Characterization of Candidate probionts isolated from human breast milk

S. Khalkhali; N. Mojgani

doi:10.14715/cmb/2017.63.5.15

Issue

Vol. 63 No. 5: Issue 5

Issue Published : July 9, 2017

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Characterization of Candidate probionts isolated from human breast milk

https://doi.org/10.14715/cmb/2017.63.5.15

S. Khalkhali

Department of Microbiology, Shiraz Branch, Islamic Azad University, Shiraz, Iran

N. Mojgani

Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Corresponding Author(s) : N. Mojgani

skh_msc@hotmail.com

Cellular and Molecular Biology, Vol. 63 No. 5: Issue 5
Article Published : May 20, 2017

Abstract

This study was designed to isolate and identify the potential probionts present in 32 healthy mothers' breast milk. Microbial culture media and 16SrRNA sequencing were used to isolate and identify the bacteria and all isolates were analyzed for their antagonistic potential, resistance to acidic pH, bile salts and survival under simulated gastric and intestinal conditions. The colonization potential was further assessed based on adherence to human enterocyte-like Caco-2 cell lines. The breast milk samples harbored significant numbers of Gram positive and catalase negative (85%) bacteria. Based on 16SrRNA sequencing, these isolates were identified as Lactobacillus casei, L.gasseri, L.fermentum, L.plantarum, Pediococcus acidilactici, and Enterococcus facieum. Among the isolates, P. acidilactici was the most frequent species (71%) present in these samples. Few Gram and catalase positive isolates, Staphylococcus aureus and S.hominiis were also observed. The isolates were viable and unviable in pH 3 and 1.5, respectively, while all isolates survived in 1.0% bile salt. As putative probionts, P.acidilactici 1C showed a significantly higher percentage of adhesion to Caco-2 cells (p< 0.05)than the other two isolates L.plantarum 7A and E.facieum 2C. Bacterial strains isolated from human breast milk were shown to have probiotic properties including anti-infective protection and may be considered as future therapeutics for infants.

Keywords

Probiotic 16SrRNA sequencing Caco-2 cell adhesion Breast milk.

Khalkhali, S., & Mojgani, N. (2017). Characterization of Candidate probionts isolated from human breast milk. Cellular and Molecular Biology, 63(5), 82–88. https://doi.org/10.14715/cmb/2017.63.5.15

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References

Albesharat R, Ehrmann MA, Korakli M, Yazaji S, Vogel RF. Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies. Systemc Applied Microbiol. 2011,34(2):148-55.
Martín R, Langa S, Reviriego C, Jimínez E, Marín ML, Xaus J, Fernández L, Rodríguez JM. Human milk is a source of lactic acid bacteria for the infant gut. J Pediat. 2003,143(6):754-8.
Heikkila MP, Saris PE. Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. J Appl Microbiol. 2003,95(3):471-8.
Makino H, Kushiro A, Ishikawa E, Muylaert D, Kubota H, Sakai T, Oishi K, Martin R, Ben Amor K, Oozeer R, Knol J, Tanaka R. Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant, determined by multilocus sequencing typing and amplified fragment length polymorphism. Appl Environ Microbiol. 2011,77(19):6788-93.
Cunningham-Rundles S, Ahrne S, Johann-Liang R, Abuav R, Dunn-Navarra AM, Grassey C, Bengmark S, Cervia JS. Effect of probiotic bacteria on microbial host defense, growth, and immune function in human immunodeficiency virus type-1 infection. Nutrients. 2011,3(12):1042-70.
Fernandez L, Langa S, Martin V, Maldonado A, Jimenez E, Martin R, Rodriguez JM. The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res. 2013,69(1):1-10.
Aslim B, Yuksekdag ZN, Sarikaya E, Beyatli Y. Determination of the bacteriocin-like substances produced by some lactic acid bacteria isolated from Turkish dairy products. Lwt-Food Sci Technol. 2005,38(6):691-4.
Mojgani N, Hussaini F, Vaseji N. Characterization of indigenous lactobacillus strains for probiotic properties. Jundishapur J Microbiol. 2015,8(2):e17523.
Sahadeva R, Leong S, Chua K, Tan C, Chan H, Tong E, Wong S, Chan H. Survival of commercial probiotic strains to pH and bile. Int Food Res J. 2011,18(4):1515-22.
Walker DK, Gilliland SE. Relationships among Bile Tolerance, Bile-Salt Deconjugation, and Assimilation of Cholesterol by Lactobacillus-Acidophilus. J Dair Sci. 1993,76(4):956-61.
Beumer RR, de Vries J, Rombouts FM. Campylobacter jejuni non-culturable coccoid cells. Int J Food Microbiol. 1992,15(1-2):153-63.
Martín V, Maldonado A, Fernández L, Rodríguez JM, Connor RI. Inhibition of human immunodeficiency virus type 1 by lactic acid bacteria from human breastmilk. Breastfeed Med. 2010,5(4):153-8.
Zacarías M, Binetti A, Laco M, Reinheimer J, Vinderola G. Preliminary technological and potential probiotic characterisation of bifidobacteria isolated from breast milk for use in dairy products. Int Dair J. 2011,21(8):548-55.
Gonzalez R, Maldonado A, Martin V, Mandomando I, Fumado V, Metzner KJ, Sacoor C, Fernandez L, Macete E, Alonso PL, Rodriguez JM, Menendez C. Breast milk and gut microbiota in African mothers and infants from an area of high HIV prevalence. PloS one. 2013,8(11):e80299.
Delgado S, Arroyo R, Jimenez E, Marin ML, del Campo R, Fernandez L, Rodriguez JM. Staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics. BMC Microbiol. 2009,9(1):82.
Sung C, Kim BG, Kim S, Joo HS, Kim PI. Probiotic potential of Staphylococcus hominis MBBL 2-9 as anti-Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman. J Appl Microbiol. 2010,108(3):908-16.
Bendali F, Hebraud M, Sadoun D. Anti-bacterial and anti-adherence activities of a probiotic strain of Lactobacillus paracasei against Listeria monocytogenes. Int J Appl Microbiol Biotech Res. 2014,2:52-63.
Coconnier M-H, Lievin V, Hemery E, Servin AL. Antagonistic activity againstHelicobacter infection in vitro and in vivo by the humanLactobacillus acidophilus strain LB. Appl Environ Microbiol. 1998,64(11):4573-80.
Hudault S, Lievin V, Bernet-Camard MF, Servin AL. Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection. Appl Environ Microbiol. 1997,63(2):513-8.
Holzapfel W. Use of starter cultures in fermentation on a household scale. Food control. 1997,8(5-6):241-58.
Jung SP, Lee KM, Kang JH, Yun SI, Park HO, Moon Y, Kim JY. Effect of Lactobacillus gasseri BNR17 on Overweight and Obese Adults: A Randomized, Double-Blind Clinical Trial. Korean J Fam Med. 2013,34(2):80-9.
Huang Y, Adams MC. In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Int J Food Microbiol. 2004,91(3):253-60.
Mandal S, Puniya AK, Singh K. Effect of alginate concentrations on survival of microencapsulated Lactobacillus casei NCDC-298. Int Dair J. 2006,16(10):1190-5.
Liong MT, Shah NP. Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. J Dair Sci. 2005,88(1):55-66.
Prasad J, Gill H, Smart J, Gopal PK. Selection and characterisation of Lactobacillus and Bifidobacterium strains for use as probiotics. Int Dair J. 1998,8(12):993-1002.
Marteau P. Safety aspects of probiotic products. Scand J Nutr. 2001,45:22-4.
Morelli L. In vitro selection of probiotic lactobacilli: a critical appraisal. Curr Issues Intest Microbiol. 2000,1(2):59-67.
Marteau P, Vesa T, Rambaud J. Lactose maldigestion. Probiotics 2: Springer; 1997. p. 65-88.
Lin WH, Hwang CF, Chen LW, Tsen HY. Viable counts, characteristic evaluation for commercial lactic acid bacteria products. Food Microbiol. 2006,23(1):74-81.
Jacobsen CN, Nielsen VR, Hayford AE, Moller PL, Michaelsen KF, Paerregaard A, Sandstrom B, Tvede M, Jakobsen M. Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl Environ Microbiol. 1999,65(11):4949-56.
Tuomola EM, Salminen SJ. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int J Food Microbiol. 1998,41(1):45-51.
Kim EY, Rafiquzzaman SM, Lee JM, Noh G, Jo GA, Lee JH, Kong IS. Structural features of glycoprotein purified from Saccharina japonica and its effects on the selected probiotic properties of Lactobacillus plantarum in Caco-2 cell. J Appli Pycol. 2015,27(2):965-73.

References

Albesharat R, Ehrmann MA, Korakli M, Yazaji S, Vogel RF. Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies. Systemc Applied Microbiol. 2011,34(2):148-55.

Martín R, Langa S, Reviriego C, Jimínez E, Marín ML, Xaus J, Fernández L, Rodríguez JM. Human milk is a source of lactic acid bacteria for the infant gut. J Pediat. 2003,143(6):754-8.

Heikkila MP, Saris PE. Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. J Appl Microbiol. 2003,95(3):471-8.

Makino H, Kushiro A, Ishikawa E, Muylaert D, Kubota H, Sakai T, Oishi K, Martin R, Ben Amor K, Oozeer R, Knol J, Tanaka R. Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant, determined by multilocus sequencing typing and amplified fragment length polymorphism. Appl Environ Microbiol. 2011,77(19):6788-93.

Cunningham-Rundles S, Ahrne S, Johann-Liang R, Abuav R, Dunn-Navarra AM, Grassey C, Bengmark S, Cervia JS. Effect of probiotic bacteria on microbial host defense, growth, and immune function in human immunodeficiency virus type-1 infection. Nutrients. 2011,3(12):1042-70.

Fernandez L, Langa S, Martin V, Maldonado A, Jimenez E, Martin R, Rodriguez JM. The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res. 2013,69(1):1-10.

Aslim B, Yuksekdag ZN, Sarikaya E, Beyatli Y. Determination of the bacteriocin-like substances produced by some lactic acid bacteria isolated from Turkish dairy products. Lwt-Food Sci Technol. 2005,38(6):691-4.

Mojgani N, Hussaini F, Vaseji N. Characterization of indigenous lactobacillus strains for probiotic properties. Jundishapur J Microbiol. 2015,8(2):e17523.

Sahadeva R, Leong S, Chua K, Tan C, Chan H, Tong E, Wong S, Chan H. Survival of commercial probiotic strains to pH and bile. Int Food Res J. 2011,18(4):1515-22.

Walker DK, Gilliland SE. Relationships among Bile Tolerance, Bile-Salt Deconjugation, and Assimilation of Cholesterol by Lactobacillus-Acidophilus. J Dair Sci. 1993,76(4):956-61.

Beumer RR, de Vries J, Rombouts FM. Campylobacter jejuni non-culturable coccoid cells. Int J Food Microbiol. 1992,15(1-2):153-63.

Martín V, Maldonado A, Fernández L, Rodríguez JM, Connor RI. Inhibition of human immunodeficiency virus type 1 by lactic acid bacteria from human breastmilk. Breastfeed Med. 2010,5(4):153-8.

Zacarías M, Binetti A, Laco M, Reinheimer J, Vinderola G. Preliminary technological and potential probiotic characterisation of bifidobacteria isolated from breast milk for use in dairy products. Int Dair J. 2011,21(8):548-55.

Gonzalez R, Maldonado A, Martin V, Mandomando I, Fumado V, Metzner KJ, Sacoor C, Fernandez L, Macete E, Alonso PL, Rodriguez JM, Menendez C. Breast milk and gut microbiota in African mothers and infants from an area of high HIV prevalence. PloS one. 2013,8(11):e80299.

Delgado S, Arroyo R, Jimenez E, Marin ML, del Campo R, Fernandez L, Rodriguez JM. Staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics. BMC Microbiol. 2009,9(1):82.

Sung C, Kim BG, Kim S, Joo HS, Kim PI. Probiotic potential of Staphylococcus hominis MBBL 2-9 as anti-Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman. J Appl Microbiol. 2010,108(3):908-16.

Bendali F, Hebraud M, Sadoun D. Anti-bacterial and anti-adherence activities of a probiotic strain of Lactobacillus paracasei against Listeria monocytogenes. Int J Appl Microbiol Biotech Res. 2014,2:52-63.

Coconnier M-H, Lievin V, Hemery E, Servin AL. Antagonistic activity againstHelicobacter infection in vitro and in vivo by the humanLactobacillus acidophilus strain LB. Appl Environ Microbiol. 1998,64(11):4573-80.

Hudault S, Lievin V, Bernet-Camard MF, Servin AL. Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection. Appl Environ Microbiol. 1997,63(2):513-8.

Holzapfel W. Use of starter cultures in fermentation on a household scale. Food control. 1997,8(5-6):241-58.

Jung SP, Lee KM, Kang JH, Yun SI, Park HO, Moon Y, Kim JY. Effect of Lactobacillus gasseri BNR17 on Overweight and Obese Adults: A Randomized, Double-Blind Clinical Trial. Korean J Fam Med. 2013,34(2):80-9.

Huang Y, Adams MC. In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Int J Food Microbiol. 2004,91(3):253-60.

Mandal S, Puniya AK, Singh K. Effect of alginate concentrations on survival of microencapsulated Lactobacillus casei NCDC-298. Int Dair J. 2006,16(10):1190-5.

Liong MT, Shah NP. Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. J Dair Sci. 2005,88(1):55-66.

Prasad J, Gill H, Smart J, Gopal PK. Selection and characterisation of Lactobacillus and Bifidobacterium strains for use as probiotics. Int Dair J. 1998,8(12):993-1002.

Marteau P. Safety aspects of probiotic products. Scand J Nutr. 2001,45:22-4.

Morelli L. In vitro selection of probiotic lactobacilli: a critical appraisal. Curr Issues Intest Microbiol. 2000,1(2):59-67.

Marteau P, Vesa T, Rambaud J. Lactose maldigestion. Probiotics 2: Springer; 1997. p. 65-88.

Lin WH, Hwang CF, Chen LW, Tsen HY. Viable counts, characteristic evaluation for commercial lactic acid bacteria products. Food Microbiol. 2006,23(1):74-81.

Jacobsen CN, Nielsen VR, Hayford AE, Moller PL, Michaelsen KF, Paerregaard A, Sandstrom B, Tvede M, Jakobsen M. Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl Environ Microbiol. 1999,65(11):4949-56.

Tuomola EM, Salminen SJ. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int J Food Microbiol. 1998,41(1):45-51.

Kim EY, Rafiquzzaman SM, Lee JM, Noh G, Jo GA, Lee JH, Kong IS. Structural features of glycoprotein purified from Saccharina japonica and its effects on the selected probiotic properties of Lactobacillus plantarum in Caco-2 cell. J Appli Pycol. 2015,27(2):965-73.

Author biographies is not available.