Skip to main content
Log in

Probiotic Characterization of Indigenous Kocuria flava Y4 Strain Isolated from Dioscorea villosa Leaves

  • Published:
Probiotics and Antimicrobial Proteins Aims and scope Submit manuscript

Abstract

This aim of the study was to isolate and screen potential probiotics from Dioscorea villosa leaves. The potential isolate Y4 was obtained from the Dioscorea villosa leaves, and its ability to grow in a medium containing high NaCl concentrations (2–10%) indicated its negative hemolytic activity. Furthermore, Y4 demonstrated inhibitory activity against human pathogens, such as Klebsiella pneumonia, Staphylococcus aureus, Citrobacter koseri, and Vibrio cholerae, as well as towards a plant pathogen isolate OR-2 (obtained from Citrus sinensis). Some biologically important functional groups of Y4 metabolites, such as sulfoxide; aliphatic ether; 1, 2, 3-trisubstituted, tertiary alcohol: vinyl ether; aromatic amine; carboxylic acid; nitro compound; alkene mono-substituted; and alcohol, were identified through FTIR analysis. The 16S rRNA sequencing and subsequent phylogenetic tree analysis indicated that Y4 and OR-2 are the closest neighbors to Kocuria flava (GenBank accession no. MT773277) and Pantoea dispersa (GenBank accession no. MT766308), respectively. The potential isolate Y4 was found to exhibit adhesion, auto-aggregation, co-aggregation, and weak biofilm activity. It also exhibited a high level of antimicrobial activity and antibiotic susceptibility. The safety of K. flava Y4 isolate, which is proposed to be a probiotic, was evaluated through acute oral toxicity test and biogenic amine production test. Moreover, the preservation potential of isolate Y4 was assessed through application on fruits under different temperatures. Thus, our results confirmed that Kocuria flava Y4 is a prospective probiotic and could also be used for the preservation of fruits.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Data Availability

Sequencing data that support the findings of this study have been deposited in GenBank with the accession numbers MT773277 and MT766308.

References

  1. De Melo Pereira GV, De Oliveira CB, Júnior AI, Thomaz-Soccol V, Soccol CR (2018) How to select a probiotic? A review and update of methods and criteria. Biotechnol Adv 36:2060–2076. https://doi.org/10.1016/j.biotechadv.2018.09.003

    Article  PubMed  Google Scholar 

  2. Markowiak P, Śliżewska K (2017) Effects of probiotics, prebiotics, and synbiotics on human health. Nutrients 9:1021. https://doi.org/10.3390/nu9091021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Prabhurajeshwar C, Chandrakanth RK (2017) Probiotic potential of Lactobacilli with antagonistic activity against pathogenic strains: an in vitro validation for the production of inhibitory substances. Biomed J 40:270–283. https://doi.org/10.1016/j.bj.2017.06.008

    Article  PubMed  PubMed Central  Google Scholar 

  4. Ayyash MM, Abdalla AK, AlKalbani NS, Baig MA, Turner MS, Liu SQ, Shah NP (2021) Invited review: Characterization of new probiotics from dairy and nondairy products—insights into acid tolerance, bile metabolism and tolerance, and adhesion capability. J Dairy Sci 104:8363–8379. https://doi.org/10.3168/jds.2021-20398

    Article  CAS  PubMed  Google Scholar 

  5. Sharma M, Wasan A, Sharma RK (2021) Recent developments in probiotics: an emphasis on Bifidobacterium. Food Biosci 41:100993. https://doi.org/10.1016/j.fbio.2021.100993

    Article  CAS  Google Scholar 

  6. Varsha KK, Maheshwari AP, Nampoothiri KM (2021) Accomplishment of probiotics in human health pertaining to immunoregulation and disease control. Clin Nutr ESPEN 44:26–37. https://doi.org/10.1016/j.clnesp.2021.06.020

    Article  PubMed  Google Scholar 

  7. Diya S, Rouf A, Jan T, Sharma P (2018) Non-dairy probiotics — an emerging trend in health care products. Int J Curr Microbiol Appl Sci 7:131–145. https://doi.org/10.20546/ijcmas.2018.710.015

  8. Siddiqee MH, Sarker H, Shurovi KM (2013) Assessment of probiotic application of lactic acid bacteria (LAB) isolated from different food items. Stamford J Microbiol 2:10–14. https://doi.org/10.3329/sjm.v2i1.15206

    Article  Google Scholar 

  9. Schoster A, Weese JS, Guardabassi L (2014) Probiotic use in horses — what is the evidence for their clinical efficacy? J Vet Intern Med 28:1640–1652. https://doi.org/10.1111/jvim.12451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lai HH, Chiu CH, Kong MS, Chang CJ, Chen CC (2019) Probiotic Lactobacillus casei: effective for managing childhood diarrhea by altering gut microbiota and attenuating fecal inflammatory markers. Nutrients 11:1150. https://doi.org/10.3390/nu11051150

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Huang C-H, Shen C-C, Liang Y-C, Jan T-R (2016) The probiotic activity of Lactobacillus murinus against food allergy. J Funct Foods 25:231–241. https://doi.org/10.1016/j.jff.2016.06.006

    Article  CAS  Google Scholar 

  12. Soleimani A, Mojarrad MZ, Bahmani F, Taghizadeh M, Ramezani M, Tajabadi-Ebrahimi M, Jafari P, Esmaillzadeh A, Asemi Z (2017) Probiotic supplementation in diabetic hemodialysis patients has beneficial metabolic effects. Kidney Int 91:435–442. https://doi.org/10.1016/j.kint.2016.09.040

    Article  CAS  PubMed  Google Scholar 

  13. Toral M, Romero M, Rodríguez-Nogales A, Jiménez R, Robles-Vera I, Algieri F, Chueca-Porcuna N, Sánchez M, de la Visitación N, Olivares M, García F (2018) Lactobacillus fermentum improves tacrolimus-induced hypertension by restoring vascular redox state and improving eNOS coupling. Mol Nutr Food Res 62:1800033. https://doi.org/10.1002/mnfr.201800033

    Article  CAS  Google Scholar 

  14. Brady C, Cleenwerck I, Venter S, Vancanneyt M, Swings J, Coutinho T (2008) Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA). Syst Appl Microbiol 31:447–460. https://doi.org/10.1016/j.syapm.2008.09.004

    Article  CAS  PubMed  Google Scholar 

  15. Toral M, Robles-Vera I, Romero M, de la Visitación N, Sánchez M, O’Valle F, Rodriguez-Nogales A, Gálvez J, Duarte J, Jiménez R (2019) Lactobacillus fermentum CECT5716: a novel alternative for the prevention of vascular disorders in a mouse model of systemic lupus erythematosus. FASEB J 33:10005–10018. https://doi.org/10.1096/fj.201900545RR

    Article  CAS  PubMed  Google Scholar 

  16. Tsai Y-T, Cheng P-C, Pan T-M (2012) The immunomodulatory effects of lactic acid bacteria for improving immune functions and benefits. Appl Microbiol Biotechnol 96:853–862. https://doi.org/10.1007/s00253-012-4407-3

    Article  CAS  PubMed  Google Scholar 

  17. Greifová Z, Kohajdová Z, Karovičová M (2006) Lactic acid fermentation of some vegetable juices. J Food Nutr Res 45:115–119

    Google Scholar 

  18. Stackebrandt E, Koch C, Gvozdiak O, Schumann P (1995) Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. Int J Syst Bacteriol 45:682–692. https://doi.org/10.1099/00207713-45-4-682

    Article  CAS  PubMed  Google Scholar 

  19. Zhou G, Luo X, Tang Y, Zhang L, Yang Q, Qiu Y, Fang C (2008) Kocuria flava sp. nov. and Kocuria turfanensis sp. nov., airborne actinobacteria isolated from Xinjiang. China Int J Syst Evol Microbiol 58:1304–1307. https://doi.org/10.1099/ijs.0.65323-0

    Article  CAS  PubMed  Google Scholar 

  20. Kim D-H, Brunt J, Austin B (2007) Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss). J Appl Microbiol 102:1654–1664. https://doi.org/10.1111/j.1365-2672.2006.03185.x

    Article  CAS  PubMed  Google Scholar 

  21. Irianto A, Austin B (2002) Probiotics in aquaculture. J Fish Dis 25:633–642. https://doi.org/10.1046/j.1365-2761.2002.00422.x

    Article  Google Scholar 

  22. Achal V, Pan X, Zhang D (2011) Remediation of copper-contaminated soil by Kocuria flava CR1, based on microbially induced calcite precipitation. Ecol Eng 37:1601–1605. https://doi.org/10.1016/j.ecoleng.2011.06.008

    Article  Google Scholar 

  23. Sharma A, Bhattacharyya KG (2005) Azadirachta indica (Neem) leaf powder as a biosorbent for removal of Cd(II) from aqueous medium. J Hazard Mater 125:102–112. https://doi.org/10.1016/j.jhazmat.2005.05.012

    Article  CAS  PubMed  Google Scholar 

  24. Gómez NC, Ramiro JM, Quecan BX, de Melo Franco BD (2016) Use of potential probiotic lactic acid bacteria (LAB) biofilms for the control of Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7 biofilms formation. Front Microbiol 7:863. https://doi.org/10.3389/fmicb.2016.00863

    Article  PubMed  PubMed Central  Google Scholar 

  25. Thirumurugan D, Vijayakumar R (2015) Characterization and structure elucidation of antibacterial compound of Streptomyces sp. ECR77 isolated from East Coast of India. Curr Microbiol 70:745–755. https://doi.org/10.1007/s00284-015-0780-3

    Article  CAS  PubMed  Google Scholar 

  26. Prasad J, Gill H, Smart J, Gopal PK (1998) Selection and characterisation of Lactobacillus and Bifidobacterium strains for use as probiotics. Int Dairy J 8:993–1002. https://doi.org/10.1016/S0958-6946(99)00024-2

    Article  Google Scholar 

  27. Gardini F, Martuscelli M, Caruso MC, Galgano F, Crudele MA, Favati F, Guerzoni ME, Suzzi G (2001) Effects of pH, temperature and NaCl concentration on the growth kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalis. Int J Food Microbiol 64:105–117. https://doi.org/10.1016/S0168-1605(00)00445-1

    Article  CAS  Google Scholar 

  28. Abdulla AA, Thikra AA, Anwar K, Saffar AL, Alahmed SG, Saffar HK, Wesawei YA (2015) Analysis of probiotic properties of Lactobacillus acidophilus from commercial yoghurt. Int J Curr Microbiol App Sci 4:548–554

    CAS  Google Scholar 

  29. Zago M, Fornasari ME, Carminati D, Burns P, Suàrez V, Vinderola G, Reinheimer J, Giraffa G (2011) Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses. Food Microbiol 28:1033–1040. https://doi.org/10.1016/j.fm.2011.02.009

    Article  CAS  PubMed  Google Scholar 

  30. Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M (2011) Evaluation of different detection methods of biofilm formation in the clinical isolates. Brazilian J Infect Dis 15:305–311. https://doi.org/10.1016/S1413-8670(11)70197-0

    Article  Google Scholar 

  31. Crow VL, Gopal PK, Wicken AJ (1995) Cell surface differences of lactococcal strains. Int Dairy J 5:45–68. https://doi.org/10.1016/0958-6946(94)P1598-8

    Article  Google Scholar 

  32. Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31:438–442. https://doi.org/10.1046/j.1365-2672.2000.00845.x

    Article  PubMed  Google Scholar 

  33. Bauer AW, Kirby WM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496

    Article  CAS  PubMed  Google Scholar 

  34. Das A, Belgaonkar P, Raman AS, Banu S, Osborne JW (2017) Bioremoval of lead using Pennisetum purpureum augmented with Enterobacter cloacae-VITPASJ1: A pot culture approach. Environ Sci Pollut Res 24:15444–15453. https://doi.org/10.1007/s11356-017-8988-3

    Article  CAS  Google Scholar 

  35. Sangeetha V, Govindarajan M, Kanagathara N, Marchewka MK, Gunasekaran S, Anbalagan G (2014) Structure and vibrational spectra of melaminium bis(trifluoroacetate) trihydrate: FT-IR, FT-Raman and quantum chemical calculations. Spectrochim Acta Part A Mol Biomol Spectrosc 125:252–263. https://doi.org/10.1016/j.saa.2014.01.018

    Article  CAS  Google Scholar 

  36. Clark JD, Gebhart GF, Gonder JC, Keeling ME, Kohn DF (1997) The 1996 guide for the care and use of laboratory animals. ILAR J 38:41–48. https://doi.org/10.1093/ilar.38.1.41

    Article  PubMed  Google Scholar 

  37. Gurría Á (2019) Organisation for Economic Co-operation and Development. Obtenido de. Available at: https://en-academic.com/dic.nsf/enwiki/120310

  38. Sirocchi V, Caprioli G, Cecchini C, Coman MM, Cresci A, Maggi F, Papa F, Ricciutelli M, Vittori S, Sagratini G (2013) Biogenic amines as freshness index of meat wrapped in a new active packaging system formulated with essential oils of Rosmarinus officinalis. Int J Food Sci Nutr 64:921–928. https://doi.org/10.3109/09637486.2013.809706

    Article  CAS  PubMed  Google Scholar 

  39. Verón HE, Di Risio HD, Isla MI, Torres S (2017) Isolation and selection of potential probiotic lactic acid bacteria from Opuntia ficus-indica fruits that grow in Northwest Argentina. LWT 84:231–240. https://doi.org/10.1016/j.lwt.2017.05.058

    Article  CAS  Google Scholar 

  40. Lim ES, Lee JE, Kim J-S, Koo OK (2017) Isolation of indigenous bacteria from a cafeteria kitchen and their biofilm formation and disinfectant susceptibility. LWT 77:376–382. https://doi.org/10.1016/j.lwt.2016.11.060

    Article  CAS  Google Scholar 

  41. Kos BV, Šušković J, Vuković S, Šimpraga M, Frece J, Matošić 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

    Article  CAS  PubMed  Google Scholar 

  42. Pérez PF, Minnaard Y, Disalvo EA, De Antoni GL (1998) Surface properties of bifidobacterial strains of human origin. Appl Environ Microbiol 64:21–26. https://doi.org/10.1128/AEM.64.1.21-26.1998

    Article  PubMed  PubMed Central  Google Scholar 

  43. Del Re B, Busetto A, Vignola G, Sgorbati B, Palenzona DL (1998) Autoaggregation and adhesion ability in a Bifidobacterium suis strain. Lett Appl Microbiol 27:307–310. https://doi.org/10.1046/j.1472-765X.1998.t01-1-00449.x

    Article  PubMed  Google Scholar 

  44. Alzawqari MH, Kermanshahi H, Moghaddam HN, Tawassoli  MH, Gilani A (2013) Impact of feed withdrawal and addition of acetic acid in drinking water during preslaughter phase on intestinal microbiota of broilers. Afr J Biotechnol 12. https://doi.org/10.5897/AJB12.2280

  45. Uzair B, Menaa F, Khan BA, Mohammad FV, Ahmad VU, Djeribi R, Menaa B (2018) Isolation, purification, structural elucidation and antimicrobial activities of kocumarin, a novel antibiotic isolated from actinobacterium Kocuria marina CMG S2 associated with the brown seaweed Pelvetia canaliculata. Microbiol Res 206:186–197. https://doi.org/10.1016/j.micres.2017.10.007

    Article  CAS  PubMed  Google Scholar 

  46. De Angelis M, Siragusa S, Berloco M, Caputo L, Settanni L, Alfonsi G, Amerio M, Grandi A, Ragni A, Gobbetti M (2006) Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding. Res Microbiol 157:792–801. https://doi.org/10.1016/j.resmic.2006.05.003

    Article  PubMed  Google Scholar 

  47. Rathore S, Salmerón I, Pandiella SS (2012) Production of potentially probiotic beverages using single and mixed cereal substrates fermented with lactic acid bacteria cultures. Food Microbiol 30:239–244. https://doi.org/10.1016/j.fm.2011.09.001

    Article  CAS  PubMed  Google Scholar 

  48. Maldonado NC, de Ruiz CS, Otero MC, Sesma F, Nader-Macías ME (2012) Lactic acid bacteria isolated from young calves — characterization and potential as probiotics. Res Vet Sci 92:342–349. https://doi.org/10.1016/j.rvsc.2011.03.017

    Article  CAS  PubMed  Google Scholar 

  49. Chander H, Batish VK, Babu S, Singh RS (1989) Factors affecting amine production by a selected strain of Lactobacillus bulgaricus. J Food Sci 54:940–942. https://doi.org/10.1111/j.1365-2621.1989.tb07917.x

    Article  CAS  Google Scholar 

  50. Corzo G, Gilliland SE (1999) Bile salt hydrolase activity of three strains of Lactobacillus acidophilus. J Dairy Sci 82:472–480. https://doi.org/10.3168/jds.S0022-0302(99)75256-2

    Article  CAS  PubMed  Google Scholar 

  51. Vizoso Pinto MG, Franz CMAP, Schillinger U, Holzapfel WH (2006) Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products. Int J Food Microbiol 109:205–214. https://doi.org/10.1016/j.ijfoodmicro.2006.01.029

    Article  CAS  PubMed  Google Scholar 

  52. Ferreira CL, Grześkowiak Ł, Collado MC, Salminen S (2011) In vitro evaluation of Lactobacillus gasseri strains of infant origin on adhesion and aggregation of specific pathogens. J Food Prot 74:1482–1487. https://doi.org/10.4315/0362-028X.JFP-11-074

    Article  PubMed  Google Scholar 

  53. Xu Y, Zhou T, Tang H, Li X, Chen Y, Zhang L, Zhang J (2020) Probiotic potential and amylolytic properties of lactic acid bacteria isolated from Chinese fermented cereal foods. Food Control 111:107057. https://doi.org/10.1016/j.foodcont.2019.107057

    Article  CAS  Google Scholar 

  54. Braun MS, Wang E, Zimmermann S, Boutin S, Wink M (2018) Kocuria uropygioeca sp. nov. and Kocuria uropygialis sp. nov., isolated from the preen glands of Great Spotted Woodpeckers ( Dendrocopos major ). Syst Appl Microbiol 41:38–43. https://doi.org/10.1016/j.syapm.2017.09.005

    Article  PubMed  Google Scholar 

  55. Vagenas N (2003) Quantitative analysis of synthetic calcium carbonate polymorphs using FT-IR spectroscopy. Talanta 59:831–836. https://doi.org/10.1016/S0039-9140(02)00638-0

    Article  CAS  PubMed  Google Scholar 

  56. Sarafin Y, Donio MBS, Velmurugan S, Michaelbabu M, Citarasu T (2014) Kocuria marina BS-15 a biosurfactant producing halophilic bacteria isolated from solar salt works in India. Saudi J Biol Sci 21:511–519. https://doi.org/10.1016/j.sjbs.2014.01.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. O’sullivan MG, Thornton G, O’sullivan GC, Collins JK (1992) Probiotic bacteria: myth or reality? Trends Food Sci Technol 3:309–314. https://doi.org/10.1016/S0924-2244(10)80018-4

    Article  CAS  Google Scholar 

  58. Donohue DC SS (1996) Safety of probiotic bacteria. Asia Pacific J Clin Nutr 5:25–8. Available at: https://apjcn.nhri.org.tw/server/apjcn/5/1/25.pdf

  59. Iannaccone PM, Jacob HJ (2009) Rats! Dis Model Mech 2:206–210. https://doi.org/10.1242/dmm.002733

  60. Alegre I, Viñas I, Usall J, Anguera M, Abadias M (2011) Microbiological and physicochemical quality of fresh-cut apple enriched with the probiotic strain Lactobacillus rhamnosus GG. Food Microbiol 28:59–66. https://doi.org/10.1016/j.fm.2010.08.006

    Article  CAS  PubMed  Google Scholar 

  61. Oliveira M, Abadias M, Colás-Medà P, Usall J, Viñas I (2015) Biopreservative methods to control the growth of foodborne pathogens on fresh-cut lettuce. Int J Food Microbiol 214:4–11. https://doi.org/10.1016/j.ijfoodmicro.2015.07.015

    Article  CAS  PubMed  Google Scholar 

  62. Alegre I, Viñas I, Usall J, Teixidó N, Figge MJ, Abadias M (2013) Control of foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas graminis. Food Microbiol 34:390–399. https://doi.org/10.1016/j.fm.2013.01.013

    Article  PubMed  Google Scholar 

  63. Kalač P (2014) Health effects and occurrence of dietary polyamines: a review for the period 2005–mid 2013. Food Chem 161:27–39. https://doi.org/10.1016/j.foodchem.2014.03.102

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors express gratitude to Dr. Anindya Bose for carrying out FT-IR analysis of our samples at the CIR facility of SPS. The authors express their gratitude towards Center for Biotechnology, Siksha O Anusandhan (Deemed to be University), Bhubaneswar, for providing lab facilities for conducting the research.

Author information

Authors and Affiliations

Authors

Contributions

The authors are equally accountable in terms of concept, design, analysis of data, and drafting the manuscript.

Corresponding author

Correspondence to Sangeeta Raut.

Ethics declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for publication

Not applicable.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Barik, A., Patel, G.D., Sen, S.K. et al. Probiotic Characterization of Indigenous Kocuria flava Y4 Strain Isolated from Dioscorea villosa Leaves. Probiotics & Antimicro. Prot. 15, 614–629 (2023). https://doi.org/10.1007/s12602-021-09877-2

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-021-09877-2

Keywords

Navigation