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DOI: http://dx.doi.org/10.20534/ESR-16-9.10-17-20
Miralimova Shakhlo Mirdjamalovna, Ogay Darya Kisenovna, Ibragimova Alina Albertovna, Sokhibnazarova Khonsuluv Abduvokhidovna, Kutlieva Guzal Djumaniyazovna. Institute of microbiology of Academy of Sciences of the Republic of Uazbekistan, E-mail: mirshakhlo@yahoo.com
Bacteriocin production by Lactobacillus plantarum 42 strain
Abstract: Bacteriocins are ribosomally synthesized antibacterial peptides secreted by certain types of bacteria and active against both closely related species, and members of other species. Currently bacteriocins are recommended for use as antimicrobial agents in the food industry and in medicine. Bacteriocin production significantly depends on several factors such as culture conditions — pH, temperature, composition of the growth medium and the growth phase of producer strain. Bacteriocins can both be released to the culture medium, and remain attached to the producer cell. Optimization of growth conditions for bacteriocin production and increase its activity is of great economic importance to reduce its production cost.
The aim of this study was to determine the localization of a bacteriocin of Lactobacillus plantarum 42, active against En-terococcus faecalis and to determine the optimal culture conditions in which there is its maximum output has been observed.
The Lactobacillus plantarum 42 strain synthesizes bacteriocin, which is active against Enterococcus faecalis, is released into a solid and a liquid nutrient medium, but found only at 10 times the concentration in MRS broth. Bacteriocin detected at early stationary growth phase (18 hours) and remains active until 76 hours after initiation of fermentation. Maximal amount of bacteriocin was detected after 48 hours of fermentation at the initial pH value of growth media 6. There was no difference in the cultivation temperatures of 30oC and 37oC for bacteriocin production. This bacteriocin proved to be a secondary metabolite.
Keywords: Lactobacillus plantarum 42; bacteriocin; Enterococcus faecalis.
Introduction
Lactic acid bacteria (LAB) secrete large amounts of antimicrobial agents, including organic acids, reuterin, diacetyl, bacteriocins, hydrogen peroxide and that are able to inhibit the growth of pathogenic microorganisms [1]. Bacteriocins are ribosomally synthesized protein, secreted by certain types of bacteria, possessing antibacterial activity against both a closely related species [2], and members of other species [3]. Currently bacteriocins are recommended for use as antimicrobial agents in the food industry and in medicine [3; 4]. Bacteriocins have different spectrum of susceptible microorganisms, including those causing food spoilage and therefore, they can serve as a natural substitute for synthetic preservatives [5]. In this connection scientific interest to search for new potential sources of these peptides has significantly increased nowadays. It was reported that bacteriocin production depends on several factors such as culture conditions — pH, temperature, composition of the culture medium and the producer strain's growth phase [6]. The bacteriocins can both be released in the culture medium [7], and remain attached to the cell producer [8]. Optimization of conditions for bacteriocin production and increase its activity is of great economic importance to reduce its production cost.
The aim of this study was to determine the localization of a Lactobacillus plantarum 42 bacteriocin, active against Enterococcus faecalis and to determine the optimal culture conditions for its maximum output.
Experimental part
Bacterial cultures. In the research the bacteriocin synthesizing Lactobacillus plantarum 42 strain was used as the test organism [9]. The strain is isolated from sauerkraut and stored in the freeze-dried stock at the laboratory of genetics of lactic acid bacteria of the Institute of microbiology of AS of Uzbekistan [10]. For using in experi-
ment bacteria were recovered by two-times subculturing in MRS (de Mann, Rogosa, Sharp) broth (HiMedia) (amount of inoculum introduced was 1%) and incubation at 37oC for 24 hours. The type strain of Enterococccus faecalis.was an indicator strain.
Determining the localization of synthesized bacteriocin. To determine whether the antimicrobial peptide secreted to the media or left attached to cell, the presence of antimicrobial activity was studied: a) in the MRS agar layer; b) in a cell-free culture medium and c) in the cell lysate. On solid agar medium bacteriocin production was studied by agar spots method described by Harris et al [11]. Breafly, overnight culture of L. plantarum, grown in MRS broth was spotted (7 ul) onto the surface of the MRS agar and cultured at 37 °C for 48 hours under anaerobic conditions to prevent the synthesis of hydrogen peroxide. L. plantarum 43 and L. plantarum ATCC strains were used for comparative analysis. To elucidate the proteinaceous nature of antimicrobial substance, proteases pepsin and proteinase K were dropped (5 ul) around grown test culture stains. The plates were then covered with a second layer of soft Brain Heart Infusion agar (BHI, HiMedia), in which 10 ml of E. faecalis in stationary growth phase was suspended. After 24 hours of incubation under aerobic conditions the presence of growth inhibition zone in the indicator cell layer and the presence of protease activity were observed.
To determine the antimicrobial activity of the culture supernatant, the recovered cells were grown for 48 hours at 30oC in MRS broth and pelleted by centrifugation at 5000 rpm for 15 minutes. The supernatant was separated, passed through a membrane filter with a pore size of 0.22 nm to remove residual cells, concentrated 10-fold by freeze drying with subsequently dissolving in a smaller amount of water. Additionally, 10% solution was prepared from the dried supernatant by dissolving 100 g in 1 ml of sterile distilled water. 10-fold concentrated nutrient MRS-broth was served as a control.
To determine the antimicrobial activity of the cell lysate, Lactobacillus plantarum 42 was recovered by two-fold subculturing on MRS medium and incubated at 30 °C for 24 hours. The culture was centri-fuged at 5000 rpm for 15 min, the cell pellet was resuspended in 300 ml of 70% isopropanol and 0.1% trifluoroacetic acid [8] and stirred on a magnetic stirrer at room temperature for 3 hours. Cell debris was removed by centrifugation at 5000 rpm for15 min and then isopropanol was evaporated from the supernatant on a rotary evaporator.
The crude protein extract from the L. plantarum 42 culture supernatant was prepared by ammonium sulfate precipitation followed by dialysis in a dialysis bag with a pore size of 1000 kDa with subsequent freeze-drying.
The antimicrobial activity of the supernatant, cell lysate and crude proteins extract was analyzed by agar well diffusion method [12].
Selection of the optimal culture conditions for maximum synthesis of bacteriocins. The time of maximal bacteriocin production was estimated by agar spot method according to the zone of indicator E. faecalis strain inhibition after 24, 36, 48, 60 and 72 hours of fermentation.
The optimal cultivation temperature was determined by cultur-ing bacteriocin producing culture at 30 °C and 37 °C and measuring indicator culture growth inhibition zone of the indicator strain in the double-layer agar.
Influence of initial pH of culture medium on bacteriocin synthesis studied when producer was grown in MRS culture medium, with initial pH 4.0, 5.0, 6.0, 7.0 and 8.0 adjusted with HCl and NaOH and the change of diameter of the indicator culture inhibition zone was observed by method described above.
In the next series of experiments the production of bacteriocin under the following fermentation conditions has been studied: 37oC temperature, while stirring on a shaker (200 rpm) in a flask with a narrow and long neck to create partial anaerobic atmosphere, at the 12, 18 24, 36 and 48 hours of cultivation time.
Changes the pH value of growth medium, amount of cells by optical density of the bacterial suspension (OD600) and the number ofviable cells in 1 ml of medium and antimicrobial activity of supernatant according to zone ofE. faecalis growth inhibition was monitored.
Time of initiation of the bacteriocin synthesis. The L. plantarum 42 culture was grown in MRS broth, then plated on the surface of MRS agar to obtain separated colonies, and after 12 hours ofgrowth plates was exposed to UV rays for 30 seconds to induce bacteriocin production [13]. Bacterial cell suspension in saline with a density of10 9 CFU/ml was prepared from surface colonies and 5% inoculum was added in a flask with 100 ml of MRS broth. Time ofinitiation ofbacteriocin synthesis was determined at 6, 12, 18, 24, 36, 48, 60 hours of fermentation by applying 7 ul of centrifuged and passed through a membrane filter with a pore size 0.22 um supernatant onto the surface of the indicator layer seeded in soft BHI agar. After cultivation at 37 °C for 24 hours the zone of growth absence in the indicator layer at the place of supernatant was dropped has been observed.
Results and discussion
The antimicrobial activity ofviable L. plantarum 42 cells, the cell lysate and its supernatant.on solid medium. It has been shown that E. faecalis has a big zone of growth inhibition around L. plantarum 42 spot (d=20 mm). The antimicrobial agent of L. plantarum 42 loses its activity against enterobacteria after treatment with pepsin and proteinase K, which is appeared as presence of growth at the pepsin and proteinase K spots on the indicator growth inhibition zone. That means the antimicrobial agent has a proteinaceous nature (Fig. 1). Other strains of L.plantarum (L.plantarum 43 and L.plantarum ATCC) have no antimicrobial activity against a given culture.
Figure 1. Antimicrobial activity of L. plantarum 42, caused by bacteriocin, against E. faecalis
1 - the zone of bacteriocin disruption by pepsin
2 - the zone of action of bacteriocin disruption by proteinase K
It is found that cell lysate has no antimicrobial activity against E. faecalis, while the culture medium exhibits antimicrobial activity caused by proteinaceous substance, but only at 10-fold concentration. The diameter of E. faecalis growth inhibition zone by 10% solution of dried supernatant and 10-fold concentrated supernatant are of13 mm and 26 mm respectively, while the concentrated MRC broth has no antagonistic activity against the indicator culture (Fig. 2).
Figure 2. Antimicrobial activity of the L. plantarum 42 culture media against E. faecalis
1. 10% solution of dried L. plantarum 42 supernatant
6. 10-times concentrated MRS broth
7. 10-times concentrated supernatant
The crude extract of L. plantarum 42 proteins obtained by ammonium sulfate precipitation at a concentrations of2.5% and 25% also showed antimicrobial activity to E. faecalis, where the diameter of the indicator culture growth inhibition zone was 17 and 25 mm, respectively.
According to the published data, most of bacteriocins are accumulated in the supernatant in sufficient amount, as their activity is found not only in the crude supernatant but also when it is diluted several times [14; 15; 16]. However, some bacteriocins such as plantaritsin F [17], lactacin B [18] and plantatsin B [19], is secreted into liquid medium in quite small quantities so it can not be detected without concentrating the supernatant. Despite this, in a solid medium accumulation of such bacteriocins are relatively effective, so the detection of presence and the activity carried out on agar medium. Studied L. plantarum 42 bacteriocin, as well as plantaricin F, lactacin B and plantacin B, is secreted mostly in a solid medium.
The culture conditions for maximum accumulation of bacteriocins. The results showed that the highest bacteriocin production is observed after 48 hours of fermentation time, and by 60 and
72 hours of cultivation, bacteriocin production decreasing. Incubation temperature (30 °C and 37 °C) does not significantly affect the synthesis of the bacteriocin (Table 1).
Table 1. - Diameter of the indicator strain growth inhibition by L. plantarum 42 depending of the time of cultivation and temperature
Temperature Cultivation time, hours
24 36 48 60 72
30 oc 12±0.3 15±0.5 20±0.5 19±0.3 18±0.2
37 0C 13±0.3 16±0.6 19±0.3 19±0.5 18±0.5
Studying the influence of pH value on bacteriocin production showed that the synthesis of the bacteriocin is maximal at pH 6, the diameter of the zone of growth inhibition was 19.6 mm, at pH 5.0 and 7.0 the significant synthesis of bacteriocin has been observ-
Table 2. - Influence of initial pH of growth media
ing and the diameter of the zone of growth inhibition is 17.6 and 17.8 mm. At pH below 5 bacteriocin production is not observed and at pH 8 it decreases (Table. 2).
on bacteriocin production by L. plantarum 42
The pH of the MRS nutrient medium The diameter of the growth inhibition zone, mm
4.0 -
5.0 18±0.5
6.0 19±0.3
7.0 18±0.5
8.0 12±0.6
The culture conditions, such as the initial pH value and temperature is an important factor for the production ofbacteriocins [20].
Determination of bacteriocin production in dynamics of growth showed that the maximum bacteriocin synthesis occurs under these conditions at 24 hours of fermentation when the maximum number of living cells (1.4x10 9) is accumulated in the medium, the diameter
Table 3. - Chronology of bacteriocinogenic L. plantarum 42 strain growth and bacteriocin synthesis
of the zone of growth inhibition of the indicator culture is 15.5 mm. In 48 hours of incubation, along with decreasing the number of living cells in the medium and accumulation of acids (the pH drops to 3.5), the diameter of the zone of growth inhibition has decreased to 14.8 mm (Table. 3).
Indicators Cultivation time, h
12 18 24 36 48
pH 4,7 4,5 3, 8 3, 5 3, 5
Optical density of bacterial suspension 2,38 2,71 2,75 2,78 3,1
The number of living cells/ml culture medium 5,5-10 8 1,2-10 9 1,4- 10 9 9,5-10 8 7, 5-10 8
Zone of bactericidal action, mm 12.2±0.48 14.5±0.45 15.5±0.45 15.1±0.14 14.8±0.59
Time of the initial bacteriocin synthesis
When L. plantarum 42 supernatant applied onto the surface layer with an indicator culture it was noted that the not all samples exhibit the inhibition of E. faecalis. Inhibition zone begins to appear in a sample taken 18 hours after initiation of growth, which corresponds to an early stationary phase of growth of the test culture. Supernatant activity is maintained up to 72 hours, after which observation was stopped. Staying active for a long time suggests that the culture does not produce extracellular proteases.
Unlike many LAB bacteriocins, bacteriocin of L. plantarum 42 is not detected in the culture supernatant before beginning the stationary growth phase. In the same growth phase the activity of bacteriocins plantaricin F [17] has been detected, while plantaricin T produced by L.plantarumLPC010, dettected only during late stationary growth phase [21]. In contrast, plantaricins A, BN, C and S [22; 23; 24; 21] — all L. plantarum bacteriocins, detected during active growth phase, as well as pediocin AsH, nisin, sakacin A and leykonocin Lcm 1 [25].
The absence of bacteriocin F activity during the exponential growth phase Paynter et al [17] explained by the following factors: it either is not synthesized in this growth phase, or it is synthesized during active growth but attached to the cell as long as the pH is reduced to a level which causes the desorption. Our study demonstrated that that the actual pH value of the media does not influence on
bacteriocin desorption since it doesn't significantly differ in 12 hours (pH=4.7), then there is no activity and in 18 hours (pH=4.5), then the activity is observed. This confirmed with the absence of antimicrobial activity of the L. plantarum 42 cells lysate. Probably bacteriocin synthesis by L.plantarum 42 begins at early stationary growth phase, which means that it is a secondary metabolite. In most cases, the synthesis of bacteriocins by LAB occurs during active growth phase [15], but some studies indicate that the LAB can synthesise bacteriocin in the stationary growth phase [17].
The culture conditions, such as the initial pH value of the media and temperature is an important factor for the production of bacteriocins [20]. Prema (2013) [26] reported that the optimal conditions for bacteriocin production by the three strains of L. plantarum (IZ, A1, F1), isolated from plant, is the MRS-medium with pH = 6.5, incubation temperature 37 °C and fermentation time is 48 hours. The same culture conditions are optimal for the production of bacteriocin by L. plantarum 42 although temperature ranging from 30oC to 37oC did not significantly affect the synthesis of bacteriocins. In another study, the synthesis of bacteriocins by L. plantarum ST194BZ [15] the optimum pH is also was higher than 4.5, while the optimum temperature for the synthesis is 30 °C. From our results and published data it can be concluded that the optimum temperature for bacteriocins production by L.plantarum is 30 °C to 37 C and optimal pH value is above 4.5.
In addition to temperature and pH the composition of the cul- Acknowledgement
ture medium is greatly influence on the synthesis of bacteriocins This research was supported by the Academy of Sciences of the Re-
[15], which will be investigated in further studies. public of Uzbekistan (Grant of the Found of Fundamental Researches
Support №Т. 4-16).
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