Antioxidant capacity and identification of radical scavenging peptides from Crema de Chiapas, Fresco and Cocido cheeses

Abstract

Bioactive peptides may positively impact bodily functions. One of these are the antioxidant peptides which are well documented for a wide variety of food matrices, mostly from plant sources. Nevertheless, information of antioxidant milk-derived peptides is still a little-known field. The present study was aimed to evaluating the antioxidant capacity (AC) in vitro of water soluble extracts < 3 kDa (WSE) from three artisanal Mexican cheeses: Crema de Chiapas (CrC), Fresco (FC) and Cocido (CC). This study was carried out for cheeses of different days of storage (0, 5, 10, 15, and 20) at 4 °C. AC was assayed to the respective WSE by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diamoniun salt (ABTS) and oxygen radical absorbance capacity (ORAC) methods and those WSE that showed the most antioxidant capacity from each cheese were analyzed by using RP-HPLC/MS to identify and characterize the novel specific peptides. All the WSE analyzed show antioxidant capacity, especially those from CrC and CC which display the highest AC at 15 days of storage. Regarding to WSE from FC, the AC was constant during storage. Identified structures reveal that these novel peptides possess high content of specific amino acids, mainly proline, valine, leucine and phenylalanine, of which it has already been shown antioxidant properties. This study demonstrate that these artisanal Mexican cheeses are sources of potential antioxidant peptides.

Graphic abstract

Introduction

Bioactive peptides are specific protein fragments which, in addition to their nutritional capabilities, have a positive impact on body’s functions or conditions and may ultimately influence health (Nielsen et al. 2017). These peptides are encrypted within the primary structure of the native protein, and may be released from their parent protein by enzymatic hydrolysis during gastrointestinal digestion, fermentation and/or maturation during food processing, or by proteolytic enzymes derived from microorganisms, animals or plants (Sánchez and Vázquez 2017). Bioactive peptides range in size from 3 to 20 amino acid residues and not only contribute to the flavor, taste, and texture but also show beneficial bioactivities such as antimicrobial, antihypertensive, immunomodulatory, opioid, and antioxidant, among others, so that they positively affect the major body systems (cardiovascular, digestive, immune and nervous) (Nielsen et al. 2017; Sánchez and Vázquez 2017).

Milk proteins are considered the most important source of bioactive peptides derived from animal origin (Mohanty et al. 2016). In addition, an increasing number of peptides have been isolated from milk, milk protein hydrolysates and many dairy products, such as fermented milks and cheeses (Aspro et al. 2018; Mohanty et al. 2016).

Cheese is one of the most important dairy products worldwide, and has been considered as an important source of a wide variety of biologically active substances (López-Expósito et al. 2012; Santiago-López et al. 2018). Numerous peptides may be released by diverse proteolytic systems involved during the cheese making and ripening, for example enzymes present naturally in milk, such as plasmin and cathepsin D, some enzymes from coagulants and others such as cell-envelope proteinases or intracellular peptidases produced by lactic acid bacteria (LAB). All these enzymes play an important role during the hydrolysis of casein and whey proteins to release bioactive peptides into the cheese matrix (Santiago-López et al. 2018).

Among the major health benefits displayed by diverse bioactive components, the antioxidant property is one of the most reported for a wide variety of food matrices, mainly by fruits and vegetables; however scarce information exists about antioxidant activity displayed by animal-derived peptides. Some animal-origin proteins have been recognized for producing antioxidant peptides, which have been derived mainly from milk proteins (Mohanty et al. 2016; Nielsen et al. 2017). Thus, since cheeses are products with a high content of proteins, it is conceivable to considerate them as a good source of these peptides (López-Expósito et al. 2012). Besides, it is widely known that cheeses are a good source of LAB producing metabolites released during fermentation showing numerous biological activities when subjected to in vitro studies and also in vivo with animal models (Aguilar-Toalá et al., 2017; Rodríguez-Figueroa et al. 2013; Santiago-López et al. 2018) which constitute a valuable tool for their assessment in forthcoming in vivo studies against different human diseases.

Since the microbial diversity greatly impacts on the enzymatic activity, novel bioactive peptides from artisanal cheeses can be produced from these complex ecosystems. Despite artisanal cheeses are generally made by farmers on small scale using raw milk and traditional techniques, they have special social prestige and are very popular worldwide, but mainly in less-developed regions, which are widely located in Mexico and Latin-American countries. Particularly in Mexico, these cheeses are consumed no only by the local population but also throughout the country because they are very appreciated by their uniqueness and tipicity (González-Córdova et al. 2016). Actually, these cheeses are part of the Mexican culture since they are essential ingredients to elaborate traditional dishes. For instance, Cocido cheese is commercialized in the north of the state of Sonora and it is smuggled through the border to the USA to prepare traditional Mexican dishes by Hispanics living in such country (Cuevas-González et al. 2017). On the other hand, these cheeses are threatened with extinction due to raw milk is not allowed by the Mexican official standards (SSA 2010) to elaborate these products and also due to the presence of imitation products in the market (Villegas et al. 2011), what compromise their microbial diversity. In consequence, since these microorganisms greatly impact on the enzymatic activity, novel bioactive peptides could cease to be produced from these complex ecosystems.

After an exhaustive revision, it was found that the identification of antioxidant peptides derived from cheeses have been scarcely reported (Meira et al. 2012; Timón et al. 2014, 2019). Some of these studies report the antioxidant activity by bioactive peptides only in Cheddar (Gupta et al. 2009; Pritchard et al. 2010), Coalho (Silva et al. 2012.), Fresco (Paul et al. 2012), Parmigiano (Bottesini et al. 2013) and Cottage (Abadía-García et al. 2013) cheeses. In consequence, it would be valuable to know whether some artisanal Mexican cheeses present antioxidant activity that may confer an extra value for playing an important role as an aid to maintenance the consumer’s health against degenerative diseases. Thus, the objective of this study was to investigate the antioxidant capacity of water-soluble extracts obtained from three artisanal Mexican cheeses at different days of storage and also identify the peptides from those fractions showing the most antioxidant activity.

Materials and methods

Materials

All the chemicals and solvents were analytical-grade reagents. 6-hydroxy-2,3,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-azinobis(2-amidinopropane) dihydrochloride (AAPH), potassium persulfate and fluorescein sodium salt were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Cheese samples

Samples were collected following the methodology reported by Cuevas-González et al. (2017) with some modifications. Three batches of samples (n = 3) of three artisanal Mexican cheeses: Crema de Chiapas cheese (CrC), Cocido cheese (CC) and Fresco cheese (FC), were collected directly from dairies, located in the states of Chiapas (CrC) and Sonora (CC and FC), Mexico. The samples were aseptically taken and placed in 710-mL Whirl–Pak sterile sampling bags (Nasco USA, Fort Atkinson, WI). Then, they were transported to the Dairy Chemistry and Biotechnology Laboratory at the Research Center for Food and Development (Centro de Investigación en Alimentación y Desarrollo, A. C. (CIAD)), located in Hermosillo, Sonora, Mexico. All the samples were stored at 4 °C and were aseptically taken at different times of storage (0, 5, 10, 15 and 20) for analyses.

Preparation of water-soluble extracts of peptides

Water-soluble extracts (WSE) of peptides present in the Cheeses were prepared according to Torres-Llanez et al. (2011) with minor modifications. Cheese samples (20 g) were thoroughly homogenized in 40 mL of deionized water by magnetic stirring (20 °C, 300 rpm for 20 min). The resulting homogenates were held at 4 °C for 1 h. The insoluble material was then separated by centrifugation (Sorvall ST 16R, Thermo Fisher Scientific, Portsmouth, NH, USA) (4 °C, 4700 × g for 30 min). The supernatants were filtered through a grass wool by using a filter paper (Whatman™ No. 42) to remove residual-suspended fat and residual solids impurities. Afterwards, the extract was ultrafiltered by using a Stirred Ultrafiltration Cell (model 8050, Millipore Corporation, Bedford, MA, USA) with a cut-off point of 3 kDa. Finally, these WSE were lyophilized with a freeze-dried (Labconco, Kansas City, MO, USA) and kept at −20 °C until analysis. The analyses were performed in triplicate.

Peptide content of each WSE was assayed by the Lowry method with a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (BSA) as standard by following the specifications of the provider. Prior to the antioxidant analyses, the WSE were adjusted to the minimum concentration showed by the samples (in this case, the samples were diluted with a PBS solution to a peptide concentration of 1 mg/mL).

Radical scavenging capacity of water-soluble extracts of peptides

The antioxidant capacity of WSE based on the radical scavenging capacity was carried out by using the ABTS (Re et al. 1999) and ORAC (Prior et al. 2005) methods with some modifications as described in Aguilar-Toalá et al. (2017). In brief, for the ABTS method, Trolox (6-hydroxy-2,3,7,8-tetramethylchroman-2-carboxylic acid) was used as a positive control. The scavenging capacity percentage of the ABTS^•+ was calculated as follows:

$${\text{ABTS}}^{·+}\text{scavenging capacity}\left(\%\right)=\left(\left[A-B\right]/A\right)\times 100,$$

where A is the absorbance in the control and B is the absorbance in the sample. The radical scavenging capacity of WSE was expressed in terms of IC₅₀ that represents the peptide concentration (mg peptides/mL WSE) in the sample required to inhibit 50% of the ABTS^•+ activity. For this purpose, a dose response curve was constructed by plotting the inhibition percentages of the ABTS^•+ as a function of their peptide concentrations (0.25, 0.5 and 1 mg/mL WSE) and the corresponding IC₅₀ values were calculated according to the resulting linear regression equation.

Reverse phase high-performance liquid chromatography analysis of water-soluble extracts and identification by tandem mass spectrometry

The fractions showing the highest AC (the lowest IC₅₀ values) were subjected to further identification of protein fragments and amino acid sequences by reverse phase high-performance liquid chromatography (RP-HPLC) by using a 1100 Series LC/MSD Trap (Agilent Technologies Inc., Waldbronn, Karlsruhe, Germany) equipped with an electrospray ionization source (LC–ESI–MS) according to the methods described by Torres-Llanez et al. (2011). Briefly, the peptide fractions (20 µL) were purified by RP-HPLC at room temperature (22 °C) at a flow rate of 0.5 mL/min using a column C18 (Extend-C18, 4.6 mm × 250 mm, 5 μm; Agilent Technologies, Santa Clara, CA). Next, selected peaks were collected using a fraction collector (Agilent 1100 Series, Analytical FC, Waldbronn, Germany), which were pooled and dried using a Centrivap concentrator (Labconco, Kansas City, MO). Finally, to identify peptides in collected samples, 1 µL was injected in C18-300SB nano-column (150 mm × 0.75 μm, 3.5 μm, Agilent Technologies Inc.) at a flow of 0.7 µL/min into the mass spectrometer via an electrospray interface using nitrogen as the nebulizing and drying gas and operated with an estimated helium pressure of 500 Pa. Peptide sequences were obtained from mass spectrometry data using the Mascot server through the UniProtKB/Swiss-Prot database.

Statistical analysis

The statistical analysis was performed using the NCSS 2007 software for Windows. All data were analyzed by one-way analysis of variance (ANOVA). Differences among means were determined at 5% level of significance by the Tukey’s test.

Results and discussion

Antioxidant capacity based on the radical scavenging capacity of water-soluble extracts of peptides

The antioxidant capacity of WSE obtained from the artisanal Mexican cheeses by the ABTS method is shown in Fig. 1, expressed as IC₅₀ values. The ABTS radical scavenging capacity indicates that peptides present in the WSE are capable to neutralize such radicals, either by direct reduction via electron transfers or by radical quenching via H atom transfer, resulting into a more stable species (Huang et al. 2005). A lower IC₅₀ value corresponds to a higher scavenging capacity.

Fig. 1.

Radical scavenging capacity of WSE by ABTS method during storage time. Different letters among days for each cheese indicate significant differences (P < 0.05)

All samples of Mexican artisanal cheeses showed antioxidant capacity. The IC₅₀ ranges (mg peptides/mL WSE) were 5.98–8.47 for CrC, 4.47–6.34 for FC and 3.91–10.86 for CC. A progressive increase in the antioxidant capacity was observed in CrC and CC according to the storage time. By the 15 days of storage, CrC showed the highest antioxidant capacity during storage with IC₅₀ values of 5.98 mg peptides/mL WSE and slightly decreased over 20 days of storage, but they were not significantly different (P > 0.05). The antioxidant activity of CC was significantly increased (P < 0.05) during the first 5 days of storage and this activity did not show significant (P > 0.05) changes in the later storage times. On the other hand, FC showed the inverse activity during storage time; this cheese showed the highest antioxidant activity during 0–10 days with 4.47–4.66 mg peptides/mL WSE and it was significantly (P < 0.05) reduced after 15 days of storage.

These results indicate that the antioxidant capacity of these cheeses is dependent of the storage time. This behavior is in agreement with studies reported by Gupta et al. (2009) and Bottesini et al. (2013). Gupta et al. (2009) found that the antioxidant capacity of Cheddar cheese, manufactured with adjunct cultures of Lactobacillus casei ssp. casei 300 and Lactobacillus paracasei ssp. paracasei 22, was dependent on the ripening time. They reported an increase of the antioxidant capacity up to 4 months of ripening; after this time, the antioxidant capacity decreased and maintained similar values until reaching 9 months of ripening. These findings contrast with previous results reported by Bottesini et al. (2013) who found the antioxidant capacity of Parmigiano-Reggiano cheese was unaffected by the ripening time (7–41 months). These authors suggested that the antioxidant capacity of this type of cheese was due to free amino acids and some peptides. However, it is also known that free amino acids are not effective antioxidants (Elias et al. 2008). Adverse to free amino acids, the effectiveness of peptides is attributed to the unique chemical and physical properties conferred by their sequence, specially the stability of the resultant peptide radicals that do not initiate or propagate oxidative reaction (Samaranayaka and Li-Chan, 2011).

The antioxidant capacity (in terms of IC₅₀ values) of the WSE of Mexican cheeses in the present study are higher than those of Coalho cheese (Silva et al. 2012) and Cheddar cheese (Gupta et al. 2009). Silva et al. (2012) found values of 7 and 10.5 mg peptides/mL extract (ABTS method) for Correntes and Sao Bento do Una Coalho cheeses, respectively. In this case, it is important to consider that such extracts were crude or total. In the present study, < 3 kDa fractions were assessed since these low molecular weight peptides are the main responsible of major contribution to the antioxidant activity. What is more, Gupta et al. (2009) reported values of IC₅₀ of 7.62 and 7.81 mg peptides/mL extract (DPPH method) in Cheddar cheese manufactured with Lactobacillus casei ssp. casei 300 and Lactobacillus paracasei ssp. paracasei 22 cultures, respectively. However, in the present work the DPPH method was not appropriate because it presented high fluctuations in the IC₅₀ values (data not shown). Actually, similar problems were reported previously in WSE from some cheeses (Abadía-García et al. 2013; Meira et al. 2012) and WSE from yogurt (Sanlidere Aloglu and Oner 2011). These fluctuations may be due to the fact that DPPH is an oil soluble free radical; thus, it is challenging for this reagent to be dissolved in an aqueous extract (Meira et al. 2012), such as WSE from the present study. Additionally to this, DPPH can serve as oxidizing substrate or as a reaction indicator molecule, which may cause spectral interference in the reader device (Sanlidere Aloglu and Oner 2011).

On the other hand, the ORAC method determines the decreasement in fluorescence of a protein (fluorescein) as a result of the loss of its conformation when it suffers oxidative damage caused by peroxyl radicals generated in biological systems (Huang et al. 2005). This method measures the ability of WSE peptides to protect the protein from oxidative damage, by radical quenching via H atom transfer. The ORAC is the only method that takes free radical action to completion and uses the area under the curve for quantification; thus, this method combines both, the percentage of inhibition and the length of inhibition, of free radical formation by antioxidants into a single quantity (Huang et al. 2005).

The antioxidant capacity determined by the ORAC method is showed in Fig. 2, expressed as µM of Trolox equivalent/mL WSE. The antioxidant capacity value ranges were 185.6–311.1, 158.8–176.1, and 159.0–223.9, for CrC, FC and CC, respectively. In general, a progressive increasement in the antioxidant capacity was observed in CrC and CC. Nevertheless, this behavior was not shown for FC, which did not show changes during storage time. CrC and CC cheeses showed in 20 days the highest antioxidant capacity with averages of 311.08 and 223.9 µM of Trolox equivalent/mL WSE, respectively and these results were significantly (P < 0.05) higher than all the preceding storage times. On the other hand, FC showed the lowest antioxidant capacity without significant changes through all the storage time with a mean value of 166.7 µM Trolox equivalent mL⁻¹ WSE.

Fig. 2.

Radical scavenging capacity of WSE by ORAC method during storage time. Different letters among days for each cheese indicate significant differences (P < 0.05)

These cheeses may work as a dynamic system matrix, which diverse proteolytic enzymes are involved such as those from milk, coagulant and LAB (Santiago-López et al. 2018) playing an important role for peptide production that are being constantly released. In consequence, some of these peptides are subsequently hydrolyzed and/or accumulated over the storage time. According to the above, it could be inferred that the peptide profile depends upon equilibrium from their formation to degradation, as well as on the cheese type.

Reverse phase high-performance liquid chromatography analysis of water-soluble extracts and identification by tandem mass spectrometry of amino acid sequences of peptides present in WSE

Protein fragments and amino acid sequences for the identified peptides in CC, FC and CrC are shown in Table 1. The WSE of these three Mexican cheeses were characterized and various fragments of casein (CN) were identified such as α_s1-CN, α_s2-CN and β-CN, which could have been released from CN since it is very susceptible to proteolysis (Yamamoto and Takano 1999). These fragments were mainly identified in WSE derived from CC and FC.

Table 1.

Peptides identified in CC, FC, and CrC cheeses and their location inside the protein sequence

CC*		FC*		CrC*
Protein fragment	Sequence	Protein fragment	Sequence	Protein fragment	Sequence
αs2-CN f(101–113)	QGPIVLNPWDQVK	αs1-CN f(14–22)	EVLNENLLR	β-LG f(15–19)	VAGTW
αs2-CN f(99–113)	LYQGPIVLNPWDQVK	αs1-CN f(24–34)	FVAPFPEVFGK	κ-CN f(35–41)	YPSYGLN
αs2-CN f(100–113)	YQGPIVLNPWDQVK	αs1-CN f(25–34)	VAPFPEVFGK	αs1-CN f(80–87)	FLDDDLTD
αs2-CN f(114–124)	RNAVPITPTLN	αs1-CN f(104–109)	YKVPQL	Lactoferrin f(135–142)	SCHTGLGR
αs1-CN f(24–34)	FVAPFPEVFGK	αs1-CN f(104–114)	YKVPQLEIVPN	Lactoferrin f(270–276)	LLSKAQE
αs1-CN f(25–34)	VAPFPEVFGK	αs2-CN f(99–113)	LYQGPIVLNNPWDQVK	Lactoferrin f(278–282)	FGKNK
αs1-CN f(26–34)	APFPEVFGK			Lactoferrin f(442–446)	ANEGL
αs1-CN f(28–34)	FPEVFGK			Lactoferrin f(515–520)	CVPNSK
αs1-CN f(14–22)	EVLNENLLR			Lactoferrin f(58–62)	AVTLD
αs1-CN f(104–109)	YKVPQL
αs1-CN f(193–199)	KTTMPLW
β-CN f(184–190)	DMPIQAF
β-CN f(193–209)	YQEPVLGPVRGPFPIIV
β-CN f(194–209)	QEPVLGPVRGPFPIIV
β-CN f(195–209)	EPVLGPVRGPFPIIV
β-CN f(203–209)	GPFPIIV

*CC (Cocido Cheese), FC (Fresco Cheese) and CrC (Crema de Chiapas Cheese)

Specifically, CC was characterized by presenting a higher number of peptides than those of CrC and FC. Thus, this WSE contained four fragments of α_s2-CN (range of 11–15 amino acid residues), seven fragments of α_s1-CN (range of 6–11 amino acid residues) and five fragments of β-CN (range of 7–17 amino acid residues). FC showed five fragments of α_s1-CN (range of 6–11 amino acid residues) and only one fragment of α_s2-CN (16 amino acid residues). Meanwhile, CrC only presented one fragment of α_s1-CN (8 amino acid residues).

Interestingly, CrC characterized by presenting different fragments of those from CC and FC since its WSE contained whey-derived peptides [β-lactoglobulin (β-LG) with 5 amino acid residues, kappa casein (κ-CN) with 7 amino acid residues and six fragments of lactoferrin (range of 5–8 amino acid residues)]. The fact that there was only one fragment of κ-CN could have been due to the low amount of this protein as well as possessing resistance of proteolysis (Addeo et al. 1994).

It is important to highlight that these whey-derived peptides have also been reported as source of antioxidant peptides (Mohanty et al. 2016; Nielsen et al. 2017). Nevertheless, it must be mentioned that it is unknown why whey-derived peptides are only present in CrC but not in CC or FC. This evidence suggests that these peptides may be produced due to the retention of whey proteins by the lack of efficiency in the draining process. Also, it is important to consider the intrinsic physicochemical composition of each cheese type, which highly depends on their microbiota and consequently a particular proteolytic activity leading a specific peptide profile released from the protein matrix. Indeed, it is well documented that CrC has high salt content (> 3%) and is produced through a prolonged acid-enzymatic coagulation of cow’s raw milk, leading to a low pH (4.5) and resulting in a highly acidic cheese (González-Córdova et al. 2016). In contrast, CC and FC are characterized by a short time of coagulation (< 1 h), are lightly acidity, and have low content of salt (< 2%) (Cuevas-González et al. 2017; González-Córdova et al. 2016).

Regarding to amino acids conforming specific bioactive peptides, it has been reported that many antioxidant peptides contain Pro (P), His (H), Cys (C), Phe (F), Trp (W) and Tyr (Y) in their sequences (Chen et al. 1995; Saito et al. 2003). The antioxidant capacity displayed of each amino acid is due to the different side chain group as phenolic (present in T and F), indolic (present in W), sulphydryl (present in C), imidazole (present in H), and pyrrolidine (present in P) that can transfer either a hydrogen atom or an electron to reactive oxygen species, forming stable molecules or less reactive species (Elias et al. 2008; Samaranayaka and Li-Chan 2011).

In the present study, most of peptides contain H, C, F, W and Y into their sequences, but mainly it was observed a special content of amino acid P in all peptide sequences. Specifically, at least one P residue was contained in 15 from 16 peptides identified in CC, in five from six peptides in FC and in two from nine peptides in CrC. These results are coincident with some studies previously reported; for example, Hernández-Ledesma et al. (2005) identified eight antioxidant peptides derived from fermented milk and found that seven contained at least one P residue. These authors suggested that the antioxidant capacity of these peptides is related to their high content of P residues. Meanwhile, Sabeena Farvin et al. (2010) observed that almost all the antioxidant peptides identified in yogurt contained at least one P residue.

In addition, the presence of Tyr (Y) at the N-terminal has also been described as determinant factor in the radical scavenging capacity in peptides (Dávalos et al. 2004; Hernández-Ledesma et al. 2007). In this sense, the following fragments: f(100–113), f(104–109) and f(193–209) in CC, f(104–109) and f(104–114) in FC and f(34–40) in CrC contain the amino acid Y at the N-terminal. As in this case, other studies have shown that the presence of Val (V) and Leu (L) at the N-terminal is a determinant factor in the radical scavenging capacity (Chen et al. 1995). In this regard, the following fragments of the present work: f(99–113) and f(25–34) in CC, f(25–34) and f(100–113) in FC and f(15–19) and f(270–276) in CrC, contain V or L at the N-terminal. Besides, the presence of Trp (W) at the C-terminal is also decisive in the radical scavenging capacity (Hernández-Ledesma et al. 2007; Saito et al. 2003). The fragments f(193–199) in CC and f(15–19) in CrC present such characteristic.

According to these results, the radical scavenging capacity of peptides from cheese WSE may be due to their amino acid-sequence conformations. Specifically, it was observed a high number of some key amino acids, mainly P, followed by V, L and F in these WSE showing higher AC. What is more, some peptides identified in the present study possess sequences with high homology with antioxidant peptides previously described in the literature. Such comparison is showed in the Table 2. From the total fragments identified in the present study in Mexican cheeses, it was found that ten sequences share structural homology with antioxidant milk-derived peptides previously reported and eight of them were derived from CC, two from FC and none from CrC. It is important to mention that only three sequences (KTTMPLW, YQGPIVLNPWDQVK and YQEPVLGPVRGPFPIIV) completely matched to milk-derived peptides already proved as antioxidants. This is important due to in previous studies confirm the findings in this work.

Table 2.

Sequences identified in the present study that share structural homology with reported antioxidant peptides

Cheeses (WSE)*	Sequence**	Previously described sequence	Reference
CC	EVLNENLLR	VLNENLLR	Hernández-Ledesma et al. (2005)
FC	EVLNENLLR	VLNENLLR	Hernández-Ledesma et al. (2005)
CC	FVAPFPEVFGK	FVAPFPE	Hernández-Ledesma et al. (2005)
FC	FVAPFPEVFGK	FVAPFPE	Hernández-Ledesma et al. (2005)
CC	KTTMPLW	KTTMPLW	Hernández-Ledesma et al. (2005)
CC	YQEPVLGPVRGPFPIIV	GPVRGPFPII GVRGPFPII	Hernández-Ledesma et al. (2005) Farvin et al. (2010)
CC	QEPVLGPVRGPFPIIV	GPVRGPFPII GVRGPFPII	Hernández-Ledesma et al. (2005), Sabeena Farvin et al. (2010)
CC	EPVLGPVRGPFPIIV	GPVRGPFPII GVRGPFPII	Hernández-Ledesma et al. (2005) Farvin et al. (2010)
CC	YQGPIVLNPWDQVK	YQGPIVLNPWDQVK	Meira et al. (2012)
CC	YQEPVLGPVRGPFPIIV	YQEPVLGPVRGPFPIIV	Meira et al. (2012), Timón et al. (2014)

*CC (Cocido Cheese), FC (Fresco Cheese) and CrC (Crema de Chiapas Cheese)

**Letters in bold represent sequences identified in the present study that have been already reported as antioxidant peptides

Nevertheless, beyond the presence or amount of key amino acids of these peptides, it is also unavoidable to consider their molecular weight, hydrophobicity, and amino acid sequence, among other characteristics that may be crucial for determining their biological activity (Zou et al. 2016). For example, peptides with lower molecular weight (< 3 kDa) have been reported as effective antioxidants compared to the higher molecular fractions (Irshad et al. 2015). On the other hand, the hydrophobicity character of peptides plays an important role in the enhancement of the antioxidant properties because it increases the accessibility of the peptide to hydrophobic cellular targets such as polyunsaturated chain fatty acids of biological membranes (Zou et al. 2016). Moreover, peptides are subjected to enzymatic degradation and absorption when consumed, hence in vivo trials should be assessed to validate these results, because the activity depends on their bioavailability.

Thus, this study provides scientific evidence supporting the fact that antioxidant peptides naturally present in these Mexican cheeses may have physiological implications helping in the maintenance the consumers good health, providing protection against degenerative diseases.

Conclusion

All the artisanal Mexican cheeses, studied in the present work, showed antioxidant capacity, being different among the cheese varieties. Particularly, the antioxidant activity of CrC and CC increased according to the storage time. The peptide fragments identified revealed high content of antioxidant amino acids in their sequences such as P, V, L, F, Y and W residues. Therefore, their consumption, in addition to meet the nutritional requirements, may provide benefits to the health of consumers. Nevertheless, further work is necessary to carry out in vivo studies to test its antioxidant effect in order to identify novel bioactive peptides responsible for such benefit. Thus, the antioxidant capacity of these cheeses revealed in this study opens up future opportunities that may allow the revalorization of genuine Mexican artisanal cheeses.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical guideline

Ethics approval was not required for this research.