Fibrinolytic bacteria of Indonesian fermented soybean: preliminary study on enzyme activity and protein profile

mthenawidjaja@yahoo.com Abstract A number of fibrinolytic enzyme producing microorganisms have been identified in various fermented food in Asia, such as from Red Oncom , Tempe , Terasi (Indonesia), Natto (Japan), Douchi , Soy paste (China), Meju , Kimchi , Jeot-gal , Tofuyo , Chungkook-jang , Soybean paste and Doen-jang (Korea). The high protein content in soybean is potential to promote the growth of proteolytic and fibrinolytic producing microbes. This study investigated several fibrinolytic bacteria, isolated from the moromi stage of the fermented soysauce production. The fibrinolytic activities of the isolates were confirmed in a fibrin plate assay, and all 3 potential isolates (K1, K2, and K3) were identified as Gram positive, rod shaped, and spore forming bacteria. Moreover, analysis of the sequences encoding 16S rRNA gene, revealed K1 as Bacillus cereus , K2 as Bacillus subtilis , and K3 as Bacillus cereus . SDS PAGE analysis demonstrated different protein profile of the cell free supernatant of isolates grown in LB media during fermentation, which ranged from 18.40-45


Introduction
Annually, cardiovascular diseases (CVDs), including stroke and heart diseases have been identified as the leading cause of death worldwide. Meanwhile, WHO data estimated about 17.9 million related deaths in 2016, which represents 31% of all global cases, and about 85% occurred due to heart attack and stroke. In addition, out of the 17 million premature demise, i.e., under the age of 70, the proportion attributed to noncommunicable diseases as reported in 2015 were 82% of low-and middle income countries, and 37% were caused by CVDs (World Health Organization, 2017). Moreover, the basic pathophysiology of stroke involves formation and attachment of fibrin clot or thrombus to the injured blood vessel walls.

Fibrinolytic bacteria of Indonesian fermented soybean: preliminary study on enzyme activity and protein profile
Fermented soysauce is one of the food products from soybean, obtained through two steps in the manufacturing process, encompassing (1) solid-state (koji fermentation) where the output is dried and (2) salt fermentation, where the dry mass is mixed with brine solution to form a mash, termed "moromi", which is further fermented within various periods of aging (range from six months to one year) (Lee et al., 2013;Zhu & Tramper, 2013). The high protein contents in the soybean is potential of being utilized as a media for growing proteolytic and fibrinolytic microorganism, and this preliminary study confirmed a strong activity from bacteria isolated at the moromi stage.

Materials and methods
In this study, the moromi of fermented soy sauce was obtained from PT. Logan Food Lombok Gandaria (Karanganyar, Central Java, Indonesia), while the bacteriological agar was purchased from Sigma, US and Luria Bertani media was procured from Scharlau, Spain. Fibrinogen sourced from bovine plasma was purchased from Sigma, US, and the API 50CHB kit for bacterial identification was obtained from BioMérieux S.A, France.

Isolation of proteolytic bacterial strain from Moromi (Afifah et al., 2014)
The process of isolating proteolytic microbe was conducted using Skim Milk Agar media (SMA), with the composition of bacteriological agar and skim milk (1.5%). In addition, a total of 1 mL of moromi was dissolved in 10 mL sterile aquadest, which was then strirred to a point of homogenity (moromi solution). Therefore, 0.1 mL was collected and spread onto the surface of SMA media, using a sterile hockey stick, which was subsequently incubated at 37 °C for 48 hours, and then the clear zone observed indicated the existance of the protease producing microbes. Furthermore, the protease colonies were isolated and purified until a pure culture in the form of a mono colony was obtained, which was then grown in slant agar, and saved as a stock in glycerol 50% for further analysis.

Enzyme production (Afifah et al., 2014)
One ose of the selected microbe was grown in 12.5 mL sterile Luria-Bertani broth media (LB), in order to obtain the optical density (OD) of 0.8 at λ 620nm . Therefore, a total of 10% (v/v) fermentation broth recorded to have reached the required value was mixed with 25 mL LB, and further incubated for 1, 2, 3, 4, and 7 days at 37 °C in the shaking incubator (120 rpm) (PolyScience, US). Furthermore, the production media with fibrinolytic protease content was taken at the appropriate time and centrifuged at 6000 g for 30 minutes at 4 °C, and then the cell free supernatant was collected as the enzyme source for subsequent analysis.

Fibrin plate assay (Astrup & Mullertz, 1952)
The Fibrinolytic bacteria were screened using a modified fibrin plate method (Astrup & Mullertz, 1952), where a total of 0.5% fibrinogen (Sigma F3879, US) solution in 50 mM sodium phosphate buffer (pH 8) was mixed with 2% agarose solution along with 0.02 mL of thrombin (Sigma T4648, US) solution (100 NIH units). These were then applied to a petri dish, and left for 1 hour at room temperature to form a fibrin clot layer. In addition, a sterile glass tube (diameter 6 mm) was used to make hole in the fibrin plate, to which twenty μL of the enzyme was dropped and incubated at 37 °C for 20 hours. Therefore, the positive control utilized the commercial fibrinolytic enzyme termed Lumbrokinase from Lumbricus rubellus (Disolf DLBS1033, Dexa Medica, Indonesia) (40 mg/mL), prepared in a 20 mM phosphate buffer. Moreover, the activity of fibrinolytic enzyme was estimated by measuring the dimensions of the clear zone on the fibrin plate.

SDS PAGE (Laemmli, 1970)
SDS-PAGE was conducted with 10% of separating and 4% stacking gel. Also, the cell free supernatant was dilluted in a sample buffer 5x (containing 60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, and 0.1% bromophenol blue (BPB)), and the gel was run at 100 V, 100 mA for 1 hour, up to the point where BPB reached the bottom. Therefore, the protein bands were visualized by staining the gel with Coomassie Blue (CBBGR) for 20 minutes, which was sequentially soaked in a destaining solution, in order to attain discoloration, and the apparent molecular mass of the protein was calculated using Broad Molecular Weight standard protein markers (ThermoFisher Scientific, US).

Protein determination (Bradford, 1976)
The protein concentration was analysed using the reagents consisting of 100 mg Coomassie Brilliant Blue (CBB) G-250 in 50 mL ethanol 95% and 100 mL phosphate acid 85% in 1L. Therefore, Bovine Serum Albumin (BSA) was used as the protein standard, and the experiments were conducted in triplicates for each measurement.

Identification of microorganisms
The identification of microorganisms followed 3 steps, which include (1) microbiology analysis i.e. Gram staining, spore staining, and morphological examination, using a microscope with 1000X magnification.

Results and discussion
Microbes usually obtained in fermented food are Gram-positive bacteria, encompassing Bacillus sp., Lactobacillus sp., Staphylococcus sp., and Streptococcus hemolyticus, which tend to be easily screened from food and the environment. There are non-toxic and beneficial microorganisms for human health, including Bacillus pumilus JB-1 from Korean Cheonggukjang, which has long been known to produce immuno-stimulant substances. Research conducted by Wei et al., (2011) reported antioxidant activity in enzymes produced by B. amyloliquefaciens LSSE-62, obtained from fermented chickpea. Juan & Chou (2010) and Lee et al. (2008) also reported similar activities in Bacillus subtilis and some fungi obtained from fermented legumes. Duc et al. (2004) stated the potential of Bacillus cereus, Bacillus clausii, and Bacillus pumilus to confer probiotic properties.
The 3 isolates (K1, K2, and K3) screened from moromi stage of soy sauce production were proven capable of producing fibrinolytic protease activity, as seen on the fibrin plate assay (Figure 1a). Specifically, K2 fermented at 1 day (K21) possessed the highest fibrinolytic activity of all, with the widest clear zone area of 1.50 cm in the fibrin plate. In addition, the measurement for K1, K2, and K3 grown for 1 day (K11, K21, and K31) were between 1.30 cm and 1.50 cm, which was higher than those fermented at 2 days (K12, K22, and K32), within the range of 0.81 cm and 1.08 cm, and 3 days (K13, K23, and K33) of 0.65 cm to 1.23 cm. Furthermore, the extracellular enzyme obtained from K13 possessed the lowest activity of about 0.65 cm. In this case the protein concentrations of all samples evaluated were within the range 0.92-0.99 mg/mL. The fibrinolytic activity of extracellular enzyme obtained from K1, K2, and K3 isolates were compared with the commercial Lumbrokinase (LK), a fibrin degrading enzyme from earthworm Lumbricus rubellus, and Figure 1b indicated a close similarity with the K21 enzyme. Afifah et al. (2014) reported the production of fibrinolytic enzymes from bacteria isolated from Indonesian fermented Gembus tempe and Red oncom. Clear zone produced on the fibrin plate using similar method were 1.35 cm and 1.45 cm, respectively. These values were less than that from the best isolate of the current study. Thus, the bacteria obtained from moromi stage of fermented soysauce, were proven capable of producing good fibrinolytic enzymes. Bacillus licheniformis, Bacillus cereus, Brevibacillus laterosporus, and Bacillus pumilus were the fibrinolytic bacteria identified from Red oncom and Gembus tempe (Afifah et al., 2014).
Based on Gram staining, spore staining, and morphological test, all of the isolates (K1, K2, and K3) were identified as rod-shaped Gram positive and spore producing bacteria ( Figure 2a). The results of biochemical tests using API 50 CHB kit specifically for Bacillus spp, identified isolate K1 as Bacillus cereus 1 (99.8%), K2 as Bacillus subtilis (97%), and K3 as Bacillus cereus (99%). Molecular identification based on 16S rRNA's gene confirmed K1 as B. cereus 1 (100%), K2 as B. subtilis (99.86%), and K3 as B. cereus (97.56%). The nucleotide sequences were deposited at GenBank, with accession number MK045762 for K1, MK652831 for K2, and MK530096 for K3, and the phylogenetic tree was constructed on the basis of the sequences as presented in Figure 2b.
Fermented foods are regarded as sources with potential fibrinolytic efficacy, as their active compounds are suspected to affect blood circulation Data on studies reporting fibrinolytic activity in various fermented foods are shown in Table 1.
The fibrinolytic activity of extracellular enzyme obtained from Bacillus cereus K1, Bacillus subtilis K2, and Bacillus cereus K3 isolate were comparable to the commercial product Lumbrokinase (LK) from earthworm (Lumbricus rubellus). This implies the useful potentials of the three new isolates (Bacillus subtilis and Bacillus cereus) in the production of fibrinolytic enzymes for health application, which in this case act as an anti thrombolytic agent.
Homeostasis is very important in the blood coagulation and fibrinolysis system because of the highly elaborated and complicated system, where both factors ought to be maintained at a balanced and well controlled state. In addition, the fibrin converted from fibrinogen during thrombosis was identified as an insoluble protein, which is stabilized by thrombin-activated factor XIII through cross-linking (Furlan, 1988). Therefore, it is critical to decrease its redundant deposits at the sites of tissue injury, in order to fully restore potency to the damaged vessel. Furthermore, the fibrinolytic system, consisting of enzymes like plasmin, tissue plasminogen activator (t-PA), plasminogen activator inhibitors (PAIs), and urokinase are identified as the body's main defense mechanism against occlusive vascular diseases (Lijnen, 2001).
Thrombolytic agents have been identified in numerous organism, ranging from earthworm, snake, centerpede, mushroom, kelp, some algae and microrganisms (Kotb, 2012;Yogesh & Halami, 2017). Hence, streptokinase from Streptococcus hemolyticus and staphylokinase from Staphylococcus aureus which act as plasminogen activator, have been adopted as thrombolytic agent's medicine to overcome thrombosis. Meanwhile, two other agents known to be effective (Kotb, 2012) include tissue-type plasminogen activator (t-PA) (Collen & Lijnen, 2004) and urokinase (Duffy, 2002), which both activate plasminogen into active plasmin, in order to properly degrade fibrin. However, in addition to being expensive, certain side effects have been reported through injection application, which is possibly due to the presence of impurities or some imune active material in the preparation. This has, therefore, ignited the quest for a research on safer sources, including traditional fermented food.
The protein profile of extracellular enzymes produced from each isolate with different fermentation time were analysed with SDS PAGE (Figure 3), using about 0.92-0.99 mg/mL concentrations loaded into the gel. Generally, protein of 20 kDa was present in almost all fermentation extract, and the elevation of fermentation time led to an increase in the protein synthesis performed by the bacterias. Therefore, the profiling illustrated the presence of only 1 or 2 protein bands on day 1 and 2, while higher protein varieties from 14 to 45 kDa, indicative of more protein bands, were found on the fermentation time of day 4 and 7. Table 2 shows the correlation between protein fractions observed in SDS PAGE profiling and proteolytic activity, pointing 20 kDa protein as responsible for the fibrinolytic effects. In addition, the strongest activity was observed on day 1 of the fermentation extract in all isolates, followed by day 2 and 3, which imply that the fibrin degrading enzyme was synthesized at the early phase of growth.

Conclusion
Based on the results, it was established that Moromi from soysauce contains fibrinolytic bacteria, as confirmed by fibrin plate analysis. Moreover, based on morphological, biochemical and molecular evaluation, K1, K2, and K3 isolates were confirmed as Gram positive, rod shaped, and spore forming Bacillus, where K1 was confirmed as Bacillus cereus, K2 Bacillus subtilis and K3 Bacillus cereus. All three isolates produced several proteins during fermentation process in the LB media.   -