If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of ChinaSchool of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of ChinaSchool of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of ChinaSchool of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of ChinaSchool of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of ChinaSchool of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China
Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis–fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126–fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.
). Compared with clinical thrombolytic drugs (urokinase and streptokinase), nattokinase as a functional food component possesses several advantages, especially its effectiveness through oral administration (
). Therefore, development of a new B. subtilis–fermented food with nattokinase activity for oral antithrombus function would be attractive. In addition to producing nattokinase, B. subtilis is known to secrete several extracellular enzymes during the fermentation process, including a milk-clotting enzyme (MCE) that can be used to replace calf rennet (
Thermal stability of chymosin or microbial coagulant in the manufacture of Malatya, a halloumi type cheese: Proteolysis, microstructure and functional properties.
Bacillus subtilis can secret some bioactive substances beneficial to human health and has been applied as a probiotic in humans. Recent clinical research reported that oral intake of B. subtilis was effective in preventing pediatric diarrhea and reducing the duration of diarrhea (
reported that peptidoglycan-associated cyclic lipopeptide (a surfactin) from B. subtilis could disrupt coronavirus (CoV) virion integrity in the HUH7 cells and reduce infection >10,000-fold, and its viricidal activity extended broadly to enveloped viruses, including SARS-CoV, MERS-CoV, influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Bacillus subtilis has been used in probiotic products for regulation of the gut microbiota (
). However, there is no report on the application of B. subtilis in fermented milk.
In this study, we screened, identified, and characterized B. subtilis JNFE0126, a strain producing both nattokinase and MCE. The enzymatic properties of nattokinase and the MCE were investigated. Then, this strain was applied to produce a yogurt-like functional food with thrombolytic activity.
MATERIALS AND METHODS
Materials
Soybean and milk were purchased from a local market. Milk fermented with Lactobacillus bulgaricus and Streptococcus thermophilus was obtained from Bright Dairy & Food Co. Enzyme inhibitors, including phenylmethylsulfonyl fluoride (PMSF; an inhibitor of serine protease and cysteine protease), phosphoramidon (an inhibitor of metallopeptidase), aprotinin (an inhibitor of trypsin and chymotrypsin), pepstatin (an acid protease inhibitor), and E-64 (a cysteine protease inhibitor) were purchased from Sangon Biotech Co. Ltd. The rat fibrinogen and thrombin were purchased from Sigma-Aldrich. All other chemical reagents were obtained from Sinopharm Chemical Reagent Co. Ltd.
Formulations of Media and Screening Plates
Activation medium (for activation of the preserved strain) consisted of 0.5% peptone, 1% glucose, 0.5% NaCl, and water; amplification medium (for shaking flask amplification of the strains) consisted of 1.0% peptone, 0.5% yeast powder, 2% glucose, 0.2% NaH2PO4, 0.4% K2HPO4, 0.5% NaCl, and water; fermentation medium (for shaking flask production of nattokinase and MCE with strains) consisted of 3% kidney bean flour, 2% sucrose, 0.2% NaH2PO4, 0.4% K2HPO4, 0.4% NaCl, 0.02% CaCl2, and water. Nattokinase-screening plates (for screening and purification of the nattokinase-producing strains) were prepared as follows: 30 mL of 1% agar was boiled for 30 min, cooled to 70°C, and added to a 10-cm diameter culture dish. Then, 100 μL of thrombin (100 U/mL) and 3 mL of fibrinogen (1% wt/vol) were added, and the mixture was stirred until a homogeneous white color formed. The mixture was stored at 4°C for formation of the firm opaque gel.
Screening of the B. subtilis Strain with Both Thrombolytic and Milk-Clotting Activity
The strains with both thrombolytic activity and milk-clotting activity (MCA) were screened from the natural straws with the procedure presented in Figure 1. The soybean fermentation was carried out following a traditional natto production process. In detail, 250 g of soybean was soaked with 300 mL of water for 18 h and then drip-dried before being steamed at 100°C for 4 h. The soybean was cooled to 60°C and transferred and tiled into petri dishes. Natural rice straws and wheat straws of 10 different origins were collected from different regions in China and used as inoculum sources for B. subtilis. Each petri dish was covered with straw and incubated at 40°C for 12 h. One piece of soy was picked from each petri dish and put on the fibrin plate for the fibrinolytic activity test. Fermented soy from 5 of the 10 soybean dishes showed fibrinolytic activity and were selected for the following strain screening.
Figure 1Procedure for screening strains with both thrombolytic activity and milk-clotting activity from naturally fermented natto.
Each selected soybean was stirred with 20 mL of NaCl solution (0.9%, wt/wt). The supernatant was diluted to an appropriate concentration (diluted 104–105 times) and smeared on the fibrin plate. The plates were cultivated at 37°C for 48 h, and large amounts of different colonies with diameters approximately 0.5 to 2 mm were observed. Bright transparent rings were observed around many colonies. We collected 200 colonies with the largest transparent rings and cultured them in amplification medium. These 200 strains were further cultured with fermentation medium using shaking flasks. The fibrinolytic activity and MCA of the culture solutions were determined after 72 h of fermentation. The mean value of enzymatic activity from 3 duplicates of the shaking flasks were calculated, and the strains with the highest mean values were selected. Specifically, 20 strains with highest MCA were first chosen for the shortlist, and the strain with highest thrombolytic activity from among these 20 strains was selected.
Subsequently, the selected strain was purified using the streak-plate method on the nattokinase-screening plate, and the monocolony with the largest transparent ring was picked for further amplification and preservation. The cryopreserved strain was activated and the culture tested for thrombolytic activity and MCA to analyze the genetic stability. No significant change in either thrombolytic activity or MCA was observed in 10 subcultures. Therefore, this strain was used in the further research.
Assay for In Vitro Thrombolytic Activity
The fibrin plates were used to evaluate the in vitro thrombolytic activity of strains, fermentation broths, and other samples. Before testing, circular holes of 1 mm in diameter were punched on fibrin plates for sampling. Samples were loaded into these holes and incubated at 37°C for 18 h; then, the diameter of the clear zone around each sample hole was measured. Thrombolytic activity was calculated based on the diameter of the clear zone according to the standard curve prepared using standard urokinase solutions. The activity unit of urokinase was used as the unit for thrombolytic activity of nattokinase.
Assay for MCA
The MCA was determined with the following procedure: reconstituted skim milk (10% skim milk powder dissolved in water) was freshly prepared, supplemented with 10 mM CaCl2, and stored overnight at 4°C for complete hydration. The pH value of the milk was adjusted to 6.0 with 1 M HCl before use. A test tube containing 10 mL of skim milk was pre-incubated at 35°C for 10 min, and then 0.5 mL of sample solution was added. All samples were diluted with 20 mM phosphate buffer at pH 6.0, and the coagulation time of the diluted enzyme was adjusted to between 1 and 3 min. The mixture was thoroughly vortexed, and the time from the addition of the sample to the formation of the first visible clot was recorded. The enzymatic activity to clot 1 mL of reconstituted skim milk (10% skim milk powder, 1% CaCl2) in 40 min was defined as 1 SU. The MCA was calculated with the following formula:
SU = (2,400 × 10 × D)/(0.5 × T),
where D = sample dilution (times diluted) and T = coagulation time (in seconds).
Identification of the Strain
The selected strain was identified with microscopic examination and 16s rDNA sequencing. The strain cells on the slides were observed using the optical microscope following Gram staining. The genetic classification of the strain was assessed on the basis of the 16S rDNA gene sequence. The DNA from the strain cells was extracted using a Genomic Mini kit (Thermo Fisher Scientific) according to the protocol for gram-positive bacteria. The 16S rDNA was amplified by PCR technique with primers fD1 (5′-AGAGTTTGATCCTGGCTCAG) and rP2 (3′-AAGGAGGTGATCCAGCCGCA). The amplified DNA was then sequenced by Sangon Biotech Co. Ltd. The 16S rDNA gene sequence was compared with those from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/nuccore/NR_112116.2) using the BLASTn service provided by the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Inhibition Assays for Classification of the 2 Enzymes
The supernatant of the culture solution was subjected to inhibition using several protease inhibitors, including 0.001 M PMSF, 0.001 M phosphoramidon, 0.001 M aprotinin, 0.001 M pepstatin, and 0.001 M E-64. Ten microliters of protease inhibitor solution was added to the supernatant of the culture solution, and then the thrombolytic activity, MCA, or both were determined.
Production and Purification of Nattokinase and MCE
Fermentation medium was inoculated with amplified B. subtilis cells and cultured at 39°C with 120 rpm rotation. The thrombolytic activity and MCA of the culture medium were determined during fermentation. The crude enzymes were extracted at the interval with highest enzymatic activity.
Ammonium sulfate fractional precipitation was used to precipitate crude nattokinase from the fermentation broth. In detail, the fermentation broth was centrifuged at 5,000 × g at 4°C for 5 min, and the supernatant was collected. Ammonium sulfate was added to the supernatant to 30% saturation and kept for 2 h. The mixture was centrifuged at 5,000 × g for 5 min, and the supernatant was collected. Then ammonium sulfate was added to the supernatant to reach 70% saturation. After 2 h, the mixture was centrifuged at 5,000 × g for 5 min, and the precipitant was collected. The precipitant was dialyzed against water with a 3-kDa dialysis membrane for three 8-h cycles. After dialysis, the crude nattokinase was separated first using a carboxymethyl cellulose fast-flow column eluted with a linear gradient of NaCl from 0 to 1 M at pH 6.5, and the thrombolytic activity of fractions collected at different NaCl concentrations were tested. The thrombolytic active peak was collected and further purified using a G75 filtration column. The high thrombolytic fraction was lyophilized and then analyzed with SDS-PAGE.
According to the inhibition results, MCE from B. subtilis JNFE 0126 was a metallopeptidase, and the purification procedure of MCE was designed on this basis. Ammonium sulfate fractional precipitation was used to precipitate crude MCE from the fermentation broth. In detail, the fermentation broth was centrifuged at 5,000 × g at 4°C for 5 min, and the supernatant was collected. Ammonium sulfate was added to the supernatant to 30% saturation and kept for 2 h. The mixture was centrifuged at 5,000 × g for 5 min, and the supernatant was collected. Ammonium sulfate was added again to reach 60% saturation. After 2 h, the mixture was centrifuged at 5,000 × g for 5 min, and the precipitant was collected. The precipitant was dialyzed against water with a 3-kDa dialysis membrane for three 8-h cycles. After dialysis, the crude MCE was separated first using a diethylaminoethyl column eluted with a linear gradient of NaCl from 0 to 1 M at pH 7.5, and the MCA of fractions collected at different NaCl concentration was tested. The milk-clotting active peak was collected and further purified using a G75 filtration column. The high milk-clotting active fraction was lyophilized. The purified enzymes were then analyzed by SDS-PAGE with 4% stacking gel and 12% separating gel. Molecular weight marker proteins (Thermo Fisher Scientific) were used as standards.
Characterization of Enzymatic Properties of Nattokinase from B. subtilis JNFE0126
Effect of Temperature on Nattokinase Activity
The solution of the purified nattokinase (0.1 mg/mL, 10 μL) was added to the fibrin plate for the thrombolytic activity test. The fibrin plates were incubated for 18 h at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65°C respectively. Then, the diameters of the transparent circles were measured and the relative activity was calculated, with the activity at optimal temperature recorded as 100%.
Effect of pH on Nattokinase Activity
Fibrin plates were prepared with buffers instead of water to attain different pH values. The buffers used included 0.05 M acetate (pH 3, 3.5, 4, 4.5, and 5), 0.05 M phosphate (pH 5, 5.5, 6, 6.5, 7, 7.5, 8), and 0.05 M Tris-HCl (pH 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12). Ten microliters of nattokinase solution (0.1 mg/mL) was added to each fibrin plate for the thrombolytic activity test. The fibrin plates were incubated for 18 h at 37°C. Then, the diameters of the transparent circles were measured and the relative activity was calculated, with the activity at optimal pH recorded as 100%.
Effect of Metal Ions on Nattokinase Activity
Both the fibrin plate and the enzyme solution were combined with corresponding salts to investigate the effect of metal ions on nattokinase activity. We added CaCl2, MnCl2, KCl, NaCl, FeCl3, CuSO4, ZnSO4, FeSO4, and MgSO4 to the fibrin plates and the enzyme solution, respectively, with the final concentration at 10 mM. Ten microliters of nattokinase solution (0.1 mg/mL) was added with the ion to the corresponding fibrin plate for thrombolytic activity test. The fibrin plates were incubated for 18 h at 37°C. Then, the diameters of the transparent circles were measured, and the relative activity was calculated; activity without ions was recorded as 100%.
Characterization of Enzymatic Properties of MCE from B. subtilis JNFE0126
The MCA test was carried out at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65°C. Relative activity was calculated, with the activity at optimal temperature recorded as 100%. Reconstituted skim milk (10% skim milk powder, 1% CaCl2) was freshly prepared, supplemented with 10 mM calcium chloride, and stored overnight at 4°C for complete hydration. The pH value of the milk was adjusted to 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 with 1 M HCl or 1 M NaOH before use. A test tube containing 10 mL of skim milk was pre-incubated at 35°C for 10 min, and then 0.5 mL of diluted enzyme solution was added. The coagulation time was recorded, and the relative MCA at different pH values were calculated. The activity at optimal pH was set as 100%. We added CaCl2, MnCl2, KCl, NaCl, FeCl3, CuSO4, ZnSO4, FeSO4, and MgSO4 to diluted MCE solution to attain final concentrations of 10 mM, respectively. Skim milk was also added with these salts to 10 mM concentration.
Application of B. subtilis JNFE0126 in Production of Fermented Milk
Milk Fermentation
Skim milk added with soybean meal and sucrose was fermented by B. subtilis JNFE0126 to produce an antithrombus fermented milk. In brief, 1,000 mL of skim milk was mixed with 40 g of sucrose and 20 g of soy protein, and then sterilized at 95°C for 10 min. The solution was cooled to 41°C, and 20 mL of amplified culture (cfu = 3 × 108/mL) broth of B. subtilis JNFE0126 was inoculated at the logarithmic growth phase. The mixture was fermented at 41°C for 12 h, and the thrombolytic activity, MCA, pH, and bacterial counts (cfu) were determined during fermentation. After 8 h of fermentation, a firm curd was formed, and no obvious whey separation was observed. In a comprehensive consideration of the thrombolytic activity and sensory properties, the fermented milk at 8 h was used for further research.
Sensory Evaluation
Twenty panelists were trained to evaluate the B. subtilis JNFE0126–fermented milk and the reference Lactobacillus bulgaricus and Streptococcus thermophilus–fermented milk. A total of 9 men and 11 women between the ages of 20 and 61 yr old completed the sensory evaluation. The panelists were trained with the defined attributes as shown in Table 1.
Sensory evaluations were privately conducted while participants were seated in a quiet area behind a privacy divider in the food sensory evaluation facilities in Jiangnan University. Tastings occurred between 1500 and 1600 h. Two samples were served in a random order. Approximately 50 mL of each sample was presented in a glass bowl with a plastic spoon. Each panelist was first required to record his or her evaluation of the overall attractiveness of the samples, using a 9-point facial hedonic scale (1 = “liked extremely,” 5 = “neither liked nor disliked,” and 9 = “disliked extremely”). Afterward, each panelist was asked to fill out a form to give scores on specific odor, taste, and texture properties of the samples. The indexes listed in Table 1 were scored with reference samples, and the unlisted indexes were scored based on personal likeness.
Rheological Test of the Fermented Milk
The rheological property of the fermented milk was tested with the parallel-round-plates (40-mm diameter, 1-mm spacing) using an AR 2000 Dynamic Shear Rheometer (TA Instruments). The samples were calibrated at 25°C for 30 min, stirred for 3 min clockwise, and then for 3 min anticlockwise. Afterward, the samples were tested with frequency scan and shear scan. Frequency scan was carried out with 0.5% constant strain, scanning from 0.1 to 10.0 Hz. Shear scan was carried out from 0 to 50 Hz in 180 s.
Storage Stability Evaluation of the Fermented Milk
After stirring, the fermented milk was transferred into 100-mL glass bottles. The fermented milk was stored at 4°C for as long as 60 d, and the thrombolytic activity was determined after 1, 2, 3, 5, 7, 10, 14, 21, 28, and 60 d of storage. The changes in appearance and flavor during storage were observed as well.
Evaluation of the Antithrombus Effect of the Fermented Milk In Vivo
Establishment of Tail Thrombosis Model
The black-tail thrombosis model experiment was carried out on mice for in vivo evaluation of thrombolytic activity of the samples as described previously (
). Kunming mice (female, 19–20 g) were obtained from the Jiangsu University Experimental Animal Center. In this study, all animal experimental protocols were approved by the University Ethics Committee for the use of experimental animals and conformed to the Guide for the Care and Use of Laboratory Animals.
Sixty mice with tail lengths of 9 to 11 cm were selected and divided into 5 groups (12 mice each); namely, the normal group, the thrombosis group, the milk group, the L. bulgaricus and S. thermophilus–fermented milk group, and the B. subtilis–fermented milk group. The normal thrombosis groups were fed their usual diet. The milk group was fed the usual diet combined with 10 mL/kg of skim milk every 12 h. The L. bulgaricus and S. thermophilus–fermented milk group was fed their usual diet combined with 10 mL/kg of the L. bulgaricus and S. thermophilus–fermented milk every 12 h. The B. subtilis–fermented milk group was fed their usual diet combined with 10 mL/kg of the B. subtilis–fermented milk every 12 h. The differentiated diets were maintained from 7 d before injection of κ-carrageenan (to induce a thrombus) until the end of the experiment. To establish the tail-thrombosis (black tail or dark tail) model, carrageenan was injected intraperitoneally at a dose of 200 mg/kg of BW for the thrombosis group, the milk group, the L. bulgaricus and S. thermophilus–fermented milk group, and the B. subtilis–fermented milk group. The length of thrombus in the tails (the black part) of all the 3 groups was measured during the following 13 d (14 d after carrageenan injection in total). Ten mice from each group were used to measure the black tail, and 2 mice from each group were used in the histological examination.
Histological Examination of the Tails
At 5 d after carrageenan injection, the mice were deeply anesthetized by intraperitoneal injection of phenobarbital sodium (200 mg/kg). The tails of the mice were taken under deep anesthesia and fixed with 4% paraformaldehyde. Transverse sections at the points of 5 cm from the distal end of the tails were prepared and stained with hematoxylin and eosin. Histological changes of the tail tissue, especially the structure of the blood vessels, were observed with under the microscope.
Statistical Analysis
All data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS 13.0 statistical software (SPSS Inc.). Two-way ANOVA was used to analyze differences between groups, and P < 0.05 was considered to be statistically significant.
RESULTS AND DISCUSSION
Screening of High Thrombolytic Activity Strain
Soybean was fermented following the traditional natto production process. The obtained natto was covered with bacterial coating and sticky silks. Fermented soybean from 5 of the 10 soybean dishes showed significant fibrinolytic activity (Figure 2A), suggesting that there were thrombolytic-active strains in the straws of these. The dishes with thrombolytic activity were selected for the following strain screening. After primary screening with fibrin plates, 200 clones with the highest thrombolytic activity, which showed large transparent circles around the colonies (Figure 2B), were picked for secondary screening. These 200 colonies with thrombolytic activity were collected and amplified using a kidney bean medium. Twenty strains with the highest milk-clotting activities were chosen for the shortlist (Figure 2C). Among these, the strain with the highest thrombolytic activity was selected for further research and named JNFE0126. No significant change in either thrombolytic activity or MCA was observed in 10 subcultures. The results indicated that a strain with both thrombolytic activity and MCA was screened from homemade natto. This strain was used for further research.
Figure 2A strain with both thrombolytic activity and milk-clotting activity was isolated from traditionally fermented natto inoculated with wheat and rice straws. (A) natto with thrombolytic activity; (B) thrombolytic-active clones isolated from leaching solution of natto; (C) milk-clotting activity test; (D) micrographs of the purified strain with positive Gram staining.
The cells of the selected strain were gram-positive bacillus (Figure 2D). The cells were mostly 2 to 3 μm long, and some of the cells were connected in chains. The 16S rDNA of the strain was sequenced and compared with that from GenBank database (https://www.ncbi.nlm.nih.gov/nuccore/NR_112116.2). The sequence was 100% identical to a B. subtilis strain in the database. In combination with the morphological and biochemical characterization, we concluded that the selected strain was a B. subtilis species.
Effect of Protease Inhibitors on Thrombolytic Activity and MCA
Culture broths with thrombolytic activity and MCA were subjected to inhibition studies using several protease inhibitors to characterize and classify the enzymes. The protease inhibitors were added to sample solutions, and thrombolytic activity and MCA were determined. The results indicated that fibrinolytic activity was inhibited by PMSF, but not affected by phosphoramidon, aprotinin, pepstatin, or E-64 (Table 2). The results agreed with former reports that nattokinase is a serine protease and can be inhibited by PMSF (
). The MCA was inhibited by 0.001 M phosphoramidon but not by 0.001 M PMSF, 0.001 M aprotinin, 0.001 M pepstatin, or 0.001 M E-64, indicating that MCE secreted from strain JNFE0126 was a metalloprotease. Metallopeptidase M4 is the most reported MCE from bacteria (
Extraction and Purification of Nattokinase and MCE
Enzyme Production Properties of B. subtilis JNFE0126 in Fermentation Medium
The enzyme production curve of the strain was investigated during 120 h of fermentation. As shown in Figure 3A, the MCA of the culture was highest after 60 h of fermentation, and reached 874.5 SU/mL. The peak value of thrombolytic activity occurred at 96 h, and reached 3,511 U/mL. Thus, crude enzyme of MCE and nattokinase were collected at 60 h and 96 h, respectively.
Figure 3Enzyme production in shake flask culture. (A) Change of enzymatic activity during fermentation. Thrombolytic activity was highest at 96 h, and milk-clotting activity was highest at 60 h (n = 3; error bars represent SD). (B) SDS-PAGE patterns of purified nattokinase (NK) and milk-clotting enzyme (MCE).
Nattokinase was purified with carboxymethyl cellulose ion exchange chromatograph and G75 gel-filtration chromatography. The specific enzymatic activity of purified nattokinase was 8,722 U/mg. As shown in Figure 3B, the molecular weight of the purified nattokinase was approximately 27 kDa, which is in accordance with the molecular weight of nattokinase reported previously (
The MCE was purified with diethylaminoethyl anion exchange and G75 gel-filtration chromatography. The specific enzymatic activity of purified MCE was 263.5 SU/mg. As shown in Figure 3B, the molecular weight of MCE was approximately 42 kDa, which was in accordance with that of a milk-clotting protease isolated from B. subtilis reported previously (
As shown in Figure 4A, the thrombolytic activity of nattokinase was very sensitive to pH change. The optimum pH of nattokinase was 7.5. The activity reduced dramatically when pH decreased, and the thrombolytic activity was almost totally inhibited at pH 4.0. The presence of thrombolytic activity at pH over 12 did not make sense as fibrin would be dissolved by strong alkali. As shown in Figure 4B, the optimum temperature of nattokinase was 42°C. The enzyme retained most of its activity at 55°C, but activity decreased dramatically at a higher temperature. As shown in Figure 4D and Figure 4E, the MCE showed optimal activity at pH 7.0 and 55°C. Between 35°C and 60°C, the change in MCA was moderate. At higher temperature, however, the MCA was lost quickly; only 26% of the original activity was retained at 65°C.
Figure 4Effect of pH (A, D), temperature (B, E), and ions (C, F) on enzymatic activity of nattokinase (A, B, C) and milk-clotting enzyme (D, E, F; MCE). n = 3; error bars represent SD.
As shown in Figure 4C, the nattokinase activity was slightly inhibited by Mg2+ and severely inhibited by Fe2+, Fe3+, Cu2+, and Zn2+. However, Ca2+, Mn2+, and Na+ slightly enhanced nattokinase activity, whereas K+ had no significant activity on nattokinase activity. Activity of MCE was significantly inactivated by Fe2+, Fe3+, and Cu2+, largely enhanced by Ca2+, Mg2+, and Mn2+, and not significantly affected by K+, Na+, or Zn2+ (Figure 4F).
According to the results above, nattokinase and MCE could maintain high activity in the milk with neutral pH and high Ca2+ concentration. However, activity of nattokinase was severely inhibited under acidic conditions. Thus, it would be improper to use B. subtilis JNFE0126 in combination with acid-producing bacteria (such as Lactobacillus) for milk fermentation. Instead, it could be used as a single starter of fermented milk due to its MCA.
Preparation and Characterization of Thrombolytic Milk Fermented by B. subtilis JNFE0126
We inoculated milk with B. subtilis JNFE0126 and incubated at 41°C. The milk was fermented for 8 h before a firm curd was formed. At this fermentation time, bacterial count of the fermented milk was 5.7 × 108 cfu/mL, and the pH was 6.10 (Figure 5) and suggested that the clotting process was induced by the MCE, rather than by acid. Whey precipitation was not observed during fermentation or after ripening. The thrombolytic activity continued to increase during fermentation. However, as the fermentation time extended to >10 h, the curd became tattered with whey precipitation, and the taste became bitter because of excessive protein hydrolysis. Thus, 8 h of fermentation time was chosen for further experiments. The fermented milk achieved a thrombolytic activity of 215.1 U/mL after 8 h of fermentation. Thrombolytic activity of this fermented milk increased during the ripening period at 4°C, reaching 223.8 U/mL after 24 h.
Figure 5The change in bacterial count (cfu), pH, and thrombolytic activity during milk fermentation (n = 3; error bars represent SD).
Physiochemical Characterization of the Fermented Milk
The fermented milk formed a curd before being stirred, and no obvious whey separation was observed (Figure 6A). After stirring, the curd turned into viscous liquid (Figure 6B). Rheological properties of the fermented milk showed typical characteristics of a pseudoplastic fluid. As shown in Figure 6C and Figure 6D, the viscosity of the fermented milk was 15.1 Pa·s, and the apparent viscosity decreased gradually as the shear frequency increased. Apparent viscosity was 14.8 Pa·s under 0.1 Hz shear, which reduced to 5.5 Pa·s under 10 Hz shear. The rheological properties of the B. subtilis–fermented milk was similar to that of the L. bulgaricus and S. thermophilus–fermented milk, which suggested that they had similar casein gel structure.
Figure 6The appearance and rheological properties of the Bacillus subtilis–fermented milk before (A) and after (B) stirring; and changes in (C) elasticity coefficient and viscosity coefficient, and (D) elastic stress and viscous stress under different shear rates.
The samples were evaluated for color, smell, taste, consistency, and overall acceptability; additionally, the panelists were asked to list any defects. In general, the fermented milk obtained with B. subtilis JNFE0126 was white and tender, with a delicate taste, instant entry, the inherent taste of fermented milk, special sweet taste, and long aftertaste. As shown in Table 3, compared with traditional fermented milk, the B. subtilis JNFE0126–fermented milk had no sour taste. The smooth, melting, and consistency scores of the B. subtilis JNFE0126–fermented milk were higher than those of the reference L. bulgaricus and S. thermophilus–fermented milk. There was no significant difference in overall odor, taste, texture, and attractiveness between the 2 groups. These results suggested that the B. subtilis JNFE0126–fermented milk was as attractive as the L. bulgaricus and S. thermophilus–fermented milk, with no disagreeable flavor.
Table 3Sensory evaluation scores (n = 20; data are shown as mean ± SD)
Sensory index
Fermented milk
Bacillus subtilis JNFE0126
Lactobacillus bulgaricus and Streptococcus thermophilus
At an almost neutral pH value, the B. subtilis–fermented milk might be more vulnerable to spoilage and pathogenic microorganisms than traditional yogurt, and cold storage would be essential for this product. The fermented milk was stored at 4°C, and the change in thrombolytic activity was tested during storage (Figure 7). The results indicated that the thrombolytic activity decreased gradually during storage. After 7 d of storage, 73.2% of the original activity was retained. After 14 d of storage, activity decreased to 66.5%. Meanwhile, no significant change was observed in the appearance or taste of the fermented milk. These results suggested that the shelf life of the product would be at least 14 d if preserved at 4°C. Thus, this product could share the same cold chain of L. bulgaricus and S. thermophilus–fermented milk.
Figure 7Changes in thrombolytic activity of the fermented milk during storage at 4°C. n = 3; error bars represent SD.
The dark-tail thrombus model was successfully established through carrageenan injection. The therapeutic effect of the fermented milk in dark-tail thrombus model mice is shown in Figure 8. The average length of the whole tail of the mice was 9.02 cm. Twenty-four hours after injection of carrageenan, most mice showed a dark part at the far end of the tail. The changes in the lengths of the black tails are shown in Figure 8F. The average lengths of the dark part at 24 h after carrageenan injection were 2.45, 2.54, 2.23, and 1.29 cm for the thrombus group, the milk group, the L. bulgaricus and S. thermophilus–fermented milk group, and the B. subtilis–fermented milk group, respectively. Subsequently, mice in the thrombus group first suffered from extension of the dark part (0–2 d), followed by tail fester (3–7 d), and, finally, the dark part fell off (8–10 d). The length of the healthy part of the tails was only 0.96 ± 0.39 cm. The milk group and the L. bulgaricus and S. thermophilus–fermented milk group showed no significant difference compared with the thrombus group. For the B. subtilis–fermented milk group, however, thrombosis development was prevented after 2 d, and the healthy part of the tails remained at 5.1 ± 1.7 cm. As shown in the tissue sections of the tails (Figure 8A2–E2), blood vessels were blocked with thrombus in the thrombus group and the milk group, whereas the vessels were clear in the B. subtilis–fermented milk group. These results suggested that oral intake of the B. subtilis–fermented milk was effective in prevention and curation of thrombosis.
Figure 8Therapeutic effect in dark-tail thrombus model mice. (A1 and A2) Healthy mouse and tissue sections of the normal tail; the vascular cavity was clear. (B1 and B2) Mice from the thrombus group at 7 d after carrageenan injection and the tissue section 5 cm away from the distal end; there was thrombus in the vascular cavity. (C1 and C2) Mice from the milk group at 7 d after carrageenan injection and the tissue section 5 cm away from the distal end; there was thrombus in the vascular cavity. (D1 and D2) Mice from the Lactobacillus bulgaricus and Streptococcus thermophilus (LB/ST)–fermented milk group at 7 d after carrageenan injection and the tissue section 5 cm away from the distal end; there was no thrombus in the vascular cavity. (E1 and E2) Mice from the B. subtilis (BS)–fermented milk group at 7 d after carrageenan injection and the tissue section 5 cm away from the distal end; there was no thrombus in the vascular cavity. (F) Changes in length of dark part of the tail (including the part fallen off) of the mice (n = 10; error bars represent SD). The asterisk represents the vascular cavity.
In this research, we selected the B. subtilis JNFE0126 strain with both thrombolytic activity and MCA from naturally fermented natto. Then, 2 enzymes were purified and characterized from the broth of this strain, which showed high thrombolytic activity and MCA, respectively. The selected strain was used to produce the thrombolytic-active fermented milk, and the overall attractiveness of the B. subtilis–fermented milk was similar to that of traditional L. bulgaricus and S. thermophilus–fermented milk. Finally, we demonstrated the thrombolytic effect of the fermented milk in vivo using the black-tail thrombosis model mice. Our results suggest that the JNFE0126 strain, which secreted both nattokinase and MCE, was an excellent starter for the thrombolytic-active fermented milk, which could have potential as a novel and functional food in the prevention of thrombosis-related cardiovascular diseases.
ACKNOWLEDGMENTS
This work was supported by the national first-class discipline program of Food Science and Technology (Beijing, China; grant number JUFSTR20180202). The authors declare that they have no conflict of interest.
REFERENCES
Ding Z.
Liu S.
Gu Z.
Zhang L.
Zhang K.
Shi G.
Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran.
Thermal stability of chymosin or microbial coagulant in the manufacture of Malatya, a halloumi type cheese: Proteolysis, microstructure and functional properties.
00701-3&id=gr1.jpg){kind=link}
00701-3&id=gr2.jpg){kind=link}
00701-3&id=gr3.jpg){kind=link}
00701-3&id=gr4.jpg){kind=link}
00701-3&id=gr5.jpg){kind=link}
00701-3&id=gr6.jpg){kind=link}
00701-3&id=gr7.jpg){kind=link}
00701-3&id=gr8.jpg){kind=link}