1. Introduction
One of the most widely consumed food is cheese in all forms. Cheese is a dairy product and the result of milk coagulation after its fermentation or acidification. It is used broadly in cooking industry and recently there have been attempts to relate cheese consumption to human health. This product belongs to the most complex, diverse and taste rich foods appreciated today. The characteristics leading to its differentiation from other foods are the various initials substances used, production process and aging. Specifically, these factors include (i) the milk used for the production of cheese, (ii) the method of coagulation used to transform the milk into a gel or coagulum (e.g., acid vs. rennet), (iii) the acidification characteristics (both rate and time), (iv) the additional steps during the cheese-making process controlling moisture levels of the young cheese (e.g., cooking temperature and pressing and salting conditions). In the ripened cheeses, a critical role for the final product is played by the ripening conditions such as temperature, relative humidity, and rates of O
2, CO
2, and NH
3, that affect the character and diversity of cheese microflora. The traits of these microorganisms and activity contribute to the final characteristics of the product, such as flavor and quality
[1][2][3]. The latter are attributed to the complex dynamics and interactions developed among the microorganisms, growth substrates and proteins in milk and the environment.
Proteolysis is present during cheese production and ripening
[4]. This phenomenon includes the hydrolysis of milk caseins as a result of chymosin action and other proteases already present in milk, such as plasmin, which originate from somatic cells or naturally present in the milk bacteria
[4]. The proteolytic enzymes produce large oligopeptides, which are the substrate for proteinases and peptidases coming from starters lactic acid bacteria (S-LAB) and nonstarters LAB (NS-LAB), to exhibit their activity
[5]. The final outcome of the hydrolysis of milk caseins is the release of 5–30 amino acids length oligopeptides
[6].
During cheese making the most uncontrollable step is the one linked with the microflora growth and their interactions
[1][2][7]. The traditional method for improving the quality characteristics of cheese is the “vaccination” with bacterial strains isolated from high quality aged cheeses
[8]. Even though the product was made in the same industry or following the same manufacturing instructions, its final characteristics demonstrate great variability
[9].
All these variants make the classification and characterization of cheeses difficult and for that reason many classification models exist. The “European” approach, which is used in Mediterranean countries, is based on the technological processes. The European legislation is very strict regarding the classification of cheese and labeling them as Protected Designation of Origin (PDO). Directives by European Union and National laws require that manufacturers label PDO cheeses only if they provide proof concerning the nature and authenticity of their products. PDO cheese adulteration is a problem that has arisen in the past few years and continues to grow, mostly because of the economic profits for the producers and the availability of milk substitutes such as milk powder and curd
[10]. To prevent counterfeit products, especially for the PDO ones, the legislation becomes even more strict and as a result the demand for new technologies concerning fraud detection in food grows
[9].
The identification of the protein amount of various samples, biological or not, is performed by means of proteomics
[11], which is the connecting link among genome, transcriptome and biological function
[12]. The most frequent proteomic methods applied in cheese analysis are 2D-gel electrophoresis followed by MALDI-TOF-MS and HPLC-MS/MS, which are fundamental tools to perform proteomics on dairy products, whereas mass spectrometry is now the most widely applied technique for accurate quantitative and qualitative protein analysis
[13], see also Table 1. Mass spectrometry-based proteomics relies on appropriate protein fractionation and protein or peptide ionization and MS-fragmentation
[14]. The fragmentation pattern of a peptide demonstrates its amino acid sequence and any post-translational modifications (PTMs). By comparing with reference databases (especially SWISS-PROT and PFAM) peptides are assigned to full-length proteins
[15]. Recently, proteomics assays have been applied in food analysis regarding microbial contamination, quality and adulterations
[16][17][18], mainly in milk
[19][20] and meat
[21][22][23][24].
This review focuses on how proteomics assays have been used in order to distinguish proteins produced during ripening or after impurity in chosen PDO cheeses. Moreover, with proteomics methods proteins that act as bioactive molecules can be specified, thus raising the economic value of the cheese. The cheeses of interest in this review are related to Mediterranean diet, which has been well studied during the past few decades because of its beneficial properties to human health and well-being. In particular, wresearchers selected Feta, Graviera Kritis, Mozzarella di Bufala Campana, Parmigiano Reggiano and Grana Padano, which originate from Greece and Italy.
8. Bioactive Peptides
In the past few years there has been a rising need in the industry for reagents that can be received through food and can contribute to improving and maintaining the status of wellness and prevent the appearance of chronic diseases. The need for wellbeing regimes can be obtained by consumption of various meat and vegetable products
[97]. Peptides are small molecules with weight less than 10 kDa and can be found naturally in foods or occur by chemical or enzymatic hydrolysis of the parent proteins
[98]. Usually peptides are latent, when encrypted into proteins, and become active, when released after proteolysis or digestion in gastrointestinal (GI) tract
[99]. They affect many physiological functions in the human body, such as gastrointestinal, cardiovascular, immune, endocrine, and nervous pathways. One of the most important, and accessible to the majority of people, sources of beneficial peptides is milk and dairy products in general
[100][101]. As previously discussed, these peptides are well protected in the protein matrix of milk, requiring proteolytic enzymes, originating from various sources, such as natural rennet or starter bacteria, for their release. A growing body of evidence indicates that milk and dairy products have unique metabolic, signaling and antimicrobial effects, apart from their high nutritional content
[102][103][104][105]. Such peptides are involved in many physiological activities, including regulation of inflammatory and immune response, signaling and metabolic process, antihypertensive, antioxidant and antimicrobial properties
[105].
According to previous studies, pasta filata cheeses (Caciocavallo and Mozzarella) have antibacterial peptides
[106]. Rizello et al.
[106] conducted in vitro simulated gastrointestinal digestion on six buffalo dairy products (Grana, ice cream, yoghurt, Mozzarella, Ricotta and Scamorza) and the isolated peptide digests were characterized by high resolution mass spectrometry, followed by a database-driven specific bioactivity assessment for each identified sequence. The study revealed a great amount of potential bioactive peptides, with characteristics promoting wellness, including antihypertensive, immunomodulatory, antimicrobial, antidiabetic, anticancer and antioxidant. The different manufacturing process of each product explained the diversity of the released peptides.
Antimicrobial peptides (AMPs) with resistance in proteolysis have a direct impact on the gut microbiome assisting to control dysbiosis, by suppressing opportunistic pathogens, such as
Helicobacter pylori [107],
Escherichia coli and
Staphylococcus aureus [108], so that the GI tract can remain healthy. The milk and dairy products from different animal breeds and species have unique compositions of bioactive peptides offering a broad range of sequences to screen for peptides with functional traits of medical and scientific interest
[109]. Tomazou et al.
[102] focused on the potential antimicrobial properties of Feta cheese, to probe for AMPs following an assessment of their stability in an intestine-like environment and they characterized the antimicrobial “load” of the proteomes of interest. Protein sequences from Feta cheese were screened using the publicly available tool AMPA
[110][111] to find sequence stretches with predicted high antimicrobial potential (i.e., low AMPA propensity). The same protein sequences were digested in silico to identify which peptides, that can actually occur in the GI tract, matched the predicted AMPA stretches. The authors stated that Feta cheese proteome had 63–64 AMPs the same with the milk from the sheep and goat breeds, used to manufacture Feta. Albeit the small proteome size of Feta cheese, the proteome presents to have unique antimicrobial properties and resistance in proteolysis AMPs in quite a large amount. Recent work has suggested that lactic acid microbes have a central role in the release of encrypted bioactive peptides during ripening
[112].
The
Lactobacillus helveticus CP790 extracellular protease hydrolyze both αs1- and β-CN of sour bovine milk resulting in the release of angiotensin converting enzyme (ACE) inhibitory peptides. These peptides exhibited antihypertensive activity in spontaneously hypertensive rats as monitored by systolic blood pressure as many studies indicated
[113][114][115][116][117]. Especially, biological activity analysis revealed great variation among samples of Parmigiano Reggiano with the average ACE-inhibitory and antioxidant activities to be elevated in the LL (low salt–low fat) group compared with the HH (high fat–high salt) group. The differences observed are attributed to factors such as milk quality, somatic cells content and extensive proteolysis by indigenous proteolytic enzymes
[118][119][120].
Although there are many ways for the presentation of inhibition of ACE, the results of Solieri et al.
[41] were in accordance with that reported by Bütikofer, Meyer, Sieber, and Wechsler
[121] for hard and semi-hard cheeses and they were comparable. According to the study
[41] the results on DPP-IV-inhibitory activity were lower than those previously reported for the peptide fraction of gouda-type cheese
[122], whereas antioxidant activity data were comparable to those already reported in the literature for PR and Cheddar cheeses’ peptide fractions
[123][124]. The authors reported for the first time 25 peptides with potential bioactive properties from the total of 40 identified peptides for Parmigiano Reggiano. The remaining 15 peptides were reported in different ripening times
[125]. In fact, 13 peptides were ACE inhibitors and already described for their ability to reduce blood pressure in vivo
[6]. In more detail, the β-casein-derived peptide KVLPVPQ, isolated from a commercial functional yogurt, demonstrated strong antihypertensive effects in spontaneously hypertensive rats
[114][126]. The peptide LHLPLP, previously found in various cheeses such as Grana Padano, Parmigiano Reggiano, Gorgonzola, and Cheddar, exhibited very low IC50 value against ACE and has been found able to decrease blood pressure in spontaneously hypertensive rats
[127][128][129][130]. Additional peptides found in all of the examined PR samples showed potent ACE-inhibitory activity, similar to the peptides NLHLPLPLL, YPFPGPIPN, and the peptide YQEPVL
[131][132][133]. Some other peptides exhibited a high inhibitory effect against ACE and in vivo antihypertensive effect. The S1-casein-derived peptide YKVPQL was detected in the majority of PR samples and has been previously identified as an in vitro and in vivo antihypertensive peptide
[129]. Similarly, the β-casein-derived peptide HLPLP has exhibited in vitro and in vivo antihypertensive effect
[134]. The lactotripeptides VPP and IPP, antihypertensive molecules in vivo in humans, were also found in all PR samples
[135]. For the ACE the same standards and peptides have been identified in Parmigiano and Grana
[131]. No information is so far available on cheeses, in which antimicrobial peptides occurred after milk proteins’ hydrolysis and interaction with cheese microflora
[136]. Knowledge of the sequence plays a fundamental role in the studies of food derived bioactive peptides and may help in the identification of the peptide source. Moreover, it can serve as the starting base for the preparation of synthetic analogues to improve the nutritional characteristics of food products. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was adopted for the identification of bioactive peptides in food matrices using a bioinformatics approach,
[137][138][139][140]. An ion trap was used to generate MS/MS and, when required, MS3 spectra, whose potential in solving structural ambiguities in peptide sequences arising from MS/MS measurements/database search has recently been proved to be very successful, leading to the identification of 45 different peptide sequences in fractions of cheese extracts displaying antimicrobial activity
[141]. All the peptides were found to be generated by the hydrolysis of milk caseins (of different mammalian species, according to the type of milk used to produce the cheese), which typically occurred in specific regions of the proteins already known for the presence of bioactive amino acid sequences.
Antithrombotic peptides are also present in milk. The mechanisms involved in blood and milk clotting are proportional, leading to the hypothesis that the C-terminal dodecapeptide of human fibrinogen γ-chain (residues 400–411) and the undecapeptide (residues 106–116) from bovine κ-CN are structurally and functionally quite similar. The casoplatelin is produced by hydrolysis of bovine k-casein with chymosin and shows antithrombotic properties
[142]. This casein-derived peptide sequence affected platelet function and inhibited both the aggregation of ADP-activated platelets and the binding of human fibrinogen λ-chain to its receptor region on the platelets’ surface
[143]. A smaller κ-CN fragment (residues 106–110), casopiastrin, was obtained from trypsin hydrolysates and exhibited antithrombotic activity by inhibiting fibrinogen binding
[143][144]. A second segment of the trypsin κ-CN fragment, residues 103–111, inhibited platelet aggregation but did not affect fibrinogen binding to the platelet receptor
[143][145][146]. Other studies concluded that bioactive peptides isolated from both casein and lactotransferrin had antithrombotic properties by affecting platelet function
[147][148]. Antithrombotic peptides have also been derived from κ-caseinoglycopeptides, that were isolated from several animal species. Bovine κ-caseinoglycopeptide, the C-terminal end of κ-CN (residues 106–169), inhibited von Willebrand factor-dependent platelet aggregation
[149]. Two antithrombotic peptides, derived from human and bovine κ-caseinoglycopeptides, have been identified in the plasma of 5d-old neonatals after breast-feeding and ingestion of cow’s milk-based formula, respectively
[150]. The C-terminal residues (106–171) of sheep κ-casein, or κ-caseinoglycopeptide, decreased thrombin- and collagen-induced platelet aggregation in a dose dependent manner
[151]. Finally, thrombin-induced platelet aggregation was inhibited by pepsin digests of sheep and human lactoferrin
[152].
Casein phosphopeptides (CPP) are the result of trypsin action inαs1-, αs2-, and β-CN
[153]. CPP are stable molecules even after gastrointestinal digestion and create a high soluble complex with calcium phosphate
[154]. Animals fed with casein diet displayed elevated capacity to absorb calcium through distal small intestines compared to animals fed with soy-based diet
[155][156][157]. Calcium physiological is absorbed through passive transport system and this is the way to supplement the human/animal organism with the calcium required for calcification
[158]. Caseinophosphopeptides inhibit caries lesions through recalcification of the dental enamel, so their application in the treatment of dental diseases has been proposed
[159].
Buffalo (
Bubalus bubalis) milk contains a lot of bioactive peptides and they have a significant role preventing various disorders
[160][161][162][163]. Bioactive peptides with antioxidant properties, regulation of oxidative stress in intestinal epithelial cells and erythrocytes, have been isolated from buffalo milk-derived products
[164][165]. Among these, MBCP, a peptide isolated after in vitro digestion of Mozzarella di Bufala Campana DOP, has shown good stability to brush border exopeptidases and a high bioavailability
[166]. In a study conducted by Tenore et al.
[165], the therapeutic potential of MBCP in inflammatory bowel disease (IBD) was discussed after submitting Caco-2 cell, which resembles colonic enterocytes
[167], in in vitro digestion conditions. In mammals, enterocytes are renewed continuously every 4–8 days through an organized cycle involving proliferation, differentiation and programmed cell death. The proliferation to differentiation transition (PDT) is a critical step in the continual renewal of a normal intestinal epithelium [166. Indeed, Caco-2 cells express tight junctions, microvilli, enzymes and transporters functionally similar to colonic enterocyte
[167]. Moreover, this cell line has the capacity to trigger a pro-inflammatory reaction in response to stimulants like TNF-α, a known mediator of gastrointestinal mucosal barrier injury
[168][169]. The results from the latter study revealed that MBCP can modulate the differentiation and permeability in Caco-2 cells stimulated with TNF-α and to attenuate inflammation and hypermotility in murine models of intestinal inflammation. Borelli et al.
[170][171] stated that intracolonic administration of DNBS induced intestinal inflammation associated to an increase of epithelial permeability, symptoms that were inverted by oral MBCP administration as demonstrated by the reduction of colon weight–colon length ratio, histological alterations, IκBα phosphorylation and of NF-κB activation associated with DNBS administration. Intestinal permeability is a predisposing factor for the development of IBD, as well as in the IBD ongoing bowel symptoms
[168][172]. Moreover, inflammation reduces barrier integrity and affects the normal intestinal permeability
[173]. Studies on Caco-2 cells have proved that MBCP restored tight junctions altered by TNF-α. Tight junctions are multiprotein complexes that maintain the intestinal barrier while regulating permeability
[174]. Intestinal permeability increased after DNBS administration and, more importantly, MBCP restored the impaired permeability. It is clinically well established that inflammation in the gut causes debilitating symptoms due to motility disturbances
[175], which were balanced after treatment with MBPC, without causing other side-effects, like constipation
[176][177]. In conclusion, the data obtained in the discussed above study indicated that MBCP, a peptide isolated from Mozzarella di Bufala Campana DOP, exerts anti-inflammatory effects both in vitro and in vivo, which is related to its beneficial activity on adherent junctions mainly during an inflammatory process and it could be used as therapeutic or supportive treatment in intestinal inflammation and possibly reducing colorectal cancer risk.
Apart from the MBCP, antimicrobial peptides are generated by digestion of buffalo Mozzarella. Characteristic examples are two ĸ-CN derived peptides (YYQQKPVA, f64-69; YYQQKPVA, f64-70) with antimicrobial activity against
E. coli ATCC 25922
[178], and two other peptides, casecidin 17 and casecidin 15, that were identical to sequences in the C-terminal of bovine β-casein (YQEPVLGPVRGPFPIIV, β-CN f208-224; YQEPVLGPVRGPFPI, β-CN f208-222). Conclusively, buffalo Mozzarella is a great source of antimicrobial peptides and it might be used as a supportive treatment to the classic antibacterial approach
[164].
Lactic acid bacteria exert the ability to downregulate the expression of virulence genes of enteropathogenic bacteria
[179][180][181][182]. In a study conducted by Ali et al.
[179], cell free spent medium (CFSM) collected from whey protein (WPI) fermented by
L. helveticus LH-2 and
L. acidophilus La-5 reduced the expression of both the hilA and ssrB genes of the
S. Typhimurium DT 104 wild strain. Among the two strains,
L. acidophilus La-5 had the most significant downregulatory effect on the virulence genes, which acts as an indicator that antivirulence capacity depends on the strain and is affected by the nature and components of CFSM. WPI fermented by La-5 contains nine unique peptides not found in LH-2-fermented or unfermented medium as the results from the same experiment revealed. The downregulatory effect of the synthetic peptide mixture shows the antivirulence effect of specific peptides produced by
L. helveticus (LH-2) and
L. acidophilus (La-5 CFSM) on the
S. Typhimurium virulence genes. In
Salmonella spp., the antivirulence activity of milk protein derived peptides is related to the presence of the oligopeptide-binding protein (OppA) gene. The undigested and fermented WPI by LH-2 and La-5 strains could be considered as a possible source of natural and functional components, which may be used to increase the biological activity of food products
[179]. The endogenous milk proteases can act synergically to LAB, which are plasmin, elastase and cathepsin D, B, and G and are still active, as the presence of peptides in unfermented WPI suggests
[183]. Their role is to release bioactive compounds. Plasmin has little or no activity toward k-casein and whey proteins
[184], so the presence of endogenous peptides from these proteins in CFSM of
L. acidophilus La-5 and
L. helveticus LH-2 could be attributed to the action of other milk proteases such as cathepsin B, D, and G
[185][186]. Dallas et al.
[187] concluded that minimal proteolysis by native milk enzymes continued to function during incubation in the heat-treated milk, when compared with that carried out by the proteases of kefir microorganisms, which were mainly
L. acidophilus and
L. helveticus.
9. Adulteration and Proteomics
Over the past few years there is a growing consumers’ movement regarding products’ originality and their distinction from their counterfeit counterparts. Moreover, consumers are interested in the correlation of food products and history. An excellent example regarding history and cheese, is Feta and its ties with ancient Greece through unique traits and characteristics. Anagnostopoulos and Tsangaris
[188] tried to gather the full protein content of Feta cheese by employing exhaustive deep-proteome analyses using LC/MS-MS. They collected Feta samples from every Greek area that produces PDO labeled Feta cheese and reported the complete list of Feta cheese proteins. The analysis of Feta contains 489 distinct proteins and eventually this method can be used as an identification tool of the authentic Greek product
[188].
Other studies tried to elucidate the caseins behavior through ripening of Feta cheese. Michaelidou et al.
[29] stated that in Feta cheese as1-CN seemed to be further hydrolyzed; a decrease has been observed during ripening. The formation of as1-CN is important because it indicates that the N-terminus of as1-CN has been hydrolyzed during the ripening of Feta cheese. Para-κ-casein is the hydrophobic part of κ-casein and is a component of the paracasein matrix in cheese curd. Chymosin or rennet are added to form the coagulum and the residuals of the enzymes have proteolytic action to the paracasein matrix, with αs1- and β-caseins being the most susceptible whereas αs2- and para-κ-casein rather resistant to their action during cheese ripening. Alexandraki and Moatsou
[189] stated that the greatest part of para-κ-casein remained intact during ripening and storage of Feta with high residual chymosin activity, although other studies indicated the opposite
[190][191]. The positive aspect in their findings is that since para-κ-casein remains intact, it can be used for the quantification of the composition of mixtures of sheep and goat cheese milk and other adulterations, even though the para-κ-casein is similar but not identical in the two species. The latter characteristic was proved by cation-exchange HPLC. In particular, sheep and goat para-κ-casein were efficiently separated, and the changes of their chromatographic areas indicated that hydrolysis happened during the early stages of ripening. Thereafter, and in accordance to the evolution of free amino groups, para-κ-caseins remained stable
[189].
One of the main consumers’ concerns is the milk origin of the PDO cheeses they are buying, which also is applied to Mozzarella di Bufala Campana. One of the cheesemaking rules is that the milk must come from Mediterranean water buffalo raised in Italy. Caseins are fully incorporated in cheese and they can create a “fingerprint” useful for the identification of milk samples origin
[10]. Mediterranean Italian buffalo is reared in Italy for centuries and is a pure breed without any crosses with animals from other countries, as being indicated by their casein polymorphism absence. The β-CN locus was found to be monomorphic
[192] and αs1-CN showed only two silent variants
[193] in Mediterranean Italian buffalo while milk samples from Romanian, Canadian, Polish and Venezuelan WB displayed additional CN variants, such as β-CN A and αs1-CN with eight internally deleted amino acid residues in positions 35–42
[192]. The new caseins variants have been detected in all the foreign countries, but not in the Mediterranean area, proving that WB herds in the Mediterranean area are pure without any crossbreeding, thus proving that the region of milk origin is significant for the PDO labeling in Mozzarella di Bufala Campana
[10].
The above-mentioned traits of WB caseins allowed Caira et al.
[10] to develop a new laboratory approach through an analytical detection method using the MALDI-TOF-MS data of signature peptides from wild and variant WB and bovine CN, thus allowing the identification of milk adulterations with non-Italian WB or bovine milk in commercial Mozzarella di Bufala Campana cheese samples
[10].
A great increase in Mozzarella di Bufala Campana production demands has been observed in the past few years, which has not been followed by an increase in Mediterranean WB milk availability
[21]. Furthermore, the price of buffalo milk varies within each year, creating an unstable economic environment for Mozzarella’s producers. In order to deal with these challenges, dairy owners turn to non-compatible with PDO rules and techniques. In more detail, they use frozen curd as a substitute for fresh milk, something highly prohibited by European Union laws, resulting in different flavor and other traits compared to the original product. To restrict this fraudulent procedure new laboratory methods were applied
[194][195], which identified the marker β-casein fragment (69–209)
[194][195][196], resulting from protein proteolysis as result of the activity of endogenous plasmin
[197][198][199], in curd. Dedicated controls in Italy from the Anti-Sophistication Nucleus for foods revealed recently this technological approach to become progressively adopted for Mozzarella DOP production
[21]. In fact, this adulteration practice allows for several stable proteolytic and lipolytic enzymes from psychrotrophic bacteria to induce non-desired coagulation of milk proteins and production of unwanted organic compounds
[200][201][202][203], affecting final Mozzarella organoleptic characteristics. Nineteen discriminant marker signals in frozen buffalo milk with respect to the fresh counterpart were linked to specific polypeptides/proteins based on literature data
[19][194][196][204][205], mass value calculations, and additional mass spectrometric investigations. Among protein/polypeptide marker signals identified by MALDI-TOF-MS profiling and worth mentioning are those related to α-lactalbumin, β-lactoglobulin and β-casein coupled with the ones associated with GLYC-AM1- and β-casein-derived fragments which possibly originated as a result of the activity of proteolytic enzymes in buffalo milk.
In view of this fact and in order to find a parameter to assess the presence of cow or ewe milk in samples of water buffalo Mozzarella cheese, attention was focused on the mass region of the whey proteins. As the molecular masses of cow, ewe and water buffalo whey proteins are very different and due to the MALDI-TOF-MS resolution in this mass region (14–19 kDa) being high enough to resolve them, lactalbumins and lactoglobulins have been used as biological markers for the evaluation of possible fraudulence in water buffalo Mozzarella cheese production. The mass difference between buffalo and ewe whey proteins is ¾78 Da for lactalbumins and ¾116 Da for lactoglobulins, therefore making it possible to use both proteins for the evaluation of ovine milk sophistication of fresh water buffalo Mozzarella cheese
[206]. The situation is very similar for the MALDI-TOF-MS analysis of Mozzarella prepared with a 90:10 mixture of water buffalo and ewe milk. Even in the case of two mass ranges presented (14–18 kDa), the mass signals of water buffalo and ovine milk proteins are well separated, and the existence of ewe milk can be readily evaluated. The study of other Mozzarella cheese samples obtained from different percentages of ewe milk added to water buffalo milk demonstrate that the response of the instrument is linear up to a limit of 2%.
The EU reference methodology to detect bovine proteins in dairy products is based on gel isoelectric focusing of g-caseins after plasminolysis (EC Regulation No. EC 273/2008) with a detection limit for bovine milk in buffalo cheese products of about 1%. However, the overlapping of species-specific bands generates complex protein profiles, thus impairing the interpretation of results. To overcome the detection of false positive responses, an additional laborious immunoblotting step may be performed
[9][207]. The phosphorylated b-casein f33-48 tryptic peptide was identified as a novel species-specific proteotypic marker whose limit of detection was three orders of magnitude lower than that declared by the methodology officially recognized by the European Commision (Reg. CE n. 273/2008). The high sensitivity of MRM-based mass spectrometry and the wide dynamic range of triple quadrupole spectrometers provide a valuable tool for the analysis of complex matrices such as dairy product
s; s
ee also Table 1 for all the proteomics methods applied to the referred cheeses.
10. Conclusions
This entreviewy is an attempt to summarize the use of proteomics methods in cheese manufacturing process, as well as in cheese per se, offering useful methods for cheese analysis. The knowledge of protein content in each PDO cheese is a useful tool in order to avoid misleading of consumers and to valorize the products. Moreover, it provides information about their bioactive peptides and antimicrobial properties ensuring the consumer’s health. The overall conclusion is that cheese is a very useful component of Mediterranean diet offering unique elements and nutrients.