Production of Siamenoside I and Mogroside IV from Siraitia grosvenorii Using Immobilized ß-Glucosidase.

Siraitia grosvenorii is a type of fruit used in traditional Chinese medicine. Previous studies have shown that the conversion of saponins was often carried out by chemical hydrolysis, which can be problematic because of the environmental hazards it may cause and the low yield it produces. Therefore, the purpose of this study is to establish a continuous bioreactor with immobilized enzymes to produce siamenoside I and mogroside IV. The results show that the immobilization process of ß-glucosidase exhibited the best relative activity with a glutaraldehyde (GA) concentration of 1.5%, carrier activation time of 1 h and binding enzyme time of 12 h. After the immobilization through GA linkage, the highest relative activity of ß-glucosidase was recorded through the reaction with the substrate at 60 °C and pH 5. Subsequently, the glass microspheres with immobilized ß-glucosidase were filled into the reactor to maintain the optimal active environment, and the aqueous solution of Siraitia grosvenorii extract was introduced by controlling the flow rate. The highest concentration of siamenoside I and mogroside IV were obtained at a flow rate of 0.3 and 0.2 mL/min, respectively. By developing this immobilized enzyme system, siamenoside I and mogroside IV can be prepared in large quantities for industrial applications.

Development and Optimization of Djulis Sourdough Bread Fermented by Lactic Acid Bacteria for Antioxidant Capacity.

This study developed a nutritionally valuable product with bioactive activity that improves the quality of bread. Djulis (Chenopodium formosanum), a native plant of Taiwan, was fermented using 23 different lactic acid bacteria strains. Lactobacillus casei BCRC10697 was identified as the ideal strain for fermentation, as it lowered the pH value of samples to 4.6 and demonstrated proteolysis ability 1.88 times higher than controls after 24 h of fermentation. Response surface methodology was adopted to optimize the djulis fermentation conditions for trolox equivalent antioxidant capacity (TEAC). The optimal conditions were a temperature of 33.5 °C, fructose content of 7.7%, and dough yield of 332.8, which yielded a TEAC at 6.82 mmol/kg. A 63% increase in TEAC and 20% increase in DPPH were observed when compared with unfermented djulis. Subsequently, the fermented djulis was used in different proportions as a substitute for wheat flour to make bread. The total phenolic and flavonoid compounds were 4.23 mg GAE/g and 3.46 mg QE/g, marking respective increases of 18% and 40% when the djulis was added. Texture analysis revealed that adding djulis increased the hardness and chewiness of sourdough breads. It also extended their shelf life by approximately 2 days. Thus, adding djulis to sourdough can enhance the functionality of breads and may provide a potential basis for developing djulis-based functional food.

Enzymatic degradation of ginkgolic acid by laccase immobilized on novel electrospun nanofiber mat.

BACKGROUND: Ginkgo biloba leaf extract contains many active ingredients that are beneficial for health. However, ginkgolic acid, one of the major components found in G. biloba extract, may cause serious allergic and toxic side effects. The purpose of this study is to immobilize the laccase system on the electrospun nylon fiber mat (NFM) to hydrolyze the ginkgolic acid in G. biloba leaf extract efficiently. RESULTS: Novel electrospinning technology successfully produced high-quality nanoscopic fiber mats made of a mixture of multi-walled carbon nanotube and nylon 6,6. Laccase that was immobilized onto the NFM exhibited much higher efficiency in the catalyzation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) than nylon 6,6 pellets. After being immobilized onto the NFM, the pH and temperature stability of laccase were significantly improved. The NFM-immobilized laccase could maintain more than 50% of its original activity even after 40 days of storage or 10 operational cycles. The kinetic parameters, including rate constant (K), the time (τ50) in which 50% of ginkgolic acid hydrolysis was reached, the time (τcomplete) required to achieve complete ginkgolic acid hydrolysis, Km and Vmax were determined, and were 0.07 ± 0.01 min-1 , 8.97 ± 0.55 min, 45.45 ± 2.79 min, 0.51 ± 0.09 mM and 0.49 ± 0.03 mM min-1 mg-1 , respectively. CONCLUSION: The result successfully demonstrated the strong potential of using novel electrospun nanofiber mats as enzyme immobilization platforms, which could significantly enhance enzyme activity and stability. © 2020 Society of Chemical Industry.

TEMPO-Oxidized Bacterial Cellulose Pellicle with Silver Nanoparticles for Wound Dressing.

Biocompatible bacterial cellulose pellicle (BCP) is a candidate for biomedical material such as wound dressing. However, due to lack of antibacterial activity, to grant BCP with the property is crucial for its biomedical application. In the present study, BCP was modified by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation using TEMPO/NaClO/NaBr system at pH 10 to form TEMPO-oxidized BCP (TOBCP) with anionic C6 carboxylate groups. The TOBCP was subsequently ion-exchanged in AgNO3 solution and silver nanoparticles (AgNP) with diameter of ∼16.5 nm were in situ synthesized on TOBC nanofiber surfaces by thermal reduction without using a reducing agent. Field emission scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectra, Fourier transform infrared spectroscopy, and thermogravimetric analysis were carried out to confirm morphology and structure of the pellicles with AgNP. The AgNP continuously released Ag+ with a rate of 12.2%/day at 37 °C in 3 days. The TOBCP/AgNP exhibited high biocompatibility according to the result of in vitro cytotoxicity test (cell viability >95% after 48 h of incubation) and showed significant antibacterial activities of 100% and 99.2% against E. coli and S. aureus, respectively. Hence, the highly biocompatible and highly antibacterial TOBCP/AgNP prepared in the present study is a promising candidate for wound dressing.

Improvement in Violacein Production by Utilizing Formic Acid to Induce Quorum Sensing in Chromobacterium violaceum.

Violacein has attracted increasing attention due to its various biological activities, such as antibacterial, antifungal, antioxidative, and antitumor effects. To improve violacein production, formic acid (FA) was added to a culture medium, which resulted in a 20% increase (1.02 g/L) compared to the no-FA-addition group (0.85 g/L). The use of a stirred-tank bioreactor system also improved violacein production (by 0.56 g/L). A quorum-sensing (QS)-related gene (cviI) was induced by FA treatment, which revealed that the mechanism induced by FA utilized regulation of the cviI gene to induce the vio gene cluster for violacein production. To analyze the antioxidative properties of the violacein produced, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) scavenging tests were conducted, and results reveal that the values of the 50% inhibitory concentration (IC50) of DPPH and ABTS were 0.286 and 0.182 g/L, respectively. Violacein also showed strong inhibitory activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In summary, this study found that the addition of formic acid can promote QS of Chromobacterium violaceum, thereby promoting the synthesis of violacein. Subsequently, the promoting effect was also evaluated in a bioreactor system. These findings will be helpful in establishing an economically beneficial production model for violacein in future work.

Extracts of Antrodia cinnamomea mycelium as a highly potent tyrosinase inhibitor.

BACKGROUND: Ganoderma has been known as a cure for diseases since ancient times, and been used as a medicinal mushroom for more than 2000 years. By many accounts, Ganoderma lucidum extracts from fruit bodies exhibited the comparable tyrosinase inhibition activity. AIMS: To validate A. cinnamomea mycelia anti-melanogenesis activity. Ethanolic extracts of A. cinnamomea mycelia were evaluated using in vitro cell-free tyrosinase assay, cell-based and zebrafish phenotype-based method. Meanwhile, safety assessment was also conducted to ensure the feasibility as the novel ingredients in cosmetic and pharmaceutic industries. METHODS: The major regulatory enzymes being in charge of cutaneous pigmentation, was investigated in both cell-free and cellular enzyme systems, and in phenotype-based zebrafish model. A high-throughput TLC in vitro screening system was introduced to perform the initial evaluation of those with anti-melanin formation activity. RESULTS: Among the fractions, 50% ethanol extracted fraction (AC_Et50_Hex) exhibited highest anti-melanin formation activity. AC_Et50_Hex (at 100 ppm) reduced 30% intracellular melanin of B16-F10 cells through suppression of tyrosinase activity and its protein expression. For animal study, not only does AC_Et50_Hex exhibited similar depigmenting efficacy to kojic acid (56.1% vs 52.3%) with lower dosage (50 ppm vs 1400 ppm), but showed less toxicity to zebrafish. CONCLUSION: A. cinnamomea mycelium extracts can be an ideal candidate/substitute for skin-whitening since kojic acid has been reported with carcinogenic effect. AC_Et50_Hex was recognized as a potential tyrosinase inhibitor throughout in vitro and in vivo analysis studies. The mass production of A. cinnamomea mycelium from agitated fermentation realizes the natural mushroom extracts for commercial application.

Enzymatic degradation of ginkgolic acids by laccase immobilized on core/shell Fe3O4/nylon composite nanoparticles using novel coaxial electrospraying process.

Enzyme immobilization can increase enzyme reusability to reduce cost of industrial production. Ginkgo biloba leaf extract is commonly used for medical purposes, but it contains ginkgolic acid, which has negative effects on human health. Here, we report a novel approach to solve the problem by degrading the ginkgolic acid with immobilized-laccase, where core/shell composite nanoparticles prepared by coaxial electrospraying might be first applied to enzyme immobilization. The core/shell Fe3O4/nylon 6,6 composite nanoparticles (FNCNs) were prepared using one-step coaxial electrospraying and can be simply recovered by magnetic force. The glutaraldehyde-treated FNCNs (FNGCNs) were used to immobilize laccase. As a result, thermal stability of the free laccase was significantly improved in the range of 60-90 °C after immobilization. The laccase-immobilized FNGCNs (L-FNGCNs) were applied to degrade the ginkgolic acids, and the rate constants (k) and times (τ50) were ~0.02 min-1 and lower than 39 min, respectively, showing good catalytic performance. Furthermore, the L-FNGCNs exhibited a relative activity higher than 0.5 after being stored for 21 days or reused for 5 cycles, showing good storage stability and reusability. Therefore, the FNGCNs carrier was a promising enzyme immobilization system and its further development and applications were of interest.

Enhanced antioxidant activity of Chenopodium formosanum Koidz. by lactic acid bacteria: Optimization of fermentation conditions.

In this study, different probiotics commonly used to produce fermented dairy products were inoculated independently for Chenopodium formosanum Koidz. fermentation. The strain with the highest level of antioxidant activity was selected and the fermentation process was further optimized via response surface methodology (RSM). Lactobacillus plantarum BCRC 11697 was chosen because, compared to other lactic acid bacteria, it exhibits increased free radical scavenging ability and can produce more phenolic compounds, DPPH (from 72.6% to 93.2%), and ABTS (from 64.2% to 76.9%). Using RSM, we further optimize the fermentation protocol of BCRC 11697 by adjusting the initial fermentation pH, agitation speed, and temperature to reach the highest level of antioxidant activity (73.5% of DPPH and 93.8% of ABTS). The optimal protocol (pH 5.55, 104 rpm, and 24.4°C) resulted in a significant increase in the amount of phenolic compounds as well as the DPPH and ABTS free radical scavenging ability of BCRC 11697 products. The IC50 of the DPPH and ABTS free radical scavenging ability were 0.33 and 2.35 mg/mL, respectively, and both protease and tannase activity increased after RSM. An increase in lower molecular weight (<24 kDa) protein hydrolysates was also observed. Results indicated that djulis fermented by L. plantarum can be a powerful source of natural antioxidants for preventing free radical-initiated diseases.