Abstract
Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abe A, Furukawa S, Watanabe S, Morinaga Y (2013) Yeasts and lactic acid bacteria mixed-specie biofilm formation is a promising cell immobilization technology for ethanol fermentation. Appl Biochem Biotechnol 171:72–79
Bacci G et al (2015) Evaluation of the performances of ribosomal database project (RDP) classifier for taxonomic assignment of 16S rRNA metabarcoding sequences generated from Illumina-Solexa NGS. J Genom 3:36–39
Borlee BR, Goldman AD, Murakami K, Samudrala R, Wozniak DJ, Parsek MR (2010) Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol Microbiol 75:827–842. https://doi.org/10.1111/j.1365-2958.2009.06991.x
Chao A, Bunge J (2002) Estimating the number of species in a Stochastic abundance model. Biometrics 58:531–539. https://doi.org/10.1111/j.0006-341X.2002.00531.x
Choi NY, Kim BR, Bae YM, Lee SY (2013) Biofilm formation, attachment, and cell hydrophobicity of foodborne pathogens under varied environmental conditions. J Korean Soc Appl Biol Chem 56:207–220. https://doi.org/10.1007/s13765-012-3253-4
Cremet L et al (2013) Comparison of three methods to study biofilm formation by clinical strains of Escherichia coli. Diagn Microbiol Infect Dis 75:252–255. https://doi.org/10.1016/j.diagmicrobio.2012.11.019
Dobson A, O’Sullivan O, Cotter PD, Ross P, Hill C (2011) High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain. FEMS Microbiol Lett 320:56–62. https://doi.org/10.1111/j.1574-6968.2011.02290.x
Fernández Ramírez MD, Smid EJ, Abee T, Nierop Groot MN (2015) Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates. Int J Food Microbiol 207:23
Gao J, Gu FY, He J, Xiao JZ, Chen QH, Ruan H, He GQ (2013) Metagenome analysis of bacterial diversity in Tibetan kefir grains. Eur Food Res Technol 236:549–556. https://doi.org/10.1007/s00217-013-1912-2
Gao W et al (2015) Microbial diversity and stability during primary cultivation and subcultivation processes of Tibetan kefir. Int J Food Sci Technol 50:1468–1476. https://doi.org/10.1111/ijfs.12801
Golowczyc MA, Mobili P, Garrote GL, Serradel MD, Abraham AG, De Antoni GL (2009) Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface protein. J Dairy Res 76:111–116. https://doi.org/10.1017/s0022029908003749
Good IJ (1953) The population frequencies of species and the estimation of population parameters. Biometrika 40:237–264
Goulter RM, Gentle IR, Dykes GA (2010) Characterisation of curli production, cell surface hydrophobicity, autoaggregation and attachment behaviour of Escherichia coli O157. Curr Microbiol 61:157–162. https://doi.org/10.1007/s00284-010-9589-2
Hirayama S, Furukawa S, Ogihara H, Morinaga Y (2012) Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11. Biochem Biophys Res Commun 419:652–655. https://doi.org/10.1016/j.bbrc.2012.02.068
Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94:981–987. https://doi.org/10.1046/j.1365-2672.2003.01915.x
Kubota H, Senda S, Nomura N, Tokuda H, Uchiyama H (2008) Biofilm formation by lactic acid bacteria and resistance to environmental stress. J Biosci Bioeng 106:381–386. https://doi.org/10.1263/jbb.106.381
Leite AMO et al (2013) Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes. J Dairy Sci 96:4149–4159. https://doi.org/10.3168/jds.2012-6263
Liu JR, Lin CW, Stone GM (2000) Effects of addition of different carbohydrates on the growth characteristics of kefir grains in soymilk. Asian-Australas J Anim Sci 13:245–248
Marsh AJ, O’Sullivan O, Hill C, Ross RP, Cotter PD (2013a) Sequence-based analysis of the microbial composition of water kefir from multiple sources. FEMS Microbiol Lett 348:79–85. https://doi.org/10.1111/1574-6968.12248
Marsh AJ, O’Sullivan O, Hill C, Ross RP, Cotter PD (2013b) Sequencing-based analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources. PLoS ONE 8:e69371. https://doi.org/10.1371/journal.pone.0069371
Monds RD, O’Toole GA (2009) The developmental model of microbial biofilms: ten years of a paradigm up for review. Trends Microbiol 17:73–87. https://doi.org/10.1016/j.tim.2008.11.001
Mozzi F, Raya RR, Vignolo GM (2010) Microbial Interactions in Kefir: A Natural Probiotic Drink. Wiley-Blackwell, Hoboken
Nielsen B, Gurakan GC, Unlu G (2014) Kefir: a multifaceted fermented dairy product. Probiot Antimicrob Proteins 6:123–135. https://doi.org/10.1007/s12602-014-9168-0
Nowak J, Cruz CD, Palmer J, Fletcher GC, Flint S (2015) Biofilm formation of the L. monocytogenes strain 15G01 is influenced by changes in environmental conditions. J Microbiol Methods 119:189–195. https://doi.org/10.1016/j.mimet.2015.10.022
Nozaka S, Furukawa S, Sasaki M, Hirayama S, Ogihara H, Morinaga Y (2014) Manganese ion increases LAB-yeast mixed-species biofilm formation. Biosci Microflora 33:79–84
O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54:49–79. https://doi.org/10.1146/annurev.micro.54.1.49
Pretzer G et al (2005) Biodiversity-based identification and functional characterization of the mannose-specific adhesin of Lactobacillus plantarum. J Bacteriol 187:6128–6136. https://doi.org/10.1128/jb.187.17.6128-6136.2005
Ruhal R et al (2015a) A multivariate approach to correlate bacterial surface properties to biofilm formation by lipopolysaccharide mutants of Pseudomonas aeruginosa. Colloids Surf B 127:182–191
Ruhal R et al (2015b) A multivariate approach to correlate bacterial surface properties to biofilm formation by lipopolysaccharide mutants of Pseudomonas aeruginosa. Colloids Surf B Biointerfaces 127:182–191. https://doi.org/10.1016/j.colsurfb.2015.01.030
Schoevers A, Britz TJ (2003) Influence of different culturing conditions on kefir grain increase. Int J Dairy Technol 56:183–187. https://doi.org/10.1046/j.1471-0307.2003.00104.x
Serra DO, Richter AM, Klauck G, Mika F, Hengge R (2013) Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm. Mbio 4:e00103–e00113. https://doi.org/10.1128/mBio.00103-13
Shannon CE, Weaver W, Wiener N (1950) The mathematical theory of communication. M.d.computing computers in medical. Practice 3:31–32
Simoes M, Simoes LC, Vieira MJ (2010) A review of current and emergent biofilm control strategies. LWT-Food Sci Technol 43:573–583. https://doi.org/10.1016/j.lwt.2009.12.008
Simpson EH (1949) Measurement of Diversity. Nature 163:688
Terada A, Hibiya K, Nagai J, Tsuneda S, Hirata A (2003) Nitrogen removal characteristics and biofilm analysis of a membrane-aerated biofilm reactor applicable to high-strength nitrogenous wastewater treatment. J Biosci Bioeng 95:170–178. https://doi.org/10.1016/s1389-1723(03)80124-x
Topaloglu N, Gulsoy M (2014) Effect of near-infrared diode laser and indocyanine green to treat infections on different wound models. In: Biophotonics: photonic solutions for better health care IV. Proc. SPIE, Brussels, p 912925
Viljoen BC (2001) The interaction between yeasts and bacteria in dairy environments. Int J Food Microbiol 69:37–44. https://doi.org/10.1016/s0168-1605(01)00570-0
Wang SY, Chen KN, Lo YM, Chiang ML, Chen HC, Liu JR, Chen MJ (2012) Investigation of microorganisms involved in biosynthesis of the kefir grain. Food Microbiol 32:274–285. https://doi.org/10.1016/j.fm.2012.07.001
Zhang Q, Yuan Y, Luo W, Zeng L, Wu Z, Zhang W (2017) Characterization of prokaryotic community diversity in new and aged pit muds from Chinese Luzhou-flavor liquor distillery. Food Sci Technol Res 23:213–220
Acknowledgements
This work was financially supported by the program of National Natural Science Foundation of China (31271906/C200204); and the Open Research Fund for Key Laboratory of Dairy Science (Northeast Agricultural University), Ministry of Education, Harbin China. The amendment suggestions of Steve S. Zeng were gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Fig. 1
Appearance of biofilm formed by (A) Control groups, (B) Lc. Lactis, (C) Leu. mesenteroides, (D) Lb. Kefiri, (E) Lb. sunkii, (F) Ac. orientalis and (G) Klu. marxianus isolated from kefir biofilm (DOCX 529 kb)
Rights and permissions
About this article
Cite this article
Han, X., Zhang, LJ., Wu, HY. et al. Investigation of microorganisms involved in kefir biofilm formation. Antonie van Leeuwenhoek 111, 2361–2370 (2018). https://doi.org/10.1007/s10482-018-1125-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-018-1125-6