Angiotensin i-converting enzyme inhibitory Activity in crust of Dry- aged Beef

Antioxidant activity, angiotensin I-converting enzyme (ACE) inhibitory activity, and protein profile of crust (the dried surface of dry-aged beef) were evaluated compared to unaged, wet-, and dry-aged beef. Antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-di-(3ethylbenzthiazoline sulfonate) radical scavenging activities, ferric reducing antioxidant power, and ferrous ion chelating activity. the crust samples showed the greatest (P < 0.05) ACE inhibitory and antioxidant activity resulting from the three different mechanisms of action (radical scavenging, nonradical redox potential activity, and metal chelating) among the treatments. Small molecular weight protein bands and small peptides (<3 kDa) indicating potent bioactivity were evident in the myofibrillar protein profile of crust samples. The lowest (P < 0.05) ACE inhibitory activity was observed in unaged beef. the results indicate that crust could be utilized in various areas as a functional ingredient possessed antioxidant and Ace inhibitory activity instead of being discarded. in addition, dry aging can use for generation of functional ingredient from beef as the regime.

Measurement of antioxidant activity. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. The meat extract was diluted 50 times with deionized distilled water (DDW), and 2 mL of the diluted samples were mixed with 2 ml of 0.2 mM DPPH in methanol 13 . After vigorous vortexing, the mixture was left in dark for 20 min at room temperature then centrifuged (Continent 512R, Hanil) at 2,268 × g for 10 min at 4 °C. The absorbance of the supernatant was measured at 517 nm using a spectrophotometer (X-ma 3100; Human Corp., Seoul, Korea). Trolox was used as the antioxidant standard. The absorbance was converted to the equivalent activity of Trolox per mL based on the standard curve and expressed as Trolox equivalent antioxidant capacity (TEAC, TEAC/ml extract). DPPH radical scavenging activity (%) was calculated as [1 − (Absorbance of sample at 517 nm/Absorbance of control at 517 nm)] × 100.
2,2′-Azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) assay. ABTS capacity of each muscle extract was measured by the degree of suppression of ABTS radical cation (ABTS.+) produced by the reaction of ABTS with potassium sulfate 14 . To obtain the ABTS. + solution, a mixture of 7.0 mM ABTS and 2.45 mM potassium persulfate (final concentration) was prepared and stored in the dark for 16 h. The solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. Fresh ABTS.+ solution was prepared for each analysis. The reaction mixture consisting of 3 mL of the ABTS.+ working solution and 20 μL sample was placed in a cuvette and incubated for 5 min at 30 °C. The mixture was centrifuged (Continent 512 R, Hanil) at 2,268 × g for 5 min. The absorbance of the supernatant was measured at 734 nm using a spectrophotometer (X-ma 3100; Human Corp.). The percentage ABTS.+ inhibition was calculated as: where AB is the absorbance of the blank and AS is the absorbance of the muscle extract. Trolox was used as the antioxidant standard as described in DPPH assay. ferric reducing antioxidant power (fRAp) assay. As a non-radical redox potential-based method, the reducing power of each muscle extract was determined 15 . Acetate buffer (300 mM, pH 3.6), 10 mM 2,4,6-tris-2,4,6-tripyridyl-2-triazine in 40 mM HCl, and 20 mM ferric chloride solution were prepared in 10:1:1 (v/v/v) portions. The mixture was incubated in a water bath maintained at 37 °C for 30 min. FRAP (3 ml) was added to 100 μl of each sample diluted 4-fold in DDW and incubated for 5 min. The mixture was centrifuged (Continent 512 R, Hanil) at 2,268 ×g for 5 min. The absorbance of the supernatant was measured at 593 nm using a spectrophotometer (X-ma 3100, Human Corp.). Increased absorbance indicated greater reducing power. ferrous ion chelating activity assay. The chelating activity of each muscle extract was determined by measuring the ferrous ion-ferrozine complex at 562 nm 16 . Briefly, 0.5 mL of muscle extract sample was added to 50 μl of FeCl 2 (2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.1 ml) and 3.2 ml of ethanol. The mixture was vortexed vigorously and left in dark at room temperature for 10 min and was then centrifuged (Continent 512R, Hanil) at 2,268 g for 5 min. The absorbance of the supernatant was measured at 562 nm using a spectrophotometer (X-ma 3100, Human Corp.). The inhibition of ferrous ion-ferozzine complex formation was calculated as: where A B ′ is the absorbance of the blank and A S ′ is the absorbance of the muscle extract.
Measurement of Ace inhibitory activity. ACE inhibitory activity of each sample was determined 17 . Ten microliters of each muscle extract was incubated with 30 μl hippuryl-L-histidyl-L-leucine (HHL, 12.5 mM in 0.1 M sodium borate buffer) at 37 °C for 10 min. Distilled water instead of sample was used as a blank and control. After incubation, 10 μl of ACE (peptidyldipeptide hydrolase from rabbit lung acetone extract) was added and the mixture incubated at 37 °C for 30 min. The enzymatic reaction was stopped by adding 50 μl of 1 N HCl. The hippuric acid generated by the action of ACE on HHL was extracted from the acidified solution into 300 μl ethyl acetate by vortex mixing for 15 s. The extract was centrifuged at 1,000 ×g for 5 min at 4 °C. Aliquots of 250 μL of each ethyl acetate layer were transferred to clean tubes and evaporated by heating at 70 °C for 1 h in a water bath.
www.nature.com/scientificreports www.nature.com/scientificreports/ The hippuric acid was redissolved in 300 μl of DDW and the amount formed was determined by the absorbance at 228 nm. ACE inhibitory activity (%) was calculated as: where A S is the absorbance of the sample, A SB is the absorbance of the sample blank, A C is the absorbance of the control, and A CB is the absorbance of the control blank. Enalapril maleate salt (0.3 mg/ml) was the control.
protein profile using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-pAGe). Myofibrillar proteins for SDS-PAGE analysis were prepared from unaged and crust sample using 0.03 M phosphate buffer (pH 7.4) and 0.1 M phosphate buffer potassium containing 0.7 M potassium iodide and 0.02% sodium azide as the isolating medium 18 . The protein concentration of isolated beef samples was determined and analyzed the isolated myofibrillar protein 19,20 . The stacking gel and separating gel contained 4.5% and 12.5% polyacrylamide, respectively, and 20 μl of meat extract was loaded onto the gel. Protein standards (Precision Plus Protein TM Unstained Standards, Bio-Rad, Hercules, CA, USA) were included in each electrophoretic run to determine molecular size. Electrophoresis was performed using the AE-6531 Mini-Slab Size Electrophoresis System (Atto Corporation, Tokyo, Japan) at 20 mA for 70 min. Gels were stained for 30 min in 0.1% Coomassie Brilliant Blue R-250 solution containing 30% methanol and 10% acetic acid, then were destained for 90 min in 30% methanol and 10% acetic acid.

Identification of peptides using quadrupole time-of-flight (Q-TOF) liquid chromatography tandem mass spectrometry (Lc/MS/MS).
The samples (1 g) were extracted from the unaged and crust samples with 9 ml of 30% acetonitrile in deionized distilled water. The extracts were centrifuged (Combi 514R, Hanil) in Amicon ® ultra centrifugal filters (Ultracel ® -3 K, Merck Millipore Ltd., Burlington, MA, USA) to obtain small peptides (<3 kDa). Then, the supernatants were injected to a Triple TOF 5600 ® Q-TOF LC/MS/MS (AB Sciex, Foster City, CA, USA) using a Ultimate 3000 RSLC HPLC (Thermo Fisher Scientific Inc., Waltham, MA, USA). Mobile phase consisted of solvent A (0.1% formic acid in double-deionized water) and solvent B (0.1% formic acid in 95% acetonitrile) and the gradient programming was as follows: Solvent A and B were set at 95 and 5% from 0 to 1 min, respectively. Solvent A was decreased at 0% from 16 to 18 min. Solvent B was increased at 40% at 16 min and kept increasing at 100% from 18 to 20 min. Solvent A was increased at 95% from 20 to 26 min. Solvent B was decreased at 5% from 21 and 26 min. The flow rate was 0.3 ml/min and the injection volume was 15 μl. The auto-sampler was set at 4 °C. The electrospray ionization (ESI) mass spectra data were acquired under positive ion mode with a source temperature of 500 °C and voltage of 5500 V. The curtain gas, ion source gas 1, and ion source gas 2 were 50, 50, and 25 psi, respectively. Two full-scan mass spectra were acquired over an m/z range of 350-1800 on the MS mode. The data were collected using Analyst TF 1.7 software and analyzed using PeakView

Results and discussion
Antioxidant activity. Radical scavenging activity. DPPH radical scavenging activity was significantly affected (P < 0.05) by the aging methods and beef portion (surface or inner) ( Fig. 1(A)). Crust protein extract exhibited the greatest values (44.7% and 0.48 TEAC/ml protein extract; P < 0.05) and wet-aged beef extract showed the lowest values (40.8% and 0.43 TEAC/mL extract; P < 0.05) in DPPH radical scavenging activity, respectively. In this study, protein extract from dry-aged beef (flesh) also showed significantly higher value than wet-aged beef and a lower DPPH radical scavenging activity compared to that from crust (P < 0.05). This result could be assumed that the presence of microorganisms 21,22 or moisture evaporation 23,24 produced or concentrated free amino acids during dry aging. As many studies agreed, amino acids or peptides produced by proteolytic enzymes of microorganisms present on the surface (crust) during dry aging since dry-aged beef is prone to microbial contamination and mold/yeast growth often occurs on the crust 21,22 . It is expected that both endogenous enzymes and exogenous proteolytic enzymes produced from microorganism act together, resulting in enhanced degradation of meat protein and production of different peptides in fresh, wet-aged, and dry-aged beef. It was described that naturally generated peptides by microorganisms can induce biological activity by producing peptides of different sizes and various free amino acids 25 . In addition, microbial fermentation can generate bioactive peptides having antioxidant activity from muscle-based foods 26 . Different molds and yeasts were found and reported from the crust of dry-aged beef 3,27 . In addition, during dry aging, moisture evaporation could lead to concentration of free amino acids showing antioxidant activity. Several studies reported that antioxidant activity of protein hydrolysates related to α-amino group content and properties of free amino acids or small peptides such as size, composition, etc. 28,29 . In addition, previous study reported more abundant aromatic amino acids, including phenylalanine, tyrosine, and tryptophan were present in dry-aged beef compared to wet-aged beef 23,24 .
Aromatic amino acids is known as strong radical scavengers since they donate a proton to electron-deficient radicals and maintain their stability with resonance structure 30 . The ABTS radical cation scavenging activity of four protein extracts were similar to the DPPH radical scavenging results (Fig. 1(B)). The greatest ABTS radical cation scavenging activity (69.7% and 43% higher than the lowest value) was observed in crust protein extract, followed by dry-aged beef. This also could be due to microbial (2020) 10:7883 | https://doi.org/10.1038/s41598-020-64861-0 www.nature.com/scientificreports www.nature.com/scientificreports/ growth on the crust previously described. The protein extracts from wet-aged and unaged beef displayed significantly lower ABTS radical cation scavenging activity, with no significant difference between them.
Ferric ion reducing power. Reducing agents act as electron donors to the reduced species and a higher value in absorbance indicates greater reducing capacity. The highest absorbance at 593 nm (0.34; P < 0.05) was observed in crust protein extract (Fig. 2(A)). Previous study presented that protein extracted from fermented sausages exhibited 2-3-time higher antioxidant activity at the end of ripening than at the initial processing stage 31 . Hence, in this study, we postulated that the direct action of microorganisms on the surface of the dry-aged meat (crust) could contribute to the generation of the reducing power capacity. The difference in absorbance among protein extracts from unaged, wet-aged, and dry-aged beef (ranging from 0.23 to 0.25) seems not to be meaningful, despite the significant difference between wet-and dry-aged beef.
Metal chelating activity. A clear difference in metal chelating activity was evident between the samples (Fig. 2(B)). Chelating activity was greatest for crust protein extract (83.9%; P < 0.05), followed by dry-aged beef (77.6%). Chelating activities were significantly lower for protein extracts from unaged and wet-aged beef (65.2% and 68.9%, respectively). According to previous studies, dry-aged beef exhibited a higher amount of the sulfur-containing amino acid methionine 23,24 , which perhaps contributed to the increased metal chelating activity in comparison with wet-aged beef. Consequently, the type of amino acid or peptide produced by proteolysis might depend on the endogenous enzymes and those from microorganisms present on the surface of the beef during aging period.
ACE inhibitory activity. ACE inhibitory activity of protein extract was affected by the aging method (wet or dry aging) and the section of dry-aged meat [internal or external (crust) sections] (Fig. 3). Significantly high and low ACE inhibitory activities were observed for protein extract from crust and unaged beef, respectively. Similarly, it was reported that peptides derived from dry-cured ham by the action of microbial endo-and exogenous enzymes presented ACE inhibitory activity 32 . The protein extract from wet-aged beef showed similar (P > 0.05) ACE inhibitory activity to unaged beef. It was described that the limited generation and/or identification of peptides with ACE inhibitory activity by endogenous proteases due to the lack of control and irregularity of endogenous proteases 33 . A recent report demonstrated that injection of thermolysin in beef loin during the marination process resulted in approximately 500-times higher ACE inhibitory activity than control (IC 50 1,206.0 vs 2.3 μg/mL) with an inhibitory effect on cancer cell viability after 3 days of storage 8 . Generation of small peptides through proteolysis by various enzymes (endo-and/or exogenous enzymes) increases the potent ACE inhibitory activity due to production of peptides with unspecified cleavage sites [34][35][36] . In addition, multi-enzymes hydrolysates derived from www.nature.com/scientificreports www.nature.com/scientificreports/ protein based ingredients exhibited higher ACE inhibitory activity compared to single-enzyme counterpart 37 . Generally, crust is more prone to protein decomposition by the aforementioned enzymes because the meat surface layer can be directly affected by microorganisms during dry aging. This assumption should be confirmed in a further study by investigating the effect of microorganisms (microbial enzymes) growing on the surface of beef on the production of ACE inhibitory peptides during dry aging. In addition, relationship between ACE inhibitory activity and the production of small peptide would be more discussed in Section of identification of peptides.
Protein profile by SDS-PAGE. Protein bands with low molecular weight (<15 kDa) were more prominently observed in aged samples compared to unaged one (Fig. 4). Especially, the crust sample had higher intensity in bands with low molecular weight among the treatments. About 5-10 kDa peptides in dry sausages exhibited up  www.nature.com/scientificreports www.nature.com/scientificreports/ to 86% of the DPPH radical scavenging activity 38 . In addition, low-molecular-weight peptides generated from protein by protease treatment exhibited higher ACE inhibitory activity compared with high-molecular-weight peptides 39 . Taken together, in this study, the generation of low molecular weight proteins may induce greater antioxidant and/or ACE inhibitory activity of crust and dry aged beef due to the proteolysis that occurs during dry aging. However, in this study, difference in appearance of protein band with <15 kDa in dry-and wet-aged samples was ambiguous. this result would be elucidated plainly based on identification of peptide and discussed in followed section.
Identification of peptides. Similar to the result from SDS-PAGE, much more number of small peptides (<3 kDa; 143 different types of peptides) from the aged and crust samples than those of unaged beef were detected, regardless of aging method (See Supplementary Table S1). On the other hand, types of peptides depended on aging method and location of dry-aged beef. Before aging, there were only 6 small peptides identified from the samples, while 52 and 64 different peptides from 19 and 25 different protein sources were found on the wet-and dry-aged samples, respectively. Interestingly, peptide containing same amino acid sequence was not generated between unaged and crust samples. This phenomenon was possibly due to presence of aforementioned microbial enzymes in crust which can more affected by microorganisms compared to edible portion. In our previous study, we found different changes in the metabolites of dry-aged beef by different microbial compositions 22 . Small peptides were more generated in dry-cured ham at final processing time causing by action of endopeptidase and exopeptidase by microorganisms during processing, comparing to those at initial time 40 . Many studies found that small peptides by enzymatic hydrolysis released and then it can increase functional property including antioxidant and ACE inhibitory activities in food 41 . In the present study, smaller peptides (<1 kDa) were observed in crust samples among the treatments, which was corresponded with specificity (lysyl-lysine residue; peptide No. 32) of previously reported peptide antioxidants 42 . According to previous study, peptides composing histidine at the C-terminus exhibited effective free radical scavenging activity 43  which can effectively act to free radicals from the lipid phase as scavenger were mostly presented in meat samples of this study.
Previous studies found that substrates having N-terminal branched-chain amino acid and C-terminal hydrophobic amino acids can bind to active site of ACE as exhibiting strong affinity 44 . In this study, total 17 types of potential inhibitory peptides were observed as indicated with asterisk in Table S1.  76, 95, 108, 133, and 135) types of the ACE inhibitory peptides were observed in unaged, wet-and dry-aged, and crust samples with partial overlap, respectively. ACE inhibitor possesses hydrophobic substituents at three C-terminal amino acids which can bind to active site of ACE 45 . Especially, presence of arginine (R), lysine (K) or proline (P) led to strong ACE inhibition activity in food protein-derived peptides at each of the three C-terminal position 46,47 . In this study, peptides (peptide 90 and 133) with aforementioned characteristic were observed in crust samples. Hence, the greatest (P < 0.05) ACE inhibition activity in crust samples was exhibited probably due to the presence of lysine and/or proline at the three C-terminal position.

conclusion
Based on this study, the excellent activities of antioxidant and ACE inhibitory activities from the crust may be due to the increases in these small peptides and sequence position of functional amino acids which were generated endo-and exopeptidases. The effect of microorganisms on proteolysis and lipolysis in the crust and edible portion of meat, and the relationship with the generation of bioactive peptides and characteristic flavors are under investigation in our laboratory. Consequently, the crust could be utilized as a functional ingredient possessing antioxidant and ACE inhibitory activity instead of disuse. In other words, dry aging can be used for generation of bioactive peptides from beef loin.