Artificial neural network modeling and optimization of ultrahigh pressure extraction of green tea polyphenols

In this study, the ultrahigh pressure extraction of green tea polyphenols was modeled and optimized by a three-layer artificial neural network. A feed-forward neural network trained with an error back-propagation algorithm was used to evaluate the effects of pressure, liquid/solid ratio and ethanol concentration on the total phenolic content of green tea extracts. The neural network coupled with genetic algorithms was also used to optimize the conditions needed to obtain the highest yield of tea polyphenols. The obtained optimal architecture of artificial neural network model involved a feed-forward neural network with three input neurons, one hidden layer with eight neurons and one output layer including single neuron. The trained network gave the minimum value in the MSE of 0.03 and the maximum value in the R² of 0.9571, which implied a good agreement between the predicted value and the actual value, and confirmed a good generalization of the network. Based on the combination of neural network and genetic algorithms, the optimum extraction conditions for the highest yield of green tea polyphenols were determined as follows: 498.8 MPa for pressure, 20.8 mL/g for liquid/solid ratio and 53.6% for ethanol concentration. The total phenolic content of the actual measurement under the optimum predicated extraction conditions was 582.4 ± 0.63 mg/g DW, which was well matched with the predicted value (597.2 mg/g DW). This suggests that the artificial neural network model described in this work is an efficient quantitative tool to predict the extraction efficiency of green tea polyphenols.     

Optimisation of supercritical fluid extraction of flavonoids from Pueraria lobata

Supercritical carbon dioxide extraction was employed to extract flavonoids from Pueraria lobata. The optimal conditions for flavonoid extraction were determined by response surface methodology. Box–Behnken design was applied to evaluate the effects of three independent variables (pressure, temperature and co-solvent amount) on the flavonoid yield of P. lobata. Correlation analysis of the mathematical-regression model indicated that a quadratic polynomial model could be employed to optimise the supercritical carbon dioxide extraction of flavonoids. From response surface plots, pressure, temperature and co-solvent amount exhibited independent and interactive effects on the extraction of flavonoids. The optimal conditions to obtain the highest flavonoid yield of P. lobata were a pressure of 20.04 MPa, a temperature of 50.24 °C and a co-solvent amount of 181.24 ml. Under these optimal conditions, the experimental values agreed with the predicted values, using analysis of variance, indicating a high goodness of fit of the model used and the success of response surface methodology for optimising supercritical carbon dioxide extraction of flavonoids from P. lobata.     

Optimization of tyrosinase inhibition activity of ultrasonic-extracted polysaccharides from longan fruit pericarp

Various ultrasonic conditions were employed to prepare polysaccharides from longan fruit pericarp (PLFP) and the Lineweaver–Burk equation was then used to determine the effect of PLFP on inhibition of tyrosinase activity. This result showed that PLFP acted as a non-competitive inhibitor of tyrosinase. The highest slope was observed for ultrasonic extraction, followed by the hot-water extraction, suggesting that the ultrasonic treatment of PLFP increased the inhibition of tyrosinase activity. Furthermore, a multilayer feed-forward neural network trained with an error back-propagation algorithm was used to evaluate the effects of ultrasonic power, time and temperature on the slope value. The trained network gave a regression coefficient (R²) of 0.98 and a mean squared error (MSE) of 0.58, implying a good agreement between the predicted value and the actual value of the slope, and confirmed a good generalization of the network. Based on the artificial neural network-genetic algorithm, the optimal ultrasonic extraction conditions to obtain the highest slope value (154.1) were determined to be 120 W, 12 min and 57 °C. Application of response surface plots showed the slope value as a function of every two factors under various ultrasonic extraction conditions, which can be observed directly. Therefore, the artificial neural network provided a model with high performance and indicated the non-linear nature of the relation between ultrasonic conditions and slope value.     

Application of response surface methodology to optimise extraction of flavonoids from fructus sophorae

Response surface methodology (RSM) based on a central composite design (CCD) was applied to optimise the extraction conditions for flavonoids from fructus sophorae with advantages in terms of resisting flavonoids during the whole process and maximising of extraction yield. Three aglycon forms of the flavonoids, namely, quercetin, kaempferol and isorhamnetin were quantified by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) to estimate extraction yield. The combined effects of independent variables were studied and the optimal extraction conditions were obtained as ethanol concentration, 74.47%; solid–liquid ratio, 17.99 ml/g; temperature, 89.13 °C; and extraction time, 2.10 h. The reliability of the method was confirmed by recovery experiments, performed under optimal conditions. Recoveries indicated that flavonoids resisted the extraction conditions. The experimental extraction yield under optimal conditions was found to be 10.459%, which was well matched with the predicted values of 10.461%.     

Extraction and characterisation of telopeptide-poor collagen from porcine lung

The aim of the investigation was to develop optimum extraction conditions of telopeptide-poor collagen from porcine lung (TPCPL). Chemical composition, histological properties of porcine lung, removal efficiency of haemoglobin by different solvents and effects of pepsin solubilisation time on TPCPL properties were evaluated. Histological observation of lung tissue revealed collagen coexists with elastin. Treatment with 0.1 M Tris–HCl/5% Triton X-100 (pH 8.2) for 12 h exhibited the highest efficiency for removal of haemoglobin. The maximal yield of TPCPL reached 6.702 g/kg by pepsin solubilisation for 72 h. SDS–PAGE indicated that type I collagen was a major component of TPCPL; FTIR spectra of all TPCPL were similar to commercial porcine collagen and absent from denatured product (gelatin) and TPCPL 72 exhibited the lowest denaturation temperature (36.4 °C) and maximum enzymatic sensitivity. Finally, the optimum pepsin solubilisation of TPCPL was controlled at 4 °C for 48 h for better structural integrity and acceptable yield.     

Response surface optimisation of aqueous enzymatic oil extraction from bayberry (Myrica rubra) kernels

Aqueous enzymatic extraction (AEE) of oil from Myrica rubra kernels was performed. The four AEE parameters namely mixed enzyme (cellulose/neutral protease = 1/2, w/w) amount, liquid/solid ratio, extraction time, and temperature, were optimised by response surface methodology. The statistical analysis indicated that the enzyme amount, liquid/solid ratio, time, and the quadratics of liquid/solid ratio, and enzyme amount, as well as the interactions between time and temperature, showed significant effects on oil yield. The optimal extraction conditions for oil yield were mixed enzyme amount, liquid/solid ratio, time, temperature as 3.17% (w/w), 4.91 ml/g, 4 h and 51.6 °C. Under the optimum conditions, the experimental oil extraction yield was 31.15%. The GC–MS analysis showed that the oil was abundant in the unsaturated fatty acids (9-octadecenoic acid and 9,12-octadecadienoic acid accounting for more than 80%), and the AEE was more efficient method to extract polyunsaturated fatty acid than the organic solvent process.     

Isolation and characterisation of collagens from the skin of largefin longbarbel catfish (Mystus macropterus)

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of largefin longbarbel catfish (Mystus macropterus) with yields of 16.8% and 28.0%, respectively, on the basis of dry weight. Both ASC and PSC contained α1 and α2 chains and the amino acid composition of collagen was close to that of calf skin type ? collagen. The intrinsic viscosities of ASC and PSC were 14.9 dl/g and 14.5 dl/g, respectively. Similar ultraviolet and FTIR spectra of ASC and PSC were observed. However, peptide maps of ASC and PSC, hydrolysed by trypsin, revealed some differences in primary structures between the two fractions. Denaturation temperatures of ASC and PSC were 32.1 °C and 31.6 °C, respectively. The higher T_m showed that it is possible to use largefin longbarbel catfish skin collagen as an alternative source of vertebrate collagens for industrial purposes.     

Isolation and characterisation of acid-solubilised collagen from the skin of Nile tilapia (Oreochromis niloticus)

Acid-solubilised collagen (ASC) was extracted from the skin of Nile tilapia (Oreochromis niloticus) and characterisation was studied. The results indicated that the yield of ASC was 39.4% on the basis of dry weight. This ASC was rich in glycine (35.6%). The amount of imino acids, proline and hydroxyproline, in ASC was 210 residues per 1000 residues. The ultraviolet (UV) absorption spectrum of ASC showed that the distinct absorption was at 220 nm. ASC showed transition curve at maximum temperature (T_max) of 32.0 °C in 0.05 M acetic acid, about 12 °C lower than that of calf skin collagen. Maximum solubility (0.75 mg/ml) in 0.5 M acetic acid was observed at pH 3. Solubility reached the minimum at pH 7. A sharp decrease in solubility was observed in 2% (w/v) NaCl or above. Biochemical studies indicated that ASC was composed of the α1α2α3 heterotrimers.     

Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus)

Extraction and some properties of pepsin-solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus) were investigated. Addition of bigeye snapper pepsin (BSP) at a level of 20 kUnits/g of defatted skin resulted in an increased content of collagen extracted from bigeye snapper skin. The yields of collagen from bigeye snapper skin extracted for 48 h with acid and with BSP were 5.31% and 18.74% (dry basis), respectively. With pre-swelling in acid for 24 h, collagen extracted with BSP at a level of 20 kUnits/g of defatted skin for 48 h had a yield of 19.79%, which was greater than that of collagen extracted using porcine pepsin at the same level (13.03%). The skin collagen was characterised to be type I with no disulfide bond. Electrophoretic study revealed slight differences in molecular weight between acid-solubilised collagen and all pepsin-solubilised collagens. The molecular weights of α1 and α2 chains in acid-solubilised collagen were estimated to be 120 and 112 kDa, respectively, whereas α1 and α2 chains of pepsin-solubilised collagens had molecular weights of 118 and 111 kDa, respectively. The result suggested that these pepsin-solubilised collagens might undergo partial cleavage in the telopeptide region by pepsin treatment. The maximum transition temperature (T_max) of acid-solubilised collagen was observed at 32.5 °C, which was slightly higher than that of pepsin-solubilised collagens (by about 1 °C). Generally, all collagens were highly solubilised in the pH range of 2–5 and sharply decreased at the neutral pH. No changes in solubility were observed in the presence of NaCl up to 3% (w/v) and the decrease was more pronounced with increasing NaCl concentration.     

Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were extracted from scale of spotted golden goatfish (Parupeneus heptacanthus) with the yields of 0.46% and 1.20% (based on dry weight basis), respectively. Both ASC and PSC were characterised as type I collagen, containing α₁ and α₂ chains. β and γ components were also found in both collagens. Based on FTIR spectra, the limited digestion by pepsin did not disrupt the triple helical structure of collagen. ASC and PSC contained glycine (336–340 residues/1000 residues) as the major amino acid and had imino acids of 186–189 residues/1000 residues. Maximal transition temperatures (T_max) were 41.58 and 41.01 °C for ASC and PSC, respectively. From zeta potential analysis, net charge of zero was found at pH 4.96 and 5.39 for ASC and PSC, respectively. Both collagens exhibited high solubility in acidic pH (2–4) and were soluble in the presence of NaCl at concentration up to 20 and 30 g/l for ASC and PSC, respectively.     

Optimization of carotenoids extraction from Rhodobacter sphaeroides

The comparison of the three methods ultrasonic assisting, grinding and HCl assisting on carotenoids extraction yield from Rhodobacter sphaeroides (R. sphaeroides) was carried out. The HCl-assisted extraction was found to be the most effective. And then, based on HCl-assisted extraction method, the effect of temperature, solvent-solid ratio and duration on the yield of carotenoids extracted from R. sphaeroides was investigated using single factor and Box-Behnken experimental design. The optimal conditions for HCl-assisted extraction of carotenoids from R. sphaeroides were found to be: temperature 30 °C, solvent-solid ratio 40 ml/g, duration 40 min. The first two extractions were enough to approximately exhaust the entire content of carotenoids in R. sphaeroides.     

Optimisation of microwave-assisted enzymatic extraction of corilagin and geraniin from Geranium sibiricum Linne and evaluation of antioxidant activity

Plant polyphenols exhibit effective bio-activities, particularly antioxidant activities. Previous studies showed that Geranium sibiricum Linne contains high levels of polyphenolic compounds such as corilagin (CG) and geraniin (GE). In this study, a microwave-assisted enzymatic extraction (MAEE) method was evaluated for the simultaneous extraction of CG and GE only with deionised water. The optimal extraction conditions were as follows: irradiation power 500 W, ratio of solvent to material 40 ml/g, irradiation temperature 33 °C, pH 5.2, amount of cellulase 3600 U/g and irradiation time 9 min. Under these conditions, the extraction yields of CG and GE achieved were 6.79 and 19.82 mg/g, which increased by 64.01% and 72.95%, respectively, as compared with the control ones. Furthermore, the antioxidant activities of crude extracts were 2.61 mmol FeSO₄/g DW and 0.118 mg/ml (IC₅₀), according to the FRAP and DPPH assays, which indicated G. sibiricum Linne possesses good potential for natural antioxidants.     

Application of response surface methodology to optimise supercritical carbon dioxide extraction of essential oil from Cyperus rotundus Linn.

Supercritical fluid extraction with carbon dioxide (SC-CO₂ extraction) was performed to isolate essential oils from the rhizomes of Cyperus rotundus Linn. Effects of temperature, pressure, extraction time, and CO₂ flow rate on the yield of essential oils were investigated by response surface methodology (RSM). The oil yield was represented by a second-order polynomial model using central composite rotatable design (CCRD). The oil yield increased significantly with pressure (p < 0.0001) and CO₂ flow rate (p < 0.01). The maximum oil yield from the response surface equation was predicted to be 1.82% using an extraction temperature of 37.6 °C, pressure of 294.4 bar, extraction time of 119.8 min, and CO₂ flow rate of 20.9 L/h.     

Optimisation of novel method for the extraction of steviosides from Stevia rebaudiana leaves

Stevioside, a diterpene glycoside, is well known for its intense sweetness and is used as a non-caloric sweetener. Its potential widespread use requires an easy and effective extraction method. Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase, pectinase and hemicellulase, using various parameters, such as concentration of enzyme, incubation time and temperature, was optimised. Hemicellulase was observed to give the highest stevioside yield (369.23 ± 0.11 μg) in 1 h in comparison to cellulase (359 ± 0.30 μg) and pectinases (333 ± 0.55 μg). Extraction from leaves under optimised conditions showed a remarkable increase in the yield (35 times) compared with a control experiment. The extraction conditions were further optimised using response surface methodology (RSM). A central composite design (CCD) was used for experimental design and analysis of the results to obtain optimal extraction conditions. Based on RSM analysis, temperature of 51-54 °C, time of 36-45 min and the cocktail of pectinase, cellulase and hemicellulase, set at 2% each, gave the best results. Under the optimised conditions, the experimental values were in close agreement with the prediction model and resulted in a three times yield enhancement of stevioside. The isolated stevioside was characterised through ¹H-NMR spectroscopy, by comparison with a stevioside standard.     

Production of theanine by Xerocomus badius (mushroom) using submerged fermentation

Theanine is a rare amino acid mainly produced by the genus Camellia, which is also found in the Basidiomycete fungus (mushroom) Xerocomus badius. Besides its favorable taste, broad potential physiological effects have been reported in the recent years, leading to the very fast growing demand for theanine worldwide. Thus a series of methods to produce theanine have been developed, including tea callus cultivation, chemical synthesis, extraction from the fresh leaves of the tea plant, and enzymatic synthesis. Different from the previous procedures, we reported here a novel protocol to produce theanine from the mushroom X. badius by means of submerged fermentation. Moreover, response surface methodology (RSM) was employed to optimize various parameters during fermentation. The statistic results indicated that the maximum theanine yield of X. badius (17.2420 mg/L) could be achieved at the optimal conditions, with glucose of 29.17 g/L, yeast extract of 6.38 g/L in a broth volume of 61.43 mL (within a flask of 250 mL) at an initial pH at 5.63. These predicted values were also validated by experiments. Taken together, these findings provided us a promising strategy for the production of theanine from X. badius in addition to the traditional preparation processes.     

Isolation of hesperidin from peels of thinned Citrus unshiu fruits by microwave-assisted extraction

Simultaneous extraction by microwave-irradiation and crystallisation were performed in the same pot of solvent of 70% (v/v) aqueous ethanol for isolation of hesperidin from thinned immature fruit peels of Citrus unshiu as refining of Citrus waste biomass. The hesperidin content in immature fruits peels was about 3.2-fold higher than that of mature fruit. After microwave-assisted extraction (MAE), the yield of hesperidin reached 58.6 mg/g, which was comparable to the amount obtained after extraction using DMSO:methanol (1:1, v/v) as a solvent for 30 min at room temperature. Heating temperature and time for isolation of hesperidin crystallites were optimised as 140 °C and 8 min by using response surface methodology. Under this optimal condition, 86.8% (47.7 mg/g) of total hesperidin was isolable by MAE and low-temperature storage (5 °C, 24 h).     

Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous)

Pepsin-solubilised collagen (PSC) was extracted from the skin of unicorn leatherjacket (Aluterus monocerous), using 0.5 M acetic acid in the presence of pepsin from albacore tuna, yellowfin tuna or porcine pepsin at a level of 20 units/g of defatted skin. Yields of 8.48 ± 0.3%, 8.40 ± 0.3% and 7.56 ± 0.4% (wet weight basis) were obtained for PSC extracted with the aid of albacore tuna pepsin (APSC), yellowfin tuna pepsin (YPSC) and porcine pepsin (PPSC), respectively. All PSCs were classified as Type I collagen containing two α₁-chains and one α₂-chain with no disulphide bond. The peptide maps of different PSCs hydrolysed by V8 protease and lysyl endopeptidase were different. ATR-FTIR spectra analysis revealed that PSC molecules had the compact triple helical structure stabilised mainly by the hydrogen bond. T_max of all PSCs (31.68–31.98 °C) shifted to lower values (29.33–29.40 °C) when dispersed in 0.05 M acetic acid, indicating the conformational changes in the collagen structure induced by acid. Relative viscosity of 0.03% PSC in 0.1 M acetic acid solution decreased progressively as the temperature increased from 4 to 52 °C, indicating thermal destabilisation or denaturation of PSC molecules. All PSCs were soluble in the pH range of 1–6 and sharply decreased at neutral pH. Decreases in solubility were observed in the presence of NaCl, especially at concentrations above 2% (w/v). Therefore, the skin of unicorn leatherjacket could serve as a potential source of collagen.     

Enhanced extraction of flavanones hesperidin and narirutin from Citrus unshiu peel using subcritical water

Flavanones including hesperidin and narirutin constitute the majority of the flavonoids that occur naturally in citrus fruits. The main purpose of this study was to extract valuable natural flavanones from agricultural by-products such as citrus peels using subcritical water extraction (SWE). Thus, the application of SWE to extraction of flavanones hesperidin and narirutin from Citrus unshiu peel was evaluated, and the effect of key operating conditions was determined by varying the extraction temperature (110–200 °C) and time (5–20 min) under high pressure (100 ± 10 atm). The maximum yields of hesperidin (72 ± 5 mg/g C. unshiu peel) and narirutin (11.7 ± 0.8 mg/g C. unshiu peel) were obtained at an extraction temperature of 160 °C for an extraction time of only 10 min. These yields accounted for approximately 99% of the total amount of these flavanones in the original material. The SWE was compared with three conventional extraction methods in terms of the extraction time and recovery yields for hesperidin and narirutin. The hesperidin yield by SWE was more than 1.9-, 3.2-, and 34.2-fold higher than those obtained by extraction methods using ethanol, methanol, or hot water, respectively, and the narirutin yield was more than 1.2-, 1.5-, and 3.7-fold higher.     

Anti-glycated activity of polysaccharides of longan (Dimocarpus longan Lour.) fruit pericarp treated by ultrasonic wave

Ultrasonic wave was used to extract the polysaccharides of longan fruit pericarp (PLFP) in this work. The anti-glycated activity of PLFP was evaluated. Through artificial neural network toolbox of MATLAB software, a mathematical model between ultrasonic conditions and anti-glycated activity was constructed. The R² and MSE (mean square error) values of the model were calculated to be 0.98 and 0.13, respectively, which suggested a good fitness of the neural network. Response surface plots showed that ultrasonic power, time and temperature had complicated and significant effects on the anti-glycated activity of PLFP. The optimal ultrasonic conditions for obtaining the highest anti-glycated activity were predicted to be 276 W, 24 min and 69 °C. The predicted anti-glycated activity was 60.4%. The experimental determinations under these conditions were not significantly different (P > 0.05) to the predicted value. It indicated the good prediction and optimisation capability of the artificial neural network.     

Collagen characteristics of farmed Atlantic salmon with firm and soft fillet texture

The possible role of collagen in texture variations among Atlantic salmon (Salmo salar L.) grown under commercial conditions at a Norwegian farm was studied. The texture was determined instrumentally as the breaking strength, and collagen and its salt (SSC), acid (ASC), pepsin (PSC) and insoluble (IF) fractions were analysed in order to determine the collagen aggregation degree. The collagen solubility and its overall amino acid (AA) composition showed no correlation to the breaking strength, but a positive correlation was observed between the breaking strength and glycine (r = 0.74) and alanine (r = 0.87) contents of the PSC fraction. Salmon with high breaking strength had higher T_peak (temperature of transition) and ΔH (enthalpy of transition), and the collagen seemed to have triple helix structures mainly stabilized by covalent associations as compared to salmon with low breaking strength. The glycosylation degree was also positively correlated to the breaking strength (r = 0.88, p ? 0.05). It is concluded that firmness of salmon muscle was not related to the total amount of collagen in the muscle, but rather higher collagen stability.