Lactic acid bacteria (LAB) is a group of Gram positive facultative anaerobic bacteria that are able to produce antagonistic molecules in their growth medium that can be used as antimicrobials and preservatives. These antagonistic properties of LAB are allied to their safe history of use in traditional fermented food products that make them very attractive as biopreservatives that can replace or allow reduction of chemical additives  . LAB is used in food biopreservation because they are safe for human consumption enjoying the status of GRAS (Generally Recognized as Safe) and are the prevalent indigenous microflora in many foods. Accordingly, a wide variety of LAB strains are routinely employed as starter cultures in the manufacture of meat, dairy, vegetable and bakery products   . One of the most important contributions of these bacteria―whether indigenous or added as starters―is the extension of shelf life of the fermented products through inhibition of the growth of spoilage and pathogenic bacteria in these foods due to competition for nutrients and the presence of the antagonistic molecules such as lactic acid, hydrogen peroxide, diacetyl and bacteriocins  . Moreover, health benefits acclaimed to be offered by LAB include production of vitamins, immunomodulation, reduction in the risk of diarrhea, and a decrease in serum cholesterol    . Among the antagonistic molecules produced by LAB are bacteriocins  which are antimicrobial peptides or proteins produced by strains of diverse bacterial species. The antimicrobial activity of this group of natural substances against foodborne pathogens, as well as spoilage bacteria, has raised considerable interest for their application in food preservation     .
Within LAB, the lactobacilli are an important group recognized for their fermentative ability as well as health and nutritional benefits  . In this group, Lactobacillus plantarum is one of the most widely distributed in nature, and is one of the most versatile species, used both as starter and probiotic   . Lb. plantarum has been isolated from various habitats, and bacteriocins have been described for strains from fermented meat products    . It is one of the most important LAB strains used for the production of fermented meat products  . Over the past few decades, there has been an increasing research interest in the development of nitrite-free meat curing systems. The principle concern with the use of nitrite for curing of meat is the eventual formation of carcinogenic N-nitrosamines  . Consumers are increasingly demanding food that is free from pathogens, with minimal processing and fewer chemical preservatives and additives. Thus biopreservation has gained increasing attention as means of naturally controlling the shelf life and safety of meat products. In recent years bacteriocins of lactic acid bacteria have attracted the attention of many investigators because of their use as a natural food preservative with probiotic capability within the human body after ingestion of food   .
Shermout is sun-dried lean beef strips widely used for prolonged storage. It has unique sensory characteristics and is very popular in Sudan and neighboring countries. It is very similar to “kaddid”  and jerky   except that no or little salt is added and the product undergoes mild fermentation by indigenous microbial flora, mainly LAB. The process is artisanal in nature, with no bacterial starters added and is usually subject to microbial deterioration. Various gram positive and gram negative bacteria like Salmonella typhi, Bacillus subtilis and staphylococci are the main causative organisms  . The objectives of this study were the isolation and identification of Lb. plantarum from local Sudanese fermented beef (shermout), characterization of the bacteriocin it produces and determination of its antibacterial activity, study of the bacteriocin kinetics and determination of the optimum growth conditions for bacteriocin production.
2. Materials and Methods
2.1. Isolation of the Bacteriocinogenic Bacterium
Ten g of traditional Sudanese fermented beef (shermout) samples were aseptically added to 90 mL sterile peptone water (10 g peptone/L distilled H2O), carefully shaken and were left to homogenize for I h. Serial decimal dilutions were prepared from the sample homogenate, and were streaked onto duplicate plates of MRS medium  to which 0.1% (w/v) nystatin had been added to inhibit fungal growth  . The streaked plates were incubated anaerobically at 30˚C for 2 - 3 days in an anaerobic jar system (GasPak; BBL Microbiology Systems, Cock- eysville, Maryland, USA) with a gas-generating kit (BR0038B, Oxoid, Hampshire, UK). The pure colonies obtained were examined for Gram reaction, catalase activity, and spore formation. A Gram-positive, catalase-negative, non- spore-forming rod was selected and identified as Lactobacillus plantarum by use of the fermentation pattern from KB009 HiCarbohydrate identification kit (HiMedia Laboratories, Mumbai, India) in conjunction with other tests which included growth at 10 and 45 C, tolerance of 6.5% NaCl, pH (4.4 and 9.6) and gas production from glucose.
2.2. Production, Partial Purification and Characterization of Supernatant from Lb. plantarum PM4
For partial purification of the cell-free supernatant (CFS), a modification of the method of ten Brink et al. (1994)  was adopted. Sensitivity of the CFS to various enzymes (proteinase K, pepsin, α-amylase) was conducted  . Production of the inhibitory factor during anaerobic growth was investigated following the technique detailed in  . The effects of heating (40˚C, 60˚C, 100˚C for 10, 30 and 60 minutes in addition to autoclaving at 121˚C and 15 psi) and different pH values (3.0, 5.0, 7.0 and 9.0) were tested according to  . The stability of the antagonistic activity of the CFS during storage (−80˚C, −20˚C, 4˚C and 35˚C for 4 weeks) was also tested. The retained activity in all tests was determined using the agar well diffusion test utilizing Staphylococcus aureus ATCC43306 as the indicator organism.
For molecular weight determination, the active moiety in the CFS was first precipitated by addition of 40% (w/v) ammonium sulfate, centrifuged (6000 rpm for 15 minutes), and the pellet and pellicle were concentrated and used for protein separation by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) using 15% acrylamide. The gel electrode assembly was placed in a Mini PROTEAN II electrophoresis chamber (BIORAD), and the protein was electrophoresed at 100 volts for about 2 h. A protein molecular weight marker (MoBiTec GmbH) with a molecular weight range of 14.0 to 116.0 KDa was included.
Staining of the gels was done by covering with Coomassie blue stain overnight and the preparation was destained with a buffer composed of 10.0 ml glacial acetic acid, 50.0 ml methanol and 100 ml deionized water.
2.3. Mode of Action
This test was conducted to find out whether the antagonistic effect of the CFS was bactericidal or bacteriostatic in nature. The procedure followed was similar to that described in  and  .
2.4. Spectrum of Activity
The inhibitory activity was tested against eight indicator bacteria, namely: S. aureus ATCC 25923, Enterococcus faecalis ATCC 10541, Escherichia coli ATCC 25922, E. coli (local isolate), Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 10031, Proteus vulgaris ATCC 6380 and Salmonella typhi ATCC 1319106. The antagonistic activity was determined by the well diffusion method in Nutrient Agar plates. Diameters of the inhibition zones were measured after 24 h of incubation at 35˚C.
2.5. Kinetics of Growth and Bacteriocin Production
The kinetics of growth and production of the antagonistic activity was investigated using the procedure described by  . MRS broth (250 mL) was inoculated with 1% of an overnight culture of the strain and incubated at 30˚C without agitation under uncontrolled pH conditions. Samples were removed at hourly intervals up to 13 h, and then at 24, 25 and 26 h from start of the investigation. Measurement of biomass by absorbance at 600 nm, pH measurement and determination of the antibacterial activity were carried out by assaying the effect of serial two-fold dilutions of the CFS on Staphylococcus aureus ATCC 43306 by the well-diffusion method. The antimicrobial titer was expressed in arbitrary units (AU/mL). One arbitrary unit was defined as the reciprocal of the highest dilution showing a clear inhibition zone around the well  .
2.6. Optimization of Growth and Bacteriocin Production
For determination of the effects of incubation temperature, pH of the growth medium and incubation period on bacteriocin production, three levels of each of these three factors were chosen and tested. The temperature levels were 25˚C, 30˚C and 37˚C, the pH values were 5.0, 5.5 and 6.0 while the incubation times were 24, 48 and 72 h. The test isolate was grown in MRS broth. Growth was measured as optical densities (O.D.) at the wavelength of 600 nm, and inhibitory activity was measured in arbitrary units (AU).
3. Results and Discussion
3.1. Isolation and Screening of the Bacteriocinogenic Bacterium
A total of 39 isolates of antagonistic LAB were obtained from Sudanese fermented beef. All isolates were Gram-positive, catalase-negative, non-spore forming rods or cocci capable of growth under anaerobic conditions, conforming to the characteristics of lactic acid bacteria  . An isolate that gave positive result in the preliminary screening (spot on lawn method) against indicator bacteria (Staphylococcus aureus ATCC 43306, Bacillus subtilis NCTC 8236) with good inhibition zone diameter (well diffusion method) was selected and identified as Lactobacillus plantarum PM4. The inhibition zone by the selected isolate was produced as early as 24 h. The isolate was rod shaped, did not grow at 10˚C but grew at 45˚C. It grew at pH 4.4 but not at pH 9.6. It did not grown in the presence of 6.5% of NaCl, and could not hydrolyse arginine. It therefore belonged to the genus Lactobacillus (homofermentative lactobacilli)     . Table 1 shows the pattern of utilization of 35 sugars by the isolate based on which it was identified as Lactobacillus plantarum PM4   .
3.2. Characterization of the Cell-Free Supernatant (CFS)
The partially purified CFS from Lb. plantarum PM4 was subjected to various treatments. Table 2 shows effects of the enzymes proteinase-k, pepsin and α- amylase on the CFS. No inhibition was produced by the CFS in presence of the protein-digesting enzymes (proteinase-k and pepsin) indicating complete destruction of the inhibitory substance in the CFS, and asserting its proteinaceus nature; while no reduction in the inhibitory activity was observed in the presence of the carbohydrate-degrading α-amylase. No inhibitory activity was shown in the uninoculated medium containing no enzyme (negative control). This suggested that the antibacterial activity was associated with bacteriocin   . Treatment with a-amylase did not affect the inhibitory activity suggesting that the CFS, similar to most other bacteriocins, was not glycosylated     .
As production of hydrogen peroxide under aerobic conditions is one of the potent defense weapons of LAB, elimination of this factor was achieved through growing the isolate under anaerobic conditions. There was no difference in inhibitory activity against indicator bacterium whether Lb. plantarum PM4 was grown under aerobic or anaerobic conditions (result not shown), indicating that the inhibitory activity was not due the production of H2O2. Figure 1 shows that the widest inhibition zone of the CFS from Lb. plantarum PM4 on Staph. aureus ATCC 43306 (20 mm) occurred at pH 5.0, declining gradually with either increase or decrease in pH value.
No reduction was observed in the inhibitory activity of the CFS on heating to 40˚C for 10 or 30 min, but a slight reduction was observed when the heating was continued for 60 minutes. However, a 7.7% reduction in activity was observed on heating at 60˚C, whether for 10, 30 or 60 minutes, while heating to 100˚C or 121˚C resulted in complete loss of the activity regardless of the length of the heating period (Figure 2). Loss of activity after heat treatment at 121˚C for 15 min has been reported  .
Table 1. Carbohydrate utilization pattern by Lb. plantarum M4.
+ = utilized; − = not utilized.
Table 2. Effect of enzymes on activity of the CFS from Lb. plantarum M4 against Staph. aureus ATCC 43306 (Inhibition zone diameters in mm).
Figure 1. Effect of pH on inhibitory activity of CFS from Lb. plantarum M4 on Staph. aureus ATCC43306.
Figure 2. Effect of heating on activity of CFS from Lb. plantarum M4.
No drop in activity of the CFS was observed on storage at −80˚C or −20˚C up to four weeks. However, at 4˚C and 35˚C a gradual decline in activity was observed starting from the second week, and by the fourth week, the retained activities were 78.6% and 77% of the starting activities at 4˚C and 35˚C, respectively (Figure 3). Bacteriocins produced by L. plantarum F1 remained fully stable after storage for 60 days at −20˚C, but declined or became undetectable after storage for 80 to 120 days at 37˚C, indicating that cold temperature may be the most appropriate preservation technique  .
Figure 4 shows bands produced by the electrophoretic separation of the protein in the Lb. plantarum PM4 CFS in comparison to a marker of standard protein molecular weights. The molecular weight of the protein was in the range of 3 - 4 KDa. This is the same as bacteriocin ST414BZ (3.7 kDa) from Lb. plantarum ST414BZ  , plantaricin 35d (4.5 kDa) produced by Lb. plantarum 35d  , bacteriocins ST28MS and ST26MS with 5.5 and 2.8 kDa, respectively  and bacteriocin BM-1 of 4638.142 Da  . This is within the range of most bacteriocins reported for the genus Lactobacillus  .
Figure 3. Effect of storage at −80˚C, -20˚C, 4˚C and 35˚C for four weeks on activity of CFS from Lb. plantarum M4.
Figure 4. Gel electrophoresis (SDS-PAGE) of the inhibitory moiety of Lb. plantarum M4 stained with Coomassie blue.
3.3. Mode of Action
On addition of the CFS from Lb. plantarum PM4 to Staphylococcus aureus ATCC 43306, growth was completely arrested, with no increase in the optical density of the treated broth culture up to five hours from the time of addition, while the optical density of the untreated broth culture rose from 0.1 to 0.39 during those five hours (Figure 5). No growth was obtained on re-culturing the treated broth culture on fresh Nutrient Agar medium indicating that it has a bactericide effect on S. aureus. This is similar to the bacteriocidal mode of activity of bacteriocin AMA-K from Lb. plantarum AMA-K  and plantaricin 35d produced by Lb. plantarum 35d  .
3.4. Spectrum of Activity
Table 3 depicts the spectrum of activity of the CFS against eight bacterial strains.
Figure 5. Mode action of CFS from Lb. plantarum M4. Arrow indicates time of addition of CFS.
Table 3. Spectrum of inhibitory activity of CFS from Lb. plantarum M4 against eight target organisms (zone diameters, mm).
It was active against both Gram-positive and Gram-negative bacteria. This spectrum of activity is similar to that reported for other plantaricins. For instance, it was reported that the bacteriocin produced by Lb. plantarum was effective against both gram positive and gram negative bacteria  . Also bacteriocin C8 from Lb. plantarum was reported to have inhibitory activity against not only many Gram-positive but also Gram-negative bacteria such as Escherichia coli  . Lb. plantarum BM-1 isolated from a traditionally fermented Chinese meat was found to produce a novel bacteriocin that is active against a wide range of gram-positive and gram-negative bacteria  .
3.5. Kinetics of Growth and Bacteriocin Production
Bacteriocin production (measured as inhibitory activity (AU)) by Lb. plantarum PM4 generally coincided with the phase of maximum growth (Figure 6). The acivity was detected after just 5 h indicating that the bacteriocin is a primary metabolite. However, maximum activity was obtained between 10 and 26 h and maximal growth occurred at the 25 h from inoculation. Similarly, it was reported
Figure 6. Kinetics of growth and bacteriocin production by Lb. plantarum M4.
 that two bacteriocins from Lb. plantarum (ST28MS and ST26MS) showed detectable levels of inhibitory activity after 5 h suggesting that the peptide is a primary metabolite. Similar results were also reported for plantaricin Y  and bacteriocin ST13BR  . Both maximum growth and inhibitory activity were obtained at pH 4.4 - 4.2. Bacteriocin production is usually observed to be proportional to growth   .
3.6. Optimization of Bacteriocin Production
Optimization of bacteriocin production by Lb. plantarum PM4 was studied in MRS broth using temperature, pH and length of incubation period (time) as variables. At pH 5.0, production of the inhibitory activity in the CFS was low (around 50 AU/mL) whether the CFS was incubated at 25˚C or 30˚C for up to 72 h. However, at 37˚C, production surged to 100 AU/mL at the first 24 h but then declined to around 50 AU/mL (Figure 7(a)). Production at pH 5.5 was generally higher than at pH 5.0, being around 100 AU/mL at the first 24 h, and the surging to 200 AU/mL at 48 h, then receding (Figure 7(b)). At pH 6.0, incubation at 25˚C for 48 h resulted in 100 AU/mL while all other incubation conditions resulted in lower yield (Figure 7(c)). Accordingly, the best combination of incubation conditions for production of the inhibitory activity by Lb. plantarum M4 appears to be at pH 5.5 for 48 h whether incubated at 25˚C, 30˚C or 37˚C.
Maximum production of bacteriocin ST13BR by Lb. plantarum ST13BR was recorded at 30˚C and not at 37˚C  . However, optimum production of bacteriocin by Lb. plantarum F12 was reported at 37˚C  . Optimal bacteriocin production (12,800 AU/mL) from Lb. plantarum AMA-K was recorded in MRS broth with an initial pH of 6.0 and 5.5  . Also, maximum activity of bacteriocin ST26MS was recorded in MRS broth with an initial pH of 5.5  . It should be noted that the titers reported for CFS are usually thousands of times lower than those reported for purified bacteriocins  . The use of bacteria isolated from meat may contribute to a better sensory quality of the meat fermented products  .
Figure 7. Activity of CFS from Lb. plantarum M4 as affected by pH and incubation period at (a) 25˚C, (b) 30˚C and (c) 37˚C.
Results of this study indicated that Lb. plantarum PM4 isolated from fermented beef (shermout) exhibited promising antimicrobial activity against both Gram positive and Gram negative bacteria, and could be used as a starter culture in the processing of fermented meat as well as biopreservative. Acidification carried out by LAB such as Lb. plantarum and the production of bacteriocins contribute, in addition to good manufacturing practices, to the inhibition of food pathogens such as Salmonella, E. coli and S. aureus and can ensure safe and improved pro- duct quality.
Conflict of Interests
The authors declare no conflict of interests whatsoever.