Proteomics as a promising biomarker in food authentication, quality and safety: A review

ABSTRACT Adulteration and mislabeling have become a very common global malpractice in food industry. Especially foods of animal origin are prepared from plant sources and intentionally mislabeled. This type of mislabeling is an important concern in food safety as the replaced ingredients may cause a food allergy or toxicity to vulnerable consumers. Moreover, foodborne pathogens also pose a major threat to food safety. There is a dire need to develop strong analytical tools to deal with related issues. In this context, proteomics stands out as a promising tool used to report the aforementioned issues. The development in the field of omics has inimitable advantages in enabling the understanding of various biological fields especially in the discipline of food science. In this review, current applications and the role of proteomics in food authenticity, safety, and quality and food traceability are highlighted comprehensively. Additionally, the other components of proteomics have also been comprehensively described. Furthermore, this review will be helpful in the provision of new intuition into the use of proteomics in food analysis. Moreover, the pathogens in food can also be identified based on differences in their protein profiling. Conclusively, proteomics, an indicator of food properties, its origin, the processes applied to food, and its composition are also the limelight of this article.

quality, food safety, and also to find new bioactive components in food (Jagadeesh et al., 2017). Proteomics being one of the omics has been extensively used in food research today (Raposo de Magalhães et al., 2020).
In current era, consumer is not only focusing the sensory attributes of the food product. Consumer demand for safe, nutritious, functional, minimal process, and low additives containing food is increasing (Saeed et al., 2021). In this context, proteomics has the promising potential to prove the reliability and safety of the food products. Proteomics are integral in tracking the product from raw materials to finished products. Furthermore the proteomics has great potential for food industry in various areas of quality, traceability, optimization, storage nutrition, and safety. However, it will take some time to adopt such sophisticated tools at industrial scale.
Proteins act as an indicator of origin, properties, and processes conducted on food (Ortea et al., 2016). Furthermore, proteomics can be used to inspect food quality which can be enhanced by improving the processes used in food production (Creydt & Fischer, 2020).
However, false claims are being made and mislabeling is done by adding another cheaper alternative ingredient instead of the main ingredient written on label (Moore et al., 2012). Different species of animals are used to make a particular product instead of the one written on the label which is a major point from a religious concern as well. This type of adulteration is a food safety threat as the replaced ingredient may cause allergies or health issues to the consumer (Spink & Moyer, 2011). Specific authentication when done with conventional methods is a time-consuming task and cannot be applied to detect adulteration of less than 5% of the product (Špoljarić et al., 2013). In this context, proteomics are an effective tool in the detection of adulterants in food (Girolamo et al., 2014).
Moreover, proteomic analysis of food is faster and gives in-depth analysis of food even at peptide level (Gallardo et al., 2013). Proteins are used as markers of different properties, compositions, and origins of food; therefore, knowledge of proteomics is used for this purpose (Erban et al., 2021). The knowledge of proteomics is applied for product traceability, authentication, and protein profiling of food especially for animal-based products (meat and dairy products) (Leitner et al., 2006;Guarino et al., 2010).
Food safety is an important health concern. Many people across the globe suffer from various foodborne illnesses every year (Bolek, 2020). Sometimes, negligence in food safety concerns can even cause death of the patient in conditions like hemolytic uremic syndrome caused by foodborne pathogens like E. coli O157:H7.
Proteomic approaches also help in the identification of microorganisms based on variations in their proteome, thus helping in the detection of different types of pathogens in food (Pavlovic et al., 2013;Shiny Matilda et al., 2020). Proteomic assays used to identify the protein must also be present in the database library. Various peptide fingerprint libraries are commercially available. One of these libraries is "spectra bank" containing mass spectral fingerprints of the pathogenic bacteria species and major spoilage causing species from seafood, and includes 120 species of interest in the food sector (Gallardo et al., 2013). Proteomic methods like HPLC and MS/ LC-MS can be used to detect and identify toxins and allergens in food (Martinović et al., 2016;Sangeetha et al., 2020).
Furthermore, high-quality products can be made by genetic improvements and studying the changes in protein structure, conformation, and posttranslational modifications (PTMs) due to different processes of food production and thus improving the production process accordingly (Pedreschi et al., 2010). Moreover, a large number of proteins are involved in development of tenderness, color, and odor in meat (Jagadeesh et al., 2017;Zapata et al., 2009). These proteins can be identified in food to enhance the quality of food.
Multiple techniques-based knowledge of proteomics has been used for the protein profiling of food, detection of foodborne pathogens, and identification of protein markers, which involve mass spectrometry (MALDI-TOF and electrospray ionization), HPLC, and gel electrophoresis. This review covers the applications of proteomics in food authentication, quality, and safety using various advanced techniques of food analysis concerned with proteomic approach.

| RE VIE W ME THODOLOGY
The literature search was carried out using scientific databases comprising Scopus, Science Direct, Google scholar, PubMed, Cochrane Library, Science Hub, and Library genesis using the following subject headings proteomics, food authentication, food safety, and biomarker using keywords: "Proteomics as analytical tool, proteomics in food authentication and food quality, adulteration, Foodborne pathogens and their identification, foodborne pathogens and toxins." The authors collected the latest available literature from primary and secondary sources.

| PROTEO MI C S
Proteomics is proteins study at a very large scale. Marc Wilkins in 1994 first use the word proteomics. A proteome is known as a complete set of proteins expressed or produced by a system or organism. Proteomics consists of six classes (Carbonaro, 2004): functional proteomics, expression proteomics, protein-protein interactions, proteome mining, posttranslational modifications, and structural proteomics. It includes the quantitative analysis of a proteome and its protein profiling. Mainly in food sector, proteomics-based techniques are used for authentication of food products for food safety by identifying foodborne pathogens based on variations in their proteome, allergens, and toxins detections, for process validation and optimization, identification of bioactive compounds in functional foods, and for identification of specie-specific biomarkers to authenticate meat and dairy products. Proteomics is an effective approach to identify protein as well as the interactions of protein with other components of foods (Kvasnicka, 2003). Mass spectrometry-based approaches like MALDI-TOF combined with gel electrophoresis and other nongel-based techniques are used in proteomic analysis of food products. Proteomics has vast application potential in different industries including food, feed, health, and medicine. Medical research has better option for diagnostic of various health maladies.
Proteomics has bright future for its wide spared use in food (safety and nutrition) and other allied fields. However, there are various constraints in use of proteomics like lack of validation, standardization, and most importantly complexity in analysis. Proteomics also helps to enhance the quality of food products by studying the effect of different processes on food proteins, thus improving the food processing line. Classification of proteomics is given in Table 1.

| WORKING PRIN CIPLE
The working principle of proteomics consists of the following important steps: (i) protein extraction; (ii) protein or peptide separation and quantification; (iii) protein identification; and (iv) data analysis and interpretation.
The protein extraction is done from the sample used for analysis (Gallardo et al., 2013). In the case of complex samples, partial purification, selective enrichment, or depletion of high abundance proteins is also done (Pedreschi et al., 2010;Surinova et al., 2011).
The separation of proteins is done using two-dimensional gel electrophoresis method. Both these separation methods are done in a bottom-up proteomic approach (Panday & Mann, 2000). The second approach is the top-down approach in which the digestion step is not done and the peptides from fragmented proteins are directly 2D electrophoresis is used and afterward followed by mass spectrometric analysis in-gel digestion of protein is done, which is also called peptide mass fingerprinting. For both these approaches, the protein to be identified is matched with the protein in the database after being subjected to the mass spectrometer. Provided that the corresponding protein is not present in the database, the most homologically related protein is matched (Gallardo et al., 2013). Proteomics workflows for bottom-up and top-down proteomics approaches are shown in Figure 1.

| E XPERIMENTAL ARE A S
Experimental areas that can adopt three key methods in proteomics based on the scientific question to be answered include qualitative, quantitative, and functional proteomics. Proteomics experimental areas, their functions and approaches are also discussed in Figure 2.

| Quantitative proteomics
The relative amount of protein in food proteomes can change mainly because of the composition of the food, the biological variability of the food components, and the technical processing of the food.
Protein concentrations are determined accurately, and quantitative proteomics requires relative quantification of specific proteins between various samples and absolute quantification. While searching for differences between different conditions based on different treatments, GM or non-GM food products, quantitative information at the protein level (absolute protein amount or the relative abundance of a particular protein between different samples) can be very helpful. Natural variations in raw materials, technical processing, and storage are common application studies on changes in the food proteome (Restani et al., 1997). Gel-based methods consist of the comparison of protein abundance determined between different samples as the spot volume and the two-dimensional electrophoresis (2-DE) separation of proteins. Using 2-D fluorescence difference gel electrophoresis (DIGE), protein quantification, authentication, and detection of different adulterants were assessed (Minden et al., 2009).
Preseparation gel base is not required in many cases and relative quantification for primary amines is achieved by using labeled mass tags (Boersema et al., 2009), such as dimethyl labeling, isobaric absolute and relative quantification tags (ITRAQ) (Ross et al., 2004), and tandem mass tags (TMT) (Thompson et al., 2003). Label-free quantification uses multiple assessment methods (Neilson et al., 2011) that take either the spectral counting based on counting the number of peptides assigned to a protein or the area under the curve based on precursor ion spectra peak area in an MS or MS experiment.
Quantitative proteomic methodologies have been greatly improved (Gallien et al., 2011) by the implementation of selected reaction monitoring experiments (SRM), a highly sensitive LC-MS or MS acquisition mode is widely used in biomedical research to validate and verify candidate biomarker proteins.

| Qualitative proteomics
The type of proteomics includes the characterization and detection of protein in food products and can include either all the proteins or specific subsets of proteins of particular interest, which is known as qualitative proteomics. Examples of qualitative proteins include glycolytic enzymes in meat or food allergens and caseins in dairy products. The two most common protein identification methods include Peptide Fragment Fingerprinting (PFF)

| Functional proteomics
Protein-to-protein interactions and protein interactions with other molecules including the effects of concerned interactions are studied in functional proteomics (Coombs, 2020;Kiemer & Cesareni, 2007).
Protein profiling activity-based probes of inhibitor screening and active enzyme levels are used in functional approaches (Serim et al., 2012). Another similar functional proteomics is activity-based proteomics, which studies the basic activities of proteins in a sample, such as inhibition and function (Elmore et al., 2021). Mass spectrometry imaging, a new imaging mode that enables proteins to be mapped within a tissue or sample section, has proven to be a tool for functional proteomics, as it can help to understand their functions by locating the different protein isoforms (Angel & Caprioli, 2013).

F I G U R E 1
Proteomics workflows for bottom-up and top-down proteomics approaches F I G U R E 2 Proteomics experimental areas, their functions, and approaches

| PROTEOMI C S APPROACHE S IN FOOD AUTHENTI C ATI ON
Consumers' demand regarding food authentication and true food labeling is becoming trendier because of health, nutrition, and religious concerns (Meijer et al., 2021). However, with increasing food demand, adulteration and false labeling have become a huge concern in the food chain and have become difficult to monitor food quality. Proteomics methods have currently been utilized as a quicker, adaptive, and high-throughput outlook for assessing the validity and traceability of species in food products due to recent developments in MS (Piñeiro et al., 2003). Therefore, proteomics has been used as a part of multiple studies for the adulteration detection, quantification, and identification in food products by identification and detection of specie-specific protein markers and the protein markers of different processes on food with the help of mass spectroscopic techniques (LC-MS, tandem mass spectrometry, and MALDI-TOF MS). These proteomicsbased techniques have been applied to milk and dairy products, meat, and plant-based foods for product traceability and authenticity (Ortea et al., 2016). MS is used in reference samples for both the identification of species-specific peptide fingerprints and the detection of certain diagnostic peptides in actual samples (Carrera et al., 2007). Proteomics tools take advantage of MS high-throughput ability to achieve rapid, reliable, and responsive detection, characterization, and quantification of peptides and proteins. The complexity of the sample treatment was not ideally suited to high-throughput analysis; proteomics-based methodologies are automated and implemented partially described species in genomic databases.

| Proteomic approaches to study proteinprotein cross-linking in food
Protein-protein cross-linking can be described as covalent bonding between intramolecular protein polypeptides or amino acid residue within intramolecular protein polypeptides (Feeney & Whitaker, 1987). Generally, cross-linking is divided into three categories that may occur in food protein: (1) natural ones that are present in raw material before processing; (2) those that are intentionally added by cross-linking reagents which could be enzymatic or chemical; and (3) those that are created by processing or environmental disruptions, such as UV exposure, heat treatment, pH, and drying changes. Cross-linking affects the nutritional (Friedman, 1999a(Friedman, , 1999b and functional (Singh, 1991) properties Secondly, proteins usually with trypsin are digested into peptides with proteases (Olsen et al., 2004). Thirdly, a combination of liquid chromatography (LC) and 2D-PAGE, the complex peptide mixture, may be isolated before being analyzed by a mass spectrometer.

| Milk and dairy products
Milk is universally important for humans for a lifetime because of the nutrients it provides to the body (Roncada et al., 2012).
Therefore, milk quality is an important factor in the determination of milk properties and safety aspects. Milk proteins play a vital key role in providing functional and structural properties to milk.

| Meat
Global meat production and consumption have increased and coupled with an ever-growing population, there is concern among governmental bodies and industries that such a high demand may be impossible to meet. Therefore, adulteration of meat with plant-

| Safety aspects of genetically modified organisms (GMOs)
The spontaneous effects of transgenesis in GMOs have raised food safety concerns. Therefore, the knowledge of proteomics has also been applied to the authentication of GMOs. Labeling requirements for GMOs have been implemented in more than 40 countries for food safety concerns and consumer knowledge (Gruère et al., 2007), therefore, it is necessary to have the inspection techniques for GM foods to provide correct information. "Substantial equivalence" is used in the inspection of the safety of GM foods (Pedreschi et al., 2010).
The properties and attributes of GM food and traditional food are compared for analysis and assessment (Cellini et al., 2004;Kuiper et al., 2001). GM food safety assessment is done mainly to prove that GM food is safe to consume and will not cause any harm to the consumer (Pedreschi et al., 2010).

| Seafood
Seafood is traded all over the world and there are bigger chances of adulteration and mislabeling of seafood due to the closely related species of fish and other kinds of seafood. Therefore, seafood authentication and origin are a huge concern to ensure product transparency. For example, an expensive species may be replaced with a of two species of mussels using 2-DE and PMF analysis. But they concluded that there can be many reasons for these variations; for example, environmental factors or genetic mutations. An assay based on MALDI-TOF was introduced for the identification of species-specific protein markers in 25 different species of fish which proved successful and also worked for fish products (Mazzeo et al., 2008). They also found that parvalbumins were the main protein markers and allergens in fish. This proved to be the fastest technique for species-specific analysis of protein markers in fish. Some proteomic approaches have also been combined with other assays for species-specific authentication of fish and fish products that are heat processed. Differentiation between species of fish including gadoid fish (Piñeiro et al., 1998), hake species (Piñeiro et al., 2001), and flatfish (Piņeiro et al., 1999) has also been done with the help of qualitative profiling of water-soluble proteins using 2-DE. The protein marker found was parvalbumin and as parvalbumin is heat resistant, this technique may also be applied for heat-processed products.
In a study using label-free and dimethyl labeling quantification, LC-MS/MS-based techniques were used to find the variations between the protein profiles of wild and farmed gilthead sea bream.
As a result of farmed fish, the variations were found in the quality of sarcoplasmic proteins, and parvalbumin was more expressed (Piovesana et al., 2016). Mazzeo and Siciliano (2016), for the authentication of fish species in their study on proteomics, reported several methods which can be used for the fishery products identification using proteomics. In their study, they concluded that MALDI-TOF molecular profiling strategies can lead to fish species identification within minutes, whereas MS proteomics techniques can not only help to identify fish species but also in the identification of major fish allergen (β-PRVBs). Hence, concluding that MS-based methods hold the potential to get authentic results in a short time.

| Proteomics in food quality
Identification and authentication of food products from farm to fork is getting huge attention from industrialists and consumers.
Knowing food composition can not only help to provide clear information to consumers but also help to improve food quality. Proteins, therefore, act as markers of food composition, origin, and processes done on food (Ortea et al., 2016). Thus, the knowledge of proteomics can help in enhancing the quality of food by optimizing the food production process, studying the effect of different processes on proteins in food, and identifying such proteins modified by processing conditions (Pedreschi et al., 2010;Renzone et al., 2021).

| Proteomics in process optimization and validation
The different processes in food production affect the quality of food and thus bring changes to the proteins. These changes help food processors to improve the production process by studying the effects of changes brought by a particular process on proteins. Each process during food production brings specific changes to particular marker proteins which act as an indicator whether the process is done properly or not. For example, product quality can be affected negatively by improper heat processing. Protein denaturation and Millard reactions are the major changes caused by heat processing.
Allergies against milk products can be induced by carbonylation of b-lactoglobulin and other milk proteins during industrial processing (Gašo-Sokač et al., 2010). Therefore, MALDI-TOF MS is used to detect these carbonylated proteins. Proteins also determine the physicochemical properties and nutritional quality of food; hence, some proteins are involved in color, odor, and tenderness of meat. Some proteins (enzymes) involved in oxidative metabolism are involved in color development of meat. Some proteins like myosin, actin, tubulin, and desmin are involved in beef tenderness (Zapata et al., 2009).
These proteins can be detected in meat to ensure the quality of meat in terms of tenderness. Meat quality depends on many different factors including the post mortem factors or modifications in meat proteins. One of the chemical degradations of proteins is dimidiation in which glutamic acid or aspartic acid are produced by hydrolysis of glutamine or asparagine, respectively; the mass spectrometric methods can be applied in the detection of such sort of protein degradation (Ortea et al., 2016;Schmid et al., 2001). Therefore, the outcome of different processing methods on food proteins can be found by the proteomic analysis of food, thus helping in modifying the production process accordingly and showing the validity of a particular process.

| Proteomics in postharvest technology
Proteomics approaches can help to improve the postharvest techniques as well. The postharvest losses of vegetables and fruits in developed countries are 10%-30% and are above about 30%-50% in developing countries per year (Legard et al., 2000;Mathabe et al., 2020). The identification of protein indicators of harvest maturity was reported by Abdi et al. (2002) and also the identification of protein indicators of horticultural quality was done (Lee et al., 2006). After harvesting, the harvest is exposed to different stressful conditions that involve cold storage and modified atmosphere storage which leads to different physiological disorders and changes in it (Chrysargyris et al., 2018). These stressful conditions also cause changes to the proteins in fruits and vegetables which act as indicators to detect the particular processes that cause such changes. Thus, they help in improving the postharvest technology.
The changes in proteins of citrus fruits upon postharvest storage were reported by Lliso et al. (2007). Low-temperature storage leads to the formation of induced proteins that are antifreeze. Currently, studies have been done on the expression of genes and accumulation of proteins in noninjured tissues of fruit during postharvest storage (Feng et al., 2016;Marondedze, 2017). Chilling injury in tomatoes revealed the presence of two thioredoxin peroxidase, cold stress proteins, and an RNA-binding protein in the noninjured part of the tomatoes (Vega-García et al., 2010). Therefore, if such proteins are manipulated, then these technologies and knowledge can benefit the frozen fruit and vegetable industry (Galindo et al., 2007).

| Role in cereals and cereal-based products
Proteomics has proved to be quite useful to improve the quality of cereals and cereal products (Alves et al., 2019). Proteins that are involved in rice quality and flavor can be identified by studying the proteomes of low-and high-quality rice cultivars (Kim et al., 2009);Bahrman et al. (2004) and Grove et al. (2009) have studied the effects of several levels of sulfur and nitrogen on gluten proteins by using proteomic approaches. Several cold-responsive proteins were identified by Yan et al. (2006) by the proteomic identification of rice leaves that were given a chilling treatment. The concentration and composition of proteins determine the quality of durum wheat pasta (De Angelis et al., 2008). In addition to this, stress conditions and temperature also affect the protein content and composition in cereals (Juhász et al., 2020). The high temperature tends to alter the composition of protein during grain filling, therefore the flour and the products resulting from such grains will have changes in their properties. Proteins that change by heat stress (Majoul et al., 2003(Majoul et al., , 2004 have been identified using proteomic methods. Yahata et al. (2005) identified heart-specific proteins that were used as markers to find cultivars that were best in the making of flour (Yahata et al., 2005).
Protein composition helps to determine flour quality (Dupont & Altenbach, 2003;Skylas et al., 2000). Therefore, heat stress during the grain filling can affect the composition of gluten proteins (Hurkman et al., 2013) which increases the size of gluten polymer.
Different proteomics techniques used in sea foods, postharvest, and cereal are discussed in Table 2.

| Proteomics in food safety
The knowledge of proteomics has also been applied to food safety.
By using proteomic techniques, food spoilage microorganisms (Gallardo et al., 2013) and different foodborne pathogens can be identified based on changes in their proteome (Carrera et al., 2020;Pavlovic et al., 2013). Food allergens have also been studied to be  (Mazzeo et al., 2006). In several studies, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) has been used for the identification of foodborne pathogens especially bacteria. Different foodborne pathogens like listeria and Escherichia coli monocytogens (Jadhav et al., 2014) can be detected and identified by this time-saving and cost-effective technique (Singhal et al., 2015). Based on the profiling of the whole bacterial proteome, MALDI-TOF MS is providing a fingerprint specific to the analyzed microorganisms in that specific time and physiological condition (Pavlovic et al., 2013). The fingerprint obtained through this method has many applications such as the characterization of subspecies, strains, and serovar and is specific to the analyzed microorganisms (Piras et al., 2016). Shiga toxin-producing

| Allergens detection
Allergens are the agents which elucidate the body responses.
Food allergens are also a big problem in food safety as there is no cure for allergy and the only way to prevent it is to avoid those foods one is allergic to. Allergens in food can be detected by proteomicsbased techniques. Carrera and colleagues (Carrera et al., 2012) used MS/MS (LIT) mass spectrometer to identify parvalbumin fish allergen in less than 2 h. The identification of allergens is also possible with mass spectrometric methods either gel-based or nongel-based HPLC combined with tandem mass spectrometry. There are six main food allergens. Ninety percent of the food hypersensitivity is due to three plant-based food allergens found in peanut, soy, and wheat (Natarajan et al., 2006;Šotkovský et al., 2008). In allergenomics, immunoblotting of IgE-reactive proteins is done using a serum of allergic patients using 2-DE (Akagawa et al., 2007). Proteolytic processing of peanut allergens (Ara H 3 and its isoallergens) has been studied using proteomics-based techniques (Piersma et al., 2005). Allergens from processed peanuts have been identified using a proteomic-based assay (Chassaigne et al., 2007). The postharvest technique known as controlled atmosphere storage has been shown to change the quantities of allergenic proteins in fruits causing birch pollen allergy (Pedreschi et al., 2007;Sancho et al., 2006). The amounts of 10 different allergens in soybean were found by Houston and colleagues using a label-free proteomic method. Proteomic approaches to assess authenticity of different food products are presented in Table 3.
As the proteomic methods applied for the identification of allergens can be gel-based methods or gel-free methods that are usually

| CON CLUS ION
Proteomics is the potential analytical approaches to classify the safety and quality changes during the storage of food commodities.
Proteomics has various applications in determining the authenticity, adulterants, and toxicity in food products. Various challenges exist in predicting the safety and quality of the products in terms of TA B L E 3 Proteomic approaches to assess authenticity of different food products accuracy. Conclusively, the application of proteomics is an emerging technology that can be helpful to ensure high-quality safe foodstuff.

ACK N OWLED G M ENT
No funding sources.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.