Korean Fermented Soybean Paste Research Articles

Reduction of aflatoxins and ochratoxin A by addition of commercial Koji during fermentation of the Korean traditional soybean paste, Doenjang

Contamination by aflatoxins and ochratoxin A of traditional Korean fermented soybean paste (Doenjang) is a serious food safety issue. In this study, we investigated the inhibitory effects of commercial Koji, a microbial starter culture used for fermentation, on mycotoxin contamination during Doenjang production at in vitro and scaled-up fermentation levels. A commercial Koji dominated by Aspergillus oryzae JI-4 exhibited strong antifungal and antimycotoxigenic activities against aflatoxigenic Aspergillus flavus and ochratoxigenic Penicillium verrucosum both in vitro and in situ. Furthermore, co-inoculation with Koji JI and mycotoxigenic fungi in the early stage of Doenjang fermentation significantly suppressed aflatoxins and ochratoxin A production by more than 93% (p < 0.05) and improved the nutritional properties and sensory qualities of the final product. These results suggest that the addition of commercial Koji could be an effective and practical approach to reduce aflatoxins and ochratoxin A contamination during the fermentation of soybean paste.

Biogenic amine reduction by food additives in Cheonggukjang, a Korean fermented soybean paste, fermented with tyramine-producing heterogeneous bacterial species

This study was conducted to mitigate the food safety risks related to biogenic amine (BA) by reducing the BA content in Cheonggukjang using applicable food additives. In in-vitro experiments, of the additives tested, tartaric acid (TA), potassium sorbate (PS), and sodium benzoate (SB) considerably inhibited tyramine production of strains of Bacillus spp. and Enterococcus faecium while less affecting their growth. In addition to these three additives, two additives, glycine (GL) and nicotinic acid (NA), reported to have significant inhibitory effects in previous studies, were applied to the Cheonggukjang fermentation with prolific tyramine-producing strains of B. subtilis and E. faecium. The content of tyramine in the Cheonggukjang samples treated with TA, PS, SB, GL, and NA was significantly reduced by 27.5%, 50.7%, 51.4%, 76.1%, and 100.0%, respectively, compared to the control sample. Additionally, the content of polyamines (putrescine, cadaverine, spermidine, and spermine) in the GL-treated sample was reduced by 42.6%–62.4%. The mode of action could be attributed to inhibiting the bacterial decarboxylase activity and/or growth. Consequently, excluding NA that interfered with Cheonggukjang fermentation, GL was the most outstanding additive with an inhibitory effect on tyramine formation in food, followed by SB and PS, all of which showed a more than 50% reduction. Therefore, the use of appropriate additives could be one of the promising strategies to avoid the food safety issues implicated in BAs in Cheonggukjang.

Complete genome sequence of Arthrobacter sp. KFRI-F3372, a strain isolated from Korean soybean paste Doenjang.

Arthrobacter sp. KFRI-F3372 is a Gram-positive bacterium with a high G + C content of 65.7%, which was isolated from Doenjang, a traditional Korean fermented soybean paste. In this report, we introduce the complete genome sequence of Arthrobacter sp. KFRI-F3372.

Biogenic amine reduction by vasoactive amine-degrading Bacillus licheniformis CH7P22 in Enterococcus faecium-contaminated Cheonggukjang, Korean fermented soybean paste

Biogenic amine reduction by vasoactive amine-degrading Bacillus licheniformis CH7P22 in Enterococcus faecium-contaminated Cheonggukjang, Korean fermented soybean paste

Korean fermented soybean paste (Doenjang) has anti-obesity and anti-hypertensive effects via the renin-angiotensin system (RAS) in high-fat diet-induced obese rats.

The health-beneficial outcomes of doenjang, a Korean fermented food have been questioned due to its high salt content; moreover, the detailed underlying mechanisms of its health beneficial effects are not fully investigated. Thus, this study aimed to investigate doenjang's anti-obesity effects, anti-hypertensive effects, and its underlying mechanisms in high-fat diet -induced obesity. Sprague-Dawley rats fed with normal diet (ND), high-fat diet (HD), HD with 8% additive salt (HDS), or HD with doenjang containing 8% salt (HDJ) for 13 weeks. Compared to HD and HDS groups, the HDJ group had lower body and epididymal fat tissue weight gain and showed hypotrophy and hypoplasia. The RAS-related mRNA levels in the adipose tissue, including Renin and Ace were downregulated in the HDJ group compared to HD and HDS groups. Additionally, HDJ groups had significant improvements in systolic blood pressure, serum RAS-associated parameters (e.g., angiotensin II and aldosterone), renal mRNA levels related to RAS (e.g., angiotensin II receptor type 1 and 2), and aldosterone-associated mRNA expressions (e.g., mineralocorticoid receptor) in the kidney of HD-induced obese rats. Most importantly, HDS and HDJ groups showed distinct outcomes regarding adipogenesis and electrolytes metabolism, even though both diets contain a high level of salt. HDS group showed a higher epididymal fat tissue weight, mass, and adipocyte size than HDJ group. In addition, compared with HDJ group, HDS group significantly decreased the release of Na+ and K+ throughout the urine and feces. The present study addresses that doenjang has anti-obesity effects and anti-hypertensive effects by activating RAS in the adipose tissue and kidney, respectively. Additionally, this study also demonstrates that salt in doenjang and the additive salt differently influences adipogenesis and electrolytes metabolism, supporting doenjang has health advantageous effects regardless of its high salt contents.

Degradation mechanism of aflatoxin B1 and aflatoxin G1 by salt tolerant Bacillus albus YUN5 isolated from ‘doenjang’, a traditional Korean food

Aflatoxins are the mycotoxins that contaminate food and feed and pose health hazards to humans and animals. Here, Bacillus albusYUN5 was isolated from doenjang (Korean fermented soybean paste) and examined for aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) degradation capabilities. The highest degradation of AFB1 (76.28 ± 0.15%) and AFG1 (98.98 ± 0.00%) was observed in the cell-free supernatant (CFS) ofB. albusYUN5, whereas negligible degradation was observed in intracellular fraction, viable cells, and cell debris. Furthermore, heat (100 °C) and proteinase K treated CFS possessed AFB1 and AFG1 degradation ability, suggesting that substances other than proteins or enzymes are responsible for the degradation. Optimal degradation of AFB1 and AFG1 by the CFS was achieved at 55 °C and 45 °C, respectively, and at pH 7–10 and salt concentration of 0–20%. Liquid chromatography–mass spectroscopy analysis of the degraded products revealed that either the difuran or lactone ring of AFB1 and lactone ring of AFG1 is the main target site by CFS of B. albus YUN5. A slightly better reduction of AFB1 and AFG1 was observed in doenjang treated with CFS and viable cells of B. albus YUN5 compared to those without CFS and B. albus YUN5 treated doenjang during one year of fermentation, suggesting the applicability of B. albus in real food.

Identification and Characterization of a Bacteriocin from the Newly Isolated Bacillus subtilis HD15 with Inhibitory Effects against Bacillus cereus.

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70°C and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

Protective Effects of Fermented Soybeans (Cheonggukjang) on Dextran Sodium Sulfate (DSS)-Induced Colitis in a Mouse Model.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease, and the incidence of IBD is increasing every year owing to changes in dietary structure. Although the exact pathogenesis of IBD is still unclear, recent evidence suggests that gut dysbiosis is closely associated with IBD pathogenesis. Cheonggukjang is a traditional Korean fermented soybean paste produced using traditional and industrial methods, and contains probiotics, which affect the gut microbiota composition. However, the protective effect of Cheonggukjang against IBD is unknown. In this study, we investigated the bacterial community structure of traditional and commercial Cheonggukjang samples, as well as the protective effect of Cheonggukjang on a dextran sulfate sodium (DSS)-induced colitis mouse model. Traditional and commercial Cheonggukjang were found to contain various type of useful probiotics in their bacterial community structure. Cheonggukjang reduced the progression of DSS-induced symptoms, such as body weight loss, colonic shortening, disease activity index, and histological changes. Further, Cheonggukjang improved the intestinal epithelial barrier integrity on DSS-induced colitis mice. In addition, Cheonggukjang suppressed the expression of proinflammatory cytokines and inflammatory mediators through the inactivation of NF-κB and MAPK signaling pathways. These results indicate that Cheonggukjang exerts protective effects against DSS-induced colitis, suggesting its possible application as a functional food for improving inflammatory diseases.

Anti-Obesity and Anti-Inflammatory Effects of the Desalted &lt;i&gt;Salicornia europaea&lt;/i&gt; L. Fermented by &lt;i&gt;Bacillus velezensis&lt;/i&gt; K26

Salicornia europaea L. (SE) as a halophyte has been widely used as a seasoned vegetable with various biological activities, including anti-inflammatory and anti-diabetic activities. In particular, the desalted SE (DSE) has been considered an attractive alternative compared to SE for its anti-obesity effect. 1-Deoxynojirimycin (1-DNJ) is one of the known α-glucosidase inhibitors and has been reported to have similar biological activities with SE. This study reports the anti-obesity and anti-inflammatory activities of DSE fermented by Bacillus velezensis K26 (BVDSE) previously isolated from Korean fermented soybean paste and elucidated to produce 1-DNJ in 3T3-L1 adipocytes and RAW264.7 cells. BVDSE (5%) did not show cytotoxicity in 3T3-L1 cells and inhibited lipid accumulation, which was decreased to 23% in the differentiated cells. BVDSE did not inhibit the release of the pro-inflammatory cytokine, TNF-α, while it remarkedly inhibited IL-6 on lipopolysaccharide-induced RAW264.7 cells. Taken together, these results indicate that the fermentation of DSE with B. velezensis K26 as a 1-DNJ producer has more significant anti-obesity and anti-inflammatory effects than even non-fermented DSE, assuming the synergistic effects by 1-DNJ production from B. velezensis K26, by which it could be highlighted that BVDSE may be considered for use as a functional food supplement.

Characterization and correlation of microbial communities and metabolite and volatile compounds in doenjang fermentation

To investigate the general fermentation features of doenjang, a traditional Korean fermented soybean paste, eleven batches of doenjang were prepared. The bacterial and fungal communities and the metabolites such as free sugars, organic acids, amino acids, and volatile compounds were analyzed during fermentation. Tetragenococcus, Aspergillus, and Debaryomyces were the most common microbes; galactose, fructose, and glucose were the major sugars; and lactate and acetate were the major organic acids. Spearman correlation analyses showed that the quantity of meju was correlated with only Pediococcus and Halomonas abundance, while solar salt concentration was correlated with the relative abundance of many microbial taxa and the amount of glycerol and total volatile compounds. The abundance of heterolactic acid bacteria, such as Tetragenococcus, Pediococcus, Weissella, and Enterococcus, was positively correlated with the levels of lactate, acetate, and ethanol, suggesting that heterolactic acid fermentation may be a major metabolism pathway during the fermentation of doenjang. The abundance of Weissella, Hyphopichia, and Wickerhamomyces was positively correlated with ethyl acetate levels, whereas the abundance of Staphylococcus and Bacillus was positively correlated with the concentration of major volatile compounds, 3-methylbutanoic acid and tetramethylpyrazine, respectively, suggesting that they may play important roles in the production of flavor compounds during fermentation.

In Vitro and In Vivo Cholesterol Reducing Ability and Safety of Probiotic Candidates Isolated from Korean Fermented Soya Beans

Hypercholesterolemia is a risk factor for cardiovascular diseases, and hence, reducing serum cholesterol levels could reduce the incidence. In this study, we ascertained the cholesterol-reducing potential of lactic acid bacteria (LAB) isolated from Korean fermented soybean paste. Live, resting, and dead cells of all the bacteria reduced cholesterol in liquid media in a strain-dependent manner. Live cells of Weissella cibaria SCCB2306, Pediococcus acidilactici SDL1402, P. acidilactici SDL1406, and Lactobacillus rhamnosus JDFM6 reduced the most cholesterol in liquid media by 78 ± 3%, 72 ± 3%, 76 ± 3%, 75 ± 5%, and 79 ± 2%, respectively. As the cholesterol levels in the media reduced, cell membrane lipids of P. acidilactici SDL1402, P. acidilactici SDL1406, and L. rhamnosus JDFM6 increased by 23.36mg/mL, 6.53mg/mL, and 8.14mg/mL, respectively, indicating that cholesterol was incorporated into the bacteria cell membranes. All the bacteria displayed bile salt hydrolase activities in a strain-dependent manner. Though all four LAB significantly reduced cholesterol levels in Caenorhabditis elegans irrespective of the order of feeding, L. rhamnosus JDFM6 reduced the most cholesterol in vivo (up to 40% of ingested cholesterol). None of the four LAB hydrolyzed mucin or gelatin and none was toxic to C. elegans. The concentrations of phenylethylamine, putrescine, cadaverine, histamine, and tyramine produced by the LAB were below the toxic limits of biogenic amines set by the European Food Safety Authority. Taken together, our results demonstrate that Weissella cibaria SCCB2306, P. acidilactici SDL1402, P. acidilactici SDL1405, and L. rhamnosus JDFM6 could be safe cholesterol-reducing probiotic candidates for preventing or managing hypercholesterolemia.

ODFM, an omics data resource from microorganisms associated with fermented foods

ODFM is a data management system that integrates comprehensive omics information for microorganisms associated with various fermented foods, additive ingredients, and seasonings (e.g. kimchi, Korean fermented vegetables, fermented seafood, solar salt, soybean paste, vinegar, beer, cheese, sake, and yogurt). The ODFM archives genome, metagenome, metataxonome, and (meta)transcriptome sequences of fermented food-associated bacteria, archaea, eukaryotic microorganisms, and viruses; 131 bacterial, 38 archaeal, and 28 eukaryotic genomes are now available to users. The ODFM provides both the Basic Local Alignment Search Tool search-based local alignment function as well as average nucleotide identity-based genetic relatedness measurement, enabling gene diversity and taxonomic analyses of an input query against the database. Genome sequences and annotation results of microorganisms are directly downloadable, and the microbial strains registered in the archive library will be available from our culture collection of fermented food-associated microorganisms. The ODFM is a comprehensive database that covers the genomes of an entire microbiome within a specific food ecosystem, providing basic information to evaluate microbial isolates as candidate fermentation starters for fermented food production.

High-throughput sequencing of the microbial community associated with the physicochemical properties of meju (dried fermented soybean) and doenjang (traditional Korean fermented soybean paste)

Doenjang, a traditional Korean soybean paste consisting of fermented meju has been consumed for centuries as a protein source. In this study, the physicochemical factors and microbial communities of samples prepared from four batches of meju and doenjang were investigated to identify the factors that affect the food quality. The water content and salt concentration were higher in doenjang than in meju, and the acidity and amino-type nitrogen varied depending on the batch. After fermentation of doenjang, the pH, salt concentration, and amino-type nitrogen increased in all batches. Enterococcus and Bacillus were present in all meju samples, whereas Enterococcus, Tetragenococcus, and Bacillus were present in all doenjang samples. The dominant fungi in meju and doenjang was Aspergillus. The abundance of certain fungi, such as Debaryomyces, Candida, Mucor, and Gibberella, varied batch to batch. The amino-type nitrogen, which determines the quality of doenjang, was correlated with the relative abundance of Aspergillus in doenjang prior to fermentation. In addition, these strains had a negative correlation with the salt concentration. This study has elucidated the composition of microbial communities in meju and doenjang, which may lead to improved quality in the production of fermented food.

The Role of Enterococcus faecium as a Key Producer and Fermentation Condition as an Influencing Factor in Tyramine Accumulation in Cheonggukjang.

The study evaluated the role of Enterococcus faecium in tyramine production and its response to fermentation temperature in a traditional Korean fermented soybean paste, Cheonggukjang. Tyramine content was detected in retail Cheonggukjang products at high concentrations exceeding the recommended limit up to a factor of 14. All retail Cheonggukjang products contained Enterococcus spp. at concentrations of at least 6 Log CFU/g. Upon isolation of Enterococcus strains, approximately 93% (157 strains) produced tyramine at over 100 µg/mL. The strains that produced the highest concentrations of tyramine (301.14–315.29 μg/mL) were identified as E. faecium through 16S rRNA sequencing. The results indicate that E. faecium is one of the major contributing factors to high tyramine content in Cheonggukjang. During fermentation, tyramine content in Cheonggukjang groups co-inoculated with E. faecium strains was highest at 45 °C, followed by 37 °C and 25 °C. The tyramine content of most Cheonggukjang groups continually increased as fermentation progressed, except groups fermented at 25 °C. At 45 °C, the tyramine content occasionally exceeded the recommended limit within 3 days of fermentation. The results suggest that lowering fermentation temperature and shortening duration may reduce the tyramine content of Cheonggukjang, thereby reducing the safety risks that may arise when consuming food with high tyramine concentrations.

Complete Genome Sequence of Bacillus velezensis Strain DKU_NT_04, Isolated from a Traditional Korean Food Made from Soybeans (Cheonggukjang).

In the present work, we report the complete genome sequence of Bacillus velezensis DKU_NT_04, isolated from cheonggukjang, which is a traditional Korean fermented soybean paste. The final genome assembly consists of a 4.328-Mbp chromosome with 4,134 coding sequences and a G+C content of 45.21%.

Effects of Enterococcus faecium and Staphylococcus succinus starters on the production of volatile compounds during doenjang fermentation

Bacterial starters Enterococcus faecium and Staphylococcus succinus were introduced into the manufacture of doenjang, a Korean fermented soybean paste, together with a fungal starter to determine the effect of bacterial starters on flavor production during doenjang fermentation. Starters cultured on soybean flakes and rice were mixed with autoclaved soybeans and then refined salt was added to 12% (w/w) to prepare doenjang samples. The doenjang samples were ripened at 20 °C and their volatile compounds were analyzed on days 0, 28, 56, and 84. The numbers of bacteria from starter cultures in the samples decreased gradually. The content of amino-type nitrogen in samples decreased as the proportion of bacterial starter culture used in doenjang preparation increased. Nineteen volatile compounds were detected in doenjang samples by gas chromatography-mass spectrometry and were subjected to principal component analysis. Addition of bacterial starters distinctively changed the flavor of doenjang compared with that prepared with a fungal starter alone. Acetic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid were important volatile compounds determining flavor development in the doenjang employing bacterial starters. Starter-specific as well as highly-produced volatile compounds determined the directionality of flavor during doenjang ripening. Raw materials used for the starter culture preparation also affected the flavor.

Safety assessment of lactic acid bacteria strains isolated from traditional Romanian fermented foods

Bacterial starters Enterococcus faecium and Staphylococcus succinus were introduced into the manufacture of doenjang, a Korean fermented soybean paste, together with a fungal starter to determine the effect of bacterial starters on flavor production during doenjang fermentation. Starters cultured on soybean flakes and rice were mixed with autoclaved soybeans and then refined salt was added to 12% (w/w) to prepare doenjang samples. The doenjang samples were ripened at 20 °C and their volatile compounds were analyzed on days 0, 28, 56, and 84. The numbers of bacteria from starter cultures in the samples decreased gradually. The content of amino-type nitrogen in samples decreased as the proportion of bacterial starter culture used in doenjang preparation increased. Nineteen volatile compounds were detected in doenjang samples by gas chromatography-mass spectrometry and were subjected to principal component analysis. Addition of bacterial starters distinctively changed the flavor of doenjang compared with that prepared with a fungal starter alone. Acetic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid were important volatile compounds determining flavor development in the doenjang employing bacterial starters. Starter-specific as well as highly-produced volatile compounds determined the directionality of flavor during doenjang ripening. Raw materials used for the starter culture preparation also affected the flavor.

Simultaneous Determination of Twenty Mycotoxins in the Korean Soybean Paste Doenjang by LC-MS/MS with Immunoaffinity Cleanup.

Doenjang, a Korean fermented soybean paste, is vulnerable to contamination by mycotoxins because it is directly exposed to environmental microbiota during fermentation. A method that simultaneously determines 20 mycotoxins in doenjang, including aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), and fumonisins (FBs) with an immunoaffinity column cleanup was optimized and validated in doenjang using LC-MS/MS. The method showed good performance in the analysis of 20 mycotoxins in doenjang with good linearity (R2 > 0.999), intra- and inter-day precision (<16%), recovery (72–112%), matrix effect (87–104%), and measurement uncertainty (<42%). The validated method was applied to investigate mycotoxin contamination levels in commercial and homemade doenjang. The mycotoxins that frequently contaminated doenjang were AFs, OTA, ZEN, and FBs and the average contamination level and number of co-occurring mycotoxins in homemade doenjang were higher than those in commercially produced doenjang.

The effect of salt concentrations on the fermentation of doenjang, a traditional Korean fermented soybean paste

Four sets of doenjang (traditional Korean fermented soybean paste) with 9%, 12%, 15%, and 18% solar salt concentrations were prepared and their pH, microbial abundances and communities, metabolites, and volatile compounds were analyzed periodically during the entire fermentation. The speeds of decrease in pH and increase in microbial abundances, representing microbial activity, were higher during early fermentation in lower (9% and 12%) salt doenjang. Microbial abundances in 15% and 18% salt doenjang were significantly lower than in the 9% and 12% salt doenjang, indicating low microbial activity. Community analysis revealed that Bacillus, Staphylococcus, and Clostridium and Aspergillus, Scopulariopsis, Fusarium, Mucor, and Penicillium, which might be derived from doenjang-meju used for preparing doenjang, were identified as major bacterial and fungal genera, respectively, in all doenjang samples. Weissella, Tetragenococcus, Oceanobacillus, and Debaryomyces, not dominant in doenjang-meju, were also identified as major groups in low salt doenjang. Metabolite analysis showed that amino acid profiles were relatively similar independent of salt concentrations and microbial growth, indicating important roles of indigenous proteases present in doenjang-meju, not microbial activity during doenjang fermentation, in amino acid production. The metabolism of free sugars to organic acids and biogenic amine production were greater in lower salt doenjang, which might be associated with the growth of microbes, particularly lactic acid bacteria. A higher level of and more diverse volatile compounds were identified in lower salt doenjang, indicating close association with microbial growth. This study provides a deeper understanding of doenjang fermentation and insight into the development of low salt doenjang.

Purification and characterization of thermostable l-asparaginase from Bacillus amyloliquefaciens MKSE in Korean soybean paste

This study aimed to produce and characterize thermostable l-asparaginase from Bacillus amyloliquefaciens MKSE, a newly screened strain from Korean fermented soybean paste. The isolated MKSE was identified by its morphological, biochemical, and physiological properties and 16S rRNA sequence. Its l-asparaginase II-like gene was expressed in Escherichia coli BL21 (DE3), by using the pET-28a (+) vector. The produced l-asparaginase (specific activity of 4.75 IU/mg) was purified by a one-step procedure, using nickel-affinity chromatography, and its molecular weight of 38.2 kDa was determined by gel electrophoresis. Its optimum pH and temperature were 8.5 and 65 °C, respectively. The enzyme was highly stable in the pH range of 3.0–11.0 for 6 h incubation, and the enzyme retained about 57% of its activity at 95 °C for 6 h. Its activity was activated in the presence of Mn2+, Mg2+, and Fe2+ while it was inhibited by Zn2+, Cu2+, PMSF, and SDS. Its Km was 1.5 mM, and its Vmax was 136.3 IU/mg. The results of this study reveal that MKSE l-asparaginase has a great potential to be used in various industries, due to its stability at high temperature and over a wide pH range.