A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America. It is an herbaceous and shrubby plant belonging to the Asteraceae family. Stevia is considered as a natural sweetener and a commercially important plant worldwide. The leaves of S. rebaudiana contain steviol glycosides (SGs) which are highly potent and non-caloric sweeteners. The sweetening property of S. rebaudiana is contributed to the presence of these high potency, calorie free steviol glycosides. SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals. SGs amount in the plant mostly varies from 8% to 10%, and the enhancement of SGs is always in demand. These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories. SGs are almost 300 times sweeter than sucrose. Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical, food and beverages industries and in international market is increasing day by day. SGs have made stevia an important part of the medicinal world as well as the food and beverage industry, but the limited production of plant material is not fulfilling the higher global market demand. Therefore, researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S. rebaudiana. This review aims to describe the emerging biotechnological strategies and approaches to understand, stimulate and enhance biosynthesis of secondary metabolites in stevia. Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.     

Ultrasonically assisted extraction of total carbohydrates from Stevia rebaudiana Bertoni and identification of extracts

In the present study, in order to maximize the yield of total carbohydrates from Stevia rebaudiana Bertoni, Stevia LUYU-131 was used as test material, and response surface methodology (RSM) was employed to optimize the ultrasound-assisted extraction condition. The results indicated the optimal extraction conditions were an extraction temperature of 68 °C, a sonic power of 60 W and an extraction time of 32 min. Using the ultrasound-assisted extraction, the yield of extracts increased by a factor of 1.5 at the lower extraction temperature (68 °C) and the extraction time (32 min) substantially shortened compared with that of classical extraction. The components analysis of crude extracts revealed that the relative amount of rebaudioside A increased in the ultrasound-assisted extracts as compared with extracts obtained by classical process, and the ultrasound-assisted extracts had better quality.     

Subcritical water extraction of steviol glycosides from Stevia rebaudiana leaves and characterization of the raffinate phase

The objectives of this work were to obtain steviol glycosides of S. rebaudiana leaves, possessing natural and noncaloric sweetener properties, using subcritical water extraction; to assess optimum extraction conditions; to determine biological activities of Stevia extracts and to characterize the raffinate phase. A Box–“Bhenken” statistical design was used to evaluate the effects of various values of temperature (100–150 °C), time (30–60 min) and flow rate (2–6 ml/min) at a pressure of 230 bar applying a solid/liquid ratio of 1:10 (m:v). The most effective parameter was temperature (p < 0.005). Optimum extraction conditions were elicited as 125 °C, 45 min, 4 ml/min flow rate which yielded 38.67 mg/g stevioside and 35.68 mg/g rebaudioside A. The total phenolic, flavonoid contents and DPPH free radical scavenging activity were found as 48.63 mg gallic acid/g extract, 29.81 mg quercetin/g extract and 92.50%, respectively. After extraction, total chlorophyll, carotenoid contents and dietary fibers were quantified as 31.91 mg/100 g, 5.71 mg/100 g and 4.98% in the raffinate phase. Hence, both extract and raffinate phases of S. rebaudiana leaves can be utilized as sources of natural sweeteners, fibers and coloring agents in the industry.     

Pulsed ultrasound-assisted extraction of natural antioxidants from mandarin (Citrus deliciosa Tenore) leaves: Experimental and modeling study

Pulsed ultrasound-assisted extraction (PUAE) of flavonoids and polyphenols from mandarin (Citrus deliciosa Tenore) leaves was examined. The response surface methodology (RSM) via face-centered central composite design (FCCD) was used to investigate the effects of extraction time (15–75?min), output amplitude (30–70%), and pH (4–10) to optimize the extraction process. The total phenolic material (TPM) and the total flavonoid material (TFM) and also the consumed energy of horn were measured as responses. Additionally, calorimetric calculations were done to evaluate the ultrasound energy dissipated into the solution. The calculated quadratic models were highly significant (p?R²) of 0.9722, 0.9805, and 0.9983. The results of the present study suggest that 65?min, 68.72?min, 15.00 extraction time, 61%, 59%, and 30% of ultrasound amplitude and 4, 6.7, and 4 pH of solution should be considered as optimal extraction conditions to get the optimum TPM (37.845?mg-GAE/g-DL), TFM (10.709?mg-CE/g-DL), and energy consumption (6130.275 Joule) for PUAE of mandarin leaves, respectively.     

Parametric optimization and modeling of batch extraction process for extraction of betulinic acid from leaves of Vitex Negundo Linn

Extraction of betulinic acid from leaves of Vitex Negundo Linn was carried out in a series of solvents (methanol, ethanol, isopropyl alcohol, and water). Methanol gave maximum yield of extraction and therefore was used to investigate influence of particle size, speed of agitation, solid loading, and temperature, etc., on the rate and recovery in a stirred batch reactor. Effective intra-particle diffusivity of the acid in each solvent at different temperatures was estimated using an unsteady state mass diffusion model. The diffusivity of betulinic acid through the cellulose matrix of leaves is in the range 1.7 x 10^?13 to 9.23 x 10^?11 m²/sec and is strongly influenced by the temperature of the extraction. The Response Surface Methodology was used to estimate optimal conditions for betulinic acid extraction.     

Ultrasound-assisted extraction of natural antioxidants from betel leaves (Piper betle): Extraction kinetics and modeling

Present study investigated the extraction kinetics of antioxidants from Piper betle by ultrasound-assisted extraction for three extraction variables: temperature, solute to solvent ratio and ethanol concentration. Based on the results of yield, total phenolic content and antioxidant activity, the optimum results were obtained at 50°C, 1:20 g/mL and 80% ethanol, respectively. Statistical coefficients of R² ≥ 0.961 and RMSE ≤ 0.508 for two-site kinetic model confirm the use of proposed models for simulation and prediction purpose. Comparison with ascorbic acid and butylated hydroxytoluene solidifies the use of Piper betle as feasible source of natural antioxidants. The presence of hydroxychavicol and eugenol was affirmed by high-performance liquid chromatography assay.     

The effect of extraction techniques on yield,extraction kinetics,and antioxidant activity of aqueous-methanolic extracts from nettle (Urtica dioica L.) leaves

Five extraction techniques, maceration, reflux, Soxhlet, Tillepape, and ultrasonic extraction, were used to obtain the extractive matter from nettle leaves. The antioxidant activity of extracts was assessed by DPPH, FRAP, and H₂O₂ test, while the total phenolic and total flavonoid content was determined according to the Folin–Ciocalteu and aluminium chloride methods, respectively. Model Ponomarev and a non-stationary diffusion model through the plant material were used for modelling extraction process. The extract obtained by Soxhlet extraction, containing higher amounts of extractive matter as well as phenolic and flavonoid compounds, showed better antioxidant activity than those obtained by other extraction techniques.     

Supercritical CO2 and low-pressure solvent extraction of mango (Mangifera indica) leaves: Global yield,extraction kinetics,chemical composition and cost of manufacturing

Low-pressure solvent extraction (LPSE) and supercritical fluid extraction (SFE) were used to obtain extracts from mango (Mangifera indica) leaves. Kinetics curves were determined for both methodologies. The extracts chemical compositions and manufacturing costs were determined for both processes. Global yield isotherms for SFE process were determined at 10–40 MPa and 313–323 K. The highest yield was 2.24% at 30 MPa and 323 K; the LPSE yield (9.3%) was almost three times higher than that of SFE (3.6%). Thin layer chromatography showed that mango leaves extracts have several classes of compounds as alkaloids, flavonoids and terpenoids, recovered by both methods. The cost of manufacturing (COM) mango leaves extracts were US$ 32/kg and US$ 92/kg for LPSE and SFE, respectively.     

Kariya (Hildegardia barteri) seed oil extraction: comparative evaluation of solvents,modeling, and optimization techniques

In this work, the effects of solid/solvent ratio (0.10–0.25?g/ml), extraction time (3–8?h), and solvent type (n-hexane, ethyl acetate, and acetone) together with their shared interactions on Kariya seed oil (KSO) yield were investigated. The oil extraction process was modeled via response surface methodology (RSM), artificial neural network (ANN) and adaptive neuro-fuzzy inference system (ANFIS) while the optimization of the three input variables essential to the oil extraction process was carried out by genetic algorithm (GA) and RSM methods. The low mean relative percent deviation (MRPD) of 0.94–4.69% and high coefficient of determination (R²) > 0.98 for the models developed demonstrate that they describe the solvent extraction process with high accuracy in this order: ANFIS, ANN, and RSM. The best operating condition (solid/solvent ratio of 0.1?g/ml, extraction time of 8?h, and acetone as solvent of extraction) that gave the highest KSO yield (32.52?wt.%) was obtained using GA-ANFIS and GA-ANN. Solvent extraction efficiency evaluation showed that ethyl acetate, n-hexane, and acetone gave maximum experimental oil yields of 19.20?±?0.28, 25.11?±?0.01, and 32.33?±?0.04?wt.%, respectively. Properties of the KSO varied based on the type of solvent used. The results of this work showed that KSO could function as raw material in both food and chemical industries.     

响应面法优化纤维素酶辅助从泡叶藻中提取泡叶藻聚糖的工艺

通过单因素实验研究纤维素酶辅助提取泡叶藻聚糖时液料比、酶添加量、酶解温度、酶解时间等关键因素对泡叶藻聚糖提取率的影响,并进一步采用Box-Behnken实验设计和响应面分析法优化其工艺参数. 结果表明,纤维素酶辅助提取泡叶藻聚糖的优化工艺条件为液料比30 mL/g、酶浓度200 IU/mL、酶解时间2.0 h、酶解温度50℃,该条件下多糖提取率为14.65%?0.73%,与模型预测值14.75%非常接近,采用响应面法对泡叶藻聚糖提取条件进行优化合理可行.     

Optimization of electrochemically synthesized Cu3(BTC)2 by Taguchi method for CO2/N2 separation and data validation through artificial neural network modeling

Cu₃(BTC)₂, a common type of metal organic framework (MOF), was synthesized through electrochemical route for CO₂ capture and its separation from N₂. Taguchi method was employed for optimization of key parameters affecting the synthesis of Cu₃(BTC)₂. The results indicated that the optimum synthesis conditions with the highest CO₂ selectivity can be obtained using 1 g of ligand, applied voltage of 25 V, synthesis time of 2 h, and electrode length of 3 cm. The single gas sorption capacity of the synthetized microstructure Cu₃(BTC)₂ for CO₂ (at 298 K and 1 bar) was a considerable value of 4.40 mmol·g⁻¹. The isosteric heat of adsorption of both gases was calculated by inserting temperature-dependent form of Langmuir isotherm model in the Clausius-Clapeyron equation. The adsorption of CO₂/N₂ binary mixture with a concentration ratio of 15/85 vol-% was also studied experimentally and the result was in a good agreement with the predicted value of IAST method. Moreover, Cu₃(BTC)₂ showed no considerable loss in CO₂ adsorption after six sequential cycles. In addition, artificial neural networks (ANNs) were also applied to predict the separation behavior of CO₂/N₂ mixture by MOFs and the results revealed that ANNs could serve as an appropriate tool to predict the adsorptive selectivity of the binary gas mixture in the absence of experimental data.     

Microwave-assisted extraction as an advanced technique for optimization of saponin yield and antioxidant potential from Phyllanthus amarus

The objective of this study was to apply microwave-assisted extraction (MAE) as an advanced technique for optimization of saponin yield and antioxidant potential from Phyllanthus amarus. The findings indicated that the optimal MAE parameters consisted of 100% methanol, irradiation time 4 s/min, extraction time 50 min, and solvent to sample ratio 50 mL/g. Under these optimal parameters, saponin content (SC), saponin extraction efficiency (SEE), and total phenolic content (TPC) of P. amarus were 229.5 mg escin equivalents (EEs)/g dried sample, 82.8%, and 40.7 mg gallic acid equivalents (GAEs)/g dried sample, respectively. The antioxidant capacity of P. amarus in terms of 2,2ˊ-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging capacity (ARSC), 2,2-diphenyl-1-picryl-hydrazil radical scavenging capacity (DRSC), and ferric reducing antioxidant power (FRAP) were 487.3, 330.6, and 233.5 mg trolox equivalents (TEs)/g dried sample, respectively. These measured values were not significantly different from the predicted values by response surface methodology (227.9 mg EE/g dried sample, 82.1%, and 39.2 mg GAE/g dried sample for SC, SEE, and TPC and 484.8, 297.3, and 226.6 mg TE/g dried sample for ARSC, DRSC, and FRAP, respectively). Hence, the optimal MAE parameters are suggested for effective extraction of saponins from P. amarus for further investigations and applications.     

Optimization of ultrasound-assisted extraction for the maximum quantity and quality of phenolics from Stachys lavandulifolia

The response surface methodology was used to evaluate the effects of extraction time, power of ultrasound, liquid to solid ratio, and solvent composition on the quantity and quality (from aspect of antioxidant activity) phenolics of Stachys lavandulifolia. The best extraction time, power of ultrasound, liquid to solid ratio, and solvent composition for both the quality and quantity of phenolics were 14 min, 300 W, 40 (v/w), and 57% methanol, respectively. Only the liquid to solid ratio was effective on the quality of phenolics. Also, the comparison between the ultrasound-assisted extraction and maceration methods showed the suitability of ultrasound-assisted extraction for extracting phenolics from this plant.     

以高产量、高产率、高生产强度为目标的发酵过程优化技术

以高产量、高底物转化率和高生产强度为目标,综合运用微生物反应计量学、生化反应和传递动力学、生物反应器工程及代谢工程理论,开发了:(1)基于微生物反应计量学的培养环境优化技术;(2)基于微生物代谢特性的分阶段培养技术;(3)基于反应动力学模型的优化技术;(4)基于代谢通量分析的优化技术;(5)基于系统观点的生物反应系统优化技术。将这些技术广泛应用于多种产品的发酵过程优化研究中并取得了成功。在此基础上总结出“简化、定量化、模型化和阶段化”的发酵过程优化基本原理,这一基本原理对提高我国发酵工业技术水平、促进生物食品产业的健康发展将起重要作用。     

Supercritical fluid extraction from dried banana peel (Musa spp., genomic group AAB): Extraction yield, mathematical modeling, economical analysis and phase equilibria

Supercritical fluid extraction from dried banana peel (Musa spp., subgroup Prata, genomic group AAB, popularly known in Brazil as Enxerto) was studied. The aspects investigated were: overall extraction curve (OEC), mass transfer modeling of the yield curves, economical analysis of the process and phase equilibrium data for the pseudo-ternary system of banana peel extract, carbon dioxide and ethanol. The extraction operating conditions evaluated were: pressure ranging from 100 bar to 300 bar, temperature from 40 to 50 °C and constant solvent flow rate of 5.0 gCO₂/min. Experimental extraction data were correlated using three kinetic models based on mass transfer equations (logistic, diffusion and Esquível models). Phase equilibrium measurements were performed using pressure from 64.9 bar to 239.9 bar and mass fraction of supercritical extract from 0.52 to 3.55 wt%. Yield results ranged from 0.6 to 6.9% d.b. (dry basis). The lowest deviation between experimental and correlated data was obtained by the Logistic model, except for the curve at 300 bar and 40 °C which was best represented by the Esquível model. The economical analysis identified the possibility to apply the supercritical fluids to obtain extracts from banana peel in an industrial scale. Phase equilibrium for the supercritical extract from banana peel with carbon dioxide modified by ethanol exhibited liquid-liquid, vapor-liquid (bubble point) and vapor-liquid-liquid phase transitions. A crossover phenomenon for the systems evaluated was observed for pressures between 200 bar and 240 bar, for both groups of assays, i.e., supercritical extraction and phase equilibrium.     

Combination of ACO-artificial neural network method for modeling of manganese and cobalt extraction onto nanometer SiO2 from water samples

In this study, modeling based on ant-colony optimization – artificial neural network have been employed to develop the model for simulation and optimization of nanometer SiO₂ for the extraction of manganese and cobalt from water samples. The pH, time, amount of SiO₂ nanoparticles and concentration of 1-(2-pyridylazo)-2-naphthol (PAN) were the input variables, while the extraction% of analytes was the output. Under the optimum conditions, the detection limits were 0.52 and 0.7 μg L^?1, for manganese and cobalt, respectively. The method was applied to the extraction of manganese and cobalt from water samples and one certified reference material.     

Extracts from pitanga (Eugenia uniflora L.) leaves: Influence of extraction process on antioxidant properties and yield of phenolic compounds

Different extraction processes were employed to extract the polyphenolic compounds from pitanga (Eugenia uniflora L.) leaves: a one-step process using water, ethanol or supercritical CO₂ as solvents, and a two-step process using supercritical CO₂ followed by either water or ethanol. The total polyphenolic compounds, total flavonoids and antioxidant activity were determined in all the extracts obtained. The process performance was evaluated with respect to three variables: global extraction yield, concentration and yield of both polyphenols and flavonoids in the extracts. For the one-step extraction, the results showed that the extraction yield increased with solvent polarity. For the two-step process, the results suggested that water was more efficient in extracting the phenolic compounds from E. uniflora when the matrix was previously extracted with scCO₂. With respect to the antioxidant activity, the ethanolic extracts obtained from both processes, using either the DPPH radical scavenging method or the β-carotene bleaching method, presented high antioxidant activities.