Comparative proteomic profiling of myofibrillar proteins in dry-cured ham with different proteolysis indices and adhesiveness

Highlights

• Instrumental adhesiveness was assessed for the first time in dry-cured ham.

• The proteolysis index is a reliable indicator of proteome degradation.

• Differential adhesiveness is dependent on the proteolysis index.

• Myosin-1, myosin-4 and actin underwent the strongest response to proteolysis.

• Novel candidate biomarkers for proteolysis and adhesiveness.

Abstract

Excessive proteolysis during dry-cured ham processing may lead to high adhesiveness and consumer dissatisfaction. The aim of this research is to identify biomarkers for proteolysis and adhesiveness. Two hundred biceps femoris porcine muscle samples from Spanish dry-cured ham were firstly evaluated for various physicochemical parameters, including their proteolysis indices and instrumental adhesiveness. Proteins of samples with extreme proteolysis indices were separated by two-dimensional electrophoresis and identified by tandem mass spectrometry (MALDI-TOF/TOF). We found that hams of higher proteolysis index had statistically significant increased adhesiveness. Proteomic analysis revealed statistically significant qualitative and quantitative differences between sample groups. Thus, protein fragments increased remarkably in samples with higher proteolysis index scores. In addition, higher proteolysis index hams showed increased degradation for a total of five non-redundant myofibrillar and sarcoplasmic proteins. However, myosin-1, α-actin and myosin-4 proteins were the biomarkers that underwent the most intense response to proteolysis and adhesiveness.

Introduction

Dry-cured ham is a high-quality food product traditionally consumed in Europe. A wide variety of physicochemical changes during the elaboration process influence the final product characteristics, such as flavor and texture (Bermúdez, Franco, Carballo, & Lorenzo, 2014). Salting and ripening are the two main steps in the elaboration process of dry-cured ham. The curing processing requires salt as preserving agent. The amount and type of salt have a significant influence on flavor, texture, color and overall quality of the final product (Paredi et al., 2013; Toldrá, Flores, & Sanz, 1997). The proteins undergo an intense proteolysis during the ripening process, which constitutes the most important enzymatic reaction regarding muscle proteins (Bermúdez et al., 2014b, Lorenzo et al., 2015). Salt content together with many other factors, such as rearing conditions (e.g., feeding, sex and slaughter age), pig line, features of raw product (initial weight, fat level and pH), type of muscle and the ripening process, have a recognized impact on protein denaturation of dry-cured hams (Škrlep et al., 2011, Théron et al., 2011).

The intensity of proteolysis during dry-cured ham processing is often measured by the proteolysis index. It is defined as the percentage of non-protein nitrogen accounting for total nitrogen. The relationship between proteolysis index and texture throughout the dry-cured ham process has been previously studied under a variety of variables, including pH, water and NaCl content, and lipid oxidation (García-Garrido et al., 1999, García-Garrido et al., 2000, Harkouss et al., 2015, Ruiz-Ramírez et al., 2006, Virgili et al., 1995). The proteolysis index of good quality Spanish dry-cured ham is considered to be between 33 and 36%, whereas in Italian ham it is between 22 and 30% (Careri et al., 1993). Myofibrillar and sarcoplasmic proteins are intensively degraded during the ripening process contributing to dry-cured ham texture and ultimate quality (Bermúdez et al., 2014b). However, myofibrillar proteins are a major fraction of the total, accounting for 65–70% muscle proteins (Lana & Zolla, 2016). Accordingly, proteolytic changes in this protein fraction are important for the development of texture and sensorial characteristics. In particular, myosin and actin are two main targets of proteolysis (Mora et al., 2011, Théron et al., 2011). However, excessive proteolysis may generate the pastiness defect, characterized by excessive softness, mushy texture and unpleasant flavors (Škrlep et al., 2011). In this regard, Morales, Arnau, Serra, Guerrero, and Gou (2008) showed that there is a close relationship between pastiness and adhesiveness (degree to which the surface of the ham slice adheres to the palate when compressed by the tongue), as described by Guerrero, Gou, and Arnau (1999). Therefore, the determination of instrumental adhesiveness could be a good indicator of pastiness level in dry-cured ham.

Proteomics has great potential to enhance our knowledge on the biochemical processes underlying the conversion of muscle into meat and identify biomarkers for meat quality traits (Lana and Zolla, 2016, Paredi et al., 2012; Paredi, Sentandreu, Mozzarelli, Fadda, Hollung, & de Almeida, 2013). In dry-cured ham, proteomic studies, generally based on one- or two-dimensional electrophoresis coupled to mass spectrometry, have tackled a wide diversity of topics. For instance, variations in quality traits, evolution of proteolysis during processing, comparative proteomics profiling of biceps femoris and semimembranosus muscles and identification of antioxidant peptides (Di Luccia et al., 2005, Mora et al., 2014, Petrova et al., 2016, Škrlep et al., 2011, Théron et al., 2011). To the best of our knowledge, however, proteome changes linked to differential adhesiveness have not been previously reported.

In this study, we undertook a comparative proteomic profiling in biceps femoris muscle from dry-cured hams with different proteolysis indices, to identify biomarkers for differential proteolytic activity and adhesiveness, using two-dimensional electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF MS).