Nattokinase Research Articles

Production of nattokinase and γ-polyglutamic acid via soybean whey fermentation by Bacillus subtilis BSNK-5 and their emulsification to form nanoemulsions

Soybean whey, a nutrient-rich byproduct generated during the production of soybean protein isolate, can only be discharged after undergoing sewage treatment, which imposes a significant economic burden on enterprises. In this study, we investigated a Bacillus subtilis BSNK-5 fermentation soybean whey process that yielded high production of nattokinase (NK) and γ-polyglutamic acid (γ-PGA), along with the fabrication of an emulsion using fermentation soybean whey, to achieve high-value conversion of soybean whey. The results showed that soybean whey can be used as a nutrient-rich substrate to promote the efficient synthesis of NK and γ-PGA by BSNK-5, with the highest yields of 9 × 104 IU/mL and 250 g/L, respectively. Meanwhile, the synthesized γ-PGA can serve as a suitable biological macromolecule to form an emulsification system that protects NK. The encapsulation efficiency of NK was over 60%, and the release of encapsulated NK was delayed in vitro. Meanwhile, the nanoemulsions remained stable when exposed to 37°C and 65°C temperatures, salt concentrations ranging from 50-150 mM, and undergoing a single freeze-thaw cycle. The development of functional thrombolytic preparations by self-emulsification protection of NK by γ-PGA produced via BSNK-5 fermentation represents an effective way to achieve high value utilization of soybean whey.

Nattokinase Attenuated Excitatory Amino Acids and Cytokines Release and Restored Cerebral Blood Flow in a Thrombolytic Focal Cerebral Ischemic Rat Model.

Nattokinase (NK), a protease enzyme present in traditional fermented Japanese food, has shown fibrinolytic properties in vitro as well as in cardiac ischemia. In the present study, the Neuroprotective effect of standardized NK was evaluated in the thrombolytic focal cerebral ischemic model. The parameters of behavioural assessment, cerebral blood flow, inflammatory mediators, excitatory amino acids, and immunohistochemistry were measured to support the NK effect. NK was administered at 150 and 300 mg/kg, and its effects were compared with streptokinase (STK) (100μl/rat). Each mg of NK contains 5.5 Units of the enzyme, which can cause lysis of the fibrin. The results indicate that 7 days of treatment of 300 mg NK restored the cerebral blood flow and prevented the release of cytokine and excitatory amino acids. Similarly, neurological scores were reduced, and grip strength increased significantly with NK treatment. The GFAP and synaptophysin staining of the hippocampus (CA1) and cerebrum have shown recovery of neurons from ischemic damage in comparison to vehicle-treated ischemic-reperfused rats. The NK (300 mg/kg) fibrinolytic effect is comparable to STK treatment. To conclude, NK, a serine protease, protects the brain from ischemic degeneration in thrombolytic cerebral ischemia. Consumption of this Japanese food might exhibit prophylactic activity.

Site-directed mutagenesis of nattokinase: Unveiling structure-function relationship for enhanced functionality

Site-directed mutagenesis was employed to investigate the structure-function relationship of nattokinase (NK) and its effect on the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK. Specific mutations (T270S, V271I, E262D, and A259T) were introduced within the nk gene, targeting regions predicted to be involved in substrate binding. The NK(E262D) mutant exhibited a significant increase in enzymatic activity (2-fold) and catalytic efficiency (2.2-fold) as assessed by N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis, compared to the wild type. In silico analysis supported these findings, demonstrating lower binding energy for the NK(E262D) mutant, suggesting stronger fibrin affinity. Thermostability assays revealed that NK(E262D) and NK(A259T) displayed exceptional stability, retaining enzyme activity at 60 °C. All mutants exhibited a broader pH tolerance range (pH 5.0–10.0) compared to the wild-type NK. The fibrinolytic activity assay revealed that the E262D mutant possessed the highest fibrinolytic activity (2414 U/mg), surpassing the wild-type. This study reported an NK variant with improved enzymatic activity, thermostability, and fibrinolytic properties.

Fermentation with Bacillus natto and Bifidobacterium improves the functional, physicochemical properties, and taste qualities of coix seed-natto

Coix seed-natto (CS-natto) is a nutritious food rich in various functional components such as nattokinase (NK), and the strains’ fermentation is crucial for enhancing its quality. This work utilized Bacillus natto GUTU09 (B9) and Bifidobacterium animalis subsp. lactis BLH1 or BLH6 to ferment soybeans that were soaked in a saccharified liquid made from CS, resulting in the preparation of CS-natto, studied the physicochemical and functional characteristics during fermentation, and analyzed the correlation of various indicators. Finally, an electronic tongue analysis was conducted on CS-natto. The results indicated that fermentation significantly increases NK and antioxidant activity in CS-natto, with NK activity in BLH6-B9 natto reaching 425.00 FU/g. Co-fermentation notably enhanced the content and composition of phenolic substances. Furthermore, the study revealed that the organic acid and soy isoflavone content in co-fermentation were significantly higher than in single-strain fermentation. Fermentation could elevate the levels of amino peptide nitrogen and soluble peptides. Additionally, the addition of Bifidobacterium exhibited a synergistic effect on the fermentation of B9 to produce natto. Correlation analysis indicated that Bifidobacterium promoted B9 to produce NK. The BLH1 encouraged the conversion of organic acids. Daidzein and daidzin were positively correlated (P<0.01), suggesting a potential mutual conversion. Finally, electronic tongue analysis indicated that co-fermentation could effectively enhance the taste. The results indicated that CS-natto could serve as an improved dietary supplement offering enhanced quality and more beneficial effects on people’s health.

Nattokinase's Neuroprotective Mechanisms in Ischemic Stroke: Targeting Inflammation, Oxidative Stress, and Coagulation.

Aims: Nattokinase (NK), a potent serine endopeptidase, has exhibited a variety of pharmacological effects, including thrombolysis, anti-inflammation, and antioxidative stress. Building on previous research highlighting NK's promise in nerve regeneration, our study investigated whether NK exerted protective effects in transient middle cerebral artery occlusion (tMCAO)-induced cerebral ischemia-reperfusion injury and the underlying mechanisms. Results: The rats were administered NK (5000, 10000, 20000 FU/kg, i.g., 7 days before surgery, once daily). We showed that NK treatment dose dependently reduced the infarction volume and improved neurological symptoms, decreased the proinflammatory and coagulation cytokines levels, and attenuated reactive oxygen species (ROS) in the infarcted area of tMCAO rats. We also found that NK could exert neuroprotective effects in a variety of vitro models, including the microglia inflammation model and neuronal oxygen-glucose deprivation/reperfusion (OGD/R) model. Notably, NK effectively countered OGD/R-induced neuron death, modulating diverse pathways, including autophagy, apoptosis, PARP-dependent death, and endoplasmic reticulum stress. Furthermore, the neuroprotection of NK was blocked by phenylmethylsulfonyl fluoride (PMSF), a serine endopeptidase inhibitor. We revealed that heat-inactive NK was unable to protect against tMCAO injury and other vitro models, suggesting NK attenuated ischemic injury by its enzymatic activity. We conducted a proteomic analysis and found inflammation and coagulation were involved in the occurrence of tMCAO model and in the therapeutic effect of NK. Innovation and Conclusion: In conclusion, these data demonstrated that NK had multifaceted neuroprotection in ischemic brain injury, and the therapeutic effect of NK was related with serine endopeptidase activity.

Construction of highly active and stable recombinant nattokinase by engineered bacteria and computational design

Nattokinase (NK) is an enzyme that has been recognized as a new potential thrombolytic drug due to its strong thrombolytic activity. However, it is difficult to maintain the enzyme activity of NK during high temperature environment of industrial production. In this study, we constructed six NK mutants with potential for higher thermostability using a rational protein engineering strategy integrating free energy-based methods and molecular dynamics (MD) simulation. Then, wild-type NK and NK mutants were expressed in Escherichia coli (E. coli), and their thermostability and thrombolytic activity were tested. The results showed that, compared with wild-type NK, the mutants Y256P, Q206L and E156F all had improved thermostability. The optimal mutant Y256P showed a higher melting temperature (Tm) of 77.4 °C, an increase of 4 °C in maximum heat-resistant temperature and an increase of 51.8 % in activity at 37 °C compared with wild-type NK. Moreover, we also explored the mechanism of the increased thermostability of these mutants by analysing the MD trajectories under different simulation temperatures.

Comparative Cardioprotective Effectiveness: NOACs vs. Nattokinase-Bridging Basic Research to Clinical Findings.

The use of non-vitamin K antagonist oral anticoagulants (NOACs) has brought a significant progress in the management of cardiovascular diseases, considered clinically superior to vitamin K antagonists (VKAs) particularly in the prevention and treatment of thromboembolic events. In addition, numerous advantages such as fixed dosing, lack of laboratory monitoring, and fewer food and drug-to-drug interactions make the use of NOACs superior to VKAs. While NOACs are synthetic drugs prescribed for specific conditions, nattokinase (NK) is a natural enzyme derived from food that has potential health benefits. Various experimental and clinical studies reported the positive effects of NK on the circulatory system, including the thinning of blood and the dissolution of blood clots. This enzyme showed not only fibrinolytic activity due to its ability to degrade fibrin, but also an affinity as a substrate for plasmin. Recent studies have shown that NK has additional cardioprotective effects, such as antihypertensive and anti-atherosclerotic effects. In this narrative review, we presented the cardioprotective properties of two different approaches that go beyond anticoagulation: NOACs and NK. By combining evidence from basic research with clinical findings, we aim to elucidate the comparative cardioprotective efficacy of these interventions and highlight their respective roles in modern cardiovascular care.

Intravenous injection of nattokinase-heparin electrostatic complex improves the therapeutic effect of advanced tumors by dissolving cancer-related thrombosis

AimsCancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors. Main methodsIn this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors. Key findingsWe observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer. SignificanceThis study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Production of Nattokinase from Bacillus amyloliquefaciens MRS18: A Bacterial Strain Isolated from Fermented Beans.

Nattokinase (NK) is a naturally occurring fibrinolytic protease enzyme obtained from the traditional Japanese food called Natto and has several uses in the pharmaceutical and medical industries. Nowadays, the most often used thrombolytic agent in the clinical field is NK, in part because it is less expensive than other thrombolytic medicines. The objective of this study is to investigate the screening, isolation and characterization of the NK enzyme-producing Bacillus strain from fermented Soya beans. The sample of fermented soya beans were tested for the presence of fibrinolytic protease- producing bacteria, followed by the screening, extraction, characterization and clot lysis assays. A total of three isolates were screened for caseinolytic activities by casein hydrolysis assay. Out of those isolates, MRS18 was found to be potent in producing the enzyme proteinase. To determine the taxonomy and phylogeny of these isolates, biochemical and molecular characterization has been carried out. Bacillus amyloliquefaciens MRS18 has been found with the highest caseinolytic activity. The clot lysing ability of the potent strain Bacillus amyloliquefaciens was found to be 61.7% after 120 min, and on further purification, by ammonium sulphate precipitation method, the lysis percentage was found to be 656% after 120 min. From the results of the present study, we concluded that Bacillus amyloliquefaciens isolated from the fermented soya beans produced an NK enzyme that exhibits immense potential to lyse blood clots.

Lipid-lowering, antihypertensive, and antithrombotic effects of nattokinase combined with red yeast rice in patients with stable coronary artery disease: a randomized, double-blinded, placebo-controlled trial.

Nattokinase (NK) and red yeast rice (RYR) are both indicated for their potential in cardiovascular disease prevention and management, but their combined effects especially in coronary artery disease (CAD) are scarcely examined. This 90-day randomized, double-blind trial aims to investigate the effect of NK and RYR supplementations on cardiometabolic parameters in patients with stable CAD. 178 CAD patients were randomized to four groups: NK + RYR, NK, RYR, and placebo. No adverse effects due to the interventions were reported. In comparisons across groups, NK + RYR showed the maximum effect in reducing triglyceride (-0.39 mmol), total cholesterol (-0.66 mmol/L), diastolic blood pressure (-7.39 mmHg), and increase in high-density lipoprotein cholesterol (0.195 mmol/L) than other groups (all p for multiple groups comparison<0.01). Both NK + RYR and NK groups had significantly better-improved lactate dehydrogenase than the others (-29.1 U/L and - 26.4 U/L). NK + RYR group also showed more potent reductions in thromboxane B2 and increases in antithrombin III compared to placebo (both p < 0.01). These improved markers suggest that combined NK and RYR may preferably alter antithrombin and COX-1 pathways, potentially reducing thrombosis risks in CAD patients. Overall, the combined NK and RYR supplementation is safe and more effective than separately in improving cardiometabolic markers among CAD patients with multiple heart medications use.

Nattokinase attenuates endothelial inflammation through the activation of SRF and THBS1

Natto contains a potent fibrinolytic enzyme called nattokinase (NK), which has thrombolytic, antihypertensive, antiatherosclerotic and lipid-lowering effects. Although NK has been recognized for its beneficial effect on humans with atherosclerotic cardiovascular disease (ASCVD), the underlying mechanisms involved in vascular inflammation-atherosclerosis development remain largely unknown. The current study aimed to explore the effects of NK on gene regulation, autophagy, necroptosis and inflammasome in vascular inflammation. The transcriptional profiles of NK in endothelial cells (ECs) by RNA sequencing (RNA-seq) revealed that NK affected THBS1, SRF and SREBF1 mRNA expression. In Q-PCR analysis, SRF and THBS1 were upregulated but SREBF1 was unaffected in ECs treated with NK. NK treatment induced autophagy and inhibited NLRP3 inflammasome and necroptosis in ECs. Furthermore, the inhibition of SRF or THBS1 by siRNA suppressed autophagy and enhanced the NLRP3 inflammasome and necroptosis. In a mouse model, NK reduced vascular inflammation by activating autophagy and inhibiting NLRP3 inflammasome and necroptosis. Our findings provide the first evidence that NK upregulates SRF and THBS1 genes, subsequently increasing autophagy and decreasing necroptosis and NLRP3 inflammasome formation to reduce vascular inflammation. Therefore, NK could serve as nutraceuticals or adjuvant therapies to reduce vascular inflammation and possible atherosclerosis progression.

Effect of nattokinase on the pathological conditions in streptozotocin induced diabetic rats

Nattokinase (NK), also known as subtilisin NAT (EC 3.4.21.62), is a serine protease produced by Bacillus subtilis natto that has anti-inflammatory and fibrinolytic properties. To study whether NK prevents the progression of pathological changes in diabetes as an inflammatory disease, we examined the effect of NK on pathological conditions in streptozotocin (STZ)-induced diabetic rats using the following parameters: fasting blood glucose (glucose), total plasma protein (TP), creatinine, histopathology of renal corpuscles and tubules, advanced glycation end products (AGEs), and C-reactive protein (CRP). STZ-administered rats were maintained on a basic diet (CE-2) as control, low-NK diet (containing 0.2 mg NK/g diet), and high-NK diet (0.6 mg NK/g diet) for 14 days. High-dose NK significantly inhibited both glycogen deposition in the renal tubules and increase in the circulating AGE levels without downregulating glucose levels. Compared with the control group, the group treated with the high-NK diet presented a significant inhibition of the increase in the circulating CRP level on day 7 after the beginning of the treatment. However, the CRP level in the NK-H group reached the same level as that in the control group on Day 14. AGEs are known to induce CRP expression in hepatocytes, but the increase in CRP levels in our animal model was independent on the circulating AGE levels. In contrast, low-dose NK did not suppress changes in these parameters. Our present study suggests that NK suppresses glycogen deposition in renal tubules in a dose-dependent manner by the downregulation of AGE formation under hyperglycaemia in the rats with STZ-induced short-term diabetes. However, it is unclear whether this downregulation is caused by intact NK or peptides derived from NK during its digestion in the digestive tract.

Nattokinase Encapsulated Nanomedicine for Targeted Thrombolysis: Development, Improved in Vivo Thrombolytic Effects, and Ultrasound/Photoacoustic Imaging.

Nattokinase (NK), a potent thrombolytic enzyme that dissolves blood clots, is highly used in the treatment of cardiovascular disorders. However, its effective delivery remains demanding because of stability and bioavailability problems owing to its high molecular weight and proteineous nature. In this research, we have developed novel NK-loaded nontargeted liposomes (NK-LS) and targeted liposomes (RGD-NK-LS and AM-NK-LS) by the reverse phase evaporation method. The physiochemical characterizations (particle size, polydispersity index, zeta potential, and morphology) were performed by a Zetasizer, SEM, TEM, and AFM. The Bradford assay and XPS analysis confirmed the successful surface conjugation of the targeting ligands. Platelet interaction studies by CLSM, photon imager optima, and flow cytometry showed significantly higher (P < 0.05) platelet binding affinity of targeted liposomes. In vitro evaluations were performed using human blood and a fibrinolysis study by CLSM imaging demonstrating the potent antithrombotic efficacy of AM-NK-LS. Furthermore, bleeding and clotting time studies revealed that the targeted liposomes were free from any bleeding complications. Moreover, the in vivo FeCl3 model on Sprague-Dawley (SD) rats using a Doppler flow meter and ultrasound/photoacoustic imaging indicated the increased % thrombolysis and potent affinity of targeted liposomes toward the thrombus site. Additionally, in vitro hemocompatibility and histopathology studies demonstrated the safety and biocompatibility of the nanoformulations.

High Production of Nattokinase via Fed-Batch Fermentation of the γ-PGA-Deficient Strain of Bacillus licheniformis

During the production of nattokinase (NK) by Bacillus species, certain by-products such as poly-γ-glutamic acid (γ-PGA) are simultaneously synthesized. The impact of γ-PGA synthesis on NK production remains unclear. In this study, we knocked out the pgsC gene, a component of the γ-PGA synthetase cluster (pgsBCA), and constructed a novel recombinant strain, Bacillus licheniformis BL11. Next, we compared the fed-batch fermentation profiles of BL11 and its parental strain BL10, conducted transcriptional analysis, and measured intracellular ATP content. We also optimized glucose-feeding strategies under varying oxygen supply conditions. Our results indicated that the utilization rates of glucose and soybean meal were both improved in the pgsC-deficient strain BL11, and NK activity was enhanced. Furthermore, the transcriptional levels of genes involved in glycolysis and the TCA cycle were relatively upregulated in BL11. The maximal NK activity reached 2522.2 FU/mL at 54 h of fermentation using a constant glucose-feeding rate of 5.0 g/(L·h) under high oxygen supply conditions. The newly developed recombinant strain B. licheniformis BL11, along with the optimized feeding strategy, shows promise for large-scale NK production.

Nattokinase enhances the sensitivity of cancer cells to oxaliplatin through mitochondrial pathway and induction of apoptosis

Combination therapy consisting of anticancer drugs has been used to decrease the adverse effects and increase the effectiveness of the drugs. The present study aimed to examine the synergistic effect and potential mechanisms of oxaliplatin and nattokinase (NK) combined treatment for BGC-823, HepG-2, HeLa, and NCI-H460 cells. NK was isolated and purified from black soybean natto by dialysis and gel filtration chromatography, which had a nattokinase activity of 600,391 U/g. MTT experiment, clone formation assay, Hoechst 33,342 staining, DCFH-DA staining, and JC-1 staining showed that NK had no cytotoxicity and oxaliplatin could inhibit cell proliferation. Oxaliplatin combined with NK could enhance its anticancer effect and lead to mitochondrial dysfunction by causing the increase of intracellular ROS, which eventually induced apoptosis. Compared to oxaliplatin alone, nattokinase in combination with oxaliplatin can effectively inhibit DNA synthesis and increase cell uptake, ultimately inhibiting cell proliferation. Transwell and angiogenesis experiments proved that the combination of NK and oxaliplatin could more effectively inhibit cell migration, invasion, and in vitro angiogenesis, thereby preventing the spread and metastasis of tumors. RT-qPCR showed that the mechanism of inhibition of cell proliferation and thus induction of apoptosis by the combination of NK and oxaliplatin may be achieved through the upregulation of BAD, BAX, and Caspase-3 genes. Molecular docking results show that NK could act as the carrier of oxaliplatin. In conclusion, the above results suggested that NK could play a synergistic anticancer role with oxaliplatin by inducing apoptosis through the mitochondrial pathway, enhance the sensitivity of cancer cells to oxaliplatin, and provide a new strategy for cancer treatment.

Protective effects of nattokinase against microvasculopathy and neuroinflammation in diabetic retinopathy.

Diabetic retinopathy (DR) is a significant global public health concern. Alternative, safe, and cost-effective pharmacologic approaches are warranted. We aimed to investigate the therapeutic potential of nattokinase (NK) for early DR and the underlying molecular mechanism. A mouse model of diabetes induced by streptozotocin was utilized and NK was administered via intravitreal injection. Microvascular abnormities were evaluated by examining the leakage from blood-retinal barrier dysfunction and loss of pericytes. Retinal neuroinflammation was examined through the assessment of glial activation and leukostasis. The level of high mobility group box 1 (HMGB1) and its downstream signaling molecules was evaluated following NK treatment. NK administration significantly improved the blood-retinal barrier function and rescued pericyte loss in the diabetic retinas. Additionally, NK treatment inhibited diabetes-induced gliosis and inflammatory response and protected retinal neurons from diabetes-induced injury. NK also improved high glucose-induced dysfunction in cultured human retinal micrangium endothelial cells. Mechanistically, NK regulated diabetes-induced inflammation partially by modulating HMGB1 signaling in the activated microglia. This study demonstrated the protective effects of NK against microvascular damages and neuroinflammation in the streptozotocin-induced DR model, suggesting that NK could be a potential pharmaceutical agent for the treatment of DR.

A single dose of oral nattokinase accelerates skin temperature recovery after cold water immersion: A double-blind, placebo-controlled crossover study

Nattokinase (NK) intake may improve blood flow; however, its effects on skin temperature, which is predominantly controlled by skin surface blood flow, are unknown. The purpose of this study was to determine the effects of a single dose of NK on changes in skin temperature after cold water immersion. A double-blinded, placebo-controlled, crossover intervention study was performed on nine healthy men. The participants were randomised to receive either a single dose of 2,000 fibrinolytic units (FU) of NK or a placebo with subsequent crossover. Two hours after supplementation, the participants immersed both hands in a water bath maintained at 10 °C for 1 min. Skin temperature, perceived coldness, cardiac output, and sympathetic nervous activity were measured before, during, and after water immersion. Two-way analysis of variance showed a significant effect of treatment interaction on the skin temperature of the middle finger, palm, and back of the right hand (p < 0.05). These findings represented that the skin temperatures of the middle finger, palm, and back of the right hand immersed in the cold water were significantly dropped due to the cold water immersion, and then recovered more quickly by NK intake than by placebo intake. The results of the current study highlight the potential implications of NK for the prevention of excessive vasoconstriction. It may be more significant for those with cold-sensitive constitution, such as women and elderly. In contrast, the acute administration of 2,000 FU of NK did not affect changes in heart rate, cardiac output, sympathetic nervous activity compared with a placebo in healthy men.

Nattokinase prevents β-amyloid peptide (Aβ1-42) induced neuropsychiatric complications, neuroinflammation and BDNF signalling disruption in mice

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disorder characterized by abnormal accumulation of extracellular β-amyloid (Aβ) plaques and neuronal damage. Although AD is typically considered a cognitive neurodegenerative disorder, almost all people diagnosed with AD develop neuropsychiatric complications at some stage in their life span. The present study investigated the effect of chronic Nattokinase (NK) administration on β-Amyloid peptide (Aβ1-42) induced neuropsychiatric conditions (depression-like behaviour, anxiety, and memory impairment) in mice. Aβ1-42 peptide injected mice demonstrated depression, anxiety, and impairment of cognitive abilities evaluated as increased immobility time in forced swim test (FST), decreased open arm time/entries in elevated plus maze (EPM) and reference and working memory error in radial arm maze (RAM) respectively with elevation in Interleukin-6 (IL-6), Tumour necrosis factor-α (TNF-α), reduction in Interleukin-10 (IL-10) and Brain-derived neurotrophic factor (BDNF) immunocontent within the hippocampus. Chronic administration of NK (50–100 mg/kg, i.p.) from day 8–27, prevented depression-like behaviour, anxiety, and memory impairment and normalized the neurochemical alteration within the hippocampus of mice injected with Aβ1-42 peptide. The effect of NK on psychiatric complications, learning, and memory was comparable to peripheral donepezil treatment. This study suggests that NK improves learning, memory impairment, and neuropsychiatric complications possibly through the downregulation of neuroinflammatory pathways and restoring BDNF signalling in AD.

Nattokinase historical sketch on experimental and clinical evidence

Nattokinase (NK) is a protease derived from food used mainly in the Japanese diet that has several properties. The main activity is related to improving fibrinolytic activities. Other activities have been demonstrated in the regulation of blood pressure by the action toward angiotensin proteases and in the antiplatelet activities. NK can be given orally and reaches its maximal concentration after 12 hours. In addition, an antithrombotic activity based on various NK activities has been proposed. First, increased fibrinolytic activity increases thrombus dissolution and/or the formation of atherosclerotic plaques; second, its enhanced antiplatelet action adds to clot dissolution. All activities have been studied in animals and humans in vitro and in vivo. Relevant adverse effects of NK therapy have not been described, however clinical experience is restricted to case series and volunteers and is not based on clinical studies, thus clinical trials are required to confirm.

Production of Nattokinase from Hemp Seed Meal by Solid-State Fermentation and Improvement of Its Nutritional Quality

Fermented foods have gained immense popularity in recent years due to their distinct flavor profile. Given the increasing demand, there is a growing focus on optimizing their nutritional quality while also reducing their costs. In this study, using a novel approach, hemp seed meal was utilized as a solid fermentation substrate to produce nattokinase (NK). Using a combination of one-factor-at-a-time experiments, Plackett–Burman design, and Box–Behnken design, the optimal fermentation conditions of Bacillus subtilis 13932 (NK-producing strain) were determined. The initial ratio of HSM (hemp seed meal) to water was 1:2.0 (v:w), the thickness of the substrate was 2.9 cm, the bacterial inoculum volume was 10% (v:w), the relative humidity was 75.2%, the temperature was set at 35 °C, and the fermentation time was 20 h. The NK activity under these conditions was measured to be 7067.12 IU/g. During fermentation, 15.15% of soluble peptides were produced, which exhibited hydroxyl radical removal ability and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical removal ability of 14.85%, down from 32.96%. Furthermore, trypsin inhibitor and urease in HSM decreased by 42.6% and 73.6%, respectively, improving the nutritional quality of HSM. Sensory evaluations indicated that HSM is expected to be a popular food, highlighting the potential of using HSM as a solid fermentation substrate for NK production.