Food Science and Biotechnology

, Volume 26, Issue 2, pp 453–459 | Cite as

Antioxidant activity and influence of Citrus byproduct extracts on adherence and invasion of Campylobacter jejuni and on the relative expression of cadF and ciaB

  • Sandra Castillo
  • Jorge Dávila-Aviña
  • Norma Heredia
  • Santos GarciaEmail author


Adherence and invasion to cells are the key processes during infection development by Campylobacter jejuni (C. jejuni). In this study, extracts from the byproducts of Citrus limon, Citrus aurantium, and Citrus medica were added to the cultures of C. jejuni, and the adherence and invasion of C. jejuni to HeLa cells and the expression of cadF and ciaB genes were analyzed. The relative expression of the genes was determined by quantitative reverse transcription PCR (qRT-PCR). The antioxidant activity was determined using spectrophotometric methods. Byproduct extracts at subinhibitory concentrations affected the adherence (reduced 2.3 to 99%) and invasion (reduced 71.3 to 99.2%) to the HeLa cells. The expression of cadF and ciaB was reduced from 66 to 99% and from 81 to 99%, respectively. The total phenolic content of the byproducts varied from 92 to 26 mg GAE/g and the total flavonoids varied from 161 to 29.29 mg QE/g. C. aurantium showed the highest percentage of radical scavenging activity (RSA, 90.1). These extracts can prove as effective alternatives for devising new strategies to control Campylobacter infections.


Campylobacter adhesion invasion citrus extract antimicrobial activity antioxidant activity 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Asakura H, Yamasaki M, Yamamoto S, Igimi S. Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni. FEMS Microbiol. Lett. 275: 278–285 (2007)CrossRefGoogle Scholar
  2. 2.
    Hänel I, Borrmann E, Müller J, Alter T. Relationships between bacterial genotypes and in vitro virulence properties of Campylobacter jejuni and Campylobacter coli isolated from turkeys. J. Appl. Microbiol. 102: 433–441 (2007)CrossRefGoogle Scholar
  3. 3.
    Hermans D, van Deun K, Martel A, van Immerseel F, Messens W, Heyndrickx M, Haesebrouck F, Pasmans F. Colonization factors of Campylobacter jejuni in the chicken gut. Vet. Res. 42: 82 (2011)CrossRefGoogle Scholar
  4. 4.
    Cróinín TÓ, Backert S. Host epithelial cell invasion by Campylobacter jejuni: Trigger or zipper mechanism? Front. Cell. Infect. Microbiol. 2: 25 (2012)Google Scholar
  5. 5.
    Dasti JI, Tareen AM, Lugert R, Zautner AE, Gross U. Campylobacter jejuni: A brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int. J. Med. Microbiol. 300: 205–211 (2010)CrossRefGoogle Scholar
  6. 6.
    Clatworthy AE, Pierson E, Hung DT. Targeting virulence: A new paradigm for antimicrobial therapy. Nat. Chem. Biol. 3: 541–548 (2007)CrossRefGoogle Scholar
  7. 7.
    Klanènik A, Piskernik S, Jeršek B, Možina SS. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. J. Microbiol. Meth. 81: 121–126 (2010)CrossRefGoogle Scholar
  8. 8.
    Singh J, Sood S, Muthuraman A. In-vitro evaluation of bioactive compounds, anti-oxidant, lipid peroxidation and lipoxygenase inhibitory potential of Citrus karna L. peel extract. J. Food Sci. Tech. Mys. 51: 67–74 (2014)CrossRefGoogle Scholar
  9. 9.
    El-aal HA, Halaweish F. Food preservative activity of phenolic compounds in orange peel extracts (Citrus sinensis L.). Lucrari Stiintifice 53: 233–240 (2010)Google Scholar
  10. 10.
    Chen ZT, Chu HL, Chyau CC, Chu CC, Duh PD. Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress. Food Chem. 135: 2119–2127 (2012)CrossRefGoogle Scholar
  11. 11.
    Yusof S, Ghazali HM, King GS. Naringin content in local citrus fruits. Food Chem. 37: 113–121 (1990)CrossRefGoogle Scholar
  12. 12.
    Nannapaneni R, Muthaiyan A, Crandall PG, Johnson MG, O’Bryan CA, Chalova VI, Callaway TR, Carroll, JA, Arthington JD, Nisbet DJ. Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains. Foodborne Pathog. Dis. 5: 695–699 (2008)CrossRefGoogle Scholar
  13. 13.
    Balasundram N, Sundram K, Samman S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem. 99: 191–203 (2006)CrossRefGoogle Scholar
  14. 14.
    Guil-Guerrero J, Ramos L, Moreno C, Zúñiga-Paredes J, Carlosama-Yepez M, Ruales P. Antimicrobial activity of plant-food by-products: A review focusing on the tropics. Livest Sci. 189: 32–49 (2016)CrossRefGoogle Scholar
  15. 15.
    Nannapaneni R, Chalova VI, Crandall PG, Ricke SC, Johnson MG, O’Bryan CA. Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions. Int. J. Food Microbiol. 129: 43–49 (2009)CrossRefGoogle Scholar
  16. 16.
    Castillo S, Heredia N, Arechiga-Carvajal E, García S. Citrus extracts as inhibitors of quorum sensing, biofilm formation and motility of Campylobacter jejuni. Food Biotechnol. 28: 106–122 (2014)CrossRefGoogle Scholar
  17. 17.
    Valtierra-Rodríguez D, Heredia NL, García S, Sánchez E. Reduction of Campylobacter jejuni and Campylobacter coli in poultry skin by fruit extracts. J. Food Protect. 73: 477–482 (2010)CrossRefGoogle Scholar
  18. 18.
    Castillo SL, Heredia N, Contreras JF, García S. Extracts of edible and medicinal plants in inhibition of growth, adherence, and cytotoxin production of Campylobacter jejuni and Campylobacter coli. J. Food Sci. 76: M421–M426 (2011)CrossRefGoogle Scholar
  19. 19.
    Sanchez E, Heredia N, Garcia S. Extracts of edible and medicinal plants damage membranes of Vibrio cholerae. Appl. Environ. Microb. 76: 6888–6894 (2010)CrossRefGoogle Scholar
  20. 20.
    Ganan M, Campos G, Muñoz R, Carrascosa AV, de Pascual-Teresa S, Martinez-Rodriguez AJ. Effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by Campylobacter. Int. J. Food Microbiol. 140: 14–18 (2010)CrossRefGoogle Scholar
  21. 21.
    Clark CG, Grant CC, Pollari F, Marshall B, Moses J, Tracz DM, Gilmour MW. Effects of the Campylobacter jejuni CJIE1 prophage homologs on adherence and invasion in culture, patient symptoms, and source of infection. BMC Microbiol. 12: 269 (2012)CrossRefGoogle Scholar
  22. 22.
    Jeon B, Itoh K, Misawa N, Ryu S. Effects of quorum sensing on flaA transcription and autoagglutination in Campylobacter jejuni. Microbiol. Immunol. 47: 833–839 (2003)CrossRefGoogle Scholar
  23. 23.
    Cloak OM, Solow BT, Briggs CE, Chen CY, Fratamico PM. Quorum sensing and production of autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in foods. Appl. Environ. Microb. 68: 4666–4671 (2002)CrossRefGoogle Scholar
  24. 24.
    Malik-Kale P, Parker CT, Konkel ME. Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression. J. Bacteriol. 190: 2286–2297 (2008)CrossRefGoogle Scholar
  25. 25.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using Real-Time Quantitative PCR and the 2 -?CT Method. Methods 25: 402–408 (2001)CrossRefGoogle Scholar
  26. 26.
    Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Method. Enzymol. 299: 152–178 (1999)CrossRefGoogle Scholar
  27. 27.
    Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64: 555–559 (1999)CrossRefGoogle Scholar
  28. 28.
    Singh S, Singh R. in vitro methods of assay of antioxidants: An overview. Food Rev. Int. 24: 392–415 (2008)CrossRefGoogle Scholar
  29. 29.
    Kedare SB, Singh R. Genesis and development of DPPH method of antioxidant assay. J. Food. Sci. Tech. Mys. 48: 412–422 (2011)CrossRefGoogle Scholar
  30. 30.
    Snelling W, Matsuda M, Moore J, Dooley J. Campylobacter jejuni. Lett. Appl. Microbiol. 41: 297–302 (2005)CrossRefGoogle Scholar
  31. 31.
    Wine E, Gareau MG, Johnson-Henry K, Sherman PM. Strain-specific probiotic (Lactobacillus helveticus) inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells. FEMS Microbiol. Lett. 300: 146–152 (2009)CrossRefGoogle Scholar
  32. 32.
    Pogaèar MŠ, Klanènik A, Bucar F, Langerholc T, Možina SS. Alpinia katsumadai extracts inhibit adhesion and invasion of Campylobacter jejuni in animal and human foetal small intestine cell lines. Phytother. Res. 29: 1585–1589 (2015)CrossRefGoogle Scholar
  33. 33.
    Castillo S, Heredia N, García S. 2 (5H)-Furanone, epigallocatechin gallate, and a citric-based disinfectant disturb quorum-sensing activity and reduce motility and biofilm formation of Campylobacter jejuni. Folia Microbiol. 60: 89–95 (2015)CrossRefGoogle Scholar
  34. 34.
    Lee KM, Kim WS, Lim J, Nam S, Youn M, Nam SW, Kim Y, Kim SH, Park W, Park S. Antipathogenic properties of green tea polyphenol epigallocatechin gallate at concentrations below the MIC against enterohemorrhagic Escherichia coli O157:H7. J. Food Protect. 72: 325–331 (2009)CrossRefGoogle Scholar
  35. 35.
    Zou Z, Xi W, Hu Y, Nie C, Zhou Z. Antioxidant activity of Citrus fruits. Food Chem. 196: 885–896 (2016)CrossRefGoogle Scholar
  36. 36.
    Daglia M. Polyphenols as antimicrobial agents. Curr. Opin. Biotech. 23: 174–181 (2012)CrossRefGoogle Scholar
  37. 37.
    de Villiers A, Venter P, Pasch H. Recent advances and trends in the liquidchromatography–mass spectrometry analysis of flavonoids. J. Chromatogr. A 1430: 16–78 (2016)CrossRefGoogle Scholar
  38. 38.
    Xi W, Fang B, Zhao Q, Jiao B, Zhou Z. Flavonoid composition and antioxidant activities of Chinese local pummelo (Citrus grandis Osbeck.) varieties. Food Chem. 161: 230–238 (2014)CrossRefGoogle Scholar
  39. 39.
    Gorinstein S, Martýìn-Belloso O, Park YS, Haruenkit R, Lojek A, ýìž M, Caspi A, Libman, I, Trakhtenberg S. Comparison of some biochemical characteristics of different citrus fruits. Food Chem. 74: 309–315 (2001)CrossRefGoogle Scholar
  40. 40.
    Ghasemi K, Ghasemi Y, Ebrahimzadeh MA. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Pak. J. Pharm. Sci. 22: 277–281 (2009)Google Scholar
  41. 41.
    Nakajima VM, Macedo GA, Macedo JA. Citrus bioactive phenolics: Role in the obesity treatment. LWT-Food Sci. Technol. 59: 1205–1212 (2014)CrossRefGoogle Scholar
  42. 42.
    Ramful D, Bahorun T, Bourdon E, Tarnus E, Aruoma OI. Bioactive phenolics and antioxidant propensity of flavedo extracts of Mauritian citrus fruits: Potential prophylactic ingredients for functional foods application. Toxicology 278: 75–87 (2010)CrossRefGoogle Scholar
  43. 43.
    Li B, Smith B, Hossain MM. Extraction of phenolics from citrus peels: I. Solvent extraction method. Sep. Purif. Technol. 48: 182–188 (2006)CrossRefGoogle Scholar
  44. 44.
    Li B, Smith B, Hossain MM. Extraction of phenolics from citrus peels: II. Enzyme-assisted extraction method. Sep. Purif. Technol. 48: 189–196 (2006)Google Scholar
  45. 45.
    Chansiripornchai N, Sasipreeyajan J. PCR detection of four virulenceassociated genes of Campylobacter jejuni isolates from Thai broilers and their abilities of adhesion to and invasion of INT-407 cells. J. Vet. Med. Sci. 71: 839–844 (2009)CrossRefGoogle Scholar
  46. 46.
    Preeti M, Craig T, Konkel M. Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression. J. Bacteriol. 190: 2286–2297 (2008)CrossRefGoogle Scholar

Copyright information

© The Korean Society of Food Science and Technology and Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Sandra Castillo
    • 1
  • Jorge Dávila-Aviña
    • 1
  • Norma Heredia
    • 1
  • Santos Garcia
    • 1
    Email author
  1. 1.Departamento de Microbiología e Inmunología, Facultad de Ciencias BiológicasUniversidad Autónoma de Nuevo LeónSan NicolásMéxico

Personalised recommendations