Hydrolysis Conditions of Porcine Blood Proteins and Antimicrobial Effects of Their Hydrolysates

Whole porcine blood was freshly obtained immediately after slaughter and immediately used for plasma preparation. Ethylenediaminetetraacetic acid was added as anticoagulation agent to fresh porcine blood at 2 g/L and mixed well. Blood was immediately placed in ice slash and brought back to the laboratory within 30 min. Samples were centrifuged (Supra 25K, Hanil Science Industrial Co., Ltd., Incheon, Korea) at 8,000×g for 15 min at 4℃ for plasma separation. Globulin and albumin proteins were isolated from the separated plasma using a modified cold ethanol method (Cohn et al., 1946).

For the separation of globulin and albumin proteins from the plasma, the plasma separated by centrifugation was cooled in an ice water bath. Cold ethanol was then added to the plasma at a final concentration of 7.4%. Next, centrifugation was performed at 10,000×g for 20 min to remove fibrinogen and antihemophilic factor. After that, ethanol was added to the supernatant at a final concentration of 24% and centrifugation was carried out again under the same conditions (10,000×g for 20 min). The resulting precipitate (globulin) was stored at 15℃. Ethanol was added to the separated supernatant at a final concentration of 60%. The precipitate (albumin) obtained after centrifugation was stored at 15℃.

The blood proteins obtained in the previous steps were subjected to single protease hydrolysis using the following proteases: alcalase, flavourzyme, neutrase, protamex, papain, and trypsin (Novozymes, Bagsvaerd, Denmark). The characteristics of commercial enzymes used in this study were decribed in Table 1. In brief, 1 g of blood protein was dissolved in 50 mL of 25 mM sodium phosphate buffer (0.2 M monobasic sodium phosphate+0.2 M dibasic sodium phosphate), which was adjusted to pH 7.0 with 1 N NaOH and 1 N HCl. The mixture was homogenized with a homogenizer (T-25 basic, Ika Works, Wilmington, NC, USA). To determine the optimum hydrolysis time for each protease, blood protein solutions were hydrolyzed with each protease in a 50℃ water bath for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. The proteases were added at a ratio of 1% for a particular blood protein. After completion of hydrolysis, the solutions were heated in boiling water for 3 min to inactivate the proteases. The mixture was centrifuged at 8,000×g for 25 min using Supra 22K (Hanil Science Industrial, Incheon, Korea). The supernatant was stored at –20℃ until use.

DH was measured using soluble protein as an indicator. In other words, protein concentration of the supernatant of the hydrolyzed solution was measured by the Lowry method (Lowry et al., 1951). DH was calculated using the following formula:

Gram-positive bacteria B. cereus KFRI 181 and S. aureus ATCC12692 and gram-negative bacteria S. Typhimurium ATCC 14028, E. coli KFRI 836, and S. flexneri ATCC 11836 were obtained from Korea Food Research Institute (KFRI, Seongnam, Korea) and Korea National Microbiological Research Resource Center (KNMRRC, Suwon, Korea). Antimicrobial tests were carried out using disc diffusion method (Lee and Lee, 2010). Bacterial suspension (1 mL) containing 10⁸ CFU/mL was spread onto nutrient agar plates (Difco Laboratories, Detroit, MI, USA). The hydrolysates of blood proteins (albumin, glubulin, plasma) used in this antimicrobial test were those at 2 h of hydrolysis. Then 50 (20 mg/mL), 200 (80 mg/mL), and 400 (160 mg/mL) μL of each hydrolysate concentrated by air drying (dry oven, JS-lin-2500, Seoul, Korea) was added to 8 mm diameter discs placed on inoculated agar. The inoculated plates were then incubated at 37℃ for 48 h. Antimicrobial activity was assessed by measuring the zone of inhibition against the tested organisms and indicated in the following manner: –, no antimicrobial activity; ±, slight antimicrobial activity with inhibition zones of 8.1–15 mm; +, moderate antimicrobial activity with inhibition zones of 15.1–20 mm; ++, clear antimicrobial activity with inhibition zones of 20.1–25 mm; +++, strong antimicrobial activity with inhibition zones of more than 25.1 mm.

All measurements were repeated three times. Results are expressed as mean values with standard deviations. Data were statistically analyzed with ANOVA and Tukey’s multiple range test. Statistical significance was accepted at p<0.05 (SAS, 2003).

Table 1 shows DH values of porcine blood albumin according to proteolytic enzyme used and hydrolysis time. Trypsin hydrolysate of albumin exhibited the highest DH values at all hydrolysis times, followed by alcalase, papain, protamex, flavourzyme and neutrase hydrolysates. Neutrase and flavourzyme hydrolysates of albumin showed DH values of less than 1% after 5 h of hydrolysis. Trypsin hydrolysate of albumin showed the highest DH values at 1 h of hydrolysis. All DH values did not change significantly at 2 h of reaction time (Table 2). In a previous study, DH of porcine hemoglobin was reported to increase with increasing hydrolysis time (Chang et al., 2007).

Table 3 shows DH values of porcine blood globulin according to proteolytic enzyme used and hydrolysis time. As in the case of albumin, alcalase, trypsin, and papain hydrolysates of globulin showed significantly higher DH values than those produced by the other three proteases. According to the tendency of DH with increasing reaction time (Table 2), DH values of alcalase, protamex, and trypsin hydrolysates of globulin did not change significantly after 1 h of hydrolysis. Papain hydrolysate of globulin showed the highest DH value at 4 h of hydrolysis.

Table 4 shows DH values of porcine blood plasma for the six proteases and reaction times used. DH values of porcine blood plasma protein hydrolysates produced by any protease did not exceed 2% after 5 h of reaction time. Virtually no hydrolysis reaction was observed for plasma proteins when using neutrase, flavourzyme, or protamex. However, alcalase, trypsin, and papain tended to exhibit higher DH values at 2 h of hydrolysis than at other reaction times (Table 3). The DH values of albumin and globulin hydrolysates were higher than those of plasma protein hydrolysates. Alcalase, trypsin, and papain hydrolysates of blood proteins exhibited higher DH values than neutrase, protamex, or flavourzyme hydrolysates of blood proteins. In addition, the blood protein hydrolysates tended to show the highest DH values with the hydrolysis time of 2 h.

Enzymatic hydrolysis is the most common method for producing bioactive peptides from proteins. For hydrolysates to be suitable for further application, hydrolysis conditions such as the enzyme type and concentration, hydrolysis reaction time, temperature, pH, and substrate-to-enzyme ratio are very important (Najafian and Babji, 2012). Among these conditions, enzyme type is crucial to peptide production. According to Hrckova et al. (2002), alcalase is an endopeptidase capable of hydrolyzing proteins, with broad specificity for peptide binding; it prefers large uncharged residues. Verma et al. (2017) reported that when the pig liver was hydrolyzed with alcalase, trypsin and papain for 0, 2, 4, or 6 h, as the hydrolysis time increased, the DH increased. Trypsin showed significantly higher DH than alcalase and papain at more than 2 h of hydrolysis. Pig liver hydrolysate obtained from trypsin hydrolysis showed highest functional activities (antioxidant and antimicrobial) followed by papain and alcalase pig liver hydrolysates. In addtion, in a result of Hiidenhovi et al. (2005), the DH of ovomucin hydrolysed with 10 different enzymes, including alcalase, protamex, and trypsin were not different between 1 h of hydrolysis and 4 h hydrolysis. Flavourzyme possesses both endoprotease and exopeptidase activities. Its ability to release free amino acids is higher than serine endoprotease alcalase (Hrckova et al., 2002). In a previous study by Qian et al. (2007), when the tuna dark muscle hydrolysate was produced by using six hydrolytic enzymes (alcalase, neutrase, pepsin, papain, α-chymotrypsin, and trypsin), the hydrolysate produced by pepsin showed the highest anti-hypertensive activity among all hydrolysates. Ranathunga et al. (2006) reported that the pepsin hydrolysate had the highest antioxidant activity, although it exhibited the lowest DH. Although pepsin was not one of the proteases used in the present study, results of previous studies suggest that DH may vary depending on the protease used. In addition, the bioactivity of the hydrolysate can also change.

The antimicrobial effects of porcine blood protein hydrolysates on five pathogenic microorganisms (B. cereus, S. aureus, S. Typhimurium, E. coli, and S. flexneri) were tested and antimicrobial effect was shown only in B. cereus (Table 5). Hydrolysates produced by all proteases exhibited high DH values at 2 h of hydrolysis, hydrolysates at 2 h were used for antimicrobial activity tests in the present study. The hydrolysates obtained by hydrolyzing porcine albumin, globulin, and plasma proteins with the six proteases did not show any antimicrobial activity against the pathogenic bacteria S. aureus, S. Typhimurium, E. coli, or S. flexneri. However, porcine blood hydrolysates showed antibacterial effects on B. cereus. Of the hydrolysates generated from the three blood proteins, albumin hydrolysates generally showed higher antimicrobial activity than globulin and plasma hydrolysates. When 50 μL of albumin hydrolysates were added to the discs, hydrolysates by all proteases except papain showed slight antimicrobial activity. Clear antimicrobial activity was observed with the addition of 400 μL of albumin hydrolysates by all proteases except papain. Furthermore, albumin and globulin hydrolysates generated by flavourzyme, protamex, and trypsin showed higher antimicrobial activity than those generated by alcalase or neutrase. Of plasma protein hydrolysates generated by different proteases, only those generated by trypsin exhibited moderate antimicrobial activity (with addition of 200 μL to the discs).

Numerous research studies have been carried out on peptides showing antimicrobial activity, using plant and animal proteins. Among animal proteins, antimicrobial hydrolysates have been isolated and characterized from milk proteins, egg proteins, and blood and muscle hemoglobin proteins (Abdou et al., 2007; Hayes et al., 2006; Jang et al., 2008; Xu et al., 2009). In a previous study by Nedjar-Arroume et al. (2006), 1% hemoglobin solution was hydrolyzed by porcine pepsin at 3% DH, and antibacterial activity of the hemoglobin hydrolysate against nine microorganisms was examined—three gram-negative (E. coli, Shigella sonnei, and Salmonella enteritidis) and six gram-positive (Micrococcus luteus A270, Listeria innocua, Enterococcus faecalis, B. cereus, Staphylococcus saprophyticus, and Staphylococcus simulans). They found that the total hemoglobin hydrolysate exhibited antimicrobial activity against M. luteus A270, L. innocua, E. coli, and Salmonella enteritidis. In addition, 9 out of 26 fractions were found to have antibacterial activity (Nedjar-Arroume et al., 2006). The fractions mentioned above can be divided into two groups on the basis of structure. One group consists of b126–145, a107–136, and a107–141 peptides containing less than 50 amino acid residues. They have an overall positive charge owing to the presence of multiple lysine and arginine residues in addition to a substantial stretch of hydrophobic residues and a higher α-helical structure (Powers and Hancock, 2003). The second group includes a133–141 and a137–141 peptides which are small (5 and 9 amino acids) and positively charged. They show little to no presence of hydrophobic residues and a higher random coil structure. These peptides are known to possess antimicrobial activity against gram-positive and gram-negative bacteria with membrane-disruptive and non-membrane-disruptive mechanisms (Mohammad et al., 1995; Powers and Hancock, 2003). Similar findings have been reported by Daoud et al. (2005) and Hu et al. (2011). Hu et al. (2011) reported that a newly discovered peptide located in the central part of bovine α-hemoglobin presented antimicrobial activity against E. coli, S. aureus, and Candida albicans. The sequence of this bovine peptide was similar to that of peptides in sheep, deer, pigs, and humans. Although antimicrobial study using hydrolysates of porcine plasma proteins, albumin, and globulin has not yet been reported, Friedrich et al. (2000) reported that bovine albumin peptides have antibacterial activity against gram-positive bacteria including strains of Staphylococcus, Enterococcus faecalis, L. monocytogenes, and Streptococcus pyogenes. Therefore, it is expected that porcine blood albumin hydrolysates may have antimicrobial activity against B. cereus. However, in a previous study by Salampessy (2010), a hydrolysate (DH 28.2%) of leatherjacket fish (Meuschenia sp.) insoluble muscle proteins generated by bromelain—a proteolytic enzyme—at 4.3 mg/mL concentration was found to be active primarily against B. cereus and S. aureus, and the sequence of the active peptide was identified as Glu-Gln-Ile-Asp-Asn-Leu-Gln (EQIDNLQ)—an anionic peptide rich in glutamic acid and aspartic acid. However, most antimicrobial peptides reported previously are cationic peptides rich in amino acid residues such as proline, arginine, phenylalanine, glycine, and tryptophan (Abdou et al., 2007; Hayes et al., 2006; Jang et al., 2008; Xu et al., 2009). The antimicrobial activity of anionic peptides has also been reported by Lai et al. (2002). Although the hydrolysates were not structurally and chemically characterized in the present study, the study showed, for the first time to our knowledge, that hydrolysates of porcine blood plasma proteins, albumin, and globulin exhibited antibacterial activity against B. cereus. This is the first step in analyzing porcine blood plasma proteins for their functionality. Further detailed studies are needed in the future to determine factors such as the optimal hydrolysis conditions, inhibitory activity against various microorganisms, and peptide sequence.