Application of response surface methodology to improve the production of antimicrobial biosurfactants by Lactobacillus paracasei subsp. tolerans N2 using sugar cane molasses as substrate

Abstract

Background

Biosurfactants are natural surface-active compounds produced by a variety of microorganisms. The high cost of culture media limits the large-scale production and use of biosurfactants. It is therefore necessary to develop an efficient and cost-effective bioprocess to improve the yield of biosurfactants from microorganisms. In this study, the response surface method was used to optimize the production of biosurfactants by a Lactobacillus strain and the antimicrobial activity of the biosurfactants was assessed.

Results

The biosurfactant-producing strain was identified as Lactobacillus paracasei subsp. tolerans N2 after 16S rRNA gene analysis. Among the different variables studied using a Plackett–Burman statistical design, temperature and peptone and sugar cane molasses concentrations were found to be the main factors that had significant (p < 0.05) influence on biosurfactant production. The results of this study showed that molasses concentration at 59.5 g/L, peptone at 6.20 g/L and temperature of 33 °C were optimal conditions for biosurfactant production, with a maximum yield of 2.70 g/L. The biosurfactant exhibited surface tension reduction of 37.85 mN/m and antimicrobial activity expressed as inhibition diameter of 63 mm. Partial characterizations by elemental, biochemical and Fourier transmission infrared spectroscopy analysis of the biosurfactant produced revealed that it was glycolipoprotein in nature. The biosurfactant exhibited bactericidal activity against Pseudomonas aeruginosa PSB2, Pseudomonas putida PSJ1, Salmonella sp. SL2, Escherichia coli MTCC 118, Bacillus sp. BC1 and Staphylococcus aureus STP1 at concentrations ranging from 6.4 to 50 mg/mL.

Conclusion

The yield of biosurfactant was four-fold higher after optimization of media components and culture conditions using response surface methodology. The results of this study suggested that sugar cane molasses can be used as a low-cost substrate to enhance the yield of biosurfactants with antimicrobial activity.

Keywords

Lactobacillus paracasei subsp. tolerans N2 / Biosurfactants / Molasses / Optimization / Response surface methodology / Antimicrobial activity

References

[1] AACC (1999) Kjeldahl method AACC 46-12.01. 10–12. https://doi.org/10.1094/aaccintmethod-46-12.01

[2] Abalos A, Maximo F, Manresa MA, Bastida J. Utilization of response surface methodology to optimize the culture media for the production of rhamnolipids by Pseudomonas aeruginosa AT10. J Chem Technol Biotechnol, 2002, 77: 777-784.

[3] Banat IM, Makkar RS, Cameotra SS. Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol, 2000, 53: 495-508.

[4] Bas D, Boyaci IH. Modeling and optimization i: usability of response surface methodology. J Food Eng, 2007, 78: 836-845.

[5] Burkovski A. Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes. FEMS Microbiol Rev, 2003, 27: 617-628.

[6] Dehghan-Noude G, Housaindokht M, Bazzaz BSF. Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633. J Microbiol, 2005, 43: 272-276.

[7] Dellaglio F, Dicks L. Designation of ATCC 334 in place of ATCC 393 (NCDO 161) as the neotype strain of Lactobacillus casei subsp. casei and rejection of the name Lactobacillus paracasei (Collins et al., 1989). Int J Syst Bacteriol, 1991, 41: 340-342.

[8] Desai JD, Banat IM. Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev, 1997, 61: 47-64.

[9] Devesa-Rey R, Vecino X, Barral MT, Cruz JM, Moldes AB. Study of the sorption of biosurfactants from Lactobacillus Pentosus on sediments. Int Sch Sci Res Innov, 2011, 5: 403-407.

[10] Doelle MB, Doelle HW. Sugar-cane molasses fermentation by Zymomonas mobilis. Appl Microbiol Biotechnol, 1990, 33: 31-35.

[11] Dubois M, Gilles K, Hamilton J, . Colorimetric method for determination of sugars and related substances. Anal Chem, 1956, 28: 350-356.

[12] Fracchia L, Cavallo M, Allegrone G, Martinotti MG. Méndez-Vilas A. A Lactobacillus-derived biosurfactant inhibits biofilm formation of human pathogenic Candida albicans biofilm producers. Current research, technology and education topics in applied microbiology and microbial biotechnology, 2010, 2, Spain: FORMATEX, 827-837.

[13] Freitas de Oliveira DW, Lima França ÍW, Nogueira Félix AK, . Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice. Colloids Surfaces B Biointerfaces, 2013, 101: 34-43.

[14] Ghribi D, Ellouze-Chaabouni S. Enhancement of Bacillus subtilis lipopeptide biosurfactants production through optimization of medium composition and adequate control of aeration. Biotechnol Res Int, 2011, 2011: 1-6.

[15] Griffin HL, Greene RV, Cotta MA. Isolation and characterization of an alkaline protease from the marine shipworm bacterium. Curr Microbiol, 1992, 24: 111-117.

[16] Gudiña EJ, Rocha V, Teixeira JA, Rodrigues LR. Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20. Lett Appl Microbiol, 2010, 50: 419-424.

[17] Gudiña EJ, Teixeira JA, Rodrigues LR. Biosurfactant-producing Lactobacilli: screening, production profiles, and effect of medium composition. Appl Environ Soil Sci, 2011, 2011: 1-9.

[18] Gudiña EJ, Fernandes EC, Teixeira JA, Rodrigues LR. Antimicrobial and anti-adhesive activities of cell-bound biosurfactant from Lactobacillus agilis CCUG31450. RSC Adv, 2015, 5: 90960-90968.

[19] Guo XH, Kim JM, Nam HM, . Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties. Anaerobe, 2010, 16: 321-326.

[20] Hamzehlou P, Sepahy AA, Mehrabian S, Hosseini F. Production of vitamins B3, B6 and B9 by Lactobacillus isolated from traditional yogurt samples from 3 cities in Iran, winter 2016. Appl Food Biotechnol, 2018, 5(2): 107-120.

[21] Huang X, Peng K, Feng Y, . Separation and characterization of effective demulsifying substances from surface of Alcaligenes sp. S-XJ-1 and its application in water-in-kerosene emulsion. Bioresour Technol, 2013, 139: 257-264.

[22] Inès M, Dhouha G. Lipopeptide surfactants: production, recovery and pore forming capacity. Peptides, 2015, 71: 100-112.

[23] Kammoun R, Naili B, Bejar S. Application of a statistical design to the optimization of parameters and culture medium for α-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product). Bioresour Technol, 2008, 99: 5602-5609.

[24] Kumar S, Stecher G, Tamura K (2016) MEGA7 : molecular evolutionary genetics analysis version 7. 0 for bigger datasets. MBE Adv Access 1–11. https://doi.org/10.1093/molbev/msw054

[25] Li C, Bai J, Cai Z, Ouyang F. Optimization of a cultural medium for bacteriocin production by Lactococcus lactis using response surface methodology. J Biotechnol, 2002, 93: 27-34.

[26] Lobna AM, Ahmed ZAA. Identification and characterization of biosurfactants produced by Rodococcus equi and Bacillus methylotrophicus. J Biol chem Environ Sci, 2013, 8: 341-358.

[27] Makkar R, Cameotra S. An update on the use of unconventional substrates for biosurfactant production and their new applications. Appl Microbiol Biotechnol, 2002, 58: 428-434.

[28] Makkar RS, Cameotra SS, Banat IM. Advances in utilization of renewable substrates for biosurfactant production. AMB Express, 2011, 1: 5.

[29] Mbawala A, Mouafo HT, Kom RR. Antibacterial activity of Lactobacillus biosurfactants against Pseudomonas spp. isolated from fresh beef. Nov Int J Biotechnol Biosci, 2013, 2: 7-22.

[30] Morais IMC, Cordeiro AL, Teixeira GS, . Biological and physicochemical properties of biosurfactants produced by Lactobacillus jensenii—P 6A and Lactobacillus gasseri—P 65. Microb Cell Fact, 2017

[31] Mouafo TH, Mbawala A, Ndjouenkeu R. Effect of different carbon sources on biosurfactants’ production by three strains of Lactobacillus spp. Biomed Res Int, 2018, 2018: 15.

[32] Moussa TAA, Mohamed MS, Samak N. Production and characterization of di-rhamnolipid produced by Pseudomonas aeruginosa TMN. Brazilian J Chem Eng, 2014, 31: 867-880.

[33] Mulligan CN, Gibbs BF. Correlation of nitrogen-metabolism with biosurfactant production by Pseudomonas-aeruginosa. Appl Environ Microbiol, 1989, 55: 3016-3019.

[34] Najafi AR, Rahimpour MR, Jahanmiri AH, . Interactive optimization of biosurfactant production by Paenibacillus alvei ARN63 isolated from an Iranian oil well. Colloids Surf B Biointerfaces, 2011, 82: 33-39.

[35] Rodrigues L, Teixeira J, Oliveira R, Van Der Mei HC. Response surface optimization of the medium components for the production of biosurfactants by probiotic bacteria. Process Biochem, 2006, 41: 1-10.

[36] Rodrigues LR, Teixeira JA, Oliveira R. Low-cost fermentative medium for biosurfactant production by probiotic bacteria. Biochem Eng J, 2006, 32: 135-142.

[37] Saimmai A, Sobhon V, Maneerat S. Molasses as a whole medium for biosurfactants production by bacillus strains and their application. Appl Biochem Biotechnol, 2011, 165: 315-335.

[38] Saitou N, Nei M. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 1987, 4: 406-425.

[39] Salar RK, Purewal SS, Bhatti MS. Optimization of extraction conditions and enhancement of phenolic content and antioxidant activity of pearl millet fermented with Aspergillus awamori MTCC-548. Resour Technol, 2016, 2: 148-157.

[40] Sambanthamoorthy K, Feng X, Patel R, . Antimicrobial and antibiofilm potential of biosurfactants isolated from lactobacilli against multi-drug-resistant pathogens. BMC Microbiol, 2014, 14: 197.

[41] Satpute SK, Kulkarni GR, Banpurkar AG, Banat IM. Review biosurfactant/s from Lactobacilli species: properties, challenges and potential biomedical applications. J Basic Microbiol, 2016, 56: 1-19.

[42] Sayyad SA, Panda BP, Javed S, Ali M. Screening of nutrient parameters for lovastatin production by Monascus purpureus MTCC 369 under submerged fermentation using Plackett-Burman design. Res J Microbiol, 2007, 2: 601-605.

[43] Sharma D, Singh Saharan B. Simultaneous production of biosurfactants and bacteriocins by probiotic Lactobacillus casei MRTL3. Int J Microbiol, 2014

[44] Sharma D, Ansari MJ, Gupta S, . Structural characterization and antimicrobial activity of a biosurfactant obtained from Bacillus pumilus DSVP18 grown on potato peels. Jundishapur J Microbiol, 2015

[45] Song TS, Kim JY, Kim KH, . In vitro evaluation of probiotic lactic acid bacteria isolated from dairy and non-dairy environments. Food Sci Biotechnol, 2010, 19: 19-25.

[46] Telmo C, Lousada J, Moreira N. Proximate analysis, backwards stepwise regression between gross calorific value, ultimate and chemical analysis of wood. Bioresour Technol, 2010, 101: 3808-3815.

[47] Topisirovic L, Kojic M, Fira D, . Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation. Int J Food Microbiol, 2006, 112: 230-235.

[48] Vecino X, Barbosa-Pereira L, Devesa-Rey R, . Optimization of extraction conditions and fatty acid characterization of Lactobacillus pentosus cell-bound biosurfactant/bioemulsifier. J Sci Food Agric, 2015, 95: 313-320.

[49] Wei YH, Chou CL, Chang JS. Rhamnolipid production by indigenous Pseudomonas aeruginosa J4 originating from petrochemical wastewater. Biochem Eng J, 2005, 27: 146-154.

[50] Xu Q, Nakajima M, Liu Z, Shiina T. Biosurfactants for microbubble preparation and application. Int J Mol Sci, 2011, 12: 462-475.

[51] Zengin H, Baysal AH. Antibacterial and antioxidant activity of essential oil terpenes against pathogenic and spoilage-forming bacteria and cell structure-activity relationships evaluated by SEM microscopy. Molecules, 2014, 19: 17773-17798.

Funding

TWAS (IT)CSIR(2016)