Improving Formulation of Biocontrol Agents Manipulating Production Process

  • J. UsallEmail author
  • N. Teixidó
  • M. Abadias
  • R. Torres
  • T. Cañamas
  • I. Viñas
Part of the Plant Pathology in the 21st Century book series (ICPP, volume 2)


There are several reasons of the limited number of commercial available biocontrol agents, such as the difficulties in developing a shelf-stable formulated product that retains biocontrol activity. This chapter shows that it is possible to improve the formulations during the production process and describes several examples of improving liquid and dry formulations using different strategies such as grow microorganisms in a w modified media, under sublethal thermal stress conditions or preservation in isotonic solutions.

Liquid formulation of C. sake was improved growing the cells in molasses medium with a w modified to 0.98 with the addition of sorbitol and preserved with an isotonic trehalose solution. After 180 days of storage at 4°C, the viability of this formulate was 100% and the efficacy against Penicillium expansum on apples was more than 95% rot reduction.

Spray drying formulations were improved by modifying growth media or temperatures during growing period. The biocontrol agent Pantoea agglomerans grown during 48 h in NaCl 0.97 a w modified medium could increase their viability after spray drying formulation from 6% in unmodified medium to near 30% without affecting their biocontrol potential.

In contrast Candida sake cells grown in unmodified molasses medium exposed to mild heat treatments at 30°C or 33°C during mid or late-exponential or early or mid-stationary growth phases showed an increase of survival when are exposed to lethal shock at 40°C, but only a very reduced improvement after spray drying formulation.

Finally the combination of thermal and osmotic stress was studied in order to improve fluidized bed drying formulations of P.agglomerans. The results showed than using NaCl to adjust a w to 0.988 in the growth medium and increasing the temperature to 35°C during 1 h in the early stationary phase could get a good formulate with only 0.5 log reductions during fluidized bed drying process.


Liquid formulation spray drying thermal stress fluidized bed drying osmotic stress isotonic solutions candida sake pantoea agglomerans 



The authors are grateful to Spanish government (Ministerio de Ciencia y Tecnología) for grants AGL-2002-01137 and AGL-2005-02510 and to FEDER (Fondo Europeo de Desarrollo Regional) for their financial support.


  1. Abadias M, Teixidó N, Usall J, Viñas I, Magan N (2000) Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake. J Appl Microbiol 89:1009-1017CrossRefPubMedGoogle Scholar
  2. Abadias M, Teixidó N, Usall J, Viñas I, Magan N (2001) Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation. Can J Microbiol 47:123-129CrossRefPubMedGoogle Scholar
  3. Abadias M, Usall J, Teixidó N, Viñas I (2003) Liquid formulation of postharvest biocontrol agent Candida sake CPA-1 in isotonic solutions. Phytopathology 93:436-442CrossRefPubMedGoogle Scholar
  4. Abadias M, Teixidó N, Usall J, Solsona C, Viñas I (2005) Survival of the postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying. Biocontrol Sci Tech 15:835-846CrossRefGoogle Scholar
  5. Ananta E, Knorr D (2004) Evidence on the role of protein biosynthesis in the induction of heat tolerance of Lactobacillus rhamnosus GG by pressure pre-treatment. Int J Food Microbiol 96:307-313CrossRefPubMedGoogle Scholar
  6. Ang D, Liberek K, Skowyra D, Zylicz M, Georgopoulos C (1991) Biological role and regulation of the universally conserved heat-shock proteins. J Biol Chem 266:24233-24236PubMedGoogle Scholar
  7. Arnold KW, Kaspar CW (1995) Starvation- and stationary-phase-induced acid tolerance in Escherichia coli O157:H7. Appl Environ Microbiol 61:2037-2039PubMedGoogle Scholar
  8. Attfield PV, Raman A, Northcott C (1992) Constructions of Saccharomyces cerevisiae strains that accumulate relatively low concentrations of trehalose, and their application in testing the contribution of the dissacharide to stress tolerance. FEBS Microbiol Lett 94:271-276Google Scholar
  9. Beever RE, Laracy EP (1986) Osmotic adjustment in the filamentous fungus Aspergillus nidulans. J Bacteriol 168:1358-1365PubMedGoogle Scholar
  10. Boutibonnes P, Tranchard C, Hartke A, Thammavongs B, Auffray Y (1992) Is thermotolerance correlated to heat shock protein synthesis in Lactococcus lactis subsp. lactis? Int J Food Microbiol 16:227-236CrossRefPubMedGoogle Scholar
  11. Cañamás TP, Viñas I, Usall J, Magan N, Morelló JR, Teixidó N (2007) Relative importante of amino acids, glycine-betaine and ectoine síntesis in the biocontrol agent Pantoea agglomerans CPA-2 in response to osmotic, acidic and heat stress. Lett Appl Microbiol 45:6-12CrossRefPubMedGoogle Scholar
  12. Cañamás TP, Viñas I, Usall J, Magan N, Solsona C, Teixidó N (2008) Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying. J Appl Microbiol 104:767-775CrossRefPubMedGoogle Scholar
  13. Corcoran BM, Stanton C, Fitzgerald GF, Ross RP (2005) Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 71:3060-3067CrossRefPubMedGoogle Scholar
  14. Costa E, Teixidó N, Usall J, Fons E, Gimeno V, Delgado J, Viñas I (2002) Survival of Pantoea agglomerans strain CPA-2 in a spray-drying process. J Food Protect 65:185-191Google Scholar
  15. Csonka LN (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 53:121-147PubMedGoogle Scholar
  16. De Angelis M, Di Cagno R, Huet C, Crecchio C, Fox PF, Gobbetti M (2004) Heat-shock response in Lactobacillus plantarum. Appl Environ Microbiol 70:1336-1346CrossRefPubMedGoogle Scholar
  17. Droby S, Cohen L, Daus A, Weiss B, Horev B, Chalutz E, Katz H, Keren-Tzur M, Shachnai A (1998) Commercial testing of Aspire: A yeast preparation for the biological control of postharvest decay of citrus. Biol Control 12:97-101CrossRefGoogle Scholar
  18. Ellis SW, Grindle M, Lewis DH (1991) Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and -resistant strains of Neurospora crassa. Mycol Res 95:457-464CrossRefGoogle Scholar
  19. Gouesbet G, Jan G, Boyaval P (2002) Two-dimensional electrophoresis study of Lactobacillus delbrueckeii subsp. bulgaricus thermotolerance. Appl Environ Microbiol 68:1055-1063CrossRefPubMedGoogle Scholar
  20. Greenacre EJ, Brocklehurst TF, Waspe CR, Wilson DR, Wilson DG (2003) Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20°C: optimization and modelling. Appl Environ Microbiol 69:3945-3951CrossRefPubMedGoogle Scholar
  21. Hall BG (1983) Yeast thermotolerance does not require protein synthesis. J Bacteriol 156:1363-1365PubMedGoogle Scholar
  22. Hallsworth JE, Magan N (1994) Effect of carbohydrate type and concentration on polydydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi. Microbiology 140:2705-2713CrossRefGoogle Scholar
  23. Hallsworth JE, Magan N (1996) Culture age, temperature and pH affect the polyol and trehalose contents of fungal propagules. Appl Environ Microbiol 62:2435-2442PubMedGoogle Scholar
  24. Hottiger T, Boller T, Wiemken A (1989) Correlation of trehalose content and heat resistance in yeast mutants altered in the RAS / adenylate cyclase pathway: Is trehalose a thermoprotectant? FEBS Lett 255:431-434CrossRefPubMedGoogle Scholar
  25. Janisiewicz WJ, Jeffers SN (1997) Efficacy of commercial formulation of two biofungicides for control of blue mold and gray mold of apples in cold storage. Crop Protect 16:629-633CrossRefGoogle Scholar
  26. Jorgensen F, Stephens PJ, Knochel S (1995) The effect of osmotic shock and subsequent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes. J Appl Bacteriol 79:274-281Google Scholar
  27. Kets EPW, Galinski EA, de Bont JAM (1994) Carnitine: a novel compatible solute in L. plantarum. Arch Microbiol 162:243-248CrossRefGoogle Scholar
  28. Ko R, Smith LT, Smith GM (1994) Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes. J Bacteriol 176:426-431PubMedGoogle Scholar
  29. Leslie SB, Teter SA, Crowe LM, Crowe JH (1994) Trehalose lowers membrane phase transitions in dry yeast cells. Biochim Biophys Acta 1192:7-13CrossRefPubMedGoogle Scholar
  30. Mattick KL, Jorgensen F, Legan JD, Lappins-Scott HM, Humphrey TJ (2000) Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance. Appl Environ Microbiol 66:4921-4925CrossRefPubMedGoogle Scholar
  31. McAlister L, Finkelstein DB (1980) Heat-shock proteins and thermal resistance in yeast. Biochem Biophys Res Commun 93:819-824CrossRefPubMedGoogle Scholar
  32. Nunes C, Usall J, Teixidó N, Viñas I (2001) Biological control of postharvest pear diseases using a bacterium Pantoea agglomerans (CPA-2). Int J Food Microbiol 70:53-61CrossRefPubMedGoogle Scholar
  33. Nunes C, Usall J, Teixidó N, Fons E, Viñas I (2002) Postharvest biological control by Pantoea agglomerans (CPA-2) on Golden Delicious apples. J Appl Microbiol 92:247-255CrossRefPubMedGoogle Scholar
  34. O’driscoll, Cormac GM, Gahan M, Hill C (1996). Adaptative acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl Environ Microbiol 62:1693-1698PubMedGoogle Scholar
  35. Periago PM, van Schaik W, Abee T, Wouters JA (2002) Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579. Appl Environ Microbiol 68:3486-3495CrossRefPubMedGoogle Scholar
  36. Piper PW (1993) Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 11:339-356CrossRefPubMedGoogle Scholar
  37. Poirier I, Maréchal PA, Evrard C, Gervais P (1998) Escherichia coli and Lactobacillus plantarum responses to osmotic stress. Appl Microbiol Biotechnol 50:704-709CrossRefPubMedGoogle Scholar
  38. Prasad J, McJarrow P, Gopal P (2003) Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying. Appl Environ Microbiol 69:917-925CrossRefPubMedGoogle Scholar
  39. Rapoport AI, Puzyrevskaya OM, Saubenova MG (1998) Polyols and resistance of yeast to dehydration. Microbiology (NY) 57:269-271Google Scholar
  40. Rhodes DJ (1993) Formulation of biological control agents. In: Jones DG (ed) Exploitation of microorganisms. Chapman & Hall, London, pp 411-439Google Scholar
  41. Ross RP, Desmond C, Fitzgerald GF, Stanton C (2005) Overcoming the technological hurdles in the development of probiotic food. J Appl Microbiol 98:1410-1417CrossRefPubMedGoogle Scholar
  42. Sanders JW, Venema G, Kok J (1999) Environmental stress responses in Lactococcus lactis. FEMS Microbiol Rev 23:483-501CrossRefGoogle Scholar
  43. Swan TM, Watson K (1999) Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat-shock proteins and trehalose. Can J Microbiol 45:472-479CrossRefPubMedGoogle Scholar
  44. Teixeira P, Castro H, Kirby R (1994) Inducible thermotolerance in Lactobacillus bulgaricus. Lett Appl Microbiol 18:218-221CrossRefGoogle Scholar
  45. Teixeira P, Castro H, Mohacsi-Farkas C, Kirby R (1997) Identification of sites of injury in Lactobacillus bulgaricus during heat stress. J Appl Microbiol 83:219-226CrossRefPubMedGoogle Scholar
  46. Teixidó N, Viñas I, Usall J, Magan N (1998a) Control of blue mold of apples by preharvest application of Candida sake grown in media with different water activity. Phytopathology 88:960-964CrossRefPubMedGoogle Scholar
  47. Teixidó N, Viñas I, Usall J, Magan N (1998b) Improving ecological fitness and environmental stress tolerance of the biocontrol yeast Candida sake (strain CPA-1) by manipulation of intracellular sugar alcohol and sugar content. Mycol Res 102:1409-1417CrossRefGoogle Scholar
  48. Teixidó N, Usall J, Palou L, Asensio A, Nunes C, Viñas I (2001) Improving control of green and blue molds on oranges by combining Pantoea agglomerans (CPA-2) and sodium bicarbonate. Eur J Plant Pathol 107:685-694CrossRefGoogle Scholar
  49. Teixidó N, Cañamás TP, Usall J, Torres R, Magan N, Viñas I (2005) Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross protection in the biocontrol agent Pantoea agglomerans CPA-2. Lett Appl Microbiol 41:248-252CrossRefPubMedGoogle Scholar
  50. Teixidó N, Cañamás TP, Abadias M, Usall J, Solsona C, Casals C, Viñas I (2006) Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments. J Appl Microbiol 101:927-937CrossRefPubMedGoogle Scholar
  51. Tiligada E, Miligkos V, Ypsilantis E, Papamichael K, Delitheos A (1999) Molybdate induces thermotolerance in yeast. Lett Appl Microbiol 29:77-80CrossRefPubMedGoogle Scholar
  52. Usall J, Teixidó N, Fons E, Viñas I (2000) Biological control of blue mould on apple by a strain of Candida sake under several controlled atmosphere conditions. Int J Food Microbiol 58:83-92CrossRefPubMedGoogle Scholar
  53. Usall J, Teixidó N, Torres R, Ochoa de Eribe X, Viñas I (2001) Pilot test of Candida sake (CPA-1) applications to control postharvest blue mold on apple fruit. Postharvest Biol Tec 21:147-156CrossRefGoogle Scholar
  54. Van Eck JH, Prior BA, Brandt EV (1993) The water relations of growth and polyhydroxy alcohol production by ascomycetous yeasts. J Gen Microbiol 139:1047-1054Google Scholar
  55. Viñas I, Usall J, Teixidó N, Sanchis V (1998) Biological control of major postharvest pathogens on apple with Candida sake. Int J Food Microbiol 40:9-16CrossRefPubMedGoogle Scholar
  56. Watson K, Dunlop G, Cavicchioli R (1984) Mitochondrial and cytoplasmic protein syntheses are not required for heat-shock acquisition of ethanol and thermotolerance in yeast. FEBS Lett 172:299-302CrossRefPubMedGoogle Scholar
  57. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214-1222CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • J. Usall
    • 1
    Email author
  • N. Teixidó
    • 1
  • M. Abadias
    • 1
  • R. Torres
    • 1
  • T. Cañamas
    • 1
  • I. Viñas
    • 2
  1. 1.IRTA, UdL-IRTA Centre, XaRTA-PostharvestLleidaCatalonia
  2. 2.University of Lleida, UdL-IRTA Centre, XaRTA-PostharvestLleidaCatalonia

Personalised recommendations