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Characterization of the purified actinidin as a plant coagulant of bovine milk

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Abstract

In this work, actinidin was characterized in view of its possible suitability as a coagulant enzyme in the manufacturing process of cheese. The results show that actinidin does exhibit milk-clotting activity, which is correlated with the enzyme concentrations. The combined use of urea and SDS–PAGE led to the conclusion that the milk clot is clearly separated from the whey proteins and corresponds to casein coagulum. Moreover, both the enzyme dependence on pH and temperature and the stability profiles are fully suitable with the chemical–physical conditions adopted during the cheese-making procedure. The analysis of the kinetic constants as well as the electrophoretic pattern of the hydrolysis products suggests that β-casein is the preferred substrate of actinidin, whereas κ-casein seems to be hydrolyzed only in a few large fragments.

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Abbreviations

EDTA:

Ethylenediaminetetraacetic acid

Tris:

(hydroxymethyl)-aminomethane

DTT:

Dithiothreitol

TCA:

Trichloroacetic acid

CT:

Clotting time

References

  1. Fox PF, McSweeney PLH (1998) Dairy chemistry and biochemistry. Kluwer, USA

    Google Scholar 

  2. Kumar A, Grover S, Sharma J, Batish VK (2010) Chymosin and other milk coagulants: sources and biotechnological interventions. Crit Rev Biotechnol 30:243–258

    Article  CAS  Google Scholar 

  3. Tavares JFP, Baptista JAB, Marcone MF (1997) Milk-coagulating enzymes of tuna fish waste as a rennet substitute. Int J Food Sci Nutr 48:169–176

    Article  CAS  Google Scholar 

  4. Rossano R, Piraino P, D’Ambrosio A, O’Connell OF, Ungaro N, McSweeney PLH, Riccio P (2005) Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant. J Biotechnol 120:220–227

    Article  CAS  Google Scholar 

  5. Rossano R, Larocca M, Lamaina A, Viggiani S, Riccio P (2011) The hepatopancreas enzymes of the crustaceans Munida and their potential application in cheese biotechnology. LWT Food Sci Technol 44:173–180

    Article  CAS  Google Scholar 

  6. Lo Piero AR, Petrone G (1999) Purification and partial characterization of an ATP-hydrolyzing serine protease from lettuce leaves. Phytochem 51:349–356

    Article  Google Scholar 

  7. Lo Piero AR, Puglisi I, Petrone G (2002) Characterization of “Lettucine”, a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting. J Agric Food Chem 50:2439–2443

    Article  CAS  Google Scholar 

  8. Egito AS, Girardet JM, Laguna LE, Poirson C, Mollé D, Miclo L, Humbert G, Gaillard JL (2007) Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κ-casein. Int Dairy J 17:816–825

    Article  CAS  Google Scholar 

  9. Tripathi P, Tomar R, Jagannadham MV (2011) Purification and biochemical characterisation of a novel protease streblin. Food Chem 125:1005–1012

    Article  CAS  Google Scholar 

  10. Fadyloglu S (2001) Immobilization and characterization of ficin. Nahrung 2:143–146

    Article  Google Scholar 

  11. Oner MD, Akar B (1993) Separation of the proteolytic enzymes from fig tree latex and its utilization in gaziantep cheese production. LWT Food Sci Technol 26:318–321

    Article  CAS  Google Scholar 

  12. Roseiro LB, Barbosa M, Ames JM, Wilbey RA (2003) Cheesemaking with vegetable coagulants—the use of Cynara L. for the production of ovine milk cheeses. Int J Dairy Technol 56(2):76–85

    Google Scholar 

  13. Arima K, Iwasaki S, Tamura G (1967) Milk clotting enzyme from microorganisms. I. Screening test and the identification of the potent fungus. Agric Biol Chem 31:540–545

    Article  CAS  Google Scholar 

  14. Yamashita T, Higashi S, Higashi T, Machida H, Iwasaki S, Nishiyama M, Beppu T (1994) Mutation of a fungal aspartic proteinase, Mucor pusillus rennin, to decrease thermostability for use as a milk coagulant. J Biotechnol 32:17–28

    Article  CAS  Google Scholar 

  15. Merheb-Dini C, Gomes E, Boscolo M, da Silva R (2010) Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31. (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31). Food Chem 120:87–93

    Article  CAS  Google Scholar 

  16. McDowall MA (1970) Anionic proteinase from Actinidia chinensis. Eur J Biochem 14:214–221

    Article  CAS  Google Scholar 

  17. Kamphuis IG, Drenth J, Baker EN (1985) Thiol proteases. Comparative studies based on the high resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H stem bromelain. J Mol Biol 182:317–329

    Article  CAS  Google Scholar 

  18. Carne A, Moore CH (1978) The amino acid sequence of the tryptic peptides from Actinidin, a proteolytic enzyme from the fruit of Actidia chinensis. Biochem J 173:73–83

    CAS  Google Scholar 

  19. Podivinsky E, Forster RLS, Gardner RC (1989) Nucleotide sequence of actinidin, a kiwi fruit protease. Nucleic Acids Res 17:8363

    Article  CAS  Google Scholar 

  20. Varughese KI, Su Y, Cromwell D, Hasnain S, Xuong NH (1992) Crystal structure of an actinidin-E-64 complex. Biochemistry 3:5172–5176

    Article  Google Scholar 

  21. Katsaros GI, Tavantzis G, Taoukis PS (2010) Production of novel dairy products using actinidin and high pressure as enzyme activity regulator. Innov Food Sci Emerg 11:47–51

    Article  CAS  Google Scholar 

  22. Brocklehurst K, Baines BS, Malthouse JPG (1981) Differences in the interactions of the catalytic groups of the active centres of actinidin and papain. Biochem J 197:739–746

    CAS  Google Scholar 

  23. Aminlari M, Shekarforoush SS, Gheisari HR, Golestan L (2009) Effect of actinidin on the protein solubility, water holding capacity, texture, electrophoretic pattern of beef, and on the quality attributes of a sausage product. J Food Sci 74(3):C221–C226

    Article  CAS  Google Scholar 

  24. Boland MJ, Hardman MJ (1972) Kinetic studies on thiol protease from Actinidia chinensis. FEBS Lett 27(2):282–284

    Article  CAS  Google Scholar 

  25. Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  26. Uchikoba T, Kaneda M (1996) Milk-clotting activity of cucumisin, a plant serine protease from Melon fruit. Appl Biochem Biotech 56:325–330

    Article  CAS  Google Scholar 

  27. Lowry OH, Roseberough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  28. Andrews AT (1983) Proteinases in normal bovine milk and their action on caseins. J Dairy Res 50:45–55

    Article  CAS  Google Scholar 

  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 277:680–685

    Article  Google Scholar 

  30. Guillaume C, Gastaldi E, Cuq JL, Marchesseau S (2004) Rennet-induced gelation of calcium and phosphate supplemented skim milk subjected to CO2 treatment. J Dairy Sci 87:3209–3216

    Article  CAS  Google Scholar 

  31. Kappeler SR, van den Brink HJM, Rahbek-Nielsen H, Farah Z, Puhan Z, Hansen E, Johansen E (2006) Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk. Biochem Bioph Res Co 342:647–654

    Article  CAS  Google Scholar 

  32. Jolles P, Alais C, Jolles J (1963) Etude de la case’ine kappa de vache. Caracte’risation de la liaison sensible a` l’action de la pre’sure. Biochim Biophys Acta 69:511–517

    Article  CAS  Google Scholar 

  33. Macedo IQ, Faro CJ, Pires EM (1993) Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) towards bovine κ-casein. J Agr Food Chem 41(10):1537–1540

    Google Scholar 

  34. Jiang T, Chen LJ, Xue L, Chen LS (2007) Study on milk-clotting mechanism of rennet-like enzyme from glutinous rice wine: proteolytic property and the cleavage site on κ-casein. J Dairy Sci 90:3126–3133

    Article  CAS  Google Scholar 

  35. Hayaloglu AA, Guven M, Fox PF, McSweeney PLH (2005) Influence of starters on chemical, biochemical, and sensory changes in Turkish white-brined cheese during ripening. J Dairy Sci 88:3460–3474

    Article  CAS  Google Scholar 

  36. Gastaldi E, Pellegrini O, Lagaude A, Tarodo de la Fuente B (1994) Functions of added calcium in acid milk coagulation. J Food Sci 59:310–320

    Article  CAS  Google Scholar 

  37. Nàjera AI, de Renobales M, Barron LJR (2003) Effects of pH, temperature, CaCl2 and enzyme concentrations on the rennet-clotting properties of milk: a multifactorial study. Food Chem 80:345–352

    Article  Google Scholar 

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Authors and Affiliations

  1. Dipartimento di Scienze delle Produzioni Agrarie e Alimentari (DISPA), Università di Catania, Via S. Sofia 98, 95123, Catania, Italy

    Angela Roberta Lo Piero, Ivana Puglisi & Goffredo Petrone

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  1. Angela Roberta Lo Piero
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  2. Ivana Puglisi
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  3. Goffredo Petrone
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Correspondence to Angela Roberta Lo Piero.

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Lo Piero, A.R., Puglisi, I. & Petrone, G. Characterization of the purified actinidin as a plant coagulant of bovine milk. Eur Food Res Technol 233, 517–524 (2011). https://doi.org/10.1007/s00217-011-1543-4

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  • DOI: https://doi.org/10.1007/s00217-011-1543-4

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