Annals of Microbiology

, Volume 62, Issue 3, pp 1301–1309 | Cite as

An amperometric biosensor developed for detection of limonin levels in kinnow mandarin juices

Original Article


The bitterness in kinnow mandarins (also in certain other citrus juices) has been ascribed to limonin that is gradually formed during processing of the juice, which lowers the consumer’s acceptability. Thus, detection of limonin at various stages of processing is crucial for the employment of suitable debittering interventions. Owing to the lack of rapid, reliable and economical method for determining limonin levels during the juice production process, an attempt has been made to develop an amperometric microbial biosensor using a mutant (lim+) of a strain Pseudomonas putida G7, that could selectively utilize limonin as a carbon source in the presence of other sugars in kinnow mandarin juice. Analytical determination was based on the respiratory activity of this stable, lim+ auxotrophic mutant in the presence of the analyte, limonin. The temperature of 30°C, pH 6.5 and the mid-log phase cells at concentrations of 106 CFU/ml were the most optimal operating conditions. A consistent correlation between limonin concentrations of 20, 25, 30 ppm (i.e. an optimal substrate–to-cell mass concentration) and the dissolved oxygen (DO) was established. Response times of approximately 20 min for the steady-state method and 12 min for the initial slope method were recorded. The calibration curve for limonin was linear in the 15–50 ppm range of limonin. The performance of the biosensor was reproducible and remained unaffected following intermittent storage and reuse for at least 1 month. Overall, this study suggests the possible application of the developed biosensor for monitoring the limonin content in citrus juices during processing.


Limonin Amperometric biosensor Pseudomonas putida G7 Debitterring 



This work was supported to M.G. by Department of Science and Technology, under the SERC Fast Track Scheme for young scientists.


  1. An L, Niu H, Zeng H (1998) A new biosensor for rapid oxygen demand measurement. Water Environ Res 70:1070–1074CrossRefGoogle Scholar
  2. Chern LH, Heng LY, Musa A (2001) A potentiometric biosensor based on urease enzyme. Proc NSF Workshop, Kuala LumpurGoogle Scholar
  3. Das N, Prabhakar P, Kayastha AM, Srivastava RC (1997) Enzyme entrapped inside the reverse micelle in the fabrication of a new urea sensor. Biotechnol Bioeng 54:329–332PubMedCrossRefGoogle Scholar
  4. D’Souza SF (2001) Microbial biosensors (review). Biosens Bioelectron 16:337–353PubMedCrossRefGoogle Scholar
  5. Dubey RS, Upadhyay SN (2001) Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pesudomosa. sp. Biosens Bioelectron 16:995–1000PubMedCrossRefGoogle Scholar
  6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  7. Ghosh M, Ganguli A, Mallik M (2006) Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida G7 strain implicated in limonin degradation. J Microbiol 44:473–479PubMedGoogle Scholar
  8. Hasegawa S, Miyake M (1996) Biochemistry and biological functions of citrus limonoids. Food Rev Int 12:413–415CrossRefGoogle Scholar
  9. Hikuma M, Obana H, Yasuda T (1980) Amperometric determination of total assimiable sugars in fermentation broths with use of immobilized whole cells. Enzyme Microbiol Technol 2:234–238CrossRefGoogle Scholar
  10. Hsieh YL, Tseng SK, Chang YJ (2002) Nitrification using polyvinyl alcohol-immobilized nitrifying biofilm on an O2-enriching membrane. Biotechnol Lett 24:315–319CrossRefGoogle Scholar
  11. Karube I, Suzuki S (1981) Preliminary screening of mutagens with a microbial sensor. Anal Chem 53:1024–1026CrossRefGoogle Scholar
  12. Karube I, Matsunaga T, Mitsuda S, Suzuki S (1977) Microbial electrode BOD Sensor. Biotechnol Bioeng 19:1535–1547PubMedCrossRefGoogle Scholar
  13. Konig A, Riedel K (1998) A microbial sensor for detecting inhibitors of nitrification in wastewater. Biosens Bioelectron 13:869–874PubMedCrossRefGoogle Scholar
  14. Konig A, Secker J, Riedel K, Metzger JW (1997) A microbial sensor for measuring inhibitors and substrates for nitrification in wastewater. Am Lab 13:12–21Google Scholar
  15. Krajewska B, Zaborska W, Leszko M (2001) Inhibition of chitosan-immobilized urease by slow-binding inhibitors: Ni2+, F andacetohydroxamic acid. J Mol Catalysis B: Enzymes 101–109Google Scholar
  16. Krajewska B, Zaborska W, Chudy M (2004) Multi-step analysis of Hg2+ ion inhibition of jack bean urease. J Inorg Biochem 98:1160–1168PubMedCrossRefGoogle Scholar
  17. Liu J, Mattiasson B (2002) Microbial BOD sensors for wastewater analysis. Water Res 36:3786–3802PubMedCrossRefGoogle Scholar
  18. Makarenko AA, Bezverbnaya IP, Kosheleva IA, Kuvichkina TN, II’yasov PV, Reshetilov AN (2002) Development of biosensors for phenol determination from bacteria found in petroleum fields of West Siberia. Appl Biochem Microbiol 38:23–27CrossRefGoogle Scholar
  19. Miller GL (1959) Use of dinitrosalicylic acid reagent determination reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  20. Mulchandani P, Hangarter CM, Lei Y, Chen W, Mulchandani A (2005) Amperometric microbial sensor for p-nitrophenol using Moraxella sp. modifed carbon paste electrode. Biosensor Bioelectron 21:523–527CrossRefGoogle Scholar
  21. Nikolelis PD, Krull JU, Wang J, Mascini M (1997) Proceedings of the NATO Advanced research Workshop, Smolenice, SlovakiaGoogle Scholar
  22. Premi BR, Lal BB, Joshi VK (1995) Distribution pattern of bittering principles in kinnow fruit. J Food Sci 31:140–141Google Scholar
  23. Puri JS, Kothari RM, Kennedy JF (1996) Biochemical basis of bitterness in citrus fruits and biotech approaches for dibittering. Crit Rev Biotech 16:145–155CrossRefGoogle Scholar
  24. Rastogi S, Kumar A, Mehra NK, Makhijani SD, Manoharan A, Gangal V, Kumar R (2003) Development and characterization of a novel immobilized microbial membrane for rapid determination of biochemical oxygen demand load in industrial wastewater. Biosens Bioelectron 18:23–29PubMedCrossRefGoogle Scholar
  25. Reznikoff WS (2003) Tn5 as a model for understanding DNA transposition. Mol Microbiol 47:1199–1206PubMedCrossRefGoogle Scholar
  26. Riedel K, Renneberg R, Kuehn M, Scheller F (1988) A fast estimation of BOD with microbial sensors. Appl Microbiol Biotechnol 28:316–318Google Scholar
  27. Riedel K, Naumov AV, Boronin LA, Golovleva LA, Stein J, Scheller F (1991) Microbial sensors for determination of aromatics and their chloroderivatives. Part II: Determination of 3-chlorobenzoate using a Pseudomonas containing biosensors. Appl Microbiol Biotechnol 35:557–562CrossRefGoogle Scholar
  28. Tan HM, Cheong SP, Tan TC (1994) An amperometric benzene sensor using whole cell Pseudomonas putida ML2. Biosens Bioelectro 9:1–8CrossRefGoogle Scholar
  29. Vaks B, Lifshitz A (1981) Debittering of Orange juice by bacteria which degrade limonin. J Agric Food Chem 29:1258–1261CrossRefGoogle Scholar
  30. Yoo EH, Lee SY (2010) Glucose biosensors: an overview of use in clinical practice. Sensor 10:4558–4576PubMedCrossRefGoogle Scholar
  31. Zhang S, Zhao H, John R (2001) Development of a quantitative relationship between inhibition percentage and both incubation time and inhibitor concentration for inhibition biosensors — theoretical and practical considerations. Biosens Bioelectron 16:1119–1126PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag and the University of Milan 2011

Authors and Affiliations

  1. 1.Department of Biotechnology and Environmental SciencesThapar UniversityPatialaIndia

Personalised recommendations