Valorization of the peptidic fraction of cheese whey

Tese de Doutoramento em Engenharia Química e BiológicaCheese whey is a waste effluent with a hig-, polluting content that cannot disposed directly into the environment and can become an environmental and economical problem for dairy industries Cheese whey treatment for disposal can be expensive and laborious as it is highly putrescible and has a very low solid concentration. Valorisation of dairy by-products is thus of great interest for economic and environmental reasons. Bovine milk whey proteins are widely used in food formulations due to their nutritional and functional properties. In fact, whey proteins have a hig-, nutritional capacity and balanced amino acid content, particularly essential amino acids. Furthermore, major whey proteins, a.-Iactalbumin and rl-Iactoglobulin, are an important source of bioactive peptides, compounds with a health promoting potential The functional applications of whey proteins include emulsification, gelation, foaming and filler/water binder The wish of the food industry to convert waste products into value-added, high-priced commodities has inspired a !?lowing interest in the development of processes for the enhancement of whey protein functionality. Thus, the modification of whey proteins to improve their functional properties in specific food systems has become a focus of current research. One market that is in huge expansion is lhat of health-promoting foods. However, such novel products have to meet consumer acceptance, in terms of efficacy, organoleptic properties and price Therefa-e the development of health-promoting foods comprises a range of processes which need to be integrated, including optimisation of protein hydrolysis, peptide characterization, sbJdy of peptides' physical-chemical properties and interactions with other food components (Iipids, pOlysaccharides, salts and others) and establishment of a standard methodolo~ to determine biological activity in wvo. Based on the premises described in the previous para!?laphs, the work presented in this document is the result of a plan that aimed at studying the hydrolysis of whey proteins for food applications In particular, lhe research undertaken was directed to the hydrolysis of whey proteins (aiming at changing their functional properties) and to the study of rheological interactions between whey proteins/hydrolysates and galactomannans, with the final goal of obtaining new textures, with hig-, protein content or with interesting (eg bioactive) peptides that can be used in existing food formulations or in the development of new food products The hydrolysis of whey proteins was performed with the aid of enzymes, both free and immobilized in different carriers. A comparison was established for the various conditions tested based on the enzyme's activity and specificity, kinetic parameters and peptide profile of the hydrolysates produced. The gelling properties of the hydrolysates were tested and the hydrolysates were combined with a polysaccharide (Iocust bean ~m), in arder to evaluate the interaction of lhose components in terms of possible new functional properties The work performed allowed to conclude lhat the choice of lhe hydrolysis enzyme is particularly important in determining the properties of the resulting hydrolysates. Besides choosing the type of enzyme it is also important to select an adequate fa-m of the chosen enzyme with the adequate purity and treatment (for instance a treated trypsin with low chymotryptic activity) for the desired application, as different hydrolysates are achieved with different forms of the enzyme The selection of the adequate operational conditions (time, pH and temperature) also determines the composition of the resulting hydrolysate. Hig-,er reaction times lead obviously to higher degrees of hydrolysis and smaller peptides (usually more hydrophobic) and pH and temperature determine the resistance of whey proteins to the hydrolysis as well as the activity of the enzyme. Considering the enzyme immobilization procedures, the activity recovery was 100 with ali carriers except for trypsin crosslinked on zeolites, where it was satisfactory However, when a more purified enzyme from bovine pancreas was used with glyoxyl-spent grain or POS-PVA wilh glutaraldehyde, the activity retention was of 46 % and 73 % against 11 % and 9 % with crude enzyme. Thus it can be stated that trypsin was successfully immobilized on spent ~ains by multipoint covalent attachment using glycidol and on POS-PVA functionalized with glutaraldehyde Even so, the immobilized trypsin with the hi~est actillity was achieved with covalent binding through glutaraldehyde to silanized zeolite followed by crosslinking with glutaraldehyde, probably due to a positive effect of the zeolite on the enzyme activity Only trypsin immobilized on spent grain showed significant activity towards whey proteins Allhou~ trypsln immobilized on cross-Iinked zeolite NaY and trypsin covalently immobilized m POS-PVA and glutaraldehyde have shown a high activity towards a small substrate (eg BAPNA), this did not happen when whey proteins were used as substrate. Peptide profile of hydrolysates trom whey protein isolate wilh both free and immobilized on spent grain enzymes were similar, which indicates lhat spent grains can be used as carriers for trypsin to produce hydrolysates similar to those obtained with the free enzyme The contrai of the extent of the hydrolytic reaction is extremely important to ensure that a hydrolysate wilh the intended properties is obtained. The immobilizatlon allows such control by simplywithdrawing the enzyme trom the reaction medium, without the need of using high temperatures or considerable pH shifts. Furlher, immobilization also allows the reuse of the enzyme, with obllious advantages trom the economical point of view. The gelling ability of whey proteins can be changed by limited hydrolysis Depending on the environmental conditions it can either be improved or impaired. At WPC concentrations close to lhe gelling point, stronger gels with lower gelation temperatures can be achieved with limited hydrolysis ofwhey proteins. However, at hl~er protein concentrations this effect Is Impaired There is an increase of the gel strength with the increase of the protein concentration, as expected, but lhis increase is smaller for the hydrolysates than for the intact proteins. In fact, a similar increase in the protein concentration corresponds to a lower increase in the amount of protein with effective gelation ability in the case of the hydrolysates The relative importance of non-covalent interactions in the structure of whey protein gels seems to increase with the de~ee of hydroslysis. LBG alters the microstructure of whey protein gels by modifying the equilibrium between aggregation and se~egation The gelation time is aiso decreased. The volume of the protein-enriched phase decreases with the increase of the LBG concentration and the protein concentratlon probably increases within that phase The final structure of the gels is a result of the equilibrium between aggregation and se~egation and of the increase of the protein concentration on the protein-enriched phase. The behaviour of gels from whey proteins ar whey protein hydrolysates towards the presence of LBG is very similar. For whey proteins and for whey protein hydrolysates a small amount of LBG in the presence of salt leads to a big enhancement In the gel strength. The gelation process is very sensible to environmental conditions and to processing and often leads to quite coarse data. The factorial planning used in this work ailowed validating conclusions using fewer experiments lhan those needed if no planning had been used, while still getting statistical siE1J1ificance out of the results. However, as many factors are involved, the modelling of the process was not strai~tforward. A simple linear or quadratic function was generally not enou~ to accurately describe lhe system behaviour. In short, hydrolysates with many different functional, nutritional and biologlcal properties can be produced by manipulating the hydrolysis conditions and lhe source of the enzyme (alone or in combination; free or immobilized; pure or Impure; H). The introduction of a pOlyssacharide allows an even bigger range of functional properties and can be used to ad]ust the desired property to the desired appIication.União Europeia. Fundo Social Europeu - Formação Avançada de Docentes do Ensino Superio