Introduction

Microorganism produces numerous metabolites that have been determined to be either useful or harmful to human health¹. Among these, Lactobacilli are widespread lactic acid bacteria (LAB) and they are found in diverse sources including plants, dairy products, mucosal surface of the human body and other food products^2,3,4. The distinct characteristics of these species include gram-positive bacteria, thermophilic, homofermentative and non-spore-forming rods. In addition, they are also characterized by the formation of lactic acid during carbohydrate metabolism⁵. According to U.S. Food and Drug Administration (FDA), Lactobacillus is considered a “GRAS” status strain and hence can be utilized for various industrial purposes. In recent scenarios, LAB plays it vital role in pharma industries because the probiotic properties of these bacteria help to maintain gastrointestinal tract health by inhibiting penetration of pathogens and also help to promote a good immune system⁶.  

The genus Lactobacillus can resist highly acidic conditions and it also can fight against virulent pathogens. This genus also produces promising biological compounds including fatty free acids, organic acids, ammonia, hydrogen peroxide, biosurfactant and bacteriocins which are well known as low molecular weight antibacterial peptides⁷. Bacteriocins can inhibit gram-positive and gram-negative pathogens, they utilize their antimicrobial property by an intrusive targeted organism cell wall and inhibit cell wall biosynthesis which resulted in the death of the targeted organism. Food industries were effective in utilizing these kinds of bioactive compounds as bio preservative ingredients in the food system to prevent microbial pathogens. Additionally, these genera reveal better viability in acidic pH medium which effectively ferment milk during fermentation and is frequently utilized as starters/co-starters in the manufacturing of numerous dairy products⁸. A few studies also demonstrated the antimicrobial efficiency of Lactobacillus, a strain of Lactobacillus fermentum that has antibacterial activity against specific pathogenic organisms has been identified⁹. The antibacterial properties of Lactobacillus fermentum point to its potential application in starter cultures for conventionally fermented foods as a way to increase the cleanliness and safety of food products¹⁰.

Lactobacilli demonstrates a wide range of bioactive substances, this method to develop eco-friendly methods for enhancing human situations as outlined by the UN 2030 Agenda and its Sustainable Development Goals can make use of Lactobacilli and their by-products. By enhancing present methods for food production, safety, and preservation, lactobacilli and their bioactive compounds may play a significant role in ensuring that people all over the world have access to healthy food. Major health issues, such as sexual, reproductive, neonatal, and environmental disorders, can be addressed by bioactive compounds of Lactobacilli in mere future. Accordingly, the focus of this paper will be the isolation of Lactobacillus from dairy products and explore its bioactive activity compound for pharmaceutical application against infant diarrheal bacterial pathogens.

Materials and methods

Sampling and isolation of Lactobacillus strains

In the current investigation, diverse milk products including curd, yoghurt, and butter were gathered from the local market of the Thanjavur region, Tamil Nadu, India, for the isolation of Lactobacillus strains. For isolation, each of the collected samples underwent unique aseptic processing. Using pre-sterilized bacteriological saline water, one gramme of the sample was serially diluted before being spread out on MRS agar (HiMedia, Cat. No. M641) plates. Using the quadrant strike plate technique on freshly made MRS agar plates, morphologically separate colonies were purely grown after 48hrs of incubation at 37°C. The composition of the MRS agar is as follows: 10g proteose peptone, 10g HM peptone B, 5g yeast extract, 20g dextrose, 10g tween 80, 20g ammonium citrate, 5g sodium acetate, 0.1g magnesium sulphate, 0.05g manganese sulphate, 2g dipotassium hydrogen phosphate and 15g agar at final pH (at 25°C) 6.5 ± 0.2. A visual inspection for the distinctive colony morphology followed by the Gram’s staining process, the purity of the strains was determined¹¹. For later usage, all of the axenic strains were kept in lyophilized conditions.

Infant diarrheal bacterial pathogens for antimicrobial study

Vibrio cholerae, a harmful bacterium that causes newborn diarrhoea, was kindly donated by Thanjavur Medical College in Thanjavur, Tamil Nadu. For thorough antibacterial testing, this pathogenic strain was kept under lyophilized conditions. Tryptone soya broth (HiMedia, Cat. No. LQ508) was used to culture the pathogen and the OD of the culture was standardized at 10⁸ CFU/ml inoculum concentration during the antimicrobial study. The composition of the Tryptone soya broth was as follows: 17g Tryptone, 3g Soya peptone, 5g Sodium chloride, 2.5g dextrose (Glucose) and 2.5g Dipotassium hydrogen phosphate at final pH (at 25°C) 7.3±0.2. The isolated Lactobacillus strains were tested for their significant antibacterial efficacy against this indicator pathogen.

Screening of extracellular or intracellular antimicrobial compound production

In 30ml screw-cap tubes with a 10ml working volume of MRS broth, each axenic Lactobacillus strain was grown separately (HiMedia, Cat. No. M369). After 96hrs incubation, each cultured broth was spun down separately for 15min. at 3000 rpm to separate the cell pellet and supernatant. With an ultrasonicator, the cell pellet was suspended in 50ml of phosphate buffer at pH 7 and sonicated for 45 sec. at 20 KHz (Hielscher, USA) whereas the supernatant was immediately applied for the antimicrobial investigation. Further, the sonicated cell debris was removed utilizing the centrifugal conditions as indicated before. This study provides evidence for the presence of external antimicrobial molecules, intracellular antimicrobial compounds, or both in the test bacterial strains.

Antimicrobial assay

By measuring the isolated Lactobacillus strains’ percentage of antibacterial activity against the study’s indicator pathogen, Vibrio cholerae, bioactive compound synthesis was calculated. The pathogenic strain was cultivated in tryptic soy broth with the optical density of this broth culture adjusted to 0.1, which is standardized to 10⁸cfu/ml inoculum concentration as per McFarland turbidity 0.5 standards). Using a microtiter plate-based test, the antibacterial activity was evaluated¹². The test was carried out in 96-well polystyrene microtiter plates with flat bottoms and covers (Tarsons, India).

125ml of sterile, double-strength tryptone soy broth and 125ml of test sample solution were added to the column of the well plates. The effectiveness of each test sample well’s antimicrobial defences was evaluated separately. Additionally, a set of wells had no antimicrobial test samples added to them, serving as growth controls; a set of wells had known antibiotic Chloramphenicol (200mg/L) added to them, serving as a positive control; and a set of wells had neither an inoculum nor any antimicrobial test samples or antibiotic compounds added to them, serving as a negative control. The well plates were injected with 2.5 ml of prepared test pathogenic bacterium, except for the negative control wells. The well plate was incubated at 37°C for two days. After incubation, the evaluations were performed in triplicate and calculated with a microplate reader (Biotek Elx808, WI, USA) to measure the well broth’s absorbance at 600nm for each well. The following calculation was used to evaluate the percentage growth inhibition of the tested strain

Percentage growth inhibition = [(1 − (A_b/A_d)] × 100

A_b = Absorbance of well plates with test samples

A_d = Absorbance of growth control well.

Molecular identification of potential strain

The Lactobacillus strain used for the molecular identification investigation has the strongest antibacterial activity. The Roche Kit (Germany) was used to collect a 2ml bacterial culture at the mid-exponential growth phase, and DNA was extracted using this culture. A universal set of the Eubac primers includes the following: 1492R – 5′- TACG GYTA CCTT GTTA CGAC TT and 27F – 5′- AGAG TTTG ATCM TGGC TCAG -3′. Using a 5µl reaction mixer, the PCR was carried out on an Eppendorf thermal cycler with the 5µl of 10x amplification buffer, 5µl of 1.5 mM MgCl₂, 1µl of each forward and reverse primer, 1µl of dNTP as well as 0.25µl of Taq polymerase. During the amplification procedure, 35 cycles of 35s at 94°C, the 40s at 55°C, 2min at 72°C, and finally an 8-minute extension at 72°C were used. Further, 1.2% agarose gel was used for the electrophoresis analysis of the PCR results (Genei). Using a Qiagen PCR purification kit, the PCR product was purified before being sequenced with ABI Prism 377 automated sequencer (Applied Biosystems, CA, USA). The tree topologies of the potential strain were performed with bootstrap analyses using a thousand replicates, the evolutionary distances were evaluated based on the maximum neighbour-joining method¹³ as well as phylogenetic trees were inferred with the neighbour-joining technique. The evolutionary analysis was done with MEGA X software¹⁴.

Growth kinetics profile of the potential bacterium on antimicrobial compound production

The selected strain’s best period for producing the most bioactive compounds about the development of cell biomass at regular intervals of 12hrs from 0 to 144hrs was investigated. The standardisation procedure was carried out in a 1000ml conical flask using 400ml of MRS broth as the base fermentation medium and 37°C and pH 7 as the culture conditions. The inoculum was made utilising prospective strains in their exponential growth phase in the same basal fermentation medium conditions, and its OD620nm was standardized to 0.1 following McFarland turbidity standards of 0.5 and is equal to 1×10⁸cfu/ml of bacterial cell concentration.

A part of cultured broth was used to monitor the assessments, and the cell biomass and supernatant were splited with centrifugation at 3000rpm for 15min. The dry weight of cell biomass resulted from centrifugation that was hot air oven dried for 30min at 60% which was used to quantify bacterial cell growth, and the generation of bioactive compounds was directly estimated from the cell-free supernatant. Additionally, the microtiter plate-based test was used to calculate the bioactive compound’s activity against the indicator pathogen Vibrio cholerae as reported in the screening trials. The data were highlighted as mean ± standard deviation after triplicate evaluation.

Recapitulation of pH and temperature

Optimizing parameters like pH and temperature conditions play a vital role in fermentation progress for enhanced bioactive molecule production. The potential strain was standardized for maximum bioactive compound production by adopting search techniques varying one parameter at a time. The standardized parameters were fixed for the subsequent evaluations. The effect of different pH conditions between pH 6 to 10 was estimated for the effective medium optimization, similarly, various temperature conditions from 20 to 50°C were applied to the production medium for the evaluation of enhanced production.

Result and Discussion

Lactobacillus isolated from naturally fermented milk products has displayed numerous applications and it has a long history¹⁵.  In this study, different dairy products such as curd (CMP), yoghurt (YMP) and butter Milk (BMP) were used to isolate bacteria with their efficiency in bioactivity against infant diarrheal bacterial pathogens. During the isolation process through serial dilution on the MRS agar plate, 11 distinct bacterial isolates were isolated of which 4 were isolated from curd and the culture was initially labelled as CMP1, CMP2, CMP3 and CMP4. Followed by 4 isolates from yoghurt (YMP1, YMP2, YMP3 and YMP4) and 3 isolates from buttermilk including BMP1, BMP2 and BMP3. Using distinctive morphological features under visual inspection and the Gram’s staining process, the purity of the strains was determined and axenic strains were maintained. Further preliminary analysis for antimicrobial activity was tested against infant diarrheal bacterial pathogens. All the bacterial broth of 11 axenic bacterial strains were subjected to antimicrobial screening and this confirmed selected bacterial strains which produced extracellular or intracellular antimicrobial compounds (Table 1). Abdelgadir et al.¹⁶ isolated Lactobacillus fermentum from the surface of traditional fermented dairy products. Aroutcheva et al.¹⁷ and Park and Oh¹⁸ stated that these kinds of Lactobacilli strains were predominant in the human intestine and clinical studies on L. fermentum RC-14 proved that this strain shows a significant role in the microflora regulation in the human intestine¹⁹ and this reported to a dominant Lactobacilli in the vaginal tract and human intestine¹⁷.

Lactobacilli against various clinical pathogens are noted to be a promising role in the health-promoting properties²⁰. In this investigation, the antimicrobial activity results showed that the maximum inhibition activity of infant diarrheal bacterial pathogen was YMP3 with 67.8±2.1% inhibition activity and this observation was recorded at the extracellular site. Similarly, the extracellular bioactive compound of CMP2 showed 41.7±1.3% inhibition activity. CMP4 showed 32.3±0.9% inhibition activity and this was at the intracellular site (Table 1). Thus, the pure stock culture of YMP3 was maintained on agar (Fig. 1) and axenic strains broth was also maintained in strew cap bottles (Fig. 3). There are few studies which highlighted bioactive compounds of Lactobacilli bacteria towards its efficiency on antimicrobial activity. Owusu-Kwarteng et al.²¹ emphasized that bacteria isolated from fermented milk products were usually noted as safe because they can withstand low pH and they can produce antimicrobial substances. Mathara et al.²² reported the influence of Lactobacillus in the controlling proliferation of bacterial pathogens in fermented milk products. In addition, these bacteria are reported to produce a wide range of active compounds including lactic and acetic acid, hydrogen peroxide, bacteriocins, protein, fatty acids, phenyl lactic acid, retrocyclin and reuterin²³. In correlation to the present findings, fermented food products showed maximum antagonistic activity in L. plantarum strains toward clinical pathogens^{24, 25}. Few in vitro studies of antimicrobial activity against C. difficile, E. coli, Shigella spp., S. mutans, S. aureus and P. aeruginosa pathogen was also exhibited good results using Lactobacillus strains^{26, 27}.

Based on the screening of antimicrobial activity in the present study, one promising bacterial isolate YMP3 was selected for further identification. Identification was done by molecular profiling using the 16s rRNA gene sequencing method. The molecular characterization revealed that selected bacterial isolates were identified as Lactobacillus apis and they were named Lactobacillus apis YMP3. The phylogenetic tree of Lactobacillus apis YMP3 is illustrated using the neighbour–joining method (Fig. 3). The Neighbor-Joining approach was used to infer the evolutionary history¹³. In the bootstrap test (1000 repetitions), the proportion of duplicate trees that linked taxa clustered together is displayed next to the branches²⁸. The evolutionary distances were measured using the Maximum Composite Likelihood technique²⁹. Twelve nucleotide sequences were subject to this investigation. For each sequence pair, all unclear places were eliminated (pairwise deletion option). The final dataset contained 1464 locations altogether. In MEGA X, evolutionary analyses were performed¹⁴. Based on FAO and WHO guidelines, microbial identification through 16SrRNA can be recorded as a more appropriate method than other molecular characterization³⁰. According to Pogacic et al.³¹ and Tulini et al.³² 16S rRNA analysis proved as an effective method to identify bacteria which produce lactic and acetic acid; which was identified from fermented dairy products. Previous studies have supported the use of the current methodology as a practical, affordable, and appropriate method for separating Lactobacillus bacteria from conventional milk products.

In the present study, the growth kinetic profile of L. apis YMP3 was done against the growth inhibition percentage of the selected pathogen. The result elaborated that 72 to 108hrs of incubation time showed maximum growth of L. apis YMP3 and inhibition activity of selected pathogen. From the beginning of the lag phase’s incubation period until the completion of the decrease growth phase under investigation, evidence of both bacterial growth and pathogen inhibition activities was seen (Fig. 4). Nearly 60 to 70% of selected pathogen growth was inhibited at 72 to 108hrs. of incubation time. In another study, Arques et al.³³ showed that 72hr incubation, the number of Staphylococcus aureus falls to 0.46 log cfu/ml. The maximum amount of active compound production was seen after the late-log phase, according to growth and bioactive compound production profiles. During the stationary period, the level of production remained constant; Ivanova et al. ³⁴ obtained similar outcomes.

The influence of different pH ranges from 5 to 9 was tested against the growth inhibition percentage of the selected pathogen using a crude bioactive compound from L. apis YMP3. The result illustrates that pH 6.5 showed a maximum of 70% of inhibition of the selected pathogen and this was followed by pH 7 and 7.5. Based on the result, it is demonstrated that both alkaline and acidic pH doesn’t favour maximum inhibition of selected pathogen using crude bioactive compound from L. apis YMP3. Similarly, the effect of different temperatures ranging from 20 to 50°C was tested against the growth inhibition percentage of the selected pathogen using a crude bioactive compound from L. apis YMP3. At 35°C, 70% growth inhibition was observed as the maximum reduced growth. A notable observation was also recorded at 30 and 40°C and the least inhibition was recorded at 20°C. Increased and declined temperature doesn’t enhance the growth inhibition of selected pathogens. Vignolo et al.³⁵ demonstrated that a temperature of 25˚C favoured maximum bioactive compound lactation production by Lactobacillus casei. According to the study, particularly low temperatures were unfavourable for growth-enhanced production. The isolated Lactobacillus fermentum M1 demonstrated antimicrobial activity in a different investigation against E. coli ATCC 25922, which displayed the highest growth inhibitor percentage possible at a temperature of 60˚C. 

Table 1: Antimicrobial activities of axenic bacteria isolated from different milk products

Figure 1: Pure culture of potential antimicrobial compound producing L. apis YMP3 Click here to view figure

Figure 2: Pure cultured Lactobacillus strains cultured in 30ml screw cap tube containing MRS broth for the screening of antimicrobial compound   Click here to view figure

Figure 3: The evolutionary history of L. apis YMP3 using the Neighbor-Joining method   Click here to view figure

Figure 4: Growth kinetics profile as a function of time on bioactive compound production using L. apis YMP3   Click here to view figure

Figure 5: Effect of various pH on the bioactive compound production using L. apis YMP3   Click here to view figure

Figure 6: Effect of different temperatures on bioactive compound production using L. apis YMP3   Click here to view figure

Conclusion

This study hypothesises that probiotic lactic acid bacteria with notable bioactivities may be isolated from conventional dairy products. The study also suggests that the isolated bacterium, L. apis YMP3 exhibited excellent antimicrobial activity against the clinical pathogen, V. cholerae. Further, the potential strain evidenced a good response regarding the enhanced production of its antimicrobial compound from the growth optimization studies. Considering all together, this strain can be used for large-scale production which also enhances the possibilities for its purification and structural characterization of the effective antibacterial substance and extensive studies have to be achieved for drug development.

Acknowledgment

The authors very gratefully acknowledge the Department of Microbiology, Sengunthar Arts and Science College, Affliated to Periyar University, Tiruchengode – 637205, Tamil Nadu, India for providing lab facilities and supporting our research.

Conflict of Interest

The authors declare that they have no conflict of interest on publication of this article.

Funding Sources

There is no funding source.

References

1. Singh R., Kumar M., Mittal A., Mehta PK. Microbial metabolites in nutrition, healthcare and agriculture. 3 Biotech. 2017; May;7(1):15.
   CrossRef
2. Mokoena M.P. Lactic acid bacteria and their bacteriocins: classification, biosynthesis and applications against uropathogens: A Mini-Review. Molecules. 2017; Jul;22(8):1255.
   CrossRef
3. Colombo M., Castilho N.P.A., Todorov S.D. Beneficial properties of lactic acid bacteria are naturally present in dairy production.  BMC Microbiol2019; 18.
   CrossRef
4. Sánchez Óscar J., Barragán Pedro J., & Serna, Liliana. Review of Lactobacillus in the food industry and their culture media. Revista Colombiana de Biotecnología. 2019; 21(2):63-76.
   CrossRef
5. Bintsis T. Lactic acid bacteria as starter cultures: An update in their metabolism and genetics. AIMS Microbiol. 2018;665-684.
   CrossRef
6. van Zyl W.F., Deane S.M., Dicks L.M.T. Molecular insights into probiotic mechanisms of action employed against intestinal pathogenic bacteria. Gut Microbes. 2020;12(1):1831339.
   CrossRef
7. Vieco-Saiz N., Belguesmia Y., Raspoet R., Auclair E., Gancel F., Kempf I., Drider D. Benefits and inputs from lactic acid bacteria and their bacteriocins as alternatives to antibiotic growth promoters during food-animal production. Front Microbiol. 2019;10:57.
   CrossRef
8. Fenster K., Freeburg B., Hollard C., Wong C., Rønhave Laursen R. and Ouwehand A.C. The production and delivery of probiotics: A review of a practical approach. Microorganisms. 2019;7(3):83.
   CrossRef
9. Liévin-Le Moal V., Servin A.L. Anti-infective activities of Lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clin Microbiol Rev. 2014;27(2):167-99.
   CrossRef
10. Byakika S., Mukisa I.M., Byaruhanga Y.B., Muyanja C. Probiotic potential of lactic acid starter cultures isolated from a traditional fermented sorghum-millet beverage. Int J Microbiol. 2020;4:7825943.
    CrossRef
11. Harley J. Laboratory Exercises in Microbiology, 9th edition. McGraw-Hill College Division, Boston, 2013.
12. Rufino R.D., Luna J.M., Sarubbo L.A., Rodrigues L.R.M., Teixeira J.A.C. and Campos-Takaki G.M. Antimicrobial and anti-adhesive potential of a biosurfactant Rufisan produced by Candida lipolytica UCP 0988. Colloids and surfaces B: Biointerfaces. 2011;84(1):1-5.
    CrossRef
13. Saitou N. and Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 1987;4:406-425.
14. Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution.2018;35:1547-1549.
    CrossRef
15. Holzapfel W.H., Haberer P., Geisen R., Björkroth J. and Schillinger U. Taxonomy and important features of probiotic microorganisms in food and nutrition. The American journal of clinical nutrition. 2001;73(2):365s-373s.
    CrossRef
16. Abdelgadir W.S., Hamad S.H., Møller P.L. and Jakobsen M. Characterisation of the dominant microbiota of Sudanese fermented milk Rob. International Dairy Journal. 2001;11(1-2):63-70.
    CrossRef
17. Aroutcheva A.A., Simoes J.A. and Faro S. Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardnerella vaginalis. Infectious diseases in obstetrics and gynecology. 2001;9(1):33-39.
    CrossRef
18. Park K.B. and Oh S.H. Enhancement of γ-aminobutyric acid production in Chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis. Biotechnology letters. 2006;28(18):1459-1463.
    CrossRef
19. Gardiner G.E., Heinemann C., Baroja M.L., Bruce A.W., Beuerman D., Madrenas J. and Reid G. Oral administration of the probiotic combination Lactobacillus rhamnosus GR-1 and fermentum RC-14 for human intestinal applications. International Dairy Journal. 2001;12(2-3):191-196.
    CrossRef
20. Cizeikiene D., Juodeikiene G., Paskevicius A. and Bartkiene E. Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism isolated from food and their control in wheat bread. Food Control. 2013; 31(2), pp.539-545.
    CrossRef
21. Owusu-Kwarteng J., Akabanda F., Agyei D., Jespersen L. Microbial Safety of Milk Production and Fermented Dairy Products in Africa. Microorganisms. 2020;8(5):752.
    CrossRef
22. Mathara J. M., Schillinger U., Kutima P. M. Functional properties of Lactobacillus plantarum strains isolated from Maasai traditional fermented milk products in Kenya. Current Microbiology, 2008;56(4):315– 321.
    CrossRef
23. Giraffa G., Chanishvili N. and Widyastuti Y. Importance of lactobacilli in food and feed biotechnology. Research in Microbiology. 2010;161(6):480-487.
    CrossRef
24. Gupta R. and Srivastava S. Antifungal effect of antimicrobial peptides (AMPs LR14) derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage. Food Microbiology. 2014;42:1-7.
    CrossRef
25. Ryu E.H., Yang E.J., Woo E.R. and Chang H.C. Purification and characterization of antifungal compounds from Lactobacillus plantarum HD1 isolated from kimchi. Food Microbiology. 2014;41:19-26.
    CrossRef
26. Kumar A., Ghate V., Kim M.J., Zhou W., Khoo G.H. and Yuk H.G. Antibacterial efficacy of 405, 460 and 520 nm light emitting diodes on Lactobacillus plantarum, Staphylococcus aureus and Vibrio parahaemolyticus. Journal of applied microbiology, 2016;120(1):49-56.
    CrossRef
27. Ahn K.B., Baik J.E., Park O.J., Yun C.H. and Han S.H. Lactobacillus plantarum lipoteichoic acid inhibits biofilm formation of Streptococcus mutans. PLoS One. 2018;13(2):0192694.
    CrossRef
28. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;39:783-791.
    CrossRef
29. Tamura K., Nei M., and Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 2004;101:11030-11035.
    CrossRef
30. Ben Amor K., Vaughan E.E. and de Vos W.M. Advanced molecular tools for the identification of lactic acid bacteria. The Journal of nutrition. 2007;137(3):741S-747S.
    CrossRef
31. Pogacic T., Mancini A., Santarelli M., Bottari B., Lazzi C., Neviani E. and Gatti M. Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter. Food microbiology. 2013;36(2):207-215.
    CrossRef
32. Tulini F.L., Winkelstroter, L.K. and De Martinis, E.C. Identification and evaluation of the probiotic potential of Lactobacillus plantarum FT259, a bacteriocinogenic strain isolated from Brazilian semi-hard artisanal cheese. Anaerobe, 2013;22:57-63.
    CrossRef
33. Arques J.L., Rodriguez E., Gaya P., Medina M. and Nunez, M. Effect of combinations of high-pressure treatment and bacteriocin-producing lactic acid bacteria on the survival of Listeria monocytogenes in raw milk cheese. International dairy Journal. 2005;15(6-9):893-900.
    CrossRef
34. Ivanova I., Miteva V., Stefanova T.S., Pantev A., Budakov I., Danova S., Moncheva P., Nikolova I., Dousset X. and Boyaval P. Characterization of a bacteriocin produced by Streptococcus thermophilusInternational Journal of Food Microbiology, 1998;42(3):147-158.
    CrossRef
35. Vignolo G., Fadda S., De Kairuz M.N., de Ruiz Holgado A.P. and Oliver G. Control of Listeria monocytogenes in ground beef by ‘Lactocin 705’, a bacteriocin produced by Lactobacillus casei CRL 705. International Journal of Food Microbiology. 1996; 29(2-3):397-402.
    CrossRef