2020 年 26 巻 6 号 p. 771-778
Streptomyces sp. P-3 produces a novel streptomycetes elastase (SEL), which is a new member of the S1 serine protease family. First, we examined the production conditions of SEL with various parameters. Maximum enzyme production was achieved when the strain was grown in a medium containing defatted soybean meal and yeast extract at 28 °C for 44 h. Subsequently, the substrate specificities of SEL were compared to those of papain and subtilisin Carlsberg, which are commercially available enzymes for meat tenderization. The substrate specificity of SEL toward elastin was more than 12-fold higher than that of the other two enzymes. Furthermore, through the analysis of the degradation of myofibrils and elastin from beef, SEL demonstrated low activity toward myofibrils and high activity toward elastin. Physical property evaluation indicated that the SEL-treated connective tissue underwent a higher tenderization effect than tissues treated with alternative enzymes. These characteristics suggest that SEL has potential applications for improving meat tenderness without causing over tenderization.
We previously reported that a new streptomycetes elastase (SEL), produced by Streptomyces sp. P-3, showed higher activity for synthetic elastase-specific substrates than reported elastases. Furthermore, the amino acid sequence of SEL showed low homology with existing elastases. Phylogenetic analysis showed that SEL, together with a number of predicted streptomycetes proteases, make up a distinct group in the S1 serine protease family (Fujii et al., 2020). Therefore, it is of interest to characterize this novel elastase under more practical conditions and systems for industrial applications. We initially focused on the meat industry when applying this enzyme for consideration of industrialization. In the present study, we investigated the production conditions of SEL and the hydrolytic capabilities of SEL toward myofibrillar and connective tissue proteins of meat. We then compared these results to those of papain and subtilisin Carlsberg, which are commercially available proteolytic enzymes for meat tenderization (Ha et al., 2012; Ray and Rosell, 2016), focusing on connective tissue containing elastin as a main component.
Tenderness is one of the most important indicators of meat quality. Meat toughness is mainly caused by changes in myofibrillar proteins and the amount of connective tissue (Bouton et al., 1975; Christensen et al., 2013; Cross et al., 1973). Many techniques for improving meat tenderization have been applied to-date, including optimization of the marinade pH or storage temperature, mechanical tenderization, and enzymatic treatment. In the meat-processing industry, marinades of low or high pH are often used to reduce myofibrillar toughness. However, connective tissue, especially elastin, which is one of its constituents, is very stable and resistant to chemical, physical, and enzymatic alteration (Rucker and Dubick, 1984).
One of the most common approaches in meat tenderization involves the use of proteolytic enzymes. Plant enzymes, such as those found in papaya and pineapple, are frequently employed in this process (Abdel-Naeem and Mohamed, 2016). However, these enzymes have broad substrate specificities, and they therefore cause the over-degradation of myofibrillar proteins. This can lead to the deterioration of sensory characteristics, such as the texture and taste. Microbial proteases are also utilized to tenderize meat (Ryder et al., 2015; Sullivan and Calkins, 2010). In particular, proteases from the Bacillus genus have been studied and characterized extensively. Subtilisins produced by Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and some other species, have high substrate specificities for connective tissue (Takagi et al., 1992; Tsai et al., 1988). Takagi et al. (1992) reported that Bacillus sp. Ya-B produced alkaline elastase YaB (subtilisin YaB), which has high elastolytic activity and shows promise as an efficient meat tenderizer. However, in our previous study (Fujii et al., 2020), we showed that SEL had 10-fold higher activity toward Suc-Ala-Ala-Pro-Ala-pNA (an elastase-specific substrate) than subtilisin YaB.
Materials SEL was purified from Streptomyces sp. P-3 culture supernatant. Papain was purchased from Fujifilm Wako Pure Chemical Co. Ltd. (Osaka, Japan). Subtilisin Carlsberg was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Elastin-Congo red was purchased from Nacalai Tesque (Kyoto, Japan). Milk casein was purchased from Merck (Darmstadt, Germany).
SEL production Streptomyce sp. P-3 was pre-cultured in 50 mL of Glucose-Yeast extract-Defatted soybean meal (GYD) medium (0.1% (w/v) glucose, 1% (w/v) yeast extract, 0.15 (w/v)% defatted soybean meal, 0.1% (w/v) (NH4)2SO4, 0.05% (w/v) MgSO4) at 28 °C with agitation at 200 rpm for 24 h in a 300-mL Erlenmeyer flask. Then, 25 mL of this starter culture was transferred to 2.5 L of GYD medium, and cultured in a 5-L Jar Fermenter (Mitsuwa Frontech, Osaka, Japan) at 28 °C with agitation at 600 rpm and aeration at 1.0 vvm.
Enzyme preparation Purified SEL was prepared according to previously reported methods (Fujii et al., 2020). The supernatant of cultures in GYD medium was concentrated 5-fold with an ultrafiltration module, then purified using DEAE-Sepharose, CM-Sepharose, and Phenyl Sepharose column chromatography, sequentially.
Enzyme assay lastase activity was measured in a reaction mixture comprising 5 mg/mL elastin-Congo red in 0.9 mL optimal pH buffer for each enzyme. The used optimal pH buffer was 50 mM Tris-HCl (pH 7.5) for subtilisin Carlsberg, 50 mM Tris-HCl (pH 8.0) for SEL, and 50 mM potassium phosphate (pH 6.0), 2 mM EDTA, and 5 mM Cys for papain. The reaction was initiated by adding enzyme, and the solution was then shaken for 1 h at 25 °C. To stop the reaction, 0.2 mL of 0.1 M NaOH was added. Excess substrate was removed by centrifugation, and the absorbance of the supernatant was measured at 495 nm. One unit of this activity was defined as the amount of enzyme that causes an increase of 1.0 absorbance unit at 495 nm in 1 h at 25 °C.
Caseinolytic activity was determined according to the method described by Hagihara et al. (1958). The activity was measured in a reaction mixture comprising 2.5 mL of 1% casein and optimal pH buffer for each enzyme. The reaction was started by adding enzyme, and the solution was then shaken for 20 min at 37 °C; 2.5 mL of 0.4 M TCA was added to stop the reaction. One unit of this activity was defined as the amount of enzyme that causes an increase in absorbance at 660 nm equivalent to that caused by 1 µg of tyrosine per minute at 25 °C.
Determination of elastin content Elastin was quantified using LC-MS/MS by detecting desmosine, which is a specific constituent amino acid of elastin. Meat samples were sliced and hydrolyzed in 10 mL of 6 M HCl at 110 °C for 24 h in the presence of nitrogen. These samples were re-dissolved in mobile-phase solvent containing 5 mM heptafluorobutyric acid. Levels of desmosine were determined using ultra high-performance liquid chromatography (Prominence UFLC, Shimadzu, Kyoto, Japan) with an AccQ-Tag™ column (3.9 150 mm; particle size, 4 µm; Waters, MA, USA). The mass spectrometry was operated in positive ion mode, and results were obtained by selected ion recording at 526 to 481 m/z (Ma et al., 2003).
Measurement of myofibrillar degradation Myofibrillar protein extraction was performed as described by Busch et al. (1972) with a slight modification. The myofibrillar proteins were prepared using the red meat part of a short plate cut of beef. The muscle was homogenized in 5 volumes of wash buffer A (20 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 2 mM EDTA) using a mixer. The homogenate was centrifuged at 1 000 g for 10 min. Then, the sediment was washed five times with wash buffer B (same as buffer A except for EDTA being omitted). The obtained myofibril suspension was diluted to a concentration of 8 mg/mL on a dry weight basis in 50 mM Tris-HCl (pH 8.0). The suspension was mixed with each enzyme at the weight ratio of 1 600:1, and incubated at 37 °C for 30 min or 4 °C for 16 h. To stop the reaction, the sample was incubated at 95 °C for 5 min. The degradation of myofibrillar protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels as described by Laemmli (1970).
Measurement of elastin degradation The elastin was prepared from bovine aorta according to the method of Miyamoto et al. (2009). After washing the aorta with a 10% NaCl solution for 24 h at 4 °C, the tissue was cut into 5-mm sections using a blender. The sections were autoclaved at 121 °C for 10 h, then washed with distilled water. Finally, the sections were immersed in 90% ethanol and dried, at which point elastin was obtained. The suspension was adjusted to 20 mg/mL on a dry weight basis in 50 mM Tris-HCl (pH 8.0). The suspension of elastin was mixed with each enzyme at the weight ratio of 2 000:1, and incubated at 37 °C for 7 h. To stop the reaction, the sample was incubated at 95 °C for 5 min. Undegraded elastin was collected by centrifugation at 15 000 rpm for 5 min, washed twice with pure water, and prepared by freeze-dry processing at 30 °C. The degradation of elastin was determined by measuring the dry weight of the precipitate. The obtained precipitate was also analyzed using a scanning electron microscope (SEM) TM-1000 (SEM, Hitachi Co. Ltd., Tokyo, Japan).
Physical measurements of connective tissue A short plate cut of beef was purchased in blocks from a commercial processor, stored at 4 °C, and used for testing within 24 h. The short plate was trimmed according to the serratus ventralis muscle and connective tissue, and cut into rectangular sections (10 mm × 50 mm) with a thickness of 5.0 mm. All enzymes were applied to a single batch of the short plate of beef. The meat sections were marinated with each enzyme solution (adjusted to 600 µg/100 g meat) at 4 °C for 16 h. Each meat sample was heated at 220 °C for 5 min. The connective tissue was trimmed from the heated meat, and the tensile stress was measured with a rheometer (EZ-SX 500N, Shimadzu). The sample was constantly extended at a rate of 10 mm/s with a tensile plunger. Tensile stress was calculated from the maximum load applied to the sample during the tensile test until rupture with a 30-mm long specimen (Aminlari et al., 2009).
Statistical analysis Statistical analysis was performed using IBM SPSS Statistics (IBM SPSS Statistics for Windows, Version 23.0, IBM Corp., Armonk, NY, USA). The results are expressed as mean values and standard deviations. The data were subjected to one-way analysis of variance (ANOVA), followed by Tukey's test to compare the mean values. A value of P < 0.05 was considered statistically significant.
Optimization of the growth conditions for SEL production The effects of various nitrogen sources and physical parameters on SEL production are shown in Table 1. Among the various nitrogen sources tested, the addition of defatted soybean meal and yeast extract was effective for SEL production. Subsequently, the optimum initial pH and temperature of the culture were tested. The optimum pH and temperature for SEL production was 6.5 and 28 °C, respectively. Following further optimization, we cultured strain P-3 in GYD medium for 47 h. The SEL activity linearly increased for 44 h (Fig. 1), indicating that the strain produced SEL at a constant rate. The optical density (OD) of the culture reached a maximum at 16 to 23 h after the start of culture, indicating that the strain produced SEL at the later stage without cell growth. The yield of SEL reached 4.5 U/mL at 44 h. The obtained culture supernatant was purified according to the “Enzyme preparation” section, and used for characterization.
| Group | Parameters | Elastase activity (U/mL) |
|---|---|---|
| Nitrogen sources | Elastin | 0.25 |
| Gelatin | 0.12 | |
| Defatted soybean meal | 0.91 | |
| Yeast extract | 0.48 | |
| Corn steep powder | 0.16 | |
| Peptone | 0.06 | |
| Initial pH | pH 6.5 | 0.37 |
| pH 7.0 | 0.25 | |
| pH 7.5 | 0.22 | |
| pH 8.0 | 0.17 | |
| pH 9.0 | 0.14 | |
| Temperature (°C) | 24 | 0.15 |
| 28 | 0.25 | |
| 32 | 0.14 |
The strain was cultured in a medium containing 0.05% (w/v) glucose, 0.05% (w/v) yeast extract, 1% (w/v) elastin, 0.7% (w/v) K2HPO4, 0.25% (w/v) KH2PO4 at 28 °C with agitation in 120 rpm in 50 mL for 48 h. The pH of the medium was adjusted to 7.0. In the nitrogen source test, elastin in the medium was replaced with the other nitrogen sources as listed.
Growth and SEL production from Streptomyces sp. P-3
Streptomyces sp. P-3 was cultured in GYD medium at 28 °C with agitation in 600 rpm with aeration at 1.0 vvm in a 5 L-Jar fermenter. Closed circles, cell growth; triangles, elastase activity; open circles, glucose.
Elastin content of beef We analyzed the composition of the actual connective tissue of beef to investigate the localization of elastin. The short plate cut of beef, which is often used in the Japanese food industry, was used for the analysis. The short plate is an area where fat and red meat with connective tissue are alternately layered and has long been an issue of partial toughness in the Japanese food industry. The short plate was trimmed according to the serratus ventralis muscle and connective tissue, divided into red meat (Fig. 2A-RM) and connective tissue (Fig. 2A-YC, WC) sections, and the relative value of the elastin content of both was measured. Elastin was quantified by detecting desmosine, an amino acid specific to elastin, using LC-MS/MS. Consequently, elastin was detected in the yellow connective tissue (YC; Fig. 2B). This type of connective tissue does not become tender during cooking, which is considered an issue for the meat industry. We hypothesized that SEL could be a potential solution to this problem. Therefore, we evaluated the substrate specificity of the enzyme in this regard.
Analysis of elastin localization in beef
(A) Structure of short plate. RM, red meat; YC, yellow connective tissue; WC, white connective tissue. (B) Elastin content in short plate. Elastin was quantified by detecting desmosine using LC-MS/MS. The data are expressed as mean ± SD (n = 3).
Proteolytic activity of SEL for casein and elastin In our previous study, we reported that SEL displayed high elastolytic activity under neutral-alkaline conditions, at pH 7.0 to 11.0 (Fujii et al., 2020). Furthermore, SEL demonstrated higher activity toward an elastase-specific substrate than subtilisin YaB (Tsai et al., 1988). In this study, we investigated the specific activity of SEL and commercially available proteolytic enzymes for casein and elastin under optimal buffer conditions. The elastolytic activity of SEL was 4.96 U/mg, which was 2.8-fold and 18.1-fold higher than that of papain and subtilisin Carlsberg, respectively (Table 2). In contrast, the caseinolytic activity of SEL was the lowest of all activities analyzed. The elastin specificity of SEL was 19.4-fold higher than that of papain, and 12.0-fold higher than that of subtilisin Carlsberg. These results indicated that SEL has higher elastolytic activity and selectivity than the commercially available enzymes currently used for meat tenderization.
| Enzyme | Elastolytic activity (U/mg) | Caseinolytic activity (U/mg) | Elastolytic activity/Caseinolytic activity |
|---|---|---|---|
| Subtilisin Carlsberg | 1.76±0.03 | 1.70±0.01 | 1.03 |
| Papain | 0.27±0.01 | 0.43±0.06 | 0.64 |
| SEL | 4.96±0.05 | 0.40±0.02 | 12.4 |
Enzyme activities represent the mean and standard deviation of at least three observations.
Analysis of myofibrillar protein degradation We performed a myofibrillar protein degradation test under alkaline conditions (pH 8.0), which are routinely used for meat tenderization. The marinating of meat in alkaline solution indicated that the Z-line, which is a structural unit of myofibrils and connects sarcomeres, was degraded. The tenderization of red meat was caused by increasing the water retention rate due to an increase in the pH of the meat to a level higher than the isoelectric point (Oreskovich et al., 1992). Each enzyme treatment was performed assuming that the reaction at 4 °C for 16 h to be typical refrigerated marinating conditions, and a normal reaction temperature of 37 °C for 30 min to be marinating conditions. The hydrolysis of myofibrillar proteins was analyzed by SDS-PAGE (Fig. 3). The myofibrillar proteins treated with papain and subtilisin Carlsberg experienced significant degradation of the myosin heavy chain and actin (the main proteins in myofibrils) at both 4 °C and 37 °C. In contrast, when the myofibrillar proteins were treated with SEL, minor degradation of the myosin heavy chain and actin occurred at 4 °C and 37 °C, and almost no difference from the control was observed. It was previously reported that plant proteases, such as papain, have low specificity for substrates in meat and degrade the main proteins of myofibrils (Han et al., 2009; Melendo et al., 1996). In contrast, subtilisins produced by the genus Bacillus were reported to have higher substrate specificity than plant proteases, and this was reflected in the results obtained here. Although the degradation of myofibrils is an important contributor to meat tenderness, it is difficult to control the softening process, which sometimes leads to over-tenderization and a reduction in palatability (Kang and Rice, 1970). Results indicate that SEL has low reactivity with myofibrillar proteins, but higher reactivity with elastin under various temperature conditions.
SDS-PAGE of myofibrillar proteins with different protease treatments
Myofibrillar proteins were incubated at 4 °C for 16 h and 37 °C for 30 min with each protease. MHC, myosin heavy chain; C, control (without enzyme); lane 1, subtilisin Carlsberg; lane 2,papain; lane 3, SEL; lane M, molecular mass standards (Precision Protein Standard Kit, Bio-Rad).
Analysis of elastin degradation We analyzed the degradation of elastin prepared from bovine aorta with each enzyme treatment under alkaline conditions (pH 8.0) by measuring the weight of the residual elastin. The residual weight was evaluated relative to the weight of the control (i.e., sample with no enzyme added). The relative weight of samples treated with papain and subtilisin Carlsberg were 92.9% and 65.8%, respectively. In comparison, the relative weight of the SEL-treated sample was 39.8% (p < 0.005), making this the most hydrolyzed sample (Fig. 4). The degradation precipitates were then examined using SEM. No significant difference was observed between the papain treatment and control samples, and both precipitates had filamentous network structures (Fig. 5A, C). In contrast, the degradation of elastin fiber was observed in both subtilisin Carlsberg- and SEL-treated samples (Fig. 5B, D). SEL treatment caused significant disruption of the structure of elastin fiber, and many broken fragments were observed.
Residual weight of elastin with different enzyme treatments
Elastin was incubated at 37 °C for 7 h with each protease. The residual weight was evaluated relative to the weight of the control (i.e. without enzyme,). The data are expressed as mean ± SD (n = 3), error bars indicate standard deviation and the different letters indicate means that significant differ at p < 0.05 (Tukey's test), using IBM SPSS statistics (IBM Corp., NY, USA).
Scanning electron micrographs of elastin treated with different proteases
Elastin was treated with no enzyme (A), subtilisin Carlsberg (B), papain (C), and SEL (D).
Tenderization effect on beef To investigate the tenderization effect of each enzyme on the connective tissue of actual beef, we carried out a physical property test using a rheometer. Tensile stress was used for physical property evaluation, because the shear force of non-treated samples could not be measured. The results are shown in Fig. 6. The tensile stress for all enzyme-treated samples tended to decrease when compared to the control. Specifically, the stress was lower by 19.9% (p < 0.005), 12.3%, and 49.7% (p < 0.001) in subtilisin Carlsberg-, papain-, and SEL-treated samples, respectively, when compared to the control. However, no significant difference was seen in the papain-treated sample (p > 0.05). The SEL-treated sample had the lowest stress level, and it was significantly lower than that of any of the other enzyme-treated samples (p < 0.001). These results were in good agreement with those of the elastolytic activity and elastin degradation test, with SEL treatment demonstrating the greatest tenderization effect on the connective tissue proteins that constitute real meat. The structural changes in elastin fibers observed in Fig. 5 were presumed to also occur in the connective tissue during the marinating of meat, i.e., SEL broke down elastin on the surface of meat and penetrated inside. These mechanisms were considered to be a cause of the tensile stress reduction.
Effect of different proteases on connective tissue in beef during the cooking process
The beef was dipped in each enzyme solution at 4 °C for 16 h and heated at 220 °C for 5 min. Connective tissue was trimmed from cooked beef and measured for tensile stress. The data are expressed as mean ± SD (n = 8), error bars indicate standard deviation and the different letters indicate means that significant differ at p < 0.05 (Tukey's test).
Microbial proteases, especially neutral-alkaline proteases from the Bacillus genus, have been reported to have a greater hydrolyzing effect on connective tissue than plant proteases (Ha et al., 2012; Takagi et al., 1992; Qihe et al., 2006). Subtilisins are serine proteases produced by various Bacillus species, and they are widely used in various industries. The subtilisin subfamily, which includes subtilisin Carlsberg, subtilisin BPN', subtilisin E, and subtilisin YaB, has been reported to have elastase activity (Takagi et al., 1992; Tsai et al., 1986). In this study, subtilisin Carlsberg showed greater substrate specificity and degradability to connective tissue than papain. Furthermore, SEL exhibited even greater specificity and degradability than papain or subtilisin Carlsberg. Although subtilisin YaB has the highest elastolytic activity in the subtilisin subfamily, the enzyme showed lower activity than SEL. In addition, this enzyme has not yet been used industrially. Therefore, it is presumed that activity using elastin as a raw substrate would be higher again. These results showed that SEL is a proteolytic enzyme capable of specifically degrading connective tissue while preventing over-tenderization of myofibrils, unlike papain and proteases from the Bacillus genus. In our previous report, SEL and its streptomycetes orthologs constituted a novel group of the S1 serine protease family (Fujii et al., 2020). The present study indicates that this novel enzyme group has beneficial applications for connective tissue-targeted meat tenderness in the commercial meat industry. In addition, this enzyme may enable previously inedible and discarded portions of meat to become edible, which will contribute to the utilization of unused resources. Outside the meat industry, this enzyme is expected to be used in various fields and industries, such as health foods, cosmetics, and medical fields. Further applied research will be conducted in the future.
Acknowledgements We are deeply grateful to Akihiro Iida of Mitsubishi Corporation Life Sciences Limited for his supervision, advice, constructive discussions, and continued support throughout this work, and Kazutaka Uno and Takanobu Sakurai for their useful comments. We are also grateful to Yui Okabe, Osamu Tanigawa, Yukiko Sakai, and Shigeyuki Totoki for their valuable discussions and assistance.
