown to be due to the production of bacteriocins only for six of these strains (about 2 % of the tested strains). Similar low percentages of bacteriocin-producing strains have been reported by other authors for strains isolated from fresh-cut vegetable products (Yang et al. 2012), from milk and meat products (Sezer and Gu¨ven 2009), or from malt (Rouse et al. 2007). The food source and isolation media might be important for the successfully isolation of bacteriocinogenic LAB, since many bacteriocin-producing LAB have been isolated from fermented dairy products, such as yogurts and cheeses (Yang et al. 2012). In our case, three of the six bacteriocin-producing strains have been isolated from fermented dairy products. The bacteriocin-producing strains have been identified based on the 16S rRNA sequencing and four of them belong to the Lactobacillus genus. Lactobacillus is a versatile bacterial genus that has a long history with human daily life. It is also one of the important group of bacteriocin-producing LAB (Diep et al. 2009). Two of the Lactobacillus strains have been identified as Lact. amylolyticus and one as Lact. oris. Lact. amylolyticus has been described in beer malt and beer wort (Bohak et al. 1998) and it is likely to be found in other fermented cereals, such is the Romanian bors. On the other hand, Lact. oris has been described in the human oral cavity (Farrow and Collins 1988), and might be only a contaminant of bors. The strains belonging to these two species, isolated from bors, showed inhibitory activity against Lact. delbrueckii subsp. bulgaricus LMG 6901T, but also against B. cereus CBAB and B. subtilis ATCC6633. The bacteriocins isolated from these strains retained the activity against the LAB, but not against the two bacilli. The proteinaceous nature of the three bacteriocins have been proven by their
inactivation after treatment with proteinase K and pronase E. Moreover, the inhibitory activity of these bacteriocins was significantly decreased after incubation at 60 °C for only 10 min. Based on the limited inhibitory spectrum, the heat sensitivity, and the high molecular mass (about 35,000 Da, as determined by Tricine-SDS-PAGE), the bacteriocins produced by these three strains (Lact. amylolyticus P40 and P50, and Lact. oris P49) might be considered to belong to class III bacteriocins (Nes et al. 1996). Class III bacteriocins are much less studied compared with the first two classes and only few reports concerning their isolation and characterization are available (Joerger and Klaenhammer 1986; Vaughan et al. 1992; Nilsen et al. 2003). However, such bacteriocins with very narrow inhibitory spectrum might find applications as highly selective antibacterial agents, if they are able to control target bacteria specifically. They can be used in lower doses and the risk to develop resistance in nontarget bacteria is much lower (Zendo 2013). Although no data available about isolation and characterization of bacteriocins produced by Lact. amylolyticus and Lact. oris, strains belonging to these species have been shown recently to have potential probiotic effect (Pedersen et al. 2004; Ko˜ll et al. 2008; Teanpaisan et al. 2011). The fourth Lactobacillus strain was identified to be Lact. plantarum. Regarding the inhibitory spectrum and the biochemical characteristics, the bacteriocin produced by this strain could be grouped with the two bacteriocins produced by the other two strains isolated from fermented dairy products, identified as L. lactis 19.3 and Ent. durans 41.2. The bacteriocins produced by these three strains might belong to class I and/or II according to the classification of Nes et al. (1996), having a broad antimicrobial spectrum, including Bacillus sp., L. monocytogenes, and Staph. aureus (except strain Ent. durans 41.2). Recent studies showed that class IIa bacteriocins exert the highest inhibitory effect on L. monocytogenes (Sip et al. 2012). Within this class, one can find many bacteriocins produced by Lact. plantarum (van Reenen et al. 1998; Atrih et al. 2001; Ennahar et al. 1999; Todorov et al. 2007), but also some bacteriocins produced by L. lactis (Ferchichi et al. 2001). It is difficult for the moment to classify the bacteriocin produced by L. lactis 19.3; however, the very low molecular mass (between 3,496 Da and 6,512 Da, as determined by Tricine-SDS-PAGE), the broad inhibitory spectrum (27 of the 36 indicator strains were inhibited) and the stability even at alkaline pH values, are common characteristics with lacticins produced by L. lactis (Zendo 2013), belonging to class IId bacteriocins. L. lactis can be considerred as a representative LAB and a model microorganism used in the manufacture of various fermented foods. Novel bacteriocins produced by this species, different from nisin, are very important, to be used in
123
World J Microbiol Biotechnol
combination with, or as a next generation of nisin (Fujita et al. 2007). The significant thermal stability and the stability under neutral and alkaline pH conditions of the bacteriocin isolated from L. lactis 19.3 might be an advantage for the potential use as a biopreservative, for instance in combination with a thermal processing of the foods (Yang et al. 2012). The third bacteriocin of this group, produced by Ent. durans 41.2 has a similar broad inhibitory spectrum, including L. monocytogenes, Leuc. mesenteroides, Ent. faecium and Ent. faecalis, but not Pediococcus pentosaceus or Staph. aureus. The biochemical characteristics are similar with the ones of the other two bacteriocins. However, the molecular mass of this bacteriocin was estimated to about 35,000 Da, which is an unusual molecular mass for a class II bacteriocin (De Vuyst and Vandamme 1994). Further studies might reveal if this is a new bacteriocin, with unique features, and in which existing class can be assigned. The co-cultivation of various combinations of two bacteriocin-producing strains resulted, in general, in a decrease of the inhibitory activity of the CFCS. It is well known that some LAB strains, such as L. lactis, exhibit strong proteolytic action, which may cause the inactivation of the bacteriocins produced by the strain used in coculture (Sip et al. 2012). On the other hand, the suppression of bacteriocin production in mixed-species cultures of LAB has been observed by Domı´nguez-Manzano and Jime´nez-Dı´az (2013). The authors suggest that this phenomenon could be due to a regulation of the bacteriocin production exerted by each strain against the other. Therefore, it is very important to check such interactions when mixed starter cultures are going to be used in food fermentations, to avoid the negative effect on the quality of the final product (Domı´nguez-Manzano and Jime´nezDı´az 2013). Moreover, some of the isolated bacteriocins seemed to exhibit a synergistic effect. The most evident effect was shown by combinations between bacteriocins with different characteristics, produced by strains isolated from different sources (dairy and bors, respectively). It was previously postulated that the presence of receptors on cell surface plays an important role in bacteriocin specificity (Drider et al. 2006). Bacteriocins which belong to different categories and with different mode of actions are likely to exhibit synergistic effect, since they do not have to compete for the same receptors on the indicator cell surface (Vignolo et al. 2000). In conclusion, traditional fermented foods are still an important source of functional LAB. The bacteriocin-producing strains isolated in our study may find applications as protective cultures. Our results also bring important information about the biochemical characterization of the
123
bacteriocins, the effect of co-cultivation and the synergistic effect of some of these proteinaceous substances. Purification to homogeneity as well as further characterization, are currently in progress. Acknowledgments This work was supported by the grants of the Romanian National Authority for Scientific Research, CNDI–UEFISCDI, project numbers 105/2012 (PLANTLAB) and 77/2012 (PROLAB).
References Atrih A, Rekhif N, Moir AJ, Lebrihi A, Lefebvre G (2001) Mode of action, purification and amino acid sequence of plantaricin C19. Int J Food Microbiol 68:93–104 Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300 Birri DJ, Brede DA, Nes IF (2012) Salivaricin D, a novel intrinsically trypsin-resistant lantibiotic from Streptococcus salivarius 5M6c isolated from a healthy infant. Appl Environ Microbiol 78:402–410 Bohak I, Back W, Richter L, Ehrmann M, Ludwig W, Schleifer KH (1998) Lactobacillus amylolyticus sp. nov., isolated from beer malt and beer wort. Syst Appl Microbiol 21:360–364 Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71:1–20 de Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135 De Vuyst L, Leroy F (2007) Bacteriocins from lactic acid bacteria: production, purification, and food applications. J Mol Microbiol Biotechnol 13:194–199 De Vuyst L, Vandamme EJ (1994) Bacteriocins of lactic acid bacteria. Blackie Academic and Professional, London De Vuyst L, Callewaert R, Pot B (1996) Characterization of the antagonistic activity of Lactobacillus amylovorus DCE 471 and large scale isolation of its bacteriocin amylovorin L471. Syst Appl Microb 19:9–20 De Vuyst L, Avonts L, Makras E (2004) Probiotics, prebiotics and gut health. In: Remacle C, Reusens B (eds) Functional foods, ageing and degenerative disease. Woodhead Publishing, Cambridge, pp 416–482 Diep DB, Straume D, Kjos M, Torres C, Nes IF (2009) An overview of the mosaic bacteriocin pln loci from Lactobacillus plantarum. Peptides 30:1562–1574 Domı´nguez-Manzano J, Jime´nez-Dı´az R (2013) Suppression of bacteriocin production in mixed-species cultures of lactic acid bacteria. Food Control 30:474–479 Doyle MP, Steenson LR, Meng J (2013) Bacteria in food and beverage production. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes: applied bacteriology and biotechnology. Springer, Berlin, pp 241–256 Drider D, Fimland G, Hechard Y, McMullen LM, Prevost H (2006) The continuing story of class IIa bacteriocins. Microbiol Mol Biol Rev 70:564–582 Ennahar S, Sonomoto K, Ishizaki A (1999) Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation. J Biosci Bioeng 87:705–716 Farrow JAE, Collins MD (1988) Lactobacillus oris sp. nov. from the human oral cavity. Int J Syst Bacteriol 38:116–118 Ferchichi M, Frere J, Mabrouk K, Manai M (2001) Lactococcin MMFII, a novel class IIa bacteriocin produced by Lactococcus lactis MMFII, isolated from a Tunisian dairy product. FEMS Microbiol Lett 205:49–55
World J Microbiol Biotechnol Fujita K, Ichimasa S, Zendo T, Koga S, Yoneyama F, Nakayama J, Sonomoto K (2007) Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of Gram-positive bacteria. Appl Environ Microbiol 73:2871–2877 Galvin M, Hill C, Ross RP (1999) Lacticin 3147 displays activity in buffer against Gram-positive bacterial pathogens which appear insensitive in standard plate assays. Lett Appl Microbiol 28:355–358 Grosu-Tudor SS, Zamfir M, Van der Meulen R, De Vuyst L (2013) Isolation of novel homopolysaccharide producing lactic acid bacteria from Romanian raw milk and fermented dairy products. Eur Food Res Technol 237:609–615 Joerger MC, Klaenhammer TR (1986) Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J Bacteriol 167:439–446 Kemperman R, Kuipers A, Karsens H, Nauta A, Kuipers O, Kok J (2003) Identification and characterization of two novel clostridial bacteriocins, circularin A and closticin 574. Appl Environ Microbiol 69:1589–1597 Ko˜ll P, Ma¨ndar R, Marcotte H, Leibur E, Mikelsaar M, Hammarstro¨m L (2008) Characterization of oral lactobacilli as potential probiotics for oral health. Oral Microbiol Immunol 23:139–147 Leroy F, DeVuyst L (2004) Lactic acid bacteria as functional starter cultures for the food fermentation Industry. Trends Food Sci Technol 15:67–78 Macwana SJ, Muriana PM (2012) A ‘bacteriocin PCR array’ for identification of bacteriocin-related structural genes in lactic acid bacteria. J Microbiol Meth 88:197–204 Nes IF, Diep DB, Ha˚verstein IS, Brurberg MB, Eijsink V, Holo H (1996) Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Van Leeuw 70:113–128 Nilsen T, Nes IF, Holo H (2003) Enterolysin A, a cell wall-degrading bacteriocin from Enterococcus faecalis LMG2333. Appl Environ Microbiol 69:2975–2984 Parada JL, Caron CR, Medeiros ABP, Soccol CR (2007) Bacteriocins from lactic acid bacteria: purification, properties and use as biopreservatives. Braz Arch Biol Technol 50:521–542 Pedersen C, Jonsson H, Lindberg JE, Roos S (2004) Microbiological characterization of wet wheat distillers’ grain, with focus on isolation of lactobacilli with potential as probiotics. Appl Environ Microbiol 70:1522–1527 Ponce AG, Moreira MR, Del Valle CE, Roura SI (2008) Preliminary characterization of bacteriocin-like substance from lactic acid bacteria isolated from organic leafy vegetables. LWT—Food. Sci Technol 41:432–441 Reis JA, Paula AT, Casarotti SN, Penna ALB (2012) Lactic acid bacteria antimicrobial compounds: characteristics and applications. Food Eng Rev 4:124–140 Rouse S, Sun F, Vaughan A, van Sinderen D (2007) High-throughput isolation of bacteriocin-producing lactic acid bacteria, with potential application in the brewing industry. J Inst Brew 113:256–262
Scha¨gger H, von Jagow G (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379 Sezer G, Gu¨ven A (2009) Investigation of bacteriocin production capability of lactic acid bacteria isolated from foods. Lafkas Univ Vet Fak Derg 15:45–50 Sip A, Wieckowicz M, Olejnik-Schmidt A, Grajek W (2012) AntiListeria activity of lactic acid bacteria isolated from golka, a regional cheese produced in Poland. Food Control 26:117–124 Stancu MM (2012) Investigation of organic solvent resistance mechanisms in Vibrio alginolyticus IBBCt2. Water Air Soil Poll 223:4495–4505 Tagg JR, Dajani AS, Wannamaker LW (1976) Bacteriocins of gram positive bacteria. Bacteriol Rev 40:722–756 Teanpaisan R, Piwat S, Dahle´n G (2011) Inhibitory effect of oral Lactobacillus against oral pathogens. Lett Appl Microbiol 53:452–459 Todorov SD, Dicks LMT (2006) Screening for bacteriocin-producing lactic acid bacteria from boza, a traditional cereal beverage from Bulgaria. Comparison of bacteriocins. Process Biochem 41:11–19 Todorov SD, Nyati H, Meincken M, Dicks LMT (2007) Partial characterization of bacteriocin AMA-K produced by Lactobacillus plantarum AMA-K isolated from naturally fermented milk from Zimbabwe. Food Control 18:656–664 Van Reenen CA, Dicks LM, Chikindas ML (1998) Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum. J Appl Microbiol 84:1131–1137 Vaughan EE, Daly C, Fitzgerald GF (1992) Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829. J Appl Bacteriol 73:299–308 Vignolo G, Palacios J, Farias ME, Sesma F, Schillinger U, Holzapfel W, Oliver G (2000) Combined effect of bacteriocins on the survival of various Listeria species in broth and meat system. Curr Microbiol 41:410–416 Whitaker JR, Granum PE (1980) An absolute method for protein determination based on the difference in absorbance at 235 and 280 nm. Anal Biochem 109:156–159 Wouters D, Grosu-Tudor SS, Zamfir M, De Vuyst L (2013) Applicability of Lactobacillus plantarum IMDO 788 as a starter culture to control vegetable fermentations. J Sci Food Agric. doi:10.1002/jsfa.6184 Yang E, Fan L, Jiang Y, Doucette C, Fillmore S (2012) Antimicrobial activity of bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts. AMB Express 2:48–59 Zamfir M, Callewaert R, Cornea CP, Savu L, Vatafu I, De Vuyst L (1999) Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801. J Appl Microbiol 87:923–931 Zendo T (2013) Screening and characterization of novel bacteriocins from lactic acid bacteria. Biosci Biotechnol Biochem 77:893–899
123
ORIGINAL PAPER
Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods Silvia-Simona Grosu-Tudor • Mihaela-Marilena Stancu Diana Pelinescu • Medana Zamfir
•
Received: 10 January 2014 / Accepted: 14 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures. Keywords Lactic acid bacteria Bacteriocins Antibacterial effect Characterization Fermented foods
S.-S. Grosu-Tudor M.-M. Stancu M. Zamfir (&) Department of Microbiology, Institute of Biology Bucharest of the Romanian Academy, Splaiul Independentei No. 296, P.O. Box 56-53, 060031 Bucharest, Romania e-mail: [email protected] D. Pelinescu Department of Genetics, Faculty of Biology, University of Bucharest, Intrarea Portocalilor 1-3, 060101 Bucharest, Romania
Introduction Lactic acid bacteria (LAB) constitute an important group of generally recognized as safe (GRAS) microorganisms found in very diverse environments, including cereals, fruits and vegetables, milk and meat, playing an essential role in the fermentation of these substrates and for the manufacture of many fermented foods and beverages (Doyle et al. 2013). Since their discovery, LAB have been extensively studied from different perspectives, and impressive amount of data are available regarding, among others, their contribution to the nutritional and organoleptic characteristics of the final products, and to the improvement of the shelf life of fermented foods, due to the production of a wide variety of compounds (organic acids, ethanol, hydrogen peroxide, bacteriocins, antibiotic-like peptides etc.), acting in a synergistic way to prevent or eliminate microbial contamination (Reis et al. 2012). Among these, bacteriocins, produced by members of most LAB genera, including lactobacilli, lactococci, pediococci, leuconostocs, streptococci, and enterococci, have been thoroughly characterized, both biochemically and genetically (Cleveland et al. 2001; Macwana and Muriana 2012). They are ribosomally synthetized antimicrobial peptides or proteins that inhibit growth of other bacteria, usually closely related to the producing organism (De Vuyst and Leroy 2007). The antibacterial spectrum includes, however, several spoilage microoorganisms and food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus (De Vuyst and Leroy 2007; Sip et al. 2012; Zendo 2013). Based on their biochemical and molecular characterization, bacteriocins have been classified into three major classes (Nes et al. 1996): heat-stable, lanthionine containing bacteriocins (class I); small, heat-stable, non-lanthionine containing
123
World J Microbiol Biotechnol
bacteriocins (class II); and large, heat-labile antimicrobial proteins (class III). Bacteriocin production can be considered as an advantage and a functional role for LAB strains to be used in the food industry, to improve food quality and safety (De Vuyst and Leroy 2007; Parada et al. 2007). Moreover, bacteriocin production by probiotic LAB may play an important role during in vivo interactions occurring in the human gastrointestinal tract (De Vuyst et al. 2004). Naturally occurring antimicrobial compounds, such as bacteriocins, received increasing attention for food preservation due to consumers’ demand for minimally processed foods, without chemical preservatives, still safe and able to maintain good shelf life (Ponce et al. 2008). On the other hand, the emergence of bacterial resistance and multiple resistance to antibiotics represents a major public health problem and intensive efforts have been devoted to the development of alternative antimicrobial agents (Galvin et al. 1999). LAB bacteriocins may have potential applications as selective antimicrobials, with little or no effect on the normal microbiota of the host (Birri et al. 2012). Having in mind the importance and potential applications of bacteriocin-producing strains, it is understandable the permanent concern of researchers to find new LAB strains producing bacteriocins with antibacterial activity towards undesirable pathogenic or food spoilage bacteria. Functional strains with the proper physiological and metabolic features have been successfully isolated from natural habitats or from traditional fermented foods (Leroy and DeVuyst 2004). Romanian traditional fermented dairy products have been shown to be rich sources for bacteriocin and exopolysaccharide producing LAB strains (Zamfir et al. 1999; Grosu-Tudor et al. 2013). In the present study, strains isolated from fermented dairy products, but also from plant-origin materials, were used for the screening of antimicrobial activity and bacteriocin production. One of the main plant-origin source was bors, an easily produced sour liquid that results from a fermentation process of wheat bran and maize flour, used to sour the typical Romanian soups named ‘‘ciorbe’’. The information about this product are so far at empirical level, but it is well known that consumption of bors has many beneficial health effects. The article describes our results concerning the selection of some novel bacteriocin-producing LAB strains and the characterization of their antimicrobial compounds.
Materials and methods Lactic acid bacteria LAB used throughout this study have been isolated from Romanian artisan dairy products (105 strains, Grosu-Tudor
123
et al. 2013), fermented vegetables (43 strains, Wouters et al. 2013), and from other several products of plant origin, such as fresh fruits and vegetables, and bors (126 strains, results not published). MRS medium (de Man et al. 1960) was used for the isolation and subsequent growth of these strains. Strains were stored at -80 °C in MRS broth, containing 25 % (v/v) of glycerol as a cryoprotectant. To obtain fresh cultures from the freezed stocks, strains were propagated twice in MRS medium at 37 °C before the experiments. Screening of LAB for antibacterial activity Antibacterial activity was assayed by directly spotting 10 ll of the cell free culture supernatant (CFCS), obtained by centrifugation (13,0009g, 10 min, 4 °C), onto fresh lawns (soft agar media) of the indicator strains (De Vuyst et al. 1996). The bacterial strains used as indicator microorganisms for the evaluation of antimicrobial activities were as follows: Enterococcus faecalis LMG 16216, Enterococcus faecium LMG 14255, Lactobacillus brevis LMG 6906, Lactobacillus delbrueckii subsp. bulgaricus LMG 6901T, Lactobacillus helveticus 102, Lactobacillus sakei LMG 13558, Leuconostoc mesenteroides LMG 13562, Pediococcus pentosaceus LMG 13561, Bacillus cereus CBAB, Bacillus subtilis ATCC 6633, Eschercihia coli ATCC 25922, Salmonella enterica ATCC 14028, and Staphylococcus aureus ATCC 25923. LAB strains were grown in MRS medium, the two Bacillus strains in Luria– Bertani medium (Bertani 1951), while the other strains in Brain Heart Infusion medium (BHI, Merck, Darmstadt, Germany). Corresponding solid media were prepared with 1.5 % (w/v) agar and the soft agar media used for overlays were prepared with 0.7 % (w/v) agar. Evidence of bacteriocin production LAB strains showing an inhibitory effect against one or more indicator strains have been further subjected to various tests in order to establish the nature of the inhibitory compound. Strains were cultivated in MRS broth at 20, 28, 37 and 42 °C. Cells were removed by centrifugation (13,000g, 10 min, 4 °C) and the pH of the CFCS was adjusted to pH 7.0 with 1 mol l-1 NaOH. Antibacterial activity was assayed quantitatively by an agar spot test (De Vuyst et al. 1996). Briefly, serial twofold dilutions in water of the bacteriocin sample were spotted (10 ll) onto fresh indicator lawns of Lact. delbrueckii subsp. bulgaricus LMG 6901T. The activity was defined as the reciprocal of the highest dilution which demonstrated complete inhibition of the indicator lawn and was expressed in activity units (AU) per millilitre of culture medium. The specific activity was determined by dividing the
World J Microbiol Biotechnol
bacteriocin activity with the total protein concentration, as determined by the method described by Whitaker and Granum (1980). The influence of several proteolytic enzymes, such as tripsin (Sigma-Aldrich Chemie GmbH, Germany), pronase E (Merck KGaA, Darmstadt, Germany), proteinase K (Carl Roth, Karlsruhe, Germany), and pepsin (Sigma-Aldrich) was further investigated. Enzymes were dissolved in 0.2 mol l-1 sodium phosphate buffer (pH 7.5), except pepsin, which was dissolved in 0.2 mol l-1 HCl/KCl buffer (pH 2.0) and were added to the CFCS, at a final concentration of 1 mg ml-1. The inhibitory activity was tested after incubation for 2 h at 37 °C. The CFCS diluted with the buffers used to dissolve the enzymes, and enzymes solutions without CFCS served as controls. Identification of the bacteriocin-producing LAB strains
Table 1 Inhibitory spectrum of the bacteriocins isolated from Lact. amylolyticus P40 and P50, Lact. oris P49, L. lactis 19.3, Lact. plantarum 26.1, and Ent. durans 41.2 Indicator strainb
P40
P49
P50 19.3a
26.1
41.2
Lactobacillus acidophilus 5e2
-
-
-
?
±
-
Lactobacillus brevis LMG 6906
-
-
-
-
±
?
Lactobacillus brevis 403
-
-
-
??
-
-
Lactobacillus brevis 530
-
-
-
??
-
-
Lactobacillus delbrueckii subsp. bulgaricus LMG 6901T
??
?
?
??
?
?
Lactobacilus pentosus 265
-
-
-
?
-
-
Lactobacillus plantarum 788
-
-
-
?
-
±
Lactobacillus plantarum 3
-
-
-
-
-
±
Lactobacillus plantarum 198
-
-
-
?
±
-
Lactobacillus plantarum 619
-
-
-
?
-
-
Lactobacillus rhamnosus L
-
-
-
-
-
±
Lactobacillus sakei subsp. sakei LMG 13558 Leuconostoc citreum 96
-
-
-
?
-
-
-
-
-
?
?
?
Leuconostoc mesenteroides LMG 13562
-
-
-
?
-
?
Lactobacillus helveticus 102
The taxonomic affiliation of isolated bacteria was determined on the basis of their 16S rRNA sequence. PCR amplification of 16S rRNA gene, purification and sequencing of amplification products were performed as previously described by Stancu (2012). The products of the sequencing reactions were analyzed with an automatic Applied Biosystems 3500 Genetic Analyzer (Applied Biosystem/Hitachi, Tokyo, Japan). DNA sequencing runs were assembled using the BioEdit software. The new sequences of isolated bacteria were compared to those from databases using the BLAST search program.
Leuconostoc mesenteroides 197
-
-
-
±
-
?
Leuconostoc mesenteroides 355
-
-
-
?
?
-
Bacteriocins isolation and partial purification
Leuconostoc mesenteroides/ pseudomesenteroides 246 Pediococcus pentosaceus LMG13561
-
±
-
??
-
??
Enterococcus faecalis LMG 16216
-
-
-
-
-
-
Enterococcus faecalis LMG 13566
-
-
-
?
-
-
Enterococcus faecium LMG 14255
-
-
-
?
?
-
Enterococcus faecium LMG 14203
-
-
-
?
?
?
Lactococcus lactis 17
-
-
-
-
?
?
Lactococcus lactis subsp. diacetylactis 21
-
-
-
?
-
?
Bacillus cereus 53
-
-
-
?
-
-
Bacillus cereus CBAB
-
-
-
??
-
-
Bacillus subtilis ATCC 6633
-
-
-
??
-
-
Escherichia coli 159
-
-
-
?
-
-
Salmonella arizonae 18
-
-
-
?
-
-
Listeria monocytogenes ATCC 1911
-
-
-
±
-
-
The bacterial strains were grown for 12 h in MRS medium, at their optimum temperature previously determined for bacteriocin production. The CFCS were neutralized and precipitated with ammonium sulphate (40 % saturation), overnight, with gentle agitation. The floating pellicle formed during this step was collected after centrifugation (10,000g, 10 min, 4 °C), dissolved in 5 mmol l-1 potassium phosphate buffer (pH 6.5), and then extracted with 15 volumes of a mixture of chloroform/methanol (2/1, v/v). After l h at 4 °C, the white precipitate formed in the organic phase was centrifuged for 15 min at 13 000 g and resuspended in ultrapure water. Characterization of bacteriocins The heat sensitivity and pH stability of the bacteriocins were firstly tested. The concentrated bacteriocins (CFCS precipitated with ammonium sulphate) were used for these tests. Heat resistance was tested at 60 °C for 10, 30, and 60 min, at 100 °C for 10, 30, 60, and 120 min, and at 121 °C for 15 min. To test the influence of pH, the
Leuconostoc citreum 167
Staphylococcus aureus 54
-
-
-
-
-
–
Staphylococcus aureus ATCC 6538
-
-
-
-
-
-
123
World J Microbiol Biotechnol Table 1 continued Indicator strain
b
P40
P49
P50 19.3
a
26.1
41.2
Klebsiella pneumoniae 2
-
-
-
?
?
?
Lactobacillus amylolyticus P40
-
-
-
?
?
-
Lactobacillus oris P49
-
-
-
?
?
-
Lactobacillus amylolyticus P50
-
-
-
-
-
-
Lactococcus lactis 19.3
-
-
-
?
±
?
Lactobacillus plantarum 26.1 Enterococcus durans 41.2
-
-
-
??
?
?
-
-
-
?
±
?
a
?? = large inhibition zones, over 20 mm in diameter; ? = clear inhibition zones, less than 20 mm in diameter; ± = less clear inhibition zones; - = no inhibition
b
LMG Laboratorium voor Microbiologie, Universiteit Gent, Belgium, ATCC America Type Culture Collection; CBAB Centre of Applied Biochemistry and Biotechnology Bucharest, Romania
bacteriocin samples were adjusted to pH values between 2.0 and 10.0 with HCl or NaOH, and allowed to stand for one hour before testing the inhibitory activity. The requirement of disulfide bridges for bacteriocin activity was determined by measuring the activity in the presence of 10 mmol l-1 dithiothreitol (DTT) (Kemperman et al. 2003). To establish the inhibitory spectrum of the bacteriocins, 10 ll of the concentrated samples were spotted onto indicator lawns of several lactic acid bacterium strains and other Gram-positive and Gram-negative bacteria (Table 1). These lawns were prepared by propagating fresh bacterial cultures to an optical density (600 nm) of about 0.5 and adding 100 ll of the cell suspension to 3.5 ml of overlay agar (top layer). Overlaid agar plates were incubated for 24 h at the optimal growth temperature for each indicator strain. To estimate the molecular mass of the bacteriocins, tricine sodium dodecylsulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) was carried out, according to the method of Scha¨gger and von Jagow (1987). Polyacrylamide concentrations in the stacking gel and separating gel were 9.6 and 16.0 %, respectively. Electrophoresis was conducted at a constant voltage of 30 V in the stacking gel and 90 V in the separating gel. After the run (10 h), the gel containing the bacteriocin samples was washed during 5 h with sterile ultrapure water that was replaced every hour. Finally, the gel was transferred to an MRS plate and overlaid with a top layer of soft MRS agar inoculated with the indicator strain, Lact. delbrueckii subsp. bulgaricus LMG 6901T. Another part of the gel, containing both bacteriocins and the standard proteins (broad range protein molecular weight markers, Promega GmbH, Mannheim, Germany; polypeptide SDS-PAGE
123
molecular weight standards, BioRad Laboratories, Hercules CA), was stained with Coomassie Brilliant Blue. Bacteriocin assay in various combinations and in co-cultures of the producing strains Partially purified bacteriocins, extracted with chloroform/ methanol, were mixed, in different combinations of two bacteriocins. In one set of experiments combinations were made with similar amounts of proteins of each bacteriocin sample used in the mixture, while in the second set of experiments, with similar bacteriocin activities. For the cocultivation studies, MRS medium was inoculated with equal proportions of inoculum of two strains. Each of the producing strain was co-cultivated with the sensitive strain Lact. delbrueckii subsp. bulgaricus LMG 6901T, but also various combinations between the producing strains were done. Only combinations of strains without killing effect against each other, as determined by the spot on the lawn method (Tagg et al. 1976), were used. As controls, the pure cultures of the tested strains were used. Bacteriocin assay was done using the cell-free culture supernatant of the mixed/pure cultures.
Results Screening for antibacterial activity; evidence of bacteriocin production None of the 274 tested strains showed inhibitory activity against the pathogens Salm. enterica ATCC 14028, E. coli ATCC 25922 and Staph. aureus ATCC 25923, as shown from the screening of the corresponding CFCS. There were, however, 53 LAB strains which inhibited the growth of the two Bacillus strains used as indicator (results not shown). Moreover, six of these strains were able to repress the growth of one or more LAB strains used as indicator. Three of these strains (isolated from bors) were only active against Lact. delbrueckii subsp. bulgaricus LMG 6901T and the two bacilli, while the other three strains (isolated from fermented dairy products), were active against the two bacilli and several LAB indicator strains. The results are included in Table 1, presenting the inhibitory spectrum of the six LAB strains. The pH adjustment of the CFCS resulted in the loss of antibacterial activity against the two bacilli (except in the case of one strain, 19.3), but did not affect the activity of the six selected strains against the other LAB indicators. In order to establish if the inhibitory compound is of proteinaceous nature, the influence of several proteolytic enzymes on the activity towards the sensitive strain Lact. delbrueckii subsp. bulgaricus LMG 6901T was investigated
World J Microbiol Biotechnol Table 3 Influence of growth temperature on bacteriocin production
1
1
P50
7
2 3
6 4
5
Strain
41.2
20 °C
28 °C
37 °C
42 °C
Lactobacillus amylolyticus P40
n.g.
400
400
200
Lactobacillus oris P49
n.g.
200
200
n.d.
Lactobacillus amylolyticus P50
n.g.
6,400
200
200
Lactococcus lactis 19.3
25,600
25,600
400
n.g.
Lactobacillus plantarum 26.1
200
800
100
n.d.
Enterococcus durans 41.2
n.g.
n.d.
200
200
7
2
6
3 4
5
Fig. 1 Influence of proteolytic enzymes on the antibacterial activity of strains Lact. amylolyticus P50 (left) and Ent. durans 41.2 (right). 1 CFCS, 2 neutralized CFCS, 3 CFCS treated with pepsin, 4 CFCS ? HCl/KCl buffer (pH 2.0), 5 CFCS treated with proteinase K, 6 CFCS ? sodium phosphate buffer (pH 7.5), 7 CFCS ? pronase E
Inhibitory activitya (AU/ml)
n.g. no growth, n.d. not detected a
Lact. delbrueckii subsp. bulgaricus LMG 6901T was used as indicator strain
Bacteriocins isolation and purification Table 2 Taxonomic affiliation of the six bacteriocin-producing LAB strains Strain name
Closest bacterial strain name (accession number)
Percentage of nucleotide identity (%)
Lactobacillus amylolyticus P40
Lactobacillus amylolyticus strain TUST015 (KC456629.1)
93
Lactobacillus oris P49 Lactobacillus amylolyticus P50
Lactobacillus oris strain F0423 (HM596285.1) Lactobacillus amylolyticus strain TUST015 (KC456629.1)
98
Lactococcus lactis 19.3
Lactococcus lactis strain MNC26 (JQ754457.1)
87
Lactobacillus plantarum 26.1
Lactobacillus plantarum strain LS13 (JQ236619.1)
88
Enterococcus durans 41.2
Enterococcus durans strain ex14 (KF317884.1)
99
99
(Fig. 1). The inhibitory activity of all the six strains was inactivated by the treatment with proteinase K and pronase E. Additionally, the activity of strain 41.2 was also inactivated by pepsin and tripsin and the actvity of strain 26.1 was inactivated by tripsin. Based on these results, the six strains were selected to be used in further experiments, as bacteriocin-producing LAB. Identification of the LAB strains Based on the analysis of the 16S rRNA gene sequences, the six selected strains have been shown to belong to Lactobacillus, Lactococcus and Enterococcus genera. Table 2 shows the identity of these strains and the similarity to other bacterial strains from the public databases.
As a preliminary test, we have studied the influence of the incubation temperature on the growth and bacteriocin activity of the six selected strains (Table 3). For strains Lact. amylolyticus P40 and Lact. oris P49, similar bacteriocin activities were determined at 28 and 37 °C. These two strains did not grow at 20 °C, while at 42 °C the bacteriocin activity was lower, or not detectable. For the bacteriocin isolation step, these two strains were incubated at 37 °C. Strain Lact. amylolyticus P50 grew well at 37 and 42 °C, with a corresponding bacteriocin activity of 200 AU ml-1. The growth of this strain was neglictible after 48 h of incubation at 20 °C and very pour at 28 °C. The bacteriocin activity of the CFCS of the culture obtained at 28 °C was very high; however, due to the slow growth at this temperature, for the further studies we have decided to grow the strain at 37 °C. The same incubation temperature was used for strain Ent. durans 41.2, for which the bacteriocin activity was similar at 37 and 42 °C. This strain was not able to grow at 20 °C, while the bacteriocin activity at 28 °C was not detectable. On the contrary, strains L. lactis 19.3 and Lact. plantarum 26.1 showed the highest bacteriocin activities when incubated at 28 °C. At 37 °C, the inhibitory activity was much lower, while at 42 °C, the growth was very slow and no activity could be detected. For these two strains, the temperature used for the bacteriocin isolation was 28 °C. Bacteriocins were firstly concentrated by ammonium sulphate precipitation (40 % saturation) and then extracted/ precipitated with a mixture of chloroform/methanol. The activities recovered in each purification step, for each strain, are shown in Table 4. Characterization of bacteriocins The concentrated bacteriocins (ammonium sulphate precipitates) were used for a further characterization,
123
World J Microbiol Biotechnol Table 4 Bacteriocin activity recovered during the purification steps Purification stepa
Volume (ml)
P40
P49
P50
19.3
26.1
41.2
Culture supernatant
500
400
200
200
25,600
800
200
AS precipitation
20
25,600
3,200
3,200
6,553,600
6,400
1,600
C/M—bacteriocin activity
3
25,600
3,200
3,200
6,553,600
3,200
1,600
C/M—protein concentration
1.02
0.94
0.99
0.94
0.61
0.91
C/M—specific activity
25,098
3,404
3,232
6,971,915
5,246
1,758
a
AS ammonium sulphate precipitation, C/M chloroform/methanol extraction/precipitation
Table 5 Influence of heat treatment on bacteriocin activity Temperature/time
P40
P49
P50
19.3
26.1
41.2
25,600
3,200
3,200
25,600
6,400
1,600
10 min
800
400
1,600
25,600
6,400
1,600
30 min 60 min
100 0
200 100
0 0
25,600 25,600
6,400 6,400
800 800
10 min
200
200
0
12,800
6,400
800
30 min
0
100
0
12,800
6,400
800
Control 60 °C
100 °C
121 °C
Inhibitory activitya (AU/ml)
60 min
0
0
0
12,800
3,200
800
120 min
0
0
0
6,400
1,200
400
15 min
0
0
0
6,400
a
800 \100
T
Lact. delbrueckii subsp. bulgaricus LMG 6901 was used as indicator strain
regarding the heat resistance and pH stability, influence of DTT on their activity, the inhibitory spectrum, and the estimation of the molecular weight. As an exception, in the case of strain 19.3, the CFCS with pH adjusted to 7.0 was used, since the activity of this bacteriocin is very high (25,600 AU ml-1 before concentration). Based on their heat resistance, the six bacteriocins could be classified into two groups: heat resistant (strains L. lactis 19.3, Lact. plantarum 26.1, and Ent. durans 41.2) and heat sensitive (strains Lact. amylolyticus P40 and P50, and Lact. oris P49). The first group retained at least 50 % of the activity after incubation at 100 °C for 2 h (Table 5). Within this group, the bacteriocin produced by L. lactis 19.3 showed a strong heat resistance, 25 % of its activity being recovered after 15 min of incubation at 121 °C. In the second group, there are the other three bacteriocins, produced by the strains isolated from bors, strongly inactivated by the heat treatment. The activities were mostly lost after incubation at 60 °C for 30 min. The incubation at 100 °C had even stronger effect, for instance in the case of Lact. amylolyticus P50, the activity was lost after 10 min of incubation (Table 5). The three heat resistant bacteriocins were also stable in a very wide pH range (activity was detected at pH between
123
2.0 and 10.0). The other three bacteriocins kept their activity at pH between 4.0 and 10.0. DTT did not destroy the activity of any of the six bacteriocins (results not shown), indicating that, most probably, they do not contain disulfide bridges that are essential for the antibacterial activity. The inhibitory spectrum of the six bacteriocins, as determined for the concentrated samples, is presented in Table 1. Again, we could make a distinction between a group of bacteriocins with a very narrow inhibitory spectrum (restricted to only one or two LAB strains), represented by the compounds that were also heat labile, and a group with a broad inhibitory spectrum, represented by the other three bacteriocins. Within this second group, we can underline the bacteriocin produced by L. lactis 19.3, able to inhibit the growth of 27 of the 36 LAB strains used as indicators, among which B. cereus 53, B. cereus CBAB, B. subtilis ATCC 6633, Listeria monocytogenes ATCC 1911 and the two Staph. aureus strains used in this test. It is was also observed that incubation of this bacteriocin at 60 and 100 °C resulted in a faster decrease of the activity when tested against Staph. aureus ATCC 6538, compared with the activity towards Lact. delbrueckii subsp. bulgaricus LMG 6901T (results not shown). Molecular mass estimation The partially purified bacteriocins (concentrated with ammonium sulphate, followed by extraction with chloroform/methanol) were subjected to Tricine-SDS-PAGE and after migration, the gels were extensively washed and overlaied with indicator lawns. After 24 h of incubation, a clear inhibition zone could be observed for each sample (Fig. 2). In case of L. lactis 19.3 and Lact. plantarum 26.1, this zone corresponded to a very low molecular mass (between 3,496 and 6,512 Da), while for the other four bacteriocins, the molecular mass was estimated to about 35,000 Da). In some gels, for the line corresponding to strain Lact. amylolyticus P40, a second inhibition zone, very weak, corresponding to a low molecular mass (about 6,500 Da), could be detected (Fig. 2).
World J Microbiol Biotechnol
a
MM
P40
P49
P50
41.2
P40
P49
P50
41.2
50,000 35,000 25,000
15,000 10,500
MM
b
26.1
19.3
19.3
c
+41.2
19.3
26.1
19.3 +41.2
26,625 16,950
26,625
14,437 6,512 3,496
16,950 14,437
6,512
Fig. 2 Tricine-SDS-PAGE of the bacteriocins isolated from the six selected strains (gels overlaied with the indicator lawn, L. delbrueckii subsp. bulgaricus LMG 6901T—left side pictures, and stained with Coomassie Brilliant Blue—right side pictures). a Lact. amylolyticus P40, Lact. oris P49, Lact. amylolyticus P50, and Ent. durans 41.2; the arrow indicates the weak band detected in some cases in the
bacteriocin sample isolated from Lact. amylolyticus P40; b Lact. plantarum 26.1, L. lactis 19.3, and a mixture of the two bacteriocins isolated from L. lactis 19.3 and Ent. durans 41.2, showing two clear inhibition bands; c L. lactis 19.3; MM molecular weight markers (see ‘‘Materials and methods’’)
123
World J Microbiol Biotechnol Table 6 Inhibitory activity of various mixtures of bacteriocins Combination of bacteriocins
Ratioa
Individual activities (AU/ml)
P40 ? P49
1:1
12,800 ? 1,600
1:8
1,600 ? 1,600
51,200
16
P40 ? P50
1:1
12,800 ? 1,600
102,400
7
1:8
1,600 1 1,600
409,600
128
P49 ? P50
1:1
1,600 1 1,600
409,600
128
19.3 ? P40
1:1
3,278,800 ? 12,800
6,553,600
2
19.3 ? P49
1:256 1:1
12,800 ? 12,800 3,278,800 ? 1,600
102,400 6,553,600
4 2
12,800
4
Combined activity (AU/ml) 102,400
Approximate fold of increase 7
1:2048
1,600 ? 1,600
1:1
3,278,800 ? 1,600
1:2048
1,600 ? 1,600
51,200
16
1.6:1
1,600 ? 8,000
51,200
5
8:1
1,600 ? 1,600
6,400
2
26.1 ? P49
1.5:1
1,600 ? 1,066
51,200
19
1:1
1,600 ? 1,600
204,800
64
26.1 ? P50
1.5:1
1,066 1 1,600
614,400
1:1
1,600 ? 1,600
25,600
1:1
800 ? 12,800
102,400
1:16
1,600 ? 1,600
1,600
19.3 ? P50 26.1 ? P40
41.2 ? P40 41.2 ? P49
6,553,600
2
230 8 8 0.5
1:1
800 ? 1,600
1,600
0.7
1:2
800 ? 800
1,600
1
41.2 ? P50
1:1 1:2
800 ? 1,600 800 ? 800
3,200
1
19.3 ? 26.1
1:1.5
2,185,866 ? 1,600
19.3 ? 41.2 26.1 ? 41.2
1,600 409,600
1 0.2
1:2048
1,600 ? 1,600
1:1
3,278,800 ? 800
800
0.3
409,600
0.1
1:4096
800 ? 800
400
0.3
1.6:1
1,600 ? 500
1,600
0.8
1:3
800 ? 800
3,200
2
Bold values indicate the combinations of bacteriocins with the highest increase in activity compared with the cumulative activities a
Ratio was calculated to get similar protein concentrations of each bacteriocin (first set of experiments) or similar activities of each bacteriocin (second set of experiments); Lact. delbrueckii subsp. bulgaricus LMG 6901T was used as indicator strain
Bacteriocin assay in various combinations and in co-cultures of the producing strains The activities of the various mixtures of the three bacteriocins produced by the strains isolated from bors were significantly higher (up to 128 times) comparing with the cumulative activities of the single bacteriocins, especially when the combinations were made with similar bacteriocin activities (Table 6). In case of the three strains isolated from fermented dairy products, the combination of their produced bacteriocins resulted in a slight decrease of the inhibitory activity. However, some combinations among bacteriocins produced by strains isolated from
123
dairy products and bors, respectively, seemed to be more effective than the single bacteriocins. For instance, an increase of about 230 times of the activity was observed in the case of a mixture of the two bacteriocins produced by Lact. plantarum 26.1 and Lact. amylolyticus P50 (Table 6). Co-cultivation of two bacteriocin-producing strains, or in the presence of the sensitive strain resulted, in most of the cases, in a decrease of the inhibitory activity (results not shown). There was only one strain, Lact. oris P49, which seemed to produce more bacteriocins when it was co-cultivated in the presence of the sensitive strain (400 AU ml-1 compared with 100 AU ml-1 for the mono-culture).
World J Microbiol Biotechnol
Discussion The present study describes six new LAB strains isolated from Romanian traditional fermented foods that are able to produce bacteriocins with various characteristics. A large number of LAB strains (274 isolates) have been tested for the ability to produce bacteriocins. They have been isolated from various types of fermented foods, such as dairy products, fermented vegetables and bors, but also from fresh vegetables, fruits and flowers. The isolation and screening of microorganisms from natural sources has always proved to be a successful way for obtaining industrially important strains or strains with valuable medical applications (Todorov and Dicks 2006; Yang et al. 2012). However, only a limited number of strains (about 20 %) showed antimicrobial activity, especially against Bacillus strains and other LAB used as indicators. In most cases, the antimicrobial activity might be due to the production of organic acids, since the pH neutralization resulted in the loss of this effect. The antibacterial activity was shown to be due to the production of bacteriocins only for six of these strains (about 2 % of the tested strains). Similar low percentages of bacteriocin-producing strains have been reported by other authors for strains isolated from fresh-cut vegetable products (Yang et al. 2012), from milk and meat products (Sezer and Gu¨ven 2009), or from malt (Rouse et al. 2007). The food source and isolation media might be important for the successfully isolation of bacteriocinogenic LAB, since many bacteriocin-producing LAB have been isolated from fermented dairy products, such as yogurts and cheeses (Yang et al. 2012). In our case, three of the six bacteriocin-producing strains have been isolated from fermented dairy products. The bacteriocin-producing strains have been identified based on the 16S rRNA sequencing and four of them belong to the Lactobacillus genus. Lactobacillus is a versatile bacterial genus that has a long history with human daily life. It is also one of the important group of bacteriocin-producing LAB (Diep et al. 2009). Two of the Lactobacillus strains have been identified as Lact. amylolyticus and one as Lact. oris. Lact. amylolyticus has been described in beer malt and beer wort (Bohak et al. 1998) and it is likely to be found in other fermented cereals, such is the Romanian bors. On the other hand, Lact. oris has been described in the human oral cavity (Farrow and Collins 1988), and might be only a contaminant of bors. The strains belonging to these two species, isolated from bors, showed inhibitory activity against Lact. delbrueckii subsp. bulgaricus LMG 6901T, but also against B. cereus CBAB and B. subtilis ATCC6633. The bacteriocins isolated from these strains retained the activity against the LAB, but not against the two bacilli. The proteinaceous nature of the three bacteriocins have been proven by their
inactivation after treatment with proteinase K and pronase E. Moreover, the inhibitory activity of these bacteriocins was significantly decreased after incubation at 60 °C for only 10 min. Based on the limited inhibitory spectrum, the heat sensitivity, and the high molecular mass (about 35,000 Da, as determined by Tricine-SDS-PAGE), the bacteriocins produced by these three strains (Lact. amylolyticus P40 and P50, and Lact. oris P49) might be considered to belong to class III bacteriocins (Nes et al. 1996). Class III bacteriocins are much less studied compared with the first two classes and only few reports concerning their isolation and characterization are available (Joerger and Klaenhammer 1986; Vaughan et al. 1992; Nilsen et al. 2003). However, such bacteriocins with very narrow inhibitory spectrum might find applications as highly selective antibacterial agents, if they are able to control target bacteria specifically. They can be used in lower doses and the risk to develop resistance in nontarget bacteria is much lower (Zendo 2013). Although no data available about isolation and characterization of bacteriocins produced by Lact. amylolyticus and Lact. oris, strains belonging to these species have been shown recently to have potential probiotic effect (Pedersen et al. 2004; Ko˜ll et al. 2008; Teanpaisan et al. 2011). The fourth Lactobacillus strain was identified to be Lact. plantarum. Regarding the inhibitory spectrum and the biochemical characteristics, the bacteriocin produced by this strain could be grouped with the two bacteriocins produced by the other two strains isolated from fermented dairy products, identified as L. lactis 19.3 and Ent. durans 41.2. The bacteriocins produced by these three strains might belong to class I and/or II according to the classification of Nes et al. (1996), having a broad antimicrobial spectrum, including Bacillus sp., L. monocytogenes, and Staph. aureus (except strain Ent. durans 41.2). Recent studies showed that class IIa bacteriocins exert the highest inhibitory effect on L. monocytogenes (Sip et al. 2012). Within this class, one can find many bacteriocins produced by Lact. plantarum (van Reenen et al. 1998; Atrih et al. 2001; Ennahar et al. 1999; Todorov et al. 2007), but also some bacteriocins produced by L. lactis (Ferchichi et al. 2001). It is difficult for the moment to classify the bacteriocin produced by L. lactis 19.3; however, the very low molecular mass (between 3,496 Da and 6,512 Da, as determined by Tricine-SDS-PAGE), the broad inhibitory spectrum (27 of the 36 indicator strains were inhibited) and the stability even at alkaline pH values, are common characteristics with lacticins produced by L. lactis (Zendo 2013), belonging to class IId bacteriocins. L. lactis can be considerred as a representative LAB and a model microorganism used in the manufacture of various fermented foods. Novel bacteriocins produced by this species, different from nisin, are very important, to be used in
123
World J Microbiol Biotechnol
combination with, or as a next generation of nisin (Fujita et al. 2007). The significant thermal stability and the stability under neutral and alkaline pH conditions of the bacteriocin isolated from L. lactis 19.3 might be an advantage for the potential use as a biopreservative, for instance in combination with a thermal processing of the foods (Yang et al. 2012). The third bacteriocin of this group, produced by Ent. durans 41.2 has a similar broad inhibitory spectrum, including L. monocytogenes, Leuc. mesenteroides, Ent. faecium and Ent. faecalis, but not Pediococcus pentosaceus or Staph. aureus. The biochemical characteristics are similar with the ones of the other two bacteriocins. However, the molecular mass of this bacteriocin was estimated to about 35,000 Da, which is an unusual molecular mass for a class II bacteriocin (De Vuyst and Vandamme 1994). Further studies might reveal if this is a new bacteriocin, with unique features, and in which existing class can be assigned. The co-cultivation of various combinations of two bacteriocin-producing strains resulted, in general, in a decrease of the inhibitory activity of the CFCS. It is well known that some LAB strains, such as L. lactis, exhibit strong proteolytic action, which may cause the inactivation of the bacteriocins produced by the strain used in coculture (Sip et al. 2012). On the other hand, the suppression of bacteriocin production in mixed-species cultures of LAB has been observed by Domı´nguez-Manzano and Jime´nez-Dı´az (2013). The authors suggest that this phenomenon could be due to a regulation of the bacteriocin production exerted by each strain against the other. Therefore, it is very important to check such interactions when mixed starter cultures are going to be used in food fermentations, to avoid the negative effect on the quality of the final product (Domı´nguez-Manzano and Jime´nezDı´az 2013). Moreover, some of the isolated bacteriocins seemed to exhibit a synergistic effect. The most evident effect was shown by combinations between bacteriocins with different characteristics, produced by strains isolated from different sources (dairy and bors, respectively). It was previously postulated that the presence of receptors on cell surface plays an important role in bacteriocin specificity (Drider et al. 2006). Bacteriocins which belong to different categories and with different mode of actions are likely to exhibit synergistic effect, since they do not have to compete for the same receptors on the indicator cell surface (Vignolo et al. 2000). In conclusion, traditional fermented foods are still an important source of functional LAB. The bacteriocin-producing strains isolated in our study may find applications as protective cultures. Our results also bring important information about the biochemical characterization of the
123
bacteriocins, the effect of co-cultivation and the synergistic effect of some of these proteinaceous substances. Purification to homogeneity as well as further characterization, are currently in progress. Acknowledgments This work was supported by the grants of the Romanian National Authority for Scientific Research, CNDI–UEFISCDI, project numbers 105/2012 (PLANTLAB) and 77/2012 (PROLAB).
References Atrih A, Rekhif N, Moir AJ, Lebrihi A, Lefebvre G (2001) Mode of action, purification and amino acid sequence of plantaricin C19. Int J Food Microbiol 68:93–104 Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300 Birri DJ, Brede DA, Nes IF (2012) Salivaricin D, a novel intrinsically trypsin-resistant lantibiotic from Streptococcus salivarius 5M6c isolated from a healthy infant. Appl Environ Microbiol 78:402–410 Bohak I, Back W, Richter L, Ehrmann M, Ludwig W, Schleifer KH (1998) Lactobacillus amylolyticus sp. nov., isolated from beer malt and beer wort. Syst Appl Microbiol 21:360–364 Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71:1–20 de Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135 De Vuyst L, Leroy F (2007) Bacteriocins from lactic acid bacteria: production, purification, and food applications. J Mol Microbiol Biotechnol 13:194–199 De Vuyst L, Vandamme EJ (1994) Bacteriocins of lactic acid bacteria. Blackie Academic and Professional, London De Vuyst L, Callewaert R, Pot B (1996) Characterization of the antagonistic activity of Lactobacillus amylovorus DCE 471 and large scale isolation of its bacteriocin amylovorin L471. Syst Appl Microb 19:9–20 De Vuyst L, Avonts L, Makras E (2004) Probiotics, prebiotics and gut health. In: Remacle C, Reusens B (eds) Functional foods, ageing and degenerative disease. Woodhead Publishing, Cambridge, pp 416–482 Diep DB, Straume D, Kjos M, Torres C, Nes IF (2009) An overview of the mosaic bacteriocin pln loci from Lactobacillus plantarum. Peptides 30:1562–1574 Domı´nguez-Manzano J, Jime´nez-Dı´az R (2013) Suppression of bacteriocin production in mixed-species cultures of lactic acid bacteria. Food Control 30:474–479 Doyle MP, Steenson LR, Meng J (2013) Bacteria in food and beverage production. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes: applied bacteriology and biotechnology. Springer, Berlin, pp 241–256 Drider D, Fimland G, Hechard Y, McMullen LM, Prevost H (2006) The continuing story of class IIa bacteriocins. Microbiol Mol Biol Rev 70:564–582 Ennahar S, Sonomoto K, Ishizaki A (1999) Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation. J Biosci Bioeng 87:705–716 Farrow JAE, Collins MD (1988) Lactobacillus oris sp. nov. from the human oral cavity. Int J Syst Bacteriol 38:116–118 Ferchichi M, Frere J, Mabrouk K, Manai M (2001) Lactococcin MMFII, a novel class IIa bacteriocin produced by Lactococcus lactis MMFII, isolated from a Tunisian dairy product. FEMS Microbiol Lett 205:49–55
World J Microbiol Biotechnol Fujita K, Ichimasa S, Zendo T, Koga S, Yoneyama F, Nakayama J, Sonomoto K (2007) Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of Gram-positive bacteria. Appl Environ Microbiol 73:2871–2877 Galvin M, Hill C, Ross RP (1999) Lacticin 3147 displays activity in buffer against Gram-positive bacterial pathogens which appear insensitive in standard plate assays. Lett Appl Microbiol 28:355–358 Grosu-Tudor SS, Zamfir M, Van der Meulen R, De Vuyst L (2013) Isolation of novel homopolysaccharide producing lactic acid bacteria from Romanian raw milk and fermented dairy products. Eur Food Res Technol 237:609–615 Joerger MC, Klaenhammer TR (1986) Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J Bacteriol 167:439–446 Kemperman R, Kuipers A, Karsens H, Nauta A, Kuipers O, Kok J (2003) Identification and characterization of two novel clostridial bacteriocins, circularin A and closticin 574. Appl Environ Microbiol 69:1589–1597 Ko˜ll P, Ma¨ndar R, Marcotte H, Leibur E, Mikelsaar M, Hammarstro¨m L (2008) Characterization of oral lactobacilli as potential probiotics for oral health. Oral Microbiol Immunol 23:139–147 Leroy F, DeVuyst L (2004) Lactic acid bacteria as functional starter cultures for the food fermentation Industry. Trends Food Sci Technol 15:67–78 Macwana SJ, Muriana PM (2012) A ‘bacteriocin PCR array’ for identification of bacteriocin-related structural genes in lactic acid bacteria. J Microbiol Meth 88:197–204 Nes IF, Diep DB, Ha˚verstein IS, Brurberg MB, Eijsink V, Holo H (1996) Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Van Leeuw 70:113–128 Nilsen T, Nes IF, Holo H (2003) Enterolysin A, a cell wall-degrading bacteriocin from Enterococcus faecalis LMG2333. Appl Environ Microbiol 69:2975–2984 Parada JL, Caron CR, Medeiros ABP, Soccol CR (2007) Bacteriocins from lactic acid bacteria: purification, properties and use as biopreservatives. Braz Arch Biol Technol 50:521–542 Pedersen C, Jonsson H, Lindberg JE, Roos S (2004) Microbiological characterization of wet wheat distillers’ grain, with focus on isolation of lactobacilli with potential as probiotics. Appl Environ Microbiol 70:1522–1527 Ponce AG, Moreira MR, Del Valle CE, Roura SI (2008) Preliminary characterization of bacteriocin-like substance from lactic acid bacteria isolated from organic leafy vegetables. LWT—Food. Sci Technol 41:432–441 Reis JA, Paula AT, Casarotti SN, Penna ALB (2012) Lactic acid bacteria antimicrobial compounds: characteristics and applications. Food Eng Rev 4:124–140 Rouse S, Sun F, Vaughan A, van Sinderen D (2007) High-throughput isolation of bacteriocin-producing lactic acid bacteria, with potential application in the brewing industry. J Inst Brew 113:256–262
Scha¨gger H, von Jagow G (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379 Sezer G, Gu¨ven A (2009) Investigation of bacteriocin production capability of lactic acid bacteria isolated from foods. Lafkas Univ Vet Fak Derg 15:45–50 Sip A, Wieckowicz M, Olejnik-Schmidt A, Grajek W (2012) AntiListeria activity of lactic acid bacteria isolated from golka, a regional cheese produced in Poland. Food Control 26:117–124 Stancu MM (2012) Investigation of organic solvent resistance mechanisms in Vibrio alginolyticus IBBCt2. Water Air Soil Poll 223:4495–4505 Tagg JR, Dajani AS, Wannamaker LW (1976) Bacteriocins of gram positive bacteria. Bacteriol Rev 40:722–756 Teanpaisan R, Piwat S, Dahle´n G (2011) Inhibitory effect of oral Lactobacillus against oral pathogens. Lett Appl Microbiol 53:452–459 Todorov SD, Dicks LMT (2006) Screening for bacteriocin-producing lactic acid bacteria from boza, a traditional cereal beverage from Bulgaria. Comparison of bacteriocins. Process Biochem 41:11–19 Todorov SD, Nyati H, Meincken M, Dicks LMT (2007) Partial characterization of bacteriocin AMA-K produced by Lactobacillus plantarum AMA-K isolated from naturally fermented milk from Zimbabwe. Food Control 18:656–664 Van Reenen CA, Dicks LM, Chikindas ML (1998) Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum. J Appl Microbiol 84:1131–1137 Vaughan EE, Daly C, Fitzgerald GF (1992) Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829. J Appl Bacteriol 73:299–308 Vignolo G, Palacios J, Farias ME, Sesma F, Schillinger U, Holzapfel W, Oliver G (2000) Combined effect of bacteriocins on the survival of various Listeria species in broth and meat system. Curr Microbiol 41:410–416 Whitaker JR, Granum PE (1980) An absolute method for protein determination based on the difference in absorbance at 235 and 280 nm. Anal Biochem 109:156–159 Wouters D, Grosu-Tudor SS, Zamfir M, De Vuyst L (2013) Applicability of Lactobacillus plantarum IMDO 788 as a starter culture to control vegetable fermentations. J Sci Food Agric. doi:10.1002/jsfa.6184 Yang E, Fan L, Jiang Y, Doucette C, Fillmore S (2012) Antimicrobial activity of bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts. AMB Express 2:48–59 Zamfir M, Callewaert R, Cornea CP, Savu L, Vatafu I, De Vuyst L (1999) Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801. J Appl Microbiol 87:923–931 Zendo T (2013) Screening and characterization of novel bacteriocins from lactic acid bacteria. Biosci Biotechnol Biochem 77:893–899
123
