State of the Art Manufacturing of Protein Hydrolysates

  • Vijai K. PasupuletiEmail author
  • Steven Braun


The use of protein hydrolysates in microbiological media has been in existence for several decades and the basic manufacturing process of protein hydrolysates has remained the same. However, with increasing use of protein hydrolysates in specialized applications such as animal cell culture processes, the manufacturing of protein hydrolysates has dramatically improved and is still in its infancy to uncover the specific peptide, peptides and combination of individual amino acids that produce intended effects for that application. This will change as the protein hydrolysate manufacturers and end-users exchange information and work towards the common goal of developing the best protein hydrolysates for specific applications. This chapter will review the generic manufacturing of protein hydrolysates describing individual unit operations, problems faced by manufacturers and suggestions for obtaining consistent product and guidelines for the end-users in getting regulatory support and setting up reliable specifications. Finally the chapter concludes with future trends of protein hydrolysates.


Manufacturing Protein hydrolysates Downstream processing Inconsistencies Hydrolysis 


  1. Adler-Nissen JA (1979) Determination of the degree of hydrolysis of food protein hydrolysates by trinitro benzene sulfonic acid. J Agric Food Chem 27:1256–1262CrossRefGoogle Scholar
  2. Adler-Nissen JA (ed) (1986) Enzymatic hydrolysis of food proteins. Elsevier, LondonGoogle Scholar
  3. Ajinomoto Product Brochure (2009) Web site last accessed 21 Dec 2009.
  4. Backwell CFR (1998) Circulating peptides and their role in milk protein síntesis. In: Grimble GK, Backwell FRC (eds) Peptides in mammalian protein metabolism. Portland Press, London, pp 69–78Google Scholar
  5. BD Bionutrients Technical Manual (2009) Web site last accessed 21 Dec 2009.
  6. Blattner FR (1977) Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science 196:161–169CrossRefGoogle Scholar
  7. Bradstreet RB (1954) Kjeldahl method for organic nitrogen. Anal Chem 26:185–187CrossRefGoogle Scholar
  8. Brink KM, Sebranek JG (1993) Combustion method for determination of crude protein in meat and meat products: collaborative study. J AOAC Int 76:787–793Google Scholar
  9. Bucci LR, Unlu L (2000) Protein and amino acid supplements in exercise and sport. In: Wolinsky I, Driskell JA (eds) Energy-yielding macronutrients and energy metabolism in sports nutrition. CRC Press, Boca Raton, FL, pp 191–212Google Scholar
  10. Chae JH, In JM, Kim HM (1998) Process development for the enzymatic hydrolysis of food protein: effects of pre-treatment and post-treatments on degree of hydrolysis and other product characteristics. Biotechnol Bioprocess Eng 3:35–39CrossRefGoogle Scholar
  11. Choung JJ, Chabmberlain DG (1998) Circulating peptides and their role in milk protein síntesis. In: Grimble GK, Backwell FRC (eds) Peptides in mammalian protein metabolism. Portland Press, London, pp 79–90Google Scholar
  12. Cordoba X, Borda E, Puig M (2005) Soy oligopeptides in weaning nutrition. Feed Int 26:14–18Google Scholar
  13. El-Naggar AH (2006) Response of plants to natural protein hydrolysates as a nitrógeno fertilizar and chelating agent in organic agricultural systems. MS Thesis, The Royal Veterinary and Agricultural University, DenmarkGoogle Scholar
  14. Fearon WA (1920) A modified kjeldahl method for the estimation of nitrogen. Dublin J Med Sci 1:28–32Google Scholar
  15. Fres Z (1988) OPA method modified by use of N, N-dimethyl 2-mercaptoehtyl ammonium chloride as thiol component. Anal Chem 330:631–633CrossRefGoogle Scholar
  16. Ganglberger P, Obermüller B, Kainer M, Hinterleitner P, Doblhoff O, Landauer K (2005) Optimization of culture medium with the use of protein hydrolysates. Cell technology for cell products. Proceedings of the 19th ESACT meeting, Harrogate, UKGoogle Scholar
  17. Gilbert ER, Wong EA, Wrbb KE (2008) Peptide absorption and utilization: implications for animal nutrition and health. J Anim Sci 86:2135–2155CrossRefGoogle Scholar
  18. Girón-Calle J, Vioque J, Pedroche J, Alaiz M, Yust MM, Megías C, Millán F (2008) Chickpea protein hydrolysate as a substitute for serum in cell culture. Cytotechnology 57:263–272CrossRefGoogle Scholar
  19. Heidemann R, Zhang C, Qi H, Rule JL, Rozales C, Park S, Chuppa S, Ray M, Michaels J, Konstantinov K, Naveh D (2004) The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology 32:157–167CrossRefGoogle Scholar
  20. Hill RL (1965) Hydrolysis of proteins. Adv Protein Chem 20:37–107CrossRefGoogle Scholar
  21. Holownia T (2008) Production of protein hydrolysates in an enzymatic membrane reactor. Biochem Eng J 39:221–229CrossRefGoogle Scholar
  22. Ikonomou L, Bastin G, Schneider Y-J, Agathos SN (2001) Design of an efficient medium for insect cell growth and recombinant protein production. In Vitro Cell Dev Biol Anim 37:549–559CrossRefGoogle Scholar
  23. Jandik P, Cheng J, Avalovic N (2003) Amino acid analysis in protein hydrolysates using anion exchange chromatography and IPAD. Method Mol Biol 211:155–167Google Scholar
  24. John TG (1993) US Patent 5266685 - Non-bitter protein hydrolyzatesGoogle Scholar
  25. Jun LL, Chuhan-He Z, Zheng Z (2008) Analyzing molecular weight distribution of whey protein hydrolysates. Food Bioprod Process 86:1–6CrossRefGoogle Scholar
  26. Kinnersley AM, Bauer BA, Crabtree KL, Kinnersley C-Y, McIntyre JL, Daniels SE (2003) US Patent 6534446 Method to mitigate plant stressGoogle Scholar
  27. Kwon MS, Dojima T, Park EY (2005) Use of plant-derived protein hydrolysates for enhancing growth of Bombyx mori (silkworm) insect cells in suspension culture. Biotechnol Appl Biochem 41:1–7CrossRefGoogle Scholar
  28. Kwon YL, Yeul SK, Heon KK, Chun BK, Lee KH, Oh DJ, Chung N (2008) Use of soybean protein hydrolysates for promoting proliferation of human keratinocytes in serum-free medium. Biotechnol Letts 30:1931–1936CrossRefGoogle Scholar
  29. Laurant B, Loubna F (2009) Membrane processes and devices for separation of bioactive peptides. Recent Pat Biotechnol 3:61–72CrossRefGoogle Scholar
  30. Limke T (2009) Impact of ultrafiltration of hydrolysates. Gen Eng News 29:29–30Google Scholar
  31. Mazurkova NA, Kolokol’tsova TD, Nechaeva EA, Shishkina LN, Sergeev AN (2008) The use of components of plant origin in the development of production technology for live cold-adapted cultural influenza vaccine. Bull Exp Biol Med 146:144–147CrossRefGoogle Scholar
  32. Mine Engineer Product Manual (2009) Web site last accessed 21 Dec 2009.
  33. Naegeli C (1882) Cited from Hucker and Carpenter. J Inf Dis 40: 485–496, 1927. Ernhrung der neidern Pilze durch Kahlenstoff und Stickstoffuerbindenger Untermuchungen Uber Niedere. Pilze 1Google Scholar
  34. Nagodawithana T (ed) (1995) Savory flavors. Esteekay, Milwaukee, WIGoogle Scholar
  35. Nagodawithana TW (1998) Production of flavors. In: Nagodawithana TW, Reed G (eds) Nutritional requirements of commercially important microorganisms. Esteekay Associates, Milwaukee, WI, pp 298–325Google Scholar
  36. Nagodawithana TW, Nelles L, Trivedi NB (2010) Protein hydrolysates as hypoallergenic, flavors and palatants for companion animals. In: Pasupuleti VK, Demain A (eds) Protein hydrolysates in biotechnology. Springer, The NetherlandsGoogle Scholar
  37. Oliver JM, Duncan HG (2002) US Patent 6387241 Method of sterilization using ozoneGoogle Scholar
  38. Olsen D, Chang R, Willimas KE, Polarek JW (2010) The development of novel recombinant human gelatins as replacements for animal-derived gelatin in pharmaceutical applications. In: Pasupuleti VK, Demain A (eds) Protein hydrolysates in biotechnology. Springer, The NetherlandsGoogle Scholar
  39. Orue MC, Bouhallab S, Garem A (1998) Nanofiltration of amino acid and peptide solutions: mechanisms of separation. J Membrane Sci 142:225–233CrossRefGoogle Scholar
  40. Ota M, Sawa A, Nio N, Ariyoshi Y (1999) Enzymatic ligation for synthesis of single-chain analogue of monellin by transglutaminase. Biopolymers 50:193–200CrossRefGoogle Scholar
  41. Pasupuleti VK (1998) Applications of protein hydrolysates in industrial fermentations. Presented at industrial and fermentation microbiology symposium, LaCrosse, WIGoogle Scholar
  42. Pasupuleti VK (2000) Influence of protein hydrolysates on the growth of hybridomas and the production of monoclonal antibodies. Presented at the waterside conference, Miami, FLGoogle Scholar
  43. Pasupuleti VK (2001) Commercial report on protein hydrolysates and monoclonal antibodies. SAI International, Geneva, ILGoogle Scholar
  44. Pasupuleti VK (2005) Manufacturing of protein hydrolysates and bioactive peptides. Presented at the annual meeting of Institute of Food Technologists, New Orleans, LAGoogle Scholar
  45. Pasupuleti VK (2007) Overview of manufacturing, characterization and screening of protein hydrolysates for industrial media formulations. Presented at Society of Industrial Microorganisms annual meeting, Denver, COGoogle Scholar
  46. Pasupuleti VK, Schie BJ (1998) Production of enzymes. In: Nagodawithana TW, Reed G (eds) Nutritional requirements of commercially important microorganisms. Esteekay, Milwaukee, WI, pp 129–162Google Scholar
  47. Peters JV, Snell EE (1953) Peptides and bacterial growth. J Biol Chem 67:69–76Google Scholar
  48. Sheffield Product Manual (2009) Web site last accessed 21 Dec 2009.
  49. Silvestre MPC, Vieira CR, Silva MR, Carreira RL, Silva VDM, Morais HA (2009) Protein extraction and preparation of protein hydrolysates from rice with low phenylalanine content. Asian J Sci Res 2:146–154CrossRefGoogle Scholar
  50. Sorensen SPL (1908) Enzymestudien, Uber die quantitative Messung Proteolytischer Spaltungen. Die Formol Titrierung. Biochem Z 7:45–101Google Scholar
  51. Tripathi NK, Shrivastva A, Biswal CK, Lakshmana Rao PV (2009) Optimization of culture medium for production of recombinant dengue protein in Escherichia coli. Ind Biotechnol 5(3):179–183CrossRefGoogle Scholar
  52. Tsuji K, Steindler KA, Harrison SJ (1980) Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product. Appl Environ Microbiol 40:533–538Google Scholar
  53. Uchida S, Houjo M, Tochikubo M (2008) Efficient sterilization of bacteria by pulse electric field in micro-gap. J Electrostat 66:427–431CrossRefGoogle Scholar
  54. Yagasaki M (2009) Fermentation technology breakthrough for the formation of dipeptides. Presented at Nutracon 2009, Anaheim, CAGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.SAI InternationalGenevaUSA
  2. 2.Mead Johnson NutritionEvansvilleUSA

Personalised recommendations