Batch-to-batch variation and storage stability of the commercial peptidase preparation Flavourzyme in respect of key enzyme activities and its influence on process reproducibility

European Food Research and Technology - Tập 242 - Trang 1005-1012 - 2015
Michael Merz1, Daniel Appel2, Pieter Berends2, Swen Rabe2, Imre Blank2, Timo Stressler1, Lutz Fischer1
1Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany
2Nestlé Product Technology Centre, Singen, Germany

Tóm tắt

The synergy of endopeptidases and exopeptidases is the key for an efficient hydrolysis of proteins. Flavourzyme is sold as a commercial peptidase preparation from Aspergillus oryzae that exhibits various endo- and exopeptidase activities and, therefore, generates protein hydrolysates with high degrees of hydrolysis. The manufacturer (Novozymes) standardizes the enzyme preparation for one peptidase activity, determined with the marker substrate H-Leu-pNA. However, seven peptidases of Flavourzyme were recently identified and purified, and the significant contribution of six of them to wheat gluten hydrolysis was demonstrated. The knowledge about the batch-to-batch variation and storage stability of the Flavourzyme preparation regarding the other peptidase activities are still unclear, and this is important information for the usage of the enzyme preparation to gain reproducible protein hydrolysis processes. In the present study, we tested 12 Flavourzyme batches for the activity of the seven peptidases. The impact of the storage time on the peptidase activities and the magnitude of the batch-to-batch variation were investigated. In contrast to the activity determined with H-Leu-pNA as a substrate, the variations of the other peptidase activities were noticeable. The variation of the endopeptidase activity was most distinct and the activity decreased during the storage time of the preparation. The variation of the Flavourzyme composition also affected the reproducibility of a casein batch hydrolysis process, which should be taken into account for any future research and industrial application.

Tài liệu tham khảo

Berends P, Appel D, Eisele T et al (2014) Performance of enzymatic wheat gluten hydrolysis in batch and continuous processes using Flavourzyme. Food Sci Technol 58:534–540 Merz M, Eisele T, Claaßen W, Appel D, Rabe S, Stressler T, Fischer L (2015) Continuous long-term hydrolysis of wheat gluten using a principally food-grade enzyme membrane reactor system. Biochem Eng J 99:114–123 Aaslyng MD, Elmore JS, Mottram DS (1998) Comparison of the aroma characteristics of acid-hydrolyzed and enzyme-hydrolyzed vegetable proteins produced from soy. J Agric Food Chem 46:5225–5231 Imm JY, Lee CM (1999) Production of seafood flavor from red hake (Urophycis chuss) by enzymatic Hydrolysis. J Agric Food Chem 47:2360–2366 Parris N, Moreau RA, Johnston DB et al (2008) Angiotensin I converting enzyme-inhibitory peptides from commercial wet- and dry-milled corn germ. J Agric Food Chem 56:2620–2623 Boschin G, Scigliuolo GM, Resta D, Arnoldi A (2014) Optimization of the enzymatic hydrolysis of lupin (Lupinus) proteins for producing ACE-inhibitory peptides. J Agric Food Chem 62:1846–1851 Xue Z, Yu W, Liu Z, Wu M, Kou X, Wang J (2009) Preparation and antioxidative properties of a rapeseed (Brassica napus) protein hydrolysate and three peptide fractions. J Agric Food Chem 57:5287–5293 Celus I, Brijs K, Delcour JA (2007) Enzymatic hydrolysis of brewers’ spent grain proteins and technofunctional properties of the resulting hydrolysates. J Agric Food Chem 55:8703–8710 Raghavan S, Kristinsson HG, Leeuwenburgh C (2008) Radical scavenging and reducing ability of tilapia (Oreochromis niloticus) protein hydrolysates. J Agric Food Chem 56:10359–10367 Merz M, Eisele T, Berends P, Appel D, Rabe S, Blank I, Stressler T, Fischer L (2015) Flavourzyme, an enzyme preparation with industrial relevance—automated nine-step purification and partial characterization of eight enzymes. J Agric Food Chem 63:5682–5693 Merz M, Ewert J, Baur C, Appel D, Blank I, Stressler T, Fischer L (2015) Wheat gluten hydrolysis using isolated Flavourzyme peptidases: product inhibition and determination of synergistic effects using response surface methodology. J Mol Catal B Enzym 122:218–226 Novozymes (2012) Product data sheet—Flavourzyme 1000L. https://www.ulprospector.com/en/na/Food/Detail/4497/331869/Flavourzyme-1000-L. Accessed 26 May 2015 Gibbs PA, Seviour RJ, Schmid F (2000) Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit Rev Biotechnol 20:17–48 Sriranganadane D, Waridel P, Salamin K, Reichard U, Grouzmann E, Neuhaus JM, Quadroni M, Monod M (2010) Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH. J Proteome Res 9:3511–3519 Adler-Nissen J (1979) Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J Agric Food Chem 27:1256–1262 Belitz HD, Grosch W, Schieberle P (2009) Milk and dairy products. In: Belitz HD, Grosch W, Schieberle P (eds) Food chemistry. Springer, Heidelberg, pp 498–545 Iversen SL, Jørgensen MH (1995) Azocasein assay for alkaline protease in complex fermentation broth. Biotechnol Tech 9:573–576 Chrispeels MJ, Boulter D (1975) Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase. Plant Physiol 55:1031–1037 Stressler T, Eisele T, Schlayer M, Kutz-Wahl S, Fischer L (2013) Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis. PLoS One 8:e70055 Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23 Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375–379 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 Wong C, Sridhara S, Bardwell JC, Jakob U (2000) Heating greatly speeds Coomassie blue staining and destaining. Biotechniques 28:426–428 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Kilcawley K, Wilkinson M, Fox P (2002) Determination of key enzyme activities in commercial peptidase and lipase preparations from microbial or animal sources. Enzyme Microb Technol 31:310–320 Hamada JS (2000) Characterization and functional properties of rice bran proteins modified by commercial exoproteases and endoproteases. J Food Sci 65:305–310