Tofu processing wastewater as a low-cost substrate for high activity nattokinase production using Bacillus subtilis

Abstract

Background Even though tofu is a traditional Chinese food loved by Asian people the wastewater generated during the production of tofu can pollute the environment, and the treatment of this generated wastewater can increase the operating cost of the plant. In this study, the production of nattokinase could be achieved by using the nitrogen source in tofu processing wastewater (TPW) instead of using the traditional nattokinase medium. This meets the need for the low-cost fermentation of nattokinase and at the same time addresses the environmental pollution concerns caused by the wastewater. Bacillus subtilis 13,932 is, a high yielding strain of nattokinase, which is stored in our laboratory. To increase the activity of nattokinase in the tofu process wastewater fermentation medium, the medium components and culture parameters were optimized. Nattokinase with high enzymatic activity was obtained in 7 L and 100 L bioreactors when TPW was used as the sole nitrogen source catalyzed by Bacillus subtilis. Such a result demonstrates that the production of nattokinase from TPW fermentation using B. subtilis can be implemented at an industrial level. Results The peptide component in TPW is a crucial factor in the production of nattokinase. Box–Behnken design (BBD) experiments were designed to optimize various critical components, i.e., Glucose, TPW, MgSO4·7H2O, CaCl2, in nattokinase fermentation media. A maximum nattokinase activity was recorded at 37 °C, pH 7.0, 70 mL liquid medium, and 200 rpm. The highest nattokinase activities obtained from 7 to 100 L bioreactors were 8628.35 ± 113.87 IU/mL and 10,661.97 ± 72.47 IU/mL, respectively. Conclusions By replacing the nitrogen source in the original medium with TPW, there was an increase in the enzyme activity by 19.25% after optimizing the medium and culture parameters. According to the scale-up experiment from conical flasks to 100 L bioreactors, there was an increase in the activity of nattokinase by 47.89%.

Production of acetoin and its derivative tetramethylpyrazine from okara hydrolysate with Bacillus subtilis

“…× 50 mm) column; the mobile phase was double distilled water, flow rate was 0.5 mL/min, column temperature was 80 °C, injection volume was 10 μL, and a refractive index detector was used (Ding et al 2019 ). Amino acid content was determined using PITC pre-column derivatisation (Hao et al 2016 ; Li et al 2021 ), and the conditions were as follows: column model Hedera ODS-2 column (4.6 mm × 250 mm, 5 μm, Hanbon Sci. & Tech., Jiangsu, China); UV detector wavelength 254 nm; column temperature 40 °C, flow rate 1 mL/min, injection volume 10 μL; mobile phase A, 0.1 mol/L sodium acetate pH 6.5; mobile phase B, 80% acetonitrile; gradient elution, (5 min 3%B, 14 min 11%B, 17 min 21% B, 29 min 34% B, 41 min 100% B, 43 min 100% A, 47 min 100% A).…”

Biotechnology, Bioengineering and Applications of Bacillus Nattokinase

“…Li used tofu processing wastewater to replace the nitrogen source in the nattokinase fermentation medium, which reduced production costs and solved the problem of environmental pollution caused by wastewater. The experiment confirmed that the enzyme activity of nattokinase produced by Bacillus subtilis 13,932 fermentation increased by 19.25% after optimizing the medium and culture parameters; the subsequent fermentation volume amplification experiment (100 L) showed that the activity of nattokinase was increased by 47.89% [ 54 ].…”

Characterization of a Nattokinase from the Newly Isolated Bile Salt-Resistant Bacillus mojavensis LY-06

“…We evaluated the ability of different carbon sources (yeast extract, lactose, glucose, saccharose) to ferment the Bacillus mojavensis LY-06 strain to produce nattokinase and found that glucose was the best carbon source ( Figure 5 a). Various types of carbon sources such as shrimp shell wastes, maltose, lactose, and galactose have been reported to be the best carbon sources for the fermentation of nattokinase by different Bacillus species, and some low-cost carbon sources (tapioca starch) and even industrial wastes (tofu processing wastewater) can also be used as carbon sources for the fermentation of nattokinase [ 16 , 26 ]. We speculate that glucose as a monosaccharide is more easily absorbed and utilized by the Bacillus mojavensis LY-06 strain.…”

“…The by-product poly-L-glutamic acid produced in solid-state fermentation has been shown to increase the fermentation yield of nattokinase, but it also increases the risk of allergy [ 23 , 24 ]. The nattokinase liquid fermentation method overcomes the shortcomings of the solid-state fermentation method, such as low water content, poor fluidity, and difficulty in process monitoring, and is widely used in the industrial production of nattokinase [ 25 , 26 ]. At present, nattokinase has been expressed in Escherichia coli [ 27 ], Lactobacillus [ 28 ], Bacillus [ 29 ], and Pichia pastoris [ 30 ].…”