International Journal of Life science and Pharma Reviews (IJLPR)  
   
 
International Journal of Life science and Pharma Research (IJLPR)
Pharmaceutical Sciences
Volume 10 Issue 3, July - September 2020    Pages:6-13
Partial Purified, Characterization and Antibacterial Activity of Bacteriocin from Leuconostoc Mesenteroides ()

Enas Nabil Danial , Salha Hassan astour Al-Zahrani, Zahra Al-Hassan Mohammad Al-Mahmoudi
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DOI: http://dx.doi.org/10.22376/ijpbs/lpr.2019.9.3.L6-13
Abstract:
Lactic acid bacteria produces a variety of antibacterial compounds such as organic acids, diacetyl, hydrogen peroxide and bacteriocins or bactericidal proteins as byproducts of fermentation. Bacteriocins are small proteins with bactericidal or bacteriostatic activity against genetically closely related species. Some bacteriocins are peptides consisting of only 13 to 37 amino acids. Some small bacteriocins contain unusual amino acids originating from modifications of conventional amino acids after translation. Bacteriocins are natural peptides secreted by several bacteria that exert bactericidal activity against other bacterial species. Research in this topic is promoted not only by the need to develop alternatives to antibiotics and drugs that have shown non-desirable effects, but also to the capacity of some bacteriocins to inhibit saprophytic and food-borne pathogens in food stuffs. The purification of bacteriocins produced is helpful for the knowledge of the mechanism of action, structure, and other characteristics, which helps to isolate the bacteriocin biosynthetic genes. In this study, purified bacteriocins from Leuconostoc mesenteroides were described. The frequently applied techniques involve ammonium sulfate salt precipitation. The results obtained through the purification of Leuconostoc mesentroides bacteriocins carried out its antimicrobial activity, thermostable and the effected of pH. the incubation of temperature on bacteriocin activity in terms of inhibition zones. The data recorded that the bacteriocin was active in a wide range of pH, the maximum activity was observed at pH 6.0 to 7.0, but at high pH of 8.0 the activity of the bacteriocin gradually decreased. Bacteriocin could retain its antimicrobial activity partially when there was a shift to acidic or basic range. It has been found to be thermo stable in nature as it can withstand high temperatures up to 121°C, although a partial loss in the activity was observed with a continuous increase in temperature. Bacteriocins carried out for its antimicrobial activity, thermostable and the effect of pH.
Keywords: bacteriocin, purification, characteristics, Leuconostoc mesenteroides, antimicrobial activity
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1.    INTRODUCTION 

Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that produce lactic acid in two pathways: homofermentative or heterofermentative. These bacteria are found in oral cavity, skin and human digestive system 1. Lactobacillus species play an important role in gastrointestinal (GI) tract function, intestinal microbiota balance and the immune system activity by exerting a strong activity against many intestinal pathogens 2. These activities arise from the production of some specific components such as hydrogen peroxide, organic acids, inhibitory enzymes and bacteriocins 3. In recent years, bacteriocins have attracted significant attention because of their potential use as safe additives for food preservation 4 and other applications such as treatment of pathogenic diseases, cancer therapy and human health enhancement. Bacteriocins are a heterogeneous group of ribosomally-synthesized pore-forming antibacterial peptides. These antibacterial peptides can be produced by many labs such as Leuconostoc, Streptococcus, Lactococcus, Pediococcus, and Lactobacillus that enable these bacteria to inhibit the growth and activity of several pathogenic bacteria 5. Lactobacilli have many different bacteriocins with the same activity but vary in some aspects such as mode of action, genetic origin (chromosomal or plasmid) and biochemical properties. These peptides are being tested to assess their application as narrow-spectrum antibiotics and nowadays, play an effective role in processing and packaging food and have major potential as natural preservatives6&7. Based on its antibacterial activity , molecular weight, genetics and chemical properties, bacteriocins are classified in one of the four major groups: Class I (small peptide inhibitors termed as lantibiotics), Class II (small heat-stable proteins that have five subclasses), Class III (large, heat labile protein and this class has two subclasses) and Class IV bacteriocins that are defined as complex bacteriocins containing lipid or carbohydrate sections 8. Lactobacilli usually produce the highest volume of bacteriocins during the maximum cell growth, but some environmental factors such as pH, temperature, incubation period and media composition can influence the volume of bacteriocin production9&10. This study describes the purification and characterization of bacteriocin from Leuconostoc mesenteroides which has been found by extensive screening to produce a relatively high bacteriocin activity and their antimicrobial activities against pathogen microorganisms 

2.    MATERIALS AND METHODS 

2.1    Production of bacteriocin

Leuconostoc mesenteroides was propagated in MRS broth (100 ml) seeded with slant inoculum of overnight culture and incubated for 24 h at 35°C 11. After incubation, the whole broth was centrifuged at 10,000 rpm for 10 min and the cell-free supernatant was used as crude bacteriocin 12. The indicator Gram positive bacterium was S. aureus ATCC25923, while the Gram negative bacteria were E. coli ATCC25422, K. pneumonia ATCC700603, P. aeruginosa ATCC27583.

2.2    Partial purification of bacteriocin  

Ammonium salt precipitation methods were used for this purpose 13. The cell free culture supernatant (crude bacteriocin) of Leuconostoc mesenteroides was saturated with various concentrations of solid ammonium sulfate 60, 70 and 80% in 250 ml Erlenmeyer flasks after stirring on a magnetic stirrer. It was kept undisturbed at 4°C overnight stored. The pellet was collected after centrifugation at 10,000 rpm at 4°C for 30 min. The pellet was dissolved in 25ml of sodium phosphate buffer (0.05 M, pH 7.0) and dialyzed against the same buffer at 4°C overnight 12.

2.3    Bacteriocin assay

Agar well diffusion procedure described by Zhang et al 14, was used to determine the production of bacteriocin in the culture supernatant using, Gram positive bacteria -S. aureus ATCC25923, and the Gram negative bacteria - E. coli ATCC25422, K. pneumonia ATCC700603, P. aeruginosa ATCC27583. Inhibition of bacterial growth was measured as inhibition zone diameters (mm). 

2.4    Characterizations of partial purified bacteriocin 

2.4.1    Heat stability

A 5-mL aliquot of partial purified bacteriocin in different test tubes was overlaid with paraffin oil to prevent evaporation and then heated at 30, 40, 50, 60, 70, 80, 90 and 100°C for 10 and 20 mins respectively, and the effect of autoclaving condition also tested and validated. The heat-treated bacteriocin samples were then assayed for bacteriocin activity as described earlier.

2.4.2    Effect of different pH on partial purified bacteriocin activity

A 5-mL aliquot of partial purified bacteriocin was placed in test tubes and the pH values of the contents were adjusted to 3, 4, 5, 6, 7, 8, 9 and 10 individually, using either diluted NaOH or HCl (1 M NaOH or 1 M HCl solution). After allowing the samples at room temperature for 2 h the antimicrobial activities of bacteriocins were assayed as described earlier 15.

2.4.3    Effect of inhibitors enzyme on partial purified bacteriocin activity

Trypsin and pepsin concentrations (1.0 mg/ml) at pH=7 were added to the purified bacteriocin and incubated for 2 h at 35°C. After incubation, the bacteriocin activities were determined by using well diffusion method as described by 8.

3.    STATISTICAL ANALYSIS

Results are presented as the mean of three or four replicates ± standard error (SE). The statistical analyses were carried out using SPSS program (version 22). Data obtained were analyzed statistically to determine the degree of significance using one way (ANOVA) at probability level P ≤ 0.05 levels of significans.

4.    RESULTS AND DISCUSSION

4.1    Partial purification of bacteriocin 

Ammonium sulfate was used to precipitation bacteriocin that was produced by Leuconostoc mesentroides. The bacteriocin extraction was done at 70% saturation of ammonium sulfate and suspended in 0.05 mmol sodium phosphate buffer, pH=7.0. Finally, the results of the pellets weight from 100 ml were 1.50 gm (Figure 1). On the other hand, various concentrations of solid ammonium sulfate 60 and 80% ammonium sulfate didn't give the result. The result of partial purification for precipitation of bacteriocin by 70% ammonium sulfate was in agreement with Oppegard 16 whose purification bacteriocin produced by Lactobacillus plantarum isolated from cow milk used (70%) ammonium sulfate and stored at 4 °C to precipitate out the proteins. The optimal bacteriocin recovery was achieved by including ammonium sulfate precipitation. This agreed with the findings of Ivanova 17.
 

Fig 1. The pellets formatted by ammonium sulfate precipitation from crude 
     bacteriocin of Leuconostoc mesenteroides

 
4.2    Determination of inhibitory spectrum

Crude bacteriocin and partial pure bacteriocin activity were assayed by the agar-well diffusion method. The susceptibilities of various Gram-positive and Gram-negative bacteria to growth inhibition by the bacteriocin produced by Leuconostoc mesenteroides dissolved in buffer (Sodium phosphate buffer 0.05M; pH 7) are presented in (Table 1). It shows the activity of crude bacteriocin (in terms of inhibition zone diameter) from 21.66 to 23.66 mm. On the other hand, the mean diameter of inhibition zone by pure bacteriocin ranged between 25 to 27.66 mm (Figure 2). It was found that pure bacteriocin from Leuconostoc mesenteroides was higher and efficient in production of bacteriocin than crude bacteriocin (Figure 3). High level of significance was obtained with the partial pure bacteriocin. By comparison, leucocin A-UAL 187 from Leuconostoc gelidum purification was conducted by an ammonium sulfate precipitation followed by three steps of chromatography 18. On the other hand Vaughan 19 reported that inhibitory activity of bacteriocin isolated from malted barley was precipitated from cell free supernatant using (40%) ammonium sulfate saturation, and resuspended in 2 mmol sodium phosphate buffer, pH=6.0 and purified using chromatography.  Bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation) from Leuconostoc mesenteroides 406 isolated from Mongolian fermented mare’s milk 20. The culture supernatant was concentrated by ammonium sulfate precipitation (60%) of Leuconostoc mesenteroides 21.
 

Table 1. Determination of inhibitory spectrum of crude bacteriocin and partial pure bacteriocin

              production by Leuconostoc mesenteroides.

Kind of bacteriocin

Indicator microorganisms

P.aeruginosa ATCC27583

S.aureus ATCC25923

E.coli ATCC25422

K.pneumonia TCC700603

Mean diameter of inhibition zone (mm)

Crude bacteriocin

22.66

21.66

22.33

23.66

Partial pure bacteriocin

26.00

25.00

26.33

27.66

 

   Fig 2. Determination of inhibitory spectrum of the partial pure bacteriocin (A) and crude bacteriocin 
                   (B) production by Leuconostoc mesenteroides against S.aureus ATCC25923

 4.3    Effect of different temperature on the activity of the partial purified bacteriocin

The effects of different temperature on bacteriocin activity were determined using P.aeruginosa ATCC27583, S.aureus ATCC25923, E.coli ATCC25422 and K.pneumoniae ATCC700603 as indicator microorganism. A volume of 5 mL of bacteriocin in different test tubes was overlaid with paraffin oil to prevent evaporation and then inoculated at 30, 40, 50, 60, 07, 80, 90 and 100 °C for 10 and 20 min respectively. The effect of autoclaving condition was also tested. The data represented that, (Table 2) shows the incubation of temperature on bacteriocin activity in terms of inhibition zones. It has been found to be thermo stable in nature as it can withstand high temperatures up to 121°C, although a partial loss in the activity was observed with a continuous increase in temperature. Partially purified bacteriocin was found to be stable at 30°C - 40°C for 20 min, The maximum antimicrobial activity was observed at 30 °C and 40°C for 20 min.

  
Fig 3. Determination of inhibitory spectrum of crude bacteriocin and partial 
           pure bacteriocin production by Leuconostoc mesenteroides

As show in Figure (4), the loss in the activity was observed at 50°C followed by  60°C, 70°C, 80°C and 90°C for 10 min it reached 21, 19, 18, 16 and 14 mm against K.pneumoniae ATCC700603 respectively.  Some of bacteriocins produced by LAB, for example, bacteriocins produced by L. acidophilus LF221 or lacticin NK24 from Lactococcus lactis could at least partially preserve their activity, even after heating at 100 °C for 30 min (22). Heat resistance is the major characteristic of many bacteriocins and bacteriocin-like compounds produced by LAB and can vary dramatically ranging from 60 ºC or 100 ºC for more than 30 min (e.g. lactocin 27, lactocin S, carno bacteriocins A and B) to autoclaving at 121 ºC for 15-20 min (e.g. lactacin B, lactacin F, nisin etc.)23. Many of bacteriocins produced by LAB, particularly the ones of class I and class II, were described as small hydrophobic proteins containing little tertiary structure, which explains their heat stability. Other factors contributing to heat stability of the bacteriocin of LAB are stable cross-linkages, a high glycine content and occurrence of strongly hydrophobic regions 23. However, after incubation for 15 min at 121 °C, partially loss of activity took place, the diameter of inhibition zone was 11 mm against K.pneumoniae ATCC700603, while at the same condition, only 8mm of activity could be retained against Escherichia coli. The inhibitory compound produced by Leuconostoc mesentroides was considered to be heat stable at 30 to 40 ºC for 20 min but partial stable at 50 to 90 ºC for 10 min. Bacteriocin produced by Leuconostoc mesentroides was considered to be the same condition, as the activity  remained constant after heating at 121ºC for 15 minutes. Earlier studies revealed that bacteriocins produced by L. paracaseii, L. lactis, L. plantarum and L. pentosus remained active after heating till 121°C for 20 min 24. 40% loss of activity of L.mesenteroides E131 bacteriocin was determined after incubation at higher temperatures (90 and 100ºC) and the pure bacteriocin of L sakei I154 under sterilization conditions retained 40% of the initial activity after 15 min of heat treatment. Indeed, other bacteriocins isolated from L. mesenteroides or L.sakei has similar features. Mesenteric 52 is stable at pH range 4.5-7.0 and resistant at 100ºC for 15 min 25, mesenteric 5 is resistant at 100ºC for 30 min 21, and sakacin P is resistant at 100ºC for 20 min22.
 

 

Table 2. Effect of different temperature on the activity of the partial purified bacteriocin

Indicator microorganisms Mean diameter of inhibition zones (mm)

K.pneumoniae

ATCC700603

E.coli

ATCC25422

S.aureus

ATCC25923

P.aeruginosa

ATCC27583

 

Time ( after 10 min , after 20 min)

 

20min

10min

20min

10min

20min

10min

20min

10min

 

26.66

27.00

25.33

25.33

23.33

24.66

26.00

26.00

30

26.00

26.33

24.66

25.00

23.33

24.00

25.00

25.33

40

21.66

22.66

19.66

21.00

20.00

20.66

20.66

21.66

50

19.66

20.66

18.00

19.33

18.33

19.00

18.66

20.00

60

17.00

18.33

15.66

16.33

16.33

17.00

16.66

17.66

70

16.00

16.66

14.00

14.66

14.66

15.33

15.33

15.66

80

14.33

15.00

12.66

13.33

13.66

14.00

14.00

14.66

90

12.00

13.33

10.33

11.33

10.66

11.66

11.66

12.66

100

10.66

8.33

8.66

10.33

121

 


 

                  Fig 4. Effect of different temperature on the activity of the partial purified bacteriocin
                             effect of different pH values on the activity of the partial purified bacteriocin

 
A 5-mL aliquot of purified bacteriocin was taken in test tubes and the pH values of the contents were adjusted to 3–10 individually. To determine the pH stability of bacteriocin, pH values of the bacteriocin were adjusted within the range of 3 to 10 by HCl or NaOH. The data recorded in (Table 3 and Figure 5) showed that the bacteriocin was active in a wide range of pH, the maximum activity was observed at pH 6.0 to 7.0, but the high pH=8.0, the activity of the bacteriocin gradually decreased. Bacteriocin could retain its antimicrobial activity partially when there was a shift to acidic or basic range. Bacteriocin was stable at acidic and neutral pH values (from pH 4 to pH 7). This differs from other bacteriocins produced by Lactococcus lactis which are unstable at neutral pH value26, but similar to bacteriocin S 50 produced by Lactococcus lactis subspecies diacetylactis which is active in the pH range 2 to 1127. Many bacteriocins are most active at low pH .28 Stability of bacteriocin at different pH scale is a limiting factor for recommending its use in food items. Bacteriocins produced by Leuconostoc mesentroides retained their antimicrobial activity in pH range of 5.0 to 7.0, while decrease occurred at pH 3.0 and 10.0. The high and loss of pH causes the gradually decreased of bacteriocins activity. Bacteriocins differ greatly with respect to their sensitivity to inactivation by changes in pH and temperature. Many of the bacteriocins and bacteriocin-like substances produced by lactic acid bacteria are only stable at acid and neutral pH 23 and are inactivated even at a pH above 8.0 (e.g. nisin, lacto strepcins, pediocin AcH, leucocin A-UAL 187). Bacteriocins produced by L. plantarum and L. brevis OGI retained their antimicrobial activity in an acidic pH range of 2.0 to 6.0, while inactivation occurred at pH 8.0 to 12.0 29. L. mesenteroides E131 bacteriocin retains activity at a broad range of pH (4.0- 9.0), the stability at low pH is very important for its potential application in foods, such as fermented meat products, in which acidic conditions prevail. Most European fermented sausages have final pH values ranging from 4.8 to 5.030.
 

Table 3. Effect of different pH values on the activity of the partial purified bacteriocin

Indicator microorganisms

pH

K.pneumoniae

ATCC700603

E.coli

ATCC25422

S.aureus

ATCC25923

P.aeruginosa

ATCC27583

Mean diameter of inhibition zone (mm)

15.33

14.33

13.66

14.66

3

16.66

14.66

14.33

16.33

4

22.00

20.00

19.33

21.33

5

25.66

25.33

24.66

25.33

6

25.33

24.66

24.00

24.33

7

19.66

17.00

16.00

19.00

8

16.66

15.66

14.66

16.33

9

15.00

13.00

12.66

14.66

10

 

Fig 5. Effect of different pH values on the activity of the partially purified bacteriocin
        effect of inhibitors enzyme on the partial purified bacteriocin activity

 All bacteriocins compounds are of protein nature 31. To identify this nature of the antimicrobial substances, the action of proteolytic enzymes (trypsin and pepsin ) was tested; it shows the activity of bacteriocin produced by Leuconostoc mesentroides was fully inactivated by the enzyme trypsin and pepsin against all four indicator microorganisms, which indicates their proteinaceous nature (Figure 6).
 

Fig 6. Effect of partial purified bacteriocin treated with trypsin 
             against K. pneumoniae ATCC700603    

 

 

  
In this study, the sensitivity of bacteriocin produced by Leuconostoc mesenteroides from all four indicator microorganisms was fully inactivated by the enzyme trypsin and pepsin, which indicates their proteinaceous nature. It is well known that bacteriocins produced by LAB are very often sensitive to trypsin, while the sensitivity to other enzymes varies 32. These data were similar with Halil33 who observed that, bacteriocin of Leuconostoc mesenteroides subsp. cremoris was treated by a variety of enzymes (trypsin -pepsin) to verify the protein nature of the inhibitor substance. The inhibitory activity of the bacteriocin was inhibited by all the proteases employed. These data clearly showed that the antimicrobial substance is of proteinaceous nature, containing cleavage-sites suitable for the mentioned proteases. Characterization of the proteinaceous inhibitor confirmed that the antimicrobial agent produced by Leuconostoc mesenteroides subsp. cremoris was a bacteriocin according to the criteria outlined by Tagg 34. There has been a few bacteriocins studied in literature like leuconocin S produced by Leuconostoc mesenteroides 35. Leuconocin S bacteriocin produced by Leuconostoc mesenteroides isolated from retail lamb and has a broad spectrum of activity. It was active against L. monocytogenes and S. aureus. 36

5.    CONCLUSION

The application of bacteriocins as biopreservatives for vegetable food matrices started approximately 20 years ago. In these years, a lot of studies have focused on the inhibition of spoilage and/or human pathogen bacteria by bacteriocins and their application appeared as a good alternative to chemical compounds and antibiotics. Furthermore, it can be concluded that in addition to the traditional hurdle technology represented by low temperature and vacuum packaging, the exploitation of bacteriocin gentic cultures, as well as their pure bacteriocins holds a great potential for extension of shelf-life and improvement of microbiological safety of vegetable raw materials and final products.

6.    AUTHOR CONTRIBUTION STATEMENT

Mrs. Zahra Al-Hassan Mohammad Al-Mahmoudi conceptualized and gathered the data with regard to this work. Dr.Salha Hassan Mastour Al-Zahrani and Enas Nabil Danial analyzed these data and necessary inputs were given towards the designing of the manuscript. All authors discussed the methodology and results and contributed to the final manuscript.

7.    CONFLICT OF INTEREST

Conflict of interest declared none.
 

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