Proteomics as a Tool for Product and Process Validation and Optimization

Read Also

In a pioneering work, Incamps et al. (3) performed a systematic proteomic analysis along a plant-scale wet fractionation process of alfalfa biomass. The manufacturing process induces significant changes including chemical modifications, heat-shock protein responses and proteolytic degradation. It was also demonstrated that during biomass processing, especially thermal treatment, a certain level of cellular regulation is still conserved such as induction of heat shock and redoxstress proteins. Proteolytic degradation of structural proteins and other changes in meat also start during storage and the first processing step of protein-rich food of animal origin such as porcine meat (21).

Advances in protocols for food processing have resulted in a reduction of the manufacturing time and optimization of product quality. The increase of production capacity also increases the need for better process control. Software-driven computer control systems, e.g. in milk or meat processing industry have made it easier and faster to change parameters during processing and production cycles. Proteins are largely responsible for the characteristics of many food products during the manufacturing process. Physicochemical properties, such as viscosity, thermal conductivity and vapor pressure, but also nutritional and sensory properties of milk, meat and cereal-derived products depend on their protein composition and content (22). In wheat flour-derived products the optimal characteristics are determined by gluten proteins, in milk and milk products, the dominating protein is casein. The proteomic-based approach for validation of a process for production of wheat-based foods is shown in Fig. 1.

Figure 1 (ref. 22)

Because of their importance, both proteins/protein groups are well characterized (23, 24). Protein compositions of other foods such as meat and meat products, or fruit and vegetables are more complex, and the change of physicochemical properties during processing depends on more than one highly abundant protein (21,25). A significant amount of pork and beef is consumed fresh, and meat texture and juiciness are the most important of all organoleptic characteristics contributing to their quality. According to proteomic studies, the meat tenderness in both pork and beef is associated with the structural proteins such as myosin, actin, desmin and tubulin (26). In a semi-quantitative comparison, based on the comparison of intensity of different protein/peptide spots in 2D electrophoresis Laville et al. (27) identified 14 different proteins that are a kind of ‘candidate biomarker’ for shear force values of cooked meat. Further studies about the meat texture and drip loss were also performed (28). Sayd et al. (29) also showed that some proteins from sarcoplasmatic reticulum of pig muscle, especially enzymes involved in oxidative metabolism, are responsible for color development which is the next organoleptic characteristic responsible for meat quality. Muscle mitochondria are also highly sensitive to protein carbonylation. By applying a complex labeling strategy, more than 200 carbonylated proteins were detected. Other oxidative modifications such as nitrosylation and hydroxylation were also detected in many carbonylated proteins. This finding provides further evidence of the susceptibility of muscle mitochondrial proteins to oxidative damage (30). Storage and treatment during production process are also responsible for changes in fish muscle proteins, again responsible for product properties (31,32).

Technological treatment may affect the overall food quality. As demonstrated above, induction of some proteins during the early stages of the process is one of the unexpected changes. In-appropriate heat treatment of milk, meat, cereal products or fruits and vegetables can negatively influence the product quality. The main modifications induced by heat treatment are protein denaturation and the complex series of chemical reactions known as Maillard reaction. An extensive review about Maillard reaction, especially from the proteomic point of view, has recently been given (33). Specific properties of food products such as color, texture digestibility, and nutritional value can be affected by the Maillard reaction. As a consequence of involving the side aminogroup of lysine, an essential amino acid, the nutritional value of food can be impaired. Glycation of proteins in meat and meat products is a further change that can affect their quality and nutrition value. It is considered as the first step in Maillard reaction. This reaction can be controlled by modifying food composition, processing and storage conditions (33). Furthermore, the Maillard reaction between amino acids, mainly asparagine and reducing sugars such as fructose, galactose, lactose and glucose can lead to formation of harmful acrylamide in food during roasting, toasting and frying processes (34). Furthermore, carbonylation of milk proteins such as β-lactoglobulin during industrial treatment can induce allergies against milk products. Carbonylated proteins can be detected by immunoblot and identified by MALDI-TOF MS or electrospray ionization tandem mass spectrometry (ESI-MS/MS) after electrophoretic separation and in-gel digestion (35). Scaloni et al. (36) demonstrated that the protein-bound carbonyl content in heat-treated milk samples was positively correlated with the severity of the treatment. On the other hand, well-controlled Maillard reaction can also be induced to achieve specific benefits like aroma generation in baked product and to improve the physicochemical properties of whey proteins (22). Deamidation is further form of chemical degradation of proteins. In this irreversible reaction, glutamine or asparagine are hydrolyzed to glutamic acid or aspartic acid respectively. Mass spectrometric techniques can also be used for detection of this form of protein degradation (37). Posttranslational modifications (PTM) of proteins can cause further modifications during the production process. Heat-susceptible phosphorylated serine and threonine residues can yield dehydroalanine and methyl-dehydroalanine respectively. Different amino acids can also cross-react and form further artificial products, such as lysinoalanine, lanthionine, and histidinoalanine (22,38). The difference in solubility of food proteins, e.g. wheat glutenins, largely reflects their ability to form inter- or intra- molecular disulfide bonds. The newly developed online LC-MS with electron-transfer dissociation is a reliable method for determination of disulfide linkages before and during processing of protein mixtures (39). Further changes in PTMs, especially in glycosylation, are a topic of plethora of proteomic studies (40, 41). Combined with other analytical methods, proteomics gives important information about food quality and safety. Monti et al. (42) demonstrated the use of proteomic methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein identification by liquid chromatography-mass spectrometry (LC-MS/MS) together with capillary electrophoresis for determination of fatty acids and metal ion content in farmed and wild sea bass. They showed that the growth conditions induce significant biochemical and nutritional differences in food quality. In summary, mass spectrometry and mass spectrometry-based proteomics have largely expanded the knowledge of food components. These analytical technologies enable identification and characterization of food components, mainly proteins, carbohydrates and lipids and their changes during the production process and storage. Isotope labeling techniques for quantitative determination of protein-based components that are developed in the last five years can give further, quantitative evaluation and process validation, and determination of batch-to-batch variations (8,43).