Research Article
 

Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products



Mohamed Abdel Hamid Rabie, Mohamed Gamal Moustafa, ElSayed Mohamed Abdel-Wahed, Khaled Bani Al-Harby and Khaled El-Zahar
 
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ABSTRACT

Background and Objective: The biopreservation of foods via bacteriocinogenic lactic acid bacteria (LAB) isolated directly from foods is an innovative approach. Bacteriocins derived from LAB isolates may show a promising antimicrobial activities and act as barriers against spoilage and/or pathogenic microorganisms in Egyptian dairy products. Materials and Methods: Eleven lactic acid bacteria (LAB) were isolated from raw buffalo milk, raw cow milk, yoghurt, raib, kareish cheese, domiati cheese and mish cheese. The isolated LAB were identified at the species level as Pediococcus acidilactici, Streptococcus thermophilus, Lactococcus lactis subsp cremoris (Lact. lactis), Lact. plantarum, Lact. lactis subsp. lactis, Enterococcus italicus (Entero. italicus), Entero. camelliae, Lactobacillus delbrueckii subsp lactis (Lb. delbrueckii), Lb. helveticus, Lb. buchneri and Lb. fermentum. The antimicrobial effects of these LAB species and three reference LAB strains were screened using an agar spot test and an agar well diffusion assay. Results: Crude bacteriocin supernatant fluid (CBSF) samples derived from these LAB species were active against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Listeria monocytogenes and Escherichia coli. The CBSFs derived from Lact. plantarum, Lb. delbrueckii subsp. lactis and Lb. fermentum were more effective against Staph. aureus. The CBSFs derived from species such as S. thermophilus, Lact. Plantarum Lb. delbrueckii subsp lactis and Lb. fermentum were effective against Listeria monocytogenes. In addition, CBSFs from Lact. lactis subsp. cremoris, Lb. helveticus and Lb. fermentum showed antimicrobial activity against E. coli and Salmonella enterica. The importance of major parameters (temperature, pH values and sensitivity to pepsin) for bacterial life and CBSF secretion was tested. The highest antimicrobial activity was observed at 30°C and a pH of 2.0-6.5 and pepsin inhibited the antimicrobial activity of the LAB by 90.9%. Conclusion: The antimicrobial activities of the bacteriocins produced by local LAB species acted as a barrier against spoilage and/or pathogenic microorganisms in dairy products.

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Mohamed Abdel Hamid Rabie, Mohamed Gamal Moustafa, ElSayed Mohamed Abdel-Wahed, Khaled Bani Al-Harby and Khaled El-Zahar, 2018. Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products. Research Journal of Microbiology, 13: 70-83.

URL: https://scialert.net/abstract/?doi=jm.2018.70.83
 
Received: December 06, 2017; Accepted: March 02, 2018; Published: June 04, 2018



INTRODUCTION

The growing need for naturally safe and healthy food has attracted increasing interest in the application of bacteriocin-producing lactic acid bacteria (LAB), which are currently used as protective cultures for the production of fermented foods and dairy products1,2. The LAB are used in food processing because they have the ability to improve the organoleptic characteristics of food.

In recent years, extensive study has been performed with bacteriocins and bacteriocin-producing LAB strains to investigate their potential use as bio-preservatives3. The LAB are generally recognized as safe (GRAS microorganisms) and play an important role in food and feed fermentation and preservation, either as natural microflora or as starter cultures that are added under controlled conditions. The preservative effects exerted by LAB are primarily attributable to the production of organic acids (such as lactic acid), which result in a lower pH4.

Chemically, LAB bacteriocins are ribosomal proteins that exert bactericidal effects, have high molecular weights and are rapidly digested by the proteinases in the human digestive tract. LAB bacteriocins also have potential practical applications in food preservation or the prevention and treatment of Gram-positive and Gram-negative bacterial infections5-7. Study of Djadouni and Kihal8 found that the LBbb 0141 strain of LAB produced an antimicrobial compound with a wide spectrum of action that inhibited the growth of ten Gram-positive and Gram-negative indicator strains. Certain LAB produce antibiotics that affect Gram-positive pathogenic bacteria and fungi9. The in vitro potential of nine bacteriocin-producing LAB strains isolated from vacuum-packaged, cold-smoked salmon (CSS) were tested for their possible use as bio-preservative cultures against Listeria monocytogenes by Tome et al.10.

Recently, bacteriocin-producing by LAB is likely used due to their safe GRAS status use as safe additives especially in food industry as bio-preservatives11. Bacteriocins are active biologically complex protein against other bacteria, either within the same species or across genera12,13. Many bacteriocins are produced by food-grade LAB, a phenomenon that offers food scientists the potential to direct or prevent the development of specific bacterial species in food. This option is particularly useful for preservation or food safety applications, but it also has implications for the development of desirable flora in fermented food. Thus, bacteriocins are used to confer a rudimentary form of innate immunity in foodstuffs, allowing processors to extend their control over food flora long after manufacturing13.

Nisin is the first studied bacteriocin that naturally produced by Lactococcus lactis for application in food technology. It has been applied as an additive to specific foods around the world14. Some food additives have been in use to realize the effectiveness of nisin vis various spoilage and pathogenic microorganisms such as L. monocytogenes and evaluate its potential application in several food products15,16. Although several bacteriocins from LAB such as pediocin have been characterized to date but they are not currently approved as antimicrobial food additives17.

The antimicrobial activity of LAB may be attributable to the production of a number of antimicrobial substances such as lactic acid, hydrogen peroxide, diacetyl and bacteriocins18. Enterocin E-760 exerts broad antimicrobial activity against Gram-positive and Gram-negative bacteria19. The antimicrobial activity of Microgard™, either individually or in combination with nisin, was investigated against a Listeria inoculant in liquid cheese whey (LCW). Microgard™ did not reduce the initial count of the Listeria inoculant during storage at 7, 12, 20 and 25°C and it showed a response similar to that of untreated whey15. Another study by De Freitas et al.16 evaluated the effectiveness of nisaplin, a commercial product in which nisin is the active component, for decreasing the staphylococcal population in refrigerated pizza dough. Two different nisaplin treatments were able to reduce the Staphylococcus sp. count (CFU g–1) in refrigerated pizza dough and the authors suggested that nisin is a promising alternative to traditional antibiotics for controlling the survival of pathogenic microorganisms, particularly Staphylococcus, in foods such as refrigerated pizza dough.

Fresh fruits and vegetables harbour various microorganisms, some of which are psychotropic. L. monocytogenes is a pathogenic bacterium that is capable of growing at refrigerator temperatures. Moreover, it is also tolerant to acidic pH values and to salt concentrations up to 10%20,21. Therefore, it is important to seek bio-preservatives that control both spoilage and pathogenic microorganisms, including L. monocytogenes. Although several studies have indicated the presence of LAB species exerting antagonistic activities that improve the quality and safety of meat and dairy products22,23, few reports have studied fresh produce24. From the mechanism point of view, the specific cell surface receptors can be recognized when the bacteriocin gain entry into the target and then kill the cell by forming ion-permeable channels in the cytoplasmic membrane by nonspecific degradation of cellular DNA, by inhibition of protein synthesis through the specific cleavage of 16S RNA or inducing cell lysis25,26. However, LAB contaminate milk obtained from animals that have been treated with antibiotics and thus carry populations of resistant bacteria27,28. The isolation and screening of microorganisms from natural sources represents the most powerful method of obtaining useful and genetically stable strains for industrially important products29.

The aim of this study was first to determine the compatibility among selected species of isolated and identified LAB from raw buffalo milk, raw cow milk, yoghurt, raib, kareish cheese, domiati cheese and mish cheese and to identify their antagonistic activities against six pathogenic organisms in an agar spot test and an agar well diffusion assay. The second aim was to gauge the activity of bacteriocin in response to different temperatures, pH values and proteolytic enzymes.

MATERIALS AND METHODS

Materials: The dairy products (raw milk, yoghurt, raib, domiati, kareish and mish cheese) under investigation were collected from departmental stores, supermarkets and farms between 2015 and 2016.

Microorganisms: A selection of 11 species was identified according to previously identified criteria using morphological and biochemical methods30,7. Eleven species of LAB isolated from dairy products (raw milk, yoghurt, raib, domiati, kareish and mish cheese) were incubated on MRS agar31 at 37°C for 24 h and on M17 agar (Biokar, Beau Vais, France)32 at 30°C for 24 h33,34. Eleven LAB species consisted of six Gram-positive strains, S. thermophilus, Lact. lactis subsp cremoris, Lact. plantarum, Lact. lactis subsp lactis, Entero. italicus and Entero. camelliae and five Gram-positive rods, Lb. delbrueckii subsp lactis, Lb. helveticus, Lb. buchneri and Lb. fermentum.

Bacterial strains: Experiments were performed to detect the antagonistic activity of six indicator organisms, specifically Staphylococcus aureus ATCC 43300, which was kindly provided by the Department of Microbiology, Faculty of Medicine, Alexandria University and clinical species of Candida albicans, which were provided by the Department of Microbiology and Natural Products, National Research Center (NRC), Cairo, Egypt. Listeria monocytogenes NRRL1020, E. coli and Klebsiella pneumoniae were kindly provided by the Department of Microbiology, Medical Research Institute (MRI), Alexandria University, Egypt. Salmonella enterica PT4 was obtained from the Department of Food Science and Technology, Agriculture University of Athens, Greece.

Antibacterial activity: All LAB species were maintained at 4°C in MRS or M17 broth. All bacterial cultures were sub-cultured at 15 days intervals. Prior to their use in experiments, LAB back cultures were sub-cultured in MRS or M17 broth. To detect antimicrobial activity, an agar spot test and an agar well diffusion assay were performed.

Surface diffusion: Overnight cultures of 11 LAB species and the 3 reference LAB strains to be tested were grown in MRS or M17 broth at 30°C/16 h. Culture aliquots (5 μL) were spotted onto the surfaces of agar plates (MRS and M17 broth with 1.2% agar) and incubated at 37°C/24 h to allow colonies to develop. The spots on each plate were overlaid with approximately 7 mL of soft nutrient agar, soft Baird Parker agar, soft Salmonella Shigella (SS) agar, soft Listeria-selective agar or soft yeast mold (YM) agar (0.75% agar) and inoculated with 650 μL of a 10–1 dilution (105 CFU mL–1) of an overnight culture of the relevant pathogenic organism.

The plates were incubated at 37°C/24 h33,34. Clear zones around the spots were measured and inhibition reactions were scored as positive if the width of a clear zone around a LAB colony was 5 mm or larger1.

Mass diffusion: Suspensions of pathogenic cells stored at -20°C were used to inoculate nutrient agar, Baird-Parker-agar, SS-agar, Listeria-selective-agar or YM-agar growth medium at a final concentration of 105 CFU mL–1 at 45°C and poured into petri dishes, then cooled at 4°C for 2 h. After the plates solidified, four wells, each 6 mm in diameter, were cut into the agar and the base of each well was sealed with a drop of melted sterile water agar (20 g L–1 water).

The ability of a probiotic to inhibit a pathogen is considered its most important health-promoting property35. Eleven Gram-positive bacteriocin-producing LAB species were isolated from milk products. In addition, three reference LAB strains were screened and tested for their ability to produce inhibitory substances against six pathogenic organisms in a diffusion agar assay. The LAB species and reference strains were grown individually in MRS or M17 broth at 37°C/18 h. Screens were performed under anaerobic conditions using melted paraffin to avoid H2O2 formation36. Extracts of lactic cultures obtained from overnight cultures underwent one of the following treatments: (1) Centrifuged at 3000 rpm for 15 min, (2) Neutralized by adding 5 N NaOH to exclude the effects of organic acids and (3) Adjusted to pH 6.5 with 1 N NaOH and the possible inhibitory actions of H2O2 were eliminated by adding sterile catalase solution (1 mg mL–1) at 25°C/30 min. Each solution was sterilized using a millipore membrane filter (0.45 μm pore diameter) before it was loaded into wells. These extracts were designated crude bacteriocin supernatant fluid (CBSF).

Fifty microliters aliquots of CBSF from each tested LAB species and 3 reference LAB strains were added to each well in seeded plates. The plates were then incubated at 37°C/24 h. After the incubation period, clear inhibition zones appearing around each well were measured in mm. An arbitrary unit (AU) of CBSF per μL was calculated by dividing the volume of the clear zone by the volume of the well37.

Sensitivity of bacteriocins to heat treatment: The effects of temperature on bacteriocin activity for the selected microbes were determined as described previously38. Ten millilitres of CBSF from each test species (11) and the reference strains (three strains) were subjected to heat treatment in a water bath at 60°C/60, 100°C/20 and 121°C/15 min (autoclaving). CBSF antimicrobial activity against pathogenic microorganisms was then tested by using an agar well diffusion assay39.

Activity of bacteriocins at different pH values: Bacteriocin stability was tested at different pH values by adjusting the pH of the CBSF of 11 LAB and three reference strains to pH 2.0, 6.5 and 9.0 with 1 N HCL and 10 N NaOH. An agar well diffusion assay was performed to evaluate residual activity40,41.

Proteolytic enzyme inactivation: The proteolytic enzyme sensitivities of the inhibitory substances produced by 11 LAB species were tested individually by adding pepsin (Merck 7189) to the cells. Free culture supernatant at 37°C/60 min and residual supernatant activity were determined using an agar well diffusion assay as described previously by Bromberg et al.42. All measurements were performed in triplicate and average values are reported.

Statistical analysis: A one-way analysis of variance was performed to analyse the data using the General Linear Model procedure in SAS (Statistical Analysis System) 1998 (SAS Inst. Inc. Cary, NC)43. A previously described method by Duncan et al.44 was used to compare the least significant difference (LSD) of the treatments at a p-value of <0.05. Mean data values were calculated in triplicate as the mean±standard error of the mean (SEM).

RESULTS AND DISCUSSION

Antibacterial activity of LAB: The inhibition diameter was calculated for each LAB species as the average of the inhibition diameter against every pathogenic organism, as shown in Table 1. Eleven LAB species were isolated from milk products, the three LAB used as reference strains showed antimicrobial activity against one or more test microorganisms according to the agar spot test. The Gram-positive bacterium Staph. aureus was quite sensitive to bacteriocins produced by different LAB strains compared with that produced by C. albicans, which exhibited no effect. The Gram-negative bacteria L. monocytogenes, E. coli and S. enterica showed considerable sensitivity. The obtained results were consistent with studies reporting on bacteriocin-producing LAB isolated from Munster cheese45.

In Table 2, some LAB species demonstrated clear inhibitory activity against the tested pathogenic organisms. Lact. plantarum, Lb. delbrueckii subsp. lactis, Lb. fermentum, S. thermophilus, Lact. lactis subsp. lactis, Entero. camelliae and S. thermophilus TH4 showed high activity against Staph. aureus (14.69, 14.69, 14.69, 13.44, 13.44, 13.44 and 13.44 AU, respectively). However, there was less inhibition demonstrated for E. coli, S. thermophilus, Lact. plantarum, Lb. delbrueckii subsp. lactis, Lb. fermentum and S. thermophilus (7.11, 6.25, 6.25, 4.69, 7.11, 5.44 and 4.69 AU, respectively). In addition, S. thermophilus TH4 demonstrated inhibition activity against L. monocytogenes (10.02, 10.02, 10.02, 10.02, 10.02 and 10.02 AU) as well as against K. pneumoniae, as shown in Table 2. Similar antimicrobial activity results were reported previously by Schillinger and Lucke1, Carrasco et al.46, Nespolo and Brandelli47 and Kivanc et al.48. The results of this study showed that the antimicrobial activities of the bacteriocins derived from LAB species were generally greater against the tested Gram-positive bacteria than Gram-negative organisms according to the results obtained from the agar spot and agar well diffusion tests.

According to results from the agar spot test and agar well diffusion assay, all LAB species tested showed no effects against C. albicans and K. pneumoniae, while K. pneumoniae was sensitive to Bifidobacteriun bifidum (B. bifidum) (Bb12). Furthermore, there were no significant differences at p<0.05 between both methods in these presented results. In addition, the three reference strains, S. thermophilus TH4, Lb. acidophilus (CYX11-1) and B. bifidum (Bb12), gave the highest inhibition diameters and AU values compared with other tested species, as indicated in Table 2. By contrast, results reported previously by Fricourt et al.49 indicated that Lb. plantarum showed inhibitory activity in an agar well diffusion test, while another study by Kivanc50 reported that Lb. plantarum showed no inhibitory effects against Staph. aureus. Hence, there was no difference in performance between the two methods we adopted in this study.

Table 1:
Lactic acid bacteria (LAB) species isolated from different milk products and reference strains that demonstrated positive inhibition against six pathogenic organisms (strains) in an agar spot test
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products
+: Large inhibition zone (>1.0 mm), (+): Small inhibition zone (0.5-1.0 mm), Ka: Kareish cheese

Table 2:
Antagonistic activities of the identified LAB isolated from different dairy products that showed inhibition zones against six pathogenic organisms in a well diffusion assay
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products
The results are presented as the mean value±SEM*: Inhibition zone around the well, abcValues expressed that were different in the treatment are significantly different at p<0.05, AU: Arbitrary unit, 0: Not active, <5 mm: No inhibition, 5-10 mm: Low inhibition, 11-20 mm: Moderate inhibition, 21-30 mm: High inhibition

Effects of different heat treatments and pH values on bacteriocin activity: As presented in Table 3, the CBSFs produced by P. acidilactici, S. thermophilus, Lact. plantarum, Lact. lactis subsp. lactis, Lb. delbrueckii subsp. lactis, Lb. helveticus, L. buchneri, Lb. fermentum, S. thermophilus TH4, Lb. acidophilus (CYX11-1) and Bif. bifidum (Bb12) demonstrated similar heat stability levels under different heat treatments (60°C/60, 100°C/20 and 120°C/15 min) against L. monocytogenes (8.02, 10.02, 10.02, 9.0, 10.02, 9.0, 8.02, 10.02, 10.02, 8.02 and 6.25 AU, respectively). After heating the samples at 60°C/60, 100°C/20 and 121°C/15min, a loss in heat stability was observed based on the activity against Staph. aureus presented in Table 3. The CBSFs produced by P. acidilactici, S. thermophilus, Lact. plantarum, Lact. lactis subsp lactis, Lb. delbrueckii subsp lactis, Lb. buchneri, Lb. fermentum and S. thermophilus TH4 showed stability at different heat treatments against E. coli (7.11, 4.69, 7.11, 7.11, 6.25, 5.44, 7.11, 6.25 and 4.69 AU, respectively) but were not heat stable under different heat treatments against Staph. aureus. As shown in Table 3, the antimicrobial substances produced by Lact. lactis subsp lactis and Lb. buchneri demonstrated similar heat stability levels at different heat treatments against Staph. aureus (13.44 and 9.0 AU, respectively), while Lact. plantarum, Lact. lactis subsp lactis, L. helveticus and Lb. fermentum were heat stable after treatment at 60°C/60, 100°C/20 and 121°C/15 min in terms of activity against Salmonella enterica (4.69, 5.44, 7.11 and 8.02 AU, respectively). The bacteriocin produced by Lb. acidophilus (CYX11-1) was stable at 60, 100 and 121°C against both K. pneumoniae (4 AU) and L. monocytogenes (8.0 AU). This bacteriocin demonstrated no heat stability against E. coli, but its antimicrobial activity was eliminated after being autoclaved at 121°C for 15 min. The heat stability of bacteriocins produced by different LAB at 121°C for 15 min was reported earlier for Lactocin RN 78 51 and L. brevis OGI52, these results were similar to this study finding showing the loss of activity of antibacterial substances produced by Lactobacillus sp. after heat treatment at 121°C for different amounts of time. Moreover, Fatima and Mebrouk53 reported on the effects of heat and pH. The bacteriocins from two LAB strains were considered to be extremely heat stable because their antibacterial activity was not altered by heat treatment after 15 min at 121°C. With respect to temperature stability, the examined bacteriocins exhibited strong heat stability, placing them within the heat stable and low-molecular-weight group of bacteriocins. The antimicrobial activity of the extracted bacteriocin was destroyed by treatment with proteinase K because there was no inhibition zone compared with that produced by the untreated bacteriocin sample (control). Similar results were also reported by other studies Todorov et al.40, Corsetti et al.54, Hernandez et al.55, Rattanachaikunsopon and Phumkhachorn56. Furthermore, the stability of bacteriocin in response to high temperatures was reported previously by Klaenhammer57, the study identified the following four distinct classes o f LAB bacteriocins based on biochemical and genetic characterization: Lantibiotics (Class I), small, heat-stable, non-lanthionine peptides (Class II), large heat-labile proteins (Class III) and complex bacteriocins containing chemical moieties such as lipids and carbohydrates (Class IV).

Table 4 presents the pH stability of CBSFs studied over a ranged from 2.0-9.0. The results showed that the antimicrobial activity of the supernatants were highest at pH 2.0 against only Staph. aureus, L. monocytogenes, E. coli and Salmonella enterica. All examined CBSFs were completely inactive above pH 9.0. This finding was also previously reported by another study Todorov et al.40, in which various physicochemical factors appeared to affect bacteriocin production as well as activity. The data presented in Table 4 show that inhibitory activity was decreased against Staph. aureus, L. monocytogenes and E. coli only at pH 6.5. By contrast, maximum activity was observed at pH 2.0 and 6.5 and at 30°C, as shown in Table 3 and 4. These results were consistent with other studies Thanh et al.41, Parada et al.58, Cheikhyoussef et al.59,60. The resulting bacteriocin was stable over a wide pH range, which is a common feature of many bacteriocins51,53-57. Bacteriocin characterization showed that these molecules are active over certain temperature and pH ranges58. Present results were also confirmed by a previous study Fatima and Mebrouk53 that examined pH stability over a range from pH 2-12. These bacteriocins were active at pH values ranging from 2-6.0 but were reduced at higher pH values, as shown in Table 4. Thus, the observed wide range of pH tolerance indicates that these bacteriocins may be useful in acidic as well as non-acidic foods.

Sensitivity to a proteolytic enzyme: The pepsin sensitivities of the CBSFs produced by the eleven examined LAB species are presented in Table 5. In this study, most of the compounds were fully inactivated by the proteolytic enzyme, indicating their protein-based, aqueous nature. These species were completely inactivated by pepsin, particularly E. italicus, which is generally resistant to enzyme treatment. Similar results were reported previously by Bromberg et al.42, the study found that pepsin inhibited the antagonistic activity of 90% of antibacterial substances produced by LAB strains isolated from meat and meat products. Table 5 shows that pepsin inhibited the antagonistic activity of 91.91% of LAB.

Table 3:
Antagonistic activities of CBSFs secreted by LAB under different heat treatments against tested microorganisms in a well diffusion assay
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products
The results are presented as the mean value±SE. AbcValues expressed with different treatments are significantly different at p<0.05, *: Inhibition zone around the well, AU: Arbitrary unit, 0: Not active, 5 mm: No inhibition, 5-10 mm: Low inhibition, 11- 20 mm: Moderate inhibition

Table 4:
Antimicrobial activities (clear zones) of CBSFs derived from LAB grown at different pH values against the tested microorganisms in a well diffusion assay
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products
The results are presented as the mean value±SE. AbcValues expressed with different treatments are significantly different at p<0.05, *: Inhibition zone around the well, AU: Arbitrary unit, 0: Not active, 5 mm: No inhibition, 5-10 mm: Low inhibition, 11- 20 mm: Moderate inhibition

Table 5:
Sensitivities of inhibitory substances produced by 11 LAB species and 3 reference strains to treatment with a proteolytic enzyme
Image for - Bacteriocin-like Substances Produced by Specific Strains of Lactic Acid Bacteria Isolated from Milk Products

Consistent with these findings, pepsin sensitivity has been demonstrated for other bacteriocins, specifically plantaricin 35D and sakacin A61 and enterocin 416 KI1. Present results strongly suggest that the inhibitory activities of CBSFs derived from LAB species are attributable to a protein-based aqueous compound. Insignificant results at p<0.05 were obtained when comparing the agar spot and agar well diffusion methods when bacteriocin was applied to C. albicans, as shown in Table 5. In contrast to Table 5, bacteriocins were more effective against pathogenic microorganisms, some bacteriocins produced by LAB exhibited higher antimicrobial activity than the antibacterial activities of certain antibiotics, as presented in Table 5. Typically, nisin and lacticin 3147 are effective agents for treating mastitis14 and nisin is considered effective for the treatment of stomach ulcers caused by Helicobacter pylori 62. Thus, present results suggest that the bacteriocins produced by certain local LAB demonstrate potential as food bio-preservatives and probiotics that are alternatives to the usually prescribed antibiotics.

CONCLUSION

In study, 11 LAB species were isolated from different sources, namely, raw buffalo milk, raw cow milk, yoghurt, raib, kareish cheese, domiati cheese and mish cheese. The only promising CBSFs identified were derived from Lact. plantarum, Lb. delbrueckii subsp lactis and Lb. fermentum and were effective against Staph. aureus. Lact. lactis subsp cremoris, Lb. helveticus and Lb. fermentum showed the highest antimicrobial activity at 30°C, pH 2.0-6.5 and pepsin inhibited the antimicrobial activity of LAB by 90.9%. Accordingly, the bacteriocins derived from LAB isolates showed promising antimicrobial activities and may act as barriers against spoilage and/or pathogenic microorganisms in dairy products. In vitro studies are needed to determine the mechanism(s) involved in mitigating the antagonistic activities and bacteriocins produced by promising isolates.

SIGNIFICANCE STATEMENT

This study discovers the antimicrobial activity of 11LAB isolates and Lact. lactis subsp cremoris, Lb. helveticus and Lb. fermentum showed the highest antimicrobial activity up to 90.9% in Egypt milk product. This study will help the researchers to uncover the antimicrobial activity of isolates that many researchers were not able to explore. Thus, the new theory on antioxidant activity of isolates in preservation of food milk and milk products may be arrived at.

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