GSH Yield Research Articles

Optimized extraction technology of glutathione from 'Haidao 86' germ rice by response surface methodology.

Glutathione is an important functional component of 'Haidao 86', which has many important physiological functions in organisms and is widely used in medicine and other industries. In this study, the effects of four extraction methods (hot water extraction, formic acid extraction, ethanol extraction, and sulfuric acid extraction) on the yield of glutathione in 'Haidao 86' germ powders were studied by high-performance liquid chromatography, and the yield of glutathione in hot water extraction was the highest. The effects of material-liquid ratio, temperature, pH, and time on the extraction rate of glutathione from 'Haidao 86' were investigated by single-factor experiment and Box-Behnken combined experiment. The results showed that the order of influence on GSH yield was pH, temperature, material-liquid ratio, and time, and the interaction of extraction time and pH had a significant influence on glutathione yield of 'Haidao 86' germ powders. The optimum parameters for hot water extraction of glutathione from 'Haidao 86' germ powders were determined as follows: material-liquid ratio of 1:12, pH value of 2.8, temperature of 84.9°C, time of 14 min, and the extraction rate of glutathione was 139.68 mg/100 g. It provided the scientific proof for the development and industrial production of functional products of 'Haidao 86'.

Improving glutathione production by engineered Pichia pastoris: strain construction and optimal precursor feeding.

Glutathione (GSH) is a metabolite that plays an important role in the fields of pharmacy, food, and cosmetics. Thus, it is necessary to increase its production to meet the demands. In this study, ScGSH1, ScGSH2, and StGshF were heterologously expressed in Pichia pastoris GS115 to realize the dual-path synthesis of GSH in yeast. To explore the effects of ATP metabolism on the synthesis of GSH, enzymes (ScADK1, PpADK1, VsVHB) of the ATP-related metabolic pathway and the energy co-substrate sodium citrate were taken into account. We found that both ScADK1 and sodium citrate had a positive influence on the synthesis of GSH. Then, a fermentation experiment in Erlenmeyer flasks was performed using the G3-SF strain (containing ScGSH1, ScGSH2, StGshF, and ScADK1), with the highest GSH titer and yield of 999.33 ± 47.26mg/L and 91.53 ± 4.70mg/g, respectively. Finally, the fermentation was scaled up in a 5-L fermentor, and the highest titer and yield were improved to 5680mg/L and 45.13mg/g, respectively, by optimizing the addition conditions of amino acids (40mM added after 40h). Our work provides an alternative strategy by combining dual-path synthesis with energy metabolism regulation and precursor feeding to improve GSH production. Key Points • ScGSH1, ScGSH2, and StGshF were overexpressed to achieve dual-path synthesis of GSH in yeast. • ScADK1 was overexpressed, and sodium citrate was added to increase the energy supply for GSH synthesis. • The addition conditions of amino acids were optimized to realize the efficient synthesis of GSH.

Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase.

The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the first time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coefficients for 12 tested variables, yeast extract, glucose, peptone, cysteine, temperature and agitation rate had a positive significant effect on GSH production with a maximum yeild 192mg/L. The impact of kinetics of adding cysteine was investigated in 19 experiments during the growth time course (0-36h), and the maximum yield of glutathione (235mg/L) was obtained by addition of cysteine after 8h post-inoculation. The most significant variables were further explored at five levels using central composite rotatable design (CCRD), giving a maximum production of GSH (552mg/L). Using baffled flasks, the yield of GSH was increased to 730mg/L, i.e., 1.32-fold increment. The two rate-limiting genes of GSH biosynthesis "γ-glutamyl cysteine synthetase (GSH1) and GSH-synthetase (GSH2)" were amplified and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with GSH1 and GSH2 genes of S. cerevisiae. Glutathione peroxidase was purified and characterized from S. boulardii with molecular mass and subunit structure of 80kDa and 35kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-fold upon demetallization confirming its metalloproteinic identity. The GPx was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.

Characterization of polyphosphate kinases for the synthesis of GSH with ATP regeneration from AMP

Polyphosphate kinase (PPK) is important for industrial processes involving ATP regeneration. While a variety of methods have been reported for regenerating ATP from ADP, few have explored enzyme catalyzed ATP regeneration from cheaper and stable AMP. In this work, PPKs from different sources were expressed and their catalytic activity were tested at different reaction temperatures, reaction pH and with different polyphosphate (polyPn) types. The ATP regeneration system for glutathione (GSH) synthesis was established using a single PPK capable of phosphorylating AMP to synthesize ATP from AMP and short chain polyPn. GSH yield was obtained using adenosine mono-, di- and triphosphates, which confirmed the flexibility of our constructed ATP regeneration system coupled with GSH synthesis via bifunctional GSH synthase. Finally, optimization of the GSH synthesis yielded conversion value above 80 %. Overall, these results illustrate that PPK is suitable for a broader range of substrates than previously expected, and has great untapped potential for applications involving ATP regeneration.

Stimulation of in situ low intensity ultrasound on batch fermentation of Saccharomyces cerevisiae to enhance the GSH yield

AbstractIn this study, an in situ 7.5 L ultrasonic fermentation tank was used to culture Saccharomyces cerevisiae at 30°C for 36 hr to explore the effects of low intensity ultrasound on the production of glutathione (GSH). Ultrasonic conditions of 28 kHz frequency, 28 W/L power density for fermentation time of 0–12 hr, 280 W/L power density for fermentation time of 12–36 hr, 3 s of interval and fixed frequency mode were used for the set‐up and a nonsonicated set‐up served as the control for the experiment. Results showed that ultrasound treatment increased total GSH production by 15.60% from a maximum value of 45.52 mg/L in the control to a maximum value of 52.62 mg/L in the sonicated sample. Also, intracellular and extracellular GSH productions in the sonicated sample increased by 5.60% and 42.92% respectively. Regular detection of the changes of biomass yield, residual sugar, dissolved oxygen and pH showed that sonication resulted in biomass increased by 7.25%, glucose consumption rate increased by 53.08% and dissolved oxygen content decreased by 41.67%. The biomass models of sonication and nonsonication treatments were established according to growth of S. cerevisiae showed that the model fitted well with Logistic regression in which the strain specific growth rate increased by 60.95% after sonication. GSH production model agreed with Luedeking‐Piret regression in which GSH synthesis parameters of sonication, α and β increased by 1.96% and 19.64% in comparison to the nonsonicated sample. This study clearly demonstrated that ultrasonic stimulation could improve the biosynthesis of GSH during fermentation.Practical ApplicationsIn this study, the GSH was producted by microbial fermentation in in situ 7.5 L ultrasonic fermentation tank. During the fermentation, sonication was applied to increasing yield of GSH. The results show that the study provides a theoretical basis for manufacture GSH combined with sonication industrialization. The fermentation kinetic model was established, which provided experimental basis for continous fermentation.

Compared to antioxidants and polyamines, the role of maize grain-derived organic biostimulants in improving cadmium tolerance in wheat plants

Recently, the strategy of seed soaking has been successfully applied using extracts from different plant parts for healthy growth of plant under different environmental stresses. Compared to antioxidants like ascorbic acid (AsA) and glutathione (GSH) or polyamines (PAs) like spermine (SPM), spermidine (SPD), and putrescine (PUT), the effects of seed soaking using maize grain extract (MGE) on the biomass, productivity, phytohormones, and antioxidant defense system and its different components were examined with Cd2+-stressed wheat plants. In a preliminary study, seed soaking using AsA + GSH or PUT + SPD + SPM was more effective in increasing shoot fresh and dry weights, SPAD chlorophyll, and grain yield, and reducing malondialdehyde (MDA) content than individuals. In addition, MGE at 2% was more efficient than other concentrations. Therefore, they were selected for the main study. In the main study, compared to the control, seed soaking in AsA + GSH, PUT + SPD + SPM or MGE had positive effects on plant growth, yield, photosynthetic efficiency, contents and redox states of AsA and GSH, contents of PAs and plant hormones to varying degrees. Proline content and its metabolism enzymes activity, contents of soluble protein, N-compounds, soluble sugars, and α-tocopherol (α-TOC), and activities of antioxidant enzymes were not affected. However, contents of MDA and hydrogen peroxide (H2O2) were significantly reduced under normal conditions. Under Cd2+ stress (1.2 mM), along with the detrimental increases in the contents of MDA, H2O2 and Cd2+, contents of N-compounds, soluble sugars, proline content and its metabolism enzymes activities, AsA and GSH and their redox states, and polyamines, and activities of antioxidant enzymes were increased. In contrast, plant growth and yield, photosynthetic efficiency, soluble protein, and plant hormones were significantly reduced compared to the control. However, all of these attributes were significantly improved to varying degrees along with reduced contents of Cd2+, MDA, and H2O2 by seed soaking in AsA + GSH, PUT + SPD + SPM or MGE compared to the Cd2+-stressed control. Compared to AsA + GSH or PUT + SPD + SPM, seed soaking in MGE at 2% conferred the best results. Therefore, it is recommended to soak wheat seeds using MGE to improve plant growth and productivity by restricting the inhibitory influences of oxidative stress induced by Cd2+ stress.

Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters

Glutathione (GSH) is an important bioactive tripeptide and widely used in food, medicine and other industries. Recently, the bifunctional glutathione synthetase, GshF, has been applied for efficient GSH production under inducible promoter. In this study, the constitutive expression of GshF from Streptococcus sanguinis (GshFss) was investigated under four different constitutive promoters. Based on previous study, five genes in Escherichia coli JM109 were deleted to eliminate the degradation of precursors and GSH. The effects of gene knockout on the constitutive expression of GshFss and GSH production were evaluated by whole cell catalysis. Finally, the engineered strain JM03Pdel1 produced 24 mM glutathione with addition of 30 mM precursors in 5-L bioreactor fed-batch fermentation. The yield of GSH based on cysteine in JM03Pdel1was reached 80% without any inducer, which was improved by 17.3% than that in the control strain.

Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 μM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.

Microbial production of glutathione.

Glutathione (GSH) is a non-coding tripeptide thiol with several important regulative and protective functions in eukaryotes and in most prokaryotes. The primary function of GSH is to maintain the redox potential of the cell, which is directly connected to GSH concentration, and to prevent cellular damages caused by reactive oxygen species or toxic heavy metals. Due to its antioxidant character, it is widely used in pharmaceutical, cosmetic and food industry. There have been different strategies to optimize GSH yield and productivity in bacteria and yeasts by means of metabolic and evolutionary engineering, media optimization and bioprocess engineering. The fed-batch procedure with yeasts of the genera Saccharomyces and Candida is still common method for industrial production. However, for an economic bioprocess production of GSH key factors like media costs, strain performance and process scalability are essential. Beside the extraction and purification of GSH as bulk product, GSH-enriched yeast cells are used for food and beverage applications, as well. This review outlines current applications of microbially produced GSH and illustrates current developments and strategies for its production.

Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase.

Glutathione (GSH) is an important bioactive substance applied widely in pharmaceutical and food industries. Due to the strong product inhibition in the GSH biosynthetic pathway, high levels of intracellular content, yield and productivity of GSH are difficult to achieve. Recently, a novel bifunctional GSH synthetase was identified to be less sensitive to GSH. A recombinant Escherichia coli strain expressing gshF encoding the bifunctional glutathione synthetase of Streptococcus thermophilus was constructed for GSH production. In this study, efficient GSH production using this engineered strain was investigated. The cultivation process was optimized by controlling dissolved oxygen (DO), amino acid addition and glucose feeding. 36.8mM (11.3g/L) GSH were formed at a productivity of 2.06mM/h when the amino acid precursors (75mM each) were added and glucose was supplied as the sole carbon and energy source.

Three-pathway combination for glutathione biosynthesis in Saccharomyces cerevisiae.

BackgroundGlutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh1 or GshA) and glutathione synthetase (Gsh2 or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro1 or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase. The Gsh1 and Gsh2 of conventional GSH biosynthetic pathway or the bifunctional GshF reported previously have been independently modulated for GSH production. This study developed a novel three-pathway combination method to improve GSH production in Saccharomyces cerevisiae.ResultsA bifunctional enzyme GshF of Actinobacillus pleuropneumoniae was functionally expressed in S. cerevisiae and Pro1 in proline biosynthetic pathway was exploited for improving GSH yield. Moreover, two fusion proteins Gsh2-Gsh1 and Pro1-GshB were constructed to increase the two-step coupling efficiency of GSH synthesis by mimicking the native domain fusion of GshF. The engineered strain W303-1b/FGP with three biosynthetic pathways presented the highest GSH concentration (216.50 mg/L) and GSH production of W303-1b/FGP was further improved by 61.37 % when amino acid precursors (5 mM glutamic acid, 5 mM cysteine and 5 mM glycine) were fed in shake flask cultures. In batch culture process, the recombinant strain W303-1b/FGP also kept high efficiency in GSH production and reached an intracellular GSH content of 2.27 % after 24-h fermentation.ConclusionsThe engineered strains harbouring three GSH pathways displayed higher GSH producing capacity than those with individually modulated pathways. Three-pathway combinatorial biosynthesis of GSH promises more effective industrial production of GSH using S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0327-0) contains supplementary material, which is available to authorized users.

On-line specific growth rate control for improving reduced glutathione production in Saccharomyces cerevisiae

Reduced glutathione (GSH), the abundant bioactive tripeptide in most living cells, is widely used in pharmaceutical, food, and cosmetic industries. Specific growth rate (μ) is a key physiological parameter for GSH high-cell-density cultivation using microbial cell factories. Here, based on a biomass probe, an on-line μ feedback control was developed to regulate glucose feeding rate during the fed-batch phase for overproducing GSH in Saccharomyces cerevisiae. Compared with real-time μ controlled at 0.15/h, μ controlled at 0.2/h achieved yeast dry weight (120 g/L), GSH concentration (1.5 g/L), and intracellular GSH content (1.25%), which improved by 9, 150, and 129.1%, respectively. To our knowledge, this is the first report about on-line μ feedback control for GSH production. On-line μ control led to 59.38 mg/L/h of GSH productivity and 3.52 mg/g of GSH yield on glucose, which improved by 107.6 and 7.2%, respectively, in comparison with those of traditional ethanol feedback control (maintaining ethanol concentration at 1%). Taken together, the on-line μ feedback control is a promising method as an efficient alternative to conventional feed control techniques presently practiced in the GSH industry, and has the potential for the production of other valuable chemicals.

Nystatin‐enhanced glutathione production by Saccharomyces cerevisiae depends on γ‐glutamylcysteine synthase activity and K+

Glutathione (GSH), an important tripeptide compound, is widely used as a therapeutic and in the food and cosmetic industries. To improve its production yield, we added the antibiotic nystatin to a batch fermentation of Saccharomyces cerevisiae, at different concentrations and at various times. Based on the results that nystatin can effectively stimulate GSH accumulation but at the same time inhibits cell growth, a three‐point addition strategy (0.05 mg/L at 8 h, 0.25 mg/L at 16 h, and 0.5 mg/L at 20 h) was developed to maximize GSH production. As a result, a maximum yield of 237.8 mg/L was obtained, which was by 50.6% higher than without the addition of nystatin. When combining this strategy with cysteine addition, the GSH yield increased to 278.9 mg/L. Subsequently, the γ‐glutamylcysteine synthetase (γ‐GCS) activity and K+ concentration were analyzed to investigate the possible mechanism involved in the increased production. It was found that the nystatin‐induced increase in the GSH yield was associated with a higher γ‐GCS activity and K+ concentration.

Improving glutathione extraction from crude yeast extracts by optimizing aqueous two-phase system composition and operation conditions

PEG-Dextran and PEG-salt aqueous two-phase systems (ATPS) have been applied to separate glutathione (GSH) from crude yeast extracts. Single-factor experiments were carried out to determine the important factors influencing the partition coefficient and extraction yield. The effect of PEG molecular weight, phase-forming components, PEG and Dextran concentration, pH value, and temperature on the GSH partitioning behavior in ATPS was investigated. Three factors, Dextran concentration, pH value, and temperature, were confirmed to have significant influence on the partition coefficient and extraction yield. These factors were further analyzed with the aid of central composite rotatable design and response surface methodology. The optimal conditions for GSH extraction in the PEGDextran system were determined, including PEG molecular weight 6,000, 10% PEG concentration, 14% Dextran concentration, pH 5.2, and temperature 32 °C. A high extraction yield (83.55%) of GSH from crude yeast extracts was achieved under these optimized conditions. This work is very helpful for developing one efficient and cost-effective process for the separation and purification of GSH from yeast broths.

Effects of magnetic fields on biomass and glutathione production by the yeast Saccharomyces cerevisiae

The effect of magnetic fields (MF) on glutathione (GSH) production by Saccharomyces cerevisiae ATCC 7754 was studied. For this purpose, a factorial design of experiments was used to determine the influence of the time of exposure (8–16 h) and MF induction (25.0–34.3 mT), in GSH and biomass production. Additionally, control experiments (CE), without the application of MF, were performed. The results indicated the existence of favourable alterations in GSH and biomass concentrations due to the application of MF. In all experiments, the amount of biomass produced was higher than in CE and, with regard to GSH yield, in all the experiments at 24 and 48 h it was higher and in three experiments at 72 h of culture. The highest specific GSH yield (20.9 mg GSH/g biomass), GSH yield (340.0 mg/L) and biomass (16.26 g/L) were obtained using a MF induction of 25.0 mT for 16 h. These results were 16.1%, 39.0% and 19.6% higher than in the CE, respectively. Through statistical analysis it was found that the MF induction was a significant factor in GSH yield, and also it was observed that, within the range of the experimental conditions used, the lower MF induction, the higher the GSH yield.

RQ feedback control for simultaneous improvement of GSH yield and GSH content in Saccharomyces cerevisiae T65

In reduced glutathione (GSH) production by high-cell-density fed-batch cultivation of Saccharomyces cerevisiae T65, a respiratory quotient (RQ) feedback control strategy was applied to determine glucose feeding rate ( F) based on on-line off-gas monitoring in a 15-l stirred bioreactor. Glucose feed was manipulated by a classical proportional-integral-derivative (PID) controller to control RQ at its set-point. RQ controlled at 0.65 resulted in the highest GSH productivity (46.9 mg/l/h) and cell productivity (3.5 g/l/h) as well as improved GSH yield and intracellular GSH content. RQ feedback control achieved yeast dry weight (126 g/l), GSH yield (2.1 g/l), GSH content (1.67%) and GSH productivity (55.3 mg/l/h), which improved by 11.5%, 75%, 57.5% and 82.5%, respectively, compared with conventional ethanol feedback control in the GSH industry. Moreover, RQ feedback control reduced ethanol (byproduct) level to below 0.3 g/l. Advantages of RQ feedback control over the conventional ethanol feedback control in the GSH industry include lower byproduct concentration, shorter cultivation period, higher GSH content, and higher GSH yield. Hence, feedback control based on RQ is a promising method that poses as a simple and efficient alternative to conventional feed control techniques presently practiced in the GSH industry.

English

  The effects of pH values on cell growth and glutathione (GSH) production were studied in batch cultivation of Candida utilis. According to the fact that lower pH value favors cells growth but retards GSH production and higher pH value promotes GSH production while inhibits cells growth, a pH-shift strategy, optimized via simulating Gauss function, was developed. By applying two-stage pH-shift strategy of controlling pH at 5.0 for first 7.5 h and switching to 6.0 afterwards, final GSH yield and productivity reached 279 and 12.7 mg/l/h after 22 h cultivation, increased by 30 and 42%, respectively, compared to constant pH 5.5 operation. Moreover, by feeding glucose instead of sulphuric acid (H2SO4) solution to control pH, maximal GSH yield of 315 mg/l was achieved, suggesting application of pH-shift strategy for GSH overproduction as being feasible.   Key words: Batch fermentation, Candida utilis, glutathione (GSH), gauss function, pH-shift strategy.

Liquid–Liquid Extraction of GSH Using DEHAP/Octanol Reverse Micelles

In this study, di-(2-ethylhexyl) ammonium phosphate (DEHAP)/octanol reverse micellar extraction was investigated and demonstrated to be an effective method to separate GSH from yeast fermentation broth. The effect of several important factors i.e., pH, DEHAP concentration, and type & concentration of cations on both extraction and strip-extraction was investigated. The optimum operating conditions for both extraction and strip-extraction were obtained by two groups of orthogonal experiments. At the optimum operating conditions, the yield of GSH can reach 68%. In the lyophilized product, the purity of GSH was higher than 75%, all the proteins can be separated, and only a small portion of amino acids were left.

Salt-induced osmotic stress for glutathione overproduction in Candida utilis

The effect of NaCl-induced osmotic stress on glutathione (GSH) production was investigated in Candida utilis. Based on the fact that NaCl stress can enhance GSH production but inhibit cells growth simultaneously, the novel strategies of multiple osmotic stresses with different NaCl additions (0.2 mol/l at 4 h, 0.4 mol/l at 8 h, and 0.6 mol/l at 12 and 16 h) were developed for GSH overproduction. After 30 h cultivation, GSH yield reached 238 mg/l and intracellular GSH content was 2.34%, increased by 66.4% and 70.7% respectively compared to the control. Further applying the strategies to 7 l fermentor, GSH yield of 356 mg/l was achieved at 30 h, which was 65.6% higher than the control. Moreover, NaCl stress led to an increase in intracellular cysteine content and activities of γ-glutamylcysteine synthetase, GSH synthetase and GSH reductase, explaining the mechanism involved in inducing cellular GSH accumulation.

Glutathione extraction and mass transfer in di-(2-ethylhexyl) ammonium phosphate/octanol reverse micelles

Di-(2-ethylhexyl) ammonium phosphate (DEHAP)/octanol reverse micellar extraction was demonstrated to be an effective method to separate glutathione (GSH) from yeast fermentation broth. The effect of several important factors including pH, DEHAP concentration, and types and concentration of cations was investigated, and the optimum operating conditions were obtained by orthogonal experiments, for forward extraction: pH 2.5, DEHAP concentration = 0.16 mol/L, W 0 = 10, phase ratio (organic/aqueous, v/v) = 2:1, K + concentration = 0.1 mol/L, and extraction time = 15 min. For back extraction: pH 10.5, Ca 2+ concentration = 0.6 mol/L, phase ratio (aqueous/organic, v/v) = 3:1, back extraction time = 45 min. Under the optimum operating conditions, the yield of GSH can reach 65% after one forward and three back extractions. No protein and only a small portion of amino acids were detected in the lyophilized product of GSH. The study on the extraction kinetics showed that the volumetric mass transfer rates of the forward and back extraction are around 0.209 s −1 and 0.054 s −1, respectively, which is very close to the literature results of electrostatic force-based reverse micellar extraction of protein.